In vitro resistance study of rupintrivir, a novel inhibitor of human rhinovirus 3C protease.

Rupintrivir (formerly AG7088) is an irreversible inhibitor of the human rhinovirus (HRV) 3C protease that has been demonstrated to have in vitro activity against all HRVs tested, consistent with its interaction with a strictly conserved subset of amino acids in the 3C protease. The potential for resistance was studied following in vitro serial passage of HRV serotypes 14, 2, 39, and Hanks in the presence of increasing rupintrivir concentrations. HRV variants with reduced susceptibilities to rupintrivir (sevenfold for HRV 14) or with no significant reductions in susceptibility but genotypic changes (HRV 2, 39, and Hanks) were initially isolated following 14 to 40 cumulative days in culture (three to six passages). Sequence analysis of the 3C protease identified one to three substitutions in diverse patterns but with common features (T129T/A, T131T/A, and T143P/S in HRV 14; N165T in HRV 2; N130N/K and L136L/F in HRV 39; T130A in HRV Hanks). Notably, three of the four HRV variants contained a substitution at residue 130 (residue 129 in HRV 14). Continued selection in the presence of escalating concentrations of rupintrivir (40 to 72 days) resulted in the accumulation of additional mutations (A121A/V and Y139Y/H in HRV 14, E3E/G and A103A/V in HRV 2, S105T in HRV 39), with only minimal further reductions in susceptibility (up to fivefold). The ability of specific substitutions to confer resistance was examined by susceptibility testing of HRV 14 variants constructed to contain 3C protease mutations. In summary, the slow accumulation of multiple amino acid substitutions with only minimal to moderate reductions in susceptibility highlight the advantages of 3C protease as an antiviral target.

Conservation of amino acids in human rhinovirus 3C protease correlates with broad-spectrum antiviral activity of rupintrivir, a novel human rhinovirus 3C protease inhibitor.

The picornavirus 3C protease is required for the majority of proteolytic cleavages that occur during the viral life cycle. Comparisons of published amino acid sequences from 6 human rhinoviruses (HRV) and 20 human enteroviruses (HEV) show considerable variability in the 3C protease-coding region but strict conservation of the catalytic triad residues. Rupintrivir (formerly AG7088) is an irreversible inhibitor of HRV 3C protease with potent in vitro activity against all HRV serotypes (48 of 48), HEV strains (4 of 4), and untyped HRV field isolates (46 of 46) tested. To better understand the relationship between in vitro antiviral activity and 3C protease-rupintrivir binding interactions, we performed nucleotide sequence analyses on an additional 21 HRV serotypes and 11 HRV clinical isolates. Antiviral activity was also determined for 23 HRV clinical isolates and four additional HEV strains. Sequence comparison of 3C proteases (n = 58) show that 13 and 11 of the 14 amino acids that are involved in side chain interactions with rupintrivir are strictly conserved among HRV and HEV, respectively. These sequence analyses are consistent with the comparable in vitro antiviral potencies of rupintrivir against all HRV serotypes, HRV isolates, and HEV strains tested (50% effective concentration range, 3 to 183 nM; n = 125). In summary, the conservation of critical amino acid residues in 3C protease and the observation of potent, broad-spectrum antipicornavirus activity of rupintrivir highlight the advantages of 3C protease as an antiviral target.

Design and synthesis of irreversible depsipeptidyl human rhinovirus 3C protease inhibitors.

Novel tripeptidyl C-terminal Michael acceptors with an ester replacement of the P(2)-P(3) amide bond were investigated as irreversible inhibitors of the human rhinovirus (HRV) 3C protease (3CP). When screened against HRV serotype-14 the best compound was shown to have very good 3CP inhibition (k(obs)/[I]=270,000M(-1)s(-1)) and potent in vitro antiviral activity (EC(50)=7.0nM).

Inhibition of human rhinovirus-induced cytokine production by AG7088, a human rhinovirus 3C protease inhibitor.

