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).

Rapid multiserotype detection of human rhinoviruses on optically coated silicon surfaces.

BACKGROUND: More than 100 immunologically distinct serotypes of human rhinoviruses (HRV) have been discovered, making detection of surface exposed capsid antigens impractical. However, the non-structural protein 3C protease (3Cpro) is essential for viral replication and is relatively highly conserved among serotypes, making it a potential target for diagnostic testing. The thin film biosensor is an assay platform that can be formatted into a sensitive immunoassay for viral proteins in clinical specimens. The technology utilizes an optically coated silicon surface to convert specific molecular binding events into visual color changes by altering the reflective properties of light through molecular thin films. OBJECTIVE: To develop a rapid test for detection of HRV by developing broadly serotype reactive antibodies to 3Cpro and utilizing them in the thin film biosensor format. STUDY DESIGN: Polyclonal antibodies to 3Cpro were purified and incorporated into the thin film assay. The in vitro sensitivity, specificity and multiserotype cross-reactivity of the 3Cpro assay were tested. Nasal washes from naturally infected individuals were also tested to verify that 3Cpro was detectable in clinical specimens. RESULTS: The 3Cpro assay is a 28-min, non-instrumented room temperature test with a visual limit of detection of 12 pM (picomolar) 3Cpro. In terms of viral titer, as few as 1000 TCID(50) equivalents of HRV2 were detectable. The assay detected 45/52 (87%) of the HRV serotypes tested but showed no cross-reactivity to common respiratory viruses or bacteria. The thin film assay detected 3Cpro in HRV-infected cell culture supernatants coincident with first appearance of cytopathic effect. Data are also presented demonstrating 3Cpro detection from clinical samples collected from HRV-infected individuals. The assay detected 3Cpro in expelled nasal secretions from a symptomatic individual on the first day of illness. In addition, 9/11 (82%) concentrated nasal wash specimens from HRV infected children were positive in the 3Cpro test. CONCLUSION: We have described a novel, sensitive thin film biosensor for rapid detection of HRV 3Cpro. This test may be suitable for the point of care setting, where rapid HRV diagnostic test results could contribute to clinical decisions regarding appropriate antibiotic or antiviral therapy.

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.

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.

Structure-based design of irreversible, tripeptidyl human rhinovirus 3C protease inhibitors containing N-methyl amino acids.

Tripeptide-derived molecules incorporating N-methyl amino acid residues and C-terminal Michael acceptor moieties were evaluated as irreversible inhibitors of the cysteine-containing human rhinovirus 3C protease (3CP). Such compounds displayed good 3CP inhibition activity (k(obs)/[I] up to 610,000 M(-1) s(-1)) and potent in vitro antiviral properties (EC50 approaching 0.03 microM) when tested against HRV serotype-14.

Solid-phase synthesis of irreversible human rhinovirus 3C protease inhibitors. Part 1: Optimization of tripeptides incorporating N-terminal amides.

The optimization of a series of irreversible human rhinovirus (HRV) 3C protease (3CP) inhibitors is described. These inhibitors are comprised of an L-Leu-L-Phe-L-Gln tripeptide containing an N-terminal amide moiety and a C-terminal ethyl propenoate Michael acceptor. Examination of approximately 500 compounds with varying N-terminal amides utilizing solid-phase synthesis and high-throughput assay techniques is described along with the solution phase preparation of several highly active molecules. A tripeptide Michael acceptor containing an N-terminal amide derived from 5-methylisoxazole-3-carboxylic acid is shown to exhibit potent, irreversible anti-3CP activity (k(obs)/[I] = 260,000 M(-1) s(-1); type-14 3CP) and broad-spectrum antirhinoviral properties (average EC50 = 0.47 microM against four different HRV serotypes).

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 3. Structure-activity studies of ketomethylene-containing peptidomimetics.

