Structure-based virtual screening and in vitro validation of inhibitors of cyclic dinucleotide phosphodiesterases ENPP1 and CdnP

Abstract

In this paper, we describe novel inhibitors of cyclic dinucleotide phosphodiesterase enzymes from Mycobacterium tuberculosis (M.tb) (CdnP) and mammals (ENPP1). The phosphodiesterase enzymes hydrolyze cyclic dinucleotides, such as 2',3'-cyclic GMP-AMP and c-di-AMP, which are stimulator of interferon gene (STING) agonists. By blocking the hydrolysis of STING agonists, the cyclic GMP-AMP synthase (cGAS)-STING-IRF3 pathway is potentiated. There is strong evidence in tuberculosis and in cancer biology that potentiation of the cGAS-STING-IRF3 pathway leads to improved M.tb clearance and also improved antitumor responses in cancer. In addition to the identification of novel inhibitors and their biochemical characterization, we provide proof-of-concept evidence that our E-3 inhibitor potentiates the cGAS-STING-IRF3 pathway in both macrophage cell lines and also in primary human monocyte-derived macrophages.

Keywords: Mycobacterium tuberculosis; cyclic dinucleotide phosphodiesterase; host-directed therapy; immune evasion.

Fig 1

Virtual depiction of CdnP and ENPP1 enzyme ribbon structures with inhibitors docked at the active site. (A) Low and (B) high magnification of inhibitor C-13 (green) docking to the active site of CdnP. (C) Low and (D) high magnification of inhibitor E-3 (blue) docking to the active site of ENPP1.

Fig 2

Structures of selected M.tb CdnP inhibitors identified in the in silico virtual screen. (A–D) Structures, NCI reference numbers, and CdnP IC₅₀ values of four selected lead compounds that inhibit M.tb CdnP. Inhibitor C-29 showed dual activity against both CdnP and ENPP1. (E) Compounds were tested for their inhibitory potential against purified CdnP protein. AMP, the end-product of the PDE reaction against c-di-AMP, was detected by measuring relative light units (RLU) using our luminescence-based, AMP detection assay.

Fig 3

Structures of selected inhibitors of host ENPP-1 identified in the in silico virtual screen. (A–D) Structures, NCI reference numbers, and ENPP1 IC₅₀ values of four selected lead compounds that inhibit mammalian ENPP1.

Fig 4

Predicted molecular interactions of four lead inhibitors with M.tb CdnP. Molecular docking of inhibitors C-13 (IC₅₀ 9.66 µM or 4 µg/mL), C-29 (IC₅₀ 14.2 µM or 6 µg/mL), C-33 (IC₅₀ 18.8 µM or 7.3 µg/mL), and C-34 (IC₅₀ 13.1 µM or 5.3 µg/mL) with the active site of CdnP. Predicted hydrogen bonds are shown by purple arrows, and the hydrophobic surfaces of the inhibitors are shaded in gray. For the enzyme active site, acidic residues and surfaces are colored orange, basic residues are purple, hydrophobic residues are green, and hydrophilic residues and surfaces are blue.

Fig 5

Predicted molecular interactions of the four lead inhibitors with human ENPP1. Molecular docking of inhibitors E-3 (IC₅₀ 26.4 µM or 10 µg/mL), E-27 (IC₅₀ 16.3 µM or 5 µg/mL), E-37 (IC₅₀ 44.6 µM or 10 µg/mL), and E-60 (IC₅₀ 9.8 µM or 4 µg/mL) with the active site of ENPP1. Predicted hydrogen bonds are shown with purple arrows, while predicted salt bridges are shown with black arrows. Predicted pi-stacking interactions are shown by green lines, with hydrophobic surfaces of the inhibitors being shaded in gray. Predicted pi-cation interactions are shown with red lines. For the enzyme active site, acidic residues and surfaces are colored orange, basic residues are purple, hydrophobic residues are green, and hydrophilic residues and surfaces are blue.

Fig 6

Lead ENPP1 inhibitor, compound E-3, elicits enhanced cGAS-STING-IRF3 pathway activation and type I interferon release in macrophages. (A) Compound E-3 (NCI 14465) elicits enhanced IRF3 activation in RAW-Blue IRF3-SEAP reporter mouse macrophages at 24 h following 2′,3′-cGAMP transfection. Briefly, reporter macrophages were pre-treated with compound E-3 at concentrations varying from 0 to 165 mM (165 mM is 6.2× the ENPP1 IC₅₀ and 20.5 mM is 0.75× the IC₅₀) and were subsequently transfected with 1 nM 2′,3′-cGAMP using the X-tremeGENE9 transfection reagent. Culture supernatants were collected 24 h after transfection, and SEAP activity was measured by colorimetry in the presence of the QUANTI-Blue detection reagent. (B) Compound E-3 elicits elevated interferon-β (IFN-β) responses in human monocyte-derived macrophages (hMDMs) stimulated with 2′,3′-cGAMP. hMDMs were pre-treated with compound E-3 at 165 µM and subsequently transfected with 2′,3′-cGAMP (1 nM) using the X-tremeGENE9 transfection reagent. At 24 h post-transfection, culture supernatants were evaluated for IFN- β levels via ELISA. All data are presented as mean values ± S.E.M. (n = 3 independent biological replicate experiments). Statistical analyses were done using a two-tailed Student’s t-test. P-values are shown for relevant comparisons.