Periodic Reporting for period 1 - FEN INHIBITORS (Development of Kinetoplastida Flap Endonuclease Inhibitors in Search for Novel Therapeutics)
Période du rapport: 2020-02-01 au 2023-01-31
A group of parasites called Kinetoplastida, is responsible for three neglected tropical diseases: leishmaniasis, Chagas disease and human African trypanosomiasis (HAT, or African sleeping sickness) caused by various Leishmania species, Trypanosoma cruzi, and Trypanosoma brucei, respectively. HAT has been greatly reduced by a successful WHO campaign with an average of less than 1000 cases declared annually, but the impact of other Kinetoplastida on public health remains high. Approximately 1.3 million new cases of leishmaniasis are reported annually (mortality rate 30,000 p.a.). Similarly, there are ~8 million Chagas disease sufferers worldwide caused by T. cruzi with reported mortality and morbidity rates ~0.025 and ~30% respectively and over 65 million people at risk.
Treatment of leishmaniasis relies on a small number of drugs which are toxic to the host, expensive
and/or difficult to administer. Two drugs are available for treatment of Chagas disease but treatments for T. cruzi have low cure rates once the infection passes from the initial acute phase to the chronic state. The existing approved treatments for kinetoplastid diseases are widely recognised as being inadequate.
This project was an early-stage drug discovery program aiming to establish test-tube screening materials and methods, followed by library screening and hit expansion to produce inhibitors of our targets that could be used in later stage drug discovery efforts. Our target enzymes are Flap endonucleases (FENs) which process the branched DNA structures (5’ flaps) arising during DNA replication. FENs are found as independent globular proteins in eukaryotes, including parasites from this study. Inhibiting FEN enzymes in any organism tested so far will lead to organism death. Specific inhibitors with good pharmacological properties, i.e. not toxic to humans, cheap to produce and with good bioavailability in vivo, would thus make potential antiparasitic drugs.
Treatment of leishmaniasis relies on a small number of drugs which are toxic to the host, expensive
and/or difficult to administer. Two drugs are available for treatment of Chagas disease but treatments for T. cruzi have low cure rates once the infection passes from the initial acute phase to the chronic state. The existing approved treatments for kinetoplastid diseases are widely recognised as being inadequate.
This project was an early-stage drug discovery program aiming to establish test-tube screening materials and methods, followed by library screening and hit expansion to produce inhibitors of our targets that could be used in later stage drug discovery efforts. Our target enzymes are Flap endonucleases (FENs) which process the branched DNA structures (5’ flaps) arising during DNA replication. FENs are found as independent globular proteins in eukaryotes, including parasites from this study. Inhibiting FEN enzymes in any organism tested so far will lead to organism death. Specific inhibitors with good pharmacological properties, i.e. not toxic to humans, cheap to produce and with good bioavailability in vivo, would thus make potential antiparasitic drugs.
The prerequisites for an early-stage drug development project are: the recombinant proteins needed for wet-lab assays; knowledge of their molecular 3D structures, followed by acquisition of inhibitor-protein complex structures to facilitate hit expansion, that is, search for or de novo design of new better inhibiting molecules.
We successfully produced active proteins required for this project and experimentally determined their native structures using protein X-ray crystallography. Further efforts yielded a set of new structures of native proteins and proteins in complex with the natural substrate (DNA), including a crystal form that was suitable for soaking with potential inhibitors. The latter was eventually used to create structures with inhibitors.
We adopted a high throughput in-crystal screening approach using a small in-house fragment-based library for the X-ray screening experiment similar to the XChem approach used at the Diamond Light Source (the UK’s national synchrotron science facility). In combination with the new well behaving crystal form, this allowed for screening through a rather broad chemical space and yielded complex structures in an unexpected set of inhibitory molecules pre-selected by in vitro screening.
In vitro experiments on the 3 clinically relevant cultured parasites have not yet been thoroughly conducted, but we have successfully established a simpler in-house system based on the non-human-pathogen model organism Leishmania tarentolae. These experiments prove that the compounds identified in this study are able to kill the target parasite organism.
Results from this project have already shifted other projects in our research group, but have not yet been disseminated. We have produced over 10 new structures that will be published on the RCSB server once the scientific publications covering this research are prepared.
We successfully produced active proteins required for this project and experimentally determined their native structures using protein X-ray crystallography. Further efforts yielded a set of new structures of native proteins and proteins in complex with the natural substrate (DNA), including a crystal form that was suitable for soaking with potential inhibitors. The latter was eventually used to create structures with inhibitors.
We adopted a high throughput in-crystal screening approach using a small in-house fragment-based library for the X-ray screening experiment similar to the XChem approach used at the Diamond Light Source (the UK’s national synchrotron science facility). In combination with the new well behaving crystal form, this allowed for screening through a rather broad chemical space and yielded complex structures in an unexpected set of inhibitory molecules pre-selected by in vitro screening.
In vitro experiments on the 3 clinically relevant cultured parasites have not yet been thoroughly conducted, but we have successfully established a simpler in-house system based on the non-human-pathogen model organism Leishmania tarentolae. These experiments prove that the compounds identified in this study are able to kill the target parasite organism.
Results from this project have already shifted other projects in our research group, but have not yet been disseminated. We have produced over 10 new structures that will be published on the RCSB server once the scientific publications covering this research are prepared.
This research has yielded a set of new structures of the drug targets and a set of novel FEN inhibitors. Thanks to successful crystallographic experiments, we have determined a new cryptic pocket that accommodates inhibitors and thus a new mode of inhibitory activity. This has already shifted research on our project that is focused on parasites that cause malaria (Genus Plasmodium), the larger “sister” project run by a team of 5 postdocs in our group and funded by the Bill & Melinda Gates Foundation, and will change science in this area after our results have been published.
Novel, better and safer drugs are crucial in our struggle against parasites and especially neglected tropical diseases caused by flagellated protists. Our research establishing the early stage structure based drug design to find and design novel FEN inhibitors was a step in this direction.
Novel, better and safer drugs are crucial in our struggle against parasites and especially neglected tropical diseases caused by flagellated protists. Our research establishing the early stage structure based drug design to find and design novel FEN inhibitors was a step in this direction.