RESUMO
BACKGROUND: Malaria remains the world's most important devastating parasitic disease. Of the five species of Plasmodium known to infect and cause human malaria, Plasmodium falciparum is the most virulent and responsible for majority of the deaths caused by this disease. Mainstream drug therapy targets the asexual blood stage of the malaria parasite, as the disease symptoms are mainly associated with this stage. The prevalence of malaria parasite strains resistance to existing anti-malarial drugs has made the control of malaria even more challenging and hence the development of a new class of drugs is inevitable. METHODS: Screening against different drug resistant and sensitive strains of P. falciparum was performed for few bicyclic lactam-based motifs, exhibiting a broad spectrum of activity with low toxicity generated via a focussed library obtained from diversity oriented synthesis (DOS). The synthesis and screening was followed by an in vitro assessment of the possible cytotoxic effect of this class of compounds on malaria parasite. RESULTS: The central scaffold a chiral bicyclic lactam (A) and (A') which were synthesized from (R)-phenylalaninol, levulinic acid and 3-(2-nitrophenyl) levulinic acid respectively. The DOS library was generated from A and from A', by either direct substitution with o-nitrobenzylbromide at the carbon α- to the amide functionality or by conversion to fused pyrroloquinolines. Upon screening this diverse library for their anti-malarial activity, a dinitro/diamine substituted bicyclic lactam was found to demonstrate exceptional activity of >85% inhibition at 50 µM concentration across different strains of P. falciparum with no toxicity against mammalian cells. Also, loss of mitochondrial membrane potential, mitochondrial functionality and apoptosis was observed in parasite treated with diamine-substituted bicyclic lactams. CONCLUSIONS: This study unveils a DOS-mediated exploration of small molecules with novel structural motifs that culminates in identifying a potential lead molecule against malaria. In vitro investigations further reveal their cytocidal effect on malaria parasite growth. It is not the first time that DOS has been used as a strategy to identify therapeutic leads against malaria, but this study establishes the direct implications of DOS in scouting novel motifs with anti-malarial activity.
Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Lactamas/síntese química , Lactamas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Ácidos Levulínicos/síntese química , Ácidos Levulínicos/farmacologia , Testes de Sensibilidade Parasitária , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/fisiologiaRESUMO
Type-2 phosphatidic acid phosphatase (PAP2) a member of PAP2 superfamily mediates the conversion of phosphatidic acid (PA) to diacylglycerol (DAG) and thus plays a pivotal role in numerous cellular signaling processes in diverse organisms. An elevated level of intracellular PA is detrimental for the cell and induces cell death. In this study we identified and characterized a PAP2 homologue in Plasmodium falciparum, PfPAP2 and further elucidated its significance in regulation of PA homeostasis in parasite life cycle. PfPAP2 is expressed in the blood stage and harbors the canonical acid phosphatase domain (APD) with signature motifs. PfPAP2 catalyzes the dephosphorylation of PA to produce DAG and inorganic phosphate (Pi). Propranolol, a generic inhibitor of PAP2, inhibited the phosphatase activity of PfPAP2 by binding to the active site of APD domain as evident by in silico docking and confirmed by surface plasmon resonance (SPR) analysis. Inhibition of native PfPAP2 by propranolol led to rise in intracellular PA mediating disruption of intracellular PA homeostasis in parasites. The propranolol mediated inhibition of PfPAP2 directed early secretion of a micronemal Perforin like Protein, PfPLP1 leading to untimely permeabilization and host cell egress. The merozoites following premature egress were non-invasive and were attenuated to invade erythrocytes and cannot continue next cycle growth. This study demonstrates that disruption of PA homeostasis can cause growth retardation in malaria parasites, and thus its master regulator, PfPAP2, can serve as a very good molecular target for antimalarial chemotherapeutic interventions.
