Cereblon contributes to cardiac dysfunction by degrading Cav1.2α.

AIMS: Cereblon (CRBN) is a substrate receptor of the E3 ubiquitin ligase complex that was reported to target ion channel proteins. L-type voltage-dependent Ca2+ channel (LTCC) density and dysfunction is a critical player in heart failure with reduced ejection fraction (HFrEF). However, the underlying cellular mechanisms by which CRBN regulates LTCC subtype Cav1.2α during cardiac dysfunction remain unclear. Here, we explored the role of CRBN in HFrEF by investigating the direct regulatory role of CRBN in Cav1.2α activity and examining how it can serve as a target to address myocardial dysfunction. METHODS AND RESULTS: Cardiac tissues from HFrEF patients exhibited increased levels of CRBN compared with controls. In vivo and ex vivo studies demonstrated that whole-body CRBN knockout (CRBN-/-) and cardiac-specific knockout mice (Crbnfl/fl/Myh6Cre+) exhibited enhanced cardiac contractility with increased LTCC current (ICaL) compared with their respective controls, which was modulated by the direct interaction of CRBN with Cav1.2α. Mechanistically, the Lon domain of CRBN directly interacted with the N-terminal of Cav1.2α. Increasing CRBN levels enhanced the ubiquitination and proteasomal degradation of Cav1.2α and decreased ICaL. In contrast, genetic or pharmacological depletion of CRBN via TD-165, a novel PROTAC-based CRBN degrader, increased surface expression of Cav1.2α and enhanced ICaL. Low CRBN levels protected the heart against cardiomyopathy in vivo. CONCLUSION: Cereblon selectively degrades Cav1.2α, which in turn facilitates cardiac dysfunction. A targeted approach or an efficient method of reducing CRBN levels could serve as a promising strategy for HFrEF therapeutics.

Synthesis and biological evaluation of N9-cis-cyclobutylpurine derivatives for use as cyclin-dependent kinase (CDK) inhibitors.

A novel 6-aminopurine scaffold bearing an N9-cis-cyclobutyl moiety was designed using structure-based molecular design based on two known CDK inhibitors, dinaciclib and Cmpd-27. A series of novel 6-aminopurine compounds was prepared for structure-activity relationship (SAR) studies of CDK2 and CDK5 inhibitors. Among the compounds synthesized, compound 8l displayed potent CDK2 and CDK5 inhibitory activities with low nanomolar ranges (IC50=2.1 and 4.8nM, respectively) and showed moderate cytotoxicity in HCT116 colon cancer and MCF7 breast cancer cell lines. Here, we report the synthesis and evaluation of novel 6-aminopurine derivatives and present molecular docking models of compound 81 with CDK2 and CDK5.

cIAPs promote the proteasomal degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis.

Although the ubiquitin-proteasome system is believed to play an important role in the pathogenesis of familial amyotrophic lateral sclerosis (FALS), caused by mutations in Cu/Zn-superoxide dismutase 1 (SOD1), the mechanism of how mutant SOD1 protein is regulated in cells is still poorly understood. Here we have demonstrated that cellular inhibitor of apoptosis proteins (cIAPs) are specifically associated with FALS-linked mutant SOD1 (mSOD1) and that this interaction promotes the ubiquitin-dependent proteasomal degradation of mutant SOD1. By utilizing cumate inducible SOD1 cells, we also showed that knock-down or pharmacologic depletion of cIAPs leads to H2O2 induced cytotoxicity in mSOD1 expressing cells. Altogether, our results reveal a novel role of cIAPs in FALS-associated mutant SOD1 regulation.

Novel c-Met inhibitor suppresses the growth of c-Met-addicted gastric cancer cells.

BACKGROUND: c-Met signaling has been implicated in oncogenesis especially in cells with c-met gene amplification. Since 20 % of gastric cancer patients show high level of c-Met expression, c-Met has been identified as a good candidate for targeted therapy in gastric cancer. Herein, we report our newly synthesized c-Met inhibitor by showing its efficacy both in vitro and in vivo. METHODS: Compounds with both triazolopyrazine and pyridoxazine scaffolds were synthesized and tested using HTRF c-Met kinase assay. We performed cytotoxic assay, cellular phosphorylation assay, and cell cycle assay to investigate the cellular inhibitory mechanism of our compounds. We also conducted mouse xenograft assay to see efficacy in vivo. RESULTS: KRC-00509 and KRC-00715 were selected as excellent c-Met inhibitors through biochemical assay, and exhibited to be exclusively selective to c-Met by kinase panel assay. Cytotoxic assays using 18 gastric cancer cell lines showed our c-Met inhibitors suppressed specifically the growth of c-Met overexpressed cell lines, not that of c-Met low expressed cell lines, by inducing G1/S arrest. In c-met amplified cell lines, c-Met inhibitors reduced the downstream signals including Akt and Erk as well as c-Met activity. In vivo Hs746T xenograft assay showed KRC-00715 reduced the tumor size significantly. CONCLUSIONS: Our in vitro and in vivo data suggest KRC-00715 is a potent and highly selective c-Met inhibitor which may have therapeutic potential in gastric tumor with c-Met overexpression.

