A single amino acid substitution in Staphylococcus aureus dihydrofolate reductase determines trimethoprim resistance.

A single amino acid substitution, Phe98 to Tyr98, in dihydrofolate reductase (DHFR) is the molecular origin of trimethoprim (TMP) resistance in Staphylococcus aureus. This active site amino acid substitution was found in all S. aureus TMP-resistant clinical isolates tested. In order to explore the structural role of Tyr98 in TMP-resistance the ternary complexes of the chromosomal S. aureus DHFR (SaDHFR) with methotrexate (MTX) and TMP in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as well as that of mutant Phe98Tyr DHFR SaDHFR(F98Y) ternary folate-NADPH complex have been determined by X-ray crystallography. Critical evidence concerning the resistance mechanism has also been provided by NMR spectral analyses of 15N-labelled TMP in the ternary complexes of both wild-type and mutant enzyme. These studies show that the mutation results in loss of a hydrogen bond between the 4-amino group of TMP and the carbonyl oxygen of Leu5. This mechanism of resistance is predominant in both transferable plasmid-encoded and non-transferable chromosomally encoded resistance. Knowledge of the resistance mechanism at a molecular level could help in the design of antibacterials active against multi-resistant Staphylococcus aureus (MRSA), one of todays most serious problems in clinical infectology.

Pyrimido[1,6-a]benzimidazoles: a new class of DNA gyrase inhibitors.

Substituted 4-quinolone- (1, A = CH) and 1,8-naphthyrid-4-one- (1, A = N) 3-carboxylic acids are currently the only classes of clinically useful antibacterial agents exerting their activity by inhibiting the subunit A of DNA gyrase. Pyrimido[1,6-alpha]benzimidazoles 11 have been found to be a new class of inhibitors of this enzyme. The design, synthesis, and biological activity of these compounds are reported.

A 1H NMR study of the interactions and conformations of rationally designed brodimoprim analogues in complexes with Lactobacillus casei dihydrofolate reductase.

A consideration of the detailed structural information available from X-ray crystallographic and NMR studies on complexes of dihydrofolate reductase with inhibitors has led to the design of trimethoprim analogues with improved binding properties. Computer graphic techniques have been used to predict which substituent groups were required at the 3'-O position of brodimoprim (2,4-diamino-5-(3,5-dimethoxy-4-bromobenzyl)pyrimidine) to make additional interactions with the enzyme. NMR spectroscopy provided a convenient method of assessing if the analogues were binding in the predicted manner. On the basis of this approach, the C4,C6-dicarboxylic acid analogue IX was designed to interact with Arg-57 and His-28 in the enzyme, and this analogue was found to bind 3 orders of magnitude more tightly than the parent brodimoprim.

Synthesis and structure-activity relationship of (lactamylvinyl)cephalosporins exhibiting activity against staphylococci, pneumococci, and enterococci.

The synthesis and structure-activity relationships of a new class of vinylcephalosporins substituted with a lactamyl residue are described. These compounds show excellent activity against enterococci and retain the broad spectrum activity of third-generation cephalosporins such as ceftriaxone.

Solution structure of a brodimoprim analogue in its complex with Lactobacillus casei dihydrofolate reductase.

Two-dimensional (2D) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating-frame NOESY (ROESY) spectra were used to assign essentially all the protons in a 1:1 complex of Lactobacillus casei dihydrofolate reductase formed with an analogue of the antibacterial drug brodimoprim [2,4-diamino-5-(3',5'-dimethoxy-4'-bromobenzyl)pyrimidine]. The analogue has a 4,6-dicarboxylic acid side chain substituted on the 3'-O position designed to interact with the Arg 57 and His 28 residues in L. casei dihydrofolate reductase; it binds a factor of 10(3) more tightly to the enzyme than does the parent compound. Thirty-eight intermolecular and 11 intramolecular NOEs were measured involving the bound brodimoprim-4,6-dicarboxylic acid analogue. These provided the distance constraints used in conjunction with an energy minimization and simulated annealing protocol (using Discover from Biosym Ltd.) to dock the brodimoprim analogue into dihydrofolate reductase. In calculations where side chains and backbone fragments for binding-site residues were allowed flexibility, 90% of the 40 calculated structures had reasonable covalent geometry and none of them had NOE distance violations of greater than 0.36 A. The conformations of the aromatic rings in the bound ligand were well-defined in all the structures, with torsion angles tau 1 = -153 degrees +/- 4 degrees (C4-C5-C7-C1') and tau 2 = 53 degrees +/- 4 degrees (C5-C7-C1'-C2'): the aromatic rings of the ligand occupied essentially the same space in all the calculated structures (root mean square deviation value 1.83 A). Inclusion of the electrostatic interactions into the energy minimizations indicated that structures in which the 4,6-dicarboxylate group of the ligand interacts with the side chains of Arg 57 and His 28 are of low energy. Significant differences in side-chain and backbone conformations were detected between binding-site residues in the enzyme complexes with the brodimorpim analogue and methotrexate.

Trimethoprim binding to Lactobacillus casei dihydrofolate reductase: a 13C NMR study using selectively 13C-enriched trimethoprim.

We have measured the 13C chemical shifts for trimethoprim molecules selectively enriched with 13C at the 2-, 4-, 5-, 6-, and 7-positions and the p-OCH3 position in their complexes with Lactobacillus casei dihydrofolate reductase in the presence and absence of coenzyme analogues. The C2 carbon shifts indicate that the pyrimidine ring is protonated at N1 in all the complexes of trimethoprim with the enzyme and coenzymes and in each case the pyrimidine ring is binding in a similar way to that of the corresponding part of methotrexate in the enzyme-methotrexate complex. The C6 carbon of trimethoprim shows a large upfield shift in all complexes (3.51 to 4.70 ppm) but no shift in the complex of 2,4-diaminopyrimidine with the enzyme: these shifts probably arise from steric interactions between the C1' and C2' carbons and the H6 proton, which approach van der Waals contact in the folded conformation adopted by trimethoprim when bound to the enzyme. The large shift observed for C6 in all complexes indicates that the basic folded conformation is present in all of them. A comparison of the 13C shifts in the enzyme-trimethoprim-NADPH complex with those in the enzyme-trimethoprim binary complex shows substantial changes even for carbons such as C6 and p-OCH3 (0.46 and -0.36 ppm, respectively), which are remote from the coenzyme: these are caused by ligand-induced conformational changes that may involve displacement of the helix containing residues 42-49.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamics of trimethoprim bound to dihydrofolate reductase.

The conformation of a small molecule in its binding site on a protein is a major factor in the specificity of the interaction between them. In this paper, we report the use of 1H and 13C NMR spectroscopy to study the fluctuations in conformation of the anti-bacterial drug trimethoprim when it is bound to its "target," dihydrofolate reductase. 13C relaxation measurements reveal dihedral angle changes of +/- 25 degrees to +/- 35 degrees on the subnanosecond time scale, while 13C line-shape analysis demonstrates dihedral angle changes of at least +/- 65 degrees on the millisecond time scale. 1H NMR shows that a specific hydrogen bond between the inhibitor and enzyme, which is believed to make an important contribution to binding, makes and breaks rapidly at room temperature.