New Medicines for Trypanosomatidic Infections
- at least 1-2 innovative, less toxic and safer drug candidates for Trypanosomatid infections compared to existing ones,
- early phase biomarkers for efficacy prediction (overall improved efficacy and safety)
UNIVERSITA DEGLI STUDI DI MODENA E REGGIO EMILIA
Via Universita 4
Higher or Secondary Education Establishments
€ 889 824,31
Maria Paola Costi (Prof.)
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European ScreeningPort GmbH
€ 87 281,18
BERNHARD-NOCHT-INSTITUT FUER TROPENMEDIZIN
€ 502 965
INSTITUTO DE BIOLOGIA MOLECULAR E CELULAR-IBMC
€ 469 318,80
UNIVERSITA DEGLI STUDI DI SIENA
€ 340 740
€ 581 808,80
TYDOCK PHARMA S.r.l
€ 388 570
NATIONAL CENTER FOR RESEARCH
€ 158 860
Hypha Discovery Limited
€ 392 793,85
ETHNIKO IDRYMA EREVNON
€ 447 492,19
UNIVERSIDAD COMPLUTENSE DE MADRID
€ 445 484,80
€ 254 339,02
CENTRO NACIONAL DE PESQUISA EM ENERGIA E MATERIAS ASSOCIACAO PRIVADA
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FRAUNHOFER GESELLSCHAFT ZUR FOERDERUNG DER ANGEWANDTEN FORSCHUNG E.V.
€ 615 968
Grant agreement ID: 603240
1 February 2014
31 January 2017
€ 7 639 176,11
€ 5 901 411,15
UNIVERSITA DEGLI STUDI DI MODENA E REGGIO EMILIA
Towards new treatment options for trypanosomatidic infections
In countries where three diseases are endemic, patients have very few reasons to take comfort. The current chemotherapies for trypanosomatidic infections are highly toxic and often lead to relapses. Drugs used to combat Leishmaniasis have many shortcomings and are also used in animals, raising concerns over potential resistances. Chagas drugs were introduced over 40 years ago with no improvement since. The NMTRYPI (New Medicines for Trypanosomatidic Infections) project developed new prototypes for the three infections. It aimed to find less toxic and more effective compounds suitable for oral use. The project investigated the potential of a drug candidate to cure more than one trypanosomatidic infection, and it tested various compound combinations against new drug targets. ‘We developed four main compound series and tested more than 11 000 extracts from natural sources,’ says project coordinator Prof. Maria Paola Costi, of the University of Modena and Reggio Emilia. ‘Using a range of systematic strategies, we selected suitable leads for advanced animal testing and developed treatment schedules specifically for use against trypanosomatidic parasites.’ Tackling black fever Perhaps one of the most impressive project outcomes was the discovery of a drug candidate for visceral leishmaniasis, also known as black fever. The latter was validated in three animal species — mice, hamsters and dogs. Results on dogs were so promising that the animals were kept alive, adopted and are still being monitored: Now, almost 9 months after treatment, all animals are still healthy and have not shown any sign of relapse. In fact, the results were so encouraging that the team decided to proceed with preliminary exploration in monkeys. ‘We had the opportunity to test the compound in a monkey trial through a synergic collaboration with KINDRED, another FP7-funded project,’ Prof. Costi explains. Conducting tests on monkeys before phase I clinical trials has several advantages: their anatomy and physiology is close to that of humans, Leishmania infection is associated with a similar profile of parasite dissemination, and their use enables deep tissues analysis. Moreover, monkeys present a clearance mechanism for drugs similar to that found in humans. ‘Because immune-suppression is associated with the re-emergence of visceral leishmaniasis, understanding the immune response related to drug therapy is of crucial interest. Evaluating surrogate markers of immunity in experimental models allowed us to consider the factors that confer protection. We can use a new drug with immune modulatory properties or formulated with the right components to help control the disease,’ says Prof Costi. In its present form, the new drug candidate has a balance of efficacy and toxicity superior to that of the currently available oral treatment, Miltefosine. It proved to be effective against phase II HAT as well. ‘It is the first time that a compound for oral treatment can pass the brain barrier and has the potential to be used in phase II of Sleeping Sickness. This potentially opens the door to the first-ever oral combination therapy in the anti-Trypanosoma brucei field. Moreover, we have a genuine opportunity to use the same compound for more than one parasitic disease,’ says Prof Costi. In addition to the new drug candidate, the project helped identify some 20 compounds acting against T.brucei and different Leishmania species in vitro, all with suitable properties for animal testing. Administration of a combination of compounds against new targets was proposed as a proof of concept. Prof Costi is hopeful that patients will soon benefit from the project’s outcomes. ‘Development of a new drug is a long, risky and costly process, but, if everything goes well, Phase I trials could start in a three-years time approximately’. In the meantime, the consortium will keep applying for follow-up grants, either under the EU’s research framework programmes or with other funding agencies. ‘Interested stakeholders such as pharma companies are at the top of our list,’ Prof Costi concludes.
Grant agreement ID: 603240
1 February 2014
31 January 2017
€ 7 639 176,11
€ 5 901 411,15
UNIVERSITA DEGLI STUDI DI MODENA E REGGIO EMILIA
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Final Report Summary - NMTRYPI (New Medicines for Trypanosomatidic Infections)
The NMTrypI concept is based on the development of innovative anti-parasitic drug leads by using a common drug discovery platform. The multidisciplinary platform has been established by experts in their respective fields from SMEs and the public research sector in Europe and in disease-endemic countries. The approaches taken encompassed ligand-based lead optimization, structure and fragment-based drug design, phenotypic screening, including dual-target inhibition and the combined application of an investigational drug with a known drug. The innovative concept is strengthened by the identification of preclinical biomarkers by proteomic profiling of compounds to study their mechanisms of action (MoA biomarker) and efficacy (pharmacodynamic biomarkers).
The NMTrypI platform has performed on-target HTS (high throughput screening) of 6 compound libraries (4000 compounds) and lead development for 2 compounds. Compounds have been tested in mice, hamsters, and dogs as a reservoir of the visceral leishmaniasis disease, and evaluated in toxicology and safety tests (in vitro against cell strains and in vivo in animals) to assess their abilitiy to overcome current limitations in anti-trypanosomatidic therapy. One long-term efficacy study in monkeys was performed in a collaborative effort with the Kindred project (www.kindred.org). Moreover, we have used structure-based approaches to guide the synthesis of 700 compounds containing thiadiazole, benzothiazole, pteridines, alkylphospholipid and other chemical cores, out of which we identified at least some genuinely new scaffolds and chemical entities. Natural compounds libraries such as the MycodiverseTM library (11000 extracts) and over 120 plants collected in Sudan enriched the NMTrypI platform compounds base and provided a number of new chemical entities. The major strength of the Consortium lies in the complementarity of the partners’ expertise. The tightly integrated platform provided: 1 innovative, less toxic and safer drug candidate for visceral Leishmania infections compared to existing ones like miltefosine; genomic and ribose profiling data leading to drug resistance definition. Dog serum protein profile associated with a new drug lead candidate has also been characterized.
NMTrypI has progressed through 6 scientific work packages (WPs 1-6) supported by 3 transversal work packages (WPs 7-9) dedicated to project dissemination and management, and synergies with other similar projects.
Expected impacts related to the work programme. The application of the new NMTrypI approach is expected to lead to an effective and affordable treatment of Trypanosomatid Infections in disease endemic countries. The NMTrypI project may contribute to a major breakthrough towards finding a real therapy for NTDs.
Carrying out the project at an international level with the inclusion of EU and disease-endemic countries in the consortium provided a unique blend of experts to work in the field of Trypanosomatidae. The three research-intensive SMEs involved in the project could exploit the results obtained in order to generate more revenues for the companies and therefore develop their competitiveness, allowing them to gain new markets, to further develop their research and also to create more jobs.
Global international cooperation: the NMTrypI consortium has developed partnership/collaboration with international initiatives such as the collaboration with GSK on the research on the Kinetobox, with existing NTD research consortia and international programs such as the COST1307 action; other actions are ongoing and will be further developed after the project end. These organizations provide formats to enable progression of compounds through clinical trials thus filling a gap to exploitation by the large pharmaceutical companies.
NMTrypI is actively involved in participating and stimulating new initiatives acting as a seed platform for establishing a continued-extended open-access lab in Europe and at the international level.
Increase of European competitiveness: NMTrypI results has contributed to the development of new scientific knowledge needed for the development of new drugs: therefore, the competitiveness of European pharmaceutical industries could be further reinforced, especially for the SMEs participating in the project.
Socio-economic impact: the final goal of the project is a step forward in the development of new therapeutic principles for anti-trypanosomal treatment that will directly lead to a benefit for human health. The exploitation of discoveries (target specific inhibitor/compounds) will directly lead to an improvement of the quality of life in developing countries as well as in Europe. Ethical aspects of NMTrypI project. The research project involved the use of experimental animals, namely mice, hamsters, dogs and monkeys under the European national regulation.
Project Context and Objectives:
According to the WHO (link: Second WHO report on Neglected Tropical Diseases, 2013), one billion people are at risk of or are affected by Neglected Tropical Diseases (NTD), which often affect communities living in remote rural areas or in urban slums with poor living and hygiene conditions.
The dissemination of infectious diseases due to microorganisms belonging to the Trypanosomatidae family – i.e. trypanosomiasis that causes sleeping sickness, leishmaniasis and Chagas disease – is a very important issue in some areas of the World, including Africa, South America and India, where these parasites are endemic.
Research on Trypanosomatidic diseases is limited and fragmented, and national initiatives are generally weak or lacking critical mass. Problems associated with existing drugs include inefficient delivery, insufficient efficacy, excessive toxicity and increasing resistance. New drugs are urgently needed now and in the near future.
The New Medicines for Trypanosomatidic Infections - NMTrypI project aims at obtaining new candidate drugs against Trypanosomatidic infections with appropriate efficiency from the lead phase to the final preclinical phase that are more accessible to patients.
The main objective of NMTrypI project is the development of 1-2 candidate drugs for non-human primate evaluation (depending on compounds class) including pharmacokinetic (PK) and pharmacodynamic (PD) evaluation on BALB/c mice, hamsters, and testing on dogs. The candidate(s) could be promoted to the late preclinical phase or at best to early clinical phase. In order to reach this goal, the following key objectives have been addressed:
Objective 1: To optimize the activity profile, the pharmacokinetics and pharmacodynamics of the main Miltefosine / Pteridine / Benzothiazole leads on the recombinant proteins selected and parasites.
During the 36 months the project should have: a) designed compound libraries; b) developed hit to lead optimisation of pteridine, benzothiazole, thiadiazole and improved miltefosine derivatives and in particular DRUG LEAD 1 derivatives; c) performed the synthesis of the designed compounds and performed the process of iterative optimization, including fragment-based drug design; d) Identified of Lead compounds from natural product libraries.
Objective 2: To set up a library screening technology platform equipped with ad hoc designed assays and test the natural compounds and the other libraries available, followed by subsequent modifications from Hypha and NCR to obtain drug leads with high specificity/potency on the parasites.
During the 36 months, the project has to prepare natural product and known drug library, chemical diverse libraries of different origins. Phenotypic assays has to be performed. In-vitro off-target liability, toxicity, early-tox and safety studies prior to animal toxicity studies. The mentioned experiments should allow the selection of best compounds in detailed direct cell based assays and other assays.
Objective 3: To identify the best possible combination therapy, composed of a specific compound targeting PTR1 and a well-known inhibitor/established drug targeting DHFR-TS enzymes, inhibiting parasite growth.
During the 36 months, the compounds should be designed to target the two enzymes, PTR1 and DHFR of the parasites, Leishmania major and Trypanosoma brucei and Trypanosoma cruzi. Then, the Consortium should test the compounds against leishmania parasites and trypanosomatids as single agent or in combination with methotrexate or pyrimetamine. Different studies on the molecular aspects of the combination have to be performed and possibly validated in snapPK studies.
Objective 4: To understand the biological profile of the best compounds and combinations on the parasites. This includes the mechanism of action, off-target identification and drug resistance studies, and uses this information to guide lead drug identification and optimization.
During the 36 months, the project should provide the identification of targets and off-targets for lead compounds, the assessment of drug resistance potentials through the establishment of a biomarker/target identification methodology and finally provided the selection of gene regions mediating Lead drug resistance for different compounds that have demonstrated interesting biological profile fitting the Target Product Profile principle. (objective 6).Objective 5: To test the best combination of specific compounds targeting PTR1 and well known inhibitor/established drugs of DHFR-TS enzymes, the best Miltefosine analog against mice, and hamster models of the parasitic diseases and establish the ED50 and other important pharmacology indexes. Dogs will be used in the restricted cases of miltefosine derivatives and in case of compound with very good activity also the monkey trial could be performed.During the 36 months the project should provide animal studies on the compounds with the suitable biological profile.
Objective 7: To obtain, gather and share information about the process of developing the best drug leads into drug candidates using the NMTrypI platform and to build a database to collect all the results. The project can provide a useful tool for next generation drug development, which can be made publically available, when IP protection has been exploited.
Description of the work performed since the beginning of the project and the main results achieved so far
During the 36 months NMTRypI has achieved the results described hereafter.
Objective 1 is related to WP1 and WP2, and mostly focused on three activities:
a) compound library design and synthesis for thiadiazoles (130), benzothiadiazoles (80) and other core molecules of potential interests (100),
b) hit to lead optimisation of pteridine (45 compounds), improved miltefosine derivatives (50) and 130 flavonoids derivatives and flavonoid-like compounds; the Consortium synthesized over 700 hundreds compounds and optimized miltefosine derivatives and pteridines, leading to compounds with largely improved biological profiles (see below) and combination effects;
c) progressing and finalizing the early pre-clinical studies of compounds from natural product libraries.
