Proteasome inhibitors as new potent antimalarials
Project Completed
Please click to see the final report.

Introduction and Background of the Project


The proteasome is a multienzyme complex found in all eukaryotic cells that functions to maintain homeostasis of intracellular proteins. As an organism that undergoes rapid growth and cell division, the malaria parasite is highly reliant on its ubiquitin proteasome system, making the proteasome a promising target for antimalarial drug discovery.

With the previous support of the GHIT Fund (T2015-134; H2017-101), our Takeda Pharmaceuticals/ University of Melbourne/ Medicines for Malaria Venture team has already identified promising early leads for a drug discovery campaign. We screened a peptide boronate library of human proteasome inhibitors for antiparasitic activity then further characterized the hits, by comparing their activities against purified P. falciparum and human 20S proteasome and assessing their activities as inhibitors of the growth of P. falciparum and human cells. We identified three hit series and performed further exploration to define structure activity relationships, ultimately arriving at a preferred series.

Our lead ‘DAG’ series are potent inhibitors of only the two beta-5 subunit’s catalytic centers in the plasmodium proteasome. Examples also have high potency against the parasite (3D7 ED50 <10nM), selectivity over human cell lines (>100-fold), are fast-acting (equivalent to artemesinin), have good bioavailability (rat >50%) and show efficacy in the SCID mouse model of P.falciparum malaria (ED90<50mg/kg 4 x b.i.d). Data from the clinical development of Takeda’s new oral human proteasome inhibitor, ixazomib (NINLARO®) suggests that molecules in this chemical class can have pharmacological and pharmaceutical properties consistent with MMV’s Target Candidate Profile (TCP) for new antimalarial medicines. Thus, with further improvements in selectivity, we are poised to deliver a novel antimalarial drug lead that can be a valuable contribution to treatment in its own right, and in addition, potentiate the action of artemisinins and overcome artemisinin resistance.


Project objective

The Project aims: 1) To discover potent inhibitors of P. falciparum proteasome with high selectivity over the human enzyme which meet MMV’s criteria for entry into lead optimization; 2) To assess potent, specific P. falciparum proteasome inhibitors for concordance with the Malaria Target Product Profiles i.e. for treatment and/or chemoprotection.


Project design

We will pursue a H2L medicinal chemistry program supported by a well-defined test cascade to deliver early lead(s) which meet MMVs criteria for entry into lead optimisation. We need to improve selectivity over the human beta-5 subunit and to improve the half-life in order to ultimately identify safe candidates with single dose potential. Differences in the substrate binding site between the human and P. falciparum enzymes will aid the rational design of highly selective inhibitors. Furthermore, our experience with boronate inhibitors has taught us that good pharmacokinetic properties (oral bioavailablity, low clearance and long half-lives, good solubility and suitable LogP) can be obtained. The inhibitor design will take advantage of our understanding of P. falciparum 20S proteasome active site obtained from modelling and cryoEM structural studies.

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

P. falciparum causes more than 200 million cases of malaria, and about 438,000 deaths, each year – mostly of children, aged 0 - 5 years. Treatment of falciparum malaria is currently heavily reliant on the artemisinins. Thus it is extremely concerning that decreased sensitivity to this drug class has emerged in South East Asia, delaying the clearance of parasites from patients, and leading to clinical failure (~50% failure to cure in some regions) [1]. The World Health Organization has warned: "There is a limited window of opportunity to avert a regional public health disaster, which could have severe global consequences."

In response to this impending crisis, the Medicines for Malaria Venture (MMV), has declared that novel targets for antimalarial therapies need to be identified and new drugs developed. This project aims to identify compounds that have highly specific and potent activity against the P. falciparum proteasome as antimalarial drug leads.

What sort of innovation are you bringing in your project?

This project builds on the knowledge gained through many years of work at Takeda Pharmaceuticals (Gould, Dick) in developing proteasome inhibitors as anti-cancer agents. Combining that expertise with the malaria biology expertise of the Tilley lab and the malaria drug development prowess of MMV (Brand), will facilitate translation of the knowledge to the development of new antimalarials.

