Final Report Summary - KINDRED (Kinetoplastid Drug Development: strengthening the preclinical pipeline)
Executive Summary:
Executive summary
The Kinetoplastid Drug Development (KINDReD) consortium was funded for three years between September 2013 and August 2016 under the final EU Framework 7 call, FP7 HEALTH.2013.2.3.4-2: Drug development for neglected parasitic diseases. The consortium was devised to include expertise in three major chronic parasitic diseases; Leishmaniasis, Chagas Disease and African trypanosomiasis. Its overall objective was to reconsider the preclinical pipeline and conventional approaches to drug discovery in a way that would mean new, cost-effective and safe drugs reach those most in need.
From its inception KINDReD was planned to be different stand. Led by a small French SME, the project took a fresh look at the problems of drug discovery in NTDs. With the aid of a parasitology group from Portugal, a consortium was created that had on one side experts in the field with decades of experience and on the other, SMEs and academic groups with exciting technological platforms but no prior experience in neglected infectious diseases. Despite the fact that few of these groups had ever worked together before, it is a testament to the success of the consortium that the new collaborations formed during its lifetime are set to continue to explore new advances together.
Because after all KINDReD was based on ambition. Its goal was to strengthen, inform and advance the current drug development pipeline in order to achieve at least one new Phase I clinical candidate for each trypanosomatid disease studied by the end of, or shortly after, the project’s course.
An impossible and naïve approach according to some detractors and in any case difficult considering that the EU budget had to cover the research of 13 groups for three years from early compound screening to advanced preclinical studies. However, KINDReD concentrated on all points of the pipeline and has been able, thanks to the incorporation of the NHP model, to move hits to leads and leads to drug candidates. One particular and important example is that KINDReD is now championing the first new chemical entity for the treatment of Chagas disease in over 30 years. K777 a cysteine protease inhibitor, has at least equivalent efficacy to benznidazole in the acute animal model for disease. Completion of the 28-day chronic dosing in primates, revealed no drug-related toxicity or other safety issues, therefore, submission of the IND application with progression to a Phase 1 safety study in “normal healthy” volunteers is now foreseeable.
With the KINDReD Association, a charitable organisation set-up as a durable footprint of the project, all the hits, leads and drug candidates from this project, and any other which should wish to join, have a lobbying force to help ensure that the best amongst these can also arrive in the clinic one day.
Project Context and Objectives:
Kinetoplastid Drug Development: strengthening the preclinical pipeline (KINDReD)
Summary description of the project context and objectives
The trypanosomatid diseases, Leishmaniasis, Human African trypanosomiasis and Chagas disease, continue to impart a heavy toll on human health, affecting millions of people worldwide particularly in the economically poorest countries(1). The handful of treatments currently available to control this enormous health burden is limited by serious adverse effects (toxic and/or teratogenic), high costs, difficulties in administration, political instability and the often rapid selection of drug resistance(2-4).
Worldwide efforts to combat “neglected infectious diseases” from bodies such as the pharmaceutical industry, the DNDi, the European Commission, Gates Foundation and others have resulted in several potential drug candidates at the preclinical trials stage but with only a few reaching clinical trial (Phases I to IV) and a depressingly low number of new compounds reaching the market. The CNS permeable drugs fexinidazole, a 2-substituted 5-nitroimidazole and SCYX-7158, an orally available benzoxaborole, show promising efficacy with the advanced stage of HAT and recruitment of patients to the fexinidaole Phase II/III (394 patients) in 2015. (http://www.dndi.org/strengthening-capacity/hat-platform/)
Nonetheless, there has been general market inertia to translate recent scientific and technological advances into the discovery of potent, safe drug candidates against the kinetoplastid protozoan parasites a factor that we communicated around strongly during the course of KINDReD.
In this SME-led consortium which brought together several novel technologies and partners new to the domain with established world leaders in neglected infectious disease research, we aimed to take an ambitious, fresh and unconventional approach to strengthen the preclinical pipeline with the aim of predicting in advance, potential pitfalls such as toxicity or resistance at the preclinical phase to have a greater chance of turning lead candidates to drugs in a timely and cost-effective manner.
References
(1) Stuart K, Brun R, Croft S, Fairlamb A, Gurtler RE, McKerrow J et al. Kinetoplastids: related protozoan pathogens, different diseases. J Clin Invest 2008; 118(4):1301-1310.
(2) Croft SL, Seifert K, Yardley V. Current scenario of drug development for leishmaniasis. Indian J Med Res 2006; 123(3):399-410.
(3) Barrett MP, Boykin DW, Brun R, Tidwell RR. Human African trypanosomiasis: pharmacological re-engagement with a neglected disease. Br J Pharmacol 2007; 152(8):1155-1171.
(4) Renslo AR, McKerrow JH. Drug discovery and development for neglected parasitic diseases. Nat Chem Biol 2006; 2(12):701-710.
Figure 1: Word cloud representation of the KINDReD project’s ethos
Objectives of the study
The aim of KINDReD is to create a unique and powerful drug discovery platform with the common objective of advancing promising laboratory-driven discoveries into clinical utility.
• To implement a coherent (integrated) approach to populate and advance all stages of the anti-trypanosomatid drug pipeline.
• To establish an integrated global network of academic and industrial partners united by a central objective: to develop and apply innovative cutting-edge molecular and cellular tools that will enable us to populate and accelerate the preclinical pipeline for anti-kinetoplastid chemotherapeutics.
• To advance every aspect of the preclinical pipeline, by developing new technologies where necessary, from early target discovery, validation and screening through to advanced toxicology and animal testing.
• To bring forward one new Phase I clinical candidate for each trypanosomatid disease studied at the end or within a short time after the end of the project.
• In parallel, and where appropriate in partnership with likeminded bodies such as WHO, Wellcome Trust, DNDI etc. we will set up an association that will lobby industrial groups to take an active role in the further clinical development of our registered candidates (repurposed or new chemical entities) showing potency against these parasitic diseases after the conclusion of the funding period.
HAT
Human African Trypanosomiasis or
Sleeping sickness
Figure 2: Our parasites and diseases
Project Results:
A description of the main S&T results/foregrounds
In KINDReD, originally composed of 14 partners, the scientific programme was divided over 9 of a total of 12 work packages (WPs). Our infrastructure for parasite screening integrated five leading academic laboratories in Europe (Portugal, United Kingdom and Switzerland), the United States of America (California) and South America (Brazil) with high throughput screening (HTS) facilities equally distributed between all three major kinetoplastid parasites. Follow-up medicinal chemistry and ADMET expertise was spear-headed by our industrial partners in France and Spain, respectively who interacted with the academic members of the consortium to guide their efforts. The full details of the progress of each of these WPs have been provided to the European Commission, in two periodic reports and a series of key deliverables. Below is a description of the work performed the original objective of the WP and the main results.
Figure 3: the central KINDReD work flow
Physiological and Target-based Screens
The largest of the work packages, WP1 “Physiological whole parasite screening assays” led by Jim McKerrow, now from UCSD, was originally planned as the driving force of the project, screening large numbers of compounds to pass hits (IC50 ≤ 1 µM) to WP 8 (Isbaal Ramos, Innoprot) the in vitro toxicology and ADME control step built into our reinforced pipeline to pinpoint potential adverse events before attempting animal experiments. This step was designed importantly to reduce the number of animals needed in preclinical testing as we were bound in our original ethical commitment to implement the 3Rs ‘Reduction, Refinement and Replacement’ under which we had agreed on a protocol to reduce the number of mice used by 59% through pooled testing of lead compounds.
We also ensured that the molecules tested were being screened in the in vitro system that best represented the physiologically relevant form of the disease-forming parasite. Screening assays incorporated state-of-the-art image-based screening of intracellular parasites in T. cruzi and Leishmania spp. as well as reporter-based assays of T. cruzi trypomastigotes, the blood-stream form of T. brucei, and Leishmania spp. intracellular amastigotes. Several whole cell parasite assays have been developed by the KINDReD partners for this purpose WP1 combined these assay systems with advances in liquid handling robotics and automated imaging to facilitate high-throughput screening of a wide collection of compounds derived from natural and synthetic sources.
The project had always been constructed with in-built flexibility which meant that over the three-year course of the programme we could change the source of molecules screened and also welcome libraries of potential hits from groups outside the original consortium to ensure our unique resources were used to full capacity.
Positive controls used in screening were melarsoprol for T. b rhodesiense, benznidazole for T. cruzi, miltefosine for L. donovani and L. infantum and podophyllotoxin for cytotoxicity in mammalian cells. All compounds were subjected to (a) single-point high-throughput screens in the physiologically-relevant parasite assay seeking > 80% inhibition at 10 μM. Then (b) EC50 values were obtained from 16-point sigmoidal dose-response curves performed in triplicate (range 100 to 0.001 μM). (c) Hits with EC50 ≤ 5 μM and ideally < 1 μM were retested to confirm long-term cidal versus static effects on cell growth. For T. cruzi and Leishmania spp. these were performed systematically on intracellular parasites where the screen identifies compounds that give complete parasitological cure in cultures of myoblasts (T. cruzi) or macrophages (Leishmania). Each promising hit compound was systematically retested in all three parasites and cytotoxicity tested in mammalian cell culture, e.g. 72h incubation with L-6 rat skeletal myoblasts, to define selectivity.
The move of the McKerrow laboratory from San Francisco to San Diego substantially reduced the whole-cell screening capacity of the consortium which endured for over 18 months this meant that alternative sources of hits needed to be considered. One of the most valuable resources has been the natural compound libraries that KINDReD has access to and formed the basis of the work in WP4. These are primarily from Helge Bode’s laboratory, in Frankfurt, (WP4). The group works mainly with exotic natural product producers such as entomopathogenic bacteria (Photorhabdus and Xenorhabdus), which are usually underrepresented in natural product libraries. These were screened against T. cruzi, T. brucei and L. donovani by Pascal Mäser’s group at the Swiss Institute for Tropical Health and interesting hits returned to the Bode laboratory for chemical total synthesis or derivatisation of the active components.
Another source of natural compounds was the Global Health Library of marine derived natural products which also produced numerous hits that have been passed on to the other WPs to study AMETox and in vitro immunomodulatory activity (WP7). A chance encounter at an International meeting on NTDs, led to KINDReD gaining access to a unique library of plant extracts from Nigeria which has now been screened by the McKerrow group, against all three parasites. This has revealed some very interesting activities especially against T. brucei and larger quantities are being synthesised in Nigeria for chemical identification of the active components in USCD. The table below summarises the screening efforts from KINDReD.
Table 1: results from the KINDReD screening programme
Another potential source of hits that has been explored is the 1 600 strong list of FDA approved drugs. Now screened against all three parasites, this collection has shown a handful of compounds of interest for further study that will be passed along the KINDReD pipeline because their previous approval status could speed registration. The compounds have now been studied in the other WPs for AMETox and in vitro immunomodulatory activity (WP7) and snap-shot PKs have been performed where considered of interest. One of these, candicidin, is currently being tested in an animal model of cutaneous leishmaniasis in the NMTrpI consortium a model available to KINDReD as part of our “synergy” activities. Unfortunately, we still do not have the results from this study so have no update on its status. If successful, our Partner AIIMS is well-positioned to run a clinical trial in India. The results from the other FDA-approved compounds have been less interesting as although the AMETox was promising, in animal testing the efficacies were generally poor.
Additional whole-cell screens have been performed using in-house molecules (Anabela Cordeiro da Silva, Porto) and fragments from a Maybridge rule-of-three library screened against L. major and T. cruzi by Terry Smith in St Andrews.
The continued use of the CDD data base has ensured all molecules have been correctly catalogue and all our data is in one place ready for further exploitation.
