Novel Small Molecule ROCK2 Inhibitor GNS-3595 Attenuates Pulmonary Fibrosis in Preclinical Studies.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that leads to respiratory decline due to scarring and thickening of lung tissues. Multiple pathways contribute to the fibrotic process in this disease, such as inflammation, epithelial to mesenchymal transition and oxidative stress. The RhoA/ROCK signaling pathway is a key regulator of profibrotic signaling, as it affects the organization of actin-myosin and the remodeling of the extracellular matrix. ROCK1/2, a downstream effector of RhoA, is overexpressed in IPF patients and is a promising target for IPF therapy. However, due to hypotensive side effects of ROCK1/2 inhibitors, selective ROCK2 compounds are being explored. In this study, we report the discovery of GNS-3595, a potent and selective ROCK2 inhibitor that has ~80-fold selectivity over ROCK1 at physiological concentrations of ATP. GNS-3595 effectively inhibited ROCK2-mediated phosphorylation of myosin light chain (p-MLC) and reduced the expression of fibrosis-related proteins, such as collagen, fibronectin, and alpha-smooth muscle actin (α-SMA) in various in vitro cellular models. GNS-3595 also prevented transforming growth factor beta (TGF-ß)-induced fibroblast-to-myofibroblast transition (FMT). Additionally, in a bleomycin-induced mouse model of pulmonary fibrosis, therapeutic exposure to GNS-3595, suppressed lung fibrosis, stabilized body weight loss, and prevented fibrosis-induced lung weight gain. Transcriptome and protein expression analysis from lung tissues showed that GNS-3595 can revert the fibrosis-related gene expression induced by bleomycin. These results indicate that GNS-3595 is a highly potent, selective, and orally active ROCK2 inhibitor with promising therapeutic efficacy against pulmonary fibrosis.

YH25448, an Irreversible EGFR-TKI with Potent Intracranial Activity in EGFR Mutant Non-Small Cell Lung Cancer.

PURPOSE: Given that osimertinib is the only approved third-generation EGFR-TKI against EGFR activating and resistant T790M mutated non-small cell lung cancer (NSCLC), additional mutant-selective inhibitors with a higher efficacy, especially for brain metastases, with favorable toxicity profile are still needed. In this study, we investigated the antitumor efficacy of YH25448, an oral, mutant-selective, irreversible third-generation EGFR-TKI in preclinical models. EXPERIMENTAL DESIGN: Antitumor activity of YH25448 was investigated in vitro using mutant EGFR-expressing Ba/F3 cells and various lung cancer cell lines. In vivo antitumor efficacy, ability to penetrate the blood-brain barrier (BBB), and skin toxicity of YH25448 were examined and compared with those of osimertinib using cell lines and PDX model. RESULTS: Compared with osimertinib, YH25448 showed a higher selectivity and potency in kinase assay and mutant EGFR-expressing Ba/F3 cells. In various cell line models harboring EGFR activating and T790M mutation, YH25448 effectively inhibited EGFR downstream signaling pathways, leading to cellular apoptosis. When compared in vivo at equimolar concentrations, YH25448 produced significantly better tumor regression than osimertinib. Importantly, YH25448 induced profound tumor regression in brain metastasis model with excellent brain/plasma and tumor/brain area under the concentration-time curve value. YH25448 rarely suppressed the levels of p-EGFR in hair follicles, leading to less keratosis than osimertinib in animal model. The potent systemic and intracranial activity of YH25448 has been shown in an ongoing phase I/II clinical trial for advanced EGFR T790M mutated NSCLC (NCT03046992). CONCLUSIONS: Our findings suggest that YH25448 is a promising third-generation EGFR inhibitor, which may be more effective and better tolerated than the currently approved osimertinib.

Crystal Structures of Spleen Tyrosine Kinase in Complex with Two Novel 4-Aminopyrido[4,3-d] Pyrimidine Derivative Inhibitors.

Spleen tyrosine kinase (SYK) is a cytosolic non-receptor protein tyrosine kinase. Because SYK mediates key receptor signaling pathways involving the B cell receptor and Fc receptors, SYK is an attractive target for autoimmune disease and cancer treatments. To date, representative oral SYK inhibitors, including fostamatinib (R406 or R788), entospletinib (GS-9973), cerdulatinib (PRT062070), and TAK-659, have been assessed in clinical trials. Here, we report the crystal structures of SYK in complex with two newly developed inhibitors possessing 4-aminopyrido[4,3-D]pyrimidine moieties (SKI-G-618 and SKI-O-85). One SYK inhibitor (SKI-G-618) exhibited moderate inhibitory activity against SYK, whereas the other inhibitor (SKI-O-85) exhibited a low inhibitory profile against SYK. Binding mode analysis indicates that a highly potent SYK inhibitor might be developed by modifying and optimizing the functional groups that interact with Leu377, Gly378, and Val385 in the G-loop and the nearby region in SYK. In agreement with our structural analysis, one of our SYK inhibitor (SKI-G-618) shows strong inhibitory activities on the ß-hexosaminidase release and phosphorylation of SYK/Vav in RBL-2H3 cells. Taken together, our findings have important implications for the design of high affinity SYK inhibitors.

