Abstract
The Plasmodium proteasome represents a potential antimalarial drug target for compounds with activity against multiple life cycle stages. We screened a library of human proteasome inhibitors (peptidyl boronic acids) and compared activities against purified P. falciparum and human 20S proteasomes. We chose four hits that potently inhibit parasite growth and show a range of selectivities for inhibition of the growth of P. falciparum compared with human cell lines. P. falciparum was selected for resistance in vitro to the clinically used proteasome inhibitor, bortezomib, and whole genome sequencing was applied to identify mutations in the proteasome β5 subunit. Active site profiling revealed inhibitor features that enable retention of potent activity against the bortezomib-resistant line. Substrate profiling reveals P. falciparum 20S proteasome active site preferences that will inform attempts to design more selective inhibitors. This work provides a starting point for the identification of antimalarial drug leads that selectively target the P. falciparum proteasome.
Highlights
Malaria remains a major health problem, threatening hundreds of millions of people and causing ∼440 000 deaths each year.[1]Current antimalarial control is highly dependent on artemisinin-based combination therapies (ACTs), which makes the emergence of artemisinin partial resistance extremely concerning.[2−4] Decreased ACT sensitivity delays the clearance of parasites from patients and leads to clinical failure, resulting in ∼50% treatment failure in regions where resistance is entrenched, compared with ∼2% failure in regions where resistance is rare.[5,6] Replacement antimalarials are urgently needed.The proteasome is a multisubunit enzyme complex that is responsible for proteostasis and for regulating key processes such as the cell cycle
While preferential inhibition of P. falciparum β2 activity was readily achieved, we show that inhibition of P. falciparum β5 activity coupled with weaker or reversible activity against human β5 activity represents a signature that permits selective killing of P
We developed resistance to bortezomib in P. falciparum and show this is associated with mutations in the β5 active site
Summary
Malaria remains a major health problem, threatening hundreds of millions of people and causing ∼440 000 deaths each year.[1]. In an effort to confirm that the peptide boronates exert their antiplasmodial activity via direct binding to one or more proteasome subunits and to determine the residues that are important for the inhibitory activity, we selected P. falciparum for resistance to bortezomib, followed by whole genome sequencing This approach has been used successfully to identify and characterize the targets of a number of antiparasitic compounds.[30,31]. Active site probe analysis of the proteasome subunit specificity of the selected compounds in 3D7 parent and bortezomib-resistant parasites. The higher inhibitory activity of MPI-3 and MPI-4 against Pf 20S may reflect their bulky hydrophobic groups at P2 and P3, and the fact that the Pf 20S β2 site is more hydrophobic than human 20S.9 While this manuscript was under review, another study was published that used substrate profiling to design optimized vinyl sulfone-based inhibitors.[44] This work suggests that choice of an optimal electrophilic warhead can further enhance selectivity
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