Novel antifungals to treat resistant organisms

Final Report Summary - NOFUN (Novel antifungals to treat resistant organisms)

Executive Summary:
The NOFUN project was conceived with the idea of exploiting some interesting antifungal chemistries and technologies in order to move forward antifungal drug discovery. This area has been neglected by the pharmaceutical industry for many years with the result that no new classes of antifungal drug have been introduce for over a decade. This is set against a background of increased systemic fungal disease and also the recognition of the role of fungi in exacerbating chronic respiratory conditions. In addition, there has been a year on year increase in resistant fungal infections which reduces the effectiveness of the main class of systemic antifungal- the azoles.
NOFUN set its goals to advance a novel chemical class broad spectrum antifungal which was active against a new fungal target. At the start of the project the team did not know for certain the mechanism of action of the drug class. This is important because firstly it allows drug discovery scientist to assess the validity of the target with respect to the treatment of people. If the target were similar to vital processes that were present in man, then that would be less attractive to continue. Also a knowledge of the target is extremely helpful to medicinal chemists who can use the information to design better compounds more quickly. During the project the NOFUN team used advanced molecular and genetic techniques to confirm the mechanism of action of the new drug class. This mechanism was evaluated and found to be a good antifungal target present in all the common pathogenic fungal species. Knowledge of the target was then fed into the medicinal chemistry design process. At the start of the project the initial chemical hits had some interesting but sporadic activity against fungal pathogens. For example activity against the increasingly resistant Candida species C. glabrata and C. krusei was poor. Through a structure guided medical chemistry approach new molecules were designed and synthesised that had good activity against all key Candida species as well as the main pathogenic Aspergillus species. This was a significant advance in the chemistry achieved by NOFUN.
In addition to improvements in antifungal potency, NOFUN assessed the drug like or ADME properties of the new antifungal chemical class. It is vital to understand the ADME properties of a drug class as a class specific liability would prevent a new class from being developed any further.
NOFUN experts assessed the protein binding, CYP inhibition properties, metabolism, distribution and mutagenicity of representative members of the new drug class. No class related issues were found with the compounds tested which would prevent further. The main issues identified was the short time the drug remained within the body before being excreted. This potentially impacts efficacy studies although significant reduction in fungal burdens were observed in several infection models. The NOFUN project ended with the identification of active broad spectrum compounds which had good ADME properties and in vivo efficacy. These compounds can now be exploited further.
Another aim of the NOFUN project was to identify new antifungal drug targets that could be exploited downstream to further promote antifungal drug discovery. The NOFUN project has discovered and validated several new antifungal drug targets which will be further exploited. New transcription factors which control proteins responsible for drug resistance were identified along with new biochemical drug targets. Patent applications and exploitable “know how” will likely follow to support future antifungal drug discovery.

Project Context and Objectives:
Invasive fungal diseases are estimated to kill 1.5 million people each year. The incidence of mortality has risen significantly across the EU over the last 20 years due to an expansion of at risk patient populations. Given the obvious importance of these diseases it is perhaps surprising that only four classes of drug are available to treat systemic fungal infection. The azole class of antifungals provide the front line role for most disease treatment but recently resistance has emerged and it is of growing concern that levels are rising dramatically. European researchers have led the world in identifying the extent of the problem with some centres reporting itraconazole resistance in the Aspergillus species as high as 20% and in Candida, resistance to posaconazole upto 30%. Additionally the epidemiology of serious fungal infections is changing with more intrinsically resistant organisms now being seen more frequently. This represents a major problem for clinicians who are increasingly treating infections for which there is no current effective therapy.
This project brought together leading European SME’s and academics to address this problem through
the development of novel classes of antifungals and the identification of novel drug targets. Prior to the start of the project, a member of the consortium had identified potent novel broad spectrum antifungal molecules that were active against multi-resistant fungal pathogens and the intension was to qualify these as drug candidates. One of these assets, the F6 series, was already at the lead identification stage. Further work was required to optimise a lead compound that would be suitable for further development.
Another member of the consortium has also identified mitochondrial (pptB) an antifungal target.
With its wide ranging expertise across medicinal chemistry, ADMET, fungal biology, high-throughput genomics, chemical genomics and drug development the partners aimed to build and progress a broad pipeline of agents that have the potential to reach the clinic within 5 years. In order to fulfil this aim, the main objectives of this project were:
• to identify a candidate from the F6 series of compounds that would be suitable for entering phase I enabling GLP toxicology.
• to identify an antifungal candidate against pptB and take it through to an in vivo clearance toxicology study.
• to identify and validate the mechanism of action of early stage novel small molecule hits that demonstrate selective antifungal activity.
• to underpin and drive the development activities by the use of a structure-based design approach and by the provision of medicinal chemistry.

