RESUMEN

In the absence of an efficacious vaccine, chemotherapy remains crucial to prevent and treat malaria. Given its key role in haemoglobin degradation, falcilysin constitutes an attractive target. Here, we reveal the mechanism of enzymatic inhibition of falcilysin by MK-4815, an investigational new drug with potent antimalarial activity. Using X-ray crystallography, we determine two binary complexes of falcilysin in a closed state, bound with peptide substrates from the haemoglobin α and ß chains respectively. An antiparallel ß-sheet is formed between the substrate and enzyme, accounting for sequence-independent recognition at positions P2 and P1. In contrast, numerous contacts favor tyrosine and phenylalanine at the P1' position of the substrate. Cryo-EM studies reveal a majority of unbound falcilysin molecules adopting an open conformation. Addition of MK-4815 shifts about two-thirds of falcilysin molecules to a closed state. These structures give atomic level pictures of the proteolytic cycle, in which falcilysin interconverts between a closed state conducive to proteolysis, and an open conformation amenable to substrate diffusion and products release. MK-4815 and quinolines bind to an allosteric pocket next to a hinge region of falcilysin and hinders this dynamic transition. These data should inform the design of potent inhibitors of falcilysin to combat malaria.

Asunto(s)

Antimaláricos , Plasmodium falciparum , Plasmodium falciparum/enzimología , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/farmacología , Antimaláricos/química , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/antagonistas & inhibidores , Cristalografía por Rayos X , Modelos Moleculares , Microscopía por Crioelectrón , Humanos

RESUMEN

Plasmodium falciparum is the main causative agent of malaria, a deadly disease that mainly affects children under five years old. Artemisinin-based combination therapies have been pivotal in controlling the disease, but resistance has arisen in various regions, increasing the risk of treatment failure. The non-mevalonate pathway is essential for the isoprenoid synthesis in Plasmodium and provides several under-explored targets to be used in the discovery of new antimalarials. 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) is the first and rate-limiting enzyme of the pathway. Despite its importance, there are no structures available for any Plasmodium spp., due to the complex sequence which contains large regions of high disorder, making crystallisation a difficult task. In this manuscript, we use cryo-electron microscopy to solve the P. falciparum DXPS structure at a final resolution of 2.42 Å. Overall, the structure resembles other DXPS enzymes but includes a distinct N-terminal domain exclusive to the Plasmodium genus. Mutational studies show that destabilization of the cap domain interface negatively impacts protein stability and activity. Additionally, a density for the co-factor thiamine diphosphate is found in the active site. Our work highlights the potential of cryo-EM to obtain structures of P. falciparum proteins that are unfeasible by means of crystallography.

Asunto(s)

Microscopía por Crioelectrón , Plasmodium falciparum , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Pentosiltransferasa/metabolismo , Pentosiltransferasa/química , Pentosiltransferasa/genética , Pentosiltransferasa/ultraestructura , Dominios Proteicos , Modelos Moleculares , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/ultraestructura , Transferasas

RESUMEN

Dynamics of epigenetic modifications such as acetylation and deacetylation of histone proteins have been shown to be crucial for the life cycle development and survival of Plasmodium falciparum, the deadliest malaria parasite. In this study, we present a novel series of peptoid-based histone deacetylase (HDAC) inhibitors incorporating nitrogen-containing bicyclic heteroaryl residues as a new generation of antiplasmodial peptoid-based HDAC inhibitors. We synthesized the HDAC inhibitors by an efficient multicomponent protocol based on the Ugi four-component reaction. The subsequent screening of 16 compounds from our mini-library identified 6i as the most promising candidate, demonstrating potent activity against asexual blood-stage parasites (IC50Pf3D7 = 30 nM; IC50PfDd2 = 98 nM), low submicromolar activity against liver-stage parasites (IC50PbEEF = 0.25 µM), excellent microsomal stability (t1/2 > 60 min), and low cytotoxicity to HEK293 cells (IC50 = 136 µM).

