High Throughput Screening to Identify, Develop and Analyse Inositol Phosphorylceramide Synthase Inhibitors as Novel Antileishmanials

Abstract

Leishmaniasis and Human African trypanosomiasis are tropical diseases caused by kinetoplastid parasites that together affect over 12 million people, with an estimated 400 million at risk worldwide. Both are potentially fatal, yet the current treatments available are expensive and many have toxic side effects. Emerging resistance to many current drugs is also a concern; novel therapeutic agents are therefore urgently required.

One novel target for drug discovery previously identified in the group is sphingolipid synthesis. Sphingolipids are ubiquitous biomolecules found in nature and are both structural membrane components and signalling molecules. Inositol phosphorylceramide synthase (IPCS) is an essential enzyme involved in kinetoplastid sphingolipid synthesis that has no mammalian equivalent, making it an attractive drug target. Whilst specific inhibitors of the fungal IPCS are known, they are unsuitable as pharmaceuticals. The overall aim of this project was to identify novel inhibitors of this enzyme that could be further investigated as potential antikinetoplastid drugs.

The first stage involved the construction of Saccharomyces cerevisiae strains as expression systems of the kinetoplastid IPCS enzymes. The strain complemented with the Leishmania major enzyme was subsequently used in the development and optimisation of a robust high throughput screening (HTS)-compatible assay. This was used to screen the 1.8 million compound library stored at the GlaxoSmithKline research site in Tres Cantos in what is believed to be the largest screening project undertaken by an academic group to date. 500 compounds were identified as selective inhibitors of the L. major IPCS enzyme, and 216 of these were selected for additional investigation.

Further compound triage was achieved by means of a screening process involving multiple in cellulo assays against both Leishmania parasites and mammalian cells. Six compounds demonstrating both high potency and selectivity were identified. Following additional biochemical testing, the two most potent compounds were found to share a common benzazepane chemical structure. Investigation of analogues of these compounds permitted the identification of preliminary structure-activity relationship data, which identified several possible avenues for further investigation.