Further evaluation of the potential anxiolytic activity of imidazo[1,5-a][1,4]diazepin agents selective for α2/3-containing GABAA receptors.

Positive allosteric modulators of GABAA receptors transduce a host of beneficial effects including anxiolytic actions. We have recently shown that bioavailability and anxiolytic-like activity can be improved by eliminating the ester functionality in imidazo[1,5-a][1,4]diazepines. In the present series of experiments, we further substantiate the value of heterocyle replacement of the ester for potential treatment of anxiety. None of three esters was active in a Vogel conflict test in rats that detects anxiolytic drugs like diazepam. Compounds 7 and 8, ester bioisosters, were selective for alpha 2 and 3 over alpha 1-containing GABAA receptors but also had modest efficacy at GABAA alpha 5-containing receptors. Compound 7 was efficacious and potent in this anxiolytic-detecting assay without affecting non-punished responding. The efficacies of the esters and of compound 7 were predicted from their efficacies as anticonvulsants against the GABAA antagonist pentylenetetrazole (PTZ). In contrast, the related structural analog, compound 8, did not produce anxiolytic-like effects in rats despite anticonvulsant efficacy. These data thus support the following conclusions: 1) ancillary pharmacological actions of compound 8 might be responsible for its lack of anxiolytic-like efficacy despite its efficacy as an anticonvulsant 2) esters of imidazo[1,5-a][1,4]diazepines do not demonstrate anxiolytic-like effects in rats due to their low bioavailability and 3) replacement of the ester function with suitable heterocycles markedly improves bioavailability and engenders molecules with the opportunity to have potent and efficacious effects in vivo that correspond to human anxiolytic actions.

Specificity and mechanism analysis of hepatitis C virus RNA-dependent RNA polymerase.

The RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene has been expressed as a nonfusion protein in bacterial cells and purified to homogeneity using sequential chromatographic columns. The purified NS5B protein exhibited RNA-dependent RNA polymerase activity using poly(A) template and the K(m) and V(max) were determined as 8.4 microM and 1976 pmol/mg-min, respectively. This full-length NS5B protein exhibited much stronger binding affinity toward the 30-mer poly(G) than other homopolymeric RNAs of the same size. For the first time, we demonstrate that the HCV NS5B was able to bind various ribonucleotides. Using a panel of oligonucleotides varying in length, we studied the NS5B catalytic efficiency and proposed the size of the NS5B active site to be 8-10 nucleotides. The multifunctional nature of NS5B protein is also discussed and compared with other viral RNA polymerases.

Identification and characterization of human rhinovirus-14 3C protease deamidation isoform.

A purified recombinant human rhinovirus-14 3C protease preparation contained only approximately 50% active enzyme as titrated using specifically designed irreversible 3C protease inhibitors. Analysis of the purified 3C protein by isoelectric focusing showed differently charged 3C isoforms that had isoelectric points (pI) of 8.3 (55%) and 9.0 (45%), with the latter one being consistent with the predicted pI of the human rhinovirus-14 3C protein. Further analysis indicated that the pI 8.3 protein was the deamidated form of 3C, and it displayed approximately 10-fold reduced cleavage activity relative to the original 3C protease sample. Peptide mapping followed by sequence analysis revealed that a single asparagine, Asn-164, was deamidated to aspartic acid in the pI 8.3 isoform. Converting Asn-164 to Asp by site-directed mutagenesis resulted in a mutated 3C protease with extremely low activity, as seen with the pI 8.3 isoform, indicating a role of Asn-164 in substrate recognition and binding. In addition, the deamidated 3C protease was found to be present in vivo, and its abundance was related to the viral replication cycle. Moreover, mutant virus carrying Asp-164 showed reduced viability in infected cells. Taken together, our data suggest that 3C protein deamidation plays a role in the regulation of its enzymatic activity.

Expression and purification of recombinant rhinovirus 14 3CD proteinase and its comparison to the 3C proteinase.

Human rhinovirus (HRV) is a positive-stranded RNA virus with an open reading frame that encodes for a single polyprotein of about 3000 amino acids. The HRV polyprotein is proteolytically processed; eight of nine cleavages are catalyzed by the 3C and/or the 3CD proteinases. We have expressed and purified recombinant HRV14 3C and 3CD proteinases and investigated their substrate selectivity and inhibitor sensitivity. Expressed 3CD proteinase had the P1/P1' residues of the 3C/3D cleavage site mutated from Gln/Gly to Ala/Ala in order to prevent autocleavage. The 3CD proteinase activities were measured by utilization of native, chromogenic, and fluorogenic peptide substrates. The 3CD proteinase exhibited < or =15% activity, compared to 3C, toward peptidyl p-nitroanilide substrates which contain only the p-nitroaniline moiety in the prime side. The 3C and 3CD proteinases exhibited similar activities for both internally quenched fluorogenic and native peptides. These results suggest that the two enzymes have similar but slightly different substrate specificity, especially on their preference for prime side residues. Inhibitor sensitivities toward classical proteinase inhibitors were generally similar for both enzymes. Small peptidyl inhibitors, specifically designed and synthesized for HRV14 3C, also inhibited the 3CD proteinase. Taken together, our data indicate that the 3D domain of 3CD proteinase had some influence on substrate recognition, but did not have dramatic impact on its interaction with inhibitors.

