RESUMO
To contribute to the global effort to develop new antimalarial therapies, we previously disclosed initial findings on the optimization of the dihydroquinazolinone-3-carboxamide class that targets PfATP4. Here we report on refining the aqueous solubility and metabolic stability to improve the pharmacokinetic profile and consequently in vivo efficacy. We show that the incorporation of heterocycle systems in the 8-position of the scaffold was found to provide the greatest attainable balance between parasite activity, aqueous solubility, and metabolic stability. Optimized analogs, including the frontrunner compound S-WJM992, were shown to inhibit PfATP4-associated Na+-ATPase activity, gave rise to a metabolic signature consistent with PfATP4 inhibition, and displayed altered activities against parasites with mutations in PfATP4. Finally, S-WJM992 showed appreciable efficacy in a malaria mouse model and blocked gamete development preventing transmission to mosquitoes. Importantly, further optimization of the dihydroquinazolinone class is required to deliver a candidate with improved pharmacokinetic and risk of resistance profiles.
Assuntos
Antimaláricos , Plasmodium falciparum , Quinazolinonas , Antimaláricos/farmacologia , Antimaláricos/química , Antimaláricos/farmacocinética , Animais , Plasmodium falciparum/efeitos dos fármacos , Quinazolinonas/farmacologia , Quinazolinonas/química , Quinazolinonas/farmacocinética , Camundongos , Administração Oral , Relação Estrutura-Atividade , Humanos , Malária/tratamento farmacológico , Feminino , SolubilidadeRESUMO
New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.
Assuntos
Antimaláricos , Plasmodium falciparum , Plasmodium vivax , Proteômica , Proteínas de Protozoários , Antimaláricos/farmacologia , Antimaláricos/química , Plasmodium falciparum/enzimologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium vivax/enzimologia , Plasmodium vivax/efeitos dos fármacos , Humanos , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteômica/métodos , Aminopeptidases/metabolismo , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/químicaRESUMO
New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum ( Pf A-M1) and Plasmodium vivax ( Pv A-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets Pf A-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on Pf A-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of Pf A-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.
RESUMO
To combat the global burden of malaria, development of new drugs to replace or complement current therapies is urgently required. Here, we show that the compound MMV1557817 is a selective, nanomolar inhibitor of both Plasmodium falciparum and Plasmodium vivax aminopeptidases M1 and M17, leading to inhibition of end-stage hemoglobin digestion in asexual parasites. MMV1557817 can kill sexual-stage P. falciparum, is active against murine malaria, and does not show any shift in activity against a panel of parasites resistant to other antimalarials. MMV1557817-resistant P. falciparum exhibited a slow growth rate that was quickly outcompeted by wild-type parasites and were sensitized to the current clinical drug, artemisinin. Overall, these results confirm MMV1557817 as a lead compound for further drug development and highlights the potential of dual inhibition of M1 and M17 as an effective multi-species drug-targeting strategy.IMPORTANCEEach year, malaria infects approximately 240 million people and causes over 600,000 deaths, mostly in children under 5 years of age. For the past decade, artemisinin-based combination therapies have been recommended by the World Health Organization as the standard malaria treatment worldwide. Their widespread use has led to the development of artemisinin resistance in the form of delayed parasite clearance, alongside the rise of partner drug resistance. There is an urgent need to develop and deploy new antimalarial agents with novel targets and mechanisms of action. Here, we report a new and potent antimalarial compound, known as MMV1557817, and show that it targets multiple stages of the malaria parasite lifecycle, is active in a preliminary mouse malaria model, and has a novel mechanism of action. Excitingly, resistance to MMV15578117 appears to be self-limiting, suggesting that development of the compound may provide a new class of antimalarial.
Assuntos
Aminopeptidases , Antimaláricos , Plasmodium falciparum , Plasmodium vivax , Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Animais , Camundongos , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/enzimologia , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/metabolismo , Resistência a Medicamentos , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , FemininoRESUMO
Over the last two decades, global malaria cases caused by Plasmodium falciparum have declined due to the implementation of effective treatments and the use of insecticides. However, the COVID-19 pandemic caused major disruption in the timely delivery of medical goods and diverted public health resources, impairing malaria control. The emergence of resistance to all existing frontline antimalarials underpins an urgent need for new antimalarials with novel mechanisms of action. Furthermore, the need to reduce malaria transmission and/or prevent malaria infection has shifted the focus of antimalarial research towards the discovery of compounds that act beyond the symptomatic blood stage and also impact other parasite life cycle stages. Phenotypic screening has been responsible for the majority of new antimalarial lead compounds discovered over the past 10 years. This review describes recently reported novel antimalarial hits that target multiple parasite stages and were discovered by phenotypic screening during the COVID-19 pandemic. Their modes of action and targets in blood stage parasites are also discussed.
Assuntos
Antimaláricos , COVID-19 , Malária , Humanos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Pandemias , Malária/tratamento farmacológico , Malária/prevenção & controle , Plasmodium falciparumRESUMO
Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.
