Background The cGAS-STING pathway is a key mediator in boosting anti-tumor responses through activation of the type I interferon signaling cascade. Major limitations of STING agonists currently studied include poor pharmacokinetic and physicochemical properties, and risks of adverse effects due to induction of excessive cytokines following systemic administration. An alternative STING activation strategy, that may improve efficacy and safety, is to inhibit the extracellular hydrolase, ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), a negative regulator of the STING pathway, which directly hydrolyses 2′3′-cGAMP, the natural ligand for STING. Increased ENPP1 expression has been shown to be associated with reduced immune cell infiltration and poor prognosis in multiple tumor types. Inhibition of ENPP1 would prevent abolishment of the cGAS-STING-mediated immune activation, and allow for modulation of the tumor microenvironment with more T-cell infiltration and dendritic cell activation. Here, we report ISM5939 is a novel, potent, selective inhibitor of ENPP1 with oral bioavailability.

Methods ISM5939 was designed with the assistance of Chemistry42, an AI platform. An ENPP1 enzyme inhibitory activity assay was used to assess the bioactivity of ISM5939. Cellular ENPP1 inhibitory activity was assessed using a 2’,3’-cGAMP ELISA assay in the triple negative breast cancer cell line, MDA-MB-231. In vivo studies were conducted in the MC-38 and CT-26 syngeneic colorectal carcinoma mouse models.

Results ISM5939 potently inhibited ENPP1 with an IC50 of 0.63 nM in the enzymatic assay, and an EC50 of 330 nM in the MDA-MB-231 cellular assay. ISM5939 monotherapy (30 mg/kg, p.o. BID) demonstrated a 67% Tumor Growth Inhibition (TGI) in the MC38 model. Further, ISM5939 dose-dependently enhanced the efficacy of anti-PD-L1 or anti-PD-1 therapy. At 30 mg/kg, p.o. BID, ISM5939 in combination with anti-PD-L1 or anti-PD-1 exhibited synergistic effects, with TGI of 96% vs 53% with anti-PD-L1 alone, and 68% vs 28% with anti-PD-1 antibody alone. Furthermore, ISM5939 administration did not result in a significant change in body weight.

ISM5939 displayed favorable ADME properties. At 10 uM, ISM5939 showed no obvious agonistic or antagonistic effects on 44 selected targets in a mini safety panel, demonstrating its selectivity as an ENPP1 inhibitor. Further, results from a 28-day rat and dog DRF study revealed a good safety margin for ISM5939.

Conclusions ISM5939, is a novel, potent ENPP1 inhibitor that augments the effectiveness of anti-PD-L1 or anti-PD1 therapy in multiple syngeneic mouse tumor models, while exhibiting favorable safety and tolerability properties. Together, these data support further evaluation of ISM5939 as an anti-tumor agent.