Introduction and Background of the Project

Introduction

Malaria is a debilitating disease caused by Plasmodium parasites. Every year ~200 million new infections are established, causing more than 400,000 deaths. There is an urgent need to develop new antimalarial drugs that are safe, fast-acting, active against different stages and all strains, effective as a single dose and suitable for both treatment and prophylaxis. We have identified a class of nucleoside sulfamates that show potential to meet these demanding criteria. Our front-runner compounds exhibit long in vivo half-lives, very high potency against malaria parasite cultures and very low toxicity against mammalian cell lines. We have demonstrated single dose efficacy in a mouse model of human malaria.

Project objective

This project seeks to undertake Hit-to-Lead studies on our nucleoside sulfamates series to identify compounds which meet MMV’s early lead criteria for entry into lead optimization.

Specifically, the Project aims:

i) To undertake a medicinal chemistry program to improve oral bioavailability of the nucleoside sulfamate series, while maintaining selectivity and potent anti-parasitic activity.

ii) To undertake a detailed analysis of the activity of orally bioavailable nucleoside sulfamates in a testbed of assays against different life stages of Plasmodium to define the potential product profile (i.e. treatment and/or chemoprotection).

iii) To demonstrate efficacy and tolerability of orally bioavailable nucleoside sulfamates in a SCID mouse model of P. falciparum malaria.

iv) To confirm the mode of action and to understand the potential for resistance generation.

Project design

The target of the nucleoside sulfamate front-runner compound has been identified as an important protein synthesis enzyme, called tyrosine tRNA synthetase. The target enzyme is divergent from its human homologue, consistent with good selectivity of the inhibitors. We will establish biochemical assays and a well-defined test cascade to support a medicinal chemistry program to improve oral availability and deliver nucleoside sulfamates with drug-like properties that maintain potency and selectivity. We anticipate that good pharmacokinetic properties (oral bioavailablity, low clearance and long half-lives, good solubility and suitable LogP) can be obtained. Within one year of project funding, we will identify nucleoside sulfamate compounds that inhibit the growth of 3D7 P. falciparum with an IC₅₀ value less than 100 nM and show more than 25% bioavailability in rats.

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

Current antimalarial control is highly dependent on artemisinin combination therapies (ACTs). Artemisinins have very short in vivo half-lives. As a consequence, ACTs are delivered as a 3-day regimen that suffers poor compliance - driving the emergence of resistant strains. Reistance is also emerging to each of the partner drugs used in ACTs. As a consequence, high levels of treatment failure are observed [1] and there is a significant risk of contracting malaria infections that are resistant to all available drugs. Our Project aims to develop a new antimalarial drug that is safe and efficacious and suitable for both treatment and prophylaxis.

What sort of innovation are you bringing in your project?

An exciting aspect of this project is that it brings together pharmaceutical industry experts (Gould, Dick; with many years of experience in developing nucleoside sulfamates as potential anti-cancer agents), an academic malaria expert (Tilley, with structural biology and biochemistry expertise) and a Product Development Partner (Brand, Baud MMV).

A particularly innovative aspect of this work is that it will explore a novel target that is essential at all stages of parasite growth. Thus, we anticipate that a nucleoside sulfamate antimalarial has the potential to meet the MMV criterion of inhibiting all stages, all strains and all species of plasmodium.

Role and Responsibility of Each Partner

Dr Sandy Gould (Lead PI), Takeda Pharmaceuticals, will co-ordinate activties across the Project. She will also lead and coordinate the activities of a team of Takeda medicinal chemists and biochemists who will be designing and analysing new nucleoside sulfamates for this hit to lead effort.

Prof Leann Tilley (PI and Designated Development Partner representative), University of Melbourne, and her team, including Dr Stanley Xie, will contribute relevant test cascade assays, provide recombinant P. falciparum tRNA synthetase, conduct mechanism of action studies, and develop key tools, such as nucleoside sulfamate resistant parasites.

