(418c) Investigation of Small Molecule Inhibitors for OAS 1 to Enhance Gene Therapy

With unremitting efforts from generations of brilliant scientists and physicians, numerous treatments have been developed to help those who are suffering from diseases. Gene therapy is undoubtedly the jewel in the crown, with a global market that is expected to be worth $24.5 billion by 2030. However, even after decades of efforts in clinical trials, there are still major hurdles such as innate immune response shackling the further development of gene therapy. 2′-5′-Oligoadenylate synthetases (OAS) protein family is crucial for the human innate immune system, three forms of 2′,5′OAS have been identified, OAS 1, OAS 2 and OAS 3 respectively.¹ When OAS 1 and OAS 2 are activated, they can defend the human body against viral infection via the production of 2′,5′-oligoadenylate (2-5A) to activate Ribonuclease L, which will eventually degrade viral and cellular mRNAs and rRNAs.² Inhibition of OAS proteins may be beneficial for gene therapy.

In this study, we aimed to identify small molecule inhibitors against OAS 1 protein via a computer-aided drug development approach. The pipeline from our previous work was adjusted with the OAS protein (PDB ID 4IG8) as the input of the computational program Molsoft ICM, a powerful tool for ligand-protein docking study.^{3, 4, 5} Prior to docking, the protein structure was prepared by removing the co-crystallized ligand, deleting water molecules, and optimizing amino acid residues. ICM PocketFinder was employed to identify the compound binding pockets, with a particular focus on the natural ligand ATP binding pocket, and a docking box was generated to encompass that region. To ensure the reliability of the docking predictions, the docking protocol was validated through redocking co-crystalized ligand 2'-deoxy ATP into OAS receptor, which obtained scores -32.1 kcal/mol and RMSD less than 2Å. The compounds from the diverse and non-redundant ChemBridge library containing 1.4 million compounds and FDA-approved 2406 drugs were used as the source of potential inhibitors for investigation. The compounds were preprocessed for docking by standardizing formats, generating 3D conformers, and assigning appropriate partial charges and rotatable bonds. The binding poses of each ligand within the ATP binding pocket of the OAS protein were predicted. This enabled a consensus scoring approach to systematically explore the conformational space and identify energetically favorable ligand-receptor interactions. The resulting docking poses were ranked based on their consensus docking scores. Compounds within the top 0.1% of the docking score distribution were selected as potential OAS inhibitors and further analyzed for their binding mode, interactions with key residues, and drug-like properties. The top 14 candidates were finally selected and proceeded with experimental validation.

The exogenous transgene expression was reported to be negatively correlated to OAS 1 activation level⁶. Since the primary human T cells endogenously express OAS 1⁷, the in vitro assay on Jurkat T-cells was designed in this work. Inhibitor candidates at 100 uM were incubated with Jurkat T-cells for 2 hours followed by transfection with a Green Fluorescent Protein (GFP) expression plasmid. The GFP signal from experimental groups (in triplicate) was measured after 48 hours, and the GFP expression relative change to the positive control group (GFP only, no treatment) for each candidate group was calculated after subtracting the background noise (blank Jurkat cells) was subtracted. With several rounds of experiments, three candidates with 25.8%, 24.8% and 24.89% GFP expression were observed while the positive control group had a GFP expression at 21%.

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