Symptom severity in patients with human rhinovirus (HRV)-induced respiratory illness is associated with elevated levels of the inflammatory cytokines interleukin-6 (IL-6) and IL-8. AG7088 is a novel, irreversible inhibitor of the HRV 3C protease. In this study, AG7088 was tested for its antiviral activity and ability to inhibit the production of IL-6 and IL-8 in a human bronchial epithelial cell line, BEAS-2B. Infection of BEAS-2B cells with HRV 14 resulted in the production of both infectious virus and the cytokines IL-6 and IL-8. Treatment of HRV 14-infected cells with AG7088 resulted in a statistically significant (P, <0.05) dose-dependent reduction in the levels of infectious virus as well as IL-6 and IL-8 released into the cell supernatant compared to the results obtained for compound-free infected cells. AG7088 was also able to inhibit the replication of HRV 2 and 16 in BEAS-2B cells. In time-of-addition studies, AG7088 could be added as late as 14 to 26 h after HRV 14 infection of BEAS-2B cells and still result in a statistically significant (P, <0.05) reduction in the levels of infectious virus, IL-6, and IL-8 compared to the results obtained for compound-free infected cells. These findings have implications for the development of an antirhinovirus agent that may not only block virus replication but also diminish symptoms.

Structure-based design of ketone-containing, tripeptidyl human rhinovirus 3C protease inhibitors.

Tripeptide-derived molecules incorporating C-terminal ketone electrophiles were evaluated as reversible inhibitors of the cysteine-containing human rhinovirus 3C protease (3CP). An optimized example of such compounds displayed potent 3CP inhibition activity (K = 0.0045 microM) and in vitro antiviral properties (EC50=0.34 microM) when tested against HRV serotype-14.

In vitro antiviral activity of AG7088, a potent inhibitor of human rhinovirus 3C protease.

AG7088 is a potent, irreversible inhibitor of human rhinovirus (HRV) 3C protease (inactivation rate constant (k(obs)/[I]) = 1,470,000 +/- 440,000 M(-1) s(-1) for HRV 14) that was discovered by protein structure-based drug design methodologies. In H1-HeLa and MRC-5 cell protection assays, AG7088 inhibited the replication of all HRV serotypes (48 of 48) tested with a mean 50% effective concentration (EC(50)) of 0.023 microM (range, 0.003 to 0.081 microM) and a mean EC(90) of 0.082 microM (range, 0.018 to 0.261 microM) as well as that of related picornaviruses including coxsackieviruses A21 and B3, enterovirus 70, and echovirus 11. No significant reductions in the antiviral activity of AG7088 were observed when assays were performed in the presence of alpha(1)-acid glycoprotein or mucin, proteins present in nasal secretions. The 50% cytotoxic concentration of AG7088 was >1,000 microM, yielding a therapeutic index of >12,346 to >333,333. In a single-cycle, time-of-addition assay, AG7088 demonstrated antiviral activity when added up to 6 h after infection. In contrast, a compound targeting viral attachment and/or uncoating was effective only when added at the initiation of virus infection. Direct inhibition of 3C proteolytic activity in infected cells treated with AG7088 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of radiolabeled proteins, which showed a dose-dependent accumulation of viral precursor polyproteins and reduction of processed protein products. The broad spectrum of antiviral activity of AG7088, combined with its efficacy even when added late in the virus life cycle, highlights the advantages of 3C protease as a target and suggests that AG7088 will be a promising clinical candidate.

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.

Cytotoxic T lymphocyte response to hepatitis C virus-derived peptides containing the HLA A2.1 binding motif.