Asunto(s)

Antimaláricos , Inhibidores de Histona Desacetilasas , Peptoides , Plasmodium falciparum , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/síntesis química , Humanos , Antimaláricos/farmacología , Antimaláricos/química , Antimaláricos/síntesis química , Peptoides/farmacología , Peptoides/química , Peptoides/síntesis química , Relación Estructura-Actividad , Células HEK293 , Pruebas de Sensibilidad Parasitaria , Estructura Molecular , Relación Dosis-Respuesta a Droga , Histona Desacetilasas/metabolismo

RESUMEN

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.

Asunto(s)

Antimaláricos , Piperidinas , Plasmodium falciparum , Inhibidores de Proteasoma , Piperidinas/química , Piperidinas/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Animales , Antimaláricos/farmacología , Antimaláricos/química , Humanos , Ratones , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/síntesis química , Administración Oral , Complejo de la Endopetidasa Proteasomal/metabolismo , Malaria/tratamiento farmacológico , Malaria/parasitología , Amidas/química , Amidas/farmacología , Amidas/síntesis química , Malaria Falciparum/tratamiento farmacológico , Femenino , Estructura Molecular

RESUMEN

Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) is essential for egress of invasive merozoite forms of the parasite, rendering PfSUB1 an attractive antimalarial target. Here, we report studies aimed to improve drug-like properties of peptidic boronic acid PfSUB1 inhibitors including increased lipophilicity and selectivity over human proteasome (H20S). Structure-activity relationship investigations revealed that lipophilic P3 amino acid side chains as well as N-capping groups were well tolerated in retaining PfSUB1 inhibitory potency. At the P1 position, replacing the methyl group with a carboxyethyl substituent led to boralactone PfSUB1 inhibitors with remarkably improved selectivity over H20S. Combining lipophilic end-capping groups with the boralactone reduced the selectivity over H20S. However, compound 4c still showed >60-fold selectivity versus H20S and low nanomolar PfSUB1 inhibitory potency. Importantly, this compound inhibited the growth of a genetically modified P. falciparum line expressing reduced levels of PfSUB1 13-fold more efficiently compared to a wild-type parasite line.

Asunto(s)

Antimaláricos , Ácidos Borónicos , Plasmodium falciparum , Complejo de la Endopetidasa Proteasomal , Proteínas Protozoarias , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Humanos , Relación Estructura-Actividad , Ácidos Borónicos/química , Ácidos Borónicos/farmacología , Ácidos Borónicos/síntesis química , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Antimaláricos/farmacología , Antimaláricos/química , Antimaláricos/síntesis química , Péptidos/química , Péptidos/farmacología , Péptidos/síntesis química , Subtilisinas

RESUMEN

PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 µM and 7.5 µM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 µM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.

Asunto(s)

Insulisina , Plasmodium falciparum , Proteínas Protozoarias , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Insulisina/genética , Insulisina/química , Insulisina/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Especificidad por Sustrato , Insulina/química , Insulina/metabolismo , Insulina/genética , Zinc/química , Zinc/metabolismo , Genoma de Protozoos , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Expresión Génica , Clonación Molecular , Antimaláricos/química , Antimaláricos/farmacología , Ciclosporina/química , Ciclosporina/farmacología

RESUMEN

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.

Asunto(s)

Antimaláricos , Plasmodium falciparum , Plasmodium vivax , Proteómica , Proteínas Protozoarias , Antimaláricos/farmacología , Antimaláricos/química , Plasmodium falciparum/enzimología , Plasmodium falciparum/efectos de los fármacos , Plasmodium vivax/enzimología , Plasmodium vivax/efectos de los fármacos , Humanos , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteómica/métodos , Aminopeptidasas/metabolismo , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/química

RESUMEN

BACKGROUND: The malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31. METHODS: Here we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX. RESULTS: We show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity. CONCLUSIONS: Our results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity. GENERAL SIGNIFICANCE: Our findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress.

