Mark A. McKinlay

Prevention of rhinovirus and poliovirus uncoating by WIN 51711, a new antiviral drug.

WIN 51711, a potent new antipicornavirus drug, has been shown to inhibit an early event in the replication cycle of human poliovirus type 2 and human rhinovirus type 2. WIN 51711 was not virucidal and had no measurable effect on the adsorption of [3H]uridine-labeled virions to cells. When virion penetration of the plasma membrane was determined through loss of sensitivity to neutralizing antisera, WIN 51711 had no effect on poliovirus penetration, but inhibited rhinovirus penetration by 40%. In the presence of WIN 51711, exposure of neutral red-encapsidated virus-infected cells to light at 3 h postinfection resulted in a 3-log reduction in the number of infectious centers, indicating that WIN 51711 maintained the viral RNA in the encapsidated state after penetrating the cell membrane. The inhibition of uncoating by WIN 51711 in the neutral red assay was found to be concentration dependent, with a concentration of 0.03 micrograms/ml resulting in a 90% inhibition of uncoating. Sucrose gradient sedimentation of lysates from whole cells infected with [3H]uridine-labeled poliovirus showed that poliovirions remained intact in the presence of WIN 51711, but were uncoated in the absence of drug. WIN 51711 also prevented thermal inactivation of poliovirus infectivity, indicating a direct stabilizing effect of this compound on virion capsid conformation.

Smac Mimetics Activate the E3 Ligase Activity of cIAP1 Protein by Promoting RING Domain Dimerization

The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.

In vitro activity of WIN 51711, a new broad-spectrum antipicornavirus drug.

WIN 51711 (5-[7-[4-(4,5-dihydro-2-oxazolyl)phenoxy]heptyl]-3-methylisoxazole), a new antipicornavirus drug, is a potent inhibitor of human entero- and rhinoviruses at concentrations not inhibitory to HeLa cell growth. In plaque reduction assays, WIN 51711 reduced plaque formation by 9 enteroviruses and 33 rhinoviruses, with MICs of 0.004 to 0.17 and 0.004 to 6.2 micrograms/ml, respectively. Addition of WIN 51711 to infected cells at concentrations of 0.02 to 5.0 micrograms/ml reduced the yield of picornaviruses by 90%. Other RNA viruses (nonpicornaviruses) and DNA viruses were unaffected by the compound.

Birinapant (TL32711), a Bivalent SMAC Mimetic, Targets TRAF2-Associated cIAPs, Abrogates TNF-Induced NF-κB Activation, and Is Active in Patient-Derived Xenograft Models

The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient–derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion. Mol Cancer Ther; 13(4); 867–79. ©2014 AACR.

Relationship of Pleconaril Susceptibility and Clinical Outcomes in Treatment of Common Colds Caused by Rhinoviruses

ABSTRACT Pleconaril, a specific inhibitor of human picornaviruses, showed therapeutic efficacy against community-acquired colds caused by rhinoviruses in two placebo-controlled trials. Virological assessments were conducted during these trails, including virus culture and drug susceptibility testing. Nasal mucus samples collected from the enrolled patients were tested for the presence of picornavirus by reverse transcriptase PCR and culture. In total, 827 baseline nasal mucus samples were positive by virus culture (420 in the placebo group and 407 in the pleconaril group). Pleconaril treatment was associated with a more rapid loss of culturable virus. By study day 3, the number of samples positive by culture fell to 282 for the placebo-treated subjects and 202 for the pleconaril-treated subjects (P < 0.0001); and by day 6, the number of samples in the two groups positive by culture fell to 196 and 165, respectively (P = 0.07). The clinical benefit correlated strongly with the pleconaril susceptibility of the baseline virus isolate. Pleconaril-treated subjects infected with the more highly susceptible viruses (50% effective concentration ≤ 0.38 μg/ml) experienced a median 1.9- to 3.9-day reduction in symptom duration compared with that for the placebo-treated subjects. By contrast, subjects whose baseline virus isolate susceptibility was >0.38 μg/ml did not benefit from pleconaril treatment. These results indicate that the magnitude of symptomatic improvement in pleconaril-treated subjects with community-acquired colds is related to the drug susceptibility of the infecting virus, clearly linking the antiviral effects of the drug to clinical efficacy. Postbaseline virus isolates with reduced susceptibility or full resistance to pleconaril were recovered from 10.7% and 2.7% of drug-treated subjects, respectively. These patients shed low levels of virus and had no unusual clinical outcomes. Nevertheless, studies on the biologic properties and transmissibility of these variant viruses are warranted.

The novel SMAC mimetic Birinapant exhibits potent activity against human melanoma cells

Purpose: Inhibitor of apoptosis proteins (IAP) promote cancer cell survival and confer resistance to therapy. We report on the ability of second mitochondria-derived activator of caspases mimetic, birinapant, which acts as antagonist to cIAP1 and cIAP2, to restore the sensitivity to apoptotic stimuli such as TNF-α in melanomas. Experimental Design: Seventeen melanoma cell lines, representing five major genetic subgroups of cutaneous melanoma, were treated with birinapant as a single agent or in combination with TNF-α. Effects on cell viability, target inhibition, and initiation of apoptosis were assessed and findings were validated in 2-dimensional (2D), 3D spheroid, and in vivo xenograft models. Results: When birinapant was combined with TNF-α, strong combination activity, that is, neither compound was effective individually but the combination was highly effective, was observed in 12 of 18 cell lines. This response was conserved in spheroid models, whereas in vivo birinapant inhibited tumor growth without adding TNF-α in in vitro resistant cell lines. Birinapant combined with TNF-α inhibited the growth of a melanoma cell line with acquired resistance to BRAF inhibition to the same extent as in the parental cell line. Conclusions: Birinapant in combination with TNF-α exhibits a strong antimelanoma effect in vitro. Birinapant as a single agent shows in vivo antitumor activity, even if cells are resistant to single agent therapy in vitro. Birinapant in combination with TNF-α is effective in a melanoma cell line with acquired resistance to BRAF inhibitors. Clin Cancer Res; 19(7); 1784–94. ©2013 AACR.

