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P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro
H S Mancebo1, G Lee, J Flygare
1Tularik, Inc., South San Francisco, California 94080 USA.
Abstract:
To identify novel inhibitors of transcriptional activation by the HIV Tat protein, we used a combination of in vitro and in vivo Tat-dependent transcription assays to screen >100,000 compounds. All compounds identified blocked Tat-dependent stimulation of transcriptional elongation. Analysis of a panel of structurally diverse inhibitors indicated that their target is the human homolog of Drosophila positive transcription elongation factor b (P-TEFb). Loss of Tat transactivation in extracts depleted of the kinase subunit of human P-TEFb, PITALRE, was reversed by addition of partially purified human P-TEFb. Transfection experiments with wild-type or kinase knockout PITALRE demonstrated that P-TEFb is required for Tat function. Our results suggest that P-TEFb represents an attractive target for the development of novel HIV therapeutics.
Insights
Researchers screened over 100,000 compounds to find HIV Tat protein inhibitors. The study identified positive transcription elongation factor b (P-TEFb) as a key target for novel HIV therapeutics.
Area of Science:
- Molecular Biology
- Virology
- Drug Discovery
Background:
- The HIV Tat protein is crucial for viral replication by enhancing viral transcription.
- Identifying inhibitors of Tat-mediated transcriptional activation is a key strategy for developing new anti-HIV drugs.
Purpose of the Study:
- To screen a large library of compounds to identify novel inhibitors of HIV Tat transcriptional activity.
- To elucidate the molecular target of the identified inhibitors and validate its role in Tat function.
Main Methods:
- High-throughput screening of over 100,000 compounds using in vitro and in vivo Tat-dependent transcription assays.
- Biochemical analysis of inhibitor mechanisms, including depletion and reconstitution experiments with human P-TEFb (positive transcription elongation factor b).
- Functional studies using transfection assays with wild-type and kinase-deficient PITALRE (the kinase subunit of human P-TEFb).
Main Results:
- Over 100,000 compounds were screened, leading to the identification of several inhibitors of Tat-dependent transcriptional elongation.
- Structurally diverse inhibitors were found to target the human homolog of Drosophila positive transcription elongation factor b (P-TEFb).
- Tat transactivation was dependent on P-TEFb, as demonstrated by depletion and reconstitution experiments and transfection studies with PITALRE variants.
Conclusions:
- Positive transcription elongation factor b (P-TEFb) is essential for HIV Tat protein function.
- P-TEFb is a promising therapeutic target for the development of novel anti-HIV drugs.
- Inhibitors targeting P-TEFb show potential for blocking HIV replication.