Reporter gene expression from LTR-circles as tool to identify HIV-1 integrase inhibitors.

Early HIV-1 integrase inhibitors, such as compounds containing a ß-diketo acid moiety, were identified by extensive high-throughput screening campaigns. Traditionally, in vitro biochemical assays, measuring the catalytic activities of integrase, have been used for this purpose. However, these assays are confounded by the absence of cellular processes or cofactors that play a role in the integration of HIV-1 DNA in the cellular genome. In contrast to regular cell-based virus inhibition assays, which targets all steps of the viral replication cycle, a novel cellular screening assays was developed to enable the specific identification of integrase inhibitors, employing a readout that is linked with the inhibition of integrase activity. Therefore, a HIV-1 lentiviral vector equipped with the enhanced green fluorescent protein (eGFP) reporter gene was used to detect expression from extrachromosomal viral DNA (1- or 2-long terminal repeat circles), formed when integration of vector DNA into the cellular genome is prevented by an integrase inhibitor. In this assay, eGFP expression from the low residual level of transcriptional activity of extrachromosomal DNA was measured via high-throughput flow cytometry. An algorithm for analysis of eGFP expression histograms enabled the specific identification of integrase inhibitors. This assay is amenable for high throughput screening to identify inhibitors of HIV-1 integrase.

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.

Effect of naturally occurring active site mutations on hepatitis C virus NS3 protease specificity.

A comparison of the DNA sequences from all available genotypes of HCV indicate that the active site residues of the NS3 protease are strictly conserved with the exception of positions 123 and 168, which border the S(4) subsite. In genotype 3, the canonic arginine and aspartic acid have been replaced with threonine and glutamine, respectively. To determine if these differences contribute to an altered specificity, we characterized single-chain NS3 proteases from strains 1a, 1b, and 3a with peptide substrates and product inhibitors on the basis of the natural cleavage junction sequences, in addition to polyprotein substrates derived from the 1a strain. No statistically significant differences in specificity were observed. To demonstrate that the active sites were actually different, we generated and evaluated peptide substrates with unnatural extended side-chains. These studies confirmed that there are measurable differences between the NS3 proteases of genotypes 1 and 3. Specifically, a 5-fold difference in K(i) was observed between the proteases from genotypes 1 and 3 when a D-Glu occupied P(5), and a 30-fold difference was seen when this position contained a D-homoglutamate. The contribution of residues 123 and 168 toward the altered specificity was then evaluated individually by site-directed mutagenesis. These mutants showed that potency differences within this series could be attributed to the residue that occupied position 123 of the protease. Modeling these unnatural substrate/mutant protease interactions, on the basis of cocrystal structures of enzyme-substrate complexes, provides a structural basis for these observations. Proteins 2001;43:82-88.

Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3).

The T7 polymerase-based pET System is one of the most powerful and widely used prokaryotic expression systems available today. Expression of even slightly toxic gene products in BL21 (DE3), however, has been problematic due to basal expression, which leads to decreased plasmid stability and variable yields following large-scale growth and induction. Use of host strains such as BL21 (DE3) pLysS provides high stringency and consistent expression but typically at the cost of reduced protein levels upon induction. The experiments presented here suggest that catabolite repression can effectively reduce basal expression of the T7 polymerase gene in BL21 (DE3), yielding tight regulation and consistency comparable to that of BL21 (DE3) pLysS. By switching to a poor carbon source for the final growth cycles, the higher expression levels typical of BL21 (DE3) can readily be obtained upon induction.

Crystal structure of an inhibitor complex of the 3C proteinase from hepatitis A virus (HAV) and implications for the polyprotein processing in HAV.

The proteolytic processing of the viral polyprotein is an essential step during the life cycle of hepatitis A virus (HAV), as it is in all positive-sense, single-stranded RNA viruses of animals. In HAV the 3C proteinase is the only proteolytic activity involved in the polyprotein processing. The specific recognition of the cleavage sites by the 3C proteinase depends on the amino acid sequence of the cleavage site. The structure of the complex of the HAV 3C proteinase and a dipeptide inhibitor has been determined by X-ray crystallography. The double-mutant of HAV 3C (C24S, F82A) was inhibited with the specific inhibitor iodoacetyl-valyl-phenylalanyl-amide. The resulting complex had an acetyl-Val-Phe-amide group covalently attached to the S(gamma) atom of the nucleophilic Cys 172 of the enzyme. Crystals of the complex of HAV 3C (C24S, F82A) acetyl-Val-Phe-amide were found to be monoclinic, space group P2(1), having 4 molecules in the asymmetric unit and diffracting to 1.9-A resolution. The final refined structure consists of 4 molecules of HAV 3C (C24S,F82A) acetyl-Val-Phe-amide, 1 molecule of DMSO, 1 molecule of glycerol, and 514 water molecules. There are considerable conformational differences among the four molecules in the asymmetric unit. The final R-factor is 20.4% for all observed reflections between 15.0- and 1.9-A resolution and the corresponding R(free) is 29.8%. The dipeptide inhibitor is bound to the S(1)(') and S(2)(') specificity subsites of the proteinase. The crystal structure reveals that the HAV 3C proteinase possesses a well-defined S(2)(') specificity pocket and suggests that the P(2)(') residue could be an important determinant for the selection of the primary cleavage site during the polyprotein processing in HAV.