These objectives guided the work plan, however, two additional objectives were:
• to provide effective project communication outwith the consortium
• to ensure protection of intellectual property in order to allow successful commercial exploitation

Project Results:
Summary of output

Mechanism of Action of the F6 series.

A major issue with the current marketed antifungal is that they are active via only three mechanism of action. Because of the increase of resistance against the azole class this could soon be reduced to two. New drugs acting via new mechanism are urgently needed. A major output of NOFUN is the definitive confirmation that the F6 antifungal series acts via a new mechanism of action. Using a combination of approaches, we have confirmed that the F6-series acts via inhibition of lipid synthesis. Reversal assays were used to first identify the general area of activity of the compounds. These assay were then refined to specifically interrogate individual pathways. This data set strongly indicated a specific set of genes affected by the F6 series. These genes were knocked out using genetic techniques and the phenotypes characterised. Again a specific target gene was implicated. Using other gene expression techniques and pathogenicity models the exact gene targeted by the F6 chemistry was confirmed. This represents a completely new and complementary mechanism of action for systemic antifungal drugs

Chemical Development of the F6 series

During NOFUN over 350 novel compounds were designed and synthesised to target the new mechanism antifungal target. These compounds were designed to explore the chemical space around the proposed binding site of the target. Several chemical themes were followed to do this. Ultimately this led to one type of molecule showing the best activity against pathogenic fungi so the majority of the analogues designed and synthesised were variants of this type of chemistry.
Using molecular models of the target enzyme, NOFUN chemists were able to iteratively design new compounds to interact with specific residues within the target active site. This led to considerable improvements in the activity profile of the analogues. Key target resistant fungal species that previously were not very susceptible to the F6 series became susceptible. This included the often drug resistant species Candida glabrata and Candida krusei. The activity against key mould pathogens such as Aspergillus fumigatus was also maintained. The key output from the chemical development of the F6 series was a more focussed chemistry effort, which delivered a more potent antifungal series.

Key Outputs
• A number of additional very active analogues have been prepared, which exhibit good activity against both Aspergillus spp and Candida spp.
• The Structure Activity Relationship around key compounds is far better understood as a result of the work undertaken.
• A number of new and improved synthetic routes to more complex analogues and derivatives have been developed.
• New synthetic routes to core substituents have been discovered and published.
• New synthetic routes to make key compounds at larger scale have been developed for efficacy and ADMET studies.
• Extensive scaffold replacement screening has revealed the crucial pharmacophores for broad antifungal activity.

Patent Applications

A basic foundation of all drug discovery efforts is the potential to protect the intellectual property (IP) of a new drug class. Without this IP protection it would be impossible to attraction or justify the hundreds of millions of US Dollars needed to fund a drug development project to a marketed agent. A key objective of NOFUN was to secure IP protection for its novel chemistry. From the start of the project IP searches were undertaken to look for any competing chemistry which would prevent IP protection. Searches of the particular chemistry space showed relatively little completion and this allowed the chemistry to proceed along the desired pathway. Formal patent searches were undertaken when the chemistry was more developed and more analogues had been exemplified. This also proved successful and led to the filing of a new patent to protect the chemistry of NOFUN.

