Preclinical characterization of the antiviral activity of SCH 900518 (narlaprevir), a novel mechanism-based inhibitor of hepatitis C virus NS3 protease.

Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (K*(i)) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC(90)) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5x EC(90) of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies.

SCH 503034, a mechanism-based inhibitor of hepatitis C virus NS3 protease, suppresses polyprotein maturation and enhances the antiviral activity of alpha interferon in replicon cells.

Cleavage of the hepatitis C virus (HCV) polyprotein by the viral NS3 protease releases functional viral proteins essential for viral replication. Recent studies by Foy and coworkers strongly suggest that NS3-mediated cleavage of host factors may abrogate cellular response to alpha interferon (IFN-alpha) (E. Foy, K. Li, R. Sumpter, Jr., Y.-M. Loo, C. L. Johnson, C. Wang, P. M. Fish, M. Yoneyama, T. Fujita, S. M. Lemon, and M. Gale, Jr., Proc. Natl. Acad. Sci. USA 102:2986-2991, 2005, and E. Foy, K. Li, C. Wang, R. Sumpter, Jr., M. Ikeda, S. M. Lemon, and M. Gale, Jr., Science 300:1145-1148, 2003). Blockage of NS3 protease activity therefore is expected to inhibit HCV replication by both direct suppression of viral protein production as well as by restoring host responsiveness to IFN. Using structure-assisted design, a ketoamide inhibitor, SCH 503034, was generated which demonstrated potent (overall inhibition constant, 14 nM) time-dependent inhibition of the NS3 protease in cell-free enzyme assays as well as robust in vitro activity in the HCV replicon system, as monitored by immunofluorescence and real-time PCR analysis. Continuous exposure of replicon-bearing cell lines to six times the 90% effective concentration of SCH 503034 for 15 days resulted in a greater than 4-log reduction in replicon RNA. The combination of SCH 503034 with IFN was more effective in suppressing replicon synthesis than either compound alone, supporting the suggestion of Foy and coworkers that combinations of IFN with protease inhibitors would lead to enhanced therapeutic efficacy.

Hepatitis C virus NS3-4A serine protease inhibitors: use of a P2-P1 cyclopropyl alanine combination for improved potency.

Modification of the P(2) and P(1) side chains of earlier P(3)-capped alpha-ketoamide inhibitor of HCV NS3 serine protease 1 resulted in the discovery of compound 24 with about 10-fold improvement in potency.

Hepatitis C virus NS3-4A serine protease inhibitors: SAR of P'2 moiety with improved potency.

We have discovered that introduction of appropriate amino acid derivatives at P'2 position improved the binding potency of P3-capped alpha-ketoamide inhibitors of HCV NS3 serine protease. X-ray crystal structure of one of the inhibitors (43) bound to the protease revealed the importance of the P'2 moiety.

Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation.

Crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible NOS (iNOS) catalytic domains were solved in complex with the arginine substrate and an inhibitor S-ethylisothiourea (SEITU), respectively. The small molecules bind in a narrow cleft within the larger active-site cavity containing heme and tetrahydrobiopterin. Both are hydrogen-bonded to a conserved glutamate (eNOS E361, iNOS E377). The active-site residues of iNOS and eNOS are nearly identical. Nevertheless, structural comparisons provide a basis for design of isozyme-selective inhibitors. The high-resolution, refined structures of eNOS (2.4 A resolution) and iNOS (2.25 A resolution) reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.

Thermodynamic and structural analysis of phosphotyrosine polypeptide binding to Grb2-SH2.

A thermodynamic analysis using isothermal titration calorimetry (ITC) has been performed to examine the binding interaction between the SH2 (Src homology 2) domain of growth factor receptor binding protein 2 (Grb2-SH2) and one of its phosphotyrosine (pY) polypeptide ligands. Interaction of the Shc-derived phosphotyrosine hexapeptide Ac-SpYVNVQ-NH2 with Grb2-SH2 was both enthalpically and entropically favorable (DeltaH = -7.55 kcal mol-1, -TDeltaS = -1.46 kcal mol-1 , DeltaG = -9.01 kcal mol-1, T = 20 degrees C). ITC experiments using five alanine-substituted peptides were performed to examine the role of each side chain in binding. The results were consistent with homology models of the Grb2-SH2-Shc hexapeptide complex which identified several possible hydrogen bonds between Grb2-SH2 and the phosphotyrosine and conserved asparagine(+2) side chains of the Shc hexapeptide. These studies also demonstrated that the hydrophobic valine(+1) side chain contributes significantly to the favorable entropic component of binding. The thermodynamic and structural data are consistent with a Grb2-SH2 recognition motif of pY-hydrophobic-N-X (where X is any amino acid residue). The measured heat capacity of binding (DeltaCp = -146 cal mol-1 K-1) was very similar to computed values using semiempirical estimates (DeltaCp = -106 to -193 cal mol-1 K-1) derived from apolar and polar accessible surface area values calculated from several homology models of the Grb2-SH2-Shc hexapeptide complex. The homology model which most closely reproduced the measured DeltaCp value is also the model which had the lowest RMS deviation from the subsequently determined crystal structure. Calculations based on the thermodynamic data and these semiempirical estimates indicated that the binding event involves burial of nearly comparable apolar (677 A2) and polar (609 A2) surface areas.