Synthesis and testing of azaglutamine derivatives as inhibitors of hepatitis A virus (HAV) 3C proteinase.

Hepatitis A virus (HAV) 3C proteinase is a picornaviral cysteine proteinase that is essential for cleavage of the initially synthesized viral polyprotein precursor to mature fragments and is therefore required for viral replication in vivo. Since the enzyme generally recognizes peptide substrates with L-glutamine at the P1 site, four types of analogues having an azaglutamine residue were chemically synthesized: hydrazo-o-nitrophenylsulfenamides A (e.g. 16); frame-shifted hydrazo-o-nitrophenylsulfenamides B (e.g. 25-28); the azaglutamine sulfonamides C (e.g. 7, 8, 11, 12); and haloacetyl azaglutamine analogues 2 and 3. Testing of these compounds for inhibition of the HAV 3C proteinase employed a C24S mutant in which the non-essential surface cysteine was replaced with serine and which displays identical catalytic parameters to the wild-type enzyme. Sulfenamide 16 (type A) showed no significant inhibition. Sulfenamide 27 (type B) had an IC50 of ca 100 microM and gave time-dependent inactivation of the enzyme due to disulfide bond formation with the active site cysteine thiol, as demonstrated by electrospray mass spectrometry. Sulfonamide 8 (type C) was a weak competitive inhibitor with an IC50 of approximately 75 microM. The haloacetyl azaglutamine analogues 2 and 3 were time-dependent irreversible inactivators of HAV 3C proteinase with rate constants k(obs)/[I] of 680 M(-1) s(-1) and 870 M(-1) s(-1), respectively, and were shown to alkylate the active site thiol.

A continuous spectrophotometric assay for the hepatitis C virus serine protease.

The hepatitis C virus (HCV) encodes a chymotrypsin-like serine protease responsible for the processing of HCV nonstructural proteins and which is a promising target for antiviral intervention. Its relatively low catalytic efficiency has made standard approaches to continuous assay development only modestly successful. In this report, four continuous spectrophotometric substrates suitable for both high-throughput screening and detailed kinetic analysis are described. One of these substrates, Ac-DTEDVVP(Nva)-O-4-phenylazophenyl ester, is hydrolyzed by HCV protease with a second-order rate constant (kcat/Km) of 80,000 +/- 10,000 M-1 s-1. Together with its negligible rate of nonenzymatic hydrolysis under assay conditions (0.01 h-1), analysis of as little as 2 nM protease can be completed in under 10 min.

Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis.

We developed a two-stage procedure, based on the QuikChange Site-Directed Mutagenesis Protocol, that significantly expands its application to a variety of gene modification experiments. A pre-PCR, single-primer extension stage before the standard protocol allows the efficient introduction of not only point mutation but also multiple mutations and deletions and insertions to a sequence of interest.

Construction, expression, and characterization of a novel fully activated recombinant single-chain hepatitis C virus protease.

Efficient proteolytic processing of essential junctions of the hepatitis C virus (HCV) polyprotein requires a heterodimeric complex of the NS3 bifunctional protease/helicase and the NS4A accessory protein. A single-chain recombinant form of the protease has been constructed in which NS4A residues 21-32 (GSVVIVGRIILS) were fused in frame to the amino terminus of the NS3 protease domain (residues 3-181) through a tetrapeptide linker. The single-chain recombinant protease has been overexpressed as a soluble protein in E. coli and purified to homogeneity by a combination of metal chelate and size-exclusion chromatography. The single-chain recombinant protease domain shows full proteolytic activity cleaving the NS5A-5B synthetic peptide substrate, DTEDVVCCSMSYTWTGK with a Km and k(cat) of 20.0 +/- 2.0 microM and 9.6 +/- 2.0 min(-1), respectively; parameters identical to those of the authentic NS3(1-631)/NS4A(1-54) protein complex generated in eukaryotic cells (Sali DL et al., 1998, Biochemistry 37:3392-3401).