A technical innovation of this project is that inhibitor design will take advantage of a detailed understanding of P. falciparum 20S proteasome active site architecture obtained from modelling and cryoEM structural studies. Subtle differences in the substrate binding site between the human and P. falciparum enzymes will be exploited to design more selective inhibitors, in combination with more traditional medicinal chemistry approaches.

Role and Responsibility of Each Partner

Dr Larry Dick (Lead PI) will coordinate activities across the Project. He will provide expert biochemcial and drug development expertise, based on his many years in the Pharmaceutical industry.

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 purified P. falciparum proteasome, conduct mechanism of action studies, including cryoEM, and develop key tools, such as proteasome inhibitor resistant parasites.

Dr Sandy Gould (PI), Takeda Pharmaceuticals, will lead a team of Takeda medicinal chemists and biochemists who will be designing and analysing new proteasome inhibitors for this hit to lead effort.  The team will be comprised of scientists that cumulatively have several decades of experience working on inhibitors of the human proteasome and inhibitors of other targets with related chemical series.

Dr Stephen Brand, 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.

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 biochemical and parasite screen to deliver compounds that meet MMV’s early lead criteria for entry into lead optimization. Additionally, we aimed to determine the parasitological profile of plasmodium proteasome inhibitors with respect to MMV’s target candidate and product profiles and to reach an understanding of the structural and kinetic factors that influence potency and selectivity, in order to develop safe therapeutics.


2. Project design

Medicinal chemistry allied to structural studies using cryo-EM and molecular simulations were used to rationally improve potency and selectivity. Cellular and biochemical assays were used to determine potency and selectivity. 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 confirm on-target activity and inhibitor binding mode and to assess the resistance risk.


3. Results, lessons learned

We have confirmed that plasmodium proteasome inhibitors have activity across the lifecycle (i.e. TCP1,4 and 5) in all lab-adapted, drug-resistant and clinical strains examined, including P. vivax, are fast-acting in-vitro and in-vivo and have the appropriate parasitological and resistance profile for use in both treatment and chemoprotection (TPP1 and 2, MIR>10e7). We have identified a compound class that specifically targets the proteasome beta-5 subunit, shows >100-fold biochemical selectivity over the human isoform and exhibits good oral efficacy in the SCID model of malaria (parasitemia reduced to BLOQ at 4 x 25 mg/kg PO).

Analysis of the kinetics of inhibition reveals that slower formation of the dative bond between the boronate and the active site nucleophile of human 20S beta5 compared to P. falciparum (Pf) 20S beta5 underpins the selectivity of the DAG series. Cryo-EM structures and modelling studies revealed the significant challenge to achieve selectivity over the human target for compounds that exhibit a reversible covalent inhibition characteristic. 

Concerns emerged during the last six months with respect to short in vivo half-life of our compounds perhaps due to the propensity of the DAG series to de-boronate, which would also be expected to impact shelf stability. We plan to perform confirmatory tests on three boronate esters of the frontrunner to deliver a stop/go conclusion for the series, including identification of metabolites from human hepatocytes and human plasma and measurement of chemical stability and shelf stability via forced degradation studies.

Our preliminary analysis (subject to confirmation) is that limited in vivo half-life may be a characteristic of the series perhaps resulting from compound metabolism or deboronation of the boronic acid warhead. As such, it may not be possible to achieve pharmacokinetic properties consistent with MMV’s criteria for single oral dose administration. Compared to other literature series, we conclude that this DAG series remains the best chance of progression, if the pharmacokinetic properties could be improved. Should the frontrunner compound (based on potency and selectivity profile) have the prospect of good chemical/in-vivo stability we will consider profiling it as a possible lead (SCID efficacy and in-vitro/in-vivo safety). No further support for the project is currently requested from GHIT.