This work paved the way for the selection of optimal compounds active against different species of Trypanosomatidic parasites.
Almost all compounds have been evaluated in HTS on target assays after cloning, purifications, and biological characterization of 7 target proteins (PTR1s and DHFR-TS proteins and phosphorilase protein) and 2 off-targets (hTS and some hDHFR); over 70 X-ray crystal structures have been determined for the purified proteins. Two biochemical assays have been developed against PTR1 from Leishmania major and Trypanosoma brucei, Trypanosoma cruzi; 4000 synthetic compounds from internal libraries/ external collaborations and 12.000 natural extracts have been tested.
Objective 2 and 3 are related to WP3 on phenotypic screening (library screening platform) of natural product (11000 from Hypha and 134 from NCR) and known drug library 200 compounds, in-vitro off-target liability, toxicity, ADME and safety studies prior to animal toxicity studies. Synthetic compounds have been tested and around 10 ADME-Tox in vitro assays set up for compounds liabilities studies. One of the crucial objectives is to identify the best possible combination therapy, composed of a specific compound targeting PTR1 and a well-known inhibitor/established drug targeting DHFR-TS enzymes, inhibiting parasite growth. Methotrexate was adopted as the DHFR inhibitor. Flavonoids (NMT-H) and pteridines (NMT-C) compounds showed a synergic activity in combination with MTX up to a synergic index value of 3 at a concentration lower than 1.5 microMolar. HTS platform successfully tested the 60% of all compounds available. Leishmania infantum assys revealed some difficulties and remain to be tested soon.
On this basis, 4 flavonoids derivatives, 6 pteridines and 4 miltefosine derivatives and 6 different compounds have been selected for animal studies against the three parasites. The most relevant compound class identified during the 36 months of the project are miltefosine derivatives and new synthetic compoudns derivaed from the benzothiazole class. In particular, two compounds showed an important activity compared to miltefosine. Specific experiments have been performed to better understand their biology and mechanism of action, off-target identification and drug resistance studies to guide lead drug identification and optimization (objective 4 related to WP4). Differential Mass Spectrometry of Leishmania parasites treated with Miltefosine and two other compounds, one miltefosine derivative and another synthetic compound tcompared to untreated, genetic studies on miltefosine and derivatives allowed a biomarker/target identification methodology (objective 6) and provided consolidated results on the selection of gene regions mediating lead drug resistance. The Consortium also identified two novel proteins involved in pathways modulated by Miltefosine, All this work related to WP4 allowed the full achievement of the objective for the project. Serum samples taken from the dog trial studies, have been evaluated in a differential approach in which samples of treated versus untreated animals are taken and examined. This will allow the track associated with the compounds activity in vivo (dogs).
16 selected compounds progressed to PK studies, according to the criteria established for the progression hit-to-lead and lead optimization (objective 5 related to WP5). The best Miltefosine analogs drug lead 1 and drug lead 2 were tested against BALB/c mice, and hamster models infected with Leishmania infantum. PK studies on spleen, liver, bone narrow samples have been performed and demonstrated similar efficacy, but lower toxicity with respect to Miltefosine. Toxicity and new efficacy studies studies have been performed on dogs. Moreover we could test the lead candidate in monkey trial in long term treatment, in the context of the Synergy activity (see WP9).
To gather and share information about the process of developing the best drug leads into drug candidates using the NMTrypI platform we build a database to collect all the results. The project can provide a useful tool for next generation drug development which can be made publically available, when IP protection has been exploited. This work allowed the full achievements of the objectives related to WP6.
Project results and impact
NMTrypI project is expecting the following results by the end of the project:
Innovative drug concepts (Miltefosine and hybrid compounds) that could be exploited in all Trypansomatidic infections.
Development of specific bioassays (HTS platform) for the evaluation of a high number of compounds against Trypanosmatidae can accelerate the drug discovery process.
Identification of new targets/off-targets, validation of known targets for the miltefosine derivatives, antifolates: the availability of the selected compounds, Miltefosine, pteridines and Benzothiazole derivatives against trypanosmatidae allows a better understanding of the mechanism of action (MoA) and lead to a specific design of new derivatives that avoid their interactions with human target with similar biological function.
Identification of biomarker for MoA and initial drug resistance identification (on the parasites): biomarkers protein profile could help in the monitoring of the candidate drug effect on the parasite and drug resistance initial development. Biomarker initial identification will be performed.
Database including all the project data that can be accessed by external scientists under agreements and can be used for further development of the candidate drugs.
Strengthening the preclinical selection of the drug leads for progression along the pipelines: proof of principle that accurate preclinical evaluation can decrease the drug candidates liabilities and increase the success rate along the drug discovery & development process
• Improvement in drug discovery for NPDs (scientific impact): the NMTrypI results will contribute to the development of new scientific knowledge needed for the development of new drugs. NMTrypI Consortium is based on the collaboration of a multi-disciplinary team of experts; the accessibility of a wide-ranging knowledge-based platform significantly improve the chances of success.
• Increase of European competitiveness: the knowledge generated by the project will be exploited especially by the SMEs participating in NMTrypI Consortium. Further collaborations with European pharmaceutical industries could be established, thus reinforcing European competitiveness. Furthermore, the consortium reaches out to international partners (CNPEM from Brazil and NCR from North Sudan) to access new sources of knowledge and address global NTD challenges, making Europe more attractive as a location for research and innovation, and boosting industrial competitiveness.
• Improvement in global international cooperation: NMTrypI consortium will further develop partnership/collaborations with international initiatives, existing research consortia or programs on NTDs – in particular, the Geneva-based Drugs for Neglected Diseases initiative (DNDi) and the US-based Consortium for Parasitic Drug Development. These organizations provide formats to enable progression of compounds through clinical trials thus filling a gap created by the abandonment of these areas by the pharmaceutical industry. This cooperation will allow joining forces, avoiding duplication and speeding up development in an international cooperation context.
• Socio-economic impact: The NMTrypI project uses innovative compound classes suitable for combination strategy targeting three parasitic diseases. At least one compound belonging to such compound classes is expected to perform better with respect to the current drugs in therapy with higher efficacy, lower drug resistance potentials, lower toxicity, and improved safety. NMTrypI project results represent a step forward in the development of novel therapeutic principles for anti-trypanosomal treatment that will lead to a benefit for human health. The exploitation of discoveries (target specific inhibitor/ compounds) will contribute to a reduction in the high socio-economic impacts of Leishmaniasis, Human African Trypanosomiasi and Chagas disease: reduction of mortality or number of cases, reduction of healthcare expenditures and improved productivity of workers thanks to an increased work capacity.
This results will substantially improve the quality of life of patients and their families as well as in developing countries and in Europe.
WP1: Lead optimization & synthesis. Predictive ADME/Tox evaluations
The overall objective of WP1 is to generate lead compounds belonging to three compound classes.
1. Synthetic folate pathway-targeting pteridine, benzothiazole and thiadiazole derivatives,
2. Improved miltefosine analogues and
3. Natural products.
Task 1.1. Design of compound libraries. Lead optimisation of pteridine, benzothiazole, thiadiazole and improved miltefosine derivatives (HITS)
Initially, homology modelling of on- and off-target proteins of the folate pathway (PTR1, DHFR, TS, FR) and analysis of the binding properties of their active sites was performed. Subsequently, several computational docking protocols were tested and applied to the above protein targets and off-targets for thiadiazoles, benzothiazoles, pteridine-based compounds and flavonol-like compounds. On the basis of docking, new compounds or types of substituents were proposed. The following results were achieved for the different compound classes investigated. (D1.1 and MS1)
NMT-C series (Pteridine derivatives). The search for dual inhibitor-core alternatives for pteridines led to the identification of the trifluoromethyl-quinazoline moiety with alternative binding mode in PTR1. Special computational methodology was used, to overcome shortcomings in halogen treatment in standard docking force-fields. Furhermore, compound design was carried out for selective dual inhibition of parasitic DHFR and PTR1. Detailed SARs analysis for PTR1 and DHFR inhibition was performed. Docking studies were translated to ligand:receptor interaction fingerprints and were correlated with observed activities. Expanding on the observed correlation of logPo/w with anti-trypanosomal activity, correlations of in silico predicted ADME-Tox properties with anti-trypanosomal activity were determined from synthesized pteridine derivatives and embedded in the design pipeline as an activity filter.
NMT-H series (Flavonol-like compounds). A docking protocol was developed for heavily hydroxylated flavonol and flavanone scaffolds, including reference binding modes for pose selection optimization and induced fit docking methodologies, to allow receptor response upon compound binding. Models of ligand-receptor complexes were used to derive SARs. Qikprop predictions of the pharmaceutically relevant properties were performed and compared with the experimental ADME-Tox properties.
NMT-F series (thiadiazoles, benzothiazoles) and FBDD. The docking methodologies used for thiadiazole-based compounds were improved leading to a notable correlation of substrate-like binding mode with high activity against the target-enzyme. Concerning the benzothiazole derivatives, alternative docking methodologies were tested, followed by docking-based multi-step optimization to target PTR1 of Tb, Lm and Tc. Furthermore, the design of derivatives of NMT-F0121 led to a sulfonamide-based library tested by docking against TbPTR1. Subsequently, efforts were dedicated to Fragment-Based Design Approaches (FBDD) as a new source of dual inhibitors of PTR1 and DHFR. Initially, computational evaluation of fragments targeting the biopterin pocket of PTR1 (L-fragments) and an adjacent region in the active site, as cores for PTR1 and DHFR was performed. To implement a scaffold hopping approach and test the interactions of halogens with TbPTR1, benzothiazole series linked with halogenated phenyl group were designed and further optimized based on activity results. Finally, an induced-fit protocol was developed to explain the activities of quinazolinone series towards TbPTR1 and, partly, towards hTS.
NMT-A series. Optimization of specialized protocol for ADME-Tox properties prediction for dimethylamino-substituted miltefosine analogues and integration in the design of improved high lipophilic compounds . Modeling to assess binding of miltefosine-like compounds to the PH domain of human Akt1 was performed. Sequence analysis for putative parasitic Akt1 and preliminary docking of compounds to the Akt1 catalytic site (as an off –target).
Natural Products. Docking evaluation of harmine, several of its derivatives and a few natural products from HYPHA screening (e.g. cucurbiticin) as PTR1 inhibitors was performed.
Tasks 1.2.1 and 1.2.2: Synthesis of folate-pathway targeting flavanonol-like derivatives, thiadiazoles, benzothiazoles, pteridines and compounds from FBDD (UNIMORE, TYDOCK Pharma). (D1.1 and MS1)
Flavonol-like compounds (UNIMORE). 37 compounds bearing the classical flavonoid scaffold and 74 flavonol-like compounds bearing heterocyclic rings (furan, 3/4/5-methylthiophene, pyridine, quinoline, indole, 1-methylindole, benzothiophene and 1,3-benzodioxole) were synthesized to obtain SAR against PTR1. Compound NMT-H0080 is the best-in-class showing a broad spectrum antiparasitic activity (EC50 L. infantum = 1.88 μM; EC50 L. donovani = 4.6 µM; EC50 L. major = 5 µM; EC50 T. brucei = 0.8 μM; EC50 T. cruzi = 2.2 μM). Compound NMT-H0087 was the most potent and selective molecule towards T. brucei (EC50 = 0.4 μM, SI = 250).
NMT-H0080 and NMT-H0087 were selected for pharmacokinetic studies on BALB/c mice. NMT-H0080 (IV t ½ = 63.2 h, Oral t ½ = 46.9 h) and the oral bioavailability and the oral maximum concentration (Cmax) were 11.8% and 1.96 μM, respectively. Formulation of NMT-H0080 with cyclodextrins increased the oral Cmax to 3.56 μM. Based on the TLP profile of NMTrypI, we have selected compound NMT-H0080 for in vivo evaluation in mouse and hamster model of visceral and cutaneous Leishmaniasis. Towards this end 1.5 g of NMT-H0080 were prepared.
Thiadiazole and Benzothiazole derivatives (UNIMORE). 58 new thiadiazoles were synthesized as inhibitors of parasitic PTR1 in a structure-based design approach supported by protein crystallography and molecular modelling. NMT-F0097 was the most potent against TbPTR1 (IC50= 16µM). All the compounds were devoid of in vitro toxicity and their in vitro antiparasitic activity was low. Antiparasitic anctivity evaluation of compounds NMT-F0042 and NMT-F0097, in combination with MTX revealed a synergy effect. Structural studies disclosed bicyclic thiadiazole derivatives as a new privileged structural scaffold. To obtain SAR 80 different benzothiazoles were synthesized. Derivative NMT-F0188 was selected for in vivo evaluation. Pharmacokinetic studies of NMT- F0188 on BALB/c mice showed a long half-life (IV t½ = 3.5 h, Oral t½ = 40 h), and a maximum concentration (Cmax), in complex with cyclodextrin, of 12 µM, 1.5-fold the antiparasitic EC50. However, in vivo evaluation of NMT-F0188 revealed a poor antiparasitic activity against T. brucei.
Compounds based on FBDD (UNIMORE)(D1.4). Based on the chemical tractability four distinct chemical scaffolds were selected for further development.
QUINAZOLINONEs: 10 new compounds were rationally designed and synthesized. Seven crystallographic structures were obtained which support the design, reproducing the poses and retaining the key interactions of the initial binding fragments. All the compounds showed a low micromolar IC50 (1.5 – 30 µM) against TbPTR1, which represents an improvement of 200-2000 times with respect to the starting fragments. However, efficacy against parasites was poor. NMT-F0121 was the most valuable compound with TbPTR1 IC50 = 4.6 µM exhibiting a strong synergic effect in association with MTX against T. brucei (CI = 3.0). NMT-F0121 is a new starting point for the design of improved analogues.