Key Output
Patent filing

Antifungal Activity

Each time a new compound was designed and synthesised by NOFUN chemists it was tested for antifungal activity using the Minimum Inhibitory Concentration (MIC). A test panel of up to 11 different pathogenic species was used in this assessment. These species represented the most common pathogenic species of Candida and Apsergillus. These data were also used to guide the chemistry design process. Chemical modifications that did not produce better antifungal MIC results were not progressed. All NOFUN compounds were assessed this way.
Compounds with high activity were further tested using an expanded panel of organisms. This included fungal pathogens that are highly resistant to current drugs and pathogens which are very difficult to treat including Scedosporium and Fusarium. Active analogues also showed activity against these difficult to treat moulds.
A number of highly active analogues have been characterised and a ready for further development.

Key Output

• Development of activity profile for new chemical series.
• Introduction of key species into spectrum
• Activity against rare and resistant moulds

Model Systems

A key milestone in the development process is the demonstration of in vivo efficacy in appropriate infection models. A range of infection models have been used and active compounds have consistently demonstration a reduction in fungal burden in these experiments. Reduction in Candida albicans burdens occurred at certain dosages and in certain tissues types. One problem encountered was sub-optimal pharmacokinetics observed in several of the models. This is common when using model systems.

AΑ model of Candida albicans urinary tract infections was also developed under NOFUN. One compound tested showed promise in this model with efficacy demonstrated in reducing fungal burden in urine and bladder. Candida UTI infections are very common and can lead to systemic infections in some patients

Several other model systems were also developed under NOFUN to investigate resistant or rare pathogens. These included in vivo infection models have been developed for mucormycosis, Rhodotorula mucilaginosa, Cryptococcus spp. and Saccharomyces cerevisiae that can be employed to develop new antifungal series such as the F6-series.

Key Outputs

• Establishment of model systems to investigate antifungal activity.
• Demonstration of in vivo activity of new analogues
• Development of new models for rare moulds
• Development of disease specific models

Characterisation of Drug Like Properties

The behaviour of drugs in the human body can to a large degree be predicted using in vitro or test tube experiments. Thus has the advantage of being quicker, cheaper and more ethical as animal experimentation is considerably reduced and human clinical trials and not required. During the NOFUN project these “ADME” tests were used to characterise the new antifungal chemical series. Multiple compounds were tested to determine if the chemical class had any intrinsic liabilities which would prevent further development. No series specific liabilities were identified, which allowed the chemistry to focus on the improvement in general ADME properties.

Key Outputs

• Newer compounds, showed much improved ADME properties including good metabolic stability, medium to low plasma protein binding, good Caco2 permeability, low toxicity and good bioavailability.
• Key analogues are distributed to important target organs such as liver, lung, kidney and brain.
• In silico toxicology predictions on 5 different compounds indicated no intrinsic series wide issues with toxicity or mutagenicity. A single compound was flagged as a potential mutagen indicating further investigation was warranted.
• In vitro mutagenicity testing (Ames bacterial reverse mutation test) found that neither compounds were mutagenic. The compound identified as being potentially mutagenic via in silico testing was shown not to be mutagenic.
• Compounds were seen to be rapidly excreted

New Drug Targets

Drug target NpgA
We have identified a new mechanism of action for two antifungal drugs initially identified by F2G. We demonstrated that this enzyme is sufficiently dissimilar from its nearest human homologue to make it a viable drug target. This was confirmed by the low toxicity of the hit compounds against human cells. We demonstrated that target gene is a conditional essential gene in A. fumigatus.
We have determined that it is required for virulence in both insect and mammalian models of infection. We have further shown that we are able to monitor the activity of enzyme using an in vitro fluorescent polarisation assay. We have converted this assay to high throughput (384 well) format to enable small molecule screens to be conducted. We conclude that the gene is a novel target for antifungal drug discovery.

