Comparative Studies to Uncover Mechanisms of Action of N-(1,3,4-Oxadiazol-2-yl)benzamide Containing Antibacterial Agents

Drug-resistant bacterial pathogens still cause highlevels of mortality annually despite the availability of manyantibiotics. Methicillin-resistantStaphylococcus aureus(MRSA) isespecially problematic, and the rise in resistance to front-linetreatments like vancomycin and linezolid calls for new chemicalmodalities to treat chronic and relapsing MRSA infections.HalogenatedN-(1,3,4-oxadiazol-2-yl)benzamides are an interestingclass of antimicrobial agents, which have been described bymultiple groups to be effective against different bacterial pathogens.The modes of action of a fewN-(1,3,4-oxadiazol-2-yl)benzamideshave been elucidated. For example, oxadiazoles KKL-35 and MBX-4132 have been described as inhibitors oftrans-translation (aribosome rescue pathway), while HSGN-94 was shown to inhibit lipoteichoic acid (LTA). However, other similarly halogenatedN-(1,3,4-oxadiazol-2-yl)benzamides neither inhibittrans-translation nor LTA biosynthesis but are potent antimicrobial agents. Forexample,HSGN-220,-218, and-144areN-(1,3,4-oxadiazol-2-yl)benzamides that are modified with OCF3, SCF3,orSF5and haveremarkable minimum inhibitory concentrations ranging from 1 to 0.06 mu g/mL against MRSA clinical isolates and show a lowpropensity to develop resistance to MRSA over 30 days. The mechanism of action of these highly potent oxadiazoles is howeverunknown. To provide insights into how these halogenatedN-(1,3,4-oxadiazol-2-yl)benzamides inhibit bacterial growth, weperformed global proteomics and RNA expression analysis of some essential genes ofS. aureustreated withHSGN-220,-218, and-144. These studies revealed that the oxadiazolesHSGN-220,-218, and-144are multitargeting antibiotics that regulatemenaquinone biosynthesis and other essential proteins like DnaX, Pol IIIC, BirA, LexA, and DnaC. In addition, these halogenatedN-(1,3,4-oxadiazol-2-yl)benzamides were able to depolarize bacterial membranes and regulate siderophore biosynthesis and hemeregulation. Iron starvation appears to be part of the mechanism of action that led to bacterial killing. This study demonstrates thatN-(1,3,4-oxadiazol-2-yl)benzamides are indeed privileged scaffolds for the development of antibacterial agents and that subtlemodifications lead to changes to the mechanism of action.