Human cytomegalovirus proteinase: candidate glutamic acid identified as third member of putative active-site triad.

The human cytomegalovirus (HCMV) proteinase is synthesized as a 709-amino-acid precursor that undergoes at least three autoproteolytic cleavages. The mature proteinase, called assemblin, is one of the products of autoproteolysis and is composed of the first 256 amino acids of the precursor. HCMV assemblin and its homologs in other herpes group viruses contain five highly conserved domains (CD1 through CD5). An absolutely conserved serine in CD3 has been shown by site-directed mutagenesis of the simian cytomegalovirus (SCMV) and herpes simplex virus type 1 (HSV-1) enzymes and by inhibitor affinity labeling of the HSV-1 and HCMV enzymes to be the active-site nucleophile of assemblin. An absolutely conserved histidine in CD2 has also been demonstrated by site-directed mutagenesis of the SCMV and HSV-1 enzymes to be essential for proteolytic activity and has been proposed to be a second member of the catalytic triad of this serine proteinase. We report here the use of site-directed mutagenesis to investigate the active-site amino acids of HCMV assemblin. Substitutions were made for the CD3 serine and CD2 histidine residues implicated as active-site components, and for other amino acids whose influence on enzyme activity was of interest. The mutant proteinases were tested in a transient transfection assay for their ability to cleave their natural substrate, the assembly protein precursor. Results of these experiments verified that HCMV CD3 serine (Ser-132) and CD2 histidine (His-63) are essential for proteolytic activity and identified a glutamic acid (Glu-122) within CD3 that is also essential for proteolytic activity and may be conserved among all herpesvirus assemblin homologs. We suggest that CD3 Glu-122, CD3 Ser-132, and CD2 His-63 constitute the active-site triad of this serine proteinase.

Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites.

The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein, subjected to the same activation conditions as the 28-kDa proteinase, had no enzymatic activity against the HCMV M- and R-site peptide substrates.

One-step purification of recombinant human papillomavirus type 16 E7 oncoprotein and its binding to the retinoblastoma gene product.

Six derivatives of the human papillomavirus type 16 E7 gene were constructed, each of which encoded a peptide at the amino terminus of the E7 protein capable of chelating metal ions, for use in subsequent purification of the proteins by chelating peptide-immobilized metal ion affinity chromatography (CP-IMAC). Five of the six chelating peptides examined contained six amino acids, and each varied in the number of histidine and tryptophan residues. The CP-E7 proteins were expressed in Escherichia coli, and the cysteine residues were blocked in the form of S-sulfonate groups. This series of CP-E7 proteins was purified in a single chromatographic step using CP-IMAC. The efficiency of purification correlated with the number of histidine and tryptophan residues present in the CP. The purified CP-E7 proteins bound to the human retinoblastoma gene product, pRB, in in vitro co-immunoprecipitation assays and immobilized CP-E7 binding assays. The efficiency of E7-pRB binding was not altered by the presence of a CP at the N-terminus of E7 nor by the S-sulfonate groups within E7.

Isolation, chromosomal mapping, and expression of the mouse tyrosinase gene.

Using a human tyrosinase cDNA probe, we have isolated mouse tyrosinase genomic clones and used them to map the mouse tyrosinase locus and to analyze the promoter sequence of the tyrosinase gene. Southern blot analyses of DNA from somatic cell hybrids, interspecies backcross mice, and albino deletion mice have revealed that the locus for mouse tyrosinase resides at or near the albino locus on mouse chromosome 7. There were three TATA-elements, but only one CAT-element, and the CAT-element appeared to be paired with the third TATA-element, located at the position farthest upstream. Mouse tyrosinase mRNA is approximately 2.4 Kb in size. The amount of tyrosinase mRNA reflects the levels of tyrosinase activity in normal melanocytes and Cloudman S-91 melanoma cell line.

Expression characteristics of two potential T cell mediator genes.