Asunto(s)

Ácido Aspártico Endopeptidasas , Plasmodium falciparum , Proteínas Protozoarias , Plasmodium falciparum/enzimología , Plasmodium falciparum/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Ácido Aspártico Endopeptidasas/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/química , Proteolisis , Humanos , Subtilisinas/metabolismo , Dominio Catalítico , Dominios Proteicos , Malaria Falciparum/parasitología , Malaria Falciparum/metabolismo , Eritrocitos/parasitología , Eritrocitos/metabolismo

RESUMEN

The riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin utilization. To determine their mechanism of action, we generated roseoflavin-resistant parasites by in vitro evolution. Relative to wild-type, these parasites were 4-fold resistant to roseoflavin and cross-resistant to 8-aminoriboflavin. Whole genome sequencing of the resistant parasites revealed a missense mutation leading to an amino acid change (L672H) in the gene coding for a putative flavokinase (PfFK), the enzyme responsible for converting riboflavin into the cofactor flavin mononucleotide (FMN). To confirm that the L672H mutation is responsible for the phenotype, we generated parasites with the missense mutation incorporated into the PfFK gene. The IC50 values for roseoflavin and 8-aminoriboflavin against the roseoflavin-resistant parasites created through in vitro evolution were indistinguishable from those against parasites in which the missense mutation was introduced into the native PfFK. We also generated two parasite lines episomally expressing GFP-tagged versions of either the wild-type or mutant forms of PfFK. We found that PfFK-GFP localizes to the parasite cytosol and that immunopurified PfFK-GFP phosphorylated riboflavin, roseoflavin, and 8-aminoriboflavin. The L672H mutation increased the KM for roseoflavin, explaining the resistance phenotype. Mutant PfFK is no longer capable of phosphorylating 8-aminoriboflavin, but its antiplasmodial activity against resistant parasites can still be antagonized by increasing the extracellular concentration of riboflavin, consistent with it also inhibiting parasite growth through competitive inhibition of PfFK. Our findings, therefore, are consistent with roseoflavin and 8-aminoriboflavin inhibiting parasite proliferation by inhibiting riboflavin phosphorylation and via the generation of toxic flavin cofactor analogues.

Asunto(s)

Antimaláricos , Resistencia a Medicamentos , Fosfotransferasas (Aceptor de Grupo Alcohol) , Plasmodium falciparum , Riboflavina , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/enzimología , Riboflavina/farmacología , Riboflavina/análogos & derivados , Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Mutación Missense , Humanos , Malaria Falciparum/parasitología , Mutación

RESUMEN

Aminoacyl-tRNA synthetases (aaRSs) play a central role in the translation of genetic code, serving as attractive drug targets. Within this family, the lysyl-tRNA synthetase (LysRS) constitutes a promising antimalarial target. ASP3026, an anaplastic lymphoma kinase (ALK) inhibitor was recently identified as a novel Plasmodium falciparum LysRS (PfLysRS) inhibitor. Here, based on cocrystal structures and biochemical experiments, we developed a series of ASP3026 analogues to improve the selectivity and potency of LysRS inhibition. The leading compound 36 showed a dissociation constant of 15.9 nM with PfLysRS. The inhibitory efficacy on PfLysRS and parasites has been enhanced. Covalent attachment of L-lysine to compound 36 resulted in compound 36K3, which exhibited further increased inhibitory activity against PfLysRS but significantly decreased activity against ALK. However, its inhibitory activity against parasites did not improve, suggesting potential future optimization directions. This study presents a new example of derivatization of kinase inhibitors repurposed to inhibit aaRS.