The HLA class I-restricted cytotoxic T lymphocyte (CTL) response is a major defense mechanism in viral infections. It has been suggested that the CTL response may contribute to viral clearance and liver cell injury during hepatitis C virus (HCV) infection. To test this hypothesis requires an understanding of the characteristics of HCV-specific cytotoxic effector cells and identification of the target antigens to which they respond. To begin this process we stimulated peripheral blood mononuclear cells (PBMC) from a group of HLA-A2 positive patients with chronic hepatitis C with a panel of 130 HCV-derived peptides containing the HLA-A2 binding motif. Effector cells were tested for their capacity to lyse HLA-A2-matched target cells that were either sensitized with peptide or infected with a vaccinia virus construct containing HCV sequences. Using this approach we have identified nine immunogenic peptides in HCV, three of which are derived from the putative core protein, three from the nonstructural (NS) 3 domain, two from NS4 and one from NS5. Selected responses were shown to be HLA-A2 restricted, mediated by CD8+ T cells and to recognize endogenously synthesized viral antigen. Unexpectedly, peptide-specific CTL responses could also be induced in sero-negative individuals, suggesting in vitro activation of naive CTL precursors. The precursor frequency of peptide-specific CTL was 10 to 100-fold higher in infected patients compared to uninfected controls, and the responses were greatly diminished by removal of CD45 RO+ (memory) T cells. Further quantitative studies are clearly required to establish whether a correlation exists between the HCV-specific CTL response and the clinical course of this disease. Definition of the molecular targets of the human CTL response to HCV creates this opportunity, and may also contribute to the development of a T cell-based HCV vaccine.

Induction in vitro of a primary human antiviral cytotoxic T cell response.

We report herein the successful priming of human anti-viral cytotoxic T cells (CTL) in vitro using two induction strategies based on the stimulation of peripheral blood mononuclear cells isolated from uninfected donors with synthetic viral peptides. The peptides used contain HLA-A2 binding motifs and have been identified as HLA-A2-restricted CTL epitopes in patients infected by the hepatitis B and C viruses. One approach uses repetitive long-term stimulation and the other uses bulk cultures containing large numbers of naive peripheral blood mononuclear cells. Both approaches successfully induce HLA-A2-restricted CTL specific for several viral epitopes. Some CTL recognize endogenously synthesized antigen on target cells infected with recombinant vaccinia virus expressing the corresponding viral proteins. This simple technique permits easy analysis of the primary human CTL repertoire, and may be exploitable for production of specific CTL effector cells for adoptive immunotherapy and dissection of the cellular and molecular requirements for priming of naive human CTL.

Inhibition of cytolytic lymphocytes by homologous restriction factor. Lack of species restriction.

Homologous restriction factor (HRF) has been shown to inhibit complement-mediated lysis in a species-restrictive manner. Human HRF is able to block lysis by human complement but not by complement from other species. HRF has also been found in the membrane of lymphokine-activated killer (LAK) cells. When this HRF is inserted into sheep erythrocyte membranes, it is able to protect the erythrocyte from LAK cell lysis. In this report, we show that while HRF can inhibit human complement but not rat complement-mediated hemolysis, it is able to inhibit LAK cell lysis by both human and rat LAK cells. HRF is therefore a more general protective protein than has been previously thought.

Isolation of homologous restriction factor from human urine. Immunochemical properties and biologic activity.

A soluble form of homologous restriction factor (HRF-U) was isolated from normal human urine. With respect to m.w. (65,000) and immunoblotting characteristics, it resembled membrane HRF (HRF-M) that had been isolated from human E membranes. The protein exhibited limited cross-reactivity with the channel-forming proteins of C and cytotoxic lymphocytes. It inhibited reactive lysis of E by human C5b-9. Inhibition occurred at the attachment stage of C5b-7 to target cells, rather than at the C8 or C9 stage of membrane attack complex assembly which is inhibited by HRF-M. In this respect, HRF-U acts analogously to S protein of serum, but no immunochemical relationship between these two proteins was detected. HRF-U might be derived from the soluble HRF detected in cytoplasmic granules of killer lymphocytes.

