Abstract 4983: Discovery and characterization of next-generation small molecule direct STING agonists

Abstract

Accumulating evidence highlights an important role of type I interferon response in the immune surveillance mechanisms. IFNβ release by antigen-presenting cells promotes spontaneous anti-tumor CD8+ T cell priming being largely dependent on activation of Stimulator of Interferon Genes (STING). STING agonists promote regression of established tumors and generation of long-term immunologic memory in preclinical animal models. Herein we report the discovery of potent and selective, first-in-class non-nucleotide, non-macrocyclic, small molecule direct STING agonists with molecular weight below 500, structurally unrelated to known cyclic dinucleotide chemotypes with potential for systemic administration. Activation of STING pathway was monitored in THP-1 Dual reporter monocytic cell line as well as peripheral blood mononuclear cells (PBMC) or antigen presenting cells from human and mouse origin. Surface expression of the antigen-presenting cell maturation markers i.e. CD80, CD86, CD83 and HLA-DR was assessed by flow cytometry. Binding affinity was confirmed by three independent assays. RNA sequencing was performed on total RNA isolated from THP-1 cells and PBMC isolated from 2 healthy human donors. Direct binding to both mouse and human STING protein of Selvita agonists have been confirmed in biophysical binding assays (FTS, MST and FP) and by crystallography studies. The compounds have fine-tunable ADME properties with good solubility, permeability and human plasma stability. They selectively activates STING-dependent signaling in both THP-1 reporter assays and in primary cells of human and mouse origin. In addition, RNA sequencing data confirmed selectivity of the Selvita compounds. In vitro functional assays demonstrated their ability to induce cytokine responses (IFNβ, TNFα) in a panel of human peripheral blood mononuclear cell (PBMC), human monocyte derived macrophage (HMDM) and human dendritic cells samples with various STING haplotypes including refractory alleles. Additionally, the compounds efficiently induced cytokine release in mouse bone marrow-derived macrophages and dendritic cells. Pro-inflammatory cytokine profile was accompanied by up-regulation of the maturation markers, i.e. CD80, CD86, CD83 and HLA-DR, on the surface of human antigen presenting cells. These data demonstrate potent, novel, next-generation small molecule STING agonists activating STING-dependent signaling in both mouse and human immune cells to promote potential antitumor immunity. The compounds show good selectivity and in vitro ADME properties enabling further development for systemic administration as a single agent or in combinatory immunotherapies for cancer treatment. Citation Format: Monika Dobrzańska, Stefan Chmielewski, Magdalena Zawadzka, Jolanta Mazurek, Karolina Gluza, Katarzyna Wójcik-Jaszczyńska, Maciej Kujawa, Grzegorz Topolnicki, Grzegorz Ćwiertnia, Aleksandra Poczkaj, Izabela Dolata, Magdalena Mroczkowska, Agnieszka Gibas, Marcin Leś, Sylwia Sudoł, Adam Radzimierski, Kinga Michalik, Magdalena Sieprawska-Lupa, Katarzyna Banaszak, Katarzyna Wiklik, Federico Malusa, Michał Combik, Karolina Wiatrowska, Agnieszka Adamus, Lukasz Dudek, Jose Alvarez, Charles Fabritius, Anna Rajda, Maciej Rogacki, Faustyna Gajdosz, Peter Littlewood, Luigi Stasi, Krzysztof Brzózka. Discovery and characterization of next-generation small molecule direct STING agonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4983.

STING Agonists as Cancer Therapeutics

“…Selvita agonists are a group of recently developed small-molecule, non-nucleotide, non-macrocyclic STING agonists. These agonists selectively bind to both mouse and human STING proteins and have tunable properties with enhanced plasma stability and permeability, making them potential candidates for systemic delivery [ 89 ]. In vitro studies on peripheral blood mononuclear cells and the THP1 monocytic cell line show that Selvita agonists induce the expression of inflammatory cytokines and upregulate maturation markers on the surface of APCs.…”

Beyond DNA sensing: expanding the role of cGAS/STING in immunity and diseases

“…The tunable properties of Selvita activators with improved plasma stability and permeability make these activators potential candidates for systemic delivery. In vitro studies on peripheral blood mononuclear cells and the THP1 monocytic cell line indicate that Selvita activators can induce the expression of inflammatory cytokines and upregulate maturation markers on the surface of APCs (Dobrzańska et al 2019 ).…”