Drug target One
In the first period of the project, we gathered evidence to conclusively prove that lipid biosynthesis was the target of the F6 series of drugs. Specifically, we demonstrated that the antifungal activity of the F6 series was reversed using lipid extracts. We showed that overexpression of the target gene causes resistance to the F6 series and we showed the target gene was required for growth and virulence of Aspergillus fumigatus in a disseminated murine infection model.

We confirmed the mechanism of action of the F6 series using the molecular genomics tools developed in the first period. Specifically we used our chemical genomics platforms (A. fumigatus and S. cerevisiae) to show that strains with mutations in the target gene are hypersensitive to a drug from the F6 series. Additionally we have shown that the transcriptional response of A. fumigatus to a known target gene inhibitor mirrors almost exactly that of the response an F6 series drug. We have also shown that the lipid ratios within cells is disrupted by an F6 series compound consistent with the predicted mechanism of action.

So to summarise our findings relating to the mechanism of action of the F6 series:
• Mutants of the target gene are identified as having chemically induced haploinsufficincy in our chemical genomics screen.
• Overexpression of the target gene causes resistance to the F6 series.
• The F6 series reduced the intracellular lipid ratios in A. fumigatus.
• The transcriptomic response of A. fumigatus to the F6 series strongly correlates with that of known lipid metabolism inhibitors.
• Strains transcriptionally deficient for the target gene are avirulent in systemic and pulmonary models of A. fumigatus infection.

Finally, to confirm, we have discovered a good drug target that could be used for therapeutic purposes, we have demonstrated that its transcriptional suppression leads to loss of virulence of A. fumigatus in a pulmonary infection model. We have developed a low throughput assay that could be used to assess novel compounds in the series.

Drug target Two
A transcriptional regulator (TF) was identified in the first period as having altered susceptibility to a number of antifungal agents active against the sterol biosynthetic pathway. This TF is a member of the CCAAT-binding complex (CBC). Using ChIPseq analysis we have shown that the CBC binds the promoters of sterol biosynthetic genes. Notably we have identified that the CBC binds within the 34mer region of the cyp51A promoter that is duplicated in environmental azole resistant isolates of A. fumigatus (TR34). We have shown that the CBC is required for virulence of A. fumigatus suggesting that disruption of this regulator could be a route for novel antifungal drug discovery.

Drug target Three
A second TF was also identified as having altered sensitivity to drugs in the sterol biosynthetic pathway. While this information is already published in the literature we were keen to investigate the role of SrbA in clinical drug resistance (namely TR34 mediated resistance which is by far the most common mutation found in clinical azole resistant isolates of A. fumigatus) and to assess if it was a potential drug target. We have demonstrated that the sterol regulatory binding protein (srbA) of A. fumigatus binds the promoter of cyp51A at the TR34 region and is the transcriptional regulator responsible for elevated expression of the drug target in environmental resistant isolates. This marks srbA as a novel target for antifungal drug discovery.

Drug targets PacC and Cp1
In the first period we showed that the transcriptional factors HapB and PacC direct resistance to 5-flucytosine (5-FC). Our analysis of RNAseq data from hapB and pacC null mutants have indicated that this occurs via transcriptional regulation of acytosine permease (Cp1). We have further confirmed this via mutational analysis. This finding indicates that Cp1 is a novel target for combination therapy in A. fumigatus. This could be achieved by disrupting regulation of Cp1 by inhibiting the activity of HapB or PacC.

Drug Target PptB

• We have elucidated the crystal structure of the Candida albicans phosphopantetheinyl transferase PPT2 (also known as pptB in A. fumigatus) in complex with the Acyl Carrier Protein (ACP1).
• We have developed a pipeline to allow rapid screening of in silico compounds libraries against modelled or crystal structures.
• We have used the solved structure of PPT2 to predict novel inhibitors of enzyme activity using the in silico pipeline.
• We have crystallised the A. fumigatus PptB/ACP complex. We expect to solve the structure of this complex in the near future.