Crystal structure of dimeric HIV-1 capsid protein.

X-ray diffraction analysis of a human immunodeficiency virus (HIV-1) capsid (CA) protein shows that each monomer within the dimer consists of seven alpha-helices, five of which are arranged in a coiled coil-like structure. Sequence assignments were made for two of the helices, and tentative connectivity of the remainder of the protein was confirmed by the recent solution structure of a monomeric N-terminal fragment. The C-terminal third of the protein is mostly disordered in the crystal. The longest helices in the coiled coil-like structure are separated by a long, highly antigenic peptide that includes the binding site of an antibody fragment complexed with CA in the crystal. The site of binding of the Fab, the position of the antigenic loop and the site of cleavage between the matrix protein and CA establish the side of the dimer that would be on the exterior of the retroviral core.

Oxidation-reduction properties of Escherichia coli thioredoxin reductase altered at each active site cysteine residue.

Thioredoxin is a small oxidation-reduction (redox) mediator protein. Its reduction by NADPH is catalyzed by the flavoenzyme thioredoxin reductase. Site-directed mutagenesis has provided forms of the reductase in which Cys135 and Cys138 have each been changed to a serine residue (Prongay, A. J., Engelke, D. R., and Williams, C. H., Jr. (1989) J. Biol. Chem. 264, 2656-2664). Cys135 and Cys138 form the redox-active disulfide in the oxidized enzyme. The redox properties of the two altered forms of Escherichia coli thioredoxin reductase have been determined from pH 6.0 to 9.0. Photoreduction of TRR(Ser135,Cys138) produces the blue, neutral semiquinone species, which disproportionates (Kf = 0.73) to an apparent maximum of 29% of the total enzyme as the semiquinone. In contrast, the semiquinone formed on TRR(Cys135,Ser138) during a photoreductive titration does not disproportionate and 70% of the enzyme is stabilized as the semiquinione. Reductive titrations have demonstrated that 1 mol of sodium dithionite (2 electrons)/mol of FAD is required to fully reduce TRR(Ser135,Cys138) whereas 2 mol of dithionite/mol of FAD are required to fully reduce TRR(Cys135,Ser138). The oxidation-reduction midpoint potentials for the 1-electron and 2-electron reductions of TRR(Ser135,Cys138) have been determined by NADH/NAD+ titrations in the presence of a mediator, benzyl viologen. The midpoint potential for the 2-electron reduction of TRR(Ser135,Cys138) is -280 mV, at pH 7.0 and 20 degrees C. Thus, the redox potential is similar to that of the FAD/FADH2 couple in the dithiol form of wild type enzyme, -270 mV (corrected to 20 degrees C) (O'Donnell, M. E., and Williams, C. H., Jr. (1983) J. Biol. Chem. 258, 13795-13805). The delta Em/delta pH is -57.1 mV, which corresponds to a proton stoichiometry of 2 H+/2 e-.A maximum of 19% of the enzyme forms a stable semiquinone species during the titration, and the potentials for the oxidized enzyme/semiquinone couple, E2, and the semiquinone/reduced enzyme couple, E1, are -306 and -256 mV, respectively, at pH 7.0 and 20 degrees C. These studies provide evidence that the residue at position 138 exerts a greater effect on the FAD than does the residue at position 135.

Preparation and crystallization of a human immunodeficiency virus p24-Fab complex.

A recombinant form of human immunodeficiency virus capsid protein, p24, expressed in Escherichia coli has been purified to homogeneity and separated into distinct isoelectric forms. A monoclonal antibody, mAb25.4, which recognizes an epitope in the amino-terminal region of p24, has been purified to homogeneity from ascites fluid and digested with papain to produce the respective antigen-binding fragment (Fab). The Fab25.4 was purified from the digestion mixture and separated into two distinct isoelectric forms. The two Fab species were each complexed with one isoelectric form of the recombinant p24 by incubating equimolar quantities of the two proteins. Two different crystal morphologies of the p24-Fab25.4 complex were obtained by the vapor-diffusion method with 12-24% PEG 3350 as the precipitant. One of these crystal forms has unit-cell parameters of a = 92.1 A, b = 85.4 A, c = 54.0 A, alpha = gamma = 90.0 degrees and beta = 90.4 degrees and belongs to the monoclinic space group P2(1), with one molecule of the complex per asymmetric unit. These crystals strongly diffracted x-rays to at least 2.7-A resolution.

