Small Molecule Discoidin Domain Receptor Kinase Inhibitors and

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Small Molecule Discoidin Domain Receptor Kinase Inhibitors and Potential Medical Applications Miniperspective Yupeng Li,†,‡,§ Xiaoyun Lu,†,§ Xiaomei Ren,*,† and Ke Ding*,† †

State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, No. 190 Kaiyuan Avenue, Guangzhou 510530, China ‡ University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049, China ABSTRACT: Discoidin domain receptors (DDRs) are members of the transmembrane receptor tyrosine kinase (RTK) superfamily which are distinguished from others by the presence of a discoidin motif in the extracellular domain and their utilization of collagens as internal ligands. Two types of DDRs, DDR1 and DDR2, have been identified with distinct expression profiles and ligand specificities. These DDRs play important roles in the regulation of fundamental cellular process, such as proliferation, survival, differentiation, adhesion, and matrix remodeling. They have also been closely linked to a number of human diseases, including various fibrotic disorders, atherosclerosis, and cancer. As a consequence, DDRs have been considered as novel potential molecular targets for drug discovery and increasing efforts are being devoted to the identification of new small molecule inhibitors targeting the receptors. In this review, we offer a contemporary overview on the discovery of DDRs inhibitors and their potential medical application for the treatment of cancer and inflammation related disorders.

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INTRODUCTION Discoidin domain receptors (DDRs) are members of the transmembrane receptor tyrosine kinase (RTK) superfamily discovered in the early 1990s. 1 They are structurally distinguished from other RTKs by the presence of a discoidin motif in the extracellular domain. Two types of DDRs, DDR1 and DDR2, with distinct expression profiles and ligand specificities have been identified. DDR1 has five splice variants, DDR1a, DDR1b, DDR1c, DDR1d, and DDR1e, generated by alternative splicing or deletion of exons, while DDR2 has only one isoform. DDR1a, DDR1b, and DDR1c encode full-length enzymatically active receptors,1g,2 but DDR1d and DDR1e are predicted as kinase-deficient receptors because of their truncated or kinase deficient cytosolic fragment (Figure 1).3 Typical RTKs use peptide-like growth factors as ligands, but DDRs are activated by various types of triple-helical collagens which are the most abundant components of the extracellular matrix (ECM).4 Specifically, DDR1 can bind to essentially almost all types of collagens identified to date, while DDR2 shows a preference for type I, II and III fibrillar collagens, and nonfibrillar type X collagen.4,5 In addition to their ligand specificities, DDR1 and DDR2 also display distinct expression profiles. DDR1 is widely expressed in epithelial cells in lung, kidney, colon, and brain, whereas DDR2 is primarily expressed in mesenchymal cells including fibroblasts, myofibroblasts, smooth muscle cells, and chondrocytes in kidney, skin, lung, heart, and connective tissues.1 © XXXX American Chemical Society

Studies have demonstrated that both DDR1 and DDR2 are important for the regulation of fundamental cellular processes, such as proliferation, survival, differentiation, adhesion, and matrix remodeling.6 Dysregulation of the receptors is closely related to a number of human diseases, including fibrotic disorders (e.g., renal fibrosis and pulmonary fibrosis, etc.), atherosclerosis, and cancer.6 Thus, DDR1 and DDR2 have been considered as novel potential molecular targets for drug discovery, and development of small molecule DDR inhibitors may lead to new and attractive therapeutic strategies for the treatment of cancer or various inflammation-related disorders. This review provides an updated overview on the discovery of DDR inhibitors and their potential application to the treatment of cancer and inflammation-related disorders. Audiences are also encouraged to read the recent comprehensive reviews to get extensive knowledge on the biological background of DDRs.6

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STRUCTURAL DETAILS OF DDRS Similar to the structural arrangement of classic RTKs, DDR1 and DDR2 consist of three fundamental elements: an extracellular domain, a transmembrane domain, and an intracellular domain with intrinsic enzymatic activity (Figure 1). Distinctively, DDRs contain, in the extracellular region, a Received: August 10, 2014

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Figure 1. Domain arrangements of DDR1 and DDR2. DDR1a, DDR1b, DDR1c, and DDR2 transmit signals normally by encoding full-length enzymatic active receptors, while DDR1d and DDR1e are kinase-deficient receptors as a result of their truncated or inactive kinase domains.1g,2,3

share high sequence and morphological homology with that of Abl (UniProtKB, P00519) or c-Kit (UniProtKB, P10721).11 Several cocrystal structures of DDR1 kinase domain with small molecule inhibitors have been reported (Table 1) and show that DDR1 kinase domain contains loops or motifs similar to those in other RTKs, including glycine-rich loop, A-loop, DFG motif, and C-helix (Figure 3).12 To date, there has been no report of a crystal structure of DDR2 kinase domain complexed with a small molecule ligand, but homology modeling suggests that the DDR2 kinase domain may adopt a DDR1-like conformational arrangement.13

unique discoidin motif, the DS domain which exhibits high structural similarity to discoidin I protein that was originally identified as a galactose-binding lectin in the slime mode Dictyostelium discoideum.7 A discoidin-like domain (DS-like domain) and an extracellular juxtamembrane region (EJXM) are also located in the extracellular region. The structure of a cocrystal of a DDR2−collagen complex reveals an important binding trench that is circumscribed by four conserved protruding loops, L1, L2, L4, and L6, at the protein−protein interface of the DS domain and is able to interact directly with collagen (Figure 2).8 In addition to the binding epitope, there are other loops regulating collagen recognition. Replacement of five solvent-exposed residues adjacent to the binding trench of DDR2 with the corresponding DDR1 substitutions results in significant ligand-selectivity alternatives.5 The transmembrane domain (TM) of DDRs provides a linkage between their extracellular and intracellular domains. Several repeated leucine zipper motifs in this region are critical for the self-associated dimerization which is required for the phosphotransferase activity of DDR kinases.9 It is notable that the intrinsic DDR dimerization is performed without recognizing ligand, which is in contrast to the ligand-induced dimerization of most of other RTKs.9 The intracellular domain contains an unexpectedly large intracellular juxtamembrane region (IJXM) and a kinase domain with a short C-terminal tail. Following collagen binding, the tyrosine residues in the IJXM are phosphorylated to serve as docking sites to which they attract nearby adaptor proteins.10 As with many other kinases, the intrinsic enzymatic activity of DDRs is determined by their kinase domain (KD). The kinase domains of DDR1 (UniProtKB, Q08345) and DDR2 (UniProtKB, Q16832)

