RESUMEN
Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Girasa de ADN/química , Quinolinas/química , Quinolinas/farmacología , Staphylococcus aureus/enzimología , Inhibidores de Topoisomerasa II , Antibacterianos/metabolismo , Apoenzimas/química , Apoenzimas/metabolismo , Arginina/metabolismo , Ácido Aspártico/metabolismo , Sitios de Unión , Dominio Catalítico , Ciprofloxacina/química , Ciprofloxacina/metabolismo , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , División del ADN , Girasa de ADN/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Diseño de Fármacos , Resistencia a Medicamentos , Escherichia coli/enzimología , Manganeso/metabolismo , Modelos Moleculares , Conformación Proteica , Quinolinas/metabolismo , Quinolonas/química , Quinolonas/metabolismo , Relación Estructura-ActividadRESUMEN
New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a 'pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.