Activation of the STING (stimulator of interferon genes) protein by its natural ligand, cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), triggers signaling responses, inducing the release of type I interferons and other proinflammatory cytokines. STING-controlled interferon production is involved in antiviral defense as well as antitumor immunity. Pharmacological activation of STING is considered a promising therapeutic strategy for cancer.

First-generation STING agonists are cyclic dinucleotide (CDN) analogs of cGAMP. When administered systemically in animal models, they induce inflammatory cytokines equipotently in tumor and normal tissues, owing to ubiquitous STING expression. Thus, CDN-based STING agonists currently undergoing clinical trials are dosed by direct intratumor injection, which limits their application to a narrow set of tumors. To address a broad spectrum of cancers, STING agonists that are suitable for systemic administration and preferentially target tumors are needed. We identified a previously unknown compound (MSA-2) that exhibits such behavior through its distinctive mechanism of action. Moreover, MSA-2 is amenable to oral administration, a desirable delivery route because of convenience and low cost.

MSA-2 was identified in a phenotypic screen for chemical inducers of interferon-β secretion (see the figure, top). In cell-free assays, MSA-2 binds human and mouse STING. MSA-2 is orally available, manifesting similar oral and subcutaneous exposure in mice. In tumor-bearing mice, MSA-2 induced elevations of interferon-β in plasma and tumors by both routes of administration. Well-tolerated regimens of MSA-2 induced tumor regressions in mice bearing MC38 syngeneic tumors. Most mice that exhibited complete regression were resistant to reinoculation of MC38 cells, suggesting establishment of durable antitumor immunity. In tumor models that were moderately or poorly responsive to PD-1 blockade, combinations of MSA-2 and anti–PD-1 antibody were superior in inhibiting tumor growth and prolonging survival over monotherapy (see the figure, right).

Structural studies showed that MSA-2 was bound as a noncovalent dimer to STING in a “closed-lid” conformation (see the figure, left). Each bound MSA-2 interacted with both monomers of the STING homodimer (depicted in blue and orange). The simplest model that can account for all observed equilibrium and kinetic behaviors of MSA-2 is as follows: MSA-2 in solution exists as monomers and noncovalent dimers in an equilibrium that strongly favors monomers; MSA-2 monomers cannot bind STING, whereas the noncovalent MSA-2 dimers bind STING with nanomolar affinity (see the figure, center). The model was further supported by findings that covalently tethered dimers of MSA-2 analogs exhibited nanomolar affinity for STING.

Simulations and experimental analyses predicted that MSA-2, a weak acid, would exhibit substantially higher cellular potency in an acidified tumor microenvironment than normal tissue, owing to increased cellular entry and retention combined with the inherently steep MSA-2 concentration dependence of STING occupancy (see the figure, bottom). It is likely that preferential activation of STING by MSA-2 in tumors substantially contributes to the observed favorable in vivo antitumor activity and tolerability profile of this compound.

In this work, we describe the identification, in vivo antitumor properties, and mechanism of action of MSA-2, an orally available human STING agonist. MSA-2 could prove valuable for the discovery and design of human STING agonists …