During our screening of fermentation broths, culture UC 11136 was identified as producing potent inhibitor(s) of the in vitro cholesteryl ester transfer protein (CETP) reaction. Subsequent chemical isolation work identified two inhibitors of CETP produced by this culture. One of these inhibitors, U-106305, represented a novel CETP inhibitor as well as a structural class of compounds not previously reported from microbial fermentations. The structure of U-106305 was elucidated as V-isobutyl-tii(-rrtins-4,5:6,7:8,9:10,ll:12,13:16,17-hexamethylene-(£,£)-2,14-octadecadienamide by extensive NMR studies. Biogenetically, the backbone of U-106305 was found to derive from nine acetates linked in a head-to-tail fashion, while the cyclopropyl methylene carbons were derived from the methyl group of L-methionine. A biosynthetic pathway is proposed based on these findings.

Sequence-specific 1H and 15N resonance assignments have been made for all 145 non-prolyl residues and for the flavin cofactor in oxidized Desulfovibrio vulgaris flavodoxin. Assignments were obtained by recording and analyzing 1H-15N heteronuclear three-dimensional NMR experiments on uniformly 15N-enriched protein, pH 6.5, at 300 K. Many of the side-chain resonances have also been assigned. Observed medium-and long-range NOEs, in combination with 3JNH alpha coupling constants and 1HN exchange data, indicate that the secondary structure consists of a five-stranded parallel beta-sheet and four alpha-helices, with a topology identical to that determined previously by X-ray crystallographic methods. One helix, which is distorted in the X-ray structure, is non-regular in solution as well. Several protein-flavin NOEs, which serve to dock the flavin ligand to its binding site, have also been identified. Based on fast-exchange into 2H2O, the 1HN3 proton of the isoalloxazine ring is solvent accessible and not strongly hydrogen-bonded in the flavin binding site, in contrast to what has been observed in several other flavodoxins. The resonance assignments presented here can form the basis for assigning single-site mutant flavodoxins and for correlating structural differences between wild-type and mutant flavodoxins with altered redox potentials.

Unregulated or overexpressed matrix metalloproteinases (MMPs), including stromelysin, collagenase, and gelatinase, have been implicated in several pathological conditions including arthritis and cancer. Small-molecule MMP inhibitors may have therapeutic value in the treatment of these diseases. In this regard, the solution structures of two stromelysin/ inhibitor complexes have been investigated using 'H, I3C, and I5N NMR spectroscopy. Both inhibitors are members of a novel class of matrix metalloproteinase inhibitor that contain a thiadiazole group and that interact with stromelysin in a manner distinct from other classes of inhibitors. The inhibitors coordinate the catalytic zinc atom through their exocyclic sulfur atom, with the remainder of the ligand extending into the SI& side of the active site. The binding of inhibitor containing a protonated or fluorinated aromatic ring was investigated using 'H and "F NMR spectroscopy. The fluorinated ring was found to have a reduced ring-flip rate compared to the protonated version. A strong, coplanar interaction between the fluorinated ring of the inhibitor and the aromatic ring of Tyr155 is proposed to account for the reduced ring-flip rate and for the increase in binding affinity observed for the fluorinated inhibitor compared to the protonated inhibitor. Binding interactions observed for the thiadiazole class of ligands have implications for the design of matrix metalloproteinase inhibitors.

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