One in-built strengthening mechanism in the KINDReD pipeline was to test promising leads against clinical isolates in addition to the laboratory strains used in the screening platforms. This precaution was put in place as laboratory strains are known to lose virulence after multiple passages. This work was performed by our partners at AIIMS (L. donovani) and FIOCRUZ (T. cruzi). All the compounds tested to date were found to be effective on clinical isolates as well as on laboratory strains of parasites.
Alternative approaches to whole-cell screening for the identification of hits were always a major part the project. WP2, “Target identification and screening”, led by Terry Smith (USTAN) has been particularly successful in identifying numerous hits from screening several parasite protein deacetylase and kinase activities, TbUAP, TbINO1/TbIMpase 1 and 2, Tb, neutral sphingomyleinase and TbIDI. Many of these of been repurchased or synthesised and retested, validated hits have moved forward through iterative rounds of medicinal chemistry to evolve potency and/ selectivity using SAR and several are entering ADMET. These include the nitro-heterocycle-amides the vast majority, ~20, of which have a better IC50 than nifurtimox against T. brucei. Of these, one KD011 was seen as the best candidate to take forward for ADMET and animal testing.
The consortium also had access to a unique SPR array screening platform from NovAliX that features a diverse collection of library compounds (>116k compounds including >25k fragments with MW< 300Da) immobilised on gold chips. These high density chemical microarrays (9,216 sensor fields/array) enable the rapid screening of any target against one of the largest fragment libraries commercially available. Early selectivity information can be provided on the primary screening level. This platform was used to screen three of the consortium’s chosen targets. The first proteins analysed in this way was the Silent Information Regulator 2 protein, SIR2, a NAD-dependant tubulin deacetylase and which has been genetically and chemically validated as an anti-Leishmania drug target in extracellular, intracellular and animal models prior to the start of the project. The use of the purified protein in the SPR-arrays has identified several molecules of interest, one of which active against the L. infantum protein has now had its potential confirmed in high concentration bioassays at PHX and in intracellular amastigotes at the IBMC.
Our second target ribose-5-phosphate isomerase (RpiB) failed to uncover any molecules suitable for further follow-up by SPR but the thermal shift approach in St Andrews identified three fragments with activity against the parasites that may be legitimate hits to follow up.
During the course of the project the group from USTAN was also able to genetically and chemically validate drug targets in T. brucei and T. cruzi using compounds identified from their screening efforts. With the support of IBMC working in the Leishmania model we now have several new targets for future therapeutic campaigns that we have characterised functionally against all three parasites these include uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) and ribose 5-phosphate isomerase.
Structure-based drug design
Several approaches to FBDD have been developed over the past decade with widely-accepted advantages over HTS campaigns to discover lead compounds. All focus on finding a seed fragment with high ligand binding efficiency (i.e. -RTKd/Heavy atom count > 0.4). Thereafter, structure-based design coupled to iterative chemistry can allow the quick evolution of potent lead compounds with optimal ‘rule of five’ properties. This approach to drug discovery was explored in work packages 2 and 3 which brought together experts from academia and industry as shown in figure 4 below.
Our original target of interest was the sirtuin 2 protein from Leishmania, the study was enlarged to include the SIR2 proteins from all three parasites. The sirtuins proved intractable to crystallisation during the first 18-months of the project. However, in the second half of the project, SIR2 from all three parasites was successfully crystallised. Although, unfortunately the structure-based drug discovery phase that was originally planned to be performed with fragments generated e.g. from high concentration bioassays (PHX) and SPR (NOVA) was not able to be completed within the time frame of the project.
The second target chosen for crystallisation was ribose-5-phosphate isomerase (RpiB). The structure of the T. cruzi protein was already known and the proteins from L. infantum and T. brucei underwent crystallography studies. Although, unlike with sirtuin, crystals were quite readily obtained within the timeframe of the project we could not proceed to structure-based drug discovery. The structures are now known for future studies.
ADME-toxicity Studies
In vitro toxicology and ADME studies were the subject of WP 8. This was another SME-led work package, demonstrating the high-throughput technologies of the Spanish-based company Innoprot a specialist in HT drug toxicology testing. The platform acted as a funnel capable of filtering the compounds emerging from the physiological screens through a series of stringent tests to yield only the very best candidates for advanced animal testing. One of the reasons cited for the failure of modern HTS technologies to generate more new approved drugs is the early focus on potency as the major selection criterion. Often only a few of the most potent candidates will be selected for animal toxicology and ‘snapshot’ PK studies in small rodent models, inevitably reducing the chances of choosing the candidate with the best balance of efficacy, PK suitability and safety. In KINDReD we applied early high content toxicology and in vitro ADME assays to remove this emphasis. Our original plan had been to feedback information to our medicinal chemists so that compounds with good ADMETox parameters but lacking in potency can be fed back into the design process for potency improvements. However, it became apparent that we were limited in budget to perform this task effectively.
Nevertheless we were able to ensure that only compounds showing both anti-kinetoplastid potency and excellent ADMETox were tested in our animal models. This strategy therefore combined the optimal use of anti-parasite screening output with application of 3R guidelines on animal testing. One important feature of the work carried out in this WP has been to obtain data on the drugs currently used for the treatment of the three parasite diseases in order to create a data base of their ADME-tox characteristics and to be able to compare any new hit and eventual leads against these.
The “gold standards” that have been characterised in WPs 6, 7, 8 and 9 are amphotericin B and miltefosine for Leishmania and pentamidine for T. brucei and benznidazole and fexinidazole for T. cruzi. The results from WP8 show that from the compounds tested (28 candidates and 5 gold-standards) showed hepatotoxicity, nephrotoxicity and neurotoxicity median cellular cytotoxicity concentration (CC50) values 100 times lower (on average) than IC50 values. In terms of adsorption, distribution, metabolism and excretion (ADME), plasma protein binding assays for mouse and human have been completed for the compounds and compared to the gold standards. With additional secondary tests such as AMES, the compounds from KINDReD have now been ranked in relation to their ADMETox profiles and some in fact appear to have a more acceptable safety profile at this stage than some current treatments. This WP has also explored the effect of using PLGA nanoparticles on the delivery of potential anti-parasitic compounds and has shown controlled and effective delivery of BNIPDaoct and for L. infantum and T. brucei infection with significantly reduced toxicology as evidenced by PLGA-encapsulation of amphotericin B.
Further exploration of our “gold-standards” by Jérôme Estaquier at the CNRS has demonstrated that miltefosine, amphotericin B and pentamidine, depending on the concentration used at either micro- or at millimolar levels, induce mitochondrial depolarisation and cell death. Moreover, we have demonstrated that the effects are cell type dependent. In particular, we found that monocytes are especially sensitive to miltefosine and pentamidine. Because monocytes represent preferential targets for Leishmania infection, we demonstrated that infection with L. infantum modulates mitochondria metabolism exerting a regulatory effect on oxidative phosphorylation. Therefore, we are testing the impact of the drugs on monocyte metabolism as well as on the profile of OPA1. This work is also of particular importance to WP7 (Immunomodulation) led by Olindo Assis Martins Filho, in Brazil. Current guidance documents for preclinical filing of new drugs require evaluation of unintended immunomodulation and advocate additional immunotoxicity studies to characterise risk including ex vivo immunophenotyping of blood cells and in vitro immune function. Our experiments to date suggest it is of crucial interest to concomitantly monitor the immune response to assess the side effects of the drugs in relation to the nature of the cells that are sensitive to their effects.
Animal testing
Work package 9, animal studies of efficacy and toxicology was led by Anabela Cordeiro-da- Silva (IBMC). The strains and models used were the following; stationary phase promastigotes (visceral leishmaniasis virulent clone L. infantum MHOM/MA/67/ITMAP-263 and cutaneous leishmaniasis, L. major LV39 MRHO/SU/59/P), for T. cruzi trypomastigotes (the clinical strain, CAI-72, produces cardiac disease and cardiac failure similar to that seen in human Chagas disease) and for T. brucei blood strain trypomastigotes (e.g. clonal line of virulent T. b. rhodesiense STIB900). Parasites were injected i.p. at 108 parasites for Leishmania and T. cruzi or 104 for T. brucei. For Leishmania L. infantum and T. cruzi four mice from each infected group were treated after 14 days of infection by i.p. injection during three consecutive days. Parasitaemia was originally monitored two to three times a week by microscopic counting of tail vein blood in a Neubauer chamber. During the project, live-imaging was introduced to follow parasitaemia (see figure 5). Mice that were aparasitaemic for 60 days after the treatment period were considered cured.
Figure 5: In vivo imaging of BALB/c mice infected with T. brucei after 5 days of treatment. Parasite load was evaluated by subcutaneous injection of 2.4 mg of luciferin and all animals were imaged using IVIS LUMINA LT groups at day 5 post-infection are represented.
The biodistribution of the reference compounds after intraperitoneal injection has been determined in our mouse models for comparison with KINDReD lead compounds. The in vivo efficacy of several test compounds against Leishmania was found to be enhanced by encapsulation in pegylated nanoparticles. However, several compounds reaching this WP and tested against the mouse model for T. brucei and T. cruzi have unfortunately, shown to have little or no in vivo efficacy.
Our second animal model in KINDReD to complete the final stages of evaluation of a drug candidate, which has shown good safety and tolerability in the mouse model, is the non-human primate (NHP) specifically the Macaca Mulata adult male. The use of NHP as animal models for the study of human diseases (including immunological studies and drug and vaccine development studies against infectious diseases) has become increasingly important. During the course of KINDReD, the NHP model has been used to perform a 28-day chronic dosing study for K777. K777 is a protease inhibitor effective against T. cruzi for which registration to use in the treatment of Chagas disease is being sought. Originally, part of the DNDi portfolio development of this drug as halted after a poor tolerability result (see figure 6). GMP drug ready for safety testing in the NHP model (CNRS) was provided by the McKerrow laboratory and the study was performed following an FDA approved protocol. Jérôme Estaquier has shown that the drug could be safely administered to the NHP with no immediate or long-term side-effects. This successful study should complete the pre-IND dossier for FDA approval we are waiting to hear from the FDA for a date to discuss this data.
Figure 6: screen shot from the DNDi website showing their decision to discontinue K777 as a potential new treatment for Chagas disease.
The unique approach of KINDReD
Two work packages that were slightly to the side of the main direction of the work flow in KINDReD are WPs 5 and 6 looking at Chemoproteomics: deciphering drug “on” and “off” target interactions and parasite metabolism, respectively. These WPs are again an opportunity for “new blood” in terms of neglected infectious diseases to use their well- defined technologies and prior knowledge in the pursuit of the project’s aims. The chemoproteomics approach WP5, led by Gilbert Skorski, Phylogene, was intended as an early exploration of the potential for ABPP probes to assist the drug development workflow, requiring (i) implementation of existing methodologies and assessing their utility to drug development as well as (ii) the development of new ABPP probes and associated workflows. Whilst several important steps have been made towards these goals, the heavy demands on novel ABPP chemistry, as well as the fine-tuning of sophisticated technology to ‘read-out’ off-target interactions, has limited our analyses to a small number of the potential avenues that could realistically be explored over this three year project. Resveratrol probes (described in the mid-term report) were abandoned in favour of more promising 5-nitro-2-furancarboxamide derivatives, in direct collaboration with USTAN and NOVA. A phosphoproteomics pipeline to screen both the parasite and host proteomes was applied in order to identify new drugs targets. Infection by T. brucei has effects on the proteome and phosphoproteome in the mouse brain model. An impact on the immune response was shown by differential expression level but also on the brain plasticity in our phosphoproteomics experiments. This preliminary -omics study can be used as a starting point to find a new molecular targets for treatment and also getting a better understanding of the T. brucei infection mechanism. Used to label proteins after co-incubation with T. brucei cells their subsequent identification with the new probes represents an important step in understanding the mechanism of action and cellular targets for this new class of kinetoplastid inhibitors.