Self-protection of cytotoxic lymphocytes: a soluble form of homologous restriction factor in cytoplasmic granules.

A soluble form of homologous restriction factor (HRF) has been isolated from the cytoplasmic granules of human large granular lymphocytes that were cultured in the presence of recombinant interleukin 2 for 2-3 weeks. The granule-derived protein (approximately 65 kDa) is soluble in detergent-free solution and reacts with antibody produced to membrane HRF. HRF was first described as a 65-kDa membrane protein of human erythrocytes capable of inhibiting the formation of transmembrane channels by the membrane attack complex of complement. It has also been isolated from activated human lymphocytes and shown to confer upon these cells relative resistance to lysis by the membrane attack complex and by the complement component C9-related protein of human cytotoxic lymphocytes. The soluble HRF of lymphocyte granules inhibits reactive lysis of erythrocytes by the membrane attack complex of human complement. It was also found to be a potent inhibitor of (i) the cytolytic activity of the C9-related protein of human cytotoxic lymphocytes, (ii) human large granular lymphocyte cytotoxicity, and (iii) the cytotoxic activity of human CD8+ lymphocytes obtained by cell sorting from recombinant interleukin 2-activated peripheral blood mononuclear cells. It is proposed that granule-derived soluble HRF and cell surface-membrane-bound HRF are involved in the mechanism of self-protection of killer lymphocytes.

Mechanism of cytotoxicity of human large granular lymphocytes: relationship of the cytotoxic lymphocyte protein to the ninth component (C9) of human complement.

A Mr 70,000 protein was isolated from cytotoxic human large granular lymphocytes and shown to have cytotoxic activity. The protein was demonstrated to be immunochemically related to the ninth component (C9) of complement and was therefore designated C9-related protein (C9RP). This finding suggests that C9RP and C9 share homology in primary structure and have a common evolutionary ancestry. C9RP was isolated, by affinity chromatography employing anti-human C9-Sepharose, from either purified cytoplasmic granules or whole-cell lysates of cultured human large granular lymphocytes. The cells were isolated from healthy blood donors and maintained in interleukin-2-dependent cultures. The immunochemical crossreactivity of C9 with C9RP was 3-4%, using a murine anti-C9RP antiserum. Certain murine monoclonal antibodies to C9RP and to C9 inhibited killing of K562 cells by human large granular lymphocytes. Killed target cells, identified by propidium iodide staining and isolated by fluorescence-activated cell-sorting, exhibited clusters of circular membrane lesions that resembled poly(C9) in appearance. Polymerization of isolated C9RP in the presence of Ca2+ resulted in the formation of two different circular structures, one having an inner diameter of approximately equal to 60 A, and the other, of 125 A. Polymerized C9RP could be incorporated into liposomes and, as such, gave rise to channels of two different sizes. The smaller channel had a functional diameter of 50-90 A, and the bigger channel, a diameter greater than 102 A.

A C9 related channel forming protein in the cytoplasmic granules of human large granular lymphocytes.

Large granular lymphocytes (LGL) from human blood maintained in culture for 2 to 6 weeks with IL-2 were found positive in the K562 cell killing assay. The cytoplasmic granules of the LGL were isolated, lysed and the soluble proteins were passed over a Sepharose-anti-C9 column. The retained protein was eluted with NaCl and found to consist by SDS polyacrylamide gel electrophoresis of essentially one component with a molecular weight of approximately 70 000. The protein did not give a positive precipitation test with anti-human C9 by Ouchterlony analysis, but it reacted reproducibly with anti-human C9 by Western blot analysis. By ELISA the cross reaction with human C9 was less than 1%. The C9 related lymphocyte protein lacked C9 hemolytic activity, but it formed functional pores in liposomes in presence of Ca++. These results suggest that the cytoplasmic granules of human LGL that are capable of killing NK target cells contain C9 related protein which is involved in the cellular cytotoxicity reaction.