DNA-Encoded Library-Derived DDR1 Inhibitor Prevents Fibrosis and Renal Function Loss in a Genetic Mouse Model of Alport Syndrome

Abstract

The importance of Discoidin Domain Receptor 1 (DDR1) in renal fibrosis has been shown via gene knockout and use of antisense oligonucleotides; however, these techniques act via a reduction of DDR1 protein, while we prove the therapeutic potential of inhibiting DDR1 phosphorylation with a small molecule. To date, efforts to generate a selective small-molecule to specifically modulate the activity of DDR1 in an in vivo model have been unsuccessful. We performed parallel DNA encoded library screens against DDR1 and DDR2, and discovered a chemical series that is highly selective for DDR1 over DDR2. Structure-guided optimization efforts yielded the potent DDR1 inhibitor 2.45, which possesses excellent kinome selectivity (including 64-fold selectivity over DDR2 in a biochemical assay), a clean in vitro safety profile, and favorable pharmacokinetic and physicochemical properties. As desired, compound 2.45 modulates DDR1 phosphorylation in vitro as well as prevents collagen-induced activation of renal epithelial cells expressing DDR1. Compound 2.45 preserves renal function and reduces tissue damage in Col4a3^-/- mice (the preclinical mouse model of Alport syndrome) when employing a therapeutic dosing regime, indicating the real therapeutic value of selectively inhibiting DDR1 phosphorylation in vivo. Our results may have wider significance as Col4a3^-/- mice also represent a model for chronic kidney disease, a disease which affects 10% of the global population.

Figure 1

Structural diversity of enriched library members within a cluster is illustrated by selected examples taken from clusters 1–4. A list of all enriched library molecules is provided in Table S3.

Figure 2

Cocrystal structure of human DDR1 with ligand 2a (PDB code: 6FEW, resolution 1.44 Å) revealing a type II binding mode and an unoccupied hydrophobic pocket near the terminal amide group. Protein–ligand hydrogen bonds (direct and water-mediated) are highlighted as red, dashed lines. Protein residues are colored by the degree of sequence conservation with respect to the full kinome (for a description of the kinase sequence variability scores that are mapped onto the binding site, see the Methods section). For clarity of view, water molecules are removed except for the one mediating the ligand–hinge interaction.

Figure 3

Modifications around DELT starting point 2a leading to the discovery of lead DDR1 inhibitor 2.45. Structure and profile of DELT starting point 2a (left), optimized in vitro compound 2.13 (middle), and proof-of-concept compound lead DDR1 inhibitor 2.45 (right). Note that the stated IC₅₀ values are derived from a binding competition assay employing purified recombinant truncated DDR1 as described in the Methods section.

Figure 4

Cocrystal structure of human DDR1 with ligand 2.45 (PDB code: 6FEX, resolution 1.29 Å). Protein–ligand hydrogen bonds (direct and water-mediated) are highlighted as red, dashed lines. For clarity of view, water molecules are removed except for the two mediating the ligand interaction with the hinge backbone of Met 704 and the side chain of Glu 672.

Figure 5

Lead compound 2.45 is a selective DDR1 inhibitor and inhibits DDR1 phosphorylation and recruitment of SHC1 in vitro and modulates phenotype of collagen stimulated renal epithelial cells. (A) Graphical representation (“TREEspot Interaction Maps”) of selectivity profile of 2.45 tested on 468 kinase targets. Test concentration 10 μM. Kinases that bind are marked with red circles, where larger circles indicate stronger binding; K_d constants for kinases inhibited with >50% at 10 μM test concentration; selectivity score S(90) at the test concentration of 10 μM. (B) Phosphorylation in HT1080 overexpressing DDR1 or DDR2 and (C) SH2 domain complementation assay in cells overexpressing DDR1 or DDR2 treated with collagen type 2. (D) DDR1 expression in a Alport patient using an in-house raised anti-human DDR1 selective antibody. Distal tubular epithelial cell DDR1 positivity indicated by green arrowheads and parietal epithelial cell indicated with black arrowheads. (E) Quantitative RT-PCR for COL1A1, COL1A2, COL3A1, FBN1, or TGFB1 in HKC8 cells overexpressing DDR1 upon collagen type 2 stimulation. **p < 0.05, ***p < 0.001, t test.

Figure 6

Compound 2.45 reduces phosphorylated DDR1 in whole kidney lysates. Col4a3 knockout mice (Col4a3^–/–) and wild type littermates (Col4a3^+/+) were injected intraperitoneally with 2.45 (90 mg/kg) or vehicle on a daily basis. Four groups of mice were analyzed: Col4a3^+/+ (n = 8), Col4a3^+/+ 2.45 (n = 8), Col4a3^–/– (n = 6), and Col4a3^–/–2.45 (n = 10). (A) Two representative Western Blots on eluates from whole kidney lysates after immunoprecipitation ot total DDR1 are shown. Numbers 1–15 indicate individual animals. (B) Bar graph analysis showing that expression of total DDR1 increased in Col4a3 knockout mice compared to wild type mice but is not affected by 2.45 treatment, ****p < 0.0001, t test. (C) Treatment with 2.45 significantly reduces the levels of pDDR1 in Col4a3 knockout mice compared to controls, **p < 0.01, ****p < 0.0001, t test.

Figure 7

DDR1 inhibition with 2.45 preserves renal function and reduces renal fibrosis in Col4a3 deficient mice. Four groups of mice were analyzed: Col4a3^+/+ (n = 10), Col4a3^+/+ 2.45 (n = 11), Col4a3^–/– (n = 11), and Col4a3^–/–2.45 (n = 14). (A) 2.45 treatment of Col4a3 knockout mice results in a reduction in the albumin/creatinine ratio compared to control. **p < 0.023, ****p < 0.0014. (B) Serum creatinine levels are increased in Col4a3 knockout mice compared to controls but were not affected by treatment with 2.45, ****p < 0.0001, t test. (C) Graph analysis showing that serum BUN levels are significantly increased in Col4a3 knockout mice compared to wild type, whereas treatment with 2.45 prevents increases in serum BUN levels in Col4a3 knockout mice. **p < 0.005, ****p < 0.0001, t test. (D) Picro Sirius Red staining of kidney sections (4 μm) in Col4a3 knockout mice (10×). (E) Bar graph analysis of PSR staining demonstrates increased fibrosis in Col4a3 knockout mice when compared to wild type littermates. Treatment with 2.45 significantly reduces fibrosis in Col4a3 knockout mice. *p < 0.01, **p < 0.005, t test. (F) α-smooth muscle actin staining of kidney sections (4 μm) in Col4a3 knockout mice (20×). (G) Bar graph analysis of α- smooth muscle actin staining demonstrates high fibrotic lesions in Col4a3 knockout mice when compared to wild type littermates. Treatment with 2.45 significantly reduces fibrosis in Col4a3 knockout mice. *p < 0.004, **p < 0.032, t test. (H) Collagen I staining of kidney sections (4 μm) in Col4a3 knockout mice (20X). (I) Bar graph analysis of collagen I staining showed elevated expression of fibrosis in Col4a3 knockout mice when compared to wild type littermates. Treatment with 2.45 significantly reduces fibrosis in Col4a3 knockout mice. *p < 0.01, ***p < 0.0007, t test.