Potential Impact:
Impact on the general society and wider societal implications
The main purpose of the NOFUN project was two-fold. Firstly, to further develop a novel antifungal chemistry that had activity against a broad range of fungal pathogen and, secondly, to establish and validate a number of assets that could form the basis of future antifungal research and development programs. As this was primarily a research project, which ultimately has to have a commercially exploitable end point, the major impacts of the research will be felt long term. Nevertheless the NOFUN project has achieved a range of near term objectives, which can benefit the wider society.

Short-term societal impact
• Increased public awareness of fungal diseases and their impacts on their health and wellbeing. A deeper knowledge of the issues of fungal infections by society can only be beneficial and led to better compliance and treatments.
• Increased student awareness of the fungal kingdom and its benefits and dangers.
• Wider acceptance from research bodies and policy making of the importance of antifungal drug development.
• Legacy of database of antifungal chemistries. Availability of easily accessible data can stimulate research, which will increase the knowledge base.
• Further funding of the research. Net inward funding into the EU will maintain, support and create research positions, which is of considerable benefit.

Medium-term societal impact
• Publication of peer reviewed research papers and patents. The availability of quality peer reviewed research data can only stimulate further work in the field and lead to an increased understanding and body of knowledge.
• Increased collaborations between partners post project. The development of strong interdisciplinary working relationships between academia and SMEs will attract further revenue to the EU and benefit the institution both commercially and intellectually.
• Further funding of research in the area. Further research will increase the knowledge base and ultimately lead to new discoveries in the area.

Long-term societal impact
• New antifungal drugs. New antifungal agents will have a positive outcome for society and help reduce the fungal disease burden.
• Increased research into antifungal drugs. A variety of novel antifungal therapies will have an significant impact on fungal disease burden within the EU.

Economic Impact
The burden of fungal diseases within the EU has been described for many individual states and found to be much higher than previously appreciated. Recurrent VVC and chronic fungal respiratory disease have been shown to occur much more frequently than previously thought. These diseases are often unresponsive or poorly responsive to current therapies. It is widely accepted that antifungal agent and new approaches to therapy in general are required.

The progression of the antifungal programs from the NOFUN project could have a significant impact on the treatment of fungal diseases, reducing fungal burden and having a positive societal and economic impact. The prosperity of the NOFUN partners has also improved as a direct result of the NOFUN program. Individual partners have gained vital experience in the therapy area that can be leveraged to generate further income from follow on projects in this or related projects. The ability of partners to raise finances based on their experience in the NOFUN project is enhanced and has already occurred. Significant inward investment, in part to support NOFUN projects, has been received.

Dissemination Activities
• Development of the NOFUN website (www.nofunproject.org) which highlights the projects objectives and hosts the antifungals compound database.
• Participation at multiple international conferences promoting our research and educating media and fellow researches on the progress of the NOFUN science
• Publication of key research findings in peer reviewed journals providing a scientific legacy to educate, encourage and support further antifungal drug discovery efforts.
• Participation in multiple events aimed at promoting the awareness of antimicrobial resistance in general and specifically the issues and challenges of antifungal diseases and treatments. This included a number of lectures and presentations to international audiences.
• Participation in educational events focussed on educating young EU citizens on the importance of fungi in the modern world and also the disease caused by fungal pathogens.

Exploitation of results
• NOFUN partners have successfully applied for IP protection of the lead chemical series, which forms the basis of continued progression of the project.
• Two targets have been identified that warrant IP protection.
• Continuation of this project has been secured by NOFUN partners receiving further funding to carry on the development work started by NOFUN. The validated targets identified by NOFUN have been the subject of a number of grant applications, which are under review. Continuation of these projects after NOFUN is highly likely.
• All the NOFUN partners will continue to benefit from the project after it has ended. The experience and expertise gained during the project will be used on other projects and so have a continued benefit.

List of Websites:
www.nofunproject.org
NOFUN Project, Manchester Fungal Infection Group, Core Technology Facility (2.30b) University of Manchester, Manchester M13 9NT, UK +44 (0)161 275 1703

Final Report Summary - NOFUN (Novel antifungals to treat resistant organisms)

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