Construction of transforming growth factor alpha (TGF-alpha) phage library and identification of high binders of epidermal growth factor receptor (EGFR) by phage display.

TGF-alpha, a 50 amino acid growth factor containing 3 disulfide bonds, was fused to the N-terminal domain of the pIII protein of fusN, a derivative of phagemid fd-tet, to form a TGF-alpha phage. The fusion phage showed binding activity to epidermal growth factor receptor (EGFR). A library of approximately 4 x 10(7) variants of TGF-alpha was generated with substitutions of total of 10 amino acids located in the C-loop region. This C-loop subdomain of TGF-alpha consists of a small antiparallel double hairpin structure involving interactions between intra-polypeptide segments. Mutants isolated from the phage library with greatly increased binding affinity were selected through panning with A431 cells (a cell line expressing an elevated number of EGFRs). Following two rounds of stringent selection, variant phages with higher binding affinity than wild type TGF-alpha were identified and the phage DNAs were sequenced for the alignment analysis. Absolute selection at position 42 as Arg, preferential selection at position 38 and 45 as Tyr or Phe with aromatic side chain and selection at position 41 with acidic residues, were obtained. Although an amino acid residue with smaller side chain at position 35 and one with larger side chain at position 36 were preferred, the steric hindering of the structure in side chains was minimized between these adjacent amino acids.

In vitro and ex vivo inhibition of hepatitis A virus 3C proteinase by a peptidyl monofluoromethyl ketone.

Hepatitis A virus (HAV) 3C proteinase is the enzyme responsible for the processing of the viral polyprotein. Although a cysteine proteinase, it displays an active site configuration like those of the mammalian serine proteinases (Malcolm, B. A. Protein Science 1995, 4, 1439). A peptidyl monofluoromethyl ketone (peptidyl-FMK) based on the preferred peptide substrates for HAV 3C proteinase was generated by first coupling the precursor, N,N-dimethylglutamine fluoromethylalcohol, to the tripeptide, Ac-Leu-Ala-Ala-OH, and then oxidizing the product to the corresponding peptidyl-FMK (Ac-LAAQ'-FMK). This molecule was found to be an irreversible inactivator of HAV 3C with a second-order rate constant of 3.3 x 10(2) M-1 s-1. 19F NMR spectroscopy indicates the displacement of fluoride on inactivation of the enzyme by the fluoromethyl ketone. NMR spectroscopy of the complex between the 13C-labeled inhibitor and the HAV 3C proteinase indicates that an (alkylthio)methyl ketone is formed. Studies of polyprotein processing, using various substrates generated by in vitro transcription/translation, demonstrated efficient blocking of even the most rapid proteolytic events such as cleavage of the 2A-2B and 2C-3A junctions. Subsequent ex vivo studies, to test for antiviral activity, show a 25-fold reduction in progeny virus production as the result of treatment with 5 microM inhibitor 24 h post-infection.

Active site properties of the 3C proteinase from hepatitis A virus (a hybrid cysteine/serine protease) probed by Raman spectroscopy.

Although the HAV 3C proteinase is a cysteine protease, it displays an active site configuration which resembles mammalian serine proteases and is structurally distinct from the papain superfamily of thiol proteases. Given the interesting serine/cysteine protease hybrid nature of HAV 3C, we have probed its active site properties via the Raman spectra of the acyl enzyme, 5-methylthiophene acryloyl HAV 3C, using the C24S variant of the enzyme to obtain stoichiometric acylation. The Raman difference spectral data show that the major population of the acyl groups in the active site experiences electron polarization intermediate between that in the papain superfamily and that in a nonpolarizing site. This is evidenced by the values of the acyl group ethylenic stretching frequency which occur near 1602 cm(-1) in a nonpolarizing environment, at 1588 cm(-1) when bound to HAV 3C (C24S), and at 1579 cm(-1) in acyl papains. The value of the electronic absorption maximum for the HAV 3C (C24S) acyl enzyme and the deacylation rate constant fit the correlation developed for the papain superfamily, suggesting that for HAV 3C too, polarizing forces in the active site can contribute to rate acceleration via transition state stabilization. The major population in the active site is s-cis about the acyl group's C1-C2 bond, but there is a second population that is s-trans, and this secondary population is not polarized. The two populations are evidenced by the presence of two sets of marker bands for s-cis and s-trans in the Raman spectra, which occur principally in the C=C stretching region near 1600 cm(-1), in the C-C stretching region near 1100 cm(-1), and near 560 cm(-1). The positions of the acyl carbonyl features in the Raman spectra point to hydrogen-bonding strengths of 20-25 kJ mol(-1) between the C=O and H-bonding donors in the active site. The 5-methylthiophene acryloyl HAV 3C (C24S) is a relatively unreactive acyl enzyme, deacylating with a pKa of 7.1 and a rate constant of 0.000 31 s(-1) at pH 9. Unlike most other cysteine or serine protease acyl enzymes characterized by Raman spectroscopy, no changes in the Raman spectrum could be detected with changes in pH.

