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Homogeneous High-Throughput Screening Assays for HIV-1 Integrase 3ß-Processing and Strand Transfer Activities

Heath Klock

Hong Yin

Karen Wolff

Kimberly Bieza

Kirk Niswonger

Jason Matzen

Drew Gunderson

Joanna Hale

Scott Lesley

Kelli Kuhen

Jeremy Caldwell

Achim Brinker

Genomics Institute of the Novartis Research Foundation, San Diego, CA.

HIV-1 integrase (HIV-IN) is a well-validated antiviral drug target catalyzing a multistep reaction to incorporate the HIV-1 provirus into the genome of the host cell. Smallmolecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only fewspecific strand transfer inhibitors have been identified to date, and the need to screen for novel compound scaffolds persists. Here, the authors describe 2 homogeneous time-resolved fluorescent resonance energy transfer-based assays for the measurement of HIV-1 integrase 3'-processing and strand transfer activities. Both assayswere optimized for high-throughput screening formats, and a diverse library containingmore than 1million compoundswas screened in 1536-well plates for HIV-IN strand transfer inhibitors. As a result, compounds were found that selectively affect the enzymatic strand transfer reaction over 3ß processing. Moreover, several bioactivemoleculeswere identified that inhibited HIV-1 reporter virus infection in cellularmodel systems. In conclusion, the assays presented herein have proven their utility for the identification ofmechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs.

Key Words: HIV-1 • antivirals • TR-FRET • strand transfer • 3ß processing

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