Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N⁶-methyladenosine (m⁶A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m⁶A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m⁶A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine.

Significance: Antiinfectives, drugs that inhibit virulence strategies of microbial pathogens without affecting bacterial growth, may prevent hospital-acquired infections caused by antibiotic-resistant Staphylococcus aureus . We used virtual screening and synthetic optimization to identify 3,6-disubstituted triazolothiadiazole compounds as inhibitors of sortase, an enzyme that incorporates surface proteins into the staphylococcal envelope. Other Gram-positive bacteria also use sortase for protein assembly in the envelope and disease pathogenesis, suggesting that sortase inhibitors could protect high-risk patients against infection with many nosocomial pathogens.

Significance: Staphylococcus aureus is one of the most successful and adaptable human pathogens and is a major cause of hospital-acquired infections. Here we provide insight into how S . aureus uses the catabolite control protein E (CcpE) to sense its intracellular metabolic status and to regulate its virulence-associated properties. We define a key circuit of the virulence regulatory network of S . aureus and emphasize that metabolic status may be a critical element governing the virulence of this pathogen. Understanding the role of metabolites in virulence factor expression ultimately may contribute to the development of novel strategies to combat this dreaded infectious disease.