The refined crystal structure of the 3C gene product from hepatitis A virus: specific proteinase activity and RNA recognition.

The virally encoded 3C proteinases of picornaviruses process the polyprotein produced by the translation of polycistronic viral mRNA. The X-ray crystallographic structure of a catalytically active mutant of the hepatitis A virus (HAV) 3C proteinase (C24S) has been determined. Crystals of this mutant of HAV 3C are triclinic with unit cell dimensions a = 53.6 A, b = 53.5 A, c = 53.2 A, alpha = 99.1 degrees, beta = 129.0 degrees, and gamma = 103.3 degrees. There are two molecules of HAV 3C in the unit cell of this crystal form. The structure has been refined to an R factor of 0.211 (Rfree = 0.265) at 2.0-A resolution. Both molecules fold into the characteristic two-domain structure of the chymotrypsin-like serine proteinases. The active-site and substrate-binding regions are located in a surface groove between the two beta-barrel domains. The catalytic Cys 172 S(gamma) and His 44 N(epsilon2) are separated by 3.9 A; the oxyanion hole adopts the same conformation as that seen in the serine proteinases. The side chain of Asp 84, the residue expected to form the third member of the catalytic triad, is pointed away from the side chain of His 44 and is locked in an ion pair interaction with the epsilon-amino group of Lys 202. A water molecule is hydrogen bonded to His 44 N(delta1). The side-chain phenolic hydroxyl group of Tyr 143 is close to this water and to His 44 N(delta1) and may be negatively charged. The glutamine specificity for P1 residues of substrate cleavage sites is attributed to the presence of a highly conserved His 191 in the S1 pocket. A very unusual environment of two water molecules and a buried glutamate contribute to the imidazole tautomer believed to be important in the P1 specificity. HAV 3C proteinase has the conserved RNA recognition sequence KFRDI located in the interdomain connection loop on the side of the molecule diametrically opposite the proteolytic site. This segment of polypeptide is located between the N- and C-terminal helices, and its conformation results in the formation of a well-defined surface with a strongly charged electrostatic potential. Presumably, this surface of HAV 3C participates in the recognition of the 5' and 3' nontranslated regions of the RNA genome during viral replication.

Epitope mapping of prostate-specific antigen with monoclonal antibodies.

Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. We identified and characterized the epitopes of two anti-PSA monoclonal antibodies (mAbs) designated B80 and B87. The epitopes were initially mapped as nonoverlapping by developing a sandwich immunoassay to measure PSA with the two anti-PSA mAbs. The two antibodies do not cross-react with homologous pancreatic kallikrein, but recognize epitopes unique to PSA. B80 and B87 can recognize both free and complexed PSA and hence measure total PSA. Epitope scanning and bacteriophage peptide library affinity selection procedures were used to identify and locate an epitope on PSA. A possible epitope for B80 was identified as being located on or near PSA amino acid residues 50-58 (-GRH-SLFHP-). The epitope for B87 was likely on an exposed nonlinear conformational determinant, unique to PSA, and not masked by the binding of B80 or alpha 1-antichymotrypsin.

Peptide rescue of an N-terminal truncation of the Stoffel fragment of taq DNA polymerase.

