Substituted benzamide inhibitors of human rhinovirus 3C protease: structure-based design, synthesis, and biological evaluation.

A series of nonpeptide benzamide-containing inhibitors of human rhinovirus (HRV) 3C protease was identified using structure-based design. The design, synthesis, and biological evaluation of these inhibitors are reported. A Michael acceptor was combined with a benzamide core mimicking the P1 recognition element of the natural 3CP substrate. alpha,beta-Unsaturated cinnamate esters irreversibly inhibited the 3CP and displayed antiviral activity (EC(50) 0.60 microM, HRV-16 infected H1-HeLa cells). On the basis of cocrystal structure information, a library of substituted benzamide derivatives was prepared using parallel synthesis on solid support. A 1.9 A cocrystal structure of a benzamide inhibitor in complex with the 3CP revealed a binding mode similar to that initially modeled wherein covalent attachment of the nucleophilic cysteine residue is observed. Unsaturated ketones displayed potent reversible inhibition but were inactive in the cellular antiviral assay and were found to react with nucleophilic thiols such as DTT.

Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes.

Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having alpha,beta-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.

Interaction of a novel GDP exchange inhibitor with the Ras protein.

Mutated, tumorigenic Ras is present in a variety of human tumors. Compounds that inhibit tumorigenic Ras function may be useful in the treatment of Ras-related tumors. The interaction of a novel GDP exchange inhibitor (SCH-54292) with the Ras-GDP protein was studied by NMR spectroscopy. The binding of the inhibitor to the Ras protein was enhanced at low Mg2+ concentrations, which enabled the preparation of a stable complex for NMR study. To understand the enhanced inhibitor binding and the increased GDP dissociation rates of the Ras protein, the conformational changes of the Ras protein at low Mg2+ concentrations was investigated using two-dimensional 1H-15N HSQC experiments. The Ras protein existed in two conformations in slow exchange on the NMR time scale under such conditions. The conformational changes mainly occurred in the GDP binding pocket, in the switch I and the switch II regions, and were reversible. The Ras protein resumed its regular conformation after an excess amount of Mg2+ was added. A model of the inhibitor in complex with the Ras-GDP protein was derived from intra- and intermolecular NOE distance constraints, and revealed that the inhibitor bound to the critical switch II region of the Ras protein.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 1. Michael acceptor structure-activity studies.

The structure-based design, chemical synthesis, and biological evaluation of peptide-derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds incorporate various Michael acceptor moieties and are shown to irreversibly bind to HRV serotype 14 3CP with inhibition activities (kobs/[I]) ranging from 100 to 600 000 M-1 s-1. These inhibitors are also shown to exhibit antiviral activity when tested against HRV-14-infected H1-HeLa cells with EC50's approaching 0.50 microM. Extensive structure-activity relationships developed by Michael acceptor alteration are reported along with the evaluation of several compounds against HRV serotypes other than 14. A 2.0 A crystal structure of a peptide-derived inhibitor complexed with HRV-2 3CP is also detailed.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 2. Peptide structure-activity studies.

The structure-based design, chemical synthesis, and biological evaluation of various peptide-derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of an ethyl propenoate Michael acceptor moiety and a tripeptidyl binding determinant. The systematic modification of each amino acid residue present in the binding determinant as well as the N-terminal functionality is described. Such modifications are shown to provide irreversible HRV-14 3CP inhibitors with anti-3CP activities (kobs/[I]) ranging from 60 to 280 000 M-1 s-1 and antiviral EC50's which approach 0.15 microM. An optimized inhibitor which incorporates several improvements identified by the structure-activity studies is also described. This molecule displays very rapid irreversible inhibition of HRV-14 3CP (kobs/[I] = 800 000 M-1 s-1) and potent antiviral activity against HRV-14 in cell culture (EC50 = 0.056 microM). A 1.9 A crystal structure of an S-alkylthiocarbamate-containing inhibitor complexed with HRV-2 3CP is also detailed.

Design, synthesis, and evaluation of nonpeptidic inhibitors of human rhinovirus 3C protease.