Furanopyrimidines: 20 derivatives were designed and synthesized and two x-ray structures were resolved. The preliminary enzymatic activity revealed a TbPTR1 IC50 (0.2 – 3.0 μm), however these compounds suffer from poor antiparasitic activity when tested as single agents.
Triazines: Designed within a structural elaboration of the pyrimetamine core, 8 new compounds showed a low nanomolar TbPTR1 inhibition (EC50 = 150–400 nM). Three crystallographic structures were resolved which support the design, reproducing the poses and retaining the key interactions of the initial binding fragments.
Triaminopyrimidines: 25 compounds were prepared, exhibiting nanomolar TbPTR1 IC50 values (i.e NMT-F220 TbPTR1 IC50 = 0.27 µM). All the compounds of this series posses a good activity against T. brucei with EC50 (0.09 – 5 µM) and a good selectivity index (SI > 40 for the least toxic derivative). 4 compounds were subjected to SNAP-PK studies; the best performer was NMT-F0252 (Cmax 2.5 µM; t1/2 = 2.1h and AUC = 16 µM) with a 30-10x EC50 exposure for the first 5 hours after administration. However, the in vivo efficacy against T. brucei was poor.
Pteridine derivatives (TYDOCK). 20 new compounds were synthesized exhibiting IC50 < 1 nM against TbPTR1 and LmPTR1. Conversely, in vitro antiparasitic activity was low probably due to low penetration. Based on docking and crystallographyc studies, PTR1 enzymatic activity inhibition and antiparasitic activity evaluation, the synthetic efforts were focused on modulating the intrinsic lipofilicity in these compounds. 24 new compounds were synthesized. The best compound was NMT-C0131, which inhibits PTR1 enzyme at picomolar concentration and posses T. brucei EC50 of 0.5 µM. Furthemore, the TYBOX comprising 750 compounds was assembled and the compounds were tested for activity and toxicity. In particular, 19 compounds resulted to be active against L. infantum, 122 against T. brucei and 23 against T. cruzi. NMT-C0074 (a non pteridine like compound) resulted to be one of the very promising compounds to be further evaluated. However it twas not stable in PK tests and it was abandoned. A very important result was the in vitro proof of concept, for the first time, of a strong synergic effect on the parasite growth inhibition for pteridine like compounds targeting PTR1 enzymes (NMT-C0044 , NMT-C0090, NMT-C0091, NMT-C0113 and NMT-C0131) in combination with MTX, a DHFR inhibitor. The most promising compound acting as synergic inhibitor with MTX is compound NMT-C0113 (Synergy index of 0,065). When subjected to SNAP-PK NMT-C0131 presents an acceptable EC50 value during the first hours in PK tests. To overcome the poor pharmacokinetic behavior for these compounds, a drug delivery system is under evaluation.
Task 1.2.3. Synthesis of new optimized derivatives of Miltefosine (NHRF). (D1.1 and MS1) Three series (31 compounds) of ring-substituted ether phospholipid derivatives and Miltefosine analogues were synthesized and evaluated for their antiparasitic activity and cytotoxicity. DRUG LEAD 2 and DRUG LEAD 1 were very potent against L. infantum amastigotes (IC50 = 2.0 ± 0.7 µM and 1.4 ± 0.1 µM, respectively) but much less toxic than Miltefosine (SI = 39 and >71, respectively). Interestingly DRUG LEAD 1 was also active against T. brucei (IC50 = 4.47 ± 0.20 µM) exhibiting 7.6 fold higher activity than Miltefosine. 4 compounds were subjected to SNAP-PK and compound DRUG LEAD 1 was selected for further in vivo studies. In addition, SAR studies around DRUG LEAD 1 resulted in 22 new derivatives. Based on the NMTrypI TPP, compound DRUG LEAD 1 was selected for evaluation of antileishmanial activity in vivo in mice, hamsters and dogs with very promising results (c.f. D1.3 MS3). In addition, DRUG LEAD 1 was effective per os in vivo against Τ. brucei acute model of infection and also against the long lasting chronic infection model (c.f. D1.3 MS3).
Task 1.3 Identification of lead compounds from natural product libraries (HYPHA, NCR).
3048 HPLC fractions resulting from 126 extracts of Sudanese medicinal plants were provided for screening. Testing in the Tb and Lm PTR1 assays yielded high hit rates. Analysis of the active fractions by LC-MS indicates that the majority, but not all, of these active fractions contain flavonoids or other phenolics, classes of compounds that have already been investigated as PTR1 inhibitors. A subset of 20 active fractions of sufficient purity for further testing and profiling in ADME-Tox assays were identified: this will enable prioritisation of any samples meriting further investigation and fulfil MS2 (Natural product purified extracts filtered for activity and ADME-Tox properties). 37 Sudanese plant extract HPLC fraction hits against L. infantum were purified further yielding 79 compounds for primary assay testing. 10 compounds had EC50 values in the range 5-15 µM. These were mostly known compounds, some of which had been previously reported to have antiparasitic properties. Assay-guided purification activities have commenced with the selection of 14 MycoDiverse™ library hits in the Tb PTR1 assay for the first stage of HPLC fractionation. 11 of these samples also showed growth inhibitory activity against T. brucei. Small-scale assay-guided purification was conducted on a total of 72 MycoDiverseTM library phenotypic assay hits (40 T. brucei, 16 T. cruzi and 16 L. infantum). This was most successful for the Tb and Li hits and less so for the Tc hits, which were mostly weaker and/or borderline toxic. Further purification and structure elucidation work was focused on the Tb and Li hits. 7 compounds were purified from Tb hits, of which 4 were novel, mostly terpenoids. The most potent of these showed poor selectivity on secondary evaluation. 5 promising Li hit compounds with micromolar primary assay potency were being investigated as the project ended: one novel and two known compounds had been identified but two remain unidentified. The most promising PTR1 inhibitory screening hits were from plants rather than the MycoDiverseTM library. Scale-up purification yielded 10 compounds, 9 of which had been previously reported. These were mostly flavonoids, a compound class already investigated by the NMTrypI consortium (c.f. D1.2).
WP 2. Studies on purified targets. M/HTS assays for Lead development.
1. Development of assays to monitor purified target activity, (PTR1 and DHFR)
2. Use of the assays in screening of synthetic small molecule libraries (pure compounds) and natural products (mixtures).
3. Information on target-protein interaction through X-ray crystallography and other techniques.
4. Ranking of compounds for further progression to WP3 (in vitro ADME and toxicity).
Task 2.1 - Protein cloning (M1 to M30) (UNIMORE)
The objective of the task is the cloning and purification of folate dependent protozoan and human proteins like PTR1, DHFR and bifunctional DHFR-TS which are already established targets as well as other proteins that will be identified by the studies undertaken under WP4, as relevant new targets of the most active compounds.
Mutant (e.g. active site mutant) proteins have been obtained to provide control protein samples or insights on the mechanism of protein-inhibitor interaction and also to obtain crystallization of the proteins that cannot crystallize or show low stability. This was the case of T. cruzi PTR1. We were not able to obtain a protein that was stable enough to be used in HTS on target screening. While for T. brucei PTR1 and LmPTR1 we could obtain the stable protein, usable for HTS on target (WP3). During the project we have cloned 10 proteins considered targets and off-targets for our compounds (3 targets, 5 off-targets and 2 newly identfieid proteins). Ad hoc vector design and expression systems were used for protein production. The encoding sequences for the target proteins were cloned in selected vectors with and without removable tags, such as His6, in order to facilitate their purification. To improve the solubility of unsoluble proteins, the cloning strategy focused on vectors encoding removable fusion proteins, such as GST or DsbA. Expression trials were performed in different E. coli strains, investigating also different protocols for bacterial growth and protein over-expression. Although protocols of heterologous expression of DHFR, DHFR-TS, PTR1 and of the DHFR domain of DHFR-TS existed, they were largely optimized for the production of these enzymes in quantities sufficient for setting up crystallization trials and other experiments.
The necessary amount of protein for crystallization studies and for HTS recombinant protein assays studies was produced by UNISI. UNIMORE and UNISI produced the protein for detailed enzymology studies on target-inhibitor interaction of benzothiazole, thiadiazole, flavonoids and pteridine libraries. (D2.1 and MS6).
Task 2.2 - Proteins purification, structural and biological characterization.
The objective of the task was the protein purification of prioritized drug targets such as PTR1and DHFR-TS from the trypanosomatid organisms under study.
Enzymes from human organism such as Thymidylate synthases and DHFR have been prepared for assaying the specificity of the drug lead towards trypanosomiatid organisms.
Miltefosine transporter (LdMT) and the protein LdRos3 (translocation machinery) in part responsible for the uptake of Miltefosine. (Pérez-Victoria F.J.et al. J. Biol. Chem. 281, 23766) and Protein Kinases of interest involved in the mechanism of action. The kinase studies was focused on AKT protein (see WP4), but then no in deep study was performed, because it was considered not a priority compared to other targets under study. While the LdMT transporter was considered very relevant. It was identified as a protein of major interest in the mecahnism of action of Miltefosine and of our drug leads DRUG LEAD 2 and DRUG LEAD 1; it emerged from the genomic studies aimed at studying drug resistance in L. donovani. Mutations of the transporter specifically altered the activity of the two drug leads and of Miltefosine. The alterations were different for the three compounds. Some molecular modeling and crystallographic studies have then been performed. The results obtained suggest a different resistance mechanism for Miltefosine with respect to the two drug leads outcome from our studies.
Feed-back from WP4 suggested to clone and crystallize other two proteins that resulted largely overexpressed from the proteomic studies (WP4) performed on L. donovani treated and untreated with these three compounds One of them was largely characterized and 5 crystal structures were obtained as apo-enzyme with different metals. The biochemical mechanism was characterized. The role of this protein in the mecahnsim of action of the two drug leads (DRUG LEAD 2 and DRUG LEAD 1) was not well understood using molecular biology and cellular approaches (WP4).
Drug-target interaction studies through enzyme kinetics and fluorescence spectroscopy were performed on the most active compounds, to complement the information from X-ray studies. Molecular modeling was also used to complement the interpretation of the results (WP4).
Spectroscopic contributions to the NMTrypI project (Unimore)
Fluorometric characterization of three inhibitors of the Lm and Tb Ptr1 proteins. These inhibitors proved to be good fluorophores with intense emissions in the visible spectral region. Therefore, they lend themselves to label-free (or one-label) fluorescence experiments, in cellular environments as well. The first one, a flavanone called NMT- H0013, was found to emit between 400 and 600 nm. Its emission moved to shorter wavelengths upon binding to Lm PTR1. From this spectral change and the increase in anisotropy, the equilibrium binding constant of NMT- H0013 with Lm PTR1 could be determined. Inhibitor NMT-H0024 was a flavonol, poorly soluble in water; yet, its fluorescence quantum yield was extremely high and enabled a fluorescence characterization to be done even with hardly observable absorption. Another flavonol inhibitor, NMT- H0032, was more water-soluble and showed an intense emission in the 450-620 nm region. All these three compounds had emission spectra compatible with good FRET efficiencies to blue-green absorbing protein tags (e.g. fluorescein-like tags), and might therefore be employed for monitoring engagement between the inhibitor and the target in a cellular environment.
Another spectroscopic work was devoted to structurally characterize proteins involved Lm and Tb PTR1 proteins. A first problem faced was raised by literature reports on activity/inhibition experiments performed on each of the two proteins at rather widely differing pH values, 3.7 and 6 for Tb PTR1 and 4.7 and 6 for Lm PTR1. We performed both fluorescence and near-UV circular dichroism (CD) experiments with the goal of establishing whether spectroscopic evidence existed of conformational differences for each of the two proteins at the two pH values. CD results indicated negligible changes for Lm PTR1 and an increased rigidity of some tyrosines at pH 3.7 with respect to pH 6 for Tb PTR1. Steady-state emission studies results confirmed the indifference of Lm PTR1 to the pH difference. On the other hand, Tb PTR1 showed an increased relative intensity of Tyr vs Trp residues at the lower pH value, likely associated with a structural change of the protein that causes a change in the quenching efficiency of the Tyr emission by the Trp residues. Time-resolved fluorescence experiments confirmed the higher efficiency of energy transfer from Tyr to Trp residues in Lm PTR1 than in Tb PTR1, but did not show significant differences in the decay profiles of each protein at different pH values. We assume this to imply that changes in the efficiency of Tyr to Trp energy transfer efficiency resulted in full quenching of some Tyr residues.
A second study was devoted to combine the fluorescence properties of each protein to the changes in its structure. For example, a shift of the emission spectrum of a Trp residue is related with a change in its exposure to the aqueous environment; a change in its emission quantum yield and lifetime(s) is related with a change, even subtle, in its proximity to a quencher, either endogenous – charged amino or carboxy groups, an amidic function, such as a peptide group – or exogenous, such as a bound ligand that absorbs in the spectral region where Trp residues emit, i.e. 300-400 nm; changes in the emission band width, as well as in the lifetime distribution, often reveal changes in the conformational disorder around some Trp residue; finally, emission steady-state or time-resolved anisotropy changes are often related with changes in the conformational mobility of a Trp residue, mediated by a change in its environment. We have started to build up this fluorometric profile for the two proteins. Furthermore, two examples of protein/inhibitor complexes, one for each protein, have been investigated in the search for changes in the fluorometric profiles that could be related with conformational evidence on the inhibitor/target protein binding mode and its, possibly long-range, structural consequences.