Deletion of the first 289 amino acids of the DNA polymerase from Thermus aquaticus (Taq polymerase) removes the 5' to 3' exonuclease domain to yield the thermostable Stoffel polymerase fragment (Lawyer et al., 1989). Preliminary N-terminal truncation studies of the Stoffel fragment suggested that removal of an additional 12 amino acids (the Stof delta 12 mutant) had no significant effect on activity or stability, but that the further truncation of the protein (the Stof delta 47, in which 47 amino acids were deleted), resulted in a significant loss of both activity and thermostability. A 33-amino acid synthetic peptide, based on this critical region (i.e., residues 303-335 inclusive), was able to restore 85% of the Stof delta 12 activity when added back to the truncated Stof delta 47 protein as well as return the temperature optimum to that of the Stof delta 12 and Stoffel proteins. Examination of the crystal structure of Taq polymerase (Kim et al., 1995) shows that residues 302-336 of the enzyme form a three-stranded beta-sheet structure that interacts with the remainder of the protein. CD analysis of the 33-amino acid peptide indicates that the free peptide also adopts an ordered structure in solution with more than 50% beta-sheet content. These data suggest that this 33-amino acid peptide constitutes a stable beta-sheet structure capable of rescuing the truncated polymerase in a fashion analogous to the well-documented complementation of Ribonuclease S protein by the 15-residue, alpha-helical, S peptide.

The picornaviral 3C proteinases: cysteine nucleophiles in serine proteinase folds.

The 3C proteinases are a novel group of cysteine proteinases with a serine proteinase-like fold that are responsible for the bulk of polyprotein processing in the Picornaviridae. Because members of this viral family are to blame for several ongoing global pandemic problems (rhinovirus, hepatitis A virus) as well as sporadic outbreaks of more serious pathologies (poliovirus), there has been continuing interest over the last two decades in the development of antiviral therapies. The recent determination of the structure of two of the 3C proteinases by X-ray crystallography opens the door for the application of the latest advances in computer-assisted identification and design of anti-proteinase therapeutic/chemoprophylactic agents.

Peptide aldehyde inhibitors of hepatitis A virus 3C proteinase.

Picornaviral 3C proteinases are a group of closely related thiol proteinases responsible for processing of the viral polyprotein into its component proteins. These proteinases adopt a chymotrypsin-like fold [Allaire et al. (1994) Nature 369, 72-77; Matthews et al. (1994) Cell 77, 761-771] and a display an active-site configuration like those of the serine proteinases. Peptide-aldehydes based on the preferred peptide substrates for hepatitis A virus (HAV) 3C proteinase were synthesized by reduction of a thioester precursor. Acetyl-Leu-Ala-Ala-(N,N'-dimethylglutaminal) was found to be a reversible, slow-binding inhibitor for HAV 3C with a Ki* of (4.2 +/- 0.8) x 10(-8) M. This inhibitor showed 50-fold less activity against the highly homologous human rhinovirus (strain 14) 3C proteinase, whose peptide substrate specificity is slightly different, suggesting a high degree of selectivity. NMR spectrometry of the adduct of the 13C-labeled inhibitor with the HAV-3C proteinase indicate that a thiohemiacetal is formed between the enzyme and the aldehyde carbon as previously noted for peptide-aldehyde inhibitors of papain [Lewis & Wolfenden (1977) Biochemistry 16,4890-4894; Gamcsik et al. (1983) J. Am. Chem. Soc. 105, 6324-6325]. The adduct can also be observed by electrospray mass spectrometry.

Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases.

The picornavirus family includes several pathogens such as poliovirus, rhinovirus (the major cause of the common cold), hepatitis A virus and the foot-and-mouth disease virus. Picornaviral proteins are expressed by direct translation of the genomic RNA into a single, large polyprotein precursor. Proteolysis of the viral polyprotein into the mature proteins is assured by the viral 3C enzymes, which are cysteine proteinases. Here we report the X-ray crystal structure at 2.3 A resolution of the 3C proteinase from hepatitis A virus (HAV-3C). The overall architecture of HAV-3C reveals a fold resembling that of the chymotrypsin family of serine proteinases, which is consistent with earlier predictions. Catalytic residues include Cys 172 as nucleophile and His 44 as general base. The 3C cleavage specificity for glutamine residues is defined primarily by His 191. The overall structure suggests that an intermolecular (trans) cleavage releases 3C and that there is an active proteinase in the polyprotein.

Hepatitis A virus 3C proteinase: some properties, crystallization and preliminary crystallographic characterization.

Several isoforms of the wild-type and three mutant hepatitis A virus (HAV) 3C proteinases have been isolated and characterized. The active site cysteine residue (residue 172) was found to be responsible for the formation of some of these isoforms. The double mutant C24S/C172A of the HAV 3C proteinase, in which both cysteine residues have been replaced by site-directed mutagenesis, was crystallized. The crystals belong to the hexagonal space group P6(1)22 (or its enantiomorph, P6(5)22) with unit cell dimensions a = b = 65.2 A, c = 246.1 A and diffract X-rays to 2.3 A resolution.