Insilico Medicine has successfully developed ISM5939, a novel oral ENPP1 inhibitor designed using generative artificial intelligence, marking a significant advancement in cancer immunotherapy approaches. The research, published in Nature Communications, demonstrates how AI-driven drug discovery can overcome traditional challenges in STING pathway modulation.
AI-Powered Target Discovery and Drug Design
The development of ISM5939 began with Insilico's PandaOmics platform, which integrates large-scale patient omics data to identify therapeutic targets. The analysis revealed ENPP1 as a critical immune checkpoint across multiple solid tumor types, including triple-negative breast cancer, hepatocellular carcinoma, acute myeloid leukemia, ovarian carcinoma, and colorectal adenocarcinoma.
Using Chemistry42, Insilico's generative chemistry platform, researchers employed structure-based drug design starting from known ENPP1 inhibitors QS1 and QPS2. The AI platform generated diverse candidate structures based on medicinal chemistry filters, pharmacophore hypothesis rewards, and synthetic accessibility, leading to the discovery of initial hit compound ISM7516 within three months.
Through iterative optimization using Chemistry42's integrated features, including Alchemistry for binding energy calculations and ADMET prediction modules, the team developed ISM5939. The compound demonstrated superior drug-like properties, including low CYP3A4 induction, high metabolic stability, minimal hERG liability, and favorable pharmacokinetic profiles.
Exceptional Potency and Selectivity
ISM5939 exhibited remarkable inhibitory activity against ENPP1, with IC50 values of 0.63 nM for 2,3-cGAMP degradation and 9.28 nM for ATP hydrolysis under physiological conditions. This potency significantly surpassed the positive control ENPP-IN-1, which showed IC50 values of 259.0 nM and 1328.0 nM, respectively.
The compound demonstrated exceptional selectivity for ENPP1, with IC50 values for ENPP2 and ENPP3 being over 15,000 and 3,400 times higher, respectively. Comprehensive safety profiling using the ICESTP SafetyOne44 panel showed no significant agonistic or antagonistic effects on 44 clinically relevant off-targets.
Enhanced Therapeutic Index Over Direct STING Agonists
A critical advantage of ISM5939 lies in its superior safety profile compared to direct STING agonists. While traditional STING agonists like ADU-S100 induced significant release of inflammatory cytokines including IFN-α, IL-6, and TNF-α in human peripheral blood mononuclear cells, ISM5939 showed no such effects even at high concentrations.
In vivo studies confirmed these findings, with systemic administration of STING agonists diABZI and MSA-2 causing marked increases in inflammatory cytokine levels in mouse plasma, while ISM5939 treatment showed no significant cytokine induction. Additionally, unlike direct STING agonists that caused dose-dependent reduction in CD4+ and CD8+ T cell viability, ISM5939 preserved T cell populations while enhancing their antitumor functions.
Synergistic Combination Therapies
ISM5939 demonstrated significant synergistic effects when combined with existing cancer therapies. In combination with anti-PD-1 therapy in the MC38 tumor model, the compound showed superior tumor growth suppression with coefficient drug interaction values confirming synergy (CDI = 0.34-0.80).
The combination enhanced the ratio of M1 to M2 macrophages and increased CD8+ T cell to regulatory T cell ratios. Tumor-infiltrating lymphocyte analysis revealed highest CD8+ T cell infiltration and CD69 activation marker expression in mice receiving the combination regimen.
ISM5939 also synergized with chemotherapeutic agents, including cisplatin (CDI = 0.70), docetaxel (CDI = 0.86), and olaparib (CDI = 0.93). The combinations resulted in significant cGAMP accumulation in tumor microenvironments and enhanced activation of both innate and adaptive immune pathways.
Clinical Translation Potential
Preclinical toxicology studies support ISM5939's clinical development potential. Twenty-eight-day GLP toxicity studies in rats and dogs showed the compound was well-tolerated at doses up to 150 mg/kg/day and 30 mg/kg/day, respectively, with no severe adverse changes in clinical signs, body weight, or microscopic examination.
Using in vitro-in vivo extrapolation and allometric scaling analyses, researchers projected a human dose range of 70 to 358 mg/day, significantly lower than the 100 to 800 mg twice daily dosage required for RBS2418, another ENPP1 inhibitor in clinical trials.
Mechanism of Action
ISM5939 functions by inhibiting ENPP1-mediated degradation of cGAMP, thereby preserving this critical second messenger in the tumor microenvironment. The stabilized cGAMP can then activate the STING pathway in antigen-presenting cells, leading to enhanced interferon signaling and improved antitumor immunity.
Molecular docking studies revealed that ISM5939 binds to the ATP/cGAMP active site of ENPP1, with its modified imidazole[4,5-c]pyridine core forming hydrogen bonds with key amino acids and additional π-π interactions contributing to its high affinity.
The research represents Insilico Medicine's third Nature Portfolio publication in 2024, demonstrating the company's continued advancement in AI-driven drug discovery. The development of ISM5939 from project initiation to preclinical candidate nomination was completed in 12-18 months, significantly faster than the typical 2.5-4 years required in traditional drug discovery.