Work package 6 led by Alain Pruvost from the CEA is another WP that changed course during this second reporting period. Terry Smith’s group in St Andrews contributed much to this WP over the latter half of the project. In collaboration with a Canadian group, they have investigated mutations in a P-type ATPase transporter in Leishmania that lead to cross-resistance to two leading drugs by distinct mechanisms. Uptake of the potential anti-T. cruzi compound KD011 into macrophages has been investigated in collaboration with the CEA and the IBMC. Attempts have been made to gain a better understanding of the catabolic processes occurring in the lysosome of the parasites. In another approach Photeomix (PHX) have characterised proteolytic activities in leishmania promastigote cell extracts which exhibit a strong potential destroy peptide drugs.
Work package 12, synergistic activities continued to make progress towards working together. KINDReD and NMTrypI were able to exchange animal models, as close collaboration has ensured the best use of resources in the interests of advancing the kinetoplastid pipeline. The synergy meeting held in Modena in June this year was a good example of how much exceptional science and technology is contained within this European Framework call. The co-ordinators of all four projects presented an open letter to the EC to this effect and have also presented a poster at the recent COST-Action Meeting in Madrid in a similar vein.
Final results and their potential impact and use
• We have now have two potential lead candidates to move into phase I trials:
o The FDA dossier is currently under approval for the use of K777 in Chagas disease
o An EMA dossier is under construction for repurposing candicidin in cutaneous Leishmaniasis
• We are confident that our new techniques for use in the preclinical pathway especially our chemoproteomic probes and metabolism measurements will be of use to other researchers in the field including our synergy partners.
• Photeomix has launched a series of services based on the discovery of small molecule inhibitors of post-translational modification enzymes which is a direct result of their involvement in KINDReD.
• New products and services are available from Photeomix and other SME partners will no doubt follow over the coming months.
• KINDReD has uncovered rich source of new targets which are worthy of further research as are the new scaffolds, fragments and natural products we have shown to be active.
• The KINDReD Association is a new and experimental model focused initially on lobbying funding bodies to bridge the gap between late-preclinical and first-in-human trials.
• KINDReD has been unique in bringing together partners new to the field of parasite research all of whom have integrated well into the project. It is a source of new and potentially powerful partnerships in a field that perhaps has suffered from dogmatic direction. We plan to continue the open and encompassing approach we have taken to studying the pipeline and difficulties experienced in this field after this funding period finishes.
Figure 1: the central KINDReD work flow
The largest of the work packages, WP1 “Physiological whole parasite screening assays” led by Jim McKerrow, now from UCSD, was originally planned as the driving force of the project, screening large numbers of compounds to pass hits (IC50 ≤ 1 µM) to WP 8 (Isbaal Ramos, Innoprot) the in vitro toxicology and ADME control step built into our reinforced pipeline to pinpoint potential adverse events before attempting animal experiments. This step is designed also to help reduce the number of animals needed in preclinical testing. However, the McKerrow laboratory that had moved from San Francisco to San Diego in the first 18-month period still only slowly began to feed hits into the pipeline. Our alternative sources of hits continued to be the natural compound libraries that KINDReD has access to. Over this period Helge Bode’s laboratory, in Frankfurt, (WP4) sent more compounds to be screened by Pascal Mäser’s group at the Swiss Institute for Tropical Health. A chance encounter at a meeting where Jane MacDougall presented the consortium, led to KINDReD gaining access to a unique library of plant extracts from Nigeria which has now been screened by the McKerrow group, against all three parasites. This has revealed some very interesting activities especially against T. brucei and larger quantities are being synthesised in Nigeria for chemical identification of the active components in USCD. The compounds screened from a1600 strong list of FDA approved drugs in the first period of the project have now been studied in the other WPs for AMETox and in vitro immunomodulatory activity (WP7). One of these, candicidin, is currently being tested in an animal model of cutaneous leishmaniasis in the NMTrpI consortium a model available to KINDReD as part of our “synergy” activities. We hope to be able to update on its status in the final report. If successful, our Partner AIIMS is keen to run a clinical trial in India. The results from the others have been less interesting as although the AMETox was promising, in animal testing the efficacies were poor.
Alternative approaches to whole-cell screening for the identification of hits were always a major part the project and work performed in work packages 2 and 3 have shown that these are of some value. WP2, “Target identification and screening”, led by Terry Smith has been particularly successful in identifying numerous hits from screening several parasite protein deacetylase and kinase activities, TbUAP, TbINO1/TbIMpase 1 and 2, Tb, neutral sphingomyleinase and TbIDI. Many of these of been repurchased or synthesised and retested, validated hits have moved forward through iterative rounds of medicinal chemistry to evolve potency and/ selectivity using SAR and several are entering ADMET. These include the nitro-heterocycle-amides the vast majority, ~20, of which have a better IC50 than nifurtimox against T. brucei. Of these, one KD011 was seen as the best candidate to take forward for ADMET and animal testing.
The group from USTAN was also able to genetically and chemically validate drug targets in T. brucei and T. cruzi using compounds identified from their screening efforts. With the support of IBMC working in the Leishmania model we now have several new targets for future therapeutic campaigns that we have characterised functionally against all three parasites these include uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) and ribose 5-phosphate isomerase.
Work package 3, “Structure-based drug design” led by Paola Ciapetti from NovAliX, continued to showcase the novel technologies brought to the consortium by our SME partners.
Our original target of interest was the sirtuin 2 protein from Leishmania, previously described by Cordeiro-Da-Silva’s group as essential to parasite growth and as such a potential target for drug discovery. The study was enlarged to include the SIR2 proteins from all three parasites. The sirtuins proved intractable to several approaches used in this consortium during the first 18-months of the project. However, in the second half of the project SIR2 from all three parasites was successfully crystallised. Although, unfortunately the structure-based drug discovery phase that was originally planned to be performed with fragments generated e.g. from high concentration bioassays at was not able to be completed within the time frame of the project. SPR-screening of the proprietary fragment libraries at NOVA resulted in some candidate fragments against SIR2 being identified. These have now been tested in both high concentration bioassays and with macrophagic amastigotes (IBMC), and one of these appears to be promising to move along the pipeline.
The second target chosen for crystallisation was ribose-5-phosphate isomerase (RpiB). The protein from the three different parasitic species was produced and subjected to SPR fragment screening. In parallel the proteins from L. infantum and T. brucei underwent crystallography studies, while again the project could not proceed to structure-based drug discovery. The structures are now known for future studies. While SPR was unable to uncover any molecules suitable for further follow-up, the thermal shift approach in St Andrews identified three fragments with activity against the parasites that may be legitimate hits to follow up.
All compounds and molecules screened in KINDReD along with the results are entered in the consortium’s data base hosted by CDD Vault for the information of, and use by, all partners.
In vitro toxicology and ADME studies are the subject of work package 8. This is another SME-led work package, demonstrating the technologies of the Spanish-based Innoprot. One important feature of the work carried out in this project has been to obtain data on the drugs currently used for the treatment of the three parasite diseases in order to be able to compare any new hit and eventual leads against these. The “gold standards” that have been characterised in WPs 6, 7, 8 and 9 are amphotericin B and miltefosine for Leishmania and pentamidine for T. brucei and benznidazole and fexinidazole for T. cruzi. The results from WP8 show that from the compounds tested (28 candidates and 5 gold-standards) showed hepatotoxicity, nephrotoxicity and neurotoxicity median cellular cytotoxicity concentration (CC50) values 100 times lower (on average) than IC50 values. In terms of adsorption, distribution, metabolism and excretion (ADME), plasma protein binding assays for mouse and human have been completed for the compounds and compared to the gold standards. With additional secondary tests such as AMES, the compounds from KINDReD have now been ranked in relation to their ADMETox profiles and some in fact appear to have a more acceptable safety profile at this stage than some current treatments. This WP has also explored the effect of using PLGA nanoparticles on the delivery of potential anti-parasitic compounds and has shown controlled and effective delivery of BNIPDaoct and for L. infantum and T. brucei infection with significantly reduced toxicology as evidenced by PLGA-encapsulation of amphotericin B.
Further exploration of our “gold-standards” by Jérôme Estaquier at the CNRS has demonstrated that miltefosine, amphotericin B and pentamidine, depending on the concentration used at either micro- or at millimolar levels, induce mitochondrial depolarisation and cell death. Moreover, we have demonstrated that the effects are cell type dependent. In particular, we found that monocytes are especially sensitive to miltefosine and pentamidine. Because monocytes represent preferential targets for Leishmania infection, we demonstrated that infection with L. infantum modulates mitochondria metabolism exerting a regulatory effect on oxidative phosphorylation. Therefore, we are testing the impact of the drugs on monocyte metabolism as well as on the profile of OPA1. This work is also of particular importance to WP7 (Immunomodulation) led by Olindo Assis Martins Filho, in Brazil. Our experiments to date suggest it is of crucial interest to concomitantly monitor the immune response to assess the side effects of the drugs in relation to the nature of the cells that are sensitive to their effects.
Work package 9, animal studies of efficacy and toxicology is led by Anabela Cordeiro-da- Silva. The biodistribution of the reference compounds has been determined after intraperitoneal injection for comparison with KINDReD lead compounds. The in vivo efficacy of several test compounds against Leishmania was found to be enhanced by encapsulation in pegylated nanoparticles. However, several other compounds reaching this WP and tested against the mouse model for T. brucei and T. cruzi have unfortunately, shown to have little or no in vivo efficacy. The NHP model has been used to complete the dossier for FDA approval for the protease inhibitor K777, which has been shown to have potential as new therapeutic agent against Chagas disease. Jérôme Estaquier working with the drug produced by the McKerrow laboratory showed that the drug could be safely administered to the NHP with no immediate or long-term side-effects. This data has been used to complete the approval procedure for the FDA.
Two work packages that are slightly to the side of the main direction of the work flow in KINDReD are WPs 5 and 6 looking at Chemoproteomics: deciphering drug “on” and “off” target interactions and parasite metabolism, respectively. These WPs are again an opportunity for “new blood” in terms of neglected infectious diseases to use their well- defined technologies and prior knowledge in the pursuit of the project’s aims. The chemoproteomics approach WP5, led by Gilbert Skorski, Phylogene, was intended as an early exploration of the potential for ABPP to assist the drug development workflow, requiring (i) implementation of existing methodologies and assessing their utility to drug development as well as (ii) the development of new ABPP probes and associated workflows. Whilst several important steps have been made towards these goals, the heavy demands on novel ABPP chemistry, as well as the fine-tuning of sophisticated technology to ‘read-out’ off-target interactions, has limited our analyses to a small number of the potential avenues that could realistically be explored over this three year project. Resveratrol probes (described in the mid-term report) were abandoned in favour of more promising 5-nitro-2-furancarboxamide derivatives, in direct collaboration with USTAN and NOVA. Used to label proteins after co-incubation with T. brucei cells their subsequent identification with the new probes represents an important step in understanding the MOA and cellular targets for this new class of kinetoplastid inhibitors.
Work package 6 is led by Alain Pruvost from the CEA is another WP that changed course during this second reporting period. Terry Smith’s group in St Andrews contributed much to this WP over the second reporting period. In collaboration with Marc Ouellette they have investigated mutations in a P-type ATPase transporter in Leishmania that lead to cross-resistance to two leading drugs by distinct mechanisms. With the CEA and the IBMC, uptake of the potential anti-T. cruzi compound KD011 into macrophages has been investigated. Also they have attempted to gain a better understanding of the catabolic processes occurring in the lysosome of the parasites while PHX have characterised proteolytic activities in leishmania promastigote cell extracts which exhibit the strong potential destroy peptide drugs.
Of the remaining three work packages, WP 11 is concerned with management activities that are shared by St Andrews the project co-ordinator and Photeomix, the scientific co-ordinator. While at the management of the consortium has continued to be challenging the working relationship is excellent and no problem has yet proved insurmountable, thanks in part to the excellent support from our EU project office.