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BIOLOGICAL FUNCTIONS OF DDRS AND MEDICAL IMPLICATIONS 1. Roles in Development. Both DDR1 and DDR2 have been demonstrated to be crucial for the regulation of developmental and physiological processes.6,16 Generally, DDR1 is important for the biogenesis of multiple organs, whereas DDR2 is one of the key players for skeletetogenesis. DDR1-null mice were characterized to be smaller stature than normal littermates and lactational defect in pregnant female animals, but further investigation is needed to confirm the results.17 Defects in kidney and inner ear architecture were also observed for mice with lack of DDR1.18 DDR1 knockout mice display severe auditory function decrease and progressive morphological alterations,18b while the animals only show a localized matrix overproduction in the glomerular basement and no chronic renal disease development was observed.18c In transgenic mice deleting collagen II, the lack of endochondrial bone or epiphyseal growth plate in long bones has been reported.19 In view of the fact that DDR2 selectively recognizes B

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Figure 2. Structural features of DDRs (PDB code 2WUH). (A) Overview structure of a DDR2−collagen cocrystal complex. (B) Binding details of DDR2 with collagen at the interface of the DS domain. Residues in the first chain of collagen are indicated in orange (V20, NLE21, and O24). Residues in the middle chain of collagen are shown in orange with asterisks (O23*, O24*, and O27*), and residues in DDR2 DS domain are in black (W52, D69, R105, I112, E113, M117, S173, M174, and N175). (C) Sequence alignment of DDR1 with DDR2.5,8

collagen II for activation, it would appear that DDR2 is important for chondrocyte proliferation,19 and this is further supported by the developmental defects such as dwarfism and shortened long bones exhibited in DDR2 (−/−) knockout mice.20 Moreover, transgenic mice with DDR2 overexpression were reported to display increased body length.21 In addition, DDR2 deletion or mis-sense mutation was shown to be closely related to autosomal recessive growth disorders such as Smallie (Slie) and human spondylo-meta-epiphyseal dysplasia which features short limbs and abnormal calcifications (SMED-SL).22 2. Regulation of Fundamental Cellular Processes. DDRs are highly implicated in fundamental cellular processes, including cell proliferation, migration, adhesion, and matrix remodeling. For example, DDR1a is important for promotion of leukocyte migration in three-dimensional collagen lattices.23 Dominant negative DDR1 inhibits neurite outgrowth both in vitro and in vivo, indicating a unique function of DDR1 in axon formation during cerebellar cortex histogenesis.24 DDR2 overexpression contributes greatly to the MMP-2 mediated proliferation and invasion of hepatic stellate cells,25 while DDR1 deletion leads to impaired ability of adhesion and migration.26 It has been demonstrated that DDR2 is required for normal fibroblast spreading and migration independent of adhesion ligand and collagen activation,27 and it plays a local and essential role in the proliferation of chondrocytes.28 Studies also suggest that DDR2 function is essential to the membrane dynamics which control the mechanical attachment of fibroblasts to the 3D collagen matrices.29 3. Correlation with Human Diseases. Dysregulation of DDR1 and DDR2 have been linked to a number of human diseases, including fibrotic disorders (e.g., renal fibrosis,

Table 1. Reported Crystal Structures of DDR−Ligand Complexes PDB code

DDR−ligand complex

refs

4AG4 3ZOS 4BKJ 4CKR 2WUH 2Z4F

DDR1/Fab DDR1/ponatinib (7) DDR1/imatinib (3) DDR1/DDR1-IN-1 (18) DDR2/collagen solution structure of DDR2 DS domain

8b 12 12 14 8a 15

Figure 3. Structure overview of cocrystal of DDR1 with drug 7 (PDB code 3ZOS).12

C

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Figure 4. Domain distribution of somatic mutations of mutated DDR1 (red) and DDR2 (blue) identified in samples of NSCLC, AML, and SMED patients. Circles indicate “gain-of-function” mutants. The loss-of-function mutants are labeled as triangles, while mutants with undefined functions are labeled as squares.47−51

pulmonary fibrosis, and liver fibrosis), atherosclerosis, arthritis, and various types of cancer.6 For example, DDR1 overexpression has been identified as a novel biomarker of epithelial ovarian cancer.30 Clinicopathological parameter analysis in nonsmall-cell lung carcinoma patients (NSCLC) reveals a significant correlation between DDR1 overexpression and lymph node metastasis (p = 0.001), and DDR1 could be a prognostic marker for NSCLC patients.31 A global phosphoproteomic survey of tyrosine kinase activity in lung cancer cells identified DDR1 as an additional new activated kinase implicated in the tumorigenesis of NSCLC.32 Other studies also have demonstrated that high expression levels and/or mutations of DDR1 are frequently detected in multiple cancer cell lines and primary tumor tissues from lung,31,32,47 breast,33 brain,34 ovary,30 head and neck,35 liver,36 pancreas,37 prostate,38 and others.39 Silencing DDR1 by siRNA has been shown to reduce metastatic activity in lung cancer models and enhance chemosensitivity to genotoxic drugs in breast cancer cells.40 High level of DDR2 has also been determined in NSCLC,31d,47 nasopharyngeal carcinomas,35 thyroid carcinomas,35,41 and Hodgkin’s lymphoma, etc.42 Multivariate analysis of data from human breast cancer patients shows that DDR2 is a favorable independent predictor of recurrence and outcome for primary breast cancers.43 DDR2 overexpression also contributes greatly to metastasis, invasion, and migration of prostate cancer, and head and neck squamous cell carcinoma.44 Further mode of action (MOA) studies suggest that DDR2 can facilitate breast cancer metastasis via stabilization of the SNAIL1 protein.45 Downregulation or a host deficiency of DDR2 was demonstrated to obviously suppress the metastasis and tumor angiogenesis of various human cancers.46 Furthermore, “gain-of-function” mutations of DDR1 and/or DDR2 have been identified in various types of cancer cells and primary human NSCLC cancer tissues (Figure 4).47 Somatic mutations of DDR1 in lung neoplasms and cancer cells such as G1486T and A496S may contribute significantly to the development of lung cancer. 47a High-throughput DNA sequence analysis has revealed several novel somatic mutations of DDR1 such as N502S, A533S, and A803V in acute myeloid leukemia (AML), but additional studies are required to define the roles that these mutants play in AML pathogenesis.48 Some other mutations, including W385C, F866Y, F824W for DDR1 and R105S, N456S for DDR2 have also been reported in various cancer cells.11,31d More significantly, a recent Sanger sequencing of 20 primary lung squamous cell carcinoma (SCC) samples demonstrated that DDR2 is associated with one of the most frequently mutated genes. The corresponding mutations

