Pharmacological activation of the STING (stimulator of interferon genes)–controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-β secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti–PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

Phosphodiester linkages, including those that join the nucleotides of DNA, are highly resistant to spontaneous hydrolysis. The rate of water attack at the phosphorus atom of phosphodiesters is known only as an upper limit, based on the hydrolysis of the dimethyl phosphate anion. That reaction was found to proceed at least 99% by C-O cleavage, at a rate suggesting an upper limit of 10 ؊15 s ؊1 for P-O cleavage of phosphodiester anions at 25°C. To evaluate the rate enhancement produced by P-O cleaving phosphodiesterases such as staphylococcal nuclease, we decided to establish the actual value of the rate constant for P-O cleavage of a simple phosphodiester anion. In dineopentyl phosphate, C-O cleavage is sterically precluded so that hydrolysis occurs only by P-O cleavage. Measurements at elevated temperatures indicate that the dineopentyl phosphate anion undergoes hydrolysis in water with a t 1/2 of 30,000,000 years at 25°C, furnishing an indication of the resistance of the internucleotide linkages of DNA to water attack at phosphorus. These results imply that staphylococcal nuclease (k cat ‫؍‬ 95 s ؊1 ) enhances the rate of phosphodiester hydrolysis by a factor of Ϸ10 17 . In alkaline solution, thymidylyl-3 -5 -thymidine (TpT) has been reported to decompose 10 5 -fold more rapidly than does dineopentyl phosphate. We find however that TpT and thymidine decompose at similar rates and with similar activation parameters, to a similar set of products, at pH 7 and in 1 M KOH. We infer that the decomposition of TpT is initiated by the breakdown of thymidine, not by phosphodiester hydrolysis.DNA hydrolysis ͉ DNA stability ͉ nuclease ͉ rate enhancement ͉ phosphate ester P hosphoric acid diesters are, in general, exceedingly unreactive in water (1-3), so that the phosphodiester linkages that join the nucleotides of DNA are highly resistant to spontaneous hydrolysis. By extrapolation of earlier model experiments at elevated temperatures, the uncatalyzed hydrolysis of dimethyl phosphate in neutral solution was found to proceed with an estimated rate constant of Ϸ2 ϫ 10 Ϫ13 s Ϫ1 at 25°C, corresponding to a half-time of 140,000 years. That reaction was found to proceed at least 99% by C-O cleavage, suggesting an upper limit of Ϸ1 ϫ 10 Ϫ15 s Ϫ1 at 25°C on the rate constant for spontaneous P-O cleavage of a phosphodiester anion, the reaction that is catalyzed by many phosphodiesterases (4).More recently, a rate constant of 6 ϫ 10 Ϫ7 s Ϫ1 has been reported for the decomposition of thymidylyl-3Ј-5Ј-thymidine (TpT) at 80°C in 1 M KOH (5). Extrapolation of the results obtained earlier for dimethyl phosphate hydrolysis in neutral solution (4), to 80°C, would indicate a rate Ϸ10 5 -fold slower. That discrepancy might indicate a major role for catalysis by hydroxide, but the hydrolysis of another dialkyl phosphodiester, bis-3-(4-carboxyphenyl)neopentyl phosphate (Np* 2 P), in which ␥-branching of the leaving alcohol prevents C-O cleavage (Fig. 1), also proceeds Ϸ10 5 -fold more slowly in 1 M KOH (6).In an effort to resolve that discre...

To estimate the relative importance of alternate routes of spontaneous degradation of DNA and the rate enhancements produced by enzymes catalyzing these reactions, rate constants and thermodynamic activation parameters for the degradation of 2'-deoxynucleosides at 25 degrees C were determined by extrapolation of rates observed in the temperature range between 90 and 200 degrees C in neutral phosphate buffer. Rates of deamination of 2'-deoxycytidine, 1-methylcytosine, and cytidine were found to be identical within experimental error (t1/2 approximately 20 years, 37 degrees C). Rate constants for deamination of 2'-deoxyadenosine and 2'-deoxyguanosine, which could not be determined directly because of rapid glycoside cleavage, were estimated by assuming that methyl replacement should generate reasonable model substrates. The rates of deamination of 9-methyladenine and 9-methylguanine were found to be similar to each other (t1/2 approximately 6000 years, 37 degrees C) and approximately 10(2)-fold slower than the rates of glycoside cleavage in 2'-deoxyadenosine and 2'-deoxyguanosine. The deamination of 2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxycytidine led to accelerated rates of glycoside cleavage. In the exceptional case of 2'-deoxycytidine, deamination and glycoside hydrolysis proceed at very similar rates at all temperatures. Glycoside cleavage proceeds with half-times ranging from 4 years for 2'-deoxyinosine to 40 years for 2'-deoxycytidine (37 degrees C). The rate enhancements produced by DNA glycosylases, estimated by comparison with the rates of these uncatalyzed reactions, are found to be substantially smaller than those produced by deaminases and staphylococcal nuclease.

Drugging large protein pockets is a challenge due to the need for higher molecular weight ligands, which generally possess undesirable physicochemical properties. In this communication, we highlight a strategy leveraging small molecule active site dimers to inhibit the large symmetric binding pocket in the STING protein. By taking advantage of the 2:1 binding stoichiometry, maximal buried interaction with STING protein can be achieved while maintaining the ligand physicochemical properties necessary for oral exposure. This mode of binding requires unique considerations for potency optimization including simultaneous optimization of protein−ligand as well as ligand−ligand interactions. Successful implementation of this strategy led to the identification of 18, which exhibits good oral exposure, slow binding kinetics, and functional inhibition of STING-mediated cytokine release.