Homogeneous high-throughput screening assays for HIV-1 integrase 3beta-processing and strand transfer activities.
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. Small molecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only few specific 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 assays were optimized for high-throughput screening formats, and a diverse library containing more than 1 million compounds was 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 3beta processing. Moreover, several bioactive molecules were identified that inhibited HIV-1 reporter virus infection in cellular model systems. In conclusion, the assays presented herein have proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs.
Wang
Yu
Y
Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA.
Klock
Heath
H
Yin
Hong
H
Wolff
Karen
K
Bieza
Kimberly
K
Niswonger
Kirk
K
Matzen
Jason
J
Gunderson
Drew
D
Hale
Joanna
J
Lesley
Scott
S
Kuhen
Kelli
K
Caldwell
Jeremy
J
Brinker
Achim
A
D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D019427:HIV Integrase / Q000235:genetics* / Q000378:metabolism*; D019428:HIV Integrase Inhibitors / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D008957:Models, Genetic; D010713:Phosphatidylcholines / Q000494:pharmacology; D013997:Time Factors
D000998:Antiviral Agents; D019428:HIV Integrase Inhibitors; D010713:Phosphatidylcholines; C044819:1,2-diarachidonoyl-glycero-3-phosphocholine; D019427:HIV Integrase