Task 2.3 - X-ray crystal structure determination
Crystallographic studies to determine the binding modes of the most promising inhibitors of the trypanosomal enzymes, with the aim of informing the design and guiding the synthesis of improved drug leads. The binding between lead compounds and the off-target proteins identified in WP4 will be determined and enter the process of driving the design of lead molecules with improved specificity towards the target enzymes.
UNISI determined 75 X-ray crystallographic structures of the complexes between the best lead compounds and the recombinant proteins produced in Task 2.1 and 2.2.
Four off-targets proteins identified in WP4 from proteomic studies were obtained in Tasks 2.1 and 2.2 and were crystallized as apo-enzyme or with the appropriate inhibitor, and their structures determined. Recombinant proteins were crystallized utilising automated facilities available and crystals screened for diffraction at home X-ray sources prior to high resolution data collection at synchrotron sources such as the ESRF (Grenoble), Elettra (Trieste), Diamond Light Source (Oxfordshire,U.K) and the EMBL Hamburg outstation at DESY (Hamburg). The majority of unliganded enzymes, as well as the inhibitor complexes, were solved by molecular replacement (MR) methods. In the case of the newly discovered protein from proteomic studies, we used different metals ito obtain the crystals. Crystals of ligand complexes were prepared either by exposing pre-existing crystals to inhibitor (soaking techniques) or by screening protein/ligand mixtures for new crystallisation conditions (co-crystallization techniques). The results are reported in D2.2 and D2.3 and MS7 and MS8.
Task 2.4 - Biochemical assay development and compound evaluation
A previous collaboration between ESP-Unimore as part of the COST-CM0801 action “New drugs for neglected diseases”, allowed a microtitre plate absorbance based assays, for purified parasitic PTR1s, to be successfully evaluated at the ESP facility. These assays were further adapted into additional assay formats that are less susceptible to compound interference; an assay to monitor purified parasite DHFR activity have been developed (MS4, MS5). When appropriate, the mechanism of action of compounds, including time-dependence inhibition and stoichiometry, have been determined. Specificity with respect to folate dependent human proteins was evaluated. Proteins were provided by UNIMORE and UNISI in Tasks 2.1 and 2.2. The Fraunhofer have screened various batches of compounds and natural product extracts (HYPHA MycoDiverseTM library and African medicinal plants extracts from Sudan) as part of the regular HTS screening against the Lm-PTR1 and Tb-PTR1 enzymes. By the end of the NMTrypI project, 10 tranches of samples have been screened and the data uploaded onto SEEK database. A total of 4000 compounds were screened against Lm PTR1 and TbPTR1. All screening data analysis (single compound concentration and dose-response studies) were performed using ActivityBase XE. A total of 271 data set have been analysed during the lifetime of the NMTrypI project which includes screening of LmPTR1, TbPTR1, mitochondrial toxicity, hERG, A549 cell-line, WI38 cell-line, Aurora B kinase, CYP450 1A2, CYP450 2C19, CYP450 2C9, CYP450 2D6, CYP450 3A4, as well as data analysis on behalf of CNPEM (NMTrypI consortium partner). This has led to the generation of approximately 20,000 data points that have been processed using ActivityBase XE, with approximately 10,000 of these being from the screening of the HYPHA MycoDiverseTM library and the remainder being from synthetic compounds and data analysis of the screening output from CNPEM. Each screening experiment was performed followed established SOPs and appropriate reference compounds were evaluated in parallel with test compounds on each occasion. (D2.1 MS7 and MS8). We have obtained and hit rate around 30% within the TbPTR1 inhibition studies and less than 10% within LmPTR1 inhibition studies.
WP3. Phenotypic screening. M/HTS assays, in vitro liability, ADME and toxicity evaluation.
1. Development of functional phenotypic assays.
2. Development of compatible assays for targets, their use in screening against natural products (mixtures) and known drug libraries.
3. Ranking of compounds for further progression based on activities of compound properties to include suitable secondary assays and in vitro off-target liability, ADME and toxicity data.
Task 3.1 - Natural product purified extracts filtered for activity and ADME-Tox properties.
(HYPHA) (D3.1 D3.2 and M9, M10)
Following the screening of the natural product libraries in the target assays which comprised screening of Hypha’s MycoDiverseTM library of fractions and extracts from fungal fermentations (11,000 samples) and a library of semi-purified HPLC fractions generated from extracts of Sudanese medicinal plants (3,312 samples from 137 extracts), the same samples were tested in phenotypic assays against T. brucei, T. cruzi-infected U2OS cells and L. infantum-infected THP-1 cells. The primary screening hit rates for both libraries were highest for the T. brucei assay (14.8% for the MycoDiverseTM and 19.6% for the plant fractions), lower for T. cruzi (2.3% and 9.4%) and lowest for L. infantum (1.1% and 1.2%). For the MycoDiverseTM library hits the most promising samples were selected for assay-guided purification studies based on anti-parasitic potency and producing organism diversity after confirmation and dose-response profiling. Small-scale assay-guided purification was conducted on a total of 72 MycoDiverseTM library phenotypic assay hits (40 T. brucei, 16 T. cruzi and 16 L. infantum). This was most successful for the T. brucei and L. infantum hits and less so for the T. cruzi hits, which were mostly weaker and/or borderline toxic. Further purification and structure elucidation work was focused on the T. brucei and L. infantum hits. Seven compounds were purified from T. brucei hits, of which 4 were novel (HD257-1, HD215-1, HD361-1 and HD1158-1), mostly terpenoids. Known compounds identified included antiamoebins, efrapeptins and tenuazonic acid. The most potent of the novel compounds (HD257-1) showed poor selectivity on secondary evaluation. Five promising L. infantum hit compounds with micromolar primary assay potency were being investigated as the project ended: one novel (HD227-2) and two known compounds (cardinalisamide A and hyphodermin C or D) had been identified but two remain unidentified.
37 Sudanese plant extract HPLC fraction hits against L. infantum were purified further yielding 79 compounds for primary assay testing. Out of these, 10 compounds had EC50 values in the range 5-15 µM. These were mostly known compounds, some of which had been previously reported to have antiparasitic properties and included carnosic acid, guieranone A, penicilloside E (a pregnane glycoside), acetyl-11-keto-β-boswellic acid and 12,16-dihydroxy-7,13-labdadien-15,16-olide. The plant extract hits in the T. brucei and T. cruzi assays were not investigated due to the numbers involved for the T. brucei hits and the lack of selectivity of the T. cruzi hits, although a small number of candidates for further study from the latter group were identified, after counterscreening of selected hits against a small panel of both tumoral and non-tumoral cell lines, at the end of the project.
The most promising PTR1 inhibitory screening hits were from plants rather than the MycoDiverseTM library. Scale-up purification yielded 10 compounds, 9 of which had been previously reported. These were mostly flavonoids or phenolics, compound classes already investigated by the NMTrypI consortium, including rosmarinic acid, cerasidin, pinocembrin and 2’’-O-β-D-apiofuranosyl-6-C-β-D-glucopyranosyl-4’,5,7,8-tetrahydroxyflavone, 7,8-dimethyl ether. Compounds from other classes identified included harmine and 5-acetylcucurbitacin I-3-O-β-D-glucoside and these were investigated in molecular modelling PTR1 docking studies. The discovery of Lead compounds from natural product libraries were postponed from M24 to M30 due to delays in completing screening and follow-on effects on assay-guided purification and hit compound identification.
Task 3.2 Phenotypic assays (Leader: CNPEM) (D3.1 and MS9-MS11)
One of the objectives of NMTrypI was to set up a screening platform for trypanosomatids using phenoptypic assays, for the screening and discovery of active molecules with antiparasitic activity of both natural and synthetic origin.
A phenotypic screening platform for trypanosomatids was developed and optimized during the duration of the project. The main advantages of this screening platform was to enable the parallel screening of both synthetic and natural product compound libraries, resulting in the discovery of new compounds with antiparasitic activity against either one of more of the three parasites, Trypanosoma brucei, T. cruzi and L. infantum. Of note, the assay development process resulted in improved and more robust phenotypic screening assays for T. cruzi and L. infantum. These improved cell-based screening assays will likely contribute to the discovery of molecules that are more likely to have antiparasitic activity in a physiological, diseased environment. These assays were used to screen natural products and syntethic compound libraries.
Natural products provide a vast range of bioactive chemical diversity and many anti-infective agents and other medicines have a natural origin, including some anti-parasitic drugs. The natural products discovery element of the NMTrypI programme has been based on the largest anti-trypanosomal and anti-leishmanial screening programme using modern high-throughput phenotypic and target-based assays to explore natural product libraries ever undertaken. This was achieved in a unique collaboration of scientific teams working at four different institutions based in three different continents: the National Center for Research, Khartoum, Sudan; Fraunhofer-IME ScreeningPort, Hamburg, Germany; Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, Brazil; and Hypha Discovery Ltd., Uxbridge, UK. The libraries tested were Hypha’s MycoDiverseTM library of fractions and extracts from fermentations of higher and insect-associated fungi (11,000 samples) and a library of semi-purified HPLC fractions generated from extracts of Sudanese medicinal plants (3312 samples from 137 extracts). They were tested in phenotypic assays against Trypanosoma brucei, Trypanosoma cruzi-infected U2OS cells and Leishmania infantum-infected THP-1 cells as well as in target-based assays against T. brucei and L. major pteridine reductase 1 (PTR1) enzymes. The main focus for screening hit follow-up and assay-guided purification was the phenotypic assay hits. The primary screening hit rates for both MycoDiverseTM libraries and the plant extract HPLC fractions were high for T. brucei, lower for T. cruzi and lowest for L. infantum.
Seventy two of the most promising MycoDiverseTM library samples active in the phenotypic assays (hits), prioritised by anti-parasitic potency and producing organism diversity, were subjected to a small scale assay-guided purification to link anti-parasitic activity to chromatographic peaks and physico-chemically characterise the active components by HPLC with diode array UV-visible and mass spectrometric detection. This was most successful for the T. brucei and L. infantum hits and less so for the T. cruzi hits. Further investigations were focused on the T. brucei and L. infantum hits. Fermentations producing potentially novel compounds with promising antiparasitic activity were reproduced at larger scale and the active compounds purified for structure elucidation. Seven compounds were purified from T. brucei hits, of which four were novel, predominantly terpenoids. Five promising L. infantum hit compounds with µM primary assay potency were being investigated as the project ended: one novel and two known compounds had been identified but two remain unidentified. Identification and evaluation of the most promising anti-parasitic compounds identified from the MycoDiverseTM library will continue at Hypha beyond the end of the NMTrypI project.
Investigation of Sudanese plant extract-derived phenotypic assay hits focussed on antileishmanial activity. Ten compounds were purified with EC50 values in the range 5-15 µM. These were mostly known compounds, some of which had been previously reported to have antiparasitic properties.
The NMtrypI natural product screening campaign has been successful in identifying new anti-parasitic agents with promising properties but these need further evaluation.
A 2000 synthetic compounds, provided by NMTrypI parterns, were also screened in the phenotypic screening platform. Active hits were selected for confirmation in dose-response, generating half-max inhibition of infection values (EC50) for determination of compounds’ potency. Overall, several tested compound presented promising antiparasitic activity in the phenotypic assays, with EC50s compatible with a hit profile (i.e. below 10 μM), and these will be further explored as starting points for lead optimization and the discovery of new chemical entities with selective antiparasitic activity.
The most advanced hits (small molecule, synthetic compounds from leading series) with anti-T. cruzi activity were screened against a panel of T. cruzi strains. Some of the compounds showed broad spectrum activity against the T. cruzi strains, indicating that these compounds show potential to become new leads for antichagasic drug discovery. Overall we obtained the following hit rate for compounds that showed inhibition percentage of parasite growth >80% at 50 μM: Tbrucei: 26% NMT e 6.5% Tybox, Leishmania: 7,4% NMT e 0.5% Tybox,Tcruzi: 11% NMT e 1% Tybox.
Task 3.3 - In vitro off-target liability, toxicity, ADME and safety studies prior to animal toxicity studies (Leader: ESP) (D3.2 D3.3 MS10-MS11)
The Fraunhofer have screened 10 Tranche of synthesized and natural product extracts (HYPHA MycoDiverseTM library and African medicinal plants from Sudan) as part of the regular SAR screening against the Lm-PTR1 and Tb-PTR1 enzymes. All data data uploaded onto SEEK. All screening data analysis (single compound concentration and dose-response studies) were performed using ActivityBase XE. A total of 271 data set have been analysed during the lifetime of the NMTRYPI project which includes screening of mitochondrial toxicity, hERG, A549 cell-line, WI38 cell-line, Aurora B kinase, CYP450 1A2, CYP450 2C19, CYP450 2C9, CYP450 2D6, CYP450 3A4, as well as data analysis on behalf of CNPEM (NMTrypI consortium partner).
Task 3.4 - Selection of best compounds in detailed, direct cell-based assays (Leader: BNI) (D3.2 D3.3 MS10, MS11)
Laboratory scale testing of activity was performed on 441 compounds from different origins: synthesis (NMT-F, NMT-H, NMT-Ty, NMT-C, NMT-A) and Hypha natural compounds. These compounds at a single dose of 10 µM, were tested against bloodstream forms of Trypanosoma brucei brucei using a resazurin based assay, Leishmania infantum infected THP1 cells with parasites overexpressing luciferase. Toxicity evaluation was also performed using MTT assay on the host cell for the Leishmania assay, THP1, enabling an estimation of the selectivity index. The compounds that presented anti-parasitic activity > 70% (were further evaluated for EC50 determination and selectivity index evaluation, SI>10). The EC50 was determined for 139 compounds for T. brucei and 23 for Leishmania. This enabled the prioritization of compounds with SI>10, enabling the identification of 58 for T. brucei and 20 for Leishmania that were considered as hits. These hits were prioritized for subsequent animal testing (WP5). For Leishmania the best hit presented low micromolar potency and SI>70 (DRUG LEAD 1) while for T. brucei sub-micromolar potency and SI>250 (NMT-H0087).