Correction: Crystal Structure of Pim1 Kinase in Complex with a Pyrido[4,3-D]Pyrimidine Derivative Suggests a Unique Binding Mode.

[This corrects the article DOI: 10.1371/journal.pone.0070358.].

Superior Efficacy and Selectivity of Novel Small-Molecule Kinase Inhibitors of T790M-Mutant EGFR in Preclinical Models of Lung Cancer.

The clinical utility of approved EGFR small-molecule kinase inhibitors is plagued both by toxicity against wild-type EGFR and by metastatic progression in the central nervous system, a disease sanctuary site. Here, we report the discovery and preclinical efficacy of GNS-1486 and GNS-1481, two novel small-molecule EGFR kinase inhibitors that are selective for T790M-mutant isoforms of EGFR. Both agents were effective in multiple mouse xenograft models of human lung adenocarcinoma (T790M-positive or -negative), exhibiting less activity against wild-type EGFR than existing approved EGFR kinase inhibitors (including osimertinib). In addition, GNS-1486 showed superior potency against intracranial metastasis of EGFR-mutant lung adenocarcinoma. Our results offer a preclinical proof of concept for new EGFR kinase inhibitors with the potential to improve therapeutic index and efficacy against brain metastases in patients. Cancer Res; 77(5); 1200-11. ©2017 AACR.

Crystal structures of spleen tyrosine kinase in complex with novel inhibitors: structural insights for design of anticancer drugs.

Spleen tyrosine kinase (SYK) is a cytosolic nonreceptor protein tyrosine kinase that mediates key signal transduction pathways following the activation of immune cell receptors. SYK regulates cellular events induced by the B-cell receptor and Fc receptors with high intrinsic activity. Furthermore, SYK has been regarded as an attractive target for the treatment of autoimmune diseases and cancers. Here, we report the crystal structures of SYK in complex with seven newly developed inhibitors (G206, G207, O178, O194, O259, O272, and O282) to provide structural insights into which substituents of the inhibitors and binding regions of SYK are essential for lead compound optimization. Our kinase inhibitors exhibited high inhibitory activities against SYK, with half-maximal inhibitory concentrations (IC50 ) of approximately 0.7-33 nm, but they showed dissimilar inhibitory activities against KDR, RET, JAK2, JAK3, and FLT3. Among the seven SYK inhibitors, O272 and O282 exhibited highly specific inhibitions against SYK, whereas O194 exhibited strong inhibition of both SYK and FLT3. Three inhibitors (G206, G207, and O178) more efficiently inhibited FLT3 while still substantially inhibiting SYK activity. The binding mode analysis suggested that a highly selective SYK inhibitor can be developed by optimizing the functional groups that facilitate direct interactions with Asn499. DATABASE: The atomic coordinates and structure factors for human SYK are in the Protein Data Bank under accession codes 4XG2 (inhibitor-free form), 4XG3 (G206), 4XG4 (G207), 5GHV (O178), 4XG6 (O194), 4XG7 (O259), 4XG8 (O272), and 4XG9 (O282).

Highly potent and selective pyrazolylpyrimidines as Syk kinase inhibitors.

A series of pyrazolylpyrimidine scaffold based Syk inhibitors were synthesized and evaluated for their biological activities and selectivity. Lead optimization efforts provided compounds with potent Syk inhibition in both enzymatic and TNF-α release assay.

G-749, a novel FLT3 kinase inhibitor, can overcome drug resistance for the treatment of acute myeloid leukemia.

Aberrant activations of Fms-like tyrosine receptor kinase (FLT) 3 are implicated in the pathogenesis of 20% to 30% of patients with acute myeloid leukemia (AML). G-749 is a novel FLT3 inhibitor that showed potent and sustained inhibition of the FLT3 wild type and mutants including FLT3-ITD, FLT3-D835Y, FLT3-ITD/N676D, and FLT3-ITD/F691L in cellular assays. G-749 retained its inhibitory potency in various drug-resistance milieus such as patient plasma, FLT3 ligand surge, and stromal protection. Furthermore, it displayed potent antileukemic activity in bone marrow blasts from AML patients regardless of FLT3 mutation status, including those with little or only minor responses to AC220 or PKC412. Oral administration of G-749 yielded complete tumor regression and increased life span in animal models. Thus, G-749 appears to be a promising next-generation drug candidate for the treatment of relapsed and refractory AML patients with various FLT3-ITD/FLT3-TKD mutants and further shows the ability to overcome drug resistance.

