HTL for selective Plasmodium proteasome inhibitors as new potent resistance-reversing antimalarials
Project Completed
Please click to see the final report.

Introduction and Background of the Project


The proteasome is an enzyme that degrades denatured proteins in cells and is responsible for maintaining 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. Proteasome inhibitors have been shown to have powerful antimalarial activity as single agents in vitro, but so far the right combination of selectivity over the human enzyme and drug-like properties have not been realized.

With the previous support of the GHIT Fund (T2015-134 project), the Takeda/ University of Melbourne/ Medicines for Malaria team have already identified promising start points 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. So far we have validated hits that potently inhibit parasite growth and show modest selectivity for inhibition of the growth of P. falciparum compared with human cell lines. Active site profiling was used to gain further insights into the molecular determinants of activity. 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 we are poised to deliver a novel antimalarial drug lead that can both 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; 2) To assess potent, specific P. falciparum proteasome inhibitors for concordance with the Malaria Target Candidate Profiles; 3) To deliver P. falciparum proteasome inhibitors which meet the GHIT and MMV early lead criteria.


Project design

We will undertake a medicinal chemistry program supported by a well-defined test cascade to deliver potent and selective proteasome inhibitors with good drug-like properties. We propose to expand and optimize our initial boronate hits into several sub-series that have potent activity against the plasmodium proteasome but weak binding and short residence times on the human target. Ultimately we will need to find the right balance of selectivity. Some level of binding to the human proteasome may in fact be beneficial as it could help to extend the half life without causing significant inhibition of the human target and the resulting problems with tolerability. The inhibitor design will take advantage of our understanding of P. falciparum 20S proteasome active site preferences obtained from a library of discrete tripeptide substrates. Clear differences were observed in substrate specificity between the human and P. falciparum enzymes, which we will exploit to design more selective inhibitors. This work has led us to focus particularly on the residues at P1 and P3 positions. 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.

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 years1. 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 Asia2, delaying the clearance of parasites from patients, and leading to clinical failure (~50% failure to cure in some regions)3-5. Even more worryingly, the first indigenous African K13 mutation was recently reported6. 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?

An exciting aspect of this project is that it brings together pharmaceutical industry experts (Dick, Gould) and an academic parasitology expert (Tilley) with a Product Development Partner (Brand, Baud MMV). Taking as a basis the knowledge gained through many years of work at Takeda Oncology in developing proteasome inhibitors as anti-cancer agents and combining it with the malaria biology expertise of the Tilley lab and the malaria drug development prowess of MMV, we will translate that knowledge to the development of new antimalarials.

A technical innovation of our project is the determination of a proteasome inhibitor "signature" that is associated with highly specific anti-plasmodium activity. We anticipate that this chemical "signature" will comprise tight interaction with both 2 and5 subunits of the P. falciparum proteasome, but weak and reversible interaction with the mammalian proteasome. This aspect of the work will be important in the selection of drug candidates that will be both effective and safe for use in malaria patients.

Role and Responsibility of Each Partner

Larry Dick, Takeda Oncology, will coordinate the activities of 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.

Leann Tilley, University of Melbourne, will contribute relevant test cascade assays, provide P. falciparum proteasome, conduct mechanism of action studies, and develop key tools, such as the bortezomib resistant parasites.

Sandy Gould, Takeda Oncology, will lead the medicinal chemistry design efforts and coordinate the synthesis of new compounds.

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.

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

Others (including references if necessary)

1. Geneva:_World_Health_Organisation. World Malaria Report 2017.  (2017).

2. Tun, K. M. et al. Lancet Infect Dis (2015).

3. Phyo, A. P. et al. Lancet 379, 1960-1966 (2012).

4. Spring, M. D. et al. Lancet Infect Dis 15, 683-691 (2015).

5. Ashley, E. A. et al. N Engl J Med 371, 411-423 (2014).

6. Lu, F. et al. N Engl J Med (2017).

Final Report

1. Project objective

Our principal goal was to develop selective hits from a biochemical and parasite screen to deliver compounds which 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 which 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 with and without wash-out to determine potency and selectivity. Studies involving the generation of resistant mutants were used to confirm on-target activity and inhibitor binding mode, to show that proteasome inhibitors reverse artemisinin resistance 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), 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). As a result of efforts to optimize three boronic acid hit series we have identified one class which specifically targets the proteasome beta-5 subunit, shows >100-fold selectivity over the human isoform, has encouraging pharmacokinetics (F>50% in rat, t1/2 >5h), and has demonstrated good oral efficacy in the SCID model of malaria (parasitemia reduced to BLOQ at 4 x 25mg/kg b.i.d.). Compared to literature series we believe that this DAG series has the best chance of progression. Based on cryo-EM structures we have generated hypotheses for the origins of selectivity which will inform continued efforts to improve selectivity. We have learnt that it is a significant challenge to measure and define the necessary level of selectivity over the human target of compounds which have a reversible covalent inhibition characteristic.