An integrated chemogenomic approach exploring and exploiting prolyl-tRNA synthetase as target for next-generation malaria and cancer therapies

Abstract

Malaria is an infectious disease caused by Plasmodium parasites and remains one of the world’s most pressing public health challenges with nearly half of the world’s population at risk, causing over 200 million cases and more than 600,000 deaths per year. The failure to develop a highly efficacious and long-lasting malaria vaccine combined with the incessant development and proliferation of resistance to all approved antimalarial drugs, including artemisinin-based combination therapy (ACT), jeopardizes both acute treatment and containment efforts. Our current antimalarial chemotherapies all share a small set of targets, which leaves them susceptible to cross-resistance, and most drugs are only useful for acute treatment because their activity is limited to the asexual blood stage (ABS). Thus, there is a dire need for new antimalarial therapies targeting novel pathways that are essential for multiple life-cycle stages for primary prophylaxis and transmission blocking, in addition to acute treatment. In recent years, aminoacyl-tRNA synthetase (aaRS) enzymes, including prolyl-tRNA synthetase (ProRS), have emerged as attractive targets for malaria chemotherapy. ProRS is an essential enzyme found in all living cells that synthesizes prolyl-tRNA for use by the ribosome during translation. The aggressive proliferation of Plasmodium parasites depends upon rapid protein translation, making ProRS a promising target. This was validated in vivo by halofuginone, one of the most potent known antimalarials (in vitro ABS EC50 = 0.5 nM), whose functional mechanism of action was recently shown to be inhibition of Plasmodium falciparum cytoplasmic ProRS (PfcProRS) by the Mazitschek and Wirth laboratories (henceforth our group). Subsequent studies by our group also revealed for the first time halofuginone is active against the liver stage and that halofuginol, an analog of halofuginone, is a single dose cure in an in vivo liver stage malaria model. However, our groups studies that revealed PfcProRS as the functional target of halofuginone also showed that Plasmodium parasites rapidly develop halofuginone resistance mediated by elevated intracellular proline, and eventually by PfcProRSL482H/F mutations that disrupt the proline pocket and are hypothesized to rely upon the elevated proline. This resistance overcomes the therapeutic window for halofuginone and has hindered clinical development. Thus, novel ProRS inhibitor chemotypes are highly sought but this has been stymied by the lack of robust yet sensitive biochemical assays for profiling inhibitors of ProRS, and more generally for aminoacyl-tRNA synthetase (aaRS) enzymes. In a complementary collaboration with the Whitmann lab, our group identified the prolyl-tRNA synthetase activity of the bifunctional glutamyl-prolyl-tRNA synthetase (HsGluProRS) as the mechanistic target of halofuginone in humans, where it has been shown to have chemotherapeutic, antifibrotic, immunomodulatory agent, and more recently antiviral activity. Thus, less selective compounds are attractive for use in humans where the inhibition of the ProRS activity of the bifunctional glutamyl-prolyl-tRNA synthetase (HsGluProRS) has been studied for chemotherapeutic, antifibrotic, immunomodulatory agent, and more recently antiviral activity. To overcome the limitations in systematically profiling ProRS inhibitors, we have developed a novel single-step biochemical assay platform for Plasmodium and human ProRS that enables quantitative inhibitor profiling with unprecedented sensitivity and flexibility (Chapter 2). Employing the CoraFluor technology that our group developed, our assay utilizes homogeneous time-resolved Förster resonance energy transfer (TR-FRET) based ligand displacement to quantitatively determine inhibitor binding modes and binding kinetics. This assay is high throughput screening compatible, and we have screened multiple libraries to identify several novel ProRS inhibitor chemotypes that are structurally diverse and exhibit favorable biochemical selectivity. We have also generalized our assay using an alternative TR-FRET tracer scaffold enables the generalization to additional aaRS isoforms from diverse bacterial and eukaryotic species (Chapter 5). Guided by this TR-FRET assay, we have developed a diverse set of high-affinity ProRS inhibitors and solved the co-crystal structures of representative inhibitor-target complexes (Chapter 3). Several compounds, including the first triple-site ligands for aaRS enzymes that simultaneously engage all three substrate-binding pockets, exhibit potent dual-stage activity against Plasmodium parasites and display good cellular host selectivity. These novel ProRS inhibitors inform the requirements to overcome existing resistance mechanisms and do not exhibit cross resistance with halofuginone. Further, we show that parasites do not develop rapid resistance to a representative proline-uncompetitive ProRS inhibitor, NCP26. Lastly, we developed a concise synthetic approach towards halofuginone, halofuginol, additional triple-site ProRS ligands with diverse linkers (Chapter 4). Together, our data promise to accelerate the rational development of ProRS-targeted anti-malarial therapies and establishes a strong framework for the development of aaRS inhibitors for other isoforms and diseases.