In vitro and in vivo activities of WIN 54954, a new broad-spectrum antipicornavirus drug.

WIN 54954 (5-[5-[2,6-dichloro-4-(4,5-dihydro-2-oxazolyl)phenoxy]pentyl]-3- methylisoxazole) is a new member of the class of broad-spectrum antipicornavirus compounds known to bind in a hydrophobic pocket within virion capsid protein VP1. In plaque reduction assays, WIN 54954 reduced plaque formation of 50 of 52 rhinovirus serotypes (MICs ranged from 0.007 to 2.2 micrograms/ml). A concentration of 0.28 microgram/ml was effective in inhibiting 80% of the 52 serotypes tested (EC80). WIN 54954 was also effective in inhibiting 15 commonly isolated enteroviruses, with an EC80 of 0.06 microgram/ml. Furthermore, WIN 54954 was effective in reducing the yield of two selected enteroviruses in cell culture by 90% at concentrations approximately equal to their MICs. The therapeutic efficacy of intragastrically administered WIN 54954 was assessed in suckling mice infected with coxsackievirus A-9 or echovirus type 9 (Barty) 2.5 days prior to initiation of therapy. Single daily doses of 2 and 100 mg/kg protected 50% of the mice from developing paralysis (PD50) following infection with coxsackievirus A-9 and echovirus-9, respectively. At the PD50 doses for these two viruses, levels of WIN 54954 in serum were maintained above the in vitro MICs for a significant portion of the dosing interval. The dose-dependent reduction in viral titers observed in coxsackievirus A-9-infected mice correlated well with the therapeutic dose response. The potency and spectrum of WIN 54954 make it a potentially useful compound for the treatment of human enterovirus and rhinovirus infections.

Inhibition of poliovirus uncoating by disoxaril (WIN 51711)

Disoxaril [WIN 51711, 5-[7-[4(4,5-dihydro-2-oxazolyl)phenoxy]heptyl]-3- methylisoxazole] inhibits the replication of polioviruses types 1 and 2 in HeLa cells by stabilizing the virus capsid, which results in the inhibition of the pH-dependent viral uncoating in endosomes and/or lysosomes. As shown by electron microscopy the virus entered into the cell by receptor-mediated endocytosis via coated pits and coated vesicles into endosomes irrespective of the presence or absence of the compound. Measurements of viral RNA synthesis showed that disoxaril completely inhibited the arrival of viral RNA in the cytoplasm for new RNA synthesis only when the inocula were preincubated with disoxaril for 15 min at 37 degrees at 0.3 microgram disoxaril/ml for poliovirus type 1 and 0.03 microgram disoxaril/ml for poliovirus type 2. Simultaneous addition of the compound and virus resulted in reduced inhibition of viral RNA synthesis. The inhibitory effect of the compound could be partially reversed up to 25 min p.i. if the compound was eluted from the cells.

Three-dimensional structures of drug-resistant mutants of human rhinovirus 14.

Mutants of human rhinovirus 14 were isolated and characterized by searching for resistance to compounds that inhibit viral uncoating. The portions of the RNA that code for amino acids that surround the antiviral compound binding site were sequenced. X-ray analysis of two of these mutants, 1188 Val----Leu and 1199 Cys----Tyr, shows that these were single-site substitutions which would sterically hinder drug binding. Differences in the resistance of mutant viruses to various antiviral compounds may be rationalized in terms of the three-dimensional structures of these mutants. Predictions of the structures of mutant rhinovirus 14 with the substitutions 1188 Val----Leu, 1199 Cys----Tyr and 1199 Cys----Trp in VP1 were made using a molecular dynamics technique. The predicted structure of the 1199 Cys----Tyr mutant was consistent with the electron density map, while the 1188 Val----Leu prediction was not. Large (up to 1.4 A) conformational differences between native rhinovirus 14 and the 1199 Cys----Tyr mutant occurred in main-chain atoms near the mutation site. These changes, as well as the orientation of the 1199 tyrosine side-chain, were correctly predicted by the molecular dynamics calculation. The structure of the predicted 1199 Cys----Trp mutation is consistent with the drug-resistant properties of this virus.

Smac mimetic reverses resistance to TRAIL and chemotherapy in human urothelial cancer cells.

Purpose: Inhibitors of apoptosis proteins (IAPs) have been shown to contribute to resistance of neoplastic cells to chemotherapy and to biologic antineoplastic agents. Consequently, new agents are being developed targeting this family of proteins. In a panel of bladder cancer cell lines, we evaluated a Smac mimetic that antagonizes several IAPs for its suitability for bladder cancer therapy. Experimental design: A panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-A alone, TRAIL alone, chemotherapy alone, compound-A plus TRAIL, and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally, siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting. Results: Single-agent compound-A had little effect, but compound-A augmented TRAIL- and chemotherapy-induced apoptosis. Immunoblotting showed that combination treatment with compound-A and TRAIL resulted in cleavage of procaspase-3 and procaspase-7, activation of which irreversibly commits cells to apoptosis. Immunoprecipitation of XIAP showed displacement of active caspase-3 fragments from XIAP, supporting the proposed mechanism of action. Furthermore, siRNA-mediated silencing of XIAP similarly sensitized these cells to apoptosis. Conclusion: Our results suggest that targeting of XIAP with the Smac mimetic compound-A has the potential to augment the effects of a variety of chemotherapeutic and biologic therapies in bladder cancer.