RESUMEN
Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na(+) levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na(+) homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.
Asunto(s)
Antimaláricos/farmacología , ATPasas Transportadoras de Calcio/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Isoquinolinas/farmacología , Malaria/tratamiento farmacológico , Modelos Moleculares , Plasmodium/efectos de los fármacos , Antimaláricos/farmacocinética , ATPasas Transportadoras de Calcio/genética , Senescencia Celular/efectos de los fármacos , Descubrimiento de Drogas , Resistencia a Medicamentos/genética , Eritrocitos/efectos de los fármacos , Citometría de Flujo , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Ensayos Analíticos de Alto Rendimiento , Isoquinolinas/farmacocinética , Estructura MolecularRESUMEN
We previously discovered and validated a class of piperidinyl ureas that regulate defective in cullin neddylation 1 (DCN1)-dependent neddylation of cullins. Here, we report preliminary structure-activity relationship studies aimed at advancing our high-throughput screen hit into a tractable tool compound for dissecting the effects of acute DCN1-UBE2M inhibition on the NEDD8/cullin pathway. Structure-enabled optimization led to a 100-fold increase in biochemical potency and modestly increased solubility and permeability as compared to our initial hit. The optimized compounds inhibit the DCN1-UBE2M protein-protein interaction in our TR-FRET binding assay and inhibit cullin neddylation in our pulse-chase NEDD8 transfer assay. The optimized compounds bind to DCN1 and selectively reduce steady-state levels of neddylated CUL1 and CUL3 in a squamous cell carcinoma cell line. Ultimately, we anticipate that these studies will identify early lead compounds for clinical development for the treatment of lung squamous cell carcinomas and other cancers.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Proteínas Cullin/antagonistas & inhibidores , Proteína NEDD8/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Cristalografía por Rayos X , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Analíticos de Alto Rendimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares/tratamiento farmacológico , Modelos Moleculares , Conformación Molecular , Proteína NEDD8/metabolismo , Unión Proteica , Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidoresRESUMEN
Phenotypic whole-cell screening in erythrocytic cocultures of Plasmodium falciparum identified a series of dihydroisoquinolones that possessed potent antimalarial activity against multiple resistant strains of P. falciparum in vitro and show no cytotoxicity to mammalian cells. Systematic structure-activity studies revealed relationships between potency and modifications at N-2, C-3, and C-4. Careful structure-property relationship studies, coupled with studies of metabolism, addressed the poor aqueous solubility and metabolic vulnerability, as well as potential toxicological effects, inherent in the more potent primary screening hits such as 10b. Analogues 13h and 13i, with structural modifications at each site, were shown to possess excellent antimalarial activity in vivo. The (+)-(3S,4S) enantiomer of 13i and similar analogues were identified as the more potent. On the basis of these studies, we have selected (+)-13i for further study as a preclinical candidate.