Discovery of novel tetrahydroisoquinoline-containing pyrimidines as ALK inhibitors.

Exploration of the two-position side chain of pyrimidine in LDK378 with tetrahydroisoquinolines (THIQs) led to discovery of 8 and 17 as highly potent ALK inhibitors. THIQs 8 and 17 showed encouraging in vitro and in vivo xenograft efficacies, comparable with those of LDK378. Although THIQ analogs (8a-o and 17a-i) prepared were not as active as their parent compounds, both 8 and 17 have significant inhibitory activities against various ALK mutant enzymes including G1202R, indicating that this series of compounds could be further optimized as useful ALK inhibitors overcoming the resistance issues found from crizotinib and LDK378.

Development of potent ALK inhibitor and its molecular inhibitory mechanism against NSCLC harboring EML4-ALK proteins.

Here, we show the newly synthesized and potent ALK inhibitor having similar scaffold to KRCA-0008, which was reported previously, and its molecular mechanism against cancer cells harboring EML4-ALK fusion protein. Through ALK wild type enzyme assay, we selected two compounds, KRCA-0080 and KRCA-0087, which have trifluoromethyl instead of chloride in R2 position. We characterized these newly synthesized compounds by in vitro and in vivo assays. Enzyme assay shows that KRCA-0080 is more potent against various ALK mutants, including L1196M, G1202R, T1151_L1152insT, and C1156Y, which are seen in crizotinib-resistant patients, than KRCA-0008 is. Cell based assays demonstrate our compounds downregulate the cellular signaling, such as Akt and Erk, by suppressing ALK activity to inhibit the proliferation of the cells harboring EML4-ALK. Interestingly, our compounds induced strong G1/S arrest in H3122 cells leading to the apoptosis, which is proved by PARP-1 cleavage. In vivo H3122 xenograft assay, we found that KRCA-0080 shows significant reduction in tumor size compared to crizotinib and KRCA-0008 by 15-20%. Conclusively, we report a potent ALK inhibitor which shows significant in vivo efficacy as well as excellent inhibitory activity against various ALK mutants.

Synthesis and evaluation of novel 2,4-diaminopyrimidines bearing bicyclic aminobenzazepines for anaplastic lymphoma kinase (ALK) inhibitor.

A series of novel 2,4-diaminopyrimidine compounds bearing bicyclic aminobenzazepine were synthesized and evaluated for their anti-ALK activities. The activities of these compounds were confirmed in both enzyme- and cell-based ALK assays. Amongst compounds synthesized, KRCA-0445 showed very promising results in pharmacokinetic study and in vivo efficacy study with H3122 xenograft mouse model.

Discovery of quinolinone derivatives as potent FLT3 inhibitors.

Recently some fms-like tyrosine kinase 3 (FLT3) inhibitors have shown good efficacy in acute myeloid leukemia (AML) patients. In an effort to develop anti-leukemic drugs, we investigated quinolinone derivatives as novel FLT3 inhibitors. Two substituted quinolinones, KR65367 and KR65370 were subjected to FLT3 kinase activity assay and showed potent inhibition against FLT3 kinase activity in vitro, with IC50 of 2.7 and 0.57 nM, respectively. As a measure of selectivity, effects on the activity of other kinases were also tested. Both compounds have negligible activity against Met, Ron, epidermal growth factor receptor, Aurora A, Janus kinase 2, and insulin receptor; with IC50 greater than 10 µM. KR compounds showed strong growth inhibition in MV4;11 AML cells and increased the apoptotic cell death in flow cytometric analyses. A decrease in STAT5 phosphorylation by KR compounds was observed in MV4;11 cells. Furthermore, in vitro evaluation of compounds structurally related to KR65367 and KR65370 showed a good structure-activity relationship.

4-Substituted quinazoline derivatives as novel EphA2 receptor tyrosine kinase inhibitors.

Erythropoietin-producing hepatocellular receptor tyrosine kinase subtype A2 (EphA2) is an attractive therapeutic target for suppressing tumor progression. In our efforts to discover novel small molecules to inhibit EphA2, a class of compound based on 4-substituted quinazoline containing 7-(morpholin-2-ylmethoxy) group was identified as a novel hit by high throughput screening campaign. Structural modification of parent quinazoline scaffolds by introducing substituents on aniline displayed potent inhibitory activities toward EphA2.

Discovery of substituted 6-pheny-3H-pyridazin-3-one derivatives as novel c-Met kinase inhibitors.

We report a series of phenyl substituted pyridazin-3-ones substituted with morpholino-pyrimidines. The SAR of the phenyl was explored and their c-Met kinase and cell-based inhibitory activity toward c-Met driven cell lines were evaluated. Described herein is a potent c-Met inhibitor by structural modification of the parent morpholino-pyridazinone scaffold, with particular focus on the phenyl and pyrimidine substituents.

Design and synthesis of novel 3-(benzo[d]oxazol-2-yl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine derivatives as selective G-protein-coupled receptor kinase-2 and -5 inhibitors.

G-protein-coupled receptor kinase (GRK)-2 and -5 are emerging therapeutic targets for the treatment of cardiovascular disease. In our efforts to discover novel small molecules to inhibit GRK-2 and -5, a class of compound based on