The design, synthesis, and biological evaluation of reversible, nonpeptidic inhibitors of human rhinovirus (HRV) 3C protease (3CP) are reported. A novel series of 2,3-dioxindoles (isatins) were designed that utilized a combination of protein structure-based drug design, molecular modeling, and structure-activity relationship (SAR). The C-2 carbonyl of isatin was envisioned to react in the active site of HRV 3CP with the cysteine responsible for catalytic proteolysis, thus forming a stabilized transition state mimic. Molecular-modeling experiments using the apo crystal structure of human rhinovirus-serotype 14 (HRV-14) 3CP and a peptide substrate model allowed us to design recognition features into the P1 and P2 subsites, respectively, from the 5- and 1-positions of isatin. Attempts to optimize recognition properties in the P1 subsite using SAR at the 5-position were performed. In addition, a series of ab initio calculations were carried out on several 5-substituted isatins to investigate the stability of sulfide adducts at C-3. The inhibitors were prepared by general synthetic methods, starting with commercially available 5-substituted isatins in nearly every case. All compounds were tested for inhibition of purified HRV-14 3CP. Compounds 8, 14, and 19 were found to have excellent selectivity for HRV-14 3CP compared to other proteolytic enzymes, including chymotrypsin and cathepsin B. Selected compounds were assayed for antiviral activity against HRV-14-infected HI-HeLa cells. A 2.8 A cocrystal structure of derivative 19 covalently bound to human rhinovirus-serotype 2 (HRV-2) 3CP was solved and revealed that the isatin was situated in essentially the same conformation as modeled.

A microtechnique for neutrophil respiratory burst oxidase in a cell-free system--characterization of oxidase activation system.

1. A microtechnique for quantitating human neutrophil NADPH oxidase in a cell-free system is described. 2. This spectrophotometric discontinuous (fixed time) method is less material-consuming than existing methods and is more useful for experiments in which superoxide production by neutrophils must be measured in a large number of samples. 3. Measurement of NADPH oxidase using the new method can be accomplished in a final vol of 0.15 ml. 4. In the assay, neutrophil membranes solubilized with deoxycholate were incubated for 3 min with cytosolic fractions, magnesium, sodium dodecyl sulfate, and cytochrome c in the absence of NADPH to preincubate the oxidase before the addition of the reducing agent. 5. The reaction was started by adding NADPH and 2 min later terminated by adding superoxide dismutase. 6. The apparent Km for NADPH obtained by the new method was almost the same as that by the authorized method (39.2 +/- 3.1 SD vs 36.8 +/- 1.6). Activation of neutrophil NADPH oxidase was characterized using the new assay method.

The respiratory burst oxidase of human neutrophils. Further studies of the purified enzyme.

A superoxide-forming oxidase from activated human neutrophil membranes was solubilized by two slightly different methods, then purified by "dye-affinity" chromatography. Kinetic studies of the purified preparations gave Vmax values of 5-10 mumol of O-2/min/mg of protein, and Km values for NADH and NADPH that were in reasonable agreement with values determined previously using particulate and crude solubilized preparations of the respiratory burst oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed prominent bands at 67, 48, and 32 kDa, together with some minor contaminants, whereas gel electrophoresis under non-denaturing conditions gave a single major band that when eluted and re-electrophoresed in the presence of sodium dodecyl sulfate showed bands at 67, 48, 32 kDa. We believe that all three bands represent oxidase components. The flavin content of the purified enzyme was 20.4 +/- 2.0 S.E. pmol of FAD/microgram of protein, whereas heme averaged 0.1 +/- 0.02 pmol/microgram and ubiquinone could not be detected. Assuming that the enzyme is composed of one 67-kDa subunit, one 48-kDa subunit, and one 32-kDa subunit (i.e. that its molecular mass is approximately 150 kDa), it can be calculated to have a turnover number of 700-1500 min-1, in agreement with a value reported previously for oxidase in a particulate O-2-forming system (Cross, A. R., Parkinson, J. F., and Jones, O. T. G. (1985) Biochem. J. 226, 881-884), and to contain the following quantities of redox carriers (mol/mol): FAD, 3.0; heme, 0.015; ubiquinone, less than 0.06. It remains to be determined whether this preparation represents the complete respiratory burst oxidase or is only the pyridine nucleotide dehydrogenating component of a more complex enzyme.