Work package 10 “regulatory affairs” led by Jane MacDougall, Photeomix, also covers standardisation of protocols and dissemination activities. Throughout this reporting period, dissemination activities continued to be closely monitored and communicated KINDReD public website www.kindred-fp7.com. Internal consortium updates have been maintained by the use of Basecamp, a web-based communication platform with encrypted access to consortium members only. The Scientific Co-ordinator, Photeomix has continued to present the consortium at International meetings.
The KINDReD Association is fully operational and now has over 50 members, including members of the public and members from all four FP7-funded projects in neglected infectious disease. The Association is an excellent means of dissemination for KINDReD’s activities but also importantly stands as a focal point to ensure the momentum from this FP7 call moves forwards. The Association is active on social media as well as more conventional channels so that interest remains to continue funding of this work and ensure our most advanced candidates do finally reach the clinic.
During this period the consortium beneficiaries published a further 21 scientific articles related to their work carried out under KINDReD to add to the 16 from the first 18-months. This latter list follows this summary. Numerous articles will continue to be published as the consortium members continue to complete the work of the consortium.
Work package 12, synergistic activities continued to make progress towards working together. KINDReD and NMTrypI were able to exchange animal models, as close collaboration has ensured the best use of resources in the interests of advancing the kinetoplastid pipeline. The synergy meeting held in Modena in June this year was a good example of how much exceptional science and technology is contained within this European Framework call. The co-ordinators of all four projects presented an open letter to the EC to this effect and have also presented a poster at the recent COST-Action Meeting in Madrid in a similar vein.
Potential Impact:
The potential impact of KINDReD
Impact on world health and quality of life
Protozoan parasites are amongst the most common infectious agents in the tropics and subtropics. The respective diseases are a major cause of morbidity and mortality in many developing countries and have serious consequences for socio-economic development in these regions. Primary infections are not the only concern; secondary infections in HIV-infected and immuno-compromised patients also pose grave health risks. A major problem associated with these diseases is their prevalence in third world countries which are the least well-equipped to develop new drugs and invest in R&D.
• Leishmaniasis is a worldwide disease, affecting 88 countries. The annual incidence is estimated at 1-1.5 million cases of CL and 500 000 cases of VL. The overall prevalence of the disease is 12 million people and the population at risk is 350 million.
• Chagas disease occurs throughout Mexico and Central and Southern America, and continues to pose a serious threat to health in many countries of the region. The overall prevalence of human T. cruzi infection is estimated at 16-18 million cases with an annual burden of 196 million € in healthcare costs. Approximately 120 million people, i.e. 25% of the inhabitants of Latin America, are at risk of contracting the infection.
• For African trypanosomiasis, estimates indicate that over 60 million in 250 foci are associated with the risk of contracting the disease, and there are about 300 000 new cases every year. However, less than 4 million people are under surveillance and only about 40 000 are diagnosed and treated, due to difficulty of diagnosis and remoteness of affected areas. These figures are relatively small compared to other tropical diseases, but African trypanosomiasis, without intervention, has the propensity to develop into epidemics, making it a major public health problem with a case fatality rate in untreated patients of 100%.
Control of trypanosomatid protozoan family is a major challenge not only in the third world, but increasingly in the western hemisphere, including Europe as migration and climate change move both people and the disease-bearing vectors, respectively into previously infected areas. The KINDReD consortium has responded to this global threat through the development of two new treatments for trypanosomal diseases. These are a completely novel reagent against Chagas disease and a potential therapy from repurposing candicidin for use in cutaneous Leishmaniasis that ultimately will convey significant benefits for human health in countries where these diseases are endemic.
Impact on the anti-trypanosomatid drug pipeline- turning pipe dreams into clinical reality
Our screening, drug design and lead optimisation strategies have amassed a comprehensive portfolio of fully characterised drug leads, with emphasis placed from the beginning of the project to having candidates for early Phase I clinical trials. Our understanding of the fact that the regulatory approval process is a long, expensive and meticulous procedure of controlled clinical testing, with a low average success rate led us to perform extensive preclinical toxicity testing including introducing novel ‘critical path initiatives’ such as the introduction of preclinical genomic and chemoproteomic profiling. By passing the current treatments available to treat these diseases through the same platforms we have built a comprehensive picture of ADME Tox profiles that are acceptable to further development.
All our promising lead compounds will be carefully scrutinised for cellular markers of oxidative stress, mitochondrial damage, genotoxicity, DNA damage, apoptosis, cell viability and morphological dysfunction, again compared directly with current treatments. This way we have a clear preclinical profile for any future registration dossier.
Novel chemoproteomics techniques using active site probes have been developed to signal unwanted drug-protein interactions in host cells, potentially allowing identification and modification of off-target effects by further iterative drug remodelling. This work is still in a preliminary stage but is worthy of further investigation.
Impact on innovation and competitiveness of the larger pharmaceutical market
One of the major distinctions of the multi-disciplinary KINDReD consortium from other consortia has been the integration of multiple technologies on a common platform. The success of our innovative drug development procedures is equally applicable to the larger pharmaceutical market. Within this consortium we were driven by a need for cost-effective research, procedures to be developed by our members, such as differential drug fragment
-selectivity-profiling and chemoproteomic ‘off-target’-toxicology-profiling, these techniques have the potential to increase preclinical research efficiency, reduce drug candidate attrition and reinforce the competitiveness of the European pharmaceutical market in general. Some of the effective targets discovered by the consortium may also be investigated independently for clinical utility in other infectious or chronic diseases, such as malaria or cancer.
The linear decline in pharmaceutical research productivity over the past six decades suggests that new knowledge, technologies and increased resources, however powerful, do not translate directly into the approval of more new drugs. This is particularly the case observed with the adoption of high throughput screening techniques. The research productivity paradox was treated in the US Food and Drug Administration’s landmark ‘Critical Path Initiative’ launched in 2004 which called for an investigation into efficiency improvements that could be made at all stages in the development path for new medicines. It is here that the most recent technology developments can have their biggest impact on productivity. Tools developed specifically to evaluate the efficacy and safety of medicines at early stages in the process can improve pharmaceutical research productivity allowing informed decisions can be made earlier in the process. The KINDReD consortium has put the development and testing of such tools at the heart of the preclinical development of antiparasitic agents. Indeed, is a fascinating and sobering thought that a FP7-led initiative, gathering experts together to tackle neglected parasitic diseases that plague some the world’s poorest nations, could provide solutions central to the revitalisation one of the world’s most wealthy industries.
Impact on European SMEs
Another focus central to the KINDReD initiative was the strength of the SME involvement, each one of the which took a leading role in the work packages. Individual European countries encourage SMEs to perform internal research into new products and services aided by regional, national and tax credit initiatives. It has long been recognised that these activities are at the heart of the European economy, where SMEs account for 99% of private sector businesses and employ two thirds of the European workforce. The 2008 Small Businesses Act for Europe created a policy framework that recognises the central role of SMEs in the European economy. This ‘think small first’ policy can be applied equally to SMEs in the biotechnology sector, where SMEs bring innovation and expertise but can suffer from being a small player in the larger market. To flourish, SMEs need to become more competitive and more entrepreneurial. KINDReD offered a risk sharing environment for SMEs to develop the innovative products that underscore their business activities whilst contributing to the greater effort of eradicating the global burden of kinetoplastid parasite diseases. This initiative provides SMEs with both financial and intellectual support within a European framework of focused drug centred research. Each SME has used their research efforts to strengthen their market competiveness by expanding their research capabilities, developing their contract research and product ranges and developing new expertise in previously untapped areas.
Impact on ICPC countries
Eradication of neglected infectious diseases in poor countries is a global health priority, a decision supported by the leaders of the world’s richest economies at several G8 summits and recently endorsed by the London Declaration on Neglected Tropical Diseases inspired by the World Health Organisation’s 2020 roadmap on NTDs. Two of the partners in the KINDReD consortium are from ICPC countries of low income (Brazil) and low-middle income (India) where kinetoplastid diseases are endemic. Biotechnology efforts are underway in these countries to develop diagnostic and medicinal products. These efforts will be assisted by strengthening their existing research infrastructure and challenging the problem internally rather than relying only on external academic/bio-pharmaceutical efforts. Thus KINDReD has had a major impact by focusing funding on ICPC research groups, helping them employ teams of researchers working in clinical environments at the source of the health burden. Their researchers have benefited from the application of many of the technological advances of the KINDReD programme and they are ready to help us with the regulatory procedures needed to be followed in order to perform clinical trials in these countries.
Impact on future EU-directed neglected infectious disease research.
The EU has long been a leader in the initiatives against neglected infectious disease supporting NID initiatives throughout several framework programmes. For these initiatives to achieve a major impact by Horizon 2020, the challenge to provide a link between prior efforts and the establishment of a clinical trial must be met. The KINDReD programme has shown how the scientific community can come together to make this a reality, leaving a permanent footprint through the establishment of clear operating procedures and workflows that will facilitate effective preclinical research throughout the current programme and beyond. The founding of these procedures in written, open access form will have a major impact on future research initiatives. Furthermore, research into neglected infectious diseases having demonstrable applications to the general pharmaceutical pipeline should aid healthcare funders and stakeholders with future funding initiatives.
Impact on society in general.
Society should be aware of how the taxpayer’s money is spent on issues of great health and
socioeconomic importance. In addition to the consortium agreement, we have created an independent non-profit association to convey information relating to KINDReD’s activities to the research community and the public in general, to leave a durable footprint at the end of the 3 year project and to ensure the necessary continuity for the clinical development of the most promising lead compounds and the associated regulatory approval processes. Our activities on social media have had positive returns particularly from people living in endemic areas and we are continuing to communicate our progress to them even although the initial funding period is over.
Main dissemination activities and exploitation of results
Our dissemination measures were run by WP10. Throughout the period of the grant, dissemination activities have been closely monitored and communicated as much as possible via the KINDReD public website. The majority of partners have been active in disseminating their activities in KINDReD at different meetings both National and International with poster and oral presentations as shown in the lists below.
The Scientific Co-ordinator partner 4 (PHX) has presented KINDReD at several International meetings and discussed with several major parties interested in NTDs including the Wellcome Trust and the French MTN network.
Members of the consortium have continued to publish numerous scientific articles with 30 peer reviewed articles shown here. Work is still on-going to publish more articles and numerous publications citing KINDReD are already planned and will continue to appear from the work performed in KINDReD. Wherever possible publications are in line with the Commission’s wishes Open Access.
The KINDReD Association, an organisation of charitable status, has been set up, initially to support the KINDReD consortium. The Association has been promoting the research undertaken by the KINDReD consortium with links to press-releases and social media and provides a durable footprint of the FP7 funded research. It operates under open-source principles, providing a community-driven forum (1) to share data and views on research objectives and results, (2) provides access to materials and techniques of the KINDReD consortium for NID drug development, (3) has established metrics to judge the success of project goals versus the international NID objectives and (4) to collaborate across organisational boundaries.
It has will continue to produce press-releases targeting the local, national and international news media. Social media including popular resources such as Twitter and Facebook provide information and link the KINDReD website and press-releases with associations and blogs.
Our work through synergy with the other four projects financed under the FP7- call HEALTH 2013.2.3.4-2 Drug Development for Neglected Parasitic Diseases we are combining our efforts to continue dissemination of the EU’s contribution to funding neglected infectious disease research by amongst other channels the KINDReD Association which has the potential to continue the research funded here for many years to come, ensuring that new, affordable treatments are provided to those in the most need.
Project public website
www.kindred-fp7.com
List of Websites:
http://kindred-fp7.com/
Contact details: Dr Jane MacDougall, Photeomix, 34 rue Carnot, 93160 Noisy le Grand, France email: jmacdougall@photeomix.com.
