Fighting the Good Fight: Structure Based Design and Biological Investigation of Allosteric OAS1 Inhibitors for Mitigating Inborn Errors of Immunity Restricted; Files Only

Abstract

      To prevent the spread of viruses, interferons are released by immune cells—activating the immunoregulatory enzyme, oligoadenylate synthetase 1 (OAS1). When viral dsRNA is present in the cytosol, OAS1 and dsRNA bind to form a complex—initiating a conformational change which allows OAS1 to produce 2’-5’-oligoadenylate molecules (2-5A). These 2-5A molecules in turn stimulate the dimerization of RNase L promoters, activating its nuclease function and ultimately degrading the viral dsRNA. OAS1-GoF mutations have been discovered in patients presenting symptoms of inborn errors of immunity (IEI), resulting in excessive degradation of the host’s own genetic material. These OAS1-GoF variants arise from single-point mutations within the amino acid sequence, folding the protein in a manner capable of producing 2-5A molecules without the necessity of pathogenic dsRNA. Currently, the only treatment for these genetic disorders is allogeneic hematopoietic cell transplantation (HCT)—a costly and risky procedure. In an effort to derive new therapeutic practices for the management of IEI, our lab, with the aid of collaborators, has set out on a journey to develop a small molecule inhibitor of OAS1-GoF mutants. In this quest, the benzimidazole-imidazole compound 13 was developed, with inhibiting enzymatic activity up to 80%. To discover an even more potent inhibitor, I undertook a structure-activity-relationship (SAR) campaign of compound 13 that explored the necessity of the benzimidazole, the optimal linker length between the benzimidazole and imidazole, and both the size and stereo configuration of the alkyl substituent on the linker. My results were that the R enantiomer of each analog had a slightly lower IC50 than the S counterpart, suggesting there may be a preference for the R enantiomer in the pocket. Additionally, it became apparent that there was no direct relationship between linker-length and binding affinity of the inhibitor. Lastly, swapping the benzimidazole with a phenyl group approved to be detrimental to the binding, signifying the importance of the benzimidazole. These findings will help us in the ongoing effort of designing an inhibitor of OAS1-GoF mutants with maximum effectivity.

Table of Contents

1. Introduction                                                                                                         1

1.1. Interferon induced OAS1 pathway                                                                          1

1.2. OAS1 activation and 2-5A production                                                                    5        

1.3. Mutations of OAS1 and associated disorders                                                        6

1.4. OAS1 docking studies and HTS screening                                                              8

1.5. Project goals: Structure Activity Relationship (SAR) campaign                        10

2. Results                                                                                                                               12

2.1. Generic synthetic strategy for #13 and sequential analogs                                12

2.2. Demystifying chirality necessity and determining optimal linker length       14

2.3. Determining optimal stereoconfiguration and linker substituent bulk           16

2.4. Exploring the necessity of the benzimidazole for pip-pi stacking      21

3. Conclusion           23

4. Experimental        26

5. References            75                             

About this Master's Thesis

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School	Laney Graduate School
Department	Chemistry
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Chemistry, Biochemistry Chemistry, Organic
Keyword	imidazole OAS1 inborn errors of immunity benzimidazole amino acid SAR drug discovery
Committee Chair / Thesis Advisor	William Wuest, Emory University
Committee Members	Laura Ackerman-Biegasiewicz, Emory University Simon Blakey, Emory University

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