Asunto(s)

Quinasa de Linfoma Anaplásico , Antimaláricos , Lisina-ARNt Ligasa , Plasmodium falciparum , Inhibidores de Proteínas Quinasas , Plasmodium falciparum/enzimología , Plasmodium falciparum/efectos de los fármacos , Lisina-ARNt Ligasa/antagonistas & inhibidores , Lisina-ARNt Ligasa/metabolismo , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/metabolismo , Quinasa de Linfoma Anaplásico/genética , Antimaláricos/farmacología , Antimaláricos/química , Relación Estructura-Actividad , Humanos , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética

RESUMEN

Chemoenzymatic synthesis of non-natural terpenes using the promiscuous activity of terpene synthases allows for the expansion of the chemical space of terpenoids with potentially new bioactivities. In this report, we describe protocols for the preparation of a novel aphid attractant, (S)-14,15-dimethylgermacrene D, by exploiting the promiscuity of (S)-germacrene D synthase from Solidago canadensis and using an engineered biocatalytic route to convert prenols to terpenoids. The method uses a combination of five enzymes to carry out the preparation of terpenoid semiochemicals in two steps: (1) diphosphorylation of five or six carbon precursors (prenol, isoprenol and methyl-isoprenol) catalyzed by Plasmodium falciparum choline kinase and Methanocaldococcus jannaschii isopentenyl phosphate kinase to form DMADP, IDP and methyl-IDP, and (2) chain elongation and cyclization catalyzed by Geobacillus stearothermophilus (2E,6E)-farnesyl diphosphate synthase and S. canadensis (S)-germacrene D synthase to produce (S)-germacrene D and (S)-14,15-dimethylgermacrene D. Using this method, new non-natural terpenoids are readily accessible and the approach can be adopted to produce different terpene analogs and terpenoid derivatives with potential novel applications.

Asunto(s)

Transferasas Alquil y Aril , Terpenos , Terpenos/metabolismo , Terpenos/química , Transferasas Alquil y Aril/metabolismo , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Plasmodium falciparum/enzimología , Animales , Biocatálisis , Especificidad por Sustrato , Áfidos/enzimología

RESUMEN

While often undetected and untreated, persistent seasonal asymptomatic malaria infections remain a global public health problem. Despite the presence of parasites in the peripheral blood, no symptoms develop. Disease severity is correlated with the levels of infected red blood cells (iRBCs) adhering within blood vessels. Changes in iRBC adhesion capacity have been linked to seasonal asymptomatic malaria infections, however how this is occurring is still unknown. Here, we present evidence that RNA polymerase III (RNA Pol III) transcription in Plasmodium falciparum is downregulated in field isolates obtained from asymptomatic individuals during the dry season. Through experiments with in vitro cultured parasites, we have uncovered an RNA Pol III-dependent mechanism that controls pathogen proliferation and expression of a major virulence factor in response to external stimuli. Our findings establish a connection between P. falciparum cytoadhesion and a non-coding RNA family transcribed by Pol III. Additionally, we have identified P. falciparum Maf1 as a pivotal regulator of Pol III transcription, both for maintaining cellular homeostasis and for responding adaptively to external signals. These results introduce a novel perspective that contributes to our understanding of P. falciparum virulence. Furthermore, they establish a connection between this regulatory process and the occurrence of seasonal asymptomatic malaria infections.

Asunto(s)

Malaria Falciparum , Plasmodium falciparum , ARN Polimerasa III , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidad , Plasmodium falciparum/enzimología , Virulencia , ARN Polimerasa III/metabolismo , ARN Polimerasa III/genética , Humanos , Malaria Falciparum/parasitología , Eritrocitos/parasitología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Adhesión Celular , Regulación de la Expresión Génica