Executive summary
The Kinetoplastid Drug Development (KINDReD) consortium was funded for three years between September 2013 and August 2016 under the final EU Framework 7 call, FP7 HEALTH.2013.2.3.4-2: Drug development for neglected parasitic diseases. The consortium was devised to include expertise in three major chronic parasitic diseases; Leishmaniasis, Chagas Disease and African trypanosomiasis. Its overall objective was to reconsider the preclinical pipeline and conventional approaches to drug discovery in a way that would mean new, cost-effective and safe drugs reach those most in need.
From its inception KINDReD was planned to be different stand. Led by a small French SME, the project took a fresh look at the problems of drug discovery in NTDs. With the aid of a parasitology group from Portugal, a consortium was created that had on one side experts in the field with decades of experience and on the other, SMEs and academic groups with exciting technological platforms but no prior experience in neglected infectious diseases. Despite the fact that few of these groups had ever worked together before, it is a testament to the success of the consortium that the new collaborations formed during its lifetime are set to continue to explore new advances together.
Because after all KINDReD was based on ambition. Its goal was to strengthen, inform and advance the current drug development pipeline in order to achieve at least one new Phase I clinical candidate for each trypanosomatid disease studied by the end of, or shortly after, the project’s course.
An impossible and naïve approach according to some detractors and in any case difficult considering that the EU budget had to cover the research of 13 groups for three years from early compound screening to advanced preclinical studies. However, KINDReD concentrated on all points of the pipeline and has been able, thanks to the incorporation of the NHP model, to move hits to leads and leads to drug candidates. One particular and important example is that KINDReD is now championing the first new chemical entity for the treatment of Chagas disease in over 30 years. K777 a cysteine protease inhibitor, has at least equivalent efficacy to benznidazole in the acute animal model for disease. Completion of the 28-day chronic dosing in primates, revealed no drug-related toxicity or other safety issues, therefore, submission of the IND application with progression to a Phase 1 safety study in “normal healthy” volunteers is now foreseeable.
With the KINDReD Association, a charitable organisation set-up as a durable footprint of the project, all the hits, leads and drug candidates from this project, and any other which should wish to join, have a lobbying force to help ensure that the best amongst these can also arrive in the clinic one day.
Project Context and Objectives:
Kinetoplastid Drug Development: strengthening the preclinical pipeline (KINDReD)
Summary description of the project context and objectives
The trypanosomatid diseases, Leishmaniasis, Human African trypanosomiasis and Chagas disease, continue to impart a heavy toll on human health, affecting millions of people worldwide particularly in the economically poorest countries(1). The handful of treatments currently available to control this enormous health burden is limited by serious adverse effects (toxic and/or teratogenic), high costs, difficulties in administration, political instability and the often rapid selection of drug resistance(2-4).
Worldwide efforts to combat “neglected infectious diseases” from bodies such as the pharmaceutical industry, the DNDi, the European Commission, Gates Foundation and others have resulted in several potential drug candidates at the preclinical trials stage but with only a few reaching clinical trial (Phases I to IV) and a depressingly low number of new compounds reaching the market. The CNS permeable drugs fexinidazole, a 2-substituted 5-nitroimidazole and SCYX-7158, an orally available benzoxaborole, show promising efficacy with the advanced stage of HAT and recruitment of patients to the fexinidaole Phase II/III (394 patients) in 2015. (http://www.dndi.org/strengthening-capacity/hat-platform/)
Nonetheless, there has been general market inertia to translate recent scientific and technological advances into the discovery of potent, safe drug candidates against the kinetoplastid protozoan parasites a factor that we communicated around strongly during the course of KINDReD.
In this SME-led consortium which brought together several novel technologies and partners new to the domain with established world leaders in neglected infectious disease research, we aimed to take an ambitious, fresh and unconventional approach to strengthen the preclinical pipeline with the aim of predicting in advance, potential pitfalls such as toxicity or resistance at the preclinical phase to have a greater chance of turning lead candidates to drugs in a timely and cost-effective manner.
References
(1) Stuart K, Brun R, Croft S, Fairlamb A, Gurtler RE, McKerrow J et al. Kinetoplastids: related protozoan pathogens, different diseases. J Clin Invest 2008; 118(4):1301-1310.
(2) Croft SL, Seifert K, Yardley V. Current scenario of drug development for leishmaniasis. Indian J Med Res 2006; 123(3):399-410.
(3) Barrett MP, Boykin DW, Brun R, Tidwell RR. Human African trypanosomiasis: pharmacological re-engagement with a neglected disease. Br J Pharmacol 2007; 152(8):1155-1171.
(4) Renslo AR, McKerrow JH. Drug discovery and development for neglected parasitic diseases. Nat Chem Biol 2006; 2(12):701-710.
Figure 1: Word cloud representation of the KINDReD project’s ethos
Objectives of the study
The aim of KINDReD is to create a unique and powerful drug discovery platform with the common objective of advancing promising laboratory-driven discoveries into clinical utility.
• To implement a coherent (integrated) approach to populate and advance all stages of the anti-trypanosomatid drug pipeline.
• To establish an integrated global network of academic and industrial partners united by a central objective: to develop and apply innovative cutting-edge molecular and cellular tools that will enable us to populate and accelerate the preclinical pipeline for anti-kinetoplastid chemotherapeutics.
• To advance every aspect of the preclinical pipeline, by developing new technologies where necessary, from early target discovery, validation and screening through to advanced toxicology and animal testing.
• To bring forward one new Phase I clinical candidate for each trypanosomatid disease studied at the end or within a short time after the end of the project.
• In parallel, and where appropriate in partnership with likeminded bodies such as WHO, Wellcome Trust, DNDI etc. we will set up an association that will lobby industrial groups to take an active role in the further clinical development of our registered candidates (repurposed or new chemical entities) showing potency against these parasitic diseases after the conclusion of the funding period.
HAT
Human African Trypanosomiasis or
Sleeping sickness
Figure 2: Our parasites and diseases
Project Results:
A description of the main S&T results/foregrounds
In KINDReD, originally composed of 14 partners, the scientific programme was divided over 9 of a total of 12 work packages (WPs). Our infrastructure for parasite screening integrated five leading academic laboratories in Europe (Portugal, United Kingdom and Switzerland), the United States of America (California) and South America (Brazil) with high throughput screening (HTS) facilities equally distributed between all three major kinetoplastid parasites. Follow-up medicinal chemistry and ADMET expertise was spear-headed by our industrial partners in France and Spain, respectively who interacted with the academic members of the consortium to guide their efforts. The full details of the progress of each of these WPs have been provided to the European Commission, in two periodic reports and a series of key deliverables. Below is a description of the work performed the original objective of the WP and the main results.
Figure 3: the central KINDReD work flow
Physiological and Target-based Screens
The largest of the work packages, WP1 “Physiological whole parasite screening assays” led by Jim McKerrow, now from UCSD, was originally planned as the driving force of the project, screening large numbers of compounds to pass hits (IC50 ≤ 1 µM) to WP 8 (Isbaal Ramos, Innoprot) the in vitro toxicology and ADME control step built into our reinforced pipeline to pinpoint potential adverse events before attempting animal experiments. This step was designed importantly to reduce the number of animals needed in preclinical testing as we were bound in our original ethical commitment to implement the 3Rs ‘Reduction, Refinement and Replacement’ under which we had agreed on a protocol to reduce the number of mice used by 59% through pooled testing of lead compounds.
We also ensured that the molecules tested were being screened in the in vitro system that best represented the physiologically relevant form of the disease-forming parasite. Screening assays incorporated state-of-the-art image-based screening of intracellular parasites in T. cruzi and Leishmania spp. as well as reporter-based assays of T. cruzi trypomastigotes, the blood-stream form of T. brucei, and Leishmania spp. intracellular amastigotes. Several whole cell parasite assays have been developed by the KINDReD partners for this purpose WP1 combined these assay systems with advances in liquid handling robotics and automated imaging to facilitate high-throughput screening of a wide collection of compounds derived from natural and synthetic sources.
The project had always been constructed with in-built flexibility which meant that over the three-year course of the programme we could change the source of molecules screened and also welcome libraries of potential hits from groups outside the original consortium to ensure our unique resources were used to full capacity.
Positive controls used in screening were melarsoprol for T. b rhodesiense, benznidazole for T. cruzi, miltefosine for L. donovani and L. infantum and podophyllotoxin for cytotoxicity in mammalian cells. All compounds were subjected to (a) single-point high-throughput screens in the physiologically-relevant parasite assay seeking > 80% inhibition at 10 μM. Then (b) EC50 values were obtained from 16-point sigmoidal dose-response curves performed in triplicate (range 100 to 0.001 μM). (c) Hits with EC50 ≤ 5 μM and ideally < 1 μM were retested to confirm long-term cidal versus static effects on cell growth. For T. cruzi and Leishmania spp. these were performed systematically on intracellular parasites where the screen identifies compounds that give complete parasitological cure in cultures of myoblasts (T. cruzi) or macrophages (Leishmania). Each promising hit compound was systematically retested in all three parasites and cytotoxicity tested in mammalian cell culture, e.g. 72h incubation with L-6 rat skeletal myoblasts, to define selectivity.
The move of the McKerrow laboratory from San Francisco to San Diego substantially reduced the whole-cell screening capacity of the consortium which endured for over 18 months this meant that alternative sources of hits needed to be considered. One of the most valuable resources has been the natural compound libraries that KINDReD has access to and formed the basis of the work in WP4. These are primarily from Helge Bode’s laboratory, in Frankfurt, (WP4). The group works mainly with exotic natural product producers such as entomopathogenic bacteria (Photorhabdus and Xenorhabdus), which are usually underrepresented in natural product libraries. These were screened against T. cruzi, T. brucei and L. donovani by Pascal Mäser’s group at the Swiss Institute for Tropical Health and interesting hits returned to the Bode laboratory for chemical total synthesis or derivatisation of the active components.
Another source of natural compounds was the Global Health Library of marine derived natural products which also produced numerous hits that have been passed on to the other WPs to study AMETox and in vitro immunomodulatory activity (WP7). A chance encounter at an International meeting on NTDs, led to KINDReD gaining access to a unique library of plant extracts from Nigeria which has now been screened by the McKerrow group, against all three parasites. This has revealed some very interesting activities especially against T. brucei and larger quantities are being synthesised in Nigeria for chemical identification of the active components in USCD. The table below summarises the screening efforts from KINDReD.
Table 1: results from the KINDReD screening programme
Another potential source of hits that has been explored is the 1 600 strong list of FDA approved drugs. Now screened against all three parasites, this collection has shown a handful of compounds of interest for further study that will be passed along the KINDReD pipeline because their previous approval status could speed registration. The compounds have now been studied in the other WPs for AMETox and in vitro immunomodulatory activity (WP7) and snap-shot PKs have been performed where considered of interest. One of these, candicidin, is currently being tested in an animal model of cutaneous leishmaniasis in the NMTrpI consortium a model available to KINDReD as part of our “synergy” activities. Unfortunately, we still do not have the results from this study so have no update on its status. If successful, our Partner AIIMS is well-positioned to run a clinical trial in India. The results from the other FDA-approved compounds have been less interesting as although the AMETox was promising, in animal testing the efficacies were generally poor.
Additional whole-cell screens have been performed using in-house molecules (Anabela Cordeiro da Silva, Porto) and fragments from a Maybridge rule-of-three library screened against L. major and T. cruzi by Terry Smith in St Andrews.
The continued use of the CDD data base has ensured all molecules have been correctly catalogue and all our data is in one place ready for further exploitation.
