The structure of human P450 2C9 complexed with flurbiprofen was determined to 2.0 Å by x-ray crystallography. In contrast to other structurally characterized P450 2C enzymes, 2C5, 2C8, and a 2C9 chimera, the native catalytic domain of P450 2C9 differs significantly in the conformation of the helix F to helix G region and exhibits an extra turn at the N terminus of helix A. In addition, a distinct conformation of the helix B to helix C region allows Arg-108 to hydrogen bond with Asp-293 and Asn-289 on helix I and to interact directly with the carboxylate of flurbiprofen. These interactions position the substrate for regioselective oxidation in a relatively large active site cavity and are likely to account for the high catalytic efficiency exhibited by P450 2C9 for the regioselective oxidation of several anionic non-steroidal anti-inflammatory drugs. The structure provides a basis for interpretation of a number of observations regarding the substrate selectivity of P450 2C9 and the observed effects of mutations on catalysis.P450 2C9 is one of three human microsomal cytochrome P450s (CYPs) 1 in subfamily 2C that contribute extensively to the hepatic metabolism of therapeutic drugs. The P450 2C9 locus is polymorphic leading to a diminished capacity to clear specific drugs in genetically affected individuals. For P450 2C9 substrates, such as warfarin or phenytoin, that have low therapeutic margins of safety, diminished metabolic capacity because of genetic polymorphisms or drug-drug interactions can lead to toxicity at normal therapeutic doses (1). P450 2C9 has also been implicated in the synthesis of arachidonic acid epoxides in extrahepatic tissues where they regulate blood pressure (2). Like other P450 subfamilies, the 2C enzymes share roughly 70% or greater amino acid identity. However, the 2C genes have duplicated and diverged rapidly as mammalian species evolved, leading to different numbers of enzymes in various species and highly divergent substrate selectivities. This diversity reflects high rates of non-synonymous substitutions that often alter residues that line the active site cavity and determine substrate selectivity.Human P450s 2C9 and 2C19 are closely related with roughly 91% amino acid identity. Although they exhibit distinct substrate selectivities, residues predicted to line the active site cavity, based on the published structures of other mammalian P450s (3-6), do not differ between the two enzymes. This suggests that conformation changes are likely to underlie differences in the substrate selectivities of P450s 2C9 and 2C19 and that the structure(s) of one or both will differ from those published previously. This is supported by studies of chimeric enzymes generated from P450s 2C9 and 2C19 that have generally identified amino acid residues that are predicted to reside outside the substrate binding cavity as determinants of their distinct catalytic properties (7-9). P450 2C9 exhibits a selectivity for the oxidation of relatively small, lipophilic anions such as the non-steroidal anti-inflammator...

The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with β-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum β-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant staphylococci.

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Modern medicine is founded on the discovery of penicillin and subsequent small molecules that inhibit bacterial peptidoglycan (PG) and cell wall synthesis. However, the discovery of new chemically and mechanistically distinct classes of PG inhibitors has become exceedingly rare, prompting speculation that intracellular enzymes involved in PG precursor synthesis are not 'druggable' targets. Here, we describe a β-lactam potentiation screen to identify small molecules that augment the activity of β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) and mechanistically characterize a compound resulting from this screen, which we have named murgocil. We provide extensive genetic, biochemical, and structural modeling data demonstrating both in vitro and in whole cells that murgocil specifically inhibits the intracellular membrane-associated glycosyltransferase, MurG, which synthesizes the lipid II PG substrate that penicillin binding proteins (PBPs) polymerize and cross-link into the cell wall. Further, we demonstrate that the chemical synergy and cidality achieved between murgocil and the β-lactam imipenem is mediated through MurG dependent localization of PBP2 to the division septum. Collectively, these data validate our approach to rationally identify new target-specific bioactive β-lactam potentiation agents and demonstrate that murgocil now serves as a highly selective and potent chemical probe to assist our understanding of PG biosynthesis and cell wall biogenesis across Staphylococcal species.

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With the emergence and rapid spreading of NDM-1 and existence of clinically relevant VIM-1 and IMP-1, discovery of pan inhibitors targeting metallo-beta-lactamases (MBLs) became critical in our battle against bacterial infection. Concurrent with our fragment and high-throughput screenings, we performed a knowledge-based search of known metallo-beta-lactamase inhibitors (MBLIs) to identify starting points for early engagement of medicinal chemistry. A class of compounds exemplified by 11, discovered earlier as B. fragilis metallo-beta-lactamase inhibitors, was selected for in silico virtual screening. From these efforts, compound 12 was identified with activity against NDM-1 only. Initial exploration on metal binding design followed by structureguided optimization led to the discovery of a series of compounds represented by 23 with a pan MBL inhibition profile. In in vivo studies, compound 23 in combination with imipenem (IPM) robustly lowered the bacterial burden in a murine infection model and became the lead for the invention of MBLI clinical candidates.

Pyrido[4,3-d]pyrimidin-4(3H)-one (1) was prepared by reacting 2-trifluoromethyl-4-iodo-nicotinic acid (2) with amidine 9a catalyzed by Pd(2)(dba)(3) and Xantphos, followed by cyclization effected with HBTU and subsequent demethylation using PhBCl(2). The amidine arylation method was found applicable for the syntheses of quinazolin-4(3H)-ones. Thus, reaction of 2-bromo or 2-iodo benzoate esters with amdidines afforded substituted quinazolin-4(3H)-ones in 44-89% yields.