Compounds of the NMT-F and NMT-H series were tested against T.cruzi-infected HG39 cells and against L. donovani-infected macrophages to select leads (BNI). A PCR method was used in this case. Compound NMT-H0080 was identified as lead compound for further development. Lead compounds DRUG LEAD 2, DRUG LEAD 1, NMT-C0074 and miltefosine were tested against macrophages infected with 3 L. donovani isolates, 1 L. braziliensis isolate, and 2 antimony-resistant L. donovani strains. NMT-C0074 showed the most consistent activity against all tested strains of Leishmania.
Combination studies. The dual targeting of (pteridine reductase 1) PTR1 and (dihydrofolate reductase) DHFR in trypanosomatids has been suggested as a possible therapeutical approach. Several molecules are capable of potentiating the activity of the known DHFR inhibitor MTX. Moreover, chemical entities with no intrinsic anti-parasitic activity, were able to increase the inhibitory activity of MTX in T. brucei. Another mechanism based combination is known in the anti-infective therapy, the beta-lactamase inhibition and the beta-lactam antibiotics.
We address the mechanism associated with synergism performing cellular biochemical studies using two pteridines derivatives that are TbPTR1 inhibitors with sub-nanomolar potency, and the traditional DHFR inhibitor MTX, that represents the best known tool for our proof of concept studies. We performed time-dependent, concentration-dependent reversal (using natural substrate of PTR1 and DHFR) inhibition studies. Our approaches demonstrated that PTR1 and DHFR enzymes are involved in the combination efficacy and give a proof of concept validation in vitro of the combination tool mechanism. More studies are now needed to validate the concept in vivo.
WP4 Drug Leads: mechanism of action, biomarkers, and resistance
1. Identification of biomarkers and targets/off-targets for lead drugs
2. Identification of drug resistance genes for drug leads
3. Computational studies of interactions with targets identified in (1) to guide iterative optimization
Task 4.1 Identification of targets and off-targets for lead compounds: Biomarker and target identification
4.1.1 - Comparative Proteomics
188.8.131.52 Proteomic studies on miltefosine and its derivates (NMT-A-0001, NMT-A-0002) (Reported in Deliverable D4.1 D4.2 and D4.5 MS12)
With the aim of identifying a protein subset characterizing the cellular activity of Miltefosine (MIL) and of 2 lead compounds we have performed:
• Mass Spectrometry studies to identify proteomic changes induced by IC50 drug treatment.
• Bioinformatics data interpretation and analysis to expand the core set of identified proteins.
Whole cell protein extracts of untreated L. donovani, and L. donovani treated with IC50 of test compounds and controls, were produced at BNI and shipped to UNIMORE for mass spectrometric analyses. 141 proteins were found differentially expressed by at least one drug treatments (ratio value ≤ 0.6 or ≥ 1.5); of these 35 proteins were similarly modulated by the three drug treatments whereas 106 were differently modulated by the three drug treatments. For 80 proteins out of 141, information on their function could be retrieved from available bioinformatics databases; the other, 61 proteins, were defined as uncharacterized proteins. We grouped the identified proteins into 7 functional groups (UniProt http://www.uniprot.org/; InterPro 1 protein synthesis, folding, stability and degradation: 26 proteins; (2) energy production: 11 proteins; (3) folate pathway: 5 proteins; (4) motility: 4 proteins; (5) transport across membranes: 10 proteins; (6) biosynthesis, amino-acid catabolism, kinase or phosphatase activity: 15 proteins; (7) signal pathways, surface glycoproteins, cell cycle regulation, DNA transcription, binding and regulation: 13 proteins. The modulated proteins were also combined with proteins reported in the literature as connected with Miltefosine mechanism of action; the complete set of proteins were entered into a StringDB analysis to give a more complete view. Results from this study lead to the identification of a protein profile associated with the mechanism of action of Miltefosine and the two drug leads (DRUG LEAD 2, DRUG LEAD 1). Two main proteins have been identified as largely overexpressed and therefore were structurally and functionally characterized (WP2). Validation of their role within the mechansim of action of the compounds remains to be achieved.
184.108.40.206 Target(s) identification for compound NMT-H0080.
We identified a new lead (named NMT-H0080) showing low microMolar EC50 towards Leishmania infantum, L. donovani and L. major. Thus, NMT-H0080 is a potent antileishmanial agent active towards both visceral and cutaneous Leishmaniasis. The compound profile shows that the molecule displays an activity similar to Miltefosine, one of the drug currently used to treat Leishmania infections, but a lower toxicity (see preliminary results). Structure-activity relationship studies have been carried out to explore the importance of the different chemical features of NMT-H0080 for the antileishmanial activity.
With the aim to develop a method to understand the NMT-H0080 mechanism of action and to identify the compound protein target/s, the following experiment sets were performed. First (i), a fluorescence-based experimental approach for the identification of the cellular target(s) combined with protein target identification though non-denaturing gel electrophoresis ; secondly (ii) mass-spectrometry proteomic investigation of the biological processes involved in the mechanism of action of the compound in treated L. donovani parasites.
i) Fluorescence-based experimental approaches for the identification of the cellular target(s).
Two compounds, NMT-H0080 and NMT-H0087, were highly fluorescent and their emission spectra shifted markedly to the red upon binding to a protein (we tested BSA and HSA), with a shift of the maximum from 440 (blue) to 530 nm (green). We observed a similar emission spectral shift upon treating NMT-H0080 solutions with pathogenic cell extracts. With the aim of identifying the intracellular interactors of these compounds, hence highlighting their mechanism of action, we took advantage of this feature to set up a test based on almost non-denaturing electrophoretic separation of cellular extracts treated with NMT- H0080 and the real-time observation of green emission from SDS page bands containing complexes of the fluorescent inhibitor with its strongest cellular interactors. Our preliminary tests have given encouraging results and this research will be continued in the near future.
Some observations during in vivo tests have raised doubts on the chemical stability of NMT-H0080. We have thus performed a spectroscopic (absorption/emission) analysis of the stability of the compound in different conditions. We have established its stability in organic solvents and have observed a decrease in absorption/emission in water. This took place in a time-scale of several tens of minutes but was strongly accelerated by nitrogen bubbling in the sample cell. This suggests that, rather than undergoing a chemical degradation, this compound tends to aggregate (or self-organize) at the gas/water interface, consistently with its amphiphilic chemical nature. Indeed, addition of HSA reversed the process causing a recovery of the absorption/emission of the compound, likely through its binding at some hydrophobic cleft of the protein.
ii) L. donovani parasites were treated, or not, for 24 hours with IC50 concentration of NMT-H0080, then whole cell protein extracts were prepared for mass spectrometry analysis, performed at Prof. Hartmut Schlüter’s lab, University Medical Centre Hamburg – Eppendorf, Department of Clinical Chemistry, in order to obtain differentially expressed proteins after the NMT-H0080 treatment. 406 proteins were identified and quantified and 17 modulated proteins were obtained: 7 proteins are down regulated and 10 proteins are up regulated.
Preliminary results suggest that proteasomal proteins may have a major role and worth further investigations.
220.127.116.11 Mass spectrometry proteome analysis of serum samples from dogs treated with DRUG LEAD 1.
The search for a proteomic signature characterizing the action of compound DRUG LEAD 1 was performed in serum samples from dog trial (WP5), to obtain a pre-clinical pharmacodynamics biomarker of the drug lead activity. This signature will serve as translational tool from the pre-clinical to the clinical study and is defined as “companion diagnostic”. The dogs were treated with DRUG LEAD 1 and blood samples were collected in non-invasive way at the appropriate time (see WP5-.Task 5.3 Dog testing). This work gave preliminary information about the potential to identify relevant proteins in the dogs serum ableto serve as biomarker to monitor the drug efficacy. We were able to identify 31 proteins as a whole where 3 proteins are more relevant. This work will be further optimised and finalized soon after the project end. Ethical requirements were followed.
Deliverables D.4.1 D.4.2 and Milestone MS12 were achieved and progress was reported therein.
Proteomic studies on dog serum samples from Leishmania infantum trial on DRUG LEAD 1 was perfromed. Preliminary results have been obtained, but the study is not finalized yet.
4.1.2 - Protein synthesis profiling
To complement the mass-spectrometry-based proteome analysis which has limited sensitivity and cannot distinguish between protein synthesis rate, proteins stability and protein modifications, we implemented the newly described Ribosome Profiling analysis (Vasquez et al., 2014, Nucleic Acids Res 42. 3623ff) to monitor protein synthesis rates after exposure to three lead compounds. In short, poly-ribosomes were isolated from parasites treated, or not, with lead compounds and treated with nuclease to degrade non-ribosome-bound RNA tracts. The nuclease-protected RNA fragments were then subjected to RNA-Seq using Illumina Next-Seq technology. Resulting sequences were then subjected to semi-quantitative alignment analysis against the Leishmania infantum genome database to deduce the relative ribosome density for each coding region. Genes with low ribosome density (< 0.100) in the solvent control were excluded and induction/repression > 3-fold was recorded. Between 64 and 200 proteins showed induced synthesis under the three drug leads and were compared with proteins identified by mass spectrometry.
Task 4.1.3 - Tagged lead affinity precipitation
Two lead compounds were synthesised with biotin tags for streptavidin-affinity chromatography-based target identification (Task 4.1.3). To verify functionality of the tagged compound, dose-effect analysis was performed on the growth of Leishmania donovani in vitro. No IC50 could be established for either tagged compound; the tagged compounds were inactive up to 50 µM. In vitro pull-down using recombinant proteins corresponding to suspecteded targets (see Tasks 4.11.) was performed. No binding of recombinant proteins to the tagged compounds was observed. Leishmania whole cell lysates were mixed with tagged compounds and precipitation was performed. Precipitated proteins were analysed by SDS-PA gel electrophoresis and high sensitivity silver nitrate staining. No bands specific for the tagged compound could be observed. Explanations: i) the tag abrogates target affinity, ii) the tag precludes lead uptake by parasites, iii) the suspected target proteins are not bound directly by the lead compounds.
Task 4.2 - Identification of drug resistance genes for drug leads
4.2.1. - Spontaneous resistance against drug leads
Leishmania donovani parasites (promastigote in liquid culture) were selected at IC50 for solvent, control and lead compounds in duplicate biological samples. Selection and passage were performed for 3 months until the selected populations grew at equal rates to untreated controls. The selected populations (in duplicate) were subjected to genomic DNA purification followed by Deep Sequencing of the entire genomes (~30-fold coverage) to identify genetic changes under drug lead challenge. Given the a natural genetic variation between L. donovani strains and the L. infantum genome database, analysis of single nucleotide polymorphisms (SNPs), insertions and deletions (INDELs) and other variations had to be restricted to known candidate genes while gene copy number analyses and chromosome amplifications were analysed genome-wide.
Deliverable D4.3 and Milestone MS 14 were achieved and progress was reported therein.
4.2.2 - Cos-Seq mapping of dominant resistance gene loci
The Cos-Seq strategy makes use of cosmid library-based functional cloning combined with batch sequencing of selected cosmids and was described recently (Gazanion et a, 2016; Tejera-Nevado et al., 2016). Parasites transfected with the cosmids from a Leishmania donovani genomic DNA library were selected in vitro either as intracellular amastigotes in mouse macrophages, or as promastigotes in axenic culture. Using intracellular amastigotes, the more natural system, has limited selective effects due to the low number of generations that can be achieved (Tejera Nevado et al., 2016). Selection of transgenic promastigotes identified a highly limited number of cosmid species representing a small number of gene regions in the Leishmania genome.
Milestone MS13 was achieved and progress was reported therein.
Task 4.3 - Computational studies of interactions of targets identified in (4.1) to guide iterative optimization
The main achievements are
(1) Computational binding site analysis for models of on-target and off-target proteins for the design and discovery of selective anti-parasitic compounds targeting the folate pathway.
(2) Models of proteins overexpressed in L. donovani upon administration of DRUG LEAD 1 or playing a role in the MoA of miltefosine or the NMT-A compounds, providing insights into putative mechanisms.
The binding properties of known targets of the parasitic folate pathway were extensively characterized for the design of selective compounds Further folate-pathway proteins were investigated by modelling.
• The binding pocket of human folate receptor β, a potential folate/antifolate transporter, was analyzed with the aim of optimizing its targeting by pteridine derivatives and other compounds.
• Based on the association of human AKT-1 kinase with apoptosis, we investigated whether this protein could be exploited for anti-kinetoplastid drug discovery. We also attempted to identify putative Akt1 kinases in the parasites and evaluate their sequence and structural relationship to human Akt1 kinase. Further mechanistic and structural data would be needed to pursue Akt1 further for anti-parasitics.
• On the basis of the knowledge that dinitroaniline-herbicides affects parasitic alpha-tubulin, the leishmanial tubulin was proposed as a target for dinitroaniline-ether-phospholipid hybrids. The leishmanial tubulin was modeled and the interaction with the respective NMT-A series of compounds was investigated by docking yielding insights into putative binding sites.
• Computational methods were applied to investigate selected newly identified protein targets from mass-spectrometry and proteomics experiments. Sequence analyses, secondary structure prediction and, if the templates were available, homology modelling and docking was performed to study the possible interactions with the substrates and inhibitors.
