Validation of the Proteasome as a Therapeutic Target in Plasmodium Using an Epoxyketone Inhibitor with Parasite-Specific Toxicity

Abstract

Summary The Plasmodium proteasome has been suggested to be a potential anti-malarial drug target, however toxicity of inhibitors has prevented validation of this enzyme in vivo. We report here a screen of a library of 670 analogs of the recently FDA approved inhibitor, carfilzomib, to identify compounds that selectively kill parasites. We identified one compound, PR3, that has significant parasite killing activity in vitro but dramatically reduced toxicity in host cells. We found that this parasite-specific toxicity is not due to selective targeting of the Plasmodium proteasome over the host proteasome, but instead is due to a lack of activity against one of the human proteasome subunits. Subsequently, we used PR3 to significantly reduce parasite load in P. berghei infected mice without host toxicity, thus validating the proteasome as a viable anti-malarial drug target.

Artemisinin Action and Resistance in Plasmodium falciparum

“…Toxicity and regulatory issue may make it difficult to reposition a clinically used proteasome inhibitor. Earlier efforts to develop Plasmodium -specific proteasome inhibitors [114–116] now benefit from a recent tour de force , in which a collaboration led by Dr. Matt Bogyo (Stanford University) applied cryo-electron microscopy and single particle analysis to solve the structure of the P. falciparum 20S proteasome to a resolution of 3.6 Å. Combined with substrate profiling, this provides valuable information regarding active site architecture that can be used to drive optimal inhibitor design [92].…”

Identifying the Minimal Enzymes Required for Biosynthesis of Epoxyketone Proteasome Inhibitors

“…In particular, the strongly electrophilic carbonyl and epoxide groups are highly susceptible to a double nucleophilic attack by the N‐terminal threonine of the 20S proteasome, leading to the irreversible formation of a morpholino ring . Consequently, these natural products have inspired the current development of anticancer, anti‐inflammatory, and antiparasitic drugs with a terminal epoxyketone warhead . One such example is the synthetic epoxomicin analogue, carfilzomib (Scheme ), which has been used as a treatment for relapsed and refractory multiple myeloma …”

“…[15] PathwayA postulates the introduction of as ingle methyl group at C2, followed by the reductionoft he carboxylica cid to an olefin that can then undergo epoxidation,w hereas pathway Bp roposes dimethylation at C2, followed by decarboxylation of the carboxylic acid and subsequente poxide installation. [15] To distinguish between these two possibilities, we fed [1,2-13 C]sodium acetate to cultures of JL11, the highest producing strain, and compared the www.chembiochem.org resulting 13 Ci ncorporation to that from unlabeled and [1-13 C]sodium acetate feeding (Scheme2 and Figure S9-10).A lthought he titerso fl abeled 3 and 4 were insufficientf or NMR analysis, LC-HRMS analysis of 3 and 4 from the extracts of cultures fed with [1,[2][3][4][5][6][7][8][9][10][11][12][13] C]sodium acetate showed as ignificant increase in the ratio of the M+ +1p eak intensity to the M+ + peak intensity compared to that of cultures fed with unlabeled and [1-13 C]sodium acetate (FiguresS9a nd S10). Further HRMS/MS analyses and examination of the isotope distribution of fragments with and withoutt he epoxyketone group indicated that this increase is the result of as ingle 13 Ca tom from doubly labeled acetate being incorporated as the C2 carbon of 3 and 4 after decarboxylation (Scheme 2a nd Figure S9-10).…”

“…[7][8][9] Consequently,t hese natural products have inspired the current development of anticancer,a nti-inflammatory,a nd antiparasitic drugs with at erminal epoxyketone warhead. [10][11][12] One such example is the synthetic epoxomicin analogue,c arfilzomib (Scheme 1), which has been used as at reatment for relapsed and refractory multiple myeloma. [13][14] Given the potency andu nique structure of these peptidyl epoxyketone natural products, there has been great interest in the biosynthetic origins of these compounds.…”

Eine strukturelle Evaluierung medizinalchemischer Strategien gegen Wirkstoffresistenzen

“…[326] Die Verwendung einer aktivitätsbasierten Sonde in P. -falciparum-Kulturen, die mit selektiven, bekannten Inhibitoren der humanen b1-, b2und b5-Untereinheiten vorbehandelt waren, lieferte wichtige Einblicke in das 20S-Proteasom. [326] Die Verwendung einer aktivitätsbasierten Sonde in P. -falciparum-Kulturen, die mit selektiven, bekannten Inhibitoren der humanen b1-, b2und b5-Untereinheiten vorbehandelt waren, lieferte wichtige Einblicke in das 20S-Proteasom.…”