The structure-based design, chemical synthesis, and biological evaluation of various ketomethylene-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of a peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The ketomethylene-containing inhibitors typically display slightly reduced 3CP inhibition activity relative to the corresponding peptide-derived molecules, but they also exhibit significantly improved antiviral properties. Optimization of the ketomethylene-containing compounds is shown to provide several highly active 3C protease inhibitors which function as potent antirhinoviral agents (EC90 = <1 microM) against multiple virus serotypes in cell culture.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 4. Incorporation of P1 lactam moieties as L-glutamine replacements.

The structure-based design, chemical synthesis, and biological evaluation of various human rhinovirus (HRV) 3C protease (3CP) inhibitors which incorporate P1 lactam moieties in lieu of an L-glutamine residue are described. These compounds are comprised of a tripeptidyl or peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The P1-lactam-containing inhibitors display significantly increased 3CP inhibition activity along with improved antirhinoviral properties relative to corresponding L-glutamine-derived molecules. In addition, several lactam-containing compounds exhibit excellent selectivity for HRV 3CP over several other serine and cysteine proteases and are not appreciably degraded by a variety of biological agents. One of the most potent inhibitors (AG7088, mean antirhinoviral EC90 approximately 0.10 microM, n = 46 serotypes) is shown to warrant additional preclinical development to explore its potential for use as an antirhinoviral agent.

Tripeptide aldehyde inhibitors of human rhinovirus 3C protease: design, synthesis, biological evaluation, and cocrystal structure solution of P1 glutamine isosteric replacements.

The investigation of tripeptide aldehydes as reversible covalent inhibitors of human rhinovirus (HRV) 3C protease (3CP) is reported. Molecular models based on the apo crystal structure of HRV-14 3CP and other trypsin-like serine proteases were constructed to approximate the binding of peptide substrates, generate transition state models of P1-P1' amide cleavage, and propose novel tripeptide aldehydes. Glutaminal derivatives have limitations since they exist predominantly in the cyclic hemiaminal form. Therefore, several isosteric replacements for the P1 carboxamide side chain were designed and incorporated into the tripeptide aldehydes. These compounds were found to be potent inhibitors of purified HRV-14 3CP with Kis ranging from 0.005 to 0.64 microM. Several have low micromolar antiviral activity when tested against HRV-14-infected H1-HeLa cells. The N-acetyl derivative 3 was also shown to be active against HRV serotypes 2, 16, and 89. High-resolution cocrystal structures of HRV-2 3CP, covalently bound to compounds 3, 15, and 16, were solved. These cocrystal structures were analyzed and compared with our original HRV-14 3CP-substrate and inhibitor models.

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.

Structure and functional relationships in human pur H.

1. The human pur H (ATIC) gene encoding a bifunctional protein, hPurH, which carries the penultimate and final enzymatic activities of the purine nucleotide synthesis pathway, AICARFT & IMPCH, has been cloned and sequenced. The gene product, hPurH has been overexpressed in E. coli, purified to homogeneity and crystallized. 2. The human pur H gene lies on chromosome 2, between band q34 and q35. There is at least one intron of 278 bp near the 5' end. 3. Truncation mutant studies demonstrate two non-overlapping functional domains in the protein arranged as indicated in Figure 5. The existence of a linker or interaction region between the catalytic domains remains to be established. 4. Cleland-type kinetic inhibition experiments indicate that the AICARFT reaction is of the ordered, sequential type with the reduced folate cofactor binding first. 5. The reaction has a broad pH optimum in the alkaline range, with a maximum at about pH 8.2. 6. Preliminary transient phase kinetic studies show the presence of a "burst" indicating that a late step in the reaction sequence is rate limiting. 7. A PurH crystal structure is that of a dimer, with a putative single binding site for the reduced folate cofactor formed using elements from each of the monomer subunits. Probable binding sites for AICAR and FAICAR can be identified on each monomer. 8. Equilibrium sedimentation studies show hPurH apoprotein to be a monomer:dimer equilibrium mixture with a kD of 0.55 uM. 9. The crystal structure has permitted identification of a number of candidate amino acid residues likely to be involved in catalysis and/or substrate binding. Among these, we have thus far completed studies on two, Lysine 265 and Histidine 266. These appear to be critically involved in the AICARFT reaction, although whether their role(s) are in catalysis or binding remains to be determined.