• For putative L. donovani lead target proteins, over-expressed in the mass-spectrometry study upon treatment with leads, homology models were constructed, and a docking analysis of the compound and fragments thereof was performed. Structural investigation of the compound moiety interactions in available crystal structures was performed to search for moiety binding motifs.
• The putative lead transporter likely involved in compound transport, was found and analysed.
Deliverable D4.4 and Milestone MS15 have been achieved and progress is reported therein
WP5 Animal models and candidate drug evaluation. PK, toxicity and safety studies
1. To determine the efficacy of non toxic and safe selected Leads (WP2) against laboratory models of trypanosomatid infection (Trypanosoma spp, Leishmania spp)
2. To study the efficacy and PK of 2 compounds (WP 5.1) against L.infantum infection in dogs.
Task 5.1. Animal evaluation on 2 types of models.
Subtask 5.1.a. Evaluation of the efficacy and safety of the lead candidates on T. cruzi, T. brucei, L. major, L. infantum infections in mice.
Work package 5 evaluated the in vivo activity on three distinct infections (by the parasites, Leishmania, Trypanosoma brucei and Trypanosoma cruzi) with 6 distinct infection models (state of the art in vivo imaging of infected animals (for T. brucei and T. cruzi) - Leishmania: BALB/c, Hamster and dog; T. brucei: BALB/c acute and chronic; T. cruzi: BALB/c) and the toxicity of the compounds that were selected from the other work packages. The work package 5 tested 18 different compounds in vivo. The tests involved PK evaluation, in vivo activity evaluation and toxicity evaluation The most significant results from this work package concern the demonstration of greater in vivo activity of DRUG LEAD 1 over the parental molecule miltefosine in three distinct animals models for Leishmaniasis (BALB/c, Hamster and Dog). This data was supported by PK data that fully support the therapeuthical options taken. The compound DRUG LEAD 1 presented oral availability (as determined by SNAP-PK) and also was bio-accumulated in the disease relevant target organs, spleen and liver. Also significantly, no major toxicity was found during the in vivo assays (as determined by evaluation of general health, blood works and routine biochemistry analysis - hepatic and renal parameters) in the three infection models. Also a comparative non-regulatory toxicity assays in BALB/c mice suggest that miltefosine in more toxic than DRUG LEAD 1 (maximum tolerated dose in a 7 day toxicity assay was 50 mg/kg for miltefosine and 100 mg/kg for DRUG LEAD 1). Therefore the data on DRUG LEAD 1 fully support it as a more active and less toxic alternative to miltefosine for the treatment of Leishmaniasis. DRUG LEAD 1 also showed in vivo activity for a second parasite (T. brucei). This activity was shown by full clearance of infection in an acute BALB/c T. brucei brucei infection model using a daily treatment of 50 mg/kg (per os) for seven consecutive days. The parental compound miltefosine showed no in vivo activity in the same treatment schemes. DRUG LEAD 1 also proved active another mice model that is representative of an acute infection with dissemination to the central nervous system. The daily treatment with 50 mg/kg for 13 day lead to a significant decrease of infection and increase in animal survival. Significantly this decrease was also found in the central nervous system as seen by the decrease of parasite signal in the brain. Moreover the continuous overtime decrease of the signal during the treatment of the animals suggests that treatment optimization might enable full clearance of the infection. Altogether DRUG LEAD 1 is effective in the BALB/c infection model for T. brucei and can cross the blood brain barrier being a possible therapeutical option for HAT. DRUG LEAD 1 was also tested in the T. cruzi model of infection but showed no activity (unlike the parental molecule miltefosine that presents some activity).
Another major axis of the project was the evaluation of several compounds from distinct chemical families that were designed as PTR1 inhibitors. Most the compounds from this series that arrived to the in vivo testing presented problems with availability in the plasma as determined by SNAP-PK studies, still a few presented oral availability but the PK parameters were not suggestive of in vivo activity in any of the animal models. The only exception was NMT-F252. This compound active against T. brucei in low nanomolar range (EC50 = 90 nM) presented a PK profile that due to the high potency was adjusted for in vivo treatment. The bi-daily treatment with 20 mg/kg was not capable of diminishing the infection or increase the survival of the animals in the acute T. brucei infection model..
Subtask 5.1.b Evaluation of the efficacy of drug candidates in L. infantum infections in hamsters
A compounds. Short term treatment
Antileishmanial efficacy and biodistribution of DRUG LEAD 2 and DRUG LEAD 1 was tested in experimentally infected hamsters in an early infection model. For comparative purposes miltefosine-treated hamsters were also included. Hamsters were infected with 108 promastigotes of a local strain of Leishmania infantum. Infection was allowed to progress for 60 days. Animals were treated with 20 mg/kg lw, 5 days. Ten days after the last treatment day the hamsters were euthanized, organs obtained and samples taken. Immediate adverse effects were more evident in miltefosine-treated hamsters. No variations were observed in alkaline phosphatase, creatinine and urea levels miltefosine (90.5±7.06) although more disperse.
A compounds. Long term treatment
Results previously obtained in both BALB/c mice (PK, efficacy) and short-treatments in hamster (biodistribution, efficacy) prompted us to evaluate the efficacy in hamsters of a long treatment (21 days) with DRUG LEAD 1 (10 mg/kg/day) (9 animals) compared to two dosages of Miltefosine (10 or 20 mg/kg/day) (8 + 5 animals, respectively). Untreated control hamsters (8 animals) and uninfected + untreated (5 hamsters) were included. As efficacy criteria live weight, short-term effects, pathology (lesions in vivo and post-mortem), toxicity (liver and renal markers) and reduction of Leishmania burden in spleen and liver were considered. Results showed that no differences were present in the spleen size and live weight/spleen weight ratio on DRUG LEAD 1 and the higher dose of Miltefosine. No relevant toxicological effects were present with any of the treatments. Parasitic burden in the spleen was reduced below the detection limit of the technique in 8 out of 9 hamsters treated with DRUG LEAD 1 (10 mg/kg) whereas a double dose of Miltefosine (20 mg/kg) was needed to get a comparable efficacy (5/5). On these grounds it was considered that at the same therapeutic regime (10 mg/kg/day, 21 days) DRUG LEAD 1 was superior to Miltefosine.
Based on the PK results obtained in BALB/c mice with NMT-H080 and NMT-H087 with an apparent increase of bioavailability of NMT-H080 using cyclodextrins (CDX) a proof of concept was carried out to test the antileishmanial efficacy of oral NMT-H080 with 30% or 50% CDX (20 mg/kg, twice a day, 10 days) in a chronic model of L.infantum infection in hamster compared to Miltefosine (20 mg/kg/day, 10 days). Hamsters (54) were infected (IV, 108 promastigotes) and the infection was allowed to progress for 16 weeks. Infected animals were treated with NMT-H0080 in 8% DMSO (40 mg/kg/day, 10 days), NMT-H080 +30% CDX with the dosage, NMT-H080 + 50% CDX with the same dosage, Miltefosine (20 mg/kg/day), 30% CDX, 50% CDX, 8% DMSO. In addition an uninfected control group was included. Results showed that oral administration of NMT-H080 or CDX did not induce any noticeable adverse effects, no significant alterations of transaminases (AST, ALT) or hepatic and renal functionality markers (Creatinine, alkaline phosphatase or urea). NMT-H080 (40 mg/kg/day, 10 days) only reduced slightly the parasite burden (25.8-35.7%:spleen; 19-20.8%: liver) of infected animals whereas Miltefosine-treated hamster showed 97.3% reduction in the spleen and 81.2% reduction in the liver. In addition CDX formulation did not increase the antileishmanial efficacy of oral NMT-H080.
Within the context of synergy with KINDRED consortium, a compound, J-02, was similarly tested in hamster (chronic infection, dosage). The compound did not show any notable activity against chronic leishmaniasis in hamster.
Task 5.2. PK studies on spleen, liver, bone marrow samples
Pharmacology and biodistribution of promising molecules were determined using the Snapshot method in BALB/c mice. Analyses were carried out with MC-MS/MS. Molecules studied belonged to 3 families: A compounds (DRUG LEAD 2, DRUG LEAD 1, NNMT-A0004 and NMT-A0021); H compounds (NMT-H005, NMT-H0024, NMT-H0080 and NMT-H0087); C compounds (NMT-C0044, NMT-C0090 and NMT-C0091). Determined parameters in plasma (Cmax, t1/2, AUC) and biodistribution in target organs for Leishmania allowed to select 2 compounds from the A family and and 2 molecules from H family to be tested in hamster. Pharmacological properties from H compounds showed a low bioavailability and an improvement was attempted with cyclodextrins. A compounds were studied in hamster and the most promising molecule, DRUG LEAD 1, also in dogs. Plasma PK parameters and biodistribution in target organs of this molecule were consistent with the antileishmanial efficacy observed.
Task 5.3. Dog testing
Beagle dogs (24), females, 4-5 months old, negative for L.infantum (IFAT) were housed until they reached 9-10 months age: 20 dogs were inoculated (IV) with 108 amastigotes of a recebt isolate of L. infantum. Four animals were kept as uninfected+ untreated control dogs. Along the infection the animals were daily observed and subjected to a complete clinical examination, blood sampling and weighing every 2 weeks. Monitoring included clinical and lesion score, immune response (IFAT), Biochemical and haematological profile (46 parameters). Inoculation was successful and by 12 weeks p.i. all animals showed clinical signs and lesions and IFAT titres corresponding to leishmaniasis (>1/40). In addition popliteal lymph node smears allowed the detection of parasites in all inoculated dogs. Four months p.i. the infected animals were divided in a stratified manner (weight, clinical score, IFAT) onto 4 groups: G1 (6 dogs) treated with DRUG LEAD 1 (2 mg/kg/day, 28 days); G2 (6 dogs) with DRUG LEAD 1 (10 mg/kg/day, 28 days); G3 (4 dogs) with Milteforan® [Miltefosine] (2 mg/kg/day, 28 days) and G4 (4 dogs) treated with the vehicle. All medications were given per os (PO). Assessment of efficacy included: reduction of clinical signs and lesions, improvement of haematological and biochemical parameters and parasitological evaluation of the animals. Results obtained showed the absence of notable post-treatment adverse effects except for reversible diarrhoea and sialorrhoea (nausea). Treatment elicited a recovery of weight of infected animals although slowed in the group treated with Miltefosine. Clinical score of treated animals, 1 week post treatment, diminished in all cases, particularly and more homogeneously when the animals received DRUG LEAD 1 (both doses).Treatment with DRUG LEAD 1 and Miltefosine normalized the values of proteinemia, albuminemia after 5 weeks post treatment. Animals treated with DRUG LEAD 1 showed a more rapid (1 week post treatment) normalization of these parameters. In addition, drug lead 1 elicited a reduction of immunoglobulins from 1 week pt onwards whereas 2 dogs treated with Miltefosine (50%) even after 5 weeks were out of the physiological range. Infected dogs had a mode IFAT titre of 1/1280 (week 16 pi), Five weeks pt DRUG LEAD 1 elicited a 2 to 4-fold decrease and 2 animals (high & low dose) were IFAT negative (<1/40). Three out of 4 Miltefosine.-treated dogs, after 5 weeks, had IFAT titres ≥1/1280. All inoculated dogs were positive before treatment by lymph node punction. Three weeks after treatment, DRUG LEAD 1 high dose treated had apparently cleared the infection in the organ (0/5) whereas Miltefosine treated animals were positive. Bone marrow samplings showed that 2 dogs from DRUG LEAD 1 high dose and DRUG LEAD 1 low dose were negative. DRUG LEAD 1 administered per os, under our conditions, was superior to that achieved by the standard oral treatment for canine leishmaniasis (Milteforan®) based on clinicopathological, immunological ad parasitological parameters. Samplings carried out 4 months after the end the treatment (spleen, lymph nodes) confirmed the superior antileishmanial efficacy of DRUG LEAD 1 compared to Miltefosine.
Conclusion and dogs follow up
There is extensive information previously published on natural and experimental dog leishmaniasis with L. infantum. Moreover, 1) along our experiment a thorough follow-up of the animals was carried out; 2) substantial improvement of the clinical status of the animals treated, especially those medicated with DRUG LEAD 1 was evident; 3) significant reduction of parasite burden in target organs was found in dogs treated with DRUG LEAD 1. These results and the consideration of 3Rs principles in animal experimentation supported the decision of avoiding euthanasia as end-point of the experiment. Consequently, animals were subjected to minimal invasion surgery to sample target organs. Those animals with more severe clinical signs were treated and all Beagle dogs were given for adoption. This strategy was supported by the Regional Government (Comunidad de Madrid) and will be considered a model for future experiments with dogs. In addition it earned us a distinction from an Animal Protection Society and, scientifically, will allow a long-term (up to 2 years) follow-up of the animals.
WP6: Database design and data management. Collection of data from all WPs for easy data handling
1. Designing and setting up the data management support for the project
2. Performing data management for the project, more data integration and analysis.
NMTrypI SEEK, Synergy SEEK systems for sharing data within the NMTrypI project and in the FP7 synergy projects, respectively
Data management for the project
Data sharing policy
In collaboration with UNIMORE set up of a replacement website for the Interface Europe-operated NMTrypI web site.
Together with UNIMORE we set up the general public web site at fp7-nmtrypi.eu. This became necessary because of the fact that Interface Europe were unable to maintain their web site due to their exit from the NMTrypI Consortium. The design of the current version is being improved by HITS staff member Marcel Petrov at no cost for the consortium.
The SEEK data management system used for NMTrypI, is co-developed between the University of Manchester and HITS. It has been built as a tool for cataloguing and publicizing the results of large research projects with a broad diversity of experimental data and methods. SEEK is successfully used as core data management system in several national and international research projects and is partly funded by the transnational FAIRDOM project, as well as the German Network for Bioinformatics Infrastructure and a BBSRC Synthetic Biology Centre in Manchester, UK. SEEK is free and open source software under the BSD license.
