Identification of Small Molecules Disrupting the Ubiquitin Proteasome System in Malaria

Abstract

The Ubiquitin Proteasome System (UPS) is one of the main proteolytic pathways in eukaryotic cells, playing an essential role in key cellular processes such as cell cycling and signal transduction. Changes in some of the components of this pathway have been implicated in various conditions, including cancer and infectious diseases such as malaria. The success of therapies based on proteasome inhibitors has been shown in human clinical trials. In addition to its proven tractability, the essentiality of the Plasmodium falciparum UPS underlines its potential as a source of targets to identify new antimalarial treatments. Two assays, previously developed 14,747 P. falciparum inhibition growth 996 544 increased the levels ub-proteins Dose response 100 µM (1/3) n=2 AlphaLISA Dose response 100 µM (1/3) n=2 DELFIA Single shot 10 µM n=2 AlphaLISA 311 validated • PfpIC50>4 (β2 and β1) Asym max>70% • Human pIC50<4 Inactive Asym max<50% 16 6 Human proteasome assays P. falciparum selective P. falciparum proteasome assays 100 µM (1/3) n=2 % inhibition Pf26S>50% P. falciparum pIC50>4 NON-PROTEASOME INHIBITORS PROTEASOME INHIBITORS pIC50 Pf26S<4 % inhibition Pf26S<50% 169

Cell communication and protein degradation: All in one parasitic package

“…Such a task is not trivial, given the high level of conservation of proteasome active sites in eukaryotes (Xie et al, 2019). However, progress in this direction has already been made with the design of compounds that show specificity against P. falciparum (Lin et al, 2023;Mata-Cantero et al, 2019;Stokes et al, 2019) and Trypanosoma brucei proteasomes (Steverding et al, 2011), and we expect that additional selective compounds will arise.…”

High Throughput Screening to Identify Selective and Nonpeptidomimetic Proteasome Inhibitors As Antimalarials

“…Of these hits, 10 out of 11 had a carboxylic acid or its tetrazole bioisostere, and one compound possesses a sulphonamide group (SI table S1) which is present in proteasome inhibitors already published. 13 Hit Expansion and Characterization of Selective Hits. A total of 1033 analogues of the 11 primary more selective hits were identified within the GSK compound collection (Figure 2 and SI table S1) and arranged in clusters.…”

“…Treatment with proteasome inhibitors has been previously reported to lead to a build-up of intracellular poly ubiquitin. 13,29 Similarly, DHA, which damages proteins and compromises proteasome activity, 28 leads to a build-up of poly ubiquitin (Figure 6B, S5B). Here, we show that treatment of P. falciparum with 100 μM GW012X results in a similar increase in the level of polyubiquitylated protein (Figure 6B, S5B), a further indication that GW012X exerts its antimalarial activity by inhibiting proteasome activity.…”

“…Different proteasome inhibitors have been used to demonstrate the essentiality of the P. falciparum UPS at all stages of its development cycle: hepatic and blood (sexual and asexual) as well as mosquito stages. − Moreover, proteasome inhibitors strongly synergise artemisinin-mediated killing of P. falciparum in vitro and P.…”

“…We previously identified cyanamides, which are nonpeptide inhibitors, with selectivity for the trypsin-like and caspase-like activities of the P. falciparum proteasome . They were inactive against the human proteasome, but they were also inactive against the chymotrypsin-like activity of the parasite proteasome and exhibited only a moderate parasite killing profile compared to compounds that inhibit the chymotrypsin-like activity.…”