Work

Molecular characterisation of the inhibitor didehydro-Cortistatin A with the HIV-1 Tat protein

Year: 2017

Type: article

Abstract: Background: The HIV-1 Tat protein binds the HIV mRNA transactivation response (TAR) element, recruits cellular transcriptional cofactors, and exponentially amplifies viral mRNA expression.There are no clinically available anti-Tat drugs.We reported that didehydro-Cortistatin A (dCA) inhibits Tat and viral production from integrated proviruses at nanomolar concentrations.dCA promotes a state of sustained latency, from which transcriptional reactivation is drastically diminished.Here, we characterized the interaction of Tat with dCA.Methods: Isothermal titration calorimetry (ITC) determined the affinity of dCA for Tat.Structure-activity relationship (SAR) studies of dCA and 11 analogs synthesized, combined with molecular modeling using AutoDock Vina of Tat NMR data, revealed interacting chemicals groups of dCA mainly with Tat's basic patch residues.dCA specificity for Tat's basic domain was evaluated by electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP).We excluded interaction of dCA with proteins with similar basic domains (Hexim-1 and HIV-1 Rev), using immunofluorescence, qPCR and ChIP.Tat's basic domain is also responsible for the stability of the protein, and dCA's stabilization of Tat was demonstrated by Trp fluorescence and enzymatic degradation assays.Results: Nanomolar affinity of dCA for Tat was determined by ITC.SAR revealed the importance of the nitrogen atom (position and orientation) in the isoquinoline group and the cycloheptene ring of dCA, in the interaction with Tat's basic (K 50 , K 51 , R 53 and R 55 ) and N-terminal (P 3 and E 2 ) regions.We demonstrated that dCA disrupts Tat-TAR interaction and stabilizes Tat structure, potentially useful in Tat crystallization efforts.Conclusions: We characterized the molecular determinants for the activity of dCA on Tat, which is invaluable for the clinical development of the Cortistatin A pharmacophore, and for the development of Tat inhibitors.

Source: Journal of Virus Eradication

DOI: 10.1016/s2055-6640(20)30679-8