include L63V, I120M, D125Y in the discoidin domain, C580Y, I638F, T765P, G774E/V in the kinase domain, and the missense mutations L239R, G253C, G505S, etc. Cancer cells with L239R and I638F mutations are selectively sensitive to DDR2 siRNAs and to a nonselective DDR2 inhibitor dasatinib (2).47d Two SCC patients with DDR2S768R mutation exhibit significant shrinkage of tumor size after treatment with drug 2.49 However, a recent proteomic study controversially suggested that the L63V and G505S mutants as well as wild type DDR2 might play a tumor-suppressive role in cancer cells induced by fibrillar collagen, indicating the complex role of DDR2 in lung cancer.50 In addition, mutation of N-glycosylation sites such as N211Q in DDR1 and N213Q in DDR2 that dysregulate the cell-surface expression of DDRs in their high molecular mass form, which is a functional isoform causing tyrosine autophosphorylation upon collagen stimulation, has also been identified.51 In addition to their significant involvement in human cancer, DDR1 and DDR2 are also closely related to fibrosis, atherosclerosis, and other inflammatory disorders or matrix degradations. Activation of DDR1 and DDR2 in human smooth muscle cells (SMCs) is important for enhancement of the activation of MMP, thus regulating extracellular matrix (ECM) remodeling in obstructive diseases of lungs and blood vessels.52 DDR1 deletion has been reported to alleviate bleomycin-induced lung inflammation and pulmonary fibrosis by blocking p38 MAPK activation.53 Use of DDR1/COL4A3 double-knockout models suggests that the collagen−DDR1 interaction plays an important role in the progressive renal fibrosis associated with Alport syndrome.18b,54a Further investigations also demonstrated that genetic deletion and/or antisense inhibition of DDR1 expression prevents the development of renal inflammation and fibrosis, blunted proteinuria, and preserved renal structure in different renal disease models (i.e., hypertensive nephropathy,54b tubular interstitial54c and glomerular nephritis54d), suggesting that pharmacological blockade of DDR1 may be an attractive strategy for efficient treatment of chronic kidney disease, an incurable today pathology. Silencing DDR2 expression was also demonstrated to decrease alcohol-induced liver injury and fibrosis in an early stage alcoholic liver disease mouse model.55 In view of its tissue distribution in articular cartilage, DDR2 reduction was clearly proved to attenuate the degeneration of osteoarthritis in knee joints.56 Studies also demonstrate that collagen I evoked DDR2 activation strongly promotes the release of inflammatory cytokines and co-stimulatory molecules via the NF-κB and JNK pathways in human monocyte derived dendritic cells.57 Consequently, the small molecule DDR inhibitor LCB 03-0110 D

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Figure 5. Strategies for the development of novel DDR inhibitors.

DDR1 and DDR2. More importantly, compounds 7rh (17)67 and 1814 were recently discovered to inhibit selectively the kinase functions of DDR1, providing important research probes to further validate DDRs as new potential targets for drug discovery.

(13) was reported to significantly promote the wound healing process with no hypertrophic scar formation.58 Most recently, DDR2 point mutations such as T713I, I726R, R752C, and E113K have been reported to be causative genetic defects for the autosomal recessive human growth disorder SMED-SL.22

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NATURAL ANTIBIOTIC 1 The protein−protein recognition of a triple-helical collagen with the DS domains of DDRs triggers a cascade of DDR signal transduction. Disruption of the DDR−collagen interaction represents a promising strategy to selectively suppress the DDR signal cascade. Using a self-constructed model of sf9 cells expressing DDR2 on type I collagen-coated wells, Yang et al. screened a drug compound library with 1040 chemicals and successfully identified the natural antibiotic 1 as an antagonist of the DDR2−collagen interaction (Figure 6).59 This

