Mammalian deubiquitinating enzyme inhibitors display in vitro and in vivo activity against malaria parasites and potentiate artemisinin action

Current malaria control efforts rely significantly on artemisinin combinational therapies which have played massive roles in alleviating the global burden of the disease. Emergence of resistance to artemisinins is therefore, not just alarming but requires immediate intervention points such as development of new antimalarial drugs or improvement of the current drugs through adjuvant or combination therapies. Artemisinin resistance is primarily conferred by Kelch13 propeller mutations which are phenotypically characterised by generalised growth quiescence, altered haemoglobin trafficking and downstream enhanced activity of the parasite stress pathways through the ubiquitin proteasome system (UPS). Previous work on artemisinin resistance selection in a rodent model of malaria, which we and others have recently validated using reverse genetics, has also shown that mutations in deubiquitinating enzymes, DUBs (upstream UPS component) modulates susceptibility of malaria parasites to both artemisinin and chloroquine. The UPS or upstream protein trafficking pathways have, therefore, been proposed to be not just potential drug targets, but also possible intervention points to overcome artemisinin resistance. Here we report the activity of small molecule inhibitors targeting mammalian DUBs 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 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 ART resistance can be overcome. Combinations of DUB inhibitors anticipated to target different DUB activities and downstream 20s 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. Graphical abstract

In addition, ART induced pleiotropic targeting is also known to activate ER stress and the unfolded 124 protein response (UPR) which allow parasites to survive drug assault by rapidly turning over 125 damaged proteins while employing cell repair mechanisms. [6][7]13 ART resistant parasites (Kelch13 126 mutants) are indeed associated with an upregulation of genes involved in these cellular stress 127 response pathways. 14 Meanwhile, parallel functional and localisation studies have also revealed that 128 Kelch13 co-localises with multiple UPR components, proteins specific to the ER and mitochondria as 129 well as intracellular vesicular trafficking Rab GTPases. [15][16] Central to the activity of the UPR is the

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To assay for in vitro activity of DUB inhibitors in malaria parasites, short term PB culture assays and 177 PF Sybergreen I ® culture assays were employed. The PB 820 and PF 3D7 lines were initially screened 178 to determine susceptibility to inhibitors and antimalarials with known activity in malaria parasites;

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To explore interactions between DUB inhibitors, and their potential synergy, b-AP15, a highly    (Table 1)

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after DHA incubation further abrogates GFP intensity illustrating that b-AP15 compromises UPS 306 activity in tandem with DHA, which would make them suitable partner drugs. In the PF 3D7 line, pre-307 incubation of ~0-3 hour old rings with b-AP15 at IC50 or half IC50 for 3 hours followed by DHA 308 treatment for 4 hours markedly impacts parasite viability (5 and 1.6 fold respectively) compared to 309 DMSO exposed parasites, while pre-exposing the parasites to b-AP15 at 4x IC50 is almost entirely 310 lethal to the parasites ( Figure 4B). Meanwhile, pre-exposure of 3D7 or an ART resistant Kelch13 311 C580Y line to epoxomicin at IC50 or 0.2x IC50 followed by DHA also significantly impacted parasite    In conclusion, our work confirms DUBs as potential druggable candidates in malaria parasites. Drug 463 discovery programs take a long time, with for example a minimum of five years required to take a 464 lead compound to a clinical candidate in malaria. [52][53] The emergent resistance to ACTs, a paucity in 465 the number of antimalarial drugs in the developmental pipeline and a lack of scalable pipelines for 466 drug discovery in other human malaria parasites such as P. vivax and P. ovale, 53 all necessitates 467 both radical as well as alternative approaches to identify new drugs and drug targets. As DUBs are 468 already being actively explored as anticancer agents with candidate inhibitors already entering 469 clinical trials, 54 antimalarial drug discovery programs could take advantage to structurally improve 470 or re-purpose such entities not just as potential drug targets in malaria, but also as combinational 471 partners to ARTs to overcome the spectre of resistance.  were gassed for 30 seconds with a special gas mix of 5% CO2, 5% O2, 90% N2 and incubated for 22-531 24 hours at 37 0 C with gentle shaking, conditions that allow for development of ring stage parasites 532 to mature schizonts. Drug assays to determine in vitro growth inhibition during the intraerythrocytic 533 stage were performed in these standard short-term cultures as previously described. 29-30 Briefly, 1 534 ml of infected blood with a non-synchronous parasitaemia of 3-5% was collected from an infected 535 mouse and cultured for 22-24 hours in 120 ml of schizont culture media. Schizonts were enriched 536 from the cultures by Nycodenz density flotation as previously described 58 followed by immediate 537 injection into a tail vein of a naive mouse. Upon IV injection of schizonts, they immediately rupture    infected with PB 820 line from which blood was obtained when the parasitaemia was between 2-5%.

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Donor blood was diluted in rich PBS following which ~10 5 parasites were inoculated by IP into four

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ART and b-AP15 were prepared at 12.5mg/ml and 1mg/ml respectively in 1:1 mixture of DMSO and