RESUMEN

The human malaria parasite Plasmodium falciparum (P. falciparum) continues to pose a significant public health challenge, leading to millions of fatalities globally. Halofuginone (HF) has shown a significant anti-P. falciparum effect, suggesting its potential as a therapeutic agent for malaria treatment. In this study, we synthesized a photoaffinity labeling probe of HF to identify its direct target in P. falciparum. Our results reveal that ubiquitin carboxyl-terminal hydrolase 3 (PfUCHL3) acts as a crucial target protein of HF, which modulates parasite growth in the intraerythrocytic cycle. In particular, we discovered that HF potentially forms hydrogen bonds with the Leu10, Glu11, and Arg217 sites of PfUCHL3, thereby inducing an allosteric effect by promoting the embedding of the helix 6' region on the protein surface. Furthermore, HF disrupts the expression of multiple functional proteins mediated by PfUCHL3, specifically those that play crucial roles in amino acid biosynthesis and metabolism in P. falciparum. Taken together, this study highlights PfUCHL3 as a previously undisclosed druggable target of HF, which contributes to the development of novel anti-malarial agents in the future.

Asunto(s)

Antimaláricos , Piperidinas , Plasmodium falciparum , Quinazolinonas , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Quinazolinonas/química , Quinazolinonas/farmacología , Quinazolinonas/metabolismo , Piperidinas/química , Piperidinas/farmacología , Piperidinas/metabolismo , Antimaláricos/farmacología , Antimaláricos/química , Humanos , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Proteómica

RESUMEN

The human malaria parasite Plasmodium falciparum genome is among the most A + T rich, with low complexity regions (LCRs) inserted in coding sequences including those for proteins targeted to its essential relict plastid (apicoplast). Replication of the apicoplast genome (plDNA), mediated by the atypical multifunctional DNA polymerase PfPrex, would require additional enzymatic functions for lagging strand processing. We identified an apicoplast-targeted, [4Fe-4S]-containing, FEN/Exo (PfExo) with a long LCR insertion and detected its interaction with PfPrex. Distinct from other known exonucleases across organisms, PfExo recognized a wide substrate range; it hydrolyzed 5'-flaps, processed dsDNA as a 5'-3' exonuclease, and was a bipolar nuclease on ssDNA and RNA-DNA hybrids. Comparison with the rodent P. berghei ortholog PbExo, which lacked the insertion and [4Fe-4S], revealed interspecies functional differences. The insertion-deleted PfExoΔins behaved like PbExo with a limited substrate repertoire because of compromised DNA binding. Introduction of the PfExo insertion into PbExo led to gain of activities that the latter initially lacked. Knockout of PbExo indicated essentiality of the enzyme for survival. Our results demonstrate the presence of a novel apicoplast exonuclease with a functional LCR that diversifies substrate recognition, and identify it as the candidate flap-endonuclease and RNaseH required for plDNA replication and maintenance.

Asunto(s)

Apicoplastos , Plasmodium falciparum , Apicoplastos/metabolismo , Apicoplastos/genética , Plasmodium falciparum/genética , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/química , Exonucleasas/metabolismo , Exonucleasas/genética , Replicación del ADN , Animales , Mutagénesis Insercional , Especificidad de la Especie , Humanos , ADN/metabolismo , ADN/química

RESUMEN

Apicomplexan parasites are the primary causative agents of many human diseases, including malaria, toxoplasmosis, and cryptosporidiosis. These opportunistic pathogens undergo complex life cycles with multiple developmental stages, wherein many key steps are regulated by phosphorylation mechanisms. The genomes of apicomplexan pathogens contain protein kinases from different groups including tyrosine kinase-like (TKL) family proteins. Although information on the role of TKL kinases in apicomplexans is quite limited, recent studies have revealed the important role of this family of proteins in apicomplexan biology. TKL kinases in these protozoan pathogens show unique organization with many novel domains thus making them attractive candidates for drug development. In this mini review, we summarize the current understanding of the role of TKL kinases in human apicomplexan pathogens' (Toxoplasma gondii, Plasmodium falciparum and Cryptosporidium parvum) biology and pathogenesis.

