RESUMEN
Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.
Asunto(s)
Enfermedad de Chagas/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Farnesil Difosfato Farnesil Transferasa/antagonistas & inhibidores , Terapia Molecular Dirigida/métodos , Tripanocidas/uso terapéutico , Animales , Chlorocebus aethiops , Cristalografía por Rayos X , Difosfonatos/química , Difosfonatos/metabolismo , Difosfonatos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Farnesil Difosfato Farnesil Transferasa/química , Farnesil Difosfato Farnesil Transferasa/metabolismo , Humanos , Modelos Moleculares , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , Unión Proteica , Quinuclidinas/química , Quinuclidinas/metabolismo , Quinuclidinas/farmacología , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Tripanocidas/química , Tripanocidas/metabolismo , Tripanocidas/farmacología , Trypanosoma cruzi/enzimología , Células VeroRESUMEN
Guanine deaminase, a key enzyme in nucleotide metabolism, catalyzes the hydrolytic deamination of guanine to xanthine. The first guanine deaminase crystal from Bacillus subtilis was grown in the absence or presence of the inhibitor hypoxanthine in 30% polyethylene glycol 4000, 0.2 M ammonium acetate and 0.1 M sodium citrate pH 6.5. The crystals belong to space group C222(1), with unit-cell parameters a = 84.91, b = 90.90, c = 80.19 angstroms, with one dimer per asymmetric unit. The crystals diffract X-rays to beyond 1.2 angstroms resolution and an initial atomic model has been built based on selenomethionyl multiwavelength anomalous data at 2 angstroms resolution. Unexpectedly, this is the first domain-swapped structure in the cytidine deaminase superfamily.