DDR INHIBITORS Given their significant contributions to the establishment and progression of human cancer and inflammation related disorders, DDRs are considered to be legitimate molecular targets for the discovery of new drugs. A variety of strategies have been adopted to develop inhibitors targeting the new class of kinases (Figure 5). For instance, the natural antibiotic actinomycin D (1) has been shown to effectively disrupt the DDR2−collagen interaction by binding to the DS domain of DDR2, the determinate region of the protein−protein interaction.59 Monoclonal antibodies including Fab 3E3,8b 48B3,26b and H-12660 have also been developed to bind to the DS-like domain of DDR1. It has been demonstrated that the phosphorylation of tyrosines located in the intracellular juxtamembrane of DDRs and the accumulation of signal adaptor proteins in this region are regulated by tyrosine kinases such as PDGFR, TrkA, Shc, and Src.61 Small molecule inhibitors of these kinases have been reported to interfere indirectly with the signaling pathway of DDRs. In addition, the phosphatase inhibitor pervanadate was also reported to induce rapid and significant autophosphorylation of DDR1 in the absence of ligand stimuli, implying that a phosphatase activator may suppress activation of DDRs.62 As members of the RTK superfamily, the enzymatic activity of DDRs is determined by their KDs. Consequently, small molecular inhibitors targeting the cytosolic kinase domain represent one of the most promising strategies for suppression of the biological functions of DDRs. This has been extensively validated by the inhibitors of many other RTKs such as BcrAbl,63 EGFR,64 or BTK.65 Because of the structural similarities in kinase domains,11 a number of well recognized kinase inhibitors such as the Bcr-Abl inhibitors 2, 3, nilotinib (4),11,68 bafetinib (5),69 and 7,12 the B-Raf inhibitor sorafenib (9), the VEGFR inhibitor pazopanib (10), the c-Met/VEGFR-2 inhibitor foretinib (11), and the p38 MAPK inhibitor BIRB796 (12)66 were found potently to inhibit the functions of both

Figure 6. Natural antibiotic 1, a novel DDR2−collagen interaction blocker.

compound selectively inhibits the activation of DDR2 stimulated by collagen I in HEK293 cells but in a parallel assay has little effect on the activation of DDR1b and also EFGR and IR kinases. However, the relatively weak inhibitory activity of compound 1 (IC50 = 9.0 μM in HEK293 cells) may restrict its further application as a biological tool for research in DDRs. E

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Figure 7. Known kinase inhibitors with DDR inhibitory effects.

Figure 8. Cocrystal structure of drugs 3 (A, PDB code 4BKJ) and 7 (B, PDB code 3ZOS) with DDR1 kinase domain. The ligands are shown in stick model colored by C in purple, N in blue, and O in red, while the protein is shown in cartoon model. The yellow dash line indicates a hydrogen bond.

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NONSELECTIVE INHIBITION OF DDRS BY KNOWN KINASE INHIBITORS A sequence alignment of human DDRs reveals that in the ATP binding domain they share ∼61% sequence identity with BcrAbl.11 Jarai et al. successfully developed a TR-FRET biochemical assay to identify three well-recognized type I or type II Bcr-Abl inhibitors 2, 3, and 4 which potently inhibit DDR1b and DDR2 with IC50 values of 0.5, 337, 43 nM and 1.4, 675, 55 nM, respectively.11 Further cell based investigations confirmed that the compounds potently suppress collageninduced autophosphorylation of DDR1b and DDR2 and inhibit MCP-1 release in monocytic cells in a DDR1 dependent manner.11 The strong DDR inhibitory effects of compounds 2, 3, and 4 were also independently validated by a global chemical proteomics approach. 68 In addition, drug 2 has been

demonstrated to display highly promising therapeutic efficacy in lung cancer cells harboring “gain-of-function” mutations of DDR2.47 In a clinical investigation two squamous cell carcinoma (SCC) patients with a DDR2S768R mutation were shown to have significant shrinkage of their tumors after the treatment with drug 2.49 Other type II Bcr-Abl inhibitors such as 5, 6, 7, and GZD824 (8) were also reported to display strong DDR inhibition (Figure 7).69 X-ray structures of cocrystals of DDR1 with drugs 3 and 7 have been reported and demonstrate that the compounds bind to inactive DDR1 with a type II binding mode similar to that of Bcr-Abl kinase (Figure 8).12 Upon binding of the inhibitor, a hydrophobic pocket is induced by extruding the C-helix and twisting the DFG motif in DDR1. Both of the inhibitors form a strong hydrogen bonding network with the E672, M704, V763, F

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Figure 9. Selective DDR1/DDR2 kinase inhibitors.

EphA3, EphB4, VEGF receptor 2, misshapen/niks-related kinase 1, c-Abl, and RET. Further study is needed to demonstrate the correlation of the therapeutic efficacy to its DDR inhibition.

H764, and D784 residues of the protein, and other favorable van der Waals contacts are formed in the allosteric pocket. Although compound 7 fails to enjoy the key hydrogen bond interaction with the T701 gatekeeper residue which contributes greatly to the binding of drug 3 with DDR1, a strong hydrogenbond interaction is formed between M704 residue with the imidazo[1, 2-b]pyridazine moiety in 7, and this compensates for the loss of potency. In addition to the reported Bcr-Abl inhibitors, several other kinase inhibitors displaying potent DDR inhibitory effects have also been identified (Figure 7).66 For instance, drug 9, a B-Raf/ VGFR dual inhibitor approved for advanced hepatocellular carcinoma, was recently reported to bind to DDR1 and DDR2 with Kd values of 1.5 and 6.6 nM, respectively. A VEGFR inhibitor, 10 also exhibits off-target binding to DDR2 with a Kd value of 57 nM. Molecule 11 is a c-Met/VEGFR-2 dual inhibitor, but a kinase selectivity profiling study revealed that it binds potently to DDR1 with a Kd value of 0.2 nM. A reported p38 MAPK inhibitor 12 also tightly binds to DDR1 and DDR2 with Kd values of 1.9 and 33 nM, respectively. Recently, a thienopyridine, compound 13, was identified as a novel small molecule DDR inhibitor by random screening of a compound library followed by medicinal chemical optimization.58 This compound selectively suppresses the activated tyrosine kinase activity of DDR2 with an IC50 value of 6.0 nM, although it is clearly less potent with the unactivated kinase, with which its IC50 value is 145 nM. It also inhibits collageninduced DDR1b or DDR2 receptor autophosphorylation with IC50 values of 164 and 171 nM, respectively, in HEK293 cells which stably overexpress either DDR1b (HEK293-DDR1b) or DDR2 (HEK293-DDR2). Further biological investigation reveals that 13, by inactivation of α-smooth muscle actin, potently inhibits the proliferation and migration of primary dermal fibroblasts induced by transforming growth factor β1 (TGFβ1) and type I collagen, FAK, and Akt1. The compound also dose-dependently inhibits lipopolysaccharide (LPS) induced cell migration and the syntheses of inflammatory factors like nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and tumor necrosis factor α (TNFα) in J774A.1 macrophage cells. In a rabbit ear wound healing model, topical treatment of 13 potently reduces the hypertrophic scar formation without delaying the wound closing process, suggesting the therapeutic potential of DDR inhibitors to treat diseases related to fibroinflammation. However, in a kinase profiling assay against 60 kinases, the compound displays broad inhibition against a panel of 20 tyrosine kinases including Btk, Syk, Tie2, FLT1, FLT3, FLT4,

