RESUMEN
The single-celled apicomplexan parasite Plasmodium falciparum is responsible for the majority of deaths due to malaria each year. The selection of drug resistance has been a recurring theme over the decades with each new drug that is developed. It is therefore crucial that future generations of drugs are explored to tackle this major public health problem. Cyclic GMP (cGMP) signaling is one of the biochemical pathways that is being explored as a potential target for new antimalarial drugs. It has been shown that this pathway is essential for all of the key developmental stages of the complex malaria parasite life cycle. This gives hope that targeting cGMP signaling might give rise to drugs that treat disease, block its transmission and even prevent the establishment of infection. Here we review previous work that has been carried out to develop and optimize inhibitors of the cGMP-dependent protein kinase (PKG) which is a critical regulator of the malaria parasite life cycle.
RESUMEN
One of the attractive properties of artemisinins is their extremely fast-killing capability, quickly relieving malaria symptoms. Nevertheless, the unique benefits of these medicines are now compromised by the prolonged parasite clearance times and the increasing frequency of treatment failures, attributed to the increased tolerance of Plasmodium falciparum to artemisinin. This emerging artemisinin resistance threatens to undermine the effectiveness of antimalarial combination therapies. Herein, we describe the medicinal chemistry efforts focused on a cGMP-dependent protein kinase (PKG) inhibitor scaffold, leading to the identification of novel chemical entities with very potent, similar to artemisinins, fast-killing potency against asexual blood stages that cause disease, and activity against gametocyte activation that is required for transmission. Furthermore, we confirm that selective PKG inhibitors have a slow speed of kill, while chemoproteomic analysis suggests for the first time serine/arginine protein kinase 2 (SRPK2) targeting as a novel strategy for developing antimalarial compounds with extremely fast-killing properties.
Asunto(s)
Antimaláricos/farmacología , Artemisininas/química , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/química , Antimaláricos/metabolismo , Artemisininas/metabolismo , Artemisininas/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Canal de Potasio ERG1/antagonistas & inhibidores , Canal de Potasio ERG1/metabolismo , Humanos , Concentración 50 Inhibidora , Mutagénesis Sitio-Dirigida , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Solubilidad , Relación Estructura-Actividad , Tiazoles/químicaRESUMEN
Antimalarial drug resistance compels the quest for new compounds that target alternative pathways to current drugs. The Plasmodium cyclic GMP-dependent protein kinase (PKG) has essential functions in all of the major life cycle developmental stages. An imidazopyridine PKG inhibitor scaffold was previously shown to clear P. falciparum infection in a rodent model in vivo and blocked transmission to mosquitoes providing proof of concept for this target. To find new classes of PKG inhibitors to serve as alternative chemical starting points, we performed a high-throughput screen of the GSK Full Diversity Collection using recombinant P. falciparum PKG. We developed a robust enzymatic assay in a 1536-well plate format. Promising compounds were then tested for activity against P. falciparum asexual blood stage growth, selectivity and cytotoxicity. By using a scoring system we selected the 66 most promising PKG inhibitors (comprising nine clusters and seven singletons). Among these, thiazoles were the most potent scaffold with mid-nanomolar activity on P. falciparum blood stage and gamete development. Using Kinobeads profiling we identified additional P. falciparum protein kinases targeted by the thiazoles that mediate a faster speed of the kill than PKG-selective compounds. This scaffold represents a promising starting point to develop a new antimalarial.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Plasmodium falciparum/crecimiento & desarrollo , Inhibidores de Proteínas Quinasas/farmacología , Tiazoles/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Ensayos Analíticos de Alto Rendimiento , Humanos , Estadios del Ciclo de Vida/efectos de los fármacos , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Inhibidores de Proteínas Quinasas/química , Proteínas Protozoarias/metabolismo , Tiazoles/químicaRESUMEN
The cyclic nucleotides 3', 5'-cyclic adenosine monophosphate (cAMP) and 3', 5'-cyclic guanosine monophosphate (cGMP) are intracellular messengers found in most animal cell types. They usually mediate an extracellular stimulus to drive a change in cell function through activation of their respective cyclic nucleotide-dependent protein kinases, PKA and PKG. The enzymatic components of the malaria parasite cyclic nucleotide signalling pathways have been identified, and the genetic and biochemical studies of these enzymes carried out to date are reviewed herein. What has become very clear is that cyclic nucleotides play vital roles in controlling every stage of the complex malaria parasite life cycle. Our understanding of the involvement of cyclic nucleotide signalling in orchestrating the complex biology of malaria parasites is still in its infancy, but the recent advances in our genetic tools and the increasing interest in signalling will deliver more rapid progress in the coming years.
Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Plasmodium/metabolismo , Transducción de Señal , Proteínas Quinasas Reguladas por Nucleótidos Cíclicos/genética , Proteínas Quinasas Reguladas por Nucleótidos Cíclicos/metabolismo , Estadios del Ciclo de Vida , Plasmodium/crecimiento & desarrollo , Plasmodium/patogenicidad , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Peptidic α-ketoamides have been developed as inhibitors of the malarial protease PfSUB1. The design of inhibitors was based on the best known endogenous PfSUB1 substrate sequence, leading to compounds with low micromolar to submicromolar inhibitory activity. SAR studies were performed indicating the requirement of an aspartate mimicking the P1' substituent and optimal P1-P4 length of the non-prime part. The importance of each of the P1-P4 amino acid side chains was investigated, revealing crucial interactions and size limitations.
Asunto(s)
Amidas/farmacología , Péptidos/química , Proteínas Protozoarias/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/farmacología , Subtilisinas/antagonistas & inhibidores , Amidas/síntesis química , Amidas/química , Relación Dosis-Respuesta a Droga , Simulación del Acoplamiento Molecular , Estructura Molecular , Proteínas Protozoarias/metabolismo , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/química , Relación Estructura-Actividad , Subtilisinas/metabolismoRESUMEN
The etiological agent of the most dangerous form of malaria, Plasmodium falciparum, has developed resistance or reduced sensitivity to the majority of the drugs available to treat this deadly disease. Innovative antimalarial therapies are therefore urgently required. P. falciparum serine protease subtilisin-like protease 1 (PfSUB1) has been identified as a key enzyme for merozoite egress from red blood cells and invasion. We present herein the rational design, synthesis, and biological evaluation of novel and potent difluorostatone-based inhibitors. Our bioinformatic-driven studies resulted in the identification of compounds 1a, b as potent and selective PfSUB1 inhibitors. The enzyme/inhibitor interaction pattern herein proposed will pave the way to the future optimization of this class of promising enzyme inhibitors.
Asunto(s)
Diseño de Fármacos , Oligopéptidos/farmacología , Plasmodium falciparum/enzimología , Inhibidores de Proteasas/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Subtilisinas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Conformación Molecular , Oligopéptidos/síntesis química , Oligopéptidos/química , Plasmodium falciparum/efectos de los fármacos , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , Subtilisinas/metabolismoRESUMEN
The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Interacciones Huésped-Parásitos/fisiología , Merozoítos/fisiología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/metabolismo , Transducción de Señal/fisiología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Orgánulos/metabolismoRESUMEN
Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) is a serine protease that plays key roles in the egress of the parasite from red blood cells and in preparing the released merozoites for the subsequent invasion of new erythrocytes. The development of potent and selective PfSUB1 inhibitors could pave the way to the discovery of potential antimalarial drugs endowed with an innovative mode of action and consequently able to overcome the current problems of resistance to established chemotherapies. Through the screening of a proprietary library of compounds against PfSUB1, we identified hydrazone 2 as a hit compound. Here we report a preliminary investigation of the structure-activity relationships for a class of PfSUB1 inhibitors related to our identified hit.
Asunto(s)
Antimaláricos/química , Hidrazonas/química , Plasmodium falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Quinolinas/química , Inhibidores de Serina Proteinasa/química , Subtilisinas/antagonistas & inhibidores , Antimaláricos/síntesis química , Antimaláricos/farmacología , Hidrazonas/síntesis química , Hidrazonas/farmacología , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Quinolinas/síntesis química , Quinolinas/farmacología , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-Actividad , Subtilisinas/metabolismoRESUMEN
Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1.