Structure-aided development of small molecule inhibitors of ENPP1, the extracellular phosphodiesterase of the immunotransmitter cGAMP

Abstract

Cancer cells initiate an innate immune response by synthesizing and exporting the small molecule immunotransmitter cGAMP, which activates the anti-cancer Stimulator of Interferon Genes (STING) pathway in the host. An extracellular enzyme, ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), hydrolyzes cGAMP and negatively regulates this anti-cancer immune response. Small molecule ENPP1 inhibitors are much needed as tools to study basic biology of extracellular cGAMP and as investigational cancer immunotherapy drugs. Here, we surveyed structure-activity relationships around a series of cell-impermeable and thus extracellular-targeting phosphonate inhibitors of ENPP1. Additionally, we solved the crystal structure of an exemplary phosphonate inhibitor to elucidate the interactions that drive potency. This study yielded several best-in-class compounds with Ki < 2 nM and excellent physicochemical and pharmacokinetic properties. Finally, we demonstrate that an ENPP1 inhibitor delays tumor growth in a breast cancer mouse model. Together, we have developed ENPP1 inhibitors that are excellent tool compounds and potential therapeutics. J.A.C., M.S., and L.L. designed the study. J.A.C., Y.A.S., and R.E.M. performed enzyme assays and analyzed the data. V.B. performed mouse experiments. J.A.B. and D.F. determined the crystal structure. J.A.C., M.S., and L.L. wrote the paper. All authors discussed the findings and commented on the manuscript. Competing interestsM.S. and L.L. are scientific cofounders of Angarus Therapeutics, which has exclusive licensing rights to patents PCT/US2018/50018 and PCT/US2020/015968. J.A.C., V.B., M.S., and L.L. are inventors on patents PCT/US2018/50018 and PCT/US2020/015968.

Probing pathophysiology of extracellular cGAMP with substrate-selective ENPP1

“…Previous studies showed that mutating key nucleotide-binding site residues inhibits ATP hydrolysis (Kato et al, 2012) ( Fig. 1c ), and we previously demonstrated that blocking the nucleotide-binding site with small molecule inhibitors blocks cGAMP hydrolysis (Carozza et al, 2020b). Therefore, we turned our attention to residues adjacent to the guanosine portion of cGAMP.…”

“…Mice were injected subcutaneously with 5 mg/kg cGAMP diluted in 100 μL PBS. After 30 min, the mice were anesthetized with isoflurane and 50 μL of blood was collected retro-orbitally and immediately supplemented with ~20 μM of ENPP1 inhibitor STF-1623 (Carozza et al, 2020b) to stop cGAMP degradation by ENPP1 post-draw. The blood was placed in heparin-coated microtainers (BD) and spun at 2,000 x g for 15 min, and the resulting plasma layer was collected.…”

Heparins are potent inhibitors of ectonucleotide pyrophosphatase/phospho-diesterase-1 (NPP1) – a promising target for the immunotherapy of cancer

“…This cyclic dinucleotide is a natural agonist of the stimulator of interferon genes (STING), which is crucial for the induction of a type I interferon response by the innate immune system (15)(16)(17). Inhibiting the hydrolysis of immunostimulatory ATP and cGAMP and the subsequent production of immunosuppressive, cancer-promoting adenosine has been proposed as a novel strategy to treat cancer and infections (18).…”

ATP and cancer immunosurveillance

“…Additional approaches that may be harnessed to target adenosine signaling in the TME encompass the use of CD38 or ENPP1 blockers and strategies that revert tumor hypoxia (e.g., respiratory hyperoxygenation) (Hatfield et al , 2014; Hatfield & Sitkovsky, 2020). However, CD38‐specific agents (including the FDA‐approved monoclonal antibody daratumumab) are currently used with the aim of eradicating CD38‐expressing myeloma cells (Facon et al , 2019), and ENPP1 blockers are still in preclinical development (Carozza et al , 2020). Similarly, hyperoxygenation has been employed in the past to improve radiosensitivity (because the ability of radiation therapy to cause DNA damage in cancer cell depends on local oxygen tension) but is no longer used for this purpose.…”