Stephan K. Grant

Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-β-lactamase

BACKGROUNDnHigh level resistance to carbapenem antibiotics in gram negative bacteria such as Bacteroides fragilis is caused, in part, by expression of a wide-spectrum metallo-beta-lactamase that hydrolyzes the drug to an inactive form. Co-administration of metallo-beta-lactamase inhibitors to resistant bacteria is expected to restore the antibacterial activity of carbapenems.nnnRESULTSnBiphenyl tetrazoles (BPTs) are a structural class of potent competitive inhibitors of metallo-beta-lactamase identified through screening and predicted using molecular modeling of the enzyme structure. The X-ray crystal structure of the enzyme bound to the BPT L-159,061 shows that the tetrazole moiety of the inhibitor interacts directly with one of the two zinc atoms in the active site, replacing a metal-bound water molecule. Inhibition of metallo-beta-lactamase by BPTs in vitro correlates well with antibiotic sensitization of resistant B. fragilis.nnnCONCLUSIONSnBPT inhibitors can sensitize a resistant B. fragilis clinical isolate expressing metallo-beta-lactamase to the antibiotics imipenem or penicillin G but not to rifampicin.

Development of Novel Assays for Proteolytic Enzymes Using Rhodamine-Based Fluorogenic Substrates

Components within synthetic chemical and natural product extract libraries often interfere with fluorescence-based assays. Fluorescence interference can result when the intrinsic spectral properties of colored compounds overlap with the fluorescent probes. Typically, fluorescence-based protease assays use peptide amidomethylcoumarin derivatives as substrates. However, because many organic compounds absorb in the ultraviolet region, they can interfere with coumarin-based fluorescence assays. Red-shifted fluorescent dyes such as peptidyl rhodamine derivatives are useful because there is generally less interference from organic compounds outside the ultraviolet wavelengths. In this report, rhodamine-based fluorogenic substrates, such as bis-(Leu)2-Rhod110 and bis-(Ala-Pro)2-Rhod110, were developed for leucine aminopeptidase and dipeptidyl aminopeptidase. Novel, tandem rhodamine substrates such as Ala-Pro-Rhod110-Leu were designed with 2 protease cleavage sites and used to assay 2 proteases in a multiplex format. General endpoint high-throughput screening (HTS) assays were also developed for leucine aminopeptidase, dipeptidyl aminopeptidase, and trypsin that incorporated both amidomethylcoumarin and rhodamine-based fluorogenic substrates into a single screening format. These dual-substrate assays allowed for the successful screening of the LOPAC™ collection and natural product extracts despite high levels of fluorescence interference.

A high quality nuclear magnetic resonance solution structure of peptide deformylase from Escherichia coli: Application of an automated assignment strategy using GARANT

The NMR structure of the peptide deformylase (PDF) (1–150) from Escherichia coli, which is an essential enzyme that removes the formyl group from nascent polypeptides and represents a potential target for drug discovery, was determined using 15N/13C doubly labeled protein. Nearly completely automated assignment routines were employed to assign three-dimensional triple resonance, 15N-resolved and 13C-resolved NOESY spectra using the program GARANT. This assignment strategy, demonstrated on a 17xa0kDa protein, is a significant advance in the automation of NMR data assignment and structure determination that will accelerate future work. A total of 2302 conformational constraints were collected as input for the distance geometry program DYANA. After restrained energy minimization with the program X-PLOR the 20 best conformers characterize a high quality structure with an average of 0.43xa0Å for the root-mean-square deviation calculated from the backbone atoms N, Cα and C′, and 0.81 Å for all heavy atoms of the individual conformers relative to the mean coordinates for residues 1 to 150. The globular fold of PDF contains two α-helices comprising residues 25–40, 125–138, six β-strands 57–60, 70–77, 85–88, 98–101, 105–111, 117–123 and one 310 helix comprising residues 49–51. The C-terminal helix contains the HEXXH motif positioning a zinc ligand in a similar fashion to other metalloproteases, with the third ligand being cysteine and the fourth presumably a water. The three-dimensional structure of PDF affords insight into the substrate recognition and specificity for N-formylated over N-acetylated substrates and is compared to other PDF structures.

Inhibition of nitric oxide synthase by benzoxazolones

Abstract A series of benzoxazolones has been synthesized using modifications of literature methods. The synthetic benzoxazolone analogs, along with commercially available analogs, were evaluated as inhibitors of nitric oxide synthases (NOS). Structure-activity relationships are also discussed.

Characterization of Inducible Nitric-oxide Synthase by Cytochrome P-450 Substrates and Inhibitors INHIBITION BY CHLORZOXAZONE

Nitric-oxide synthases (NOS, EC 1.14.13.39) are heme-containing enzymes that catalyze the formation of nitric oxide from L-Arg. General cytochrome P-450 inhibitors and cytochrome P-450 isoform-selective substrates and inhibitors were used to characterize the activity of recombinant human inducible NOS (iNOS). Classical cytochrome P-450 ligands such as the mechanism-based inactivator 1-aminobenzotriazole did not inhibit iNOS. Of a panel of 30 human cytochrome P-450 isoform-selective substrates and inhibitors, only chlorzoxazone, a cytochrome P-450 2E1 (CYP2E1) substrate, showed any significant inhibition of iNOS activity. Chlorzoxazone was not a substrate for iNOS but was a potent competitive inhibitor with respect to L-Arg with Ki = 3.3 ± 0.7 μM. The binding of chlorzoxazone to iNOS and human and rat liver microsomal cytochrome P-450 induced a high spin, type I spectra, which was reversed by imidazole. Although the binding of chlorzoxazone to iNOS and its inhibition of iNOS activity suggest some similarity between iNOS and CYP2E1 activity, other CYP2E1 substrates and inhibitors including zoxazolamine were not inhibitors of iNOS. Overall, iNOS activity is distinctly different from the major cytochrome P-450 enzymes in human liver microsomes.

Expression and Immunoaffinity Purification of Human Inducible Nitric-oxide Synthase INHIBITION STUDIES WITH 2-AMINO-5,6-DIHYDRO-4H-1,3-THIAZINE

Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37°C and contained 1.15 ± 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret λmax at 396 nm with a shoulder at 460 nm and contained 0.28-0.64 mol of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (Ki = 22.6 ± 1.9 nM), reversed the type II difference spectrum induced by imidazole (Kd = 17.7 nM), and altered the CO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.

Inactivation of HIV-1 protease by a tripeptidyl epoxide

Abstract (2S,3R,4S)-N-[N-(N-benzyloxycarbonyl)-L-phenylalanyl]-L-alanyl-1-phenyl-2-amino-3,4-epoxy-6-methylheptane, a tripeptidyl epoxide analogue of peptide substrates of the retroviral protease of the human immunodeficiency virus-1, is a potent, active-site directed, irreversible inactivator of this enzyme. (2S, 3R, 4S)-N-[N-(N-benzyloxycarbonyl)-L-phenylalanyl]-L-alanyl-1-phenyl-2-amino-3,4-epoxy-6-methylheptane, a tripeptidyl epoxide analogue of peptide substrates of the retroviral protease of the human immunodeficiency virus-1, is a potent, active-site directed, irreversible inactivator of this enzyme.