Mammalian Deubiquitinating Enzyme Inhibitors Display in Vitro and in Vivo Activity against Malaria Parasites and Potentiate Artemisinin Action

Abstract

The ubiquitin proteasome system (UPS) is an emerging drug target in malaria due to its essential role in the parasite’s life cycle stages as well its contribution to resistance to artemisinins. Polymorphisms in the Kelch13 gene of Plasmodium falciparum are primary markers of artemisinin resistance and among other things are phenotypically characterized by an overactive UPS. Inhibitors targeting the proteasome, critical components of the UPS, display activity in malaria parasites and synergize artemisinin action. Here we report the activity of small molecule inhibitors targeting mammalian deubiquitinating enzymes, DUBs (upstream UPS components), in malaria parasites. We show that generic DUB inhibitors can block intraerythrocytic development of malaria parasites in vitro and possess antiparasitic activity in vivo and can be used in combination with additive to synergistic effect. We also show that inhibition of these upstream components of the UPS can potentiate the activity of artemisinin in vitro as well as in vivo to the extent that artemisinin resistance can be overcome. Combinations of DUB inhibitors anticipated to target different DUB activities and downstream proteasome inhibitors are even more effective at improving the potency of artemisinins than either inhibitors alone, providing proof that targeting multiple UPS activities simultaneously could be an attractive approach to overcoming artemisinin resistance. These data further validate the parasite UPS as a target to both enhance artemisinin action and potentially overcome resistance. Lastly, we confirm that DUB inhibitors can be developed into in vivo antimalarial drugs with promise for activity against all of human malaria and could thus further exploit their current pursuit as anticancer agents in rapid drug repurposing programs.

The newly discovered role of endocytosis in artemisinin resistance

“…Nonsynonymous mutations in four proteins, K13, 17,18,95 AP-2µ, 96 UBP1, 51,96,97 and coronin, 44 as well as deletions or knockdown of several others 51,76,77 have been shown to cause ART resistance in vitro. K13 mutations are responsible for ART resistance in most patients.…”

“…49 It has further been suggested that an elevated stress response is required as a second component in addition to reduced endocytosis to survive DHA exposure. 71 Inhibitors affecting the stress response, like deubiquitinase inhibitors and proteasome inhibitors, or increasing stress therefore synergize with ART 49,97,125 and make Plasmodium-specific proteasome inhibitors, which are also potent as monotherapy, promising candidates for new partner drugs. [126][127][128][129][130] 3.5 | Genetic background and geographic distribution…”

“…Mutations in these proteins, for example, AP-2μ and UBP1, could therefore result in reduced susceptibility to ART throughout the life cycle. 42,43,97,98 Resistance caused by these mutations might not be overcome by prolonged administration of ART and might additionally reduce the susceptibility to other drugs.…”

“…However, mutations causing such multidrug resistance have so far conferred lower levels of resistance to ART than K13 mutations, 42,43,51,97,98 probably because of the negative effect mutations in these genes have on later stages, restricting the possible reduction of endocytosis. Additionally, it was suggested that even strong effects on the IC 50 would not have a large effect on survival because the short half-life of ART in the blood reduces the benefit of full resistance (which increases IC 50 ) compared with the partial resistance caused by K13 mutations.…”

Current status of experimental models for the study of malaria

“…Experimental tractability of RMPs could also mean such mutant parasites could be assessed for transmission fitness in the absence and or presence of drug pressure. These mutant RMPs are also offering opportunities to assess the ex vivo and in vivo efficacy of antimalarial agents which can be used as combinational partners with ARTs to offset resistance (Simwela et al ., 2020 b , 2021 ).…”