Enzymatic Preparation of 2 5 ,3 5 Cyclic Dinucleotides, Their Binding Properties to Stimulator of Interferon Genes Adaptor Protein, and Structure Activity Correlations

Abstract

Cyclic dinucleotides are second messengers in the cyclic GMP AMP synthase cGAS stimulator of interferon genes STING pathway, which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 TBK1 interferon regulatory factor 3 IRF3 and inhibitor of nuclear factor amp; 954;B I amp; 954;B kinase IKK nuclear factor amp; 954;B NF amp; 954;B signaling cascades. In this work, we describe an enzymatic preparation of 2 amp; 8242; 5 amp; 8242;,3 amp; 8242; 5 amp; 8242; cyclic dinucleotides 2 amp; 8242;3 amp; 8242;CDNs with use of cyclic GMP AMP synthases cGAS from human, mouse, and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide 5 amp; 8242; triphosphate NTP analogues and use them to prepare 33 2 amp; 8242;3 amp; 8242;CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next, we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells PBMCs and evaluate their cytotoxic effect on monocytes. Additionally, we report X ray crystal structures of two new CDNs bound to STING protein and discuss structure activity relationship by using quantum and molecular mechanical QM MM computational modelin