Asunto(s)

Apicomplexa , Cryptosporidium parvum , Plasmodium falciparum , Proteínas Protozoarias , Toxoplasma , Humanos , Toxoplasma/enzimología , Toxoplasma/genética , Cryptosporidium parvum/enzimología , Cryptosporidium parvum/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Apicomplexa/enzimología , Apicomplexa/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/química , Fosforilación

RESUMEN

Plasmodium falciparum aminoacyl tRNA synthetases (PfaaRSs) are potent antimalarial targets essential for proteome fidelity and overall parasite survival in every stage of the parasite's life cycle. So far, some of these proteins have been singly targeted yielding inhibitor compounds that have been limited by incidences of resistance which can be overcome via pan-inhibition strategies. Hence, herein, for the first time, we report the identification and in vitro antiplasmodial validation of Mitomycin (MMC) as a probable pan-inhibitor of class 1a (arginyl(A)-, cysteinyl(C), isoleucyl(I)-, leucyl(L), methionyl(M), and valyl(V)-) PfaaRSs which hypothetically may underlie its previously reported activity on the ribosomal RNA to inhibit protein translation and biosynthesis. We combined multiple in silico structure-based discovery strategies that first helped identify functional and druggable sites that were preferentially targeted by the compound in each of the plasmodial proteins: Ins1-Ins2 domain in Pf-ARS; anticodon binding domain in Pf-CRS; CP1-editing domain in Pf-IRS and Pf-MRS; C-terminal domain in Pf-LRS; and CP-core region in Pf-VRS. Molecular dynamics studies further revealed that MMC allosterically induced changes in the global structures of each protein. Likewise, prominent structural perturbations were caused by the compound across the functional domains of the proteins. More so, MMC induced systematic alterations in the binding of the catalytic nucleotide and amino acid substrates which culminated in the loss of key interactions with key active site residues and ultimate reduction in the nucleotide-binding affinities across all proteins, as deduced from the binding energy calculations. These altogether confirmed that MMC uniformly disrupted the structure of the target proteins and essential substrates. Further, MMC demonstrated IC50 < 5 µM against the Dd2 and 3D7 strains of parasite making it a good starting point for malarial drug development. We believe that findings from our study will be important in the current search for highly effective multi-stage antimalarial drugs.

Asunto(s)

Aminoacil-ARNt Sintetasas , Antimaláricos , Reposicionamiento de Medicamentos , Mitomicina , Plasmodium falciparum , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Aminoacil-ARNt Sintetasas/genética , Antimaláricos/farmacología , Antimaláricos/química , Mitomicina/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Simulación del Acoplamiento Molecular

RESUMEN

Target-based approaches have traditionally been used in the search for new anti-infective molecules. Target selection process, a critical step in Drug Discovery, identifies targets that are essential to establish or maintain the infection, tractable to be susceptible for inhibition, selective towards their human ortholog and amenable for large scale purification and high throughput screening. The work presented herein validates the Plasmodium falciparum mRNA 5' triphosphatase (PfPRT1), the first enzymatic step to cap parasite nuclear mRNAs, as a candidate target for the development of new antimalarial compounds. mRNA capping is essential to maintain the integrity and stability of the messengers, allowing their translation. PfPRT1 has been identified as a member of the tunnel, metal dependent mRNA 5' triphosphatase family which differs structurally and mechanistically from human metal independent mRNA 5' triphosphatase. In the present study the essentiality of PfPRT1 was confirmed and molecular biology tools and methods for target purification, enzymatic assessment and target engagement were developed, with the goal of running a future high throughput screening to discover PfPRT1 inhibitors.