RESUMO
Natural products offer an abundant source of diverse novel scaffolds that inspires development of next generation anti-malarials. With this vision, a library of scaffolds inspired by natural biologically active alkaloids was synthesized from chiral bicyclic lactams with steps/scaffold ratio of 1.7:1. On evaluation of library of scaffolds for their growth inhibitory effect against malaria parasite we found one scaffold with IC50 in low micro molar range. It inhibited parasite growth via disruption of Na+ homeostasis. P-type ATPase, PfATP4 is responsible for maintaining parasite Na+ homeostasis and is a good target for anti-malarials. Molecular docking with our scaffold showed that it fits well in the binding pocket of PfATP4. Moreover, inhibition of Na+-dependent ATPase activity by our potent scaffold suggests that it targets parasite by inhibiting PfATP4, leading to ionic imbalance. However how ionic imbalance attributes to parasite's death is unclear. We show that ionic imbalance caused by scaffold 7 induces autophagy that leads to onset of apoptosis in the parasite evident by the loss of mitochondrial membrane potential (ΔΨm) and DNA degradation. Our study provides a novel strategy for drug discovery and an insight into the molecular mechanism of ionic imbalance mediated death in malaria parasite.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Indóis/química , Indóis/farmacologia , Malária/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Malária Falciparum/tratamento farmacológico , Modelos Moleculares , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
Eukaryotic Armadillo (ARM) repeat proteins are multifaceted with prominent roles in cell-cell adhesion, cytoskeletal regulation and intracellular signaling among many others. One such ARM repeat containing protein, ARM Repeats Only (ARO), has recently been demonstrated in both Toxoplasma (TgARO) and Plasmodium (PfARO) parasites to be targeted to the rhoptries during the late asexual stages. TgARO has been implicated to play an important role in rhoptry positioning i.e. directing the rhoptry towards the apical end of the parasite. Here, we report for the first time that PfARO exhibits a DNA binding property and a dynamic sub-cellular localization between the nucleus (early schizont) and rhoptry (late schizont) during the different stages of the asexual blood-stage life cycle. PfARO possesses a putative nuclear export signal (NES) and the nucleo-apical shuttling was sensitive to Leptomycin B (LMB) suggesting that the nuclear export was mediated by CRM1. Importantly, PfARO specifically bound an A-T rich DNA sequence of the P. falciparum Gyrase A (PfgyrA) gene, suggesting that the DNA binding specificity of PfARO is likely due to the AT-richness of the probe. This is a novel functional characteristic that has not been reported previously for any P. falciparum ARM containing protein and suggests a putative role for PfARO in gene regulation. This study describes for the first time a conserved P. falciparum ARM repeat protein with a high degree of functional versatility.
Assuntos
Proteínas do Domínio Armadillo/metabolismo , Núcleo Celular/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , beta Catenina/metabolismo , Animais , Anticorpos Antiprotozoários/metabolismo , Especificidade de Anticorpos/efeitos dos fármacos , Proteínas do Domínio Armadillo/química , Biomarcadores/metabolismo , Núcleo Celular/efeitos dos fármacos , Sequência Conservada , DNA/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Ácidos Graxos Insaturados/farmacologia , Imunofluorescência , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Espectrometria de Massas , Parasitos/efeitos dos fármacos , Parasitos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/imunologia , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/química , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Herein we have reported design, synthesis and in vitro biological evaluation of a library of bicyclic lactams that led to the discovery of compounds 6 and 7 as a novel class of α-glucosidase inhibitors. They inhibited α-glucosidase (yeast origin) in a mixed type of inhibition with an IC50 of â¼150 nM. Molecular docking studies further substantiated screening results. Interestingly phenotypic screening of this library against the human malaria parasite revealed 7 as a potent antiplasmodial agent.
Assuntos
Antimaláricos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Glucosidases/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Antimaláricos/síntese química , Antimaláricos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glucosidases/metabolismo , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Saccharomyces cerevisiae/enzimologia , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Histone lysine methyltransferase (HKMT) are histone-modifying enzymes that catalyze the transfer of methyl groups to lysine and arginine residues of histone protein. HKMTs have been involved in transcriptional regulation of various proteins in organisms. Malaria parasite also has HKMT, which plays a major role in parasite development and pathogenesis and also in regulation of various biological process and pathways. Our aim is to study fundamental biology of key molecules involved in the survival of Plasmodium falciparum and use these to develop efficient synthetic peptides and chemical compounds. As a first step in this direction, we computationally predicted the three-dimensional structure of HKMT of P. falciparum (PfHKMT) by using iterative threading assembly refinement. The PfHKMT three-dimensional model was validated using PROCHECK and docked with known HKMT inhibitor Bix01294 using Autodock. Our initial results are encouraging and indicate that structural analysis of PfHKMT could be important in developing novel synthetic molecules against malaria.