Unlocking Cancer Immunotherapy: The Potential of Nucleotide Analogs as STING Agonists

Immunotherapy has become a significant strategy in cancer treatment, but resistance is still a challenge. Studies indicate that activating the Stimulator of Interferon Genes (STING) pathway can trigger type I Interferon production, leading to tumor cell apoptosis and stimulating an adaptive immune response, which is beneficial for cancer treatment. The development of STING pathway activators is thus crucial.

Our team has utilized structure-guided drug design with the aid of published crystal structures of cyclic dinucleotides bound to STING. A library of nucleotide compounds was synthesized and screened for their ability to induce Interferon regulatory factor (IRF), Interferon-stimulated gene 54 (ISG54), and NF-KB using reporter assays. The initial screening was conducted using the HEK293 cell line, and active compounds were further tested in PBMCs and THP1 cells. The induction of IRF and NF-kB was measured by luminescence changes, and the EC50 values were determined using Xlfit software. Lead STING agonists were evaluated for binding affinity using Differential Scanning Fluorimetry and for their ability to induce pathogen recognition receptors (PRRs), ISGs, and Programmed Death Ligands 1 & 2 (PDL1, PDL2) genes through quantitative RT-PCR. Apoptosis-inducing activity was assessed using the Caspase-Glo 3/7 Assay, and in vitro anti-tumor activity was evaluated through high-content imaging or the Cell titer Glo Cytotoxicity Assay.

We identified several potent and selective STING agonists through in vitro assays and Structure Activity Relationship (SAR) studies. A lead nucleotide compound, SB 11285, demonstrated high potency in inducing IRF and NF-kB, selective apoptosis of human monocyte leukemic cell lines, and expression of various PRRs, ISGs, PDL1, and PDL2. SB 11285 also showed significant in vitro anti-tumor activity across multiple tumor cell lines.

In conclusion, we have discovered potent first-in-class STING agonists that selectively induce IFN, NF-KB, ISGs, and PRRs, and cause apoptosis in tumor cell lines. The lead compound SB 11285 possesses strong immune-modulating and anti-tumor properties and is progressing towards further preclinical studies for potential use in immuno-oncology.