Crystal structure of pim1 kinase in complex with a pyrido[4,3-d]pyrimidine derivative suggests a unique binding mode.

Human Pim1 kinase is a serine/threonine protein kinase that plays important biological roles in cell survival, apoptosis, proliferation, and differentiation. Moreover, Pim1 is up-regulated in various hematopoietic malignancies and solid tumors. Thus, Pim1 is an attractive target for cancer therapeutics, and there has been growing interest in developing small molecule inhibitors for Pim1. Here, we describe the crystal structure of Pim1 in complex with a newly developed pyrido[4,3-d]pyrimidine-derivative inhibitor (SKI-O-068). Our inhibitor exhibits a half maximum inhibitory concentration (IC50) of 123 (±14) nM and has an unusual binding mode in complex with Pim1 kinase. The interactions between SKI-O-068 and the Pim1 active site pocket residue are different from those of other scaffold inhibitor-bound structures. The binding mode analysis suggests that the SKI-O-068 inhibitor can be improved by introducing functional groups that facilitate direct interaction with Lys67, which aid in the design of an optimized inhibitor.

Synthesis and structure-activity relationship of aminopyridines with substituted benzoxazoles as c-Met kinase inhibitors.

A series of hydroxybenzoxazole derivatives was synthesized, and their c-Met kinase inhibitory activity was evaluated. Described herein is a potent c-Met inhibitor by structural modification of the parent benzoxazole scaffold, with particular focus on the hydroxyl substituent of the benzoxazole moiety.

Preclinical metabolism of LB42908, a novel farnesyl transferase inhibitor, and its effects on the cytochrome P450 isozyme activities.

Metabolism of LB42908, a novel farnesyl transferase inhibitor, was investigated for preclinical development. In vitro hepatic metabolism of LB42908 gave rise to at least 9 metabolites via phase I biotransformation pathways, which were characterized by HPLC-UV, LC-MS, and LC-MS/MS analyses. N-Dealkylation was shown to be a major phase I metabolic pathway. Species-specific in vitro metabolism of LB42908 was studied in liver fractions of rat, dog, monkey, and human. Order of metabolic stability is human≈dog>rat≈monkey in both S9 and microsomal fractions. Tissue-specific metabolism of LB42908 in various tissue homogenates of rats demonstrated that the liver was the major organ responsible for phase I metabolism of LB42908. The results from both qualitative and quantitative metabolism studies such as metabolic profiling and metabolic clearance indicated that dog would be the animal model of choice for preclinical toxicology studies. In addition, LB42908 was a potent CYP3A4 inhibitor in human liver microsomes and induced the activities of several CYP isozymes, implying that it has the potential for drug-drug interactions. Repeated dosing of LB42908 in rats did not significantly affect its own metabolism, indicating that long-term administration of LB42908 would not alter its pharmacokinetic profiles.

Evaluation of a multi-kinase inhibitor KRC-108 as an anti-tumor agent in vitro and in vivo.

Kinases have been studied as potential cancer targets because they play important roles in the cellular signaling of tumors. A number of small molecules targeting kinases are prescribed in clinics and many kinase inhibitors are being evaluated in the clinical phase. Previously, we discovered a series of aminopyridines substituted with benzoxazole as orally active c-Met kinase inhibitors. One of the compounds, KRC-108, has been evaluated as an anti-cancer agent in vitro and in vivo. A kinase panel assay exhibited that KRC-108 is a potent inhibitor of Ron, Flt3 and TrkA as well as c-Met. Moreover, KRC-108 inhibited oncogenic c-Met M1250T and Y1230D more strongly than wild type c-Met. The anti-proliferative activity of KRC-108 was measured by performing a cytotoxicity assay on a panel of cancer cell lines. The GI(50) values (i.e., 50% inhibition of cell growth) for KRC-108 ranged from 0.01 to 4.22 µM for these cancer cell lines. KRC-108 was also effective for the inhibition of tumor growth in human HT29 colorectal cancer and NCI-H441 lung cancer xenograft models in athymic BALB/c nu/nu mice. This molecule should serve as a useful lead for inhibitors targeting kinases and may lead to new therapeutics for the treatment of cancer.

Phosphatase of regenerating liver-3 promotes migration and invasion by upregulating matrix metalloproteinases-7 in human colorectal cancer cells.

