(M1330-04-23) The Search for HIV Latency Reversing Selective HDAC3 Inhibitors Through Virtual Screening

Purpose: There are numerous antiretroviral therapies (ARTs) developed for treating HIV, lowering the viral load to undetectable levels and thereby inhibiting transmission of the disease. However, HIV remains incurable due to the presence of latent HIV reservoirs that are not treatable with the current ART¹. In addition, the life-long use of ART by patients results in chronic side effects and the development of drug-resistant mutations, thus a cure is needed². Herein, we report the use of Computer-Aided Drug Design (CADD) approaches to discover selective histone deacetylase 3 allosteric inhibitors (HDAC3Is) which are latency-reversing agents (LRAs) that can activate latent HIV reservoirs³. Activating the latent reservoirs while inhibiting viral replication with ART, via the “shock and kill” method, is a promising approach to eliminating latent HIV and potentially curing HIV¹. Although there are class I HDACIs on the market, they are not isoform-selective due to the structural homology across the catalytic sites of class I HDACs⁴, resulting in drug-induced adverse effects such as nausea, diarrhea, and anemia⁵. To overcome the challenge of selectivity, instead of targeting the highly conserved catalytic sites, our novel approach takes advantage of the presence of un-conserved regions of the allosteric corepressor site of HDAC3 to design small molecules that would selectively bind that site using various CADD approaches. HDAC3 requires exclusive complexation with the SMRT/NCoR corepressor to carry out its catalytic function, whereas its class I homologs form various complexes with other common corepressors⁴. We hypothesize that inhibition of HDAC3-SMRT/NCoR complexation by small-molecule(s) will result in enzyme inactivation (Graphic 1).

Methods: Our in-house and ZINC chemical libraries were screened using the Virtual Screening (VS) Workflow of Glide docking (Schrödinger Maestro)^6-8. The HDAC3 protein structure (PDB ID: 4A69) and ligands were preprocessed prior to docking. In addition, the co-crystallized inositol tetraphosphate (IOP) ligand was used as a control ligand to validate the docking protocol, and its docking score was used as a cut-off for identifying potential hits through VS. Additionally, selected hit molecules were docked across class I HDAC complexes (HDAC1:MTA1 complex – PDB ID: 5ICN, HDAC1:MIDEAS complex – PDB ID: 6Z2J) to predict their potential selectivity to bind HDAC3. Select hit molecules were experimentally validated using a fluorogenic inhibitor screening assay from which we calculated IC₅₀ values using GraphPad Prism 9.

Results: The Glide docking approach successfully regenerated the co-crystalized pose of IOP (indicated by the low RMSD value of 1.419 Å) with a docking score of -7.247 kcal/mol. Thus far, VS of our in-house chemical library yielded an HDAC3 inhibitor (S521051) with an IC₅₀ of approximately 5.11 µM (Graphic 2). The docking score of S521051 was -10.235 kcal/mol for the HDAC3:NCoR complex compared to -6.391 kcal/mol and -6.851 kcal/mol for the HDAC1:MTA1 complex and HDAC1:MIDEAS complex respectively, conferring possible selectivity for HDAC3 (Graphic 3).

Conclusion: Through virtual screening workflow using Glide docking, we were able to identify an allosteric inhibitor for HDAC3 with an IC₅₀ of 5.11 µM. Our search for novel allosteric HDAC3I scaffolds, and optimization of S521051 to improve its potency is ongoing.

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Acknowledgements: We acknowledge the financial support from Concordia University Wisconsin School of Pharmacy to fund the research and the technical support from Schrödinger Inc. to assist with the application of software tools. We also acknowledge collaborative support from Dr. Michael Pickart and Dr. Robert Burlarge.

Vorinostat and S521051 bound to HDAC3 at the Orthosteric catalytic and the Allosteric co-repressor binding domain respectively.

Fluorogenic HDAC3 screen of S521051 with inhibitor control Trichostatin A (TSA)

Comparative Glide docking results of S521501 against the corepressor sites of class I HDACs