Lipid targeted compounds that inhibit virus-cell membrane fusion, targeting the virus membrane.
Advances in antiviral therapeutics have allowed for effective management of specific viral infections, most notably human immunodeficiency virus (HIV). Yet, the one-bug-one-drug paradigm of drug discovery is insufficient to meet the looming threat of emerging and re-emerging viral pathogens that endangers global human and livestock health. This underscores the need for broad-spectrum antivirals that act on multiple viruses based on some commonality in their viral life cycle, rather than on specific viral proteins.
We recently described a small molecular compound that inhibited the entry of all lipid-enveloped viruses tested (ME Wolf et al, PNAS, 2010). LJ001 is a membrane-binding compound with broad-spectrum antiviral activity in vitro. LJ001 acts on the virus, and not the cell, inhibiting enveloped virus infection at the level of entry. LJ001 is non-cytotoxic at antiviral concentrations, yet had the remarkable property of inhibiting all enveloped viruses tested, including those of global biomedical and biosecurity importance such as HIV, HCV, Influenza, Ebola, henipaviruses, bunyaviruses, arenaviruses, and poxviruses. LJ001 is not virolytic, does not act as a "detergent", and LJ001-treated virions are still able to bind to their receptors. LJ001 was lipophilic, and could bind to both viral and cellular membranes. Yet, it inhibited virus-cell but not cell-cell fusion. This puzzling dichotomy was illuminated when studies with lipid biosynthesis inhibitors indicated that LJ001 was indeed cytotoxic when the ability of a cell to repair and turnover its membranes is compromised. Thus, we posited that the antiviral activity of LJ001 relies on exploiting the physiological difference between inert viral membranes and biogenic cellular membranes with reparative capabilities.
Our latest studies have identified the molecular target of LJ001 and its precise molecular mechanism of action. LJ001 acts as a membrane-binding photosensitizer that induces singlet oxygen mediated modifications of specific phospholipid components of viral membranes. This results in changes in the biophysical properties of viral membranes that are not conducive for the membrane curvature dynamics required for productive viral-cell fusion. Importantly, these biophysical changes are not found in biogenic cellular membranes treated with antiviral concentrations of the compound, likely due to multiple endogenous mechanisms that protect lipids against oxidative damage.
Structure Activity Relations (SAR) studies based on this mechanistic understanding led to novel second-generation compounds with markedly enhanced antiviral potencies as a result of improved photochemical and photophysical properties. This is a multi-disciplinary trans-national project requiring expertise ranging from molecular and animal virology, to membrane biophysics and lipidomics, to medicinal and photo- chemistry.
Current projects that will extend these studies include:
- Developing new strategies for improved control and activation of these antiviral compounds in vivo.
- Using the unique properties of these photosensitizers for more in-depth study of the viral-fusion process e.g. capturing and visualizing fusion intermediates using cryo-electron tomography studies of virus-cell fusion trapped just before membrane merger, or dissecting the fine mechanics of class I-III fusion by regulating the time of light-induced inactivation.
Bioactive Proteasome Inhibitors
Our basic studies on Nipah virus budding led us to discover that ubiquitin-regulated nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M) protein is critical for matrix budding function. Inhibiting ubiquitination of NiV-M by proteasome inhibitors such as Bortozemib, which is FDA-approved for oncologic indications, led to nuclear retention of NiV-M, and abrogation of NiV-M budding. Live NiV budding and replication is exquisitely sensitive to Bortozemib with an in vitro IC50 (~2.7 nM) that is about 100-fold lower than the peak plasma concentration (~300 nM) that can be achieved in patients.
The ubiquitin proteasome pathway (UPP) is implicated in the lifecycle of multiple viruses. The UPP regulates a wide array of protein function and cellular processes and many viruses are known to manipulate the host cell UPP to enable replication, egress and immune evasion. Many proteasome inhibitors (PSM Inbs) have a negative impact on viral infections in vitro, but the involvement of the UPP in multiple cellular functions, coupled with the lack of potency, specificity or in vivo stability of the first generation PSM Inbs, discouraged the consideration that PSM INbs could be developed safely as an antiviral therapeutic. In 2003, the FDA approval of the first PSM Inb, Bortezomib, for the treatment of multiple myeloma, provided proof-of-principle that PSM Inbs can be developed with acceptable toxicology profile and good pharmacokinetics/ bioavailability. This has sparked the development of many second generation PSM Inbs with improved potency, selectivity, and bioavailability - several of which are already in Phase I-III trials for oncologic applications.
Excitingly, we have now obtained in vitro preliminary data showing that Bortezomib can also inhibit, to varying degrees, the replication of multiple Category A-C pathogens: Filoviridae (Ebola), Paramyxoviridae (Nipah), Bunyaviridae (Rift Valley fever), Flaviviridae (Russian-Spring-Summer encephalitis), and Arenaviridae (Junin). These data indicate that multiple viral families, though clearly not all, are susceptible to proteasome inhibition, and suggest that the anti-viral efficacy of Bortezomib and/or 2G-PSM Inbs should be evaluated against a broader spectrum of viruses.
This project can be fairly described as discovery driven science. Our immediate primary goal is to empirically evaluate and re-purpose these bioavailable PSM Inbs as potential broad-spectrum antivirals for infections caused by acutely pathogenic viral agents such as those classified as NIAID Category A-C pathogens. Based on the results, we will then elucidate the mechanisms underlying the differential efficacy of the various proteasome inhibitors against distinct viral families.