Cytoplasmic isoleucyl tRNA synthetase as an attractive multistage antimalarial drug target

Abstract

Development of antimalarial compounds into clinical candidates remains costly and arduous without detailed knowledge of the target. As resistance increases and treatment options at various stages of disease are limited, it is critical to identify multistage drug targets that are readily interrogated in biochemical assays. Whole-genome sequencing of 18 parasite clones evolved using thienopyrimidine compounds with submicromolar, rapid-killing, pan–life cycle antiparasitic activity showed that all had acquired mutations in the P. falciparum cytoplasmic isoleucyl tRNA synthetase (cIRS). Engineering two of the mutations into drug-naïve parasites recapitulated the resistance phenotype, and parasites with conditional knockdowns of cIRS became hypersensitive to two thienopyrimidines. Purified recombinant P. vivax cIRS inhibition, cross-resistance, and biochemical assays indicated a noncompetitive, allosteric binding site that is distinct from that of known cIRS inhibitors mupirocin and reveromycin A. Our data show that Plasmodium cIRS is an important chemically and genetically validated target for next-generation medicines for malaria.

Identification of an inhibitory pocket in falcilysin provides a new avenue for malaria drug development

“…Indeed, CQ been shown previously to induce dimerization of arginyl-tRNA ligase rendering the enzyme inactive (Jain et al, 2016). Moreover, tRNA ligases are being considered by several antimalarial development programs as high value drug targets (Herman et al, 2015; Hewitt et al, 2017; Istvan et al, 2023; Jain et al, 2017; Jain et al, 2015; Jain et al, 2016; Khan et al, 2013; Milne et al, 2022; Sharma et al, 2022; Sonoiki et al, 2016; Xie et al, 2022). Our MS-CETSA now links tRNA ligases with quinoline drug’s MOA suggesting their higher potential.…”

“…The mutations resulting from these selections are good indicators of proteins and/or pathways affected by the drugs and as such, provide important additional information regarding their MOA. This method has been successful in identifying or reconfirming antimalarial drug targets, such as the proteasome (Xie et al, 2018), thymidylate synthase, farnesyltransferase, dipeptidyl aminopeptidase 1, aminophospholipid-transporting P-type ATPase (Cowell et al, 2018), protein kinase CLK3 (Alam et al, 2019), acetyl-coenzyme A synthetase (Summers et al, 2022) and isoleucil tRNA synthetase (Istvan et al, 2023). Upon identification of such genes, complementary studies are typically conducted to discern the actual drug/compound targets from factors of resistance (Luth et al, 2018).…”

“…Upon identification of such genes, complementary studies are typically conducted to discern the actual drug/compound targets from factors of resistance (Luth et al, 2018). Such studies include metabolic profiling of drug-treated parasites (Alam et al, 2019; Allman et al, 2016; Cowell et al, 2018; de Vries et al, 2022; Gisselberg et al, 2018; Istvan et al, 2023; Murithi et al, 2020; Summers et al, 2022), modelling and crystallography studies (Istvan et al, 2023; Summers et al, 2022; Xie et al, 2022), and CRISPR-based allelic replacement studies (Istvan et al, 2023; Summers et al, 2022; Xie et al, 2022). In the case where resistant parasites cannot be selected using IVIEWGA, chemoproteomics approaches are typically considered.…”

Translation inhibition efficacy does not determine thePlasmodium bergheiliver stage antiplasmodial efficacy of protein synthesis inhibitors

“…It is notable that the two aaRS inhibitors, LysRS-IN-2 and MMV019266 display the same profound difference in speed-of-effect/kill as observed in PRR assays where 10x growth inhibition EC 50 s were tested. LysRS-IN-2 is slower to kill mixed P. falciparum ABS cultures than even atovaquone, the slowest-acting reference compound, while MMV019266 and similar thienopyrimidines kill as quickly as artemisinin, the fastest-acting reference compound ( 26, 28 ). LysRS-IN-2 was the most reversible compound we tested, and yet still able to reduce merosome release by ∼50% at both 5x- and 10x translation inhibition EC 50 , while MMV019266 exerted much greater antiplasmodial effects, reducing developmental success to ∼ 5% at both 5x- and 10x EC 50 , with substantial developmental delay.…”

“…MMV019266, a multistage active Malaria Box compound (25), which was identified in our previous study as a Plasmodium-specific translation inhibitor (17), is likely to target the cytoplasmic isoleucyl-tRNA synthetase, as has been demonstrated for several structurally related thienopyrimidines (26). Using a parasite reduction ratio (PRR) assay (27), 10xEC50 MMV019266 was shown to kill the majority of P. falciparum ABS parasites after 24 h of treatment (26), a rapid rate-of-kill similar to that of the fast-acting reference antimalarial artesunate. This rapid ABS rate-of-kill of MMV019266 is in marked contrast to that of DDD107498, which does not reduce the viable parasite population at all after a 24 h 10xEC50 treatment (5), and has a PRR similar to that of atovaquone, the slowest acting reference antimalarial in the assay.…”