Phosphatase of regenerating liver (PRL)-3, a member of a subgroup of protein tyrosine phosphatases that can stimulate the degradation of the extracellular matrix, is over-expressed in metastatic colorectal cancer (CRC) relative to primary tumors. To determine whether PRL-3-induced enhancement of migration and invasion is dependent on the expression of matrix metalloproteinases (MMPs), PRL-3 was expressed in DLD-1 human CRC cells. The motility, migration and invasion characteristics of the cells were examined, and metastasis to the lung was confirmed in a nude mouse using PRL-3-overexpressing DLD-1 cells [DLD-1 (PRL-3)]. Migration and invasion of the cells were inhibited by phosphatase and farnesyltransferase inhibitors. Expression of MMPs was enhanced 3- to 10-fold in comparison to control cells, and migration and invasion were partially inhibited by small interfering RNA (siRNA) knockdown of MMP-2, -13 or -14. Importantly, siRNA knockdown of MMP-7 completely inhibited the migration and invasion of DLD-1 (PRL-3) cells, whereas overexpression of MMP-7 increased migration. The expression of MMP-7 was also downregulated by phosphatase and farnesyltransferase inhibitors. It was found that PRL-3 induced MMP-7 through oncogenic pathways including PI3K/AKT and ERK and that there is a relationship between the expression of PRL-3 and MMP-7 in human tumor cell lines. The expression of MMP-13 and -14 was very sensitive to the inhibition of farnesyltransferase; however, the migration and invasion of DLD-1 (PRL-3) cells did not strongly depend on the expression of MMP-13 or -14. These results suggest that the migration and invasion of PRL-3-expressing CRC cells depends primarily on the expression of MMP-7.

Design and synthesis of triazolopyridazines substituted with methylisoquinolinone as selective c-Met kinase inhibitors.

A series of triazolopyridazines substituted with methylisoquinolinone were designed and synthesized. Some of the triazolopyridazines strongly inhibited c-Met kinase and showed good anti-proliferative activity against a panel of c-Met-amplified gastric cancer cell lines (MKN-45, SNU-5 and Hs746T).

Discovery of aminopyridines substituted with benzoxazole as orally active c-Met kinase inhibitors.

We report the synthesis and biological evaluation of aminopyridines substituted with benzoxazole. The SAR of the aminopyridines was explored to improve the inhibitory activity against c-Met and to decrease hERG affinity. These studies led to the discovery of amide 24 which showed good c-Met inhibitory potency, low affinity to hERG and favorable pharmacokinetic properties in rats.

Prognostic significance of c-Met expression in glioblastomas.

BACKGROUND: The authors investigated whether expression of c-Met protein in glioblastomas is associated with overall survival and biologic features representing tumor invasiveness in patients with glioblastomas. METHODS: Paraffin-embedded specimens of glioblastomas from 62 patients treated in a single institution were assessed by immunohistochemical (IHC) analysis of c-Met expression. On the basis of the clinical data for these patients, the association between c-Met expression and clinicobiologic features representing tumor invasiveness was analyzed. RESULTS: c-Met overexpression was detected in 29.0% (18 of 62) of glioblastomas. In patients with c-Met overexpression, the median survival was 11.7 months (95% confidence interval [95% CI], 9.9 months-13.5 months), compared with a median survival of 14.3 months (95% CI, 7.6 months-21.0 months) for patients whose tumors had no or little expression of c-Met (P=.031). On the radiographic analysis, 9 of 18 patients (50%) with tumors overexpressing c-Met demonstrated invasive and multifocal lesions on the initial magnetic resonance images, whereas only 9 of 44 patients (20.5%) with tumors that expressed no or little c-Met demonstrated these features (P=.030). Using immunohistochemistry, we also found a significant association between c-Met expression and matrix metalloproteinase-2,-9 (P=.020 and P=.013). Furthermore, Myc overexpression was found to be closely correlated with c-Met overexpression on IHC analysis (P=.004). CONCLUSIONS: The authors suggest that c-Met overexpression is associated with shorter survival time and poor treatment response in glioblastomas, the mechanism for which is elevated tumor invasiveness on the molecular and clinical phenotypes. This implies that more effective therapeutic strategies targeting c-Met receptors may have important clinical implication.

Synthesis, SAR, and X-ray structure of novel potent DPPIV inhibitors: oxadiazolyl ketones.

Synthesis of a novel series of DPPIV inhibitors with 1,2,4- and 1,3,4-oxadiazolyl ketone derivatives and its structure-activity relationships are discussed. Compound 18h showed good inhibitory activity against DPPIV and favorable pharmacokinetic properties. In vivo pharmacodynamic efficacy and co-crystal structure of compound 18h with DPPIV is also described.

The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells.

LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21(CIP1/WAF1), RhoB and EGFR, that can explain the differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21(CIP1/WAF1) and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21(CIP1/WAF1) and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells.

Hydantoin derivatives as non-peptidic inhibitors of Ras farnesyl transferase.

1,3,5,5-Tetrasubstituted 2,4-imidazolinedione (hydantoin) derivatives were evaluated as Ftase inhibitors. Potent Ftase inhibitors without thiol or peptide were obtained in three steps.