Asunto(s)

Antimaláricos , Descubrimiento de Drogas , Plasmodium falciparum , Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/enzimología , Descubrimiento de Drogas/métodos , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos , ARN Mensajero/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , Inhibidores Enzimáticos/farmacología , Ácido Anhídrido Hidrolasas

RESUMEN

Malaria is a severe disease that presents a significant threat to human health. As resistance to current drugs continues to increase, there is an urgent need for new antimalarial medications. Aminoacyl-tRNA synthetases (aaRSs) represent promising targets for drug development. In this study, we identified Plasmodium falciparum tyrosyl-tRNA synthetase (PfTyrRS) as a potential target for antimalarial drug development through a comparative analysis of the amino acid sequences and three-dimensional structures of human and plasmodium TyrRS, with particular emphasis on differences in key amino acids at the aminoacylation site. A total of 2141 bioactive compounds were screened using a high-throughput thermal shift assay (TSA). Okanin, known as an inhibitor of LPS-induced TLR4 expression, exhibited potent inhibitory activity against PfTyrRS, while showing limited inhibition of human TyrRS. Furthermore, bio-layer interferometry (BLI) confirmed the high affinity of okanin for PfTyrRS. Molecular dynamics (MD) simulations highlighted the stable conformation of okanin within PfTyrRS and its sustained binding to the enzyme. A molecular docking analysis revealed that okanin binds to both the tyrosine and partial ATP binding sites of the enzyme, preventing substrate binding. In addition, the compound inhibited the production of Plasmodium falciparum in the blood stage and had little cytotoxicity. Thus, okanin is a promising lead compound for the treatment of malaria caused by P. falciparum.

Asunto(s)

Antimaláricos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Plasmodium falciparum , Tirosina-ARNt Ligasa , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Tirosina-ARNt Ligasa/antagonistas & inhibidores , Tirosina-ARNt Ligasa/metabolismo , Humanos , Antimaláricos/farmacología , Antimaláricos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Sitios de Unión , Unión Proteica , Animales , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología

RESUMEN

Transketolases (TKs) are key enzymes of the pentose phosphate pathway, regulating several other critical pathways in cells. Considering their metabolic importance, TKs are expected to be conserved throughout evolution. However, Tittmann et al. (J Biol Chem, 2010, 285(41): 31559-31570) demonstrated that Homo sapiens TK (hsTK) possesses several structural and kinetic differences compared to bacterial TKs. Here, we study 14 TKs from pathogenic bacteria, fungi, and parasites and compare them with hsTK using biochemical, bioinformatic, and structural approaches. For this purpose, six new TK structures are solved by X-ray crystallography, including the TK of Plasmodium falciparum. All of these TKs have the same general fold as bacterial TKs. This comparative study shows that hsTK greatly differs from TKs from pathogens in terms of enzymatic activity, spatial positions of the active site, and monomer-monomer interface residues. An ubiquitous structural pattern is identified in all TKs as a six-residue histidyl crown around the TK cofactor (thiamine pyrophosphate), except for hsTK containing only five residues in the crown. Residue mapping of the monomer-monomer interface and the active site reveals that hsTK contains more unique residues than other TKs. From an evolutionary standpoint, TKs from animals (including H. sapiens) and Schistosoma sp. belong to a distinct structural group from TKs of bacteria, plants, fungi, and parasites, mostly based on a different linker between domains, raising hypotheses regarding evolution and regulation.

Asunto(s)

Evolución Molecular , Transcetolasa , Transcetolasa/metabolismo , Transcetolasa/química , Transcetolasa/genética , Humanos , Cristalografía por Rayos X , Biología Computacional/métodos , Modelos Moleculares , Dominio Catalítico , Plasmodium falciparum/enzimología , Secuencia de Aminoácidos , Conformación Proteica

RESUMEN

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.

Asunto(s)

Péptidos , Plasmodium falciparum , Proteínas Protozoarias , Ubiquitina Tiolesterasa , Plasmodium falciparum/enzimología , Plasmodium falciparum/metabolismo , Plasmodium falciparum/efectos de los fármacos , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/genética , Humanos , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/antagonistas & inhibidores , Antimaláricos/farmacología , Antimaláricos/química , Ubiquitina/metabolismo , Malaria Falciparum/parasitología , Malaria Falciparum/tratamiento farmacológico