RESUMO
Malaria remains a global health concern as drug resistance threatens treatment programs. We identified a piperidine carboxamide (SW042) with anti-malarial activity by phenotypic screening. Selection of SW042-resistant Plasmodium falciparum (Pf) parasites revealed point mutations in the Pf_proteasome ß5 active-site (Pfß5). A potent analog (SW584) showed efficacy in a mouse model of human malaria after oral dosing. SW584 had a low propensity to generate resistance (minimum inoculum for resistance [MIR] >109) and was synergistic with dihydroartemisinin. Pf_proteasome purification was facilitated by His8-tag introduction onto ß7. Inhibition of Pfß5 correlated with parasite killing, without inhibiting human proteasome isoforms or showing cytotoxicity. The Pf_proteasome_SW584 cryoelectron microscopy (cryo-EM) structure showed that SW584 bound non-covalently distal from the catalytic threonine, in an unexplored pocket at the ß5/ß6/ß3 subunit interface that has species differences between Pf and human proteasomes. Identification of a reversible, species selective, orally active series with low resistance propensity provides a path for drugging this essential target.
Assuntos
Antimaláricos , Piperidinas , Plasmodium falciparum , Inibidores de Proteassoma , Piperidinas/química , Piperidinas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Animais , Antimaláricos/farmacologia , Antimaláricos/química , Humanos , Camundongos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/síntese química , Administração Oral , Complexo de Endopeptidases do Proteassoma/metabolismo , Malária/tratamento farmacológico , Malária/parasitologia , Amidas/química , Amidas/farmacologia , Amidas/síntese química , Malária Falciparum/tratamento farmacológico , Feminino , Estrutura MolecularRESUMO
Current malaria treatments are threatened by drug resistance, and new drugs are urgently needed. In a phenotypic screen for new antimalarials, we identified (S)-SW228703 ((S)-SW703), a tyrosine amide with asexual blood and liver stage activity and a fast-killing profile. Resistance to (S)-SW703 is associated with mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) and P. falciparum acetyl CoA transporter (PfACT), similarly to several other compounds that share features such as fast activity and liver-stage activity. Compounds with these resistance mechanisms are thought to act in the ER, though their targets are unknown. The tyramine of (S)-SW703 is shared with some reported PfCARL-associated compounds; however, we observed that strict S-stereochemistry was required for the activity of (S)-SW703, suggesting differences in the mechanism of action or binding mode. (S)-SW703 provides a new chemical series with broad activity for multiple life-cycle stages and a fast-killing mechanism of action, available for lead optimization to generate new treatments for malaria.
Assuntos
Antimaláricos , Malária Falciparum , Malária , Humanos , Antimaláricos/farmacologia , Antimaláricos/química , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Malária Falciparum/tratamento farmacológico , Malária/tratamento farmacológico , Fígado , Aminas/metabolismoRESUMO
The methylerythritol phosphate (MEP) pathway is an essential metabolic pathway found in malaria parasites, but absent in mammals, making it a highly attractive target for the discovery of novel and selective antimalarial therapies. Using high-throughput screening, we have identified 2-phenyl benzo[d]isothiazol-3(2H)-ones as species-selective inhibitors of Plasmodium spp. 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD), the third catalytic enzyme of the MEP pathway. 2-Phenyl benzo[d]isothiazol-3(2H)-ones display nanomolar inhibitory activity against P. falciparum and P. vivax IspD and prevent the growth of P. falciparum in culture, with EC50 values below 400 nM. In silico modeling, along with enzymatic, genetic and crystallographic studies, have established a mechanism-of-action involving initial non-covalent recognition of inhibitors at the IspD binding site, followed by disulfide bond formation through attack of an active site cysteine residue on the benzo[d]isothiazol-3(2H)-one core. The species-selective inhibitory activity of these small molecules against Plasmodium spp. IspD and cultured parasites suggests they have potential as lead compounds in the pursuit of novel drugs to treat malaria.
Assuntos
Antimaláricos/farmacologia , Benzotiazóis/farmacologia , Colina-Fosfato Citidililtransferase/química , Malária Falciparum/prevenção & controle , Plasmodium falciparum/efeitos dos fármacos , Plasmodium vivax/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Eritritol/análogos & derivados , Eritritol/química , Concentração Inibidora 50 , Proteínas Recombinantes/química , Fosfatos Açúcares/químicaRESUMO
There is a pressing need for new antimicrobial therapies to combat globally important drug-resistant human pathogens, including Plasmodium falciparum malarial parasites, Mycobacterium tuberculosis, and Gram-negative bacteria, including Escherichia coli. These organisms all possess the essential methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, which is not found in humans. The first dedicated enzyme of the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr), is inhibited by the phosphonic acid antibiotic fosmidomycin and its analogs, including the N-acetyl analog FR900098 and the phosphoryl analog fosfoxacin. In order to identify mutations in dxr that confer resistance to these drugs, a library of E. coli dxr mutants was screened at lethal fosmidomycin doses. The most resistant allele (with the S222T mutation) alters the fosmidomycin-binding site of Dxr. The expression of this resistant allele increases bacterial resistance to fosmidomycin and other fosmidomycin analogs by 10-fold. These observations confirm that the primary cellular target of fosmidomycin is Dxr. Furthermore, cell lines expressing Dxr-S222T will be a powerful tool to confirm the mechanisms of action of future fosmidomycin analogs.
Assuntos
Aldose-Cetose Isomerases/genética , Antibacterianos/farmacologia , Fosfomicina/análogos & derivados , Mycobacterium tuberculosis/enzimologia , Pró-Fármacos/farmacologia , Fosfomicina/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genéticaRESUMO
As resistance to current therapies spreads, novel antimalarials are urgently needed. In this work, we examine the potential for therapeutic intervention via the targeting of Plasmodium IspD (2-C-methyl-D-erythritol 4-phosphate cytidyltransferase), the second dedicated enzyme of the essential methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis. Enzymes of this pathway represent promising therapeutic targets because the pathway is not present in humans. The Malaria Box compound, MMV008138, inhibits Plasmodium falciparum growth, and PfIspD has been proposed as a candidate intracellular target. We find that PfIspD is the sole intracellular target of MMV008138 and characterize the mode of inhibition and target-based resistance, providing chemical validation of this target. Additionally, we find that the Pf ISPD genetic locus is refractory to disruption in malaria parasites, providing independent genetic validation for efforts targeting this enzyme. This work provides compelling support for IspD as a druggable target for the development of additional, much-needed antimalarial agents.