Introduction and Background of the Project

Introduction

Treatment and ultimately elimination of malaria remains a massive challenge due, mainly, to the emergence of drug-resistant strains of Plasmodium falciparum, the most lethal species in humans. It is therefore necessary to discover lead candidates unaffected by existing mechanisms of resistance to traditional antimalarial chemotypes. Additionally, while prophylaxis and transmission-blocking drugs are needed to prevent epidemics and to protect vulnerable populations, standard-of-care antimalarials do not address all of the requirements for pan-lifecycle activity. The Broad Institute, in collaboration with Eisai Ltd., has discovered a series of antimalarial compounds with a novel mechanism of action (targeting Plasmodium falciparum cytosolic phenylalanine tRNA synthetase (PfcPheRS)) (Nature, doi:10.1038/nature19804). Our unique bicyclic azetidine series exhibits potent activity both in vitro and in vivo against blood-, liver- and transmission-stage P. falciparum parasites.

Project objective

The current proposal builds on the progress made with GHIT grant (G2014-107) that delivered multiple compounds with excellent efficacy in mouse models of malaria while identifying potential safety liabilities. Enabled by program advances in chemistry and biology, improved molecules will be delivered that yield a highly desired therapeutic profile including fast acting, potent against drug-resistant strains, prophylactic and transmission-blocking activity. The goal of this proposal is to improve the safety profile of the series to yield a lead optimization candidate meeting one of the target product profiles defined by Medicines for Malaria Venture (MMV).

Project design

Our project design is to optimize the safety profile of our novel bicyclic azetidine series to yield a lead candidate that meets one of the target product profiles defined by MMV. Exploratory toxicology studies identified histological changes with an initial shortlist compound that were mitigated in follow-up analogs. Our objectives include 1) to determine the biochemical selectivity of advanced analogs against P. falciparum and human cytosolic PheRS (PfcPheRS and hcPheRS respectively) and obtain X-ray co-crystal structures with PfcPheRS and 2) to design and synthesize new analogs with greater PfcPheRS selectivity and safety while maintaining excellent in vivo efficacy.  

How can your partnership (project) address global health challenges?

While the global public health community has made significant progress in reducing mortality due to malaria, this mosquito-borne parasite still infects over 212 million people per year. Approximately 429,000 deaths were attributed to malaria in 2015, the majority (almost 70%) of them in children under the age of five. In order to further decrease the morbidity and mortality associated with malaria, a multi-pronged approach is required, including vector (mosquito) control, effective vaccines, and new chemotherapeutics that can target drug-resistant parasites, the infectious and transmission stages of the parasites (sporozoites and gametocytes, respectively), and the dormant hypnozoite stages of P. vivax and P. ovale. Medicines for Malaria Venture (MMV) has proposed the development of a Single Exposure Radical Cure and Prophylaxis (SERCaP) for the treatment of uncomplicated malaria in adults and children. The target product profile (TPP) for the ideal SERCaP would include rapid asexual blood-stage parasite reduction, transmission-blocking activity, and targeting of hypnozoites. We anticipate that a candidate from the proposed work would not only meet the requirements of at least one MMV TCP, but due to the known activity against gametocytes and liver-stage parasites, would provide a significant advantage to patients. 

What sort of innovation are you bringing in your project?

Effective eradication strategies for the treatment of malaria have been elusive, primarily owing to the complex life cycle of Plasmodium and the emergence of drug-resistant strains of P. falciparum, the most lethal Plasmodium species in humans. The majority of the current antimalarial drugs target the asexual blood stage of Plasmodium, in which they parasitize and replicate within erythrocytes. Even though liver and transmission-stage parasites do not cause malarial symptoms, prophylaxis and transmission-blocking drugs are essential for the proactive prevention of disease epidemics and to protect vulnerable populations. Unfortunately, the current antimalarial drugs do not address all of the requirements for the targeting of pan-life-cycle activity. Our unique bicyclic azetidine series exhibits potent activity both in vitro and in vivo against blood-, liver- and transmission-stage P. falciparum parasites with a novel mechanism of action (targeting Plasmodium falciparum cytosolic phenylalanine tRNA synthetase (PfcPheRS)).

Role and Responsibility of Each Partner

Broad is the designated development partner for the project. Broad will be responsible for directing the lead optimization efforts to identify the candidate with improved selectivity and safety profile. Eisai will be responsible for pharmacokinetics and safety studies to identify any key liabilities. Eisai will direct and monitor all safety studies needed for lead candidate nomination. MMV will be responsible for Pf NSG in vivo efficacy experiments, ex vivo profiling, biochemical selective efforts (hcPheRS and PfcPheRS) and endeavor to generate crystal structures of PfcPheRS with program compounds.

Others (including references if necessary)

Nature, doi:10.1038/nature19804

Final Report

1. Project objective 

A novel series of bicyclic azetidine antimalarials was discovered via phenotypic screening of the Broad Institute’s Diversity-Oriented Synthesis small-molecule collection. These compounds target the malaria parasite at all human-host stages of the lifecycle by inhibiting its cytosolic phenylalanine tRNA synthetase (cPheRS), a previously unknown mechanism of action. Early efforts in this program, supported by GHIT, delivered multiple compounds with excellent efficacy in mouse models of malaria. The goal of this project was to improve the safety profile of the series to yield a lead optimization candidate meeting one of the target product profiles defined by Medicines for Malaria Venture (MMV).

2. Project design 

This project comprised two specific aims: (1) Determine the target selectivity of advanced analogs and obtain X-ray co-crystal structures with P. falciparum and human cPheRS; (2) Design and synthesize new analogs with greater PfcPheRS selectivity and safety while maintaining excellent in vivo efficacy.

3. Results, lessons learned 

We have demonstrated that bicyclic azetidine compounds bind and inhibit the parasite enzyme with high selectivities, exceeding 1000-fold, over the human ortholog. We have solved the first co-crystal structures of bicyclic azetidines bound to their mechanistic target, Plasmodium cPheRS (Nat. Commun. 2021, 12, 343). These structures reveal a unique binding mode of bicyclic azetidines in cPheRS, distinct from those of known aminoacyl-tRNA synthetase inhibitors, and thus highlight the crucial role of Diversity-Oriented Synthesis in discovering novel mechanism-of-action probes and therapeutic candidates. We have identified shortlist compounds that exhibit good efficacy, physicochemical and pharmacokinetic (PK) properties, and in vitro and in vivo toxicity profiles. Key features of these compounds are significantly improved compared to earlier leads. In addition, efficient synthetic routes have been developed for these compounds that completely avoid the need for chromatographic separations, which results in lower projected costs of manufacturing. Lastly, experimental workflows and strategies have been established to address remaining liabilities. Our project data indicate that compounds of this chemical series and, more generally, of this mechanistic class, hold promise to fulfill the important need for a novel mechanism of action, fast-acting malaria therapeutic that can target multiple stages of the parasite lifecycle.