Daniel C. Pevear

Potent Antiviral Synergy between Monoclonal Antibody and Small-Molecule CCR5 Inhibitors of Human Immunodeficiency Virus Type 1

ABSTRACT The chemokine receptor CCR5 provides a portal of entry for human immunodeficiency virus type 1 (HIV-1) into susceptible CD4+ cells. Both monoclonal antibody (MAb) and small-molecule CCR5 inhibitors have entered human clinical testing, but little is known regarding their potential interactions. We evaluated the interactions between CCR5 MAbs, small-molecule CCR5 antagonists, and inhibitors of HIV-1 gp120, gp41, and reverse transcriptase in vitro. Inhibition data were analyzed for cooperative effects using the combination index (CI) method and stringent statistical criteria. Potent, statistically significant antiviral synergy was observed between the CCR5 MAb PRO 140 and the small-molecule CCR5 antagonists maraviroc (UK-427,857), vicriviroc (SCH-D), and TAK-779. High-level synergy was observed consistently across various assay systems, HIV-1 envelopes, CCR5 target cells, and inhibition levels. CI values ranged from 0.18 to 0.64 and translated into in vitro dose reductions of up to 14-fold. Competition binding studies revealed nonreciprocal patterns of CCR5 binding by MAb and small-molecule CCR5 inhibitors, suggesting that synergy occurs at the level of receptor binding. In addition, both PRO 140 and maraviroc synergized with the chemokine RANTES, a natural ligand for CCR5; however, additive effects were observed for both small-molecule CCR5 antagonists and PRO 140 in combination with other classes of HIV-1 inhibitors. The findings provide a rationale for clinical exploration of MAb and small-molecule CCR5 inhibitors in novel dual-CCR5 regimens for HIV-1 therapy.

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.

STABILIZATION OF HUMAN RHINOVIRUS SEROTYPE 2 AGAINST PH-INDUCED CONFORMATIONAL CHANGE BY ANTIVIRAL COMPOUNDS

Four WIN compounds with anti-picornavirus activities were tested for their ability to stabilize human rhinovirus serotype 2 (HRV-2) against low pH-induced conformational changes in vitro, as determined by specific immunoprecipitation. These results were compared to the minimal inhibitory concentration (MIC) as measured in a plaque reduction assay. A direct relationship was observed between the concentration of the compound that prevented the low pH-induced conformational changes and the MIC, indicating that stabilization is an important element in the mode of action of these drugs against HRV-2.

Characterization and Specificity of Humoral Immune Responses to Theiler's Murine Encephalomyelitis Virus Capsid Proteins

Humoral antibody responses to Theilers murine encephalomyelitis virus (TMEV) capsid proteins were examined. Rabbit antisera produced against the native BeAn strain of TMEV and against the isolated capsid proteins (VP1, VP2 and VP3) were tested for their ability to bind or neutralize virus and to inhibit the virus-induced haemagglutination of human O+ erythrocytes. Western immunoblotting analysis showed that isolated VP1, VP2 and VP3 each primed for a specific antibody response, but that native virions primed for antibodies specific for VP1 and VP2, but not VP3. Virus neutralization studies revealed that a dominant TMEV neutralizing determinant(s) lay on VP1, as did the haemagglutinating determinant. The possible location of the neutralizing epitopes are discussed on the basis of molecular modelling of the predicted amino acid sequence of TMEV from that of the closely related Mengo virus for which the three-dimensional structure is known.

Structures of four methyltetrazole-containing antiviral compounds in human rhinovirus serotype 14.

Four novel antiviral WIN compounds, that contain a methyl tetrazole ring as well as isoxazole, pyridazine or acetylfuran rings, have had their structures determined in human rhinovirus serotype 14 at 2.9 A resolution. These compounds bind in the VP1 hydrophobic pocket, but are shifted significantly towards the pocket pore when compared to previously examined WIN compounds. A putative water network at the pocket pore is positioned to hydrogen bond with these four WIN compounds, and this network can account for potency differences seen in structurally similar WIN compounds.

3-Pyridines as replacements for an isoxazole ring: The antirhinoviral activity of pyridine analogues related to disoxaril

Abstract In a series of rhinovirus inhibitors related to Disoxaril, replacement of 3-methylisoxazole with all 3 isomers of pyridine showed the 3-pyridyl isomer most similar to 3-methylisoxazole in spectrum of activity over 15 human rhinovirus serotypes.

Drugs as Molecular Tools

Chemotherapeutic agents such as acyclovir and zidovudine have been extremely useful in treating patients with herpes simplex virus infections or AIDS, respectively. However, there is another scientific use for drugs or compounds which is to use them as molecular tools in the research laboratory in order to dissect the replication of viruses and to discover new facts about viruses. For example, alpha-amanitin, an inhibitor of the cellular DNA-dependent RNA polymerase II (Pol II), is used to define whether a particular virus RNA species is synthesized with the involvement of Pol II or solely by viral polymerases. With herpesviruses, cycloheximide, an inhibitor of protein synthesis, is used to define the “immediate early” class of viral genes. If a viral RNA is synthesized in the presence of cycloheximide, then the synthesis of that viral RNA is not dependent on the synthesis of any viral protein and that viral gene can be classified as immediate early. Arabinosyl cytosine (AraC) is an anticancer/antiherpesvirus drug which inhibits DNA synthesis in cell culture systems but allows RNA and protein synthesis to proceed. One can ask whether the replication of an RNA virus is dependent on cellular DNA synthesis by determining if virus replication is sensitive to AraC. These examples demonstrate how chemotherapeutic agents can be used as molecular tools in the research laboratory.