Introduction and Background of the Project

1. Introduction

Each year, Plasmodium falciparum causes more than 200 million cases of malaria, and over 400,000 deaths, mostly in children under 5 and pregnant women. Because current antimalarial control is highly dependent on artemisinin combination therapies (ACTs), it is extremely concerning that decreased parasite sensitivity has emerged to all currently used ACTs, leading to significant failure rates in parts of Southeast Asia and more recently Africa, where partner drug resistance is also becoming evident. If resistance becomes widespread in Africa (where most deaths occur), a major health crisis is possible. In response to this impending crisis and with the eventual aim of eradicating the disease, MMV and Astellas as a collaboration partner seek to discover, develop, and deliver new drugs with novel modes of action which address resistance associated with existing therapeutics. Development of compounds which can block transmission and be used in chemoprotection/chemoprevention, in addition to acute treatment, are especially valuable to drive the eradication agenda.

Previously, supported by GHIT funding, hit compounds with antimalarial activity were identified from screening a compound library provided by Astellas. Initial profiling of the hit compounds demonstrated that they are novel, attractive start points for a Hit Validation and subsequent Hit-to-Lead project with potential to deliver new malaria drug candidates.

2. Project objective

The objective of the project is to identify at least 1 novel compound series meeting MMV Early Lead Criteria (1) that has clear potential for further development and progression to Lead Optimization.

3. Project design

The project will consist of two phases. The first 6-month phase will involve the synthesis and profiling of small set of compounds designed around each of the four hit compounds identified in the previous project. The compounds will be designed to explore both structure activity relationships (SAR) and scope for structural modification to improve the compound profile (potency, DMPK, safety, etc.). Two series will be selected for Hit-to-Lead studies. The second 18-month phase of the project will involve the optimization of the series (prioritizing the series with the greatest potential) with the goal of identifying a series with a frontrunner compound meeting the MMV Early Lead criteria.

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

MMV has worked with the wider malaria community to establish Target Candidate Profiles (TCPs) that define the attributes of the next generation antimalarials needed to not only control but also eradicate the disease (2).

Key characteristics are:

• New drugs for treatment or protection that are fast acting and have long duration.
• New drugs with efficacy against all known field resistance and a low risk of resistance generation.
• New drugs that are developable as a cheap, fixed dose combination drug with no contra-indication for use by children and women of child-bearing potential.

The initial profiling of the hit compounds identified from the high throughput screen indicates that the compounds have potential meet these criteria.

What sort of innovation are you bringing in your project?

All the compound series being explored have been prioritized based on novelty of the chemotype, life-cycle fingerprint, and the extent to which they fill strategic gaps in the MMV portfolio. Compound series will only be proposed for Lead Optimization if they fulfill the MMV Early Lead criteria (1) and are differentiated from existing series in the MMV portfolio at the time of the LO proposal. All series have novel drug-like chemotypes and mechanism of action, different from current antimalarial drugs in use or compounds in the clinical pipeline.

Role and Responsibility of Each Partner

TCGLS will be responsible for compound synthesis and, in vitro parasitology (asexual blood stage growth inhibition and rate of kill), cytotoxicity and DMPK. All other assays will be provided by MMV network test centers or the appropriate CRO. MMV will lead project strategy, medicinal chemistry (data analysis and compound design) and ensure alignment with global malaria elimination goals. Astellas will contribute to the design of new analogues and interpretation of the data using state of the art computer-aided drug design techniques.

Others (including references if necessary)

1. https://www.mmv.org/research-development/information-scientists

2. Burrows, J. N., et al. New developments in anti-malarial target candidate and product profiles. Malar. J. 16:26 (2017).

Final Report

1. Project objective

The aim of the Astellas-TCGLS project was to discover new antimalarial drug leads by identifying compounds meeting GHIT/MMV Early Lead criteria with potential for subsequent optimization towards a Late Lead.

2. Project design

In this HTLP project, the team identified a series that met the MMV Early Lead milestone criteria. During the first six-month phase, compound arrays around each of the four hits were synthesized and profiled. These arrays were designed to explore SAR and assess the potential for structural modifications to enhance the compound profile (potency, DMPK, safety, etc.). The second phase, spanning 18 months, focuses on optimizing Series 2, which showed superior potential for optimization, with the goal of identifying a frontrunner compound that meets the milestone criteria and has the potential for further development.

3. Results, lessons learned

Series 2, exemplified by MMV1964583, is a fast-acting compound with a parasite-killing rate comparable to Chloroquine. Growth inhibition of all drug-resistant strains in the AReBar assay suggests a potential novel mechanism of action or resistance. Additionally, no recrudescence was observed for MMV2214556 in the resistance risk assessment assay (Log MIR ≥ 8.3), indicating resistance-refractory properties. Compounds are compact (MW ≤ 350), highly soluble (> 100 μM at pH 7.4), and exhibit low lipophilicity (LogD_7.4 = 1 - 3). In vitro rat metabolite identification studies revealed that the amino group is the primary site of metabolism, with the methyl group as a secondary site. Substituting the 2-aminopyridine group (MMV1964583) with piperidine (MMV2214556) or azetidine (MMV2324997) improved metabolic stability (HLM CL_int,app = 5 and 3 mL/min/kg; Rat Hep CL_int,app = 41 and 22 mL/min/kg) and antiplasmodium activity (3D7 IC₅₀ = 23 nM).

While most compounds exhibited poor oral exposure due to low permeability (Papp A > B << 1×10⁶ cm/s), MMV2324045 showed significantly better permeability. This was associated with LogD_7.4 and pKa values within optimal ranges (≥ 3 and ≤ 9, respectively), enhancing absorption and resulting in greater systemic exposure and 33% oral bioavailability. The compound displayed a long half-life (t_1/2 = 9 h), attributed to its V_ss,u (186 L/kg) and rat CL_p (12 mL/min/kg). MMV2480670 also showed high oral bioavailability (27%) despite low absorption (C_max = 0.004 μM) and high rat CL_p (65 mL/min/kg). Its stability (CL_int,app = 16 mL/min/kg) may be underestimated due to solubility limitations (kinetic solubility = 9 μM). The high CL_p may be due to the amino group, suggesting potential modifications to mitigate rapid metabolism.

Figure 1. Cassette rat PK of MMV1964583, MMV2324045 and MMV2480670 (IV = 0.4 mg/kg and PO = 2 mg/kg).

Predicted human plasma MPC values for MMV2324045 and MMV2480670 aligned well with plasma exposures, indicating that a 4x50 mg/kg dose may achieve therapeutic levels.

Most Series 2 compounds (LogD_7.4 ≥ 3, pKa ≤ 9) show low potential for cross-resistance (Dd2/3D7 ≤ 3), though MMV1964583 and MMV2214556 exhibited reduced activity against Brazilian Pf field isolates, warranting further study.

MMV2324045 and MMV2480670 showed no cytotoxicity (HepG2 SI ≥ 100). MMV2480670 has a hERG IC₅₀ of ~1 μM (SI ~59), while MMV2324045 is still under assessment. Small structural changes can significantly impact hERG activity without affecting antiplasmodium activity (e.g., MMV2480769 3D7 IC₅₀ = 60 nM, SI = 217).

The SCID study for MMV2324045 is scheduled for January 27, 2025, alongside additional studies to meet MMV and GHIT Fund criteria for Early Leads. Pending strong data, the project will be submitted to GHIT for PDP funding consideration.