Homogeneous high-throughput screening assays for HIV-1 integrase 3beta-processing and strand transfer activities

Area of Science:

• Biochemistry
• Virology
• Drug Discovery

Background:

• HIV-1 integrase (HIV-IN) is a critical target for antiviral therapies, facilitating proviral DNA integration into host genomes.
• Small molecule inhibitors targeting the strand transfer step of HIV-IN are effective but limited in number, necessitating new scaffold discovery.

Purpose of the Study:

• To develop and validate novel homogeneous time-resolved FRET-based assays for measuring HIV-1 integrase 3'-processing and strand transfer activities.
• To screen a large compound library for novel HIV-1 integrase strand transfer inhibitors using high-throughput screening (HTS) formats.

Main Methods:

• Development of two homogeneous time-resolved FRET assays to quantify HIV-1 integrase enzymatic activities.
• Optimization of assays for HTS in 1536-well plates.
• Screening of a diverse library of over 1 million compounds for HIV-IN strand transfer inhibitors.

Main Results:

• Identification of compounds selectively inhibiting the enzymatic strand transfer activity over 3'-processing.
• Discovery of several bioactive molecules demonstrating inhibition of HIV-1 reporter virus infection in cellular models.
• Validation of the developed assays for identifying novel antiviral compounds.

Conclusions:

• The developed FRET-based assays are effective tools for HTS of HIV-1 integrase inhibitors.
• The identified compounds show potential as starting points for developing new anti-HIV-1 drugs.
• These assays facilitate the discovery of mechanistically novel and biologically active HIV-1 integrase inhibitors.