SEEK is a generic system that is able to store any kind of data file. It enables structuring and enriching the data after upload. Furthermore, it adapts to the workflow of the users in that data can be structured and enriched after upload. Versioning support facilitates keeping track of different versions of the same file.
The NMTrypI project consortium uses two different SEEK instances for their research data management: NMTrypI SEEK (https://nmtrypi.h-its.org/) is password protected and for project internal use only. Synergy SEEK (https://fp7h-synergy.h-its.org/) is used for publication and dissemination of research data to share the NMTrypI project results with other drug discovery projects and the scientific community. After ending the project, HITS offered to maintain both SEEK instances at least for the next ten years until January 31st, 2027.
Within the NMTrypI project additional features in SEEK were developed and implemented to meet the user requirements for the exploration and visualization of the research data and especially for the handling of chemical compound specific information.
Compound and protein specific SEEK features
The handling of compound related data and the representation of compound structures based on SMILES (Simplified Molecular-Input Line-Entry System) strings relies on compound IDs being used approximately as agreed in the consortium: Compounds are identified by strings of the form NMT-X-12345. NMT stands for NMTrypI, X is a letter that corresponds to a group within the project, and 12345 is a 5-digit number. However, among the user of the NMTrypI data management there is only weak agreement on (i) how many digits constitute the proper NMTrypI compound number, (ii) if the separation between the blocks is “-“ or “_”. Others distribute the compound ID over several columns or cell, e.g. writing only A1 and leaving “NMT-“ implicit or as legend. To overcome these problems SEEK tries to recognize in an intelligent manner the use of compounds and indexes the data accordingly. So it is possible to find the compound ID “NMT-A1” with the query “NMT_A001”.
If a compound identifier is detected in an Excel table additional functionality is connected to that compound, e.g. searching the SEEK database for all data files containing information about the selected compound. The search result includes not only a list of data files but also data preview from the Excel tables.
A compound summary report collects all data for one compound from all accessible Excel tables and displays this information in one single document. Each user gets a compound report that reflects the data they are allowed to see. A small symbol next to the compound identifier links up with the visualization of the chemical compound by drawing the chemical structure based on an available SMILES string.
One of the main requirements for the NMTrypI project partners was to be able to search all compound data in SEEK by their chemical structure. Based on the available SMILES strings within the Excel tables in SEEK we implemented a graphical representation for drawing 2D molecules as input for substructure (see Figure 4) and similarity search tools in SEEK. The results allow the user to view all the files in SEEK that contain the resulting compounds and for an exploration of the files and data relating to those particular compounds.
New features specifically implemented in SEEK for the NMTrypI project include the automatic detection of UniprotKB accession numbers based on regular expressions. These UniprotKB identifiers are highlighted in Excel tables and a popup menu is available offering links to the Uniprot database (http://www.uniprot.org/) and StringDB (http://string-db.org/).
Data visualization in SEEK
With an emphasis on data visualization, we have expanded the system such that one can select and visualize data from different sheets of the same file with an Excel-like selection, i.e. sheet1:sheet3! A1:F200 would select the cells A1 to F200 from sheets 1-3. Past and present visualization functionalities were expanded such that they could include data from different sources. That will serve as a middle step to selecting and plotting data from different Excel files.
Interactive exploration of the data can be achieved through the newly introduced Parallel Coordinates plot and the improved Heat Map function. Data can be visualized as interactive heat maps (see Figure 6) where thresholds corresponding to the colours can easily modified using sliders. The new heat map is instantly calculated and visualized.
A parallel coordinate plot allows to visualize and analyze multivariate data such that insights could be drawn from easily observed 2D patterns. The user may choose to filter the data within the plot and rearrange it in different ways. As often happens in experiments, not all parameters are always measurable. The plot allows to account for these cases and learn of their distribution in the data through the horizontal ‘missing values axis’. Both plotting functions allow for immediate data extraction by hovering on the data, interactive filtering and coloring, as well as exporting the plots as an image.
Main deviations and contingency measures
The milestones were met.
WP9: Synergic collaborative activities
Objectives for the reporting period
1. To identify and develop specific concepts concerning in vitro (cell screening) and in vivo (animal testing) candidate drugs to be shared among the three funded projects (A-PARADDISE, KINDReD, NMTrypI) focused on drug development in Neglected Parasitic Diseases.
2. To perform activities among the three projects that can help the communication, process analysis and create a larger platform for dissemination of the projects’ results.
3. To identify topics of interest for the field to be discussed at workshops and meetings
The EC has been notably successful in bringing together large, multi-national research networks. Four such consortia, with the acronyms NMTrypI (http://fp7-nmtrypi.eu) KINDReD (http://kindred-fp7.com/) AParaDDisE (http://a-paraddise.cebio.org/) and PDE4NPD (www.pde4npd .eu), involving more than 50 teams in Europe, Africa, India, South America, Australia and the USA, were funded in the last call under the Seventh Framework Programme related to drug development in neglected infectious diseases. The projects targeted the major chronic parasitic diseases Leishmaniasis, Chagas Disease, Sleeping Sickness, Schistosomiasis and Malaria. Together, these diseases affect more than a billion people worldwide, cause hundreds of thousands of deaths and are major contributors to the poverty trap in the countries concerned (Cochrane G et al. (2017) Evaluation of the impact of the European Union’s Research Funding for Poverty Related and Neglected Diseases. Final Report. Prepared for European Commission, Directorate General for Research and Innovation. (In press)) The projects were given the following specific objectives (derived from the WP9 general objectives):
- To establish a common drug discovery platform that should have the capacity to undertake screening of compound libraries, lead development.
- To test validated hit compounds in relevant animal models as well as toxicology and safety testing of new drug candidates.
The four projects pursued distinct strategies for drug discovery, involving both phenotypic screening, target-based methods and repurposing of existing approved drugs, and have all made significant progress. Based on the criteria for the selection of “hit” and “lead” compounds recently defined (Katsuno K. et al. (2015) Hit and lead criteria in drug discovery for infectious diseases of the developing world. Nat. Rev. Drug Discov. 14:751-758), well over a hundred hits have been identified of which more than ten can so far be defined as lead compounds with significant potential for further development. These numbers will grow as all the projects reach their terms. In addition, novel drug targets have been identified and validated for each of the parasites studied, homology models and crystal structures of target enzymes have been resolved and new chemical entities identified as potential drugs of interest both in NTDs and other pathologies. Importantly, as suggested by the EC at the time of funding, the four consortia have worked in close collaboration throughout the course of their projects under the umbrella of a common “Synergy” workpackage. This has enabled them to adopt a common strategy to face the challenge of ensuring that the advances made will not be lost once the current projects come to an end. These cross consortia interactions have shown clearly where current funding schemes are failing. Support is needed for developing lead compounds from in vivo animal studies, ADME-Tox and GLP/GMP registration to phase 1 clinical trials. During the project duration the activities performed by the synergy team were related to: SOP definition in typical biological experiments in vitro and in vivo, Target Product Profile definition, experiments related to compounds ADME-Tox properties evaluation, drug resistance of Leishmania infantum strains, monkey trial on DRUG LEAD 1 from NMTrypI, dissemination activities.
• As a result of the screening approaches a large number of compounds (see Table 1 in Annex) have been moved along the drug discovery pipeline, for each of the parasitic diseases targeted. More than 100 “hits” have been characterized, and selection has been performed following the suggested criteria for target product profile cut-off and definition (see Task Quality assurance of results [All partners]
• In depth early ADMET followed by testing in vitro has been carried out on the most promising of these.( Task 3. Synergy activities with Kindred as from Amendment 4)
• Several lead compounds with in vivo activity against the parasites have already been defined. Test of miltefosine derivatives against clinical resistant isolates was established to be performed with Dr.Sarma Singh.
• Technology platforms existing within the four projects.
• Due to the orphan drug designation request it has been possible to move one drug candidate (against Chagas Disease) forward to phase 1 clinical trials/FDA registration (Kindered project). 1 drug lead showing improved activity against Leishmania infantum was obtained by NMTrypI and it was tested in a monkey trial shared with Kindred. (Task 3: Animal model descriptions)
• Defintion of SOP for common processes in the drug discovery pipeline (Task 1: Protocol standardization [All Partners].
• Dissemination and data collection (Task 4: Dissemination and data collection from Task1, Task2, Task3 (all partners))
Description of the tasks.
2.1 Description of methodologies used and/or activities carried out
The four consortia have used various approaches to develop “hit” and “lead” candidates for drug development. These include:
• Phenotypic screening of extensive or focused compound libraries or natural products.
• Target-based screening (phosphodiesterases, epigenetic enzymes, dihydrofolate reductases, pteridine reductase, sirtuin etc.) including HTS and in silico screening.
• Repurposing or modifying existing approved drugs
• Extensive ADMET in a novel high throughput format to pre-select candidates for in vivo studies
• In vivo screening in animal disease models including for the most advanced candidates a NHP primate model.
• Dissemination activities and events organization.
• Databases sharing among the four project.
As a result of the synergy discussions, standardization of procedures in some araes and consensus definition of hit and lead compounds have been achieved (definition was based on published criteria).
2.2. In depth early ADMET followed by testing in vivo has been carried out on the most promising of these, and more will be tested before the end of the current projects.
The bioavailability of several hits was evaluated by snapshot-PK in BALB/c mice. These were important for early evaluation of the abundance and for prioritization of hits to be tested in the infection models. The snap-shot PK was used to evaluate the total availability of the compound after intravenous administration (1 mg/kg) and also to evaluate the oral availability (20 mg/kg). The methods for the detection of the PK parameters for all compounds have been reported in Deliverable 5.3.
2.3 Several lead compounds with in vivo activity against the parasites have already been defined.
One compound from the Kindred pipeline was tested against Leishmania major-infected mice, a model of Old World cutaneous leishmaniasis. In vitro phenotypic testing on L. major-infected mouse macrophages yielded a very good efficacy (98%), with an IC50 below microMolar, justifying the testing in vivo. Mice (BALB/c) were infected intradermally with Leishmania major strain 5ASKH (2×105 parasites/animal) and monitored twice weekly for the onset of lesion formation. Based on the preliminary results and the promising activity of the lead candidate in vitro it a further development can be justified.
2.4 Technology platforms existing within the four projects.
Technology platforms existing within the four projects that include all the expertise related to the drug discovery and development process. We have identified a few key scientists, partners of the projects, who can work as key roles in the shared platform. We planned to use the Technology table to perform some activities and disseminate an open-lab source for interested stakeholders.
- The Consortium, after a long discussion that took place in different meetings (WPleaders meeting) and in Heidelberg meeting, decided to test the best drug lead available, namely Drug lead 1, in monkey trial offered by Kindred project.
The work was included in the Amendment 4 as an additional task within the synergy WP9. The aim of the trial was to perform an head-to-head comparison between Miltefosine and Drug lead 1 in monkeys. During the time of the discussion on the topic a list of advantages/disadavantages was prepared by Jerome Estaquier (Kindred) and Josè Alunda (NMTrypI) to highlight the potential benefit and issues related to the activity.
- SOPs concerning Leishmania in vivo (1) and in vitro infection (2).
2.7 Dissemination and data collection (WP9)
Contribution to dissemination:
- Contribution to the knowledge base via publication in high-impact journals (8 from NMTrypI, of which 1 is within the synergy activity and one is just accepted in Trends in parasitology and 3 submitted, many more in preparation, some presentations at congresses specifically about the synergy activity).
- Interactions with stake holders (many MTA have been established.
- Meetings and congress organization. The congress at which the synergy meeting were presented as oral or poster presentations are reported below:
✓ Porto Synergy meeting 17/9/2014 see Appendix.
✓ Brussels 9/11/2015 meeting with the project Officer of the two projects Kindred and NMTrypI. (see Appendix)
✓ Brussels on the 22 February 2016, Hotel Colonies (Details reported below)
✓ Synergy meeting held in Modena on the 15-16 of June 2016. Website of the meeting: see Appendix.
✓ Joint Meeting COST CM1307 Action/SEFIG/ SOCEPA, Madrid 24-26 October 2016 Poster presentation (see Appendix).
2.11 Data Collection for sharing among the four consortia.
The SEEK database was proposed for storage of experimental data and reporting activity other than data elaboration using the SEEK tools (see 2nd periodic report in WP6 description).
The SEEK address for the Synergy activity is the following: https://fp7h-synergy.h-its.org/. Synergy SEEK data management system to store and manage your data. It is password protected and you need to register to have an user account there (your login name: name).
It is possible to create a project where the user can upload his/her data. Dr.Wolfgang Muller and his collaborators at HITS (Heidelberg) are also available to offer an introduction to the SEEK system via Skype video chat. Both A-Paraddise and Kindred will store their data in SEEK after the end of the project. SEEK will be available for 10 years at least at no cost.
Main deviations and contingency measures
The milestones were met.
EXPLOITATION Plan of NMtryPI Partners
Partner 1 (Unimore)
Unimore has developed a few compound classes with uniques structures showing antiparasitic activity sgsint Tbrucei, Tcruzi, Leishmania infantum and major. One compound class is stemming from the flavonoids-like compounds; the others are generated by modification of compounds designed through fragment-based drug design approaches. The two patents are the starting point to unsure a smart development for the initial leads. In particular both compounds require still some work for shading lights on the targets and mechanism of action. Being active on the three or four parasites, they represent potential tools for broad-spectrum antiparasitic agents. Moreover it is possible that a new target(s) can be identified thus opening new possibilities of parasites growth inhibition.