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SELECTIVE DDR KINASE INHIBITORS

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COMPOUND 17 AND ITS ANALOGUES

To date, several classes of DDR kinase inhibitors have been identified. Most of them however display broad inhibitory activity against a panel of many other kinases, and selective DDR1 and/or DDR2 inhibitors are highly desirable for further validation of DDRs as drug targets. Sequence alignment of human DDRs demonstrated that they share high homology with many kinases in the ATP binding domain, and this highlights the challenge of identifying selective DDR inhibitors.11 Only recently, Ding et al. reported a pyrazolopyrimidine alkyne derivative 17 which selectively binds and inhibits the enzymatic function of DDR1.67 Compound 18 has also been identified as a selective DDR1 inhibitor by Gray’s group at the Dana Farber Cancer Institute.14

On the basis of the sequence similarity of DDR1/2 and Bcr-Abl in the kinase domain, Ding et al. conducted a focused screening against an internal library containing approximately 2000 kinase inhibitors and identified N-isopropyl-4-methyl-3-(2-(pyrazolo[1,5-a]pyrimidin-6-yl)ethynyl)benzamide 14 as a new potent DDR1 inhibitor with almost the same potency to c-Kit kinase (Figure 9).67 Structural optimization by replacing the isopropyl moiety with a meta-trifluoromethyl-substituted phenyl group gave compound 15, which has an improved DDR1 inhibitory activity and a diminished effect on c-Kit kinase. In spite of the introduction of a pharmaceutically acceptably hydrophilic (4methylpiperazin-1-yl)methyl group, compound 16 did not apparently lose its inhibitory activity against DDR1; the modification clearly restored the potency against Bcr-Abl. Further replacement of the “flag methyl” group with an ethyl group yielded a highly selective DDR1 inhibitor 17. This compound potently inhibits DDR1 kinase with an IC50 value of 6.8 nM but is significantly less active against DDR2, Bcr-Abl, and c-Kit with IC50 values of 101.4, 355 and >10 000 nM, respectively. Kinase profiling analysis using an Ambit kinome screening platform reveals that compound 17 possesses an excellent selective profile for DDR1. The new inhibitor 17 tightly binds to the ATP-binding site of DDR1 with a Kd value of 0.6 nM under an active-site-dependent competition binding assay,70 while it only displays ∼65% binding rates with 14 of the G

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Figure 10. Other DDR kinases inhibitors.

enzymatic kinase assay, with IC50 values of 105 and 47 nM, respectively. They also exhibit strong blockage on the collagen induced autophosphorylation of DDR1 in U2OS cells with EC50 values of 86 and 9.0 nM, respectively (Figure 9).14 A KinomeScan profiling study was then conducted to demonstrate that inhibitor 18 displays a good selective profile on DDR1 with the S (1) score of 0.01 against 451 kinase at 1.0 μM, whereas compound 19 is considerably less selective, with an S (1) score of 0.07. Further studies demonstrated that inhibitor 18 shows significant synergy with PI3K or mTOR inhibitors to suppress the proliferation of cancer cells with DDR1 gain-of-function mutations and/or overexpression, but it is not active as single agents.14 The cocrystal X-ray structure of 18 with DDR1 confirms its type II binding mode with the protein. The indolinone motif of 18 forms two pairs of hydrogen bonds with Asp702 and Met704 in the hinge region, respectively, while the trifluoromethyl group binds deeply into a hydrophobic pocket directly adjacent to the ATP binding site. The amide group and the nitrogen in piperazine can also make additional hydrogen bond networks with Glu672, Asp784, Val763, and His764, respectively. A mutation of the gatekeeper residue T701 leads to a significant loss of potency of 18, while a G707A mutation in the hinge region results in 20- or 100-fold improvement in potency for compounds 18 and 19, respectively, further supporting the postulate of their binding to the DDR1 kinase domain.

other 395 nonmutated kinases evaluated at 100 nM which is about 160 times higher than its Kd value with DDR1. The offtarget kinases include Abl1, DDR2, EPHA8, HCK, LOK, MAK, PDGFRβ, Tie2, TRKb, TRBc, and ZAK, and the S (35) and S (10) selectivity scores are 0.035 and 0.008, respectively. Although the detail interaction of inhibitor 17 with DDR1 remains unclear, it is predicted that the compound binds to the receptor with a type II binding mode. Cell based investigation also reveals that 17 dose-dependently inhibits the activation of DDR1 and downstream signals and potently suppresses the proliferation, invasion, adhesion, and tumorigenicity of cancer cells with high level of DDR1. Preliminary pharmacokinetic studies were also conducted and demonstrated that the compound possesses promising PK profiles, with an oral bioavailability of 67.4% and T1/2 of 15.5 h.

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INHIBITOR 18 AND THE DERIVATIVES Typically, a type II kinase inhibitor contains several key structural elements that engage in crucial interactions with the protein: (1) a heterocyclic moiety binding to the adenine pocket and participating in a hydrogen bonding network with residues in the hinge region (“hinge binding part”); (2) a hydrophobic group binding the back pocket induced by the DFG-out conformation (“tail motif”); (3) a suitable linker between the “head region” and the “tail motif”.14 On the basis of this pharmacophore model, scientists in Gray’s group designed and synthesized a collection of ∼100 type II inhibitors, among which compound 18 and DDR1-IN-2 (19) displayed significant inhibitory activity against DDR1 in an H

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Figure 11. Chemical structures of new DDR inhibitors with unknown mechanism.

