Discovery and Evaluation of a Methylpyrazolopyrimidine Antibacterial Active against Methicillin-Resistant...

Methicillin-resistant Staphylococcus aureus (MRSA) is a nefarious human bacterial pathogen classified as a serious threat. MRSA strains are resistant to virtually all β-lactam antibiotics (including penicillins and cephalosporins). A common resistance mechanism to β-lactams is mediated by the function of the bla operon, which encodes a β-lactam sensor/signal transducer protein BlaR, a gene repressor BlaI, and a resistance determinant: a class A β-lactamase (BlaZ) and/or a unique penicillin-binding protein 2a (PBP2a). BlaR is responsible for sensing the presence of β-lactam antibiotics and transducing a signal to its cytoplasmic domain upon binding covalently to the β-lactam. This triggers a series of cytoplasmic events that culminate in full-blown antibiotic resistance. We have used a fluorescence-reporter assay in live S. aureus to screen two NCI compound libraries─natural product and diversity libraries─comprising 1,974 compounds for both antibacterial and antibiotic-potentiation activities. Compound 1, N⁴,N⁶-bis(4-bromophenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, emerged from these assays as a potentiator of the activity of oxacillin (a second-generation penicillin), while also exhibiting antibacterial activity of its own. The compound binds to the BlaR sensor domain (31 μM) in shutting down the bla operon, and also binds to the structurally related PBP2 and PBP2a, which are both critical targets for cell wall assembly. Scanning electron microscopy documented cell wall damage caused by compound 1 in combination with oxacillin.