Medicinal chemistry approaches to malaria drug discovery

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016

Malaria remains a major burden to global public health, causing nearly 600,000 deaths annually. Efforts to control malaria are hampered by parasite drug resistance, insecticide resistance in mosquitoes, and the lack of an effective vaccine. However antimalarial drugs are a mainstay in efforts to control and eventually eradicate this disease, thus the discovery of new antimalarials is critical. Antimalarial drug discovery is especially challenging due to the unique biology of malaria parasites, the scarcity of tools for identifying new drug targets, and the poorly understood mechanisms of action of existing antimalarials. Therefore, this work describes the use of different medicinal chemistry approaches to address unmet needs in antimalarial drug discovery. Part of this process includes ensuring that sufficient drug leads are available to prime the drug discovery pipeline, particularly those with novel modes of action in order to limit issues of cross-resistance with existing drugs. The first approach described in this thesis consists on the phenotypic-based hit to lead optimization designed to explore the antimalarial potential of the 3-piperidin-4-yl-1H-indole. The hit compound was identified in a phenotypic screen of ~2 million compounds against asexual blood stage P. falciparum. Three series of analogs were synthesized following a reagentbased diversity approach, in a total of 38 compounds, and screened for their blood stage antimalarial activity. The SAR shows that 3-piperidin-4-yl-1H-indole is intolerant to most Npiperidinyl modifications. Out of the analogs synthesized, three were active (2.19, 2.20 and 2.29). Furthermore, the (4-(1H-indol-3-yl)piperidin-1-yl)(pyridin-3-yl)methanone (2.29) showed in vitro antimalarial activity (EC50 values ∼3 μM), no cross-resistance with chloroquine, selectivity for the parasite, and lead-like properties (cLogP < 3; MW ∼300). This represents a promising new antimalarial chemotype with a potential novel mechanism of action. Further medicinal chemistry efforts are needed to improve the potency of compound 2.29 and disclose its antimalarial mechanism of action. In the second part of this work we focus on exploring the potential of aminoacyl tRNA synthetases (aaRS) as a novel class of antimalarial targets. We hypothesize that the inhibition of some but not all, P. falciparum aaRSs will result in activation of amino acid response pathways and that inhibition of this subgroup represents an attractive approach for chemotherapeuticintervention in malaria. First, a library of 21 aminoacyl-sulfamoyl adenosine (aaSA) analogs was synthetized and used as tool compounds to profile 19 of the PfaaRSs as drug targets. Among the analogs tested, L-PheSA, L-HisSA, L-AlaSA and L-ProSA were the compounds that exhibited higher antimalarial activity (EC50 against the Dd2 strain < 120 nM) and selectivity index (> 20). These results allow the prioritization of the phenylalanyl tRNA synthetase (FRS), histidyl tRNA synthetase (HRS), alanyl tRNA synthetase (ARS), and prolyl tRNA synthetase (PRS) as the top four enzymes for further exploration as drug targets in blood stage malaria. aaSA compound treatment of P. falciparum increased eIF2α phosphorylation at 100X concentration, inducing the amino acid starvation pathway through direct inhibition of the corresponding PfaaRS, with few exceptions. Moreover, analogs were also active in vitro against liver stage P. berghei, with > 99% parasite growth inhibition at the higher concentration of 10 μM. Thus, underscoring the potential of the aaRS family as an attractive novel class of antimalarial drug targets. To further explore cytoplasmic prolyl tRNA synthetase (cPRS) as an antimalarial target, which we have previously identified as the long sought biochemical target of the antimalarial halofuginone (HFG), novel HFG based inhibitors were designed to exploit additional ligandprotein interactions in the active site of cPRS, and may serve as lead compounds in the preclinical development of a mechanistically unique class of malaria drugs with activity against both liver and blood stage life cycle stages. Furthermore, we aimed to characterize the biology of cPRS inhibition and resulting amino acid starvation response. Understanding the enzymeinhibitor complex formed by the different types of inhibitors (HFG and L-ProSA) would further elucidate on the deferential effect observed on the amino acid starvation response in mammalian cells, despite targeting the same enzyme. Thus, a two-step proteomic approach to isolate the protein complex using immunoprecipitation followed by identification of its components using mass spectrometry is proposed. Despite not being able to completely establish the protocol, the results in MCF-7 cells are consistent with the model proposed, thus further work needs to be done towards increasing the amount of the enzyme-inhibitor complex isolated to meet the detection requirements of the techniques used. Finally, to address the problem of limited target identification and validation tools for novel antimalarial compounds, the third aim of this thesis investigates the use of fluorescently labeled small molecules as a novel target discovery approach in malaria drug discovery efforts. A new methodology, which originated from our efforts to synthetize MayaFluors in a one-pot, two-step approach via BODIPY-OTf intermediate, was developed to label drugs with the BODIPY fluorophore. The method allows for substitution of either one or both of the canonical fluorides on the BODIPY dye with alkoxy ligands, under mild conditions. We successfully labeled a group of small molecules. Of these, two known drugs ((+) JQ1 and hydroxychloroquine) were evaluated for their activity and cellular localization. In both cases the labeled drug presented comparable activity to the parent drug. Furthermore, the fluorescently labeled antimalarial displayed a subcellular staining pattern in mammalian cells that is consistent with accumulation in acidic vesicles in the cytoplasm. Moreover, the probe was also tested in Plasmodium falciparum cultures, where results show subcellular staining pattern that seems consistent with accumulation in the food vacuole. Despite the identified limitation concerning the solvent compatibility of the method, this approach allows direct labeling of hydroxylfunctionalized drugs, which we believe may have broad applications for rapid and specific imaging of elusive biological targets in living cells. Taken together, in this thesis multiple medicinal chemistry approaches are explored in an effort to identify novel antimalarial chemotypes that act on underexploited targets. Furthermore, these results present new opportunities for malaria drug discovery to aid efforts in malaria control and eventual eradication.