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OTHER DDR KINASE INHIBITORS With the aim of discovering novel DDR inhibitors, Rauh et al. developed a new “fluorescent labels in kinases” (FLiK) assay to successfully identify a series of pyrazolourea compounds (20− 22) as new type II inhibitors of DDR2 with the DFG-out conformation (Figure 10).13 The DDR2 inhibitory effects of the compounds were further validated by an orthogonal activity-based assay. Compounds 20, 21, and 22 potently inhibit DDR2 with IC50 values of 0.065, 0.075, and 0.018 μM, respectively. The compounds also strongly inhibit DDR1 with IC50 values of 0.068, 0.235, and 0.039 μM, respectively. Significantly, compounds 20 and 22 also exhibit strong suppression against the T654M gatekeeper mutant of DDR2 with IC50 values of 2.0 and 1.0 nM, respectively. It is noteworthy that from an in vitro investigation in DDR2dependent lung cancer cell lines, the gatekeeper mutation T654I has been implied as a primary mechanism for the acquired resistance against DDR inhibitor therapy.71 Thus, the inhibitory effects of the compounds against DDR2T654I is eagerly awaited to demonstrate their potential to overcome DDR2 mutation induced resistance. The compounds may also provide useful probes with which to decipher the orchestrated network of signaling by DDRs and their relevance to tumor biology. However, further kinase profiling study reveals that the compounds display relatively broad inhibitory spectra against a panel of protein kinases. Furthermore, since no cocrystal structures of DDR2 with type II inhibitors are currently available, extensive investigations to demonstrate that binding modes of the compounds with wild type DDR2 and the resistant mutant are highly desirable. Recently, benzamide72 and quinazolinedione73 derivatives have also been identified as novel DDR1 inhibitors (Figure 10). A benzamide-derived compound 23 was found to exhibit inhibitory activity against DDR1 kinase with an IC50 value of 0.097 μM.72 It also suppresses the proliferation of U2OS cells harboring DDR1 overexpression with an IC50 value of 0.44 μM. The conformationally restrained quinazoline 24 also strongly inhibits the kinase activity of DDR1 with an IC50 value of 0.043 μM.73 It is encouraging that compounds 23 and 24 show promising in vivo antitumor efficacies with tumor growth inhibition (TGI) rates of ∼60% after oral administration in a mouse xenograft model of DDR1 overexpressed cancer cells. Benzylurea derivatives have also been identified by the Merck pharmaceutical company as potent DDR2 inhibitors (Figure 10).74 For instance, compound 25 suppresses the enzymatic activity of DDR2 with an IC50 value of 30 nM. Further work led to the discovery of more potent compounds 26, 27, and 28 which have IC50 values of 15, 20, and 6.0 nM, respectively. Biological investigation also revealed that compound 25 induces a dose-dependent reduction in the extent of thermal

hyperalgesia but not the carrageenan (CAR) induced swelling in rats, suggesting the potential application of DDR2 inhibitors as novel pain-reducing agents. However, further evidence demonstrating that the in vivo efficacies of the compounds are related to their DDR1 or DDR2 inhibition is eagerly awaited and no kinase selectivity profile of the compounds is available.

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DDR INHIBITORS WITH UNKNOWN MECHANISM OF ACTION A family of polycyclic alkaloids 29−32 (discoipyrroles A−D, Figure 11), isolated from marine-derived Bacillus hunanensis, strain SNA-048, have recently been discovered to inhibit BR5 fibroblast migration in a 2D Matrigel matrix at 1.0 μM.75 The compounds also selectively inhibit the growth of HCC366 nonsmall-cell lung cancer cells harboring a “gain of function” mutation of DDR2 but are clearly less potent against A549 cancer cells with wild-type DDR2. Preliminary biological investigations suggest that compound 29 exhibits its biological effects through regulation of the glycosylation status of DDR2 during post-transcriptional modification. However, direct evidence demonstrating whether or how the compounds interact with DDR2 is still lacking.

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PERSPECTIVE DDR1 and DDR2 are promising targets for discovery of drugs for the treatment of human cancer or inflammation-related diseases including renal fibrosis, pulmonary fibrosis, atherosclerosis, or osteoarthritis, etc. Several classes of small molecule DDR inhibitors with different selectivity profiles have been discovered. Among these, compound 2 in an early stage of clinical investigation has shown promising benefit for a small population of SCC patients with activating mutation of DDR2. Some other compounds also show promising therapeutic potential for various human cancers, would healing promotion, and antihyperalgesic effect. However, most of the reported DDR inhibitors display broad inhibition against a panel of protein kinases, and it is thus difficult to correlate their therapeutic efficacies with their DDR inhibition. Encouragingly, a few highly selective small DDR1 inhibitors have recently been identified. Compound 18 shows great synergistic effect with inhibitors of PI3K or mTOR in cancer cell growth suppression assays.14 Combination of inhibitor 17 with the first-line chemodrugs also shows a significantly improved therapeutic benefit for pancreatic cancer and NSCLC cancer in different animal models.76 These results intimate the promising potential for DDR-based therapy. However, the studies also demonstrate that almost all of the selective DDR1 inhibitors used alone fail to achieve significant inhibition of the in vitro growth of various cancer cell lines, suggesting that DDRs may not be the driving force for proliferation of the cancer cells. More biological I