One in-built strengthening mechanism in the KINDReD pipeline was to test promising leads against clinical isolates in addition to the laboratory strains used in the screening platforms. This precaution was put in place as laboratory strains are known to lose virulence after multiple passages. This work was performed by our partners at AIIMS (L. donovani) and FIOCRUZ (T. cruzi). All the compounds tested to date were found to be effective on clinical isolates as well as on laboratory strains of parasites.
Alternative approaches to whole-cell screening for the identification of hits were always a major part the project. WP2, “Target identification and screening”, led by Terry Smith (USTAN) has been particularly successful in identifying numerous hits from screening several parasite protein deacetylase and kinase activities, TbUAP, TbINO1/TbIMpase 1 and 2, Tb, neutral sphingomyleinase and TbIDI. Many of these of been repurchased or synthesised and retested, validated hits have moved forward through iterative rounds of medicinal chemistry to evolve potency and/ selectivity using SAR and several are entering ADMET. These include the nitro-heterocycle-amides the vast majority, ~20, of which have a better IC50 than nifurtimox against T. brucei. Of these, one KD011 was seen as the best candidate to take forward for ADMET and animal testing.
The consortium also had access to a unique SPR array screening platform from NovAliX that features a diverse collection of library compounds (>116k compounds including >25k fragments with MW< 300Da) immobilised on gold chips. These high density chemical microarrays (9,216 sensor fields/array) enable the rapid screening of any target against one of the largest fragment libraries commercially available. Early selectivity information can be provided on the primary screening level. This platform was used to screen three of the consortium’s chosen targets. The first proteins analysed in this way was the Silent Information Regulator 2 protein, SIR2, a NAD-dependant tubulin deacetylase and which has been genetically and chemically validated as an anti-Leishmania drug target in extracellular, intracellular and animal models prior to the start of the project. The use of the purified protein in the SPR-arrays has identified several molecules of interest, one of which active against the L. infantum protein has now had its potential confirmed in high concentration bioassays at PHX and in intracellular amastigotes at the IBMC.
Our second target ribose-5-phosphate isomerase (RpiB) failed to uncover any molecules suitable for further follow-up by SPR but the thermal shift approach in St Andrews identified three fragments with activity against the parasites that may be legitimate hits to follow up.
During the course of the project the group from USTAN was also able to genetically and chemically validate drug targets in T. brucei and T. cruzi using compounds identified from their screening efforts. With the support of IBMC working in the Leishmania model we now have several new targets for future therapeutic campaigns that we have characterised functionally against all three parasites these include uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) and ribose 5-phosphate isomerase.
Structure-based drug design
Several approaches to FBDD have been developed over the past decade with widely-accepted advantages over HTS campaigns to discover lead compounds. All focus on finding a seed fragment with high ligand binding efficiency (i.e. -RTKd/Heavy atom count > 0.4). Thereafter, structure-based design coupled to iterative chemistry can allow the quick evolution of potent lead compounds with optimal ‘rule of five’ properties. This approach to drug discovery was explored in work packages 2 and 3 which brought together experts from academia and industry as shown in figure 4 below.
Our original target of interest was the sirtuin 2 protein from Leishmania, the study was enlarged to include the SIR2 proteins from all three parasites. The sirtuins proved intractable to crystallisation during the first 18-months of the project. However, in the second half of the project, SIR2 from all three parasites was successfully crystallised. Although, unfortunately the structure-based drug discovery phase that was originally planned to be performed with fragments generated e.g. from high concentration bioassays (PHX) and SPR (NOVA) was not able to be completed within the time frame of the project.
The second target chosen for crystallisation was ribose-5-phosphate isomerase (RpiB). The structure of the T. cruzi protein was already known and the proteins from L. infantum and T. brucei underwent crystallography studies. Although, unlike with sirtuin, crystals were quite readily obtained within the timeframe of the project we could not proceed to structure-based drug discovery. The structures are now known for future studies.
ADME-toxicity Studies
In vitro toxicology and ADME studies were the subject of WP 8. This was another SME-led work package, demonstrating the high-throughput technologies of the Spanish-based company Innoprot a specialist in HT drug toxicology testing. The platform acted as a funnel capable of filtering the compounds emerging from the physiological screens through a series of stringent tests to yield only the very best candidates for advanced animal testing. One of the reasons cited for the failure of modern HTS technologies to generate more new approved drugs is the early focus on potency as the major selection criterion. Often only a few of the most potent candidates will be selected for animal toxicology and ‘snapshot’ PK studies in small rodent models, inevitably reducing the chances of choosing the candidate with the best balance of efficacy, PK suitability and safety. In KINDReD we applied early high content toxicology and in vitro ADME assays to remove this emphasis. Our original plan had been to feedback information to our medicinal chemists so that compounds with good ADMETox parameters but lacking in potency can be fed back into the design process for potency improvements. However, it became apparent that we were limited in budget to perform this task effectively.
Nevertheless we were able to ensure that only compounds showing both anti-kinetoplastid potency and excellent ADMETox were tested in our animal models. This strategy therefore combined the optimal use of anti-parasite screening output with application of 3R guidelines on animal testing. One important feature of the work carried out in this WP has been to obtain data on the drugs currently used for the treatment of the three parasite diseases in order to create a data base of their ADME-tox characteristics and to be able to compare any new hit and eventual leads against these.
The “gold standards” that have been characterised in WPs 6, 7, 8 and 9 are amphotericin B and miltefosine for Leishmania and pentamidine for T. brucei and benznidazole and fexinidazole for T. cruzi. The results from WP8 show that from the compounds tested (28 candidates and 5 gold-standards) showed hepatotoxicity, nephrotoxicity and neurotoxicity median cellular cytotoxicity concentration (CC50) values 100 times lower (on average) than IC50 values. In terms of adsorption, distribution, metabolism and excretion (ADME), plasma protein binding assays for mouse and human have been completed for the compounds and compared to the gold standards. With additional secondary tests such as AMES, the compounds from KINDReD have now been ranked in relation to their ADMETox profiles and some in fact appear to have a more acceptable safety profile at this stage than some current treatments. This WP has also explored the effect of using PLGA nanoparticles on the delivery of potential anti-parasitic compounds and has shown controlled and effective delivery of BNIPDaoct and for L. infantum and T. brucei infection with significantly reduced toxicology as evidenced by PLGA-encapsulation of amphotericin B.
Further exploration of our “gold-standards” by Jérôme Estaquier at the CNRS has demonstrated that miltefosine, amphotericin B and pentamidine, depending on the concentration used at either micro- or at millimolar levels, induce mitochondrial depolarisation and cell death. Moreover, we have demonstrated that the effects are cell type dependent. In particular, we found that monocytes are especially sensitive to miltefosine and pentamidine. Because monocytes represent preferential targets for Leishmania infection, we demonstrated that infection with L. infantum modulates mitochondria metabolism exerting a regulatory effect on oxidative phosphorylation. Therefore, we are testing the impact of the drugs on monocyte metabolism as well as on the profile of OPA1. This work is also of particular importance to WP7 (Immunomodulation) led by Olindo Assis Martins Filho, in Brazil. Current guidance documents for preclinical filing of new drugs require evaluation of unintended immunomodulation and advocate additional immunotoxicity studies to characterise risk including ex vivo immunophenotyping of blood cells and in vitro immune function. Our experiments to date suggest it is of crucial interest to concomitantly monitor the immune response to assess the side effects of the drugs in relation to the nature of the cells that are sensitive to their effects.
Animal testing
Work package 9, animal studies of efficacy and toxicology was led by Anabela Cordeiro-da- Silva (IBMC). The strains and models used were the following; stationary phase promastigotes (visceral leishmaniasis virulent clone L. infantum MHOM/MA/67/ITMAP-263 and cutaneous leishmaniasis, L. major LV39 MRHO/SU/59/P), for T. cruzi trypomastigotes (the clinical strain, CAI-72, produces cardiac disease and cardiac failure similar to that seen in human Chagas disease) and for T. brucei blood strain trypomastigotes (e.g. clonal line of virulent T. b. rhodesiense STIB900). Parasites were injected i.p. at 108 parasites for Leishmania and T. cruzi or 104 for T. brucei. For Leishmania L. infantum and T. cruzi four mice from each infected group were treated after 14 days of infection by i.p. injection during three consecutive days. Parasitaemia was originally monitored two to three times a week by microscopic counting of tail vein blood in a Neubauer chamber. During the project, live-imaging was introduced to follow parasitaemia (see figure 5). Mice that were aparasitaemic for 60 days after the treatment period were considered cured.
Figure 5: In vivo imaging of BALB/c mice infected with T. brucei after 5 days of treatment. Parasite load was evaluated by subcutaneous injection of 2.4 mg of luciferin and all animals were imaged using IVIS LUMINA LT groups at day 5 post-infection are represented.
The biodistribution of the reference compounds after intraperitoneal injection has been determined in our mouse models for comparison with KINDReD lead compounds. The in vivo efficacy of several test compounds against Leishmania was found to be enhanced by encapsulation in pegylated nanoparticles. However, several compounds reaching this WP and tested against the mouse model for T. brucei and T. cruzi have unfortunately, shown to have little or no in vivo efficacy.
Our second animal model in KINDReD to complete the final stages of evaluation of a drug candidate, which has shown good safety and tolerability in the mouse model, is the non-human primate (NHP) specifically the Macaca Mulata adult male. The use of NHP as animal models for the study of human diseases (including immunological studies and drug and vaccine development studies against infectious diseases) has become increasingly important. During the course of KINDReD, the NHP model has been used to perform a 28-day chronic dosing study for K777. K777 is a protease inhibitor effective against T. cruzi for which registration to use in the treatment of Chagas disease is being sought. Originally, part of the DNDi portfolio development of this drug as halted after a poor tolerability result (see figure 6). GMP drug ready for safety testing in the NHP model (CNRS) was provided by the McKerrow laboratory and the study was performed following an FDA approved protocol. Jérôme Estaquier has shown that the drug could be safely administered to the NHP with no immediate or long-term side-effects. This successful study should complete the pre-IND dossier for FDA approval we are waiting to hear from the FDA for a date to discuss this data.
Figure 6: screen shot from the DNDi website showing their decision to discontinue K777 as a potential new treatment for Chagas disease.
The unique approach of KINDReD
Two work packages that were slightly to the side of the main direction of the work flow in KINDReD are WPs 5 and 6 looking at Chemoproteomics: deciphering drug “on” and “off” target interactions and parasite metabolism, respectively. These WPs are again an opportunity for “new blood” in terms of neglected infectious diseases to use their well- defined technologies and prior knowledge in the pursuit of the project’s aims. The chemoproteomics approach WP5, led by Gilbert Skorski, Phylogene, was intended as an early exploration of the potential for ABPP probes to assist the drug development workflow, requiring (i) implementation of existing methodologies and assessing their utility to drug development as well as (ii) the development of new ABPP probes and associated workflows. Whilst several important steps have been made towards these goals, the heavy demands on novel ABPP chemistry, as well as the fine-tuning of sophisticated technology to ‘read-out’ off-target interactions, has limited our analyses to a small number of the potential avenues that could realistically be explored over this three year project. Resveratrol probes (described in the mid-term report) were abandoned in favour of more promising 5-nitro-2-furancarboxamide derivatives, in direct collaboration with USTAN and NOVA. A phosphoproteomics pipeline to screen both the parasite and host proteomes was applied in order to identify new drugs targets. Infection by T. brucei has effects on the proteome and phosphoproteome in the mouse brain model. An impact on the immune response was shown by differential expression level but also on the brain plasticity in our phosphoproteomics experiments. This preliminary -omics study can be used as a starting point to find a new molecular targets for treatment and also getting a better understanding of the T. brucei infection mechanism. Used to label proteins after co-incubation with T. brucei cells their subsequent identification with the new probes represents an important step in understanding the mechanism of action and cellular targets for this new class of kinetoplastid inhibitors.
