Cardiotoxicity Associated with Nicotinamide Phosphoribosyltransferase Inhibitors in Rodents and in Rat and Human-Derived Cells Lines.

Nicotinamide phosphoribosyltransferase (NAMPT) is a pleiotropic protein that functions as an enzyme, cytokine, growth factor and hormone. As a target for oncology, NAMPT is particularly attractive, because it catalyzes the rate-limiting step in the salvage pathway to generate nicotinamide adenine dinucleotide (NAD), a universal energy- and signal-carrying molecule involved in cellular energy metabolism and many homeostatic functions. Inhibition of NAMPT generally results in NAD depletion, followed by ATP reduction and loss of cell viability. Herein, we describe NAMPT inhibitor (NAMPTi)-induced cardiac toxicity in rodents following short-term administration (2-7 days) of NAMPTi's. The cardiac toxicity was interpreted as a functional effect leading to congestive heart failure, characterized by sudden death, thoracic and abdominal effusion, and myocardial degeneration. Based on exposures in the initial in vivo safety rodent studies and cardiotoxicity observed, we conducted studies in rat and human in vitro cardiomyocyte cell systems. Based on those results, combined with human cell line potency data, we demonstrated the toxicity is both on-target and likely human relevant. This toxicity was mitigated in vitro by co-administration of nicotinic acid (NA), which can enable NAD production through the NAMPT-independent pathway; however, this resulted in only partial mitigation in in vivo studies. This work also highlights the usefulness and predictivity of in vitro cardiomyocyte assays using human cells to rank-order compounds against potency in cell-based pharmacology assays. Lastly, this work strengthens the correlation between cardiomyocyte cell viability and functionality, suggesting that these assays together may enable early assessment of cardiotoxicity in vitro prior to conduct of in vivo studies and potentially reduce subsequent attrition due to cardiotoxicity.

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.

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.

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-based design of novel, urea-containing FKBP12 inhibitors.

The structure-based design and subsequent chemical synthesis of novel, urea-containing FKBP12 inhibitors are described. These compounds are shown to disrupt the cis-trans peptidylprolyl isomerase activity of FKBP12 with inhibition constants (Ki,app) approaching 0.10 microM. Analyses of several X-ray crystal structures of FKBP12-urea complexes demonstrate that the urea-containing inhibitors associate with FKBP12 in a manner that is similar to, but significantly different from, that observed for the natural product FK506.