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Journal of Medicinal Chemistry investigations to demonstrate the precise roles of DDRs in human cancer establishment and development are highly desirable. It will be of special value to identify “activating mutation” of DDRs in the primary human cancer tissues. In addition, although biological investigation suggests that DDRs are important for a variety of inflammatory processes, there is still a lack of convincing evidence demonstrating the therapeutic application of selective DDR inhibitors against inflammation related disorders such as atherosclerosis, pulmonary fibrosis, or renal fibrosis. Nevertheless, given the fact that DDR overexpression and/or mutation contributes greatly to acquired resistance and plays critical roles in tumor invasion and metastasis for certain types of cancer,6,47 it is rational to postulate that combination of DDR inhibitors with other therapeutic agents may be a new tractable approach for novel anticancer therapy development. Despite the significant progress in DDR research area, from a medicinal chemist’s point of view, there are still many issues that are highly interesting for future investigations: (1) Is it feasible to design a selective DDR2 inhibitor over DDR1, since they share extremely high sequence identity in the ATP binding domain? A cocrystal structure will provide insight to the interaction of an inhibitor with DDR2 kinase domain. (2) Molecules selectively targeting the activating and/or drug induced DDR mutants, particularly the resistant gatekeeper mutants (i.e., DDR2T654I/M), will be of special value for future DDRs-based drug discovery. (3) DDR1/DDR2 inhibitors with preferred tissue distribution profile are highly desirable, since DDRs may function differently in various human tissues. (4) Will dual DDR1/DDR2 inhibition achieve synergistic effect or be antagonistic? (5) In vivo efficacy data of a selective DDR1 or DDR2 inhibitor are eagerly awaited for validating their therapeutic potential. (6) What are the optimal therapeutic indications and the corresponding disease models for DDR inhibitor therapies? In summary, biological investigations suggest DDRs as novel potential targets for drug discovery to treat human cancer and inflammatory disorders. A number of small molecule DDR inhibitors have been identified with different selectivity profiles, and some of these have been demonstrated to possess a promising therapeutic potential. However, given the fact that the precise role of DDRs in human diseases remains elusive, more explorations to further validate DDRs as the pharmaceutically accessible molecular targets are necessary. New selective DDR1 and/or DDR2 inhibitors are also highly valuable as research tools in future biological investigations.

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of Biomedicine and Health, Chinese Academy of Sciences. He is currently a Postdoctoral Research Fellow working in the computeraided molecular design lab at Mayo Clinic under the supervision of Prof. Yuan-ping Pang. His research mainly focuses on the discovery and optimization of inhibitors targeting key kinase receptors as well as protein−protein interactions. Xiaoyun Lu is an Associate Research Professor in Medicinal Chemistry at Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. She received her Ph.D. from China Pharmaceutical University in 2010 and then joined Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, as a Research Associate in Prof. Ke Ding’s group. She was promoted to Associate Research Professor in 2012. Her research mainly focuses on the rational design and synthesis of medicinal lead compounds targeting cancer and tuberculosis. Xiaomei Ren is an Associate Research Professor in Pharmacology at Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. She got her Bachelor and Master degrees from China Pharmaceutical University and then worked as a Research Associate in Shanghai Institutes of Biological Sciences (Chinese Academy of Sciences), the University of Michigan (Ann Arbor) and Guangzhou Institutes of Biomedicine and Health and health (Chinese Academy of Sciences). She obtained her Ph.D. from Sun Yat-Sen University in 2013 and become an Associate Professor at Guangzhou Institutes of Biomedicine and Health (Chinese Academy of Sciences) in 2014. Her research mainly focuses on biological assay development and bioactivity evaluation of new anticancer molecules. Ke Ding got his Bachelor and Master degrees from China Pharmaceutical University in 1995 and 1998, respectively, and obtained his Ph.D. degree in Bioorganic Chemistry from Fudan University in 2001. After 5-year postdoctoral training at University of Michigan, Ann Arbor, he became a Professor of Medicinal Chemistry in Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences in 2006. Dr. Ding currently serves as an editorial board member for J. Med. Chem., ACS Med. Chem. Lett., and J. Chin. Pharm. Sci. Dr. Ding’s research interests mainly focus on design and synthesis of novel bioactive lead compounds for drug discovery. He has published more than 90 publications and is co-inventor of over 40 international patents, some of which have been licensed to international pharmaceutical companies.

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ACKNOWLEDGMENTS We are thankful for a Strategic Collaboration grant between China and New Zealand from Ministry of Science and Technology of China (Grant 2014DFG32100), National Science Fund for Distinguished Young Scholars of China (Grant 81425021), and 100-Distinguished Scientist Award of Guangdong Province (Nanyue-Baijie Award) for financial support.

AUTHOR INFORMATION

Corresponding Authors

*X.R.: phone, 86-20-32015300; e-mail, [email protected]. cn. *K.D.: phone, +86-20-32015276; fax, +86-20-32015299; e-mail, [email protected].

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ABBREVIATIONS USED DDR, discoidin domain receptor; RTK, receptor tyrosine kinase; ECM, extracellular matrix; DS, discoidin; EJXM, extracellular juxtamembrane; TM, transmembrane; IJXM, intracellular juxtamembrane; KD, kinase domain; DFG, AspPhe-Gly; SCC, squamous cell carcinoma; SMC, smooth muscle cell; GBM, glomerular basement membrane; MCP-1, monocyte chemoattractant protein 1; IR, insulin receptor; TGI, tumor growth inhibition; CAR, carrageenan; SMED-SL, spondylometa-epiphyseal dysplasia with short limbs and abnormal calcifications; i-NOS, inducible nitric oxide synthase; COX-2,

Author Contributions §

Y.L. and X.L. contributed equally to this work.