Work package 6 led by Alain Pruvost from the CEA is another WP that changed course during this second reporting period. Terry Smith’s group in St Andrews contributed much to this WP over the latter half of the project. In collaboration with a Canadian group, they have investigated mutations in a P-type ATPase transporter in Leishmania that lead to cross-resistance to two leading drugs by distinct mechanisms. Uptake of the potential anti-T. cruzi compound KD011 into macrophages has been investigated in collaboration with the CEA and the IBMC. Attempts have been made to gain a better understanding of the catabolic processes occurring in the lysosome of the parasites. In another approach Photeomix (PHX) have characterised proteolytic activities in leishmania promastigote cell extracts which exhibit a strong potential destroy peptide drugs.
Work package 12, synergistic activities continued to make progress towards working together. KINDReD and NMTrypI were able to exchange animal models, as close collaboration has ensured the best use of resources in the interests of advancing the kinetoplastid pipeline. The synergy meeting held in Modena in June this year was a good example of how much exceptional science and technology is contained within this European Framework call. The co-ordinators of all four projects presented an open letter to the EC to this effect and have also presented a poster at the recent COST-Action Meeting in Madrid in a similar vein.
Final results and their potential impact and use
• We have now have two potential lead candidates to move into phase I trials:
o The FDA dossier is currently under approval for the use of K777 in Chagas disease
o An EMA dossier is under construction for repurposing candicidin in cutaneous Leishmaniasis
• We are confident that our new techniques for use in the preclinical pathway especially our chemoproteomic probes and metabolism measurements will be of use to other researchers in the field including our synergy partners.
• Photeomix has launched a series of services based on the discovery of small molecule inhibitors of post-translational modification enzymes which is a direct result of their involvement in KINDReD.
• New products and services are available from Photeomix and other SME partners will no doubt follow over the coming months.
• KINDReD has uncovered rich source of new targets which are worthy of further research as are the new scaffolds, fragments and natural products we have shown to be active.
• The KINDReD Association is a new and experimental model focused initially on lobbying funding bodies to bridge the gap between late-preclinical and first-in-human trials.
• KINDReD has been unique in bringing together partners new to the field of parasite research all of whom have integrated well into the project. It is a source of new and potentially powerful partnerships in a field that perhaps has suffered from dogmatic direction. We plan to continue the open and encompassing approach we have taken to studying the pipeline and difficulties experienced in this field after this funding period finishes.
Figure 1: the central KINDReD work flow
The largest of the work packages, WP1 “Physiological whole parasite screening assays” led by Jim McKerrow, now from UCSD, was originally planned as the driving force of the project, screening large numbers of compounds to pass hits (IC50 ≤ 1 µM) to WP 8 (Isbaal Ramos, Innoprot) the in vitro toxicology and ADME control step built into our reinforced pipeline to pinpoint potential adverse events before attempting animal experiments. This step is designed also to help reduce the number of animals needed in preclinical testing. However, the McKerrow laboratory that had moved from San Francisco to San Diego in the first 18-month period still only slowly began to feed hits into the pipeline. Our alternative sources of hits continued to be the natural compound libraries that KINDReD has access to. Over this period Helge Bode’s laboratory, in Frankfurt, (WP4) sent more compounds to be screened by Pascal Mäser’s group at the Swiss Institute for Tropical Health. A chance encounter at a meeting where Jane MacDougall presented the consortium, led to KINDReD gaining access to a unique library of plant extracts from Nigeria which has now been screened by the McKerrow group, against all three parasites. This has revealed some very interesting activities especially against T. brucei and larger quantities are being synthesised in Nigeria for chemical identification of the active components in USCD. The compounds screened from a1600 strong list of FDA approved drugs in the first period of the project have now been studied in the other WPs for AMETox and in vitro immunomodulatory activity (WP7). One of these, candicidin, is currently being tested in an animal model of cutaneous leishmaniasis in the NMTrpI consortium a model available to KINDReD as part of our “synergy” activities. We hope to be able to update on its status in the final report. If successful, our Partner AIIMS is keen to run a clinical trial in India. The results from the others have been less interesting as although the AMETox was promising, in animal testing the efficacies were poor.
Alternative approaches to whole-cell screening for the identification of hits were always a major part the project and work performed in work packages 2 and 3 have shown that these are of some value. WP2, “Target identification and screening”, led by Terry Smith has been particularly successful in identifying numerous hits from screening several parasite protein deacetylase and kinase activities, TbUAP, TbINO1/TbIMpase 1 and 2, Tb, neutral sphingomyleinase and TbIDI. Many of these of been repurchased or synthesised and retested, validated hits have moved forward through iterative rounds of medicinal chemistry to evolve potency and/ selectivity using SAR and several are entering ADMET. These include the nitro-heterocycle-amides the vast majority, ~20, of which have a better IC50 than nifurtimox against T. brucei. Of these, one KD011 was seen as the best candidate to take forward for ADMET and animal testing.
The group from USTAN was also able to genetically and chemically validate drug targets in T. brucei and T. cruzi using compounds identified from their screening efforts. With the support of IBMC working in the Leishmania model we now have several new targets for future therapeutic campaigns that we have characterised functionally against all three parasites these include uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) and ribose 5-phosphate isomerase.
Work package 3, “Structure-based drug design” led by Paola Ciapetti from NovAliX, continued to showcase the novel technologies brought to the consortium by our SME partners.
Our original target of interest was the sirtuin 2 protein from Leishmania, previously described by Cordeiro-Da-Silva’s group as essential to parasite growth and as such a potential target for drug discovery. The study was enlarged to include the SIR2 proteins from all three parasites. The sirtuins proved intractable to several approaches used in this consortium during the first 18-months of the project. However, in the second half of the project SIR2 from all three parasites was successfully crystallised. Although, unfortunately the structure-based drug discovery phase that was originally planned to be performed with fragments generated e.g. from high concentration bioassays at was not able to be completed within the time frame of the project. SPR-screening of the proprietary fragment libraries at NOVA resulted in some candidate fragments against SIR2 being identified. These have now been tested in both high concentration bioassays and with macrophagic amastigotes (IBMC), and one of these appears to be promising to move along the pipeline.
The second target chosen for crystallisation was ribose-5-phosphate isomerase (RpiB). The protein from the three different parasitic species was produced and subjected to SPR fragment screening. In parallel the proteins from L. infantum and T. brucei underwent crystallography studies, while again the project could not proceed to structure-based drug discovery. The structures are now known for future studies. While SPR was unable to uncover any molecules suitable for further follow-up, the thermal shift approach in St Andrews identified three fragments with activity against the parasites that may be legitimate hits to follow up.
All compounds and molecules screened in KINDReD along with the results are entered in the consortium’s data base hosted by CDD Vault for the information of, and use by, all partners.
In vitro toxicology and ADME studies are the subject of work package 8. This is another SME-led work package, demonstrating the technologies of the Spanish-based Innoprot. One important feature of the work carried out in this project has been to obtain data on the drugs currently used for the treatment of the three parasite diseases in order to be able to compare any new hit and eventual leads against these. The “gold standards” that have been characterised in WPs 6, 7, 8 and 9 are amphotericin B and miltefosine for Leishmania and pentamidine for T. brucei and benznidazole and fexinidazole for T. cruzi. The results from WP8 show that from the compounds tested (28 candidates and 5 gold-standards) showed hepatotoxicity, nephrotoxicity and neurotoxicity median cellular cytotoxicity concentration (CC50) values 100 times lower (on average) than IC50 values. In terms of adsorption, distribution, metabolism and excretion (ADME), plasma protein binding assays for mouse and human have been completed for the compounds and compared to the gold standards. With additional secondary tests such as AMES, the compounds from KINDReD have now been ranked in relation to their ADMETox profiles and some in fact appear to have a more acceptable safety profile at this stage than some current treatments. This WP has also explored the effect of using PLGA nanoparticles on the delivery of potential anti-parasitic compounds and has shown controlled and effective delivery of BNIPDaoct and for L. infantum and T. brucei infection with significantly reduced toxicology as evidenced by PLGA-encapsulation of amphotericin B.
Further exploration of our “gold-standards” by Jérôme Estaquier at the CNRS has demonstrated that miltefosine, amphotericin B and pentamidine, depending on the concentration used at either micro- or at millimolar levels, induce mitochondrial depolarisation and cell death. Moreover, we have demonstrated that the effects are cell type dependent. In particular, we found that monocytes are especially sensitive to miltefosine and pentamidine. Because monocytes represent preferential targets for Leishmania infection, we demonstrated that infection with L. infantum modulates mitochondria metabolism exerting a regulatory effect on oxidative phosphorylation. Therefore, we are testing the impact of the drugs on monocyte metabolism as well as on the profile of OPA1. This work is also of particular importance to WP7 (Immunomodulation) led by Olindo Assis Martins Filho, in Brazil. Our experiments to date suggest it is of crucial interest to concomitantly monitor the immune response to assess the side effects of the drugs in relation to the nature of the cells that are sensitive to their effects.
Work package 9, animal studies of efficacy and toxicology is led by Anabela Cordeiro-da- Silva. The biodistribution of the reference compounds has been determined after intraperitoneal injection for comparison with KINDReD lead compounds. The in vivo efficacy of several test compounds against Leishmania was found to be enhanced by encapsulation in pegylated nanoparticles. However, several other compounds reaching this WP and tested against the mouse model for T. brucei and T. cruzi have unfortunately, shown to have little or no in vivo efficacy. The NHP model has been used to complete the dossier for FDA approval for the protease inhibitor K777, which has been shown to have potential as new therapeutic agent against Chagas disease. Jérôme Estaquier working with the drug produced by the McKerrow laboratory showed that the drug could be safely administered to the NHP with no immediate or long-term side-effects. This data has been used to complete the approval procedure for the FDA.
Two work packages that are slightly to the side of the main direction of the work flow in KINDReD are WPs 5 and 6 looking at Chemoproteomics: deciphering drug “on” and “off” target interactions and parasite metabolism, respectively. These WPs are again an opportunity for “new blood” in terms of neglected infectious diseases to use their well- defined technologies and prior knowledge in the pursuit of the project’s aims. The chemoproteomics approach WP5, led by Gilbert Skorski, Phylogene, was intended as an early exploration of the potential for ABPP to assist the drug development workflow, requiring (i) implementation of existing methodologies and assessing their utility to drug development as well as (ii) the development of new ABPP probes and associated workflows. Whilst several important steps have been made towards these goals, the heavy demands on novel ABPP chemistry, as well as the fine-tuning of sophisticated technology to ‘read-out’ off-target interactions, has limited our analyses to a small number of the potential avenues that could realistically be explored over this three year project. Resveratrol probes (described in the mid-term report) were abandoned in favour of more promising 5-nitro-2-furancarboxamide derivatives, in direct collaboration with USTAN and NOVA. Used to label proteins after co-incubation with T. brucei cells their subsequent identification with the new probes represents an important step in understanding the MOA and cellular targets for this new class of kinetoplastid inhibitors.
Work package 6 is led by Alain Pruvost from the CEA is another WP that changed course during this second reporting period. Terry Smith’s group in St Andrews contributed much to this WP over the second reporting period. In collaboration with Marc Ouellette they have investigated mutations in a P-type ATPase transporter in Leishmania that lead to cross-resistance to two leading drugs by distinct mechanisms. With the CEA and the IBMC, uptake of the potential anti-T. cruzi compound KD011 into macrophages has been investigated. Also they have attempted to gain a better understanding of the catabolic processes occurring in the lysosome of the parasites while PHX have characterised proteolytic activities in leishmania promastigote cell extracts which exhibit the strong potential destroy peptide drugs.
Of the remaining three work packages, WP 11 is concerned with management activities that are shared by St Andrews the project co-ordinator and Photeomix, the scientific co-ordinator. While at the management of the consortium has continued to be challenging the working relationship is excellent and no problem has yet proved insurmountable, thanks in part to the excellent support from our EU project office.