Assuntos
Antimaláricos , Malária Falciparum , Terapia de Alvo Molecular , Plasmodium falciparum , Biossíntese de Proteínas , Proteínas de Protozoários , Tirosina-tRNA Ligase , Adenosina/análogos & derivados , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Cristalografia por Raios X , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Camundongos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Ácidos Sulfônicos/química , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/metabolismoRESUMO
Current best practice for the treatment of malaria relies on short half-life artemisinins that are failing against emerging Kelch 13 mutant parasite strains. Here, we introduce a liposome-like self-assembly of a dimeric artesunate glycerophosphocholine conjugate (dAPC-S) as an amphiphilic prodrug for the short-lived antimalarial drug, dihydroartemisinin (DHA), with enhanced killing of Kelch 13 mutant artemisinin-resistant parasites. Cryo-electron microscopy (cryoEM) images and the dynamic light scattering (DLS) technique show that dAPC-S typically exhibits a multilamellar liposomal structure with a size distribution similar to that of the liposomes generated using thin-film dispersion (dAPC-L). Liquid chromatography-mass spectrometry (LCMS) was used to monitor the release of DHA. Sustainable release of DHA from dAPC-S and dAPC-L assemblies increased the effective dose and thus efficacy against Kelch 13 mutant artemisinin-resistant parasites in an in vitro assay. To better understand the enhanced killing effect, we investigated processes for deactivation of both the assemblies and DHA, including the roles of serum components and trace levels of iron. Analysis of parasite proteostasis pathways revealed that dAPC assemblies exert their activity via the same mechanism as DHA. We conclude that this easily prepared multilamellar liposome-like dAPC-S with long-acting efficacy shows potential for the treatment of severe and artemisinin-resistant malaria.
Assuntos
Antimaláricos , Artemisininas , Malária Falciparum , Malária , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Artemisininas/farmacologia , Artemisininas/uso terapêutico , Artesunato/farmacologia , Artesunato/uso terapêutico , Microscopia Crioeletrônica , Resistência a Medicamentos/genética , Humanos , Lipossomos/química , Malária/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Plasmodium falciparum/genéticaRESUMO
Plasmodium falciparum causes the most lethal form of malaria. Peroxide antimalarials based on artemisinin underpin the frontline treatments for malaria, but artemisinin resistance is rapidly spreading. Synthetic peroxide antimalarials, known as ozonides, are in clinical development and offer a potential alternative. Here, we used chemoproteomics to investigate the protein alkylation targets of artemisinin and ozonide probes, including an analogue of the ozonide clinical candidate, artefenomel. We greatly expanded the list of proteins alkylated by peroxide antimalarials and identified significant enrichment of redox-related proteins for both artemisinins and ozonides. Disrupted redox homeostasis was confirmed by dynamic live imaging of the glutathione redox potential using a genetically encoded redox-sensitive fluorescence-based biosensor. Targeted liquid chromatography-mass spectrometry (LC-MS)-based thiol metabolomics also confirmed changes in cellular thiol levels. This work shows that peroxide antimalarials disproportionately alkylate proteins involved in redox homeostasis and that disrupted redox processes are involved in the mechanism of action of these important antimalarials.
Assuntos
Antimaláricos , Antimaláricos/farmacologia , Eritrócitos , Homeostase , Oxirredução , Peróxidos , Plasmodium falciparumRESUMO
Ozonide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel), are synthetic peroxide-based antimalarials with potent activity against the deadliest malaria parasite, Plasmodium falciparum. Here we used a "multi-omics" workflow, in combination with activity-based protein profiling (ABPP), to demonstrate that peroxide antimalarials initially target the haemoglobin (Hb) digestion pathway to kill malaria parasites. Time-dependent metabolomic profiling of ozonide-treated P. falciparum infected red blood cells revealed a rapid depletion of short Hb-derived peptides followed by subsequent alterations in lipid and nucleotide metabolism, while untargeted peptidomics showed accumulation of longer Hb-derived peptides. Quantitative proteomics and ABPP assays demonstrated that Hb-digesting proteases were increased in abundance and activity following treatment, respectively. Ozonide-induced depletion of short Hb-derived peptides was less extensive in a drug-treated K13-mutant artemisinin resistant parasite line (Cam3.IIR539T) than in the drug-treated isogenic sensitive strain (Cam3.IIrev), further confirming the association between ozonide activity and Hb catabolism. To demonstrate that compromised Hb catabolism may be a primary mechanism involved in ozonide antimalarial activity, we showed that parasites forced to rely solely on Hb digestion for amino acids became hypersensitive to short ozonide exposures. Quantitative proteomics analysis also revealed parasite proteins involved in translation and the ubiquitin-proteasome system were enriched following drug treatment, suggestive of the parasite engaging a stress response to mitigate ozonide-induced damage. Taken together, these data point to a mechanism of action involving initial impairment of Hb catabolism, and indicate that the parasite regulates protein turnover to manage ozonide-induced damage.