Dr Stephen Brand (PI), MMV, will provide drug discovery expertise and strategic input and will connect the team with partners in the MMV network to provide access to state-of-the-art assays and expert advice.

Dr Larry Dick, MMV consultant, will provide expert biochemcial and drug development expertise, based on his many year in the Pharmaceutical industry.

Ms Delphine Baud, MMV, will coordinate logisitics, compound management and testing in the MMV platform of assays.

The assembled international team of researchers has the necessary expertise to deliver a chemical entity from this compound class into a Lead Optimization program.

Others (including references if necessary)

[1] van der Pluijm RW, Imwong M, Chau NH, Hoa NT, Thuy-Nhien NT, Thanh NV, et al. Determinants of dihydroartemisinin-piperaquine treatment failure in Plasmodium falciparum malaria in Cambodia, Thailand, and Vietnam: a prospective clinical, pharmacological, and genetic study. Lancet Infect Dis 2019 Jul 22.

Final Report

1. Project objective

Our principal goal was to develop selective hits from a parasite screen of nucleoside sulfamates to deliver compounds that meet MMV’s early lead criteria for entry into lead optimization. We also aimed to validate the target as plasmodium cytoplasmic tyrosine tRNA synthetase (YRS). We further aimed to determine the parasitological profile of plasmodium YRS inhibitors with respect to MMV’s target candidate and product profiles and to reach an understanding of the structural and biochemical factors that influence potency and selectivity, in order to develop safe therapeutics.

2. Project design

Medicinal chemistry allied to structural studies using X-ray crystallography was used to rationally improve potency and selectivity. Cellular and biochemical assays were used to determine potency and selectivity and to understand the mechanism of inhibition. Key prototypes were evaluated in MMV’s in-vitro and in-vivo platform to determine the parasitological profile. Studies involving the generation of resistant mutants were used to validate the target as PfYRS, and to assess the resistance risk.

3. Results, lessons learned 

A series of pyrazolopyrimidine ribose sulfamates (nucleoside sulfamates) was synthesized and characterized. Compounds were identified that exhibit low nanomolar potency against blood stage cultures of all strains of P. falciparum tested, including drug resistant strains. The frontrunner compound MMV1793207 (ML471) exhibits a fast rate of kill. This indicates TCP1 potential (i.e. rapid blood stage). The compounds are equipotent against liver stages (Pf and Pb) indicating TCP4 (chemoprotection) potential. The compounds exhibit activity against sexual stages (DGFA at 170 nM) indicating TCP5 (transmission blocking) potential. The resistance development risk needs to be further explored as one frontrunner met the MMV criteria (MIR = 7) while the other was below criteria (MIR = 6).

Biochemical investigations revealed that parasite killing is caused by inhibition of protein translation. The target, PfYRS, is an adenylate-forming enzyme, i.e. has an activated adenylate intermediate. Our work showed that PfYRS catalyses a reaction between the nucleoside sulfamate and the aminoacyl-tRNA enzyme product to yield a stable conjugate of the nucleoside sulfamate with the tyrosine substrate. That conjugate binds very tightly to the enzyme and inhibits its activity. Structural and biochemical studies revealed the basis for the selective inhibition of PfYRS compared with human

MMV1793207 (ML471) exhibits >50,000x selectivity compared with a mammalian cell line. It was tested in the SCID mouse model of P. falciparum malaria as a single oral dose of 100 mg/kg. It was well tolerated and showed curative efficacy. 

The series met MMV’s early lead criteria, as determined by MMV Expert Scientific Advisory Committee.

The key challenge was the very challenging nature of the synthetic chemistry routes. The slow rate of successful synthesis of new compounds hampered efforts to explore the several options available to improving oral bioavailability. The team pursued a medicinal chemistry strategy to enhance absorption by improving permeability, increasing LogD and decreasing (or masking through internal bonding) the number of hydrogen bond donors. With additional work, we remain hopeful that compounds can be identified with rat oral bioavailability of >30%. This would put the team in a position to apply to GHIT for funding for Lead Optimization.