Structure-Aided Development of Small-Molecule Inhibitors of ENPP1, the Extracellular Phosphodiesterase of the Immunotransmitter cGAMP

Jacqueline A Carozza; Jenifer A Brown; Volker Böhnert; Daniel Fernandez; Yasmeen AlSaif; Rachel E Mardjuki; Mark Smith; Lingyin Li

doi:10.1016/j.chembiol.2020.07.007

Structure-Aided Development of Small-Molecule Inhibitors of ENPP1, the Extracellular Phosphodiesterase of the Immunotransmitter cGAMP

Cell Chem Biol. 2020 Nov 19;27(11):1347-1358.e5. doi: 10.1016/j.chembiol.2020.07.007. Epub 2020 Jul 28.

Authors

Jacqueline A Carozza¹, Jenifer A Brown², Volker Böhnert³, Daniel Fernandez⁴, Yasmeen AlSaif⁵, Rachel E Mardjuki¹, Mark Smith⁶, Lingyin Li⁷

Affiliations

¹ Department of Chemistry, Stanford University, Stanford, CA 93405, USA; Stanford ChEM-H, Stanford University, Stanford, CA 93405, USA.
² Stanford ChEM-H, Stanford University, Stanford, CA 93405, USA; Biophysics Program, Stanford University, Stanford, CA 93405, USA.
³ Stanford ChEM-H, Stanford University, Stanford, CA 93405, USA; Department of Biochemistry, Stanford University, Stanford, CA 93405, USA.
⁴ Stanford ChEM-H Macromolecular Structure Knowledge Center, Stanford University, Stanford, CA 93405, USA.
⁵ Stanford ChEM-H, Stanford University, Stanford, CA 93405, USA; Department of Biology, Stanford University, Stanford, CA 93405, USA.
⁶ Stanford ChEM-H Medicinal Chemistry Knowledge Center, Stanford, CA 93405, USA.
⁷ Stanford ChEM-H, Stanford University, Stanford, CA 93405, USA; Department of Biochemistry, Stanford University, Stanford, CA 93405, USA. Electronic address: lingyinl@stanford.edu.

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 the 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. In addition, we solved the crystal structure of an exemplary phosphonate inhibitor to elucidate the interactions that drive potency. This study yielded several best-in-class inhibitors with K_i < 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.

Keywords: 2′3′-cGAMP; ENPP1; STING; cancer immunotherapy; crystal structure; extracellular signaling; immunotransmitter; small-molecule inhibitors; structure-aided design.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
Antineoplastic Agents / chemical synthesis
Antineoplastic Agents / chemistry
Antineoplastic Agents / pharmacology*
Cell Survival / drug effects
Cells, Cultured
Dose-Response Relationship, Drug
Drug Screening Assays, Antitumor
Enzyme Inhibitors / chemical synthesis
Enzyme Inhibitors / chemistry
Enzyme Inhibitors / pharmacology*
Female
Humans
Mice
Mice, Inbred C57BL
Molecular Structure
Neoplasms, Experimental / drug therapy
Neoplasms, Experimental / metabolism
Neoplasms, Experimental / pathology
Neurotransmitter Agents / chemistry
Neurotransmitter Agents / isolation & purification
Neurotransmitter Agents / metabolism
Nucleotides, Cyclic / chemistry
Nucleotides, Cyclic / isolation & purification
Nucleotides, Cyclic / metabolism
Phosphoric Diester Hydrolases / metabolism
Pyrophosphatases / antagonists & inhibitors*
Pyrophosphatases / metabolism
Small Molecule Libraries / chemical synthesis
Small Molecule Libraries / chemistry
Small Molecule Libraries / pharmacology*
Structure-Activity Relationship

Substances

Antineoplastic Agents
Enzyme Inhibitors
Neurotransmitter Agents
Nucleotides, Cyclic
Small Molecule Libraries
cyclic guanosine monophosphate-adenosine monophosphate
Phosphoric Diester Hydrolases
ectonucleotide pyrophosphatase phosphodiesterase 1
Pyrophosphatases

Abstract

Publication types

MeSH terms

Substances

Grants and funding