Work package 10 “regulatory affairs” led by Jane MacDougall, Photeomix, also covers standardisation of protocols and dissemination activities. Throughout this reporting period, dissemination activities continued to be closely monitored and communicated KINDReD public website www.kindred-fp7.com. Internal consortium updates have been maintained by the use of Basecamp, a web-based communication platform with encrypted access to consortium members only. The Scientific Co-ordinator, Photeomix has continued to present the consortium at International meetings.
The KINDReD Association is fully operational and now has over 50 members, including members of the public and members from all four FP7-funded projects in neglected infectious disease. The Association is an excellent means of dissemination for KINDReD’s activities but also importantly stands as a focal point to ensure the momentum from this FP7 call moves forwards. The Association is active on social media as well as more conventional channels so that interest remains to continue funding of this work and ensure our most advanced candidates do finally reach the clinic.
During this period the consortium beneficiaries published a further 21 scientific articles related to their work carried out under KINDReD to add to the 16 from the first 18-months. This latter list follows this summary. Numerous articles will continue to be published as the consortium members continue to complete the work of the consortium.
Work package 12, synergistic activities continued to make progress towards working together. KINDReD and NMTrypI were able to exchange animal models, as close collaboration has ensured the best use of resources in the interests of advancing the kinetoplastid pipeline. The synergy meeting held in Modena in June this year was a good example of how much exceptional science and technology is contained within this European Framework call. The co-ordinators of all four projects presented an open letter to the EC to this effect and have also presented a poster at the recent COST-Action Meeting in Madrid in a similar vein.
Potential Impact:
The potential impact of KINDReD
Impact on world health and quality of life
Protozoan parasites are amongst the most common infectious agents in the tropics and subtropics. The respective diseases are a major cause of morbidity and mortality in many developing countries and have serious consequences for socio-economic development in these regions. Primary infections are not the only concern; secondary infections in HIV-infected and immuno-compromised patients also pose grave health risks. A major problem associated with these diseases is their prevalence in third world countries which are the least well-equipped to develop new drugs and invest in R&D.
• Leishmaniasis is a worldwide disease, affecting 88 countries. The annual incidence is estimated at 1-1.5 million cases of CL and 500 000 cases of VL. The overall prevalence of the disease is 12 million people and the population at risk is 350 million.
• Chagas disease occurs throughout Mexico and Central and Southern America, and continues to pose a serious threat to health in many countries of the region. The overall prevalence of human T. cruzi infection is estimated at 16-18 million cases with an annual burden of 196 million € in healthcare costs. Approximately 120 million people, i.e. 25% of the inhabitants of Latin America, are at risk of contracting the infection.
• For African trypanosomiasis, estimates indicate that over 60 million in 250 foci are associated with the risk of contracting the disease, and there are about 300 000 new cases every year. However, less than 4 million people are under surveillance and only about 40 000 are diagnosed and treated, due to difficulty of diagnosis and remoteness of affected areas. These figures are relatively small compared to other tropical diseases, but African trypanosomiasis, without intervention, has the propensity to develop into epidemics, making it a major public health problem with a case fatality rate in untreated patients of 100%.
Control of trypanosomatid protozoan family is a major challenge not only in the third world, but increasingly in the western hemisphere, including Europe as migration and climate change move both people and the disease-bearing vectors, respectively into previously infected areas. The KINDReD consortium has responded to this global threat through the development of two new treatments for trypanosomal diseases. These are a completely novel reagent against Chagas disease and a potential therapy from repurposing candicidin for use in cutaneous Leishmaniasis that ultimately will convey significant benefits for human health in countries where these diseases are endemic.
Impact on the anti-trypanosomatid drug pipeline- turning pipe dreams into clinical reality
Our screening, drug design and lead optimisation strategies have amassed a comprehensive portfolio of fully characterised drug leads, with emphasis placed from the beginning of the project to having candidates for early Phase I clinical trials. Our understanding of the fact that the regulatory approval process is a long, expensive and meticulous procedure of controlled clinical testing, with a low average success rate led us to perform extensive preclinical toxicity testing including introducing novel ‘critical path initiatives’ such as the introduction of preclinical genomic and chemoproteomic profiling. By passing the current treatments available to treat these diseases through the same platforms we have built a comprehensive picture of ADME Tox profiles that are acceptable to further development.
All our promising lead compounds will be carefully scrutinised for cellular markers of oxidative stress, mitochondrial damage, genotoxicity, DNA damage, apoptosis, cell viability and morphological dysfunction, again compared directly with current treatments. This way we have a clear preclinical profile for any future registration dossier.
Novel chemoproteomics techniques using active site probes have been developed to signal unwanted drug-protein interactions in host cells, potentially allowing identification and modification of off-target effects by further iterative drug remodelling. This work is still in a preliminary stage but is worthy of further investigation.
Impact on innovation and competitiveness of the larger pharmaceutical market
One of the major distinctions of the multi-disciplinary KINDReD consortium from other consortia has been the integration of multiple technologies on a common platform. The success of our innovative drug development procedures is equally applicable to the larger pharmaceutical market. Within this consortium we were driven by a need for cost-effective research, procedures to be developed by our members, such as differential drug fragment
-selectivity-profiling and chemoproteomic ‘off-target’-toxicology-profiling, these techniques have the potential to increase preclinical research efficiency, reduce drug candidate attrition and reinforce the competitiveness of the European pharmaceutical market in general. Some of the effective targets discovered by the consortium may also be investigated independently for clinical utility in other infectious or chronic diseases, such as malaria or cancer.
The linear decline in pharmaceutical research productivity over the past six decades suggests that new knowledge, technologies and increased resources, however powerful, do not translate directly into the approval of more new drugs. This is particularly the case observed with the adoption of high throughput screening techniques. The research productivity paradox was treated in the US Food and Drug Administration’s landmark ‘Critical Path Initiative’ launched in 2004 which called for an investigation into efficiency improvements that could be made at all stages in the development path for new medicines. It is here that the most recent technology developments can have their biggest impact on productivity. Tools developed specifically to evaluate the efficacy and safety of medicines at early stages in the process can improve pharmaceutical research productivity allowing informed decisions can be made earlier in the process. The KINDReD consortium has put the development and testing of such tools at the heart of the preclinical development of antiparasitic agents. Indeed, is a fascinating and sobering thought that a FP7-led initiative, gathering experts together to tackle neglected parasitic diseases that plague some the world’s poorest nations, could provide solutions central to the revitalisation one of the world’s most wealthy industries.
Impact on European SMEs
Another focus central to the KINDReD initiative was the strength of the SME involvement, each one of the which took a leading role in the work packages. Individual European countries encourage SMEs to perform internal research into new products and services aided by regional, national and tax credit initiatives. It has long been recognised that these activities are at the heart of the European economy, where SMEs account for 99% of private sector businesses and employ two thirds of the European workforce. The 2008 Small Businesses Act for Europe created a policy framework that recognises the central role of SMEs in the European economy. This ‘think small first’ policy can be applied equally to SMEs in the biotechnology sector, where SMEs bring innovation and expertise but can suffer from being a small player in the larger market. To flourish, SMEs need to become more competitive and more entrepreneurial. KINDReD offered a risk sharing environment for SMEs to develop the innovative products that underscore their business activities whilst contributing to the greater effort of eradicating the global burden of kinetoplastid parasite diseases. This initiative provides SMEs with both financial and intellectual support within a European framework of focused drug centred research. Each SME has used their research efforts to strengthen their market competiveness by expanding their research capabilities, developing their contract research and product ranges and developing new expertise in previously untapped areas.
Impact on ICPC countries
Eradication of neglected infectious diseases in poor countries is a global health priority, a decision supported by the leaders of the world’s richest economies at several G8 summits and recently endorsed by the London Declaration on Neglected Tropical Diseases inspired by the World Health Organisation’s 2020 roadmap on NTDs. Two of the partners in the KINDReD consortium are from ICPC countries of low income (Brazil) and low-middle income (India) where kinetoplastid diseases are endemic. Biotechnology efforts are underway in these countries to develop diagnostic and medicinal products. These efforts will be assisted by strengthening their existing research infrastructure and challenging the problem internally rather than relying only on external academic/bio-pharmaceutical efforts. Thus KINDReD has had a major impact by focusing funding on ICPC research groups, helping them employ teams of researchers working in clinical environments at the source of the health burden. Their researchers have benefited from the application of many of the technological advances of the KINDReD programme and they are ready to help us with the regulatory procedures needed to be followed in order to perform clinical trials in these countries.
Impact on future EU-directed neglected infectious disease research.
The EU has long been a leader in the initiatives against neglected infectious disease supporting NID initiatives throughout several framework programmes. For these initiatives to achieve a major impact by Horizon 2020, the challenge to provide a link between prior efforts and the establishment of a clinical trial must be met. The KINDReD programme has shown how the scientific community can come together to make this a reality, leaving a permanent footprint through the establishment of clear operating procedures and workflows that will facilitate effective preclinical research throughout the current programme and beyond. The founding of these procedures in written, open access form will have a major impact on future research initiatives. Furthermore, research into neglected infectious diseases having demonstrable applications to the general pharmaceutical pipeline should aid healthcare funders and stakeholders with future funding initiatives.
Impact on society in general.
Society should be aware of how the taxpayer’s money is spent on issues of great health and
socioeconomic importance. In addition to the consortium agreement, we have created an independent non-profit association to convey information relating to KINDReD’s activities to the research community and the public in general, to leave a durable footprint at the end of the 3 year project and to ensure the necessary continuity for the clinical development of the most promising lead compounds and the associated regulatory approval processes. Our activities on social media have had positive returns particularly from people living in endemic areas and we are continuing to communicate our progress to them even although the initial funding period is over.
Main dissemination activities and exploitation of results
Our dissemination measures were run by WP10. Throughout the period of the grant, dissemination activities have been closely monitored and communicated as much as possible via the KINDReD public website. The majority of partners have been active in disseminating their activities in KINDReD at different meetings both National and International with poster and oral presentations as shown in the lists below.
The Scientific Co-ordinator partner 4 (PHX) has presented KINDReD at several International meetings and discussed with several major parties interested in NTDs including the Wellcome Trust and the French MTN network.
Members of the consortium have continued to publish numerous scientific articles with 30 peer reviewed articles shown here. Work is still on-going to publish more articles and numerous publications citing KINDReD are already planned and will continue to appear from the work performed in KINDReD. Wherever possible publications are in line with the Commission’s wishes Open Access.
The KINDReD Association, an organisation of charitable status, has been set up, initially to support the KINDReD consortium. The Association has been promoting the research undertaken by the KINDReD consortium with links to press-releases and social media and provides a durable footprint of the FP7 funded research. It operates under open-source principles, providing a community-driven forum (1) to share data and views on research objectives and results, (2) provides access to materials and techniques of the KINDReD consortium for NID drug development, (3) has established metrics to judge the success of project goals versus the international NID objectives and (4) to collaborate across organisational boundaries.
It has will continue to produce press-releases targeting the local, national and international news media. Social media including popular resources such as Twitter and Facebook provide information and link the KINDReD website and press-releases with associations and blogs.
Our work through synergy with the other four projects financed under the FP7- call HEALTH 2013.2.3.4-2 Drug Development for Neglected Parasitic Diseases we are combining our efforts to continue dissemination of the EU’s contribution to funding neglected infectious disease research by amongst other channels the KINDReD Association which has the potential to continue the research funded here for many years to come, ensuring that new, affordable treatments are provided to those in the most need.
Project public website
www.kindred-fp7.com
List of Websites:
http://kindred-fp7.com/
Contact details: Dr Jane MacDougall, Photeomix, 34 rue Carnot, 93160 Noisy le Grand, France email: jmacdougall@photeomix.com.
