MMV s distinct mode of inhibition
MMV019313's distinct mode of inhibition addresses key impediments in the development of PfFPPS/GGPPS inhibitors as antimalarial drugs. First, it is the first non-bisphosphonate inhibitor of Plasmodium FPPS/GGPPS with drug-like physicochemical properties satisfying the “Rule of 5” (Van Voorhis et al., 2016). Unlike bisphosphonates, it does not mimic a charged diphosphate substrate to achieve FPPS/GGPPS inhibition. As a 14613 in the Malaria Box library, the results of >300 assays characterizing this library as part of an innovative “open-source” drug discovery effort by many groups will be a rich source of information (Allman et al., 2016, Paul et al., 2016, Ullah et al., 2017, Van Voorhis et al., 2016). Second, MMV019313 showed high selectivity for PfFPPS/GGPPS with no inhibition of human FPPS and GGPPS at up to 200 μM, minimizing the potential for mechanism-based (e.g., “on-target”) toxicity. Consistent with its lack of enzymatic inhibition, MMV019313 showed no cytotoxicity against a panel of 60 human cancer cell lines at 10 μM (Van Voorhis et al., 2016). In contrast, the most selective bisphosphonate BPH-703 identified so far showed 2.6- to 3.6-fold selectivity in our enzymatic assays, corresponding to a reported therapeutic index of 193 in growth inhibition assays (No et al., 2012). Overall, we demonstrate a novel mode of inhibition of PfFPPS/GGPPS that circumvents the inherent liabilities of previous bisphosphonate inhibitors.
MMV019313 likely binds a non-substrate site in PfFPPS/GGPPS that is either absent from or substantially different in the human homologs, accounting for its high selectivity. One possibility is the allosteric site observed in human FPPS structures. Unfortunately, this site has not been characterized in Plasmodium FPPS/GGPPS homologs, and molecular docking in the apo PvFPPS/GGPPS structure was inconclusive. Another clue is the resistance caused by the S228T variant, which could be explained by a direct contact between Ser228 and MMV019313 in a new binding pocket. But since the change from Ser to Thr is quite conservative, the addition of a methyl group, Ser228, may also be involved in conformational dynamics important for catalysis. Structural analysis of this variant enzyme may reveal altered conformational states underlying the resistance to MMV019313.
Identification of lead compounds, based on MMV019313 or alternative scaffolds, that target this new small-molecule binding pocket will accelerate antimalarial drug discovery. Optimization of potency and pharmacokinetic properties of lead compounds will be a prerequisite for testing in mouse models of Plasmodium infection. For example, we found that while MMV019313 is stable to human liver microsomal enzymes (t½ > 159 min), its t½ in mouse liver microsomes was 4 min. This metabolic instability may account for a <1-μM peak serum concentration following oral administration in mice (Van Voorhis et al., 2016). Importantly, our study has generated several tools for identifying specific PfFPPS/GGPPS inhibitors as lead compounds. The luciferase-based enzymatic assays used in our study can easily be adapted to high-throughput screening of libraries to identify analogs with (1) improved potency that (2) maintain selectivity for PfFPPS/GGPPS and (3) are dependent on residue S228 (in contrast, we found that a commercially available colorimetric assay was not sufficiently sensitive) (Crowther et al., 2011). P. falciparum strains overexpressing wild-type or the S228T variant can also be retooled as secondary cellular assays for on-target specificity. Thus, our finding provides a critical foundation for the development of specific PfFPPS/GGPPS inhibitors.
Acknowledgments We are grateful to Medicines for Malaria Venture (MMV) for providing the Malaria Box compounds and making this valuable library freely available, as well as GlaxoSmithKline for their screening efforts that first identified MMV019313 (TCMDC-123889). We would like to thank Dr. Susmitha Suresh for performing drug screens, Dr. Felice Kelly for advice on chemical mutagenesis, Dr. James Dunford (University of Oxford) for advice in developing the in vitro enzyme activity assays, and Dr. Wei Zhu and Professor Eric Oldfield (University of Illinois, Urbana-Champaign) for providing BPH-703. Funding support for this project was generously provided by the Stanford Consortium for Innovation, Design, Evaluation and Action (C-IDEA), NIH 1K08AI097239 (E.Y.), NIH 1DP5OD012119 (E.Y.), the Burroughs Wellcome Fund Career Award for Medical Scientists (E.Y.), the Burroughs Wellcome Fund Investigators in Pathogenesis of Infectious Disease (PATH) Award 1007041 (M.L.), and the Stanford School of Medicine Dean's Postdoctoral Fellowship (J.E.G.).