Evidence for direct interaction between cysteine 138 and the flavin in thioredoxin reductase. A study using flavin analogs.

The flavoenzyme thioredoxin reductase from Escherichia coli contains an oxidation-reduction active disulfide made up of Cys135 and Cys138. Mutations changing each Cys residue to a Ser residue have been effected (Prongay, A. J., engelke, D. R., and Williams, C. H., Jr. (1989) J. Biol. Chem. 264, 2656-2664). The FAD prosthetic group of each altered thioredoxin reductase has been replaced with 1-deaza-FAD (a flavin analog with carbon substituted for nitrogen at position 1), 4-thio-FAD (a flavin analog with sulfur substituted for oxygen at position 4), and 6-thiocyanato-FAD. 1-Deaza-FAD-TRR(Cys135,Ser138) has absorbance and fluorescence spectral properties similar to the oxidized form of wild type apothioredoxin reductase reconstituted with 1-deaza-FAD. The absorbance spectrum of 1-deaza-FAD-TRR(Ser135,Cys138) is similar to the spectrum of the two-electron reduced form of wild type apothioredoxin reductase reconstituted with 1-deaza-FAD, indicating that it is a mixture of two species (O'Donnell, M. E., and Williams, C. H., Jr. (1984) J. Biol. Chem. 259, 2243-2251). The spectrum of one of these species of 1-deaza-FAD-TRR(Ser135,Cys138) resembles the spectrum of oxidized 1-deaza-FAD bound to wild type apothioredoxin reductase. The other species has an absorbance spectrum with a single peak at 400 nm (epsilon 400 = 11,100 M-1 cm-1) and resembles the spectrum of a thiolate adduct at the C4a position of the 1-deaza-FAD. The equilibrium between these species is pH-dependent, with a maximum of 50% C4a-adduct formation at low pH, and is linked to pK alpha values at 8.2 and 9.3. The absorbance spectrum of 4-thio-FAD-TRR(Cys135,Ser138) resembles the spectrum of the unbound 4-thio-FAD, whereas 4-thio-FAD-TRR(Ser135,Cys138) has a spectrum indicative of a mixture of 4-thio-FAD and FAD, suggesting a reaction between the 4-position of the flavin and Cys138. The binding of 6-thiocyanato-FAD to the apoprotein of the mutated enzymes showed no evidence for a reaction between the thiols and the group at the 6-position of the flavin.

Characterization of two active site mutations of thioredoxin reductase from Escherichia coli.

Thioredoxin reductase (TRR), a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes, undergoes two sequential thiol-disulfide interchange reactions with thioredoxin during catalysis. In order to assess the catalytic role of each nascent thiol of the active site disulfide of thioredoxin reductase, the 2 cysteines (Cys-136 and Cys-139) forming this disulfide have been individually changed to serines by site-directed mutageneses of the cloned trxB gene of Escherichia coli. Spectral analyses of TRR(Ser-136,Cys-139) as a function of pH and ionic strength have revealed two pKa values associated with the epsilon 456, one of which increases from 7.0 to 8.3 as the ionic strength is increased, and a second at 4.4 which is seen only at high ionic strength. epsilon 458 of wild type TRR(Cys-136,Cys-139) and epsilon 453 of TRR(Cys-136,Ser-139) are pH-independent. A charge transfer complex (epsilon 530 = 1300 M-1 cm-1), unique to TRR(Ser-136,Cys-139), has been observed under conditions of high ammonium cation concentration (apparent Kd = 54 microM) at pH 7.6. These results suggest the assignment of Cys-139 as the FAD-interacting thiol in the reduction of thioredoxin by NADPH via thioredoxin reductase. If, as with other members of this enzyme family, the two distinct catalytic functions are each carried out by a different nascent thiol, then Cys-136 would perform the initial thiol-disulfide interchange with thioredoxin. Steady state kinetic analyses of the proteins have revealed turnover numbers of 10 and 50% of the value of the wild type enzyme for TRR(Ser-136,Cys-139) and TRR(Cys-136,Ser-139), respectively, and no changes in the apparent Km values of TR(S2) or NADPH. The finding of activity in the mutants indicates that the remaining thiol can carry out interchange with the disulfide of thioredoxin, and the resulting mixed disulfide can be reduced by NADPH via the flavin.

Energetics of the oxidase activity of NADPH-cytochrome P-450 reductase.

Oxidase activity of rat liver microsomes has been evaluated microcalorimetrically. delta H for the oxidation of reduced nicotine adenine dinucleotide phosphate was measured to be -39.33 (uncertainty 2.18) kcal/mol. The contribution of peroxide reduction and NADPH ring opening heats to this value have been considered. Calorimetric assay of the hydroxylase activity of the P-450-reductase-hydroxylase enzymatic complex has been demonstrated using the organocarbamate, Isolan (1-isopropyl-3-methyl-5-pyrazolyl dimethylcarbamate), as substrate.