A biomarker protein set identified was found to be associated with the mechanism of action of some of the most advanced compounds NMT-H0080. DRUG LEAD 1 the NMTrypI. The protein set is useful to track the compounds activity in the parasite cells and can be useful to translate the information to the final pre-clinical study in dogs in particular. While we have finalized the study of DRUG LEAD 1 in Leishmania, the study in dogs serum will be finalized in the next months after the end of the project. The BM study in dogs is not very common and the information an be important for the development of a pre-clinical biomarker with potential impact in transaltional studies.
Partner 3 (BNI):
The development and implementation of novel technologies for the project caused a significant shift in the general outlook of the research group, opening up new research opportunities in the process.
BNI scientists were authors and co-authors in - so far - 3 peer-reviewed, original papers in journals from established publishing houses.
The general outline of NMTrypI activities was posted as part of the research groups web site.
An interview for Deutschlandfunk Radio conducted in August 2014 allowed to inform the German-speaking public about the needs for new anti-parasitic drugs and the goals behind NMTrypI.
Oral and poster presentations so far have focussed on the technological side of the work to avoid compromising the IP rights.
Partner 4 (IBMC):
The project delivered a new chemical entity that is now lead compounds for Leishmaniasis and HAT. For Leishmaniasis the DRUG LEAD 1 due to its characteristics (more potent and less toxic in the models tested) as the potential to fully substitute miltefosine and the reference oral drug for the treatment of Leishmaniasis. The characteristics of DRUG LEAD 1 might translate into shorter more intense treatments that might enable a faster disease clearance. The possibility of combination is also enticing and will certainly be a possibility to exploit. Toxicological studies, particularly embryo- and fetotoxicity, of DRUG LEAD 1 would complete the safety profile of the molecule and the feasibility of its use in human medicine. Alternatively treatment of canine leishmaniasis is a possibility to be addressed. The DRUG LEAD 1 is also a lead for the treatment of HAT, the oral availability and the capacity to pass the blood brain barrier make it a promising molecule to use either in monotherapy or in combination. Overall the miltefosine derivatives in their diversity present an unexplored cache of novel chemical entities that should be exploited to address other bacterial and protozoan infections like granulomatous amoebic encephalitis and primary amoebic meningoencephalitis that have very low success rates (less than 95% survival) that have as therapeuthical option miltefosine. The data on T. brucei is also suggestive that some of the generated derivatives might not just be better options than miltefosine but might also be completely unexpected treatment options like was the case of T. brucei.
Partner 5 (UNISI)
UNISI: the availability of pure targets, off-target proteins and of the crystal structures of their complexes with molecules obtained within the project (described in the scientific results) constitute a wealth of information that will need time far exceeding the time limits of the NMTrypI project in order to be fully exploited. Furthermore, the above products of the project constitute the basis for further research projects and development.
Partner 6 (HITS):
HITS: The work at HITS has been or will be disseminated by publication in scientific journals and deposition in the SEEK database, and by presentations at scientific meetings or for the general public.
WP1/WP4 The molecular modeling work performed in NMTrypI has contributed to the discovery of new compounds with anti-parasitic activity and with potential for development for therapeutic purposes. The modeling work has also contributed insights into the mechanisms of action and resistance of antiparasitics, including their interactions with known and putative on- and off- targets. The modeling approaches developed in this project have general applicability to other drug discovery projects. Patent applications will be made regarding the parts of the work with high potential for the development of therapeutics.
WP6 The SEEK system is a data management system for the use in collaborative projects. It enables data collection, data discovery, and dissemination. The additions to SEEK made during the project will be made available in the general SEEK distribution which is open source. SEEK is widely used in international systems biology projects and the work performed in NMTrypI provides a basis for extending its usage to drug discovery projects.
Partner 7 (TYDOCK PHARMA) :
the work at TYDOCK will be disseminated by pubblication in scientific journals and deposition in SEEK database and presented in scientific meeting or for general public.
WP 1 Patent implementation in NMTrypI generates new important knowledges insights into the inhibition of PTR1 enzymes as a target for anti-parasitics lead candidates design and development.
The synthesis of new pteridine compounds (more than 40 new compounds) help to better uderstand the role of folate receptors in the transport of compounds into the parasits
For the first time, TYDOCK in collaboration with IBMC, partner into the NMTrypI project, observed and gave the proof of concept for the undisclosed synergy priniciple between Pteridine compounds and DHFR inhibitors as MTX. The assay developed in collaboration with IBMC will be proposed as a tool for service.
The synthetic work new microwave assisted synthetic methods were developed that represent a strong knowledge in teh chemistry area.
TYBOX : a collection of more than 750 in house compounds were prepared and evaluated for their anti-parasitic action. All the compounds have been characterize in erly TOX experiments. TYBOX represents an important tool with applycability to other drug discovery project.
Patent applications will be made regarding the parts of the work with high potential for the development of therapeutics.
Partner 8 NCR:
Sudan is one of largest counties in Africa. The country expand through several vegetation regions and has a great variation in ecology and soil. This has resulted in making Sudan of the richest countries of Africa in term diverse and virgin flora. Sudan contain more than 3156 species, 1137 genera and 170 families of plants. Out of this more than 400 plants are used in Sudan’s traditional medicine, which is considered to be one of the best in Africa.
because of the NMTRYPI project NCR has established the only extracts bank in Sudan. NCR collected, extracted and contributed about 120 plant extracts in NMTRYPI project. The work in this plant extract bank continues and by today the NCR extract bank contains more than 200 plant extracts. 100 more are collected and due to be extracted. And it is expected that most of the medicinal plants of Sudan will be included in the plant extract within 6 months from now. NCR is determined to provide all these plant extracts (and all necessary documentation about their traditional applications) available for all collaborative drug discovery projects.
Partner 9 (Hypha)
Hypha Discovery’s main goal within the NMTrypI project was to explore its MycoDiverseTM libraries of fungal fermentation extracts and fractions for the presence of anti-parasitic compounds useful as potential drug leads using the consortium’s phenotypic assays. A second goal was to assist in the evaluation of the Sudanese medicinal plant extracts as another natural resource for antiparasitic drug discovery. These goals are judged by Hypha to have largely been achieved, although the technical and logistical challenges involved in screening and assay-guided purification have meant that not all the purification, structure elucidation and secondary biological evaluation work has been completed within the three years of the project. Hypha will continue to investigate the most promising compounds from the MycoDiverseTM library. These are currently undergoing secondary evaluation and if their resulting activity profiles merit further investigation, Hypha will complete the scale-up production and structure elucidation of those that are yet to be identified. IP searches will be conducted to establish the scope for lead optimization and further development. Hypha will seek partners for further investigation of suitable candidates, starting within the current NMTrypI consortium, and will identify appropriate routes for funding through grant applications or via project-specific investment. Any resulting MycoDiverseTM library-derived compounds that meet the target product profile and are ready for out-licensing for preclinical/clinical development will be patented and licensing partners sought from pharmaceutical companies active in NTD development as described in the DOW. We have also benefited from participation in the NMTrypI project in a major way as the MycoDiverseTM library screening campaign has proved to be the most extensive in terms of hit-follow up and characterization that the company has undertaken, and dissemination of interim results has already been used to present Hypha’s natural products expertise and assets at international conferences. The NMTrypI screening campaign manuscript currently being prepared for publication and subsequent papers will be invaluable in marketing the company’s natural product capabilities. Our scientists have also relished the opportunity to work in this important area and enjoyed networking with other members of the consortium.
Partner 10 (NHRF)
NHRF is co-inventor in the US Patent No 8097752, “Antiprotozoal ring-substituted phospholipids”. on miltefosine derivatives as background IP. The unique assignee is MAKScientific. The role of NHRF in the project was to develop and progress along the NMTrypI pipeline the Miltefosine derivatives based on the above mentioned patent and design and synthesize new ones with improved profile.
A separate agreement was signed between MAKScientific and UNIMORE on behalf of the consortium expressing a mutual interest for further collaboration. Compound DRUG LEAD 1 is covered by the claims of US Patent No 8097752 and therefore MAKScientific will be asked to contribute to the further exploitation of the compound. As the SMEs involved in the NMTrypI project didn’t express an interest to be involved the development, European pharma industries involved in the area of antiparasitic drugs and/or International Organisations will be approached to discuss potential exploitation either in collaboration with MAKScientific (co-development) or through licensing. The general Exploitation principle will be followed by NHRF as it is clear that benefit for Europe will be preserved in the MAKScientific actions.
Concerning the new compounds not covered by the US Patent No 8097752 (foreground IP), depending on the biological results (which are still ongoing), NHRF will evaluate whether they merit patenting and a first option will be given to the SMEs involved in the NMTrypI project. In case of refusal other potential stakeholders will be approached. In parallel, grant applications will be submitted to support the further optimisation and preclinical development of the new compounds.
Partner 11: UCM
• Activity of H80 formulated with cyclodextrins against experimental visceral leishmaniasis in hamster
[Manuscript will be sent after number 7]
PK and antileishmanial efficacy: (UCM)
Int J Parasitol Drugs & Drug Resistance
Pending: H80 organ biodistribution & statistical analysis of ELISA results
• Pharmacokinetics and efficacy studies on two alkylphospholipids against experimental visceral leishmaniasis.
Pharmacology on mice: Anabela Cordeiro
Pharmacology in Hamsters and dogs: José María Alunda and his group (UCM)
[We suggest the split of this subject: “big paper” + other papers] [To be decided by the Consortium]
• Improved method to determine Miltefosine: value for pharmacological monitoring (tentative title)
(To be finalized and submitted)
Infection, PK and biodistribution
Journal Pharmaceutical & Biomedical Analysis
Analyses to be finalized and writing final version of manuscript
• Antileishmanial efficacy of an alkylphospholipid, DRUG LEAD 2, in experimental visceral leishmaniasis
PK, biodistribution and antileshmanial efficacy in hamster: (UCM)
Efficacy in mice: (IBMC)
• Antibody response in experimentally infected dogs with Leishmania infantum. Prognostic value.(tentative title) *
Infection, IFAT, ELISA (IgG1, IgG2) and Western blot.
International Journal for Parasitology
• Experimental infection of Beagle dogs with Leishmania infantum. Follow-up and surrogate marker parameters*
Journal to be decided
*These two papers only include infected (and non treated animals). As such no interference with the “big paper” is foreseen.
• “DRUG LEAD 1 is an ionic surfactant which aggregates in saline solutions of NaCl producing a homogeneous gel product. The homogeneity of the product facilitates homogeneity of dosing (variability among doses is lower). This gel was not observed at the same conditions with other similar molecules such as Miltefosine and DRUG LEAD 2”. This observation could lead to develop a solution (e.g. Maria Sammalkorpi, Mikko Karttunen and Mikko HaatajaIonic. Surfactant Aggregates in Saline Solutions: Sodium Dodecyl Sulfate (SDS) in the Presence of Excess Sodium Chloride (NaCl) or Calcium Chloride (CaCl2). J. Phys. Chem. B 2009, 113, 5863–5870).
Similar procedures have been patented (e.g. Sodium chloride solution for drug reconstitution or dilution. WO 2007053533 A2)
• Exploitation of a partially protected molecule (for the USA): maybe not so a big problem although it must be carefully considered
• Unless conclusive evidence of lack of teratogenicity (and other toxicities) from DRUG LEAD 1 for mammalians we suggest to focus on the veterinary sector.
• Super-Miltefosine (“Next Generation Miltefosine”): Offered, after SME from the Consortium and GSK (DOW), to Virbac (manufacturer of Milteforan®
• PhD degrees: 2 students
• Master Degree: 1 student
Partner 13 (CNPEM )
The main goals of CNPEM were: 1) to establish a multi-parasite screening platform for simultaneously screening against trypanosomatidic parasites of major medical importance; 2) use this screening platform to carry out the most complex natural product drug screening initiative so far undertaken for kinetoplastids; and 3) use the established screening platform in the screening of natural products and synthetic compounds for the discovery of promising starting points for drug discovery for Human African trypanosomiasis, Chagas disease and leishmaniasis. CNPEM considers that all 3 goals were successfully achieved. The screening platform will be further explored in continued efforts for drug screening for these diseases, and the assays developed during NMTrypI will be instrumental in this aspect. The results achieved during the screening campaign are being currently processed to generate at least 6 scientific papers, to be published in indexed journals, with possibly more papers being prepared in the future following identification of novel natural products with anti-T. cruzi and anti-Leishmania activity (as CNPEM will continue to offer the assays to enable natural product identification by Hypha).
Partner 14 (Fraunhofer)
The promotion of natural products with potential for application as anti-parasitic therapies will be examined within the established network of partners of this project from public research institutions and private companies. Natural product drug discovery for anti-parasitic diseases was a good reason to initiate this project with the exploitation of such compounds to a potential high added value product. Due to the time and cost intensive nature of this type of research, most pharmaceutical companies are not able to operate large screening efforts and currently seek the input of lead compounds ready for clinical testing.
List of Websites:
The website is active at http://fp7-nmtrypi.eu
Grant agreement ID: 603240
1 February 2014
31 January 2017
€ 7 639 176,11
€ 5 901 411,15
UNIVERSITA DEGLI STUDI DI MODENA E REGGIO EMILIA
Deliverables not available
Publications not available
Grant agreement ID: 603240
1 February 2014
31 January 2017
€ 7 639 176,11
€ 5 901 411,15
UNIVERSITA DEGLI STUDI DI MODENA E REGGIO EMILIA
Grant agreement ID: 603240
1 February 2014
31 January 2017
€ 7 639 176,11
€ 5 901 411,15
UNIVERSITA DEGLI STUDI DI MODENA E REGGIO EMILIA