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.

Characterization of the DNA-binding activity of HIV-1 integrase using a filter binding assay.

Based on the selective binding of proteins and DNA to distinct filter materials a double-layered dot blot radio assay was developed to evaluate the binding of DNA to HIV-1 integrase. In this assay the DNA-binding was found to be independent of Mn2+ concentration, inhibited by concentrations of Mg2+ above 5 mM, abolished by zinc chelation and inhibited by monoclonal antibodies reacting with either the N-terminal or C-terminal regions of integrase. Atomic absorption spectroscopy revealed the molar ratio between integrase and zinc to be close to 1. It is concluded that both the N-terminal and the C-terminal regions of integrase are involved in DNA-binding and that the reported double-layered dot blot radio assay is well suited for further characterization of the integrase.

Isolation of high-affinity RNA ligands to HIV-1 integrase from a random pool.

We were able to isolate high-affinity RNAs from a random pool that binds to integrase protein from the human immunodeficiency virus-type 1 using the procedure now known as SELEX. Generally, the RNAs fell into three different classes in binding buffer containing 250 mM NaCl: group I class of molecules binds integrase with a dissociation constant (Kd) on the order of 10 nM, group II molecules had a Kd of about 80 nM, and group III about 800 nM. The RNA with the highest affinity from the group I class of molecules, designated P5, was characterized using computer modeling, chemical and enzymatic probing, and deletion analysis. Our secondary structure model for this RNA suggests interactions between looped-out fixed nucleotides and nucleotides from the randomized region; a GNRA tetraloop is also in the structure. We showed that our integrase was able to process a U5 mimic in vitro. P5 competes effectively for binding with the double-stranded DNA mimic of U5 at 180 mM NaCl concentration.

Nucleotide binding by the HIV-1 integrase protein in vitro.

Recombinant human immunodeficiency virus type 1 (HIV-1) integrase was shown to bind ATP and other nucleoside triphosphates and nucleotide analogs in vitro. Cross-linking of ATP and the photoaffinity analog 8-azido-ATP to integrase occurred in a UV dose-dependent manner. Covalent binding of ATP to integrase was also achieved without UV irradiation when the nucleotide was oxidized to the 2',3'-dialdehyde derivative (oxidized ATP) prior to incubation with the protein, indicating the presence of a reactive lysine residue in the nucleotide binding region of the protein. A number of experimental observations indicate that nucleotides and DNA substrates bind at the same or overlapping site(s) on the integrase protein. For example, the binding of nucleotides or nucleotide analogs to integrase was blocked by prior incubation with DNA substrates, and the covalent cross-linking of 8-azido-ATP to integrase inhibited the DNA binding and oligonucleotide cleavage activities of the protein. Oxidized ATP inhibited the oligonucleotide cleavage activity of integrase at concentrations that had no effect on DNA binding, suggesting that oxidized nucleotides may specifically target the catalytic center of the enzyme. These studies indicate that nucleotide analogs may serve as probes for the DNA binding and catalytic sites of the enzyme and may serve as models for the design of active site inhibitors of retroviral integrase.

Inducible overexpression, purification, and active site mapping of DNA topoisomerase II from the yeast Saccharomyces cerevisiae.

Overexpression of yeast DNA topoisomerase II was achieved by placing the coding sequences of the gene TOP2 downstream of an inducible promoter PGAL1 on a multicopy plasmid. By using a simple purification procedure, milligram amounts of the enzyme of a high specific activity can be obtained from a few liters of culture. In the presence of a drug VM-26 (teniposide), more than 90% of the enzyme molecules become covalently bound to DNA upon addition of the protein denaturant sodium dodecyl sulfate. The formation of the covalent complex was used to map the tyrosine residue that becomes covalently linked to DNA when the enzyme transiently breaks DNA. After exhaustive digestion of the DNA-protein complex with trypsin, a DNA-linked peptide was purified and sequenced directly to identify Tyr-783 as the active site residue.