Identification of Inhibitors of Human and Plasmodium Falciparum Orotate Phosphoribosyltransferase and Orotidine 5'-Monophosphate Decarboxylase, and a Novel Hybrid Screening Technique

Author: Michael Roach

Roach, Michael, 2015 Identification of Inhibitors of Human and Plasmodium Falciparum Orotate Phosphoribosyltransferase and Orotidine 5'-Monophosphate Decarboxylase, and a Novel Hybrid Screening Technique, Flinders University, School of Biological Sciences

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Abstract

The enzymes of the de novo pyrimidine biosynthetic pathway are ideal drug targets for the treatment of malarial infections as, unlike the human host, the parasite is solely reliant on this pathway for survival. Furthermore, this pathway is associated with, and therefore, a drug target for a variety of diseases including cancer, autoimmune disorders and viral infections. Hence, this study focussed on the use of bioinformatics approaches to identify potential lead compounds against human and Plasmodium enzymes from this pathway. The Plasmodium falciparum de novo biosynthesis enzymes orotate phosphoribosyltransferase (OPRTase) and orotidine 5’-monophosphate decarboxylase (ODCase) were recombinantly expressed and purified. The expression and purification of P. falciparum OPRTase (PfOPRTase) was optimised in this study to an extent which enabled biochemical assay and x-ray crystallography experiments to be performed on the enzyme. Constructs were made for the recombinant expression of the bifunctional human homologue Uridine Monophosphate Synthase (HsUMPS) as well as its individual OPRTase (HsOPRTase) and ODCase (HsODCase) domains. These were successfully expressed and purified. A 3-D homology model for the structure of PfOPRTase was generated. The PfOPRTase homology model and crystal structures for P. falciparum ODCase (PfODCase), HsOPRTase and HsODCase were screened virtually by docking against approximately 1 million drug-like compounds. The hits were analysed and 19 novel, diverse compounds were selected for inhibition assays. Recombinant HsOPRTase, HsODCase, PfOPRTase and PfODCase were used in biochemical inhibition assays to characterise the 19 compounds that were identified by virtual screening for inhibition. For PfOPRTase and HsOPRTase, nine compounds showed inhibition against one or both enzymes and five were specific for one of the homologues. These inhibitors would be suitable for further development with hit to lead (H2L) drug discovery experiments. Eight very promising inhibitors for PfODCase and HsODCase were identified which led to some small scale ‘hit expansion’ H2L experiments, and further biochemical evaluation. Seven compounds successfully underwent further inhibition kinetic characterisation. One inhibitor had good potency (Ki/αKi~ 50 µM) and six inhibitors were specific for one of the homologues. Four of the inhibitors (including the most potent inhibitor) satisfy the criteria for drug-likeness and bioavailability. An alternative substrate for HsODCase was discovered and characterised. The discovery of this alternative substrate and the unexpected modes of inhibition of the identified ODCase inhibitors allowed for new insights into the structure and catalytic mechanism of this enzyme. Interconnectivity between the ODCase dimer active sites is likely. Finally, a novel method was developed for performing virtual screening on an enzyme target. This novel method is a ‘hybrid’ of structure- and ligand-based methods. The method requires knowledge of only a single known binder and a crystal structure. It is estimated to be approximately 60-fold faster than a typical structure-based approach. It outperformed a typical structure based approach with 25 % better mean bias towards actives, 45 % better mean enrichment at 1 %, whilst maintaining similar hit diversity. It performed similarly compared to a ligand-based approach for bias towards actives and enrichment at 1 % (but with far greater consistency) and 30 % better hit diversity.

Keywords: in silico screening, virtual screening, hybrid screening, drug discovery, OPRTase, ODCase, UMPS, orotate phosphoribosyltransferase, orotidine 5'-monophosphate decarboxylase, uridine monophosphate synthase, plasmodium falciparum
Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2015
School: School of Biological Sciences
Supervisor: R. Ian Menz