Rami Musharrafieh

An M2-V27A channel blocker demonstrates potent in vitro and in vivo antiviral activities against amantadine-sensitive and -resistant influenza A viruses

ABSTRACT Adamantanes such as amantadine (1) and rimantadine (2) are FDA‐approved anti‐influenza drugs that act by inhibiting the wild‐type M2 proton channel from influenza A viruses, thereby inhibiting the uncoating of the virus. Although adamantanes have been successfully used for more than four decades, their efficacy was curtailed by emerging drug resistance. Among the limited number of M2 mutants that confer amantadine resistance, the M2‐V27A mutant was found to be the predominant mutant under drug selection pressure, thereby representing a high profile antiviral drug target. Guided by molecular dynamics simulations, we previously designed first‐in‐class M2‐V27A inhibitors. One of the potent lead compounds, spiroadamantane amine (3), inhibits both the M2‐WT and M2‐V27A mutant with IC50 values of 18.7 and 0.3 &mgr;M, respectively, in in vitro electrophysiological assays. Encouraged by these findings, in this study we further examine the in vitro and in vivo antiviral activity of compound 3 in inhibiting both amantadine‐sensitive and ‐resistant influenza A viruses. Compound 3 not only had single to sub‐micromolar EC50 values against M2‐WT‐ and M2‐V27A‐containing influenza A viruses in antiviral assays, but also rescued mice from lethal viral infection by either M2‐WT‐ or M2‐V27A‐containing influenza A viruses. In addition, we report the design of two analogs of compound 3, and one was found to have improved in vitro antiviral activity over compound 3. Collectively, this study represents the first report demonstrating the in vivo antiviral efficacy of inhibitors targeting M2 mutants. The results suggest that inhibitors targeting drug‐resistant M2 mutants are promising antiviral drug candidates worthy of further development. HIGHLIGHTSCompound 3 displayed potent in vivo antiviral activity against both M2‐WT‐ and M2‐V27A‐containing influenza A viruses.Dithiane analog (16) had improved in vitro antiviral activity against M2‐WT‐ and M2‐V27A‐containing influenza A viruses.Potent M2 channel blockers with very slow reversal of channel inhibition kinetics showed potent antiviral activity.

Discovery of dapivirine, a nonnucleoside HIV-1 reverse transcriptase inhibitor, as a broad-spectrum antiviral against both influenza A and B viruses

&NA; The emergence of multidrug‐resistant influenza viruses poses a persistent threat to public health. The current prophylaxis and therapeutic interventions for influenza virus infection have limited efficacy due to the continuous antigenic drift and antigenic shift of influenza viruses. As part of our ongoing effort to develop the next generation of influenza antivirals with broad‐spectrum antiviral activity and a high genetic barrier to drug resistance, in this study we report the discovery of dapivirine, an FDA‐approved HIV nonnucleoside reverse transcriptase inhibitor, as a broad‐spectrum antiviral against multiple strains of influenza A and B viruses with low micromolar efficacy. Mechanistic studies revealed that dapivirine inhibits the nuclear entry of viral ribonucleoproteins at the early stage of viral replication. As a result, viral RNA and protein synthesis were inhibited. Furthermore, dapivirine has a high in vitro genetic barrier to drug resistance, and its antiviral activity is synergistic with oseltamivir carboxylate. In summary, the in vitro antiviral results of dapivirine suggest it is a promising candidate for the development of the next generation of dual influenza and HIV antivirals.

Chemical Genomics Approach Leads to the Identification of Hesperadin, an Aurora B Kinase Inhibitor, as a Broad-Spectrum Influenza Antiviral

Influenza viruses are respiratory pathogens that are responsible for annual influenza epidemics and sporadic influenza pandemics. Oseltamivir (Tamiflu®) is currently the only FDA-approved oral drug that is available for the prevention and treatment of influenza virus infection. However, its narrow therapeutic window, coupled with the increasing incidence of drug resistance, calls for the next generation of influenza antivirals. In this study, we discovered hesperadin, an aurora B kinase inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. Hesperadin inhibits multiple human clinical isolates of influenza A and B viruses with single to submicromolar efficacy, including oseltamivir-resistant strains. Mechanistic studies revealed that hesperadin inhibits the early stage of viral replication by delaying the nuclear entry of viral ribonucleoprotein complex, thereby inhibiting viral RNA transcription and translation as well as viral protein synthesis. Moreover, a combination of hesperadin with oseltamivir shows synergistic antiviral activity, therefore hesperadin can be used either alone to treat infections by oseltamivir-resistant influenza viruses or used in combination with oseltamivir to delay resistance evolution among oseltamivir-sensitive strains. In summary, the discovery of hesperadin as a broad-spectrum influenza antiviral offers an alternative to combat future influenza epidemics and pandemics.

In Vitro Pharmacokinetic Optimizations of AM2-S31N Channel Blockers Led to the Discovery of Slow-Binding Inhibitors with Potent Antiviral Activity against Drug-Resistant Influenza A Viruses

Influenza viruses are respiratory pathogens that are responsible for both seasonal influenza epidemics and occasional influenza pandemics. The narrow therapeutic window of oseltamivir, coupled with the emergence of drug resistance, calls for the next-generation of antivirals. With our continuous interest in developing AM2-S31N inhibitors as oral influenza antivirals, we report here the progress of optimizing the in vitro pharmacokinetic (PK) properties of AM2-S31N inhibitors. Several AM2-S31N inhibitors, including compound 10b, were discovered to have potent channel blockage, single to submicromolar antiviral activity, and favorable in vitro PK properties. The antiviral efficacy of compound 10b was also synergistic with oseltamivir carboxylate. Interestingly, binding kinetic studies (Kd, Kon, and Koff) revealed several AM2-S31N inhibitors that have similar Kd values but significantly different Kon and Koff values. Overall, this study identified a potent lead compound (10b) with improved in vitro PK properties that is suitable for the in vivo mouse model studies.

Focusing on the influenza virus polymerase complex: recent progress in drug discovery and assay development

Influenza viruses are severe human pathogens that pose persistent threat to public health. Each year more people die of influenza virus infection than that of breast cancer. Due to the limited efficacy associated with current influenza vaccines, as well as emerging drug resistance from small molecule antiviral drugs, there is a clear need to develop new antivirals with novel mechanisms of action. The influenza virus polymerase complex has become a promising target for the development of the next-generation of antivirals for several reasons. Firstly, the influenza virus polymerase, which forms a heterotrimeric complex that consists of PA, PB1, and PB2 subunits, is highly conserved. Secondly, both individual polymerase subunit (PA, PB1, and PB2) and inter-subunit interactions (PA-PB1, PB1-PB2) represent promising drug targets. Lastly, growing insight into the structure and function of the polymerase complex has spearheaded the structure-guided design of new polymerase inhibitors. In this review, we highlight recent progress in drug discovery and assay development targeting the influenza virus polymerase complex and discuss their therapeutic potentials.

Exploring Ugi-Azide Four-Component Reaction Products for Broad-Spectrum Influenza Antivirals with a High Genetic Barrier to Drug Resistance

Influenza viruses are respiratory pathogens that are responsible for seasonal influenza and sporadic influenza pandemic. The therapeutic efficacy of current influenza vaccines and small molecule antiviral drugs is limited due to the emergence of multidrug-resistant influenza viruses. In response to the urgent need for the next generation of influenza antivirals, we utilized a fast-track drug discovery platform by exploring multi-component reaction products for antiviral drug candidates. Specifically, molecular docking was applied to screen a small molecule library derived from the Ugi-azide four-component reaction methodology for inhibitors that target the influenza polymerase PAC-PB1N interactions. One hit compound 5 was confirmed to inhibit PAC-PB1N interactions in an ELISA assay and had potent antiviral activity in an antiviral plaque assay. Subsequent structure-activity relationship studies led to the discovery of compound 12a, which had broad-spectrum antiviral activity and a higher in vitro genetic barrier to drug resistance than oseltamivir. Overall, the discovery of compound 12a as a broad-spectrum influenza antiviral with a high in vitro genetic barrier to drug resistance is significant, as it offers a second line of defense to combat the next influenza epidemics and pandemics if vaccines and oseltamivir fail to confine the disease outbreak.

Profiling the in vitro drug-resistance mechanism of influenza A viruses towards the AM2-S31N proton channel blockers

&NA; The majority of human influenza A viruses currently in circulation carry the amantadine‐resistant AM2‐S31N channel mutation. We previously discovered a series of AM2‐S31N inhibitors with potent antiviral activity against both oseltamivir‐sensitive and ‐resistant influenza A viruses. To understand the drug‐resistance mechanism of AM2‐S31N inhibitors, we performed serial viral passage experiments using the influenza virus A/California/07/2009 (H1N1) to select drug‐resistant AM2 mutations against two representative AM2‐S31N channel blockers (1 and 2). Unlike amantadine, which gives rise to resistance after a single passage, compounds 1 and 2 selected for partially resistant viruses at passages 05 and 04 with a V27I and L26I mutation, respectively. This appears to suggest compounds 1 and 2 have a higher genetic barrier to resistance than amantadine at least in cell culture. Passage with a higher drug concentration of compound 2 selected higher level resistant viruses with a double mutant L26I + A30T. The mechanism of resistance and replication fitness for mutant viruses were evaluated by electrophysiology, reverse genetics, growth kinetics, and competition assays. AM2‐S31N/V27I and AM2‐S31N/L26I channels achieved similar specific proton conductance as AM2‐S31N, but the AM2‐S31N/L26I/A30T triple mutant had drastically reduced specific proton conductance. Viral replication fitness of AM2‐S31N/V27I and AM2‐S31N/L26I double mutant viruses were similar to AM2‐S31N containing viruses in cell culture. However, AM2‐S31N/L26I/A30T viruses displayed attenuated growth as well as inability to compete with AM2‐S31N viruses. The results herein offer insight regarding the resistance mechanism of AM2‐S31N inhibitors, and may help guide the design of the next‐generation of AM2‐S31N inhibitors with a higher genetic barrier to drug resistance.