Parasite FEN Inhibitors

A group of flagellated protists, 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 under 3000 of cases per year, 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 was an early-stage target-based drug discovery project aiming to establish in vitro screening materials and methods, followed by in vitro and in silico library screening and hit expansion to produce target inhibitors that could be used in later stage drug discovery efforts. Our targets are Flap endonucleases (FENs) which process the branched DNA structures (5’ flaps) arising from lagging strand 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 prerequisite for an early stage drug development project are recombinant proteins used in wet-lab assays and native structure determination efforts, 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 used on this project and acquired their native structures. 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 and was eventually used to create structures with inhibitors.

I adopted a high throughput in-crystal screening, a small in-house version of XChem, the fragment-based X-Ray screening experiment developed Diamond. 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. This had a great impact on this project as well as the larger “sister” project run by a team of 5 postdocs in our group and funded by the Bill & Melinda Gates Foundation.

In vitro experiments on the 3 clinically relevant cultured parasites have not been thoroughly conducted, but we have successfully established a simpler in-house system based on the non-human-pathogen Leishmania tarentolae. These experiments prove toxicity in living parasite organisms.

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 another project in our group that is focused on parasites that cause malaria (Genus Plasmodium).

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 successful step in this direction.