T lymphocyte subset-specific cDNA clones were recently isolated by a modified differential screening procedure. The expression patterns of two of these cDNAs, designated as 4-1BB and L2G25B, were studied in greater detail. Nucleotide sequence comparison revealed that 4-1BB was not previously recognized. Although the L2G25B sequence had been recognized recently, the function of the encoded molecule has yet to be well studied. The transcripts of the two cDNAs were inducible by concanavalin A in mouse spleen cells, cloned helper T cells (L2), cloned cytolytic T cells (L3), and cytolytic T cell hybridomas. They were also inducible with stimulation through antigen receptor (TCR), with immobilized anti-TCR antibody in cloned T cells L2, dB45, and L3. Concanavalin A inducibility was inhibited by cyclosporin A. They were not inducible by IL-2 stimulation. The expression patterns of these transcripts were similar to those of IFN-gamma, except that the level of transcripts of the two cDNAs was at least fivefold lower than that of IFN-gamma, and the peak level of expression occurred earlier. These data suggest that L2G25B and 4-1BB may represent new T cell mediators.

The structure of the mouse lymphocyte pore-forming protein perforin.

Purified murine lymphocyte pore-forming protein (PFP or perforin) was partially sequenced. Oligonucleotides synthesized on the basis of this sequence information were used to screen a murine cytotoxic T lymphocyte (CTL) cDNA library. Seven clones were obtained, two of which were sequenced, providing full-length sequence information on PFP. Murine PFP (534 a.a.) is 68% identical to human PFP. Hydropathic analysis revealed a predominantly hydrophilic protein with some hydrophobic domains, including a region (a.a. 191-251) that could contain putative membrane-spanning domains. PFP is approx. 20% identical to human C7, C8 and C9 within a region encompassing 270 a.a., confirming previous immunological cross-reactivity studies. Northern blot analysis showed that expression of PFP but not of a serine esterase transcript is enhanced in a CTL line by antigen receptor-stimulation. Southern blot analysis of mouse genomic DNA indicated that PFP is encoded as a single-copy gene with the coding region contained within 10 kilobases of genomic DNA.

Isolation and sequence analysis of serine protease cDNAs from mouse cytolytic T lymphocytes.

Three new cDNA clones (designated MCSP-1, MCSP-2, and MCSP-3) encoding mouse serine proteases were isolated from cloned cytolytic T lymphocytes (CTL) by a modified differential screening procedure. The putative mature proteins of MCSP-2 and MCSP-3 are each composed of 228 amino acids with molecular weights of 25,477 and 25,360, respectively. NH2-terminal amino acids of MCSP-2- and MCSP-3-predicted proteins were identical to those reported for granzyme E and F, respectively. The third species, MCSP-1, was closely related to the two other cDNA species but approximately 30 amino acids equivalents of the NH2-terminal portion of the cDNA were not cloned. The amino acids forming the active sites of serine proteases were well conserved among the three predicted proteins. The active site pocket residue positioned six residues before the active-site Ser184 is alanine in MCSP-1, threonine in MCSP-2, and serine in MCSP-3, indicating that both MCSP-2 and MCSP-3 may have chymotrypsin-like specificity. There are three potential asparagine-linked glycosylation sites in MCSP-1 and MCSP-3, and four in MCSP-2-deduced amino acid sequences. Amino acid comparison of MCSP-1 with four other reported serine proteases whose active site pocket residue is alanine revealed that MCSP-1 was substantially different from the other molecules, indicating that MCSP-1 may be a new member of mouse T cell serine protease family. Antibodies made against a MCSP-1 lacZ gene fusion protein stain granules of CTL and react on immunoblots with two distinct granule protein bands of 29 and 35-40 kD. Only the 35-kD species labels with [3H]DFP. Since a protease cascade may play a key role in cytolytic lymphocyte activation, our isolation of cDNAs representative of unique serine esterases should help to investigate such a cascade process.

Sequence analysis of mouse tyrosinase cDNA and the effect of melanotropin on its gene expression.

Using human tyrosinase cDNA as a probe, a mouse tyrosinase cDNA clone representing approximately 75% of the tyrosinase coding region and a mouse genomic clone which includes the tyrosinase 5' coding sequences were isolated: nucleotide and deduced amino acid sequence of the mouse tyrosinase gene were determined from these clones. The predicted amino acid sequence revealed that the mouse tyrosinase is composed of 533 amino acids with a molecular weight of 60,536. The deduced protein contains 6 potential N-glycosylation sites, two cysteine- and two histidine-rich regions which may serve as copper-binding sites, a potential signal and transmembrane sequences. The mouse and human tyrosinase nucleotide and deduced amino acid sequences are approximately 81% homologous. The level of mouse tyrosinase mRNA was elevated after stimulation of Cloudman S-91 melanoma cells with melanotropin and isobutylmethylxanthine and the level of transcript reflected that of tyrosinase activity and melanin content in the cells.