Previous research demonstrated that a small HIV protein called Nef interacts with many other proteins in infected cells to help the virus multiply and hide from the immune system. The Pitt group developed a way to track Nef activity in high-throughput drug screening protocols by linking it to an enzyme called Hck, which is activated by Nef in HIV-infected cells, explained senior author Thomas E. Smithgall, Ph.D., William S. McEllroy Professor and Chair, Department of Microbiology and Molecular Genetics.
"We reasoned that agents that prevent Nef from its usual interactions with other proteins might be able to stop HIV from replicating and infecting other cells," Dr. Smithgall said. "For this study, we devised an automated screening procedure and tested nearly 250,000 compounds to find ones that could block Nef activity."
One of the compounds they discovered, called B9, seemed particularly potent at blocking Nef. In follow-up experiments, the research team examined how B9 accomplished this and found that it could prevent two Nef molecules from interacting to form dimers as effectively as a mutation in a critical area of the protein surface. The inability of Nef to dimerize consequently impairs its function in the viral replication process.
"This pocket where B9 binds to Nef and where Nef forms a dimer indicates it's a hot spot, or Achilles heel, that could represent a new target for HIV drugs," Dr. Smithgall said. "Our test tube and cell culture experiments show that blocking this site brings HIV replication to a halt."
The team is working with medicinal chemists at the University of Pittsburgh Drug Discovery Institute (DDI) to find analogs of B9 that have therapeutic potential, and plan to assess them in animal models of HIV/AIDS.
Co-authors of the study include lead investigator Lori Emert-Sedlak, Ph.D., Purushottam Narute, Ph.D., Sherry Shu, Ph.D., and Jerrod A. Poe, Ph.D., all of the Department of Microbiology and Molecular Genetics, Pitt School of Medicine; Haibin Shi, Ph.D., Naveena Yanamala, Ph.D., John Jeff Alvarado, Ph.D., and Joanne Yeh, Ph.D., all of the Department of Structural Biology, Pitt School of Medicine; Paul Johnston, Ph.D., of DDI and the Department of Pharmaceutical Sciences, Pitt School of Pharmacy; and John Lazo, Ph.D., now of the University of Virginia.
The project was funded by National Institutes of Health grants R01 AI057083, R21 AI077444, and X01 MH083223.
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About the University of Pittsburgh School of Medicine
Likewise, the School of Medicine is equally committed to advancing the quality and strength of its medical and graduate education programs, for which it is recognized as an innovative leader, and to training highly skilled, compassionate clinicians and creative scientists well-equipped to engage in world-class research. The School of Medicine is the academic partner of UPMC, which has collaborated with the University to raise the standard of medical excellence in Pittsburgh and to position health care as a driving force behind the region's economy. For more information about the School of Medicine, seewww.medschool.pitt.edu.
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