A Biosensor for Detection of HIV Fusion Inhibitors
Miriam Gochin, Touro University, Vallejo, CA
Biomedical and Clinical Sciences
Background: Human Immunodeficiency Virus Type 1 (HIV1) envelope protein gp41 regulates viral - host cell fusion, and is an important target in the development of entry inhibitors to control HIV infection.Currently the peptide Enfuvirtide® is the only approved fusion inhibitor.Small molecule drugs that inhibit fusion could be an effective addition to the anti-HIV therapeutic arsenal.They are typically designed to disrupt the formation of the six-helix bundle conformation of fusion-competent gp41.There are large repositories of compounds in national laboratories and pharmaceutical companies that could be screened for compounds active against HIV-1 fusion,provided that a cost-effective screening procedure was available.We describe a methodology to create a solid-state biosensor that will be able to rapidly and efficiently detect compounds with potential anti-fusion properties.
Methods: Prototype non-peptide fusion drugs have mostly been targeted to a hydrophobic pocket known to exist in the trimeric coiled coil core domain (HR1) of gp41.The HR1 domain is not stable when excised from full length gp41 nor exposed in solution in intact gp41. To make it available as a target in a biochemical assay, we are developing a self-assembled monolayer (SAM) of a segment of gp41 HR1 anchored on a gold-plated quartz crystal. Integrity of the trimeric coiled coil structure will be preserved through the use of a transition metal ion coordinated to a metal-ligating N-terminal bipyridyl group. Three ligands binding tightly to the metal and hydrophobic peptide-peptide contacts should ensure the trimeric folded form of the HR1 domain.We are testing the formation and stability of the monolayer using an electrochemical quartz crystal nanobalance (EQCN) to measure mass changes at the surface due to SAM formation, and electrochemical changes due to the redox properties of the metal ion.Confirmation will be obtained using scanning electron microscopy or atomic force microscopy. The sensitivity of the physicochemical properties to the binding of inhibitors to the SAM will be tested,initially with peptides known to bind tightly to the HR1 domain. Using a flow cell, our ability to regenerate the sensor for repeated use will be tested.The product envisioned is a nanoscale sensor that could detect the presence of a binding molecule in 5-10µL of solution upon contact. The correlation between binding and fusion inhibition will be tested using a cell-based fusion assay.
Expected results: A 26-residue peptide has been designed for the initial SAM formation. It contains an N-terminal 2,2'bipyridyl group, connected to the linker sequence GQAV, followed by 21 residues of wild-type gp41 HR1 encompassing the hydrophobic pocket,anda C-terminalD-cysteineresidueforthiol adsorption on gold,while maintaining vertical positioning of the coiled coil with respect to the surface.The peptide will be ligated to metal ion CoII, NiII, FeII or RuII prior to SAM formation. We expect to observe a strong signal associated with peptide attachment using theEQCN,and acyclicvoltammogramassociated with the metal. Signals should reflect stability and consistency of the monolayer. An 18-residue peptide modeled after the gp41 HR2 domain has been developed and shown to bind with 2µM affinity to the coiled coil. Exposure of the SAM to a solution of this peptide should result in changes in mass, CV or electron transfer rate as a result of peptide binding to the SAM. Changes will be calibrated in order to establish the range and sensitivity of the method for the detection of small molecule binders.We have a cell-cell fusion assay and access to viral infectivity assays for correlation of observed binding to anti-fusion properties.