Discovery and Evaluation of a Methylpyrazolopyrimidine Antibacterial Active against Methicillin-Resistant <i>Staphylococcus aureus</i>

Methicillin-resistant <i>Staphylococcus aureus</i> (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 <i>bla</i> 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 <i>S. aureus</i> to screen two NCI compound libraries─natural product and diversity libraries─comprising 1,974 compounds for both antibacterial and antibiotic-potentiation activities. Compound <b>1</b>, <i>N</i>⁴,<i>N</i>⁶<i>-bis</i>(4-bromophenyl)-1-methyl-1<i>H</i>-pyrazolo[3,4-<i>d</i>]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 <i>bla</i> 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 <b>1</b> in combination with oxacillin.