Assuntos
Adamantano/análogos & derivados , Antimaláricos/farmacologia , Eritrócitos , Hemoglobinas/metabolismo , Compostos Heterocíclicos com 1 Anel/farmacologia , Peróxidos/farmacologia , Plasmodium falciparum/metabolismo , Compostos de Espiro/farmacologia , Adamantano/farmacologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Hemoglobinas/genética , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Plasmodium falciparum/genética , ProteômicaRESUMO
The mechanism of action of ozonide antimalarials involves activation by intraparasitic iron and the formation of highly reactive carbon-centered radicals that alkylate malaria parasite proteins. Given free intraparasitic heme is generally thought to be the iron source responsible for ozonide activation and its likely close proximity to the activated drug, we investigated heme as a possible molecular target of the ozonides. Using an extraction method optimized for solubilization of free heme, untargeted LC-MS analysis of ozonide-treated parasites identified several regioisomers of ozonide-alkylated heme, which resulted from covalent modification of the heme porphyrin ring by an ozonide-derived carbon-centered radical. In addition to the intact alkylated heme adduct, putative ozonide-alkylated heme degradation products were also detected. This study directly demonstrates ozonide modification of heme within the malaria parasite Plasmodium falciparum, revealing that this process may be important for the biological activity of ozonide antimalarials.
Assuntos
Antimaláricos/farmacologia , Heme/química , Compostos Heterocíclicos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Alquilação , Cromatografia Líquida , Compostos Heterocíclicos/química , Espectrometria de Massas , Estrutura Molecular , Plasmodium falciparum/químicaRESUMO
The ozonides are one of the most advanced drug classes in the antimalarial development pipeline and were designed to improve on limitations associated with current front-line artemisinin-based therapies. Like the artemisinins, the pharmacophoric peroxide bond of ozonides is essential for activity, and it appears that these antimalarials share a similar mode of action, raising the possibility of cross-resistance. Resistance to artemisinins is associated with Plasmodium falciparum mutations that allow resistant parasites to escape short-term artemisinin-mediated damage (elimination half-life ~1 h). Importantly, some ozonides (e.g., OZ439) have a sustained in vivo drug exposure profile, providing a major pharmacokinetic advantage over the artemisinin derivatives. Here, we describe recent progress made towards understanding ozonide antimalarial activity and discuss ozonide utility within the context of artemisinin resistance.
Assuntos
Artemisininas/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos/uso terapêutico , Malária/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Resistência a Medicamentos/genética , Humanos , Plasmodium falciparum/genéticaRESUMO
The peroxide bond of the artemisinins inspired the development of a class of fully synthetic 1,2,4-trioxolane-based antimalarials, collectively known as the ozonides. Similar to the artemisinins, heme-mediated degradation of the ozonides generates highly reactive radical species that are thought to mediate parasite killing by damaging critical parasite biomolecules. We examined the relationship between parasite dependent degradation and antimalarial activity for two ozonides, OZ277 (arterolane) and OZ439 (artefenomel), using a combination of in vitro drug stability and pulsed-exposure activity assays. Our results showed that drug degradation is parasite stage dependent and positively correlates with parasite load. Increasing trophozoite-stage parasitemia leads to substantially higher rates of degradation for both OZ277 and OZ439, and this is associated with a reduction in in vitro antimalarial activity. Under conditions of very high parasitemia (â¼90%), OZ277 and OZ439 were rapidly degraded and completely devoid of activity in trophozoite-stage parasite cultures exposed to a 3-h drug pulse. This study highlights the impact of increasing parasite load on ozonide stability and in vitro antimalarial activity and should be considered when investigating the antimalarial mode of action of the ozonide antimalarials under conditions of high parasitemia.
Assuntos
Antimaláricos/farmacologia , Compostos Heterocíclicos/farmacologia , Adamantano/análogos & derivados , Adamantano/farmacologia , Antimaláricos/química , Eritrócitos/parasitologia , Compostos Heterocíclicos com 1 Anel/farmacologia , Humanos , Peróxidos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Compostos de Espiro/farmacologia , Trofozoítos/químicaRESUMO
Fully synthetic endoperoxide antimalarials, namely, OZ277 (RBx11160; also known as arterolane) and OZ439 (artefenomel), have been approved for marketing or are currently in clinical development. We undertook an analysis of the kinetics of the in vitro responses of Plasmodium falciparum to the new ozonide antimalarials. For these studies we used a K13 mutant (artemisinin resistant) isolate from a region in Cambodia and a genetically matched (artemisinin sensitive) K13 revertant. We used a pulsed-exposure assay format to interrogate the time dependence of the response. Because the ozonides have physicochemical properties different from those of the artemisinins, assay optimization was required to ensure that the drugs were completely removed following the pulsed exposure. Like that of artemisinins, ozonide activity requires active hemoglobin degradation. Short pulses of the ozonides were less effective than short pulses of dihydroartemisinin; however, when early-ring-stage parasites were exposed to drugs for periods relevant to their in vivo exposure, the ozonide antimalarials were markedly more effective.