Southern Research - Life Sciences Contract Services - HIV Virus Protein-Protein Interactions

Human Immunodeficiency Virus Protein-Protein Interactions: Developing High-Throughput Screens for Novel Drug Targets.

Roger Ptak, Priscilla Ward, Susan Halliday, Stacy Bartram, Thomas Fletcher III and Robert Buckheit, Jr. Serquest, Frederick, MD.

Abstract

Development of drug resistance to current therapies for human immunodeficiency virus type 1 (HIV-1) infection has led to the need for additional strategies for drug treatment. The HIV-1 regulatory and structural proteins represent drug targets that have not yet been exploited. The Serquest HTS group has developed an extensive panel of assays to test for inhibitors of protein-protein interactions between HIV genes and their corresponding viral and cellular targets. This system utilizes the Promega Dual-Luciferase^TM Reporter Assay System and has been adapted to a Beckman-Coulter Core robotics platform. Finding inhibitors of the interactions of these proteins with other viral and/or cellular proteins is a novel approach to HIV-1 drug discovery. With viral resistance limiting current therapies, such compounds could prove useful in treating HIV infected individuals.

Limitations of Current HIV Therapies

Of the 15 viral proteins only two, RT and PR, are targeted by current therapies.
Drug resistance will always be a problem when targeting these enzymes.
Will never be able to eradicate virus from patients using these therapies alone (>60 years).
These therapies do not target the proteins that cause the pathogenic effects of the virus.

Use of a Beckman Coulter Core Platform to run the Promega Checkmate^TM Mammalian Two-Hybrid System

	Prepare cell suspension to deliver 10,000 cells/well in 100 ul of media.

	Incubate overnight to allow cells to adhere to plastic.

	Remove media from wells, add 100 ul fresh media/drugs.

	Incubate for 3 hours.

	Prepare DNA/CaPO4 complexes according to Promega's ProFection® protocol. Add 12 ul of transfection complexes/well.

	Shake slowly for 15 seconds to evenly distribute transfection complexes.

	Incubate for 16 hours.

	Wash plates three times with PBS.

	Add fresh media.

	Incubate for 32 hours.

	Wash plates once with PBS.

	Add lysis buffer (20 ul/well).

	Shake for 10 minutes.

	Prepare luciferase reagents, inject and read using the Dual-Luciferase^TM protocol on the TriLux.
Human Immunodeficiency Virus Life Cycle