Periodic Reporting for period 1 - FEN INHIBITORS (Development of Kinetoplastida Flap Endonuclease Inhibitors in Search for Novel Therapeutics)
Período documentado: 2020-02-01 hasta 2023-01-31
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.
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.
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.