Notes

The authors declare no competing financial interest. Biographies Yupeng Li received his B.S. degree in Traditional Chinese Medicine in 2009 at Huazhong University of Science & Technology, Tongji Medical School, and completed his Ph.D. in Medicinal Chemistry in 2014 under the supervision of Prof. Ke Ding at Guangzhou Institutes J

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receptor DDR2 is a receptor for type X collagen. Matrix Biol. 2006, 25, 355−364. (f) Agarwal, G.; Mihai, C.; Iscru, D. F. Interaction of discoidin domain receptor 1 with collagen type 1. J. Mol. Biol. 2007, 367, 443−455. (5) Xu, H.; Raynal, N.; Stathopoulos, S.; Myllyharju, J.; Farndale, R. W.; Leitinger, B. Collagen binding specificity of the discoidin domain receptors: binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1. Matrix Biol. 2011, 30, 16−26. (6) (a) Valiathan, R. R.; Marco, M.; Leitinger, B.; Kleer, C. G.; Fridman, R. Discoidin domain receptor tyrosine kinases: new players in cancer progression. Cancer Metast. Rev. 2012, 31, 295−321. (b) Vogel, W. F.; Abdulhussein, R.; Ford, C. E. Sensing extracellular matrix: an update on discoidin domain receptor function. Cell Signal 2006, 18, 1108−1116. (c) Leitinger, B. Discoidin domain receptor functions in physiological and pathological conditions. Int. Rev. Cell Mol. Biol. 2014, 310, 39−87. (d) Iwai, L. K.; Luczynski, M. T.; Huang, P. H. Discoidin domain receptors: a proteomic portrait. Cell. Mol. Life Sci. 2014, 71, 3269−3279. (e) Borza, C. M.; Pozzi, A. Discoidin domain receptors in disease. Matrix Biol. 2014, 34, 185−192. (f) Kothiwale, S.; Borza, C. M.; Lowe, W.; Meiler, J. Discoidin domain receptor 1 (DDR1) kinase as target for structure-based drug discovery. Drug Discovery Today 2014, DOI: 10.1016/j.drudis.2014.09.025. (7) (a) Kiedzierska, A.; Smietana, K.; Czepczynska, H.; Otlewski, J. Structural similarities and functional diversity of eukaryotic discoidinlike domains. Biochim. Biophys. Acta 2007, 1774, 1069−1078. (b) Carafoli, F.; Hohenester, E. Collagen recognition and transmembrane signalling by discoidin domain receptors. Biochim. Biophys. Acta 2013, 1834, 2187−2194. (8) (a) Carafoli, F.; Bihan, D.; Stathopoulos, S.; Konitsiotis, A. D.; Kvansakul, M.; Farndale, R. W.; Leitinger, B.; Hohenester, E. Crystallographic insight into collagen recognition by discoidin domain receptor 2. Structure 2009, 17, 1573−1581. (b) Carafoli, F.; Mayer, M. C.; Shiraishi, K.; Pecheva, M. A.; Chan, L. Y.; Nan, R.; Leitinger, B.; Hohenester, E. Structure of the discoidin domain receptor 1 extracellular region bound to an inhibitory Fab fragment reveals features important for signaling. Structure 2012, 20, 688−697. (9) (a) Noordeen, N. A.; Carafoli, F.; Hohenester, E.; Horton, M. A.; Leitinger, B. A transmembrane leucine zipper is required for activation of the dimeric receptor tyrosine kinase DDR1. J. Biol. Chem. 2006, 281, 22744−22751. (b) Mihai, C.; Chotani, M.; Elton, T. S.; Agarwal, G. Mapping of DDR1 distribution and oligomerization on the cell surface by FRET microscopy. J. Mol. Biol. 2009, 385, 432−445. (c) Lemmon, M. A.; Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 2010, 141, 1117−1134. (10) Lemeer, S.; Bluwstein, A.; Wu, Z.; Leberfinger, J.; Muller, K.; Kramer, K.; Kuster, B. Phosphotyrosine mediated protein interactions of the discoidin domain receptor 1. J. Proteomics 2012, 75, 3465−3477. (11) Day, E.; Waters, B.; Spiegel, K.; Alnadaf, T.; Manley, P. W.; Buchdunger, E.; Walker, C.; Jarai, G. Inhibition of collagen-induced discoidin domain receptor 1 and 2 activation by imatinib, nilotinib and dasatinib. Eur. J. Pharmacol. 2008, 599, 44−53. (12) Canning, P.; Tan, L.; Chu, K.; Lee, S. W.; Gray, N. S.; Bullock, A. N. Structural mechanisms determining inhibition of the collagen receptor DDR1 by selective and multi-targeted type II kinase inhibitors. J. Mol. Biol. 2014, 426, 2457−2470. (13) Richters, A.; Nguyen, H. D.; Phan, T.; Simard, J. R.; Grütter, C.; Engel, J.; Rauh, D. Identification of type II and III DDR2 inhibitors. J. Med. Chem. 2014, 57, 4252−4262. (14) Kim, H. G.; Tan, L.; Weisberg, E. L.; Liu, F.; Canning, P.; Choi, H. G.; Ezell, S. A.; Wu, H.; Zhao, Z.; Wang, J.; Mandinova, A.; Griffin, J. D.; Bullock, A. N.; Liu, Q.; Lee, S. W.; Gray, N. S. Discovery of a potent and selective DDR1 receptor tyrosine kinase inhibitor. ACS Chem. Biol. 2013, 8, 2145−2150. (15) Ichikawa, O.; Osawa, M.; Nishida, N.; Goshima, N.; Nomura, N.; Shimada, I. Structural basis of the collagen-binding mode of discoidin domain receptor 2. EMBO J. 2007, 26, 4168−4176.

cyclooxygenase 2; JNK, c-Jun N-terminal kinase; PDGFR, platelet-derived growth factor receptor; TrkA, tropomycinrelated kinase A; BTK, Bruton tyrosine kinase; Syk, spleen tyrosine kinase; FLT, FMS-like tyrosine kinase; ZAK, zipper and sterile α motif containing kinase Amino Acids

Ala or A, alanine; Arg or R, arginine; Asn or N, asparagines; Asp or D, aspartic acid; Cys or C, cysteine; Gln or Q, glutamine; Glu or E, glutamic acid; Gly or G, glycine; His or H, histidine; Ile or I, isoleucine; Leu or L, leucine; Lys or K, lysine; Met or M, methionine; Phe or F, phenylalanine; Pro or P, proline; Ser or S, serine; Thr or T, threonine; Trp or W, tryptophan; Tyr or Y, tyrosine; Val or V, valine

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DOI: 10.1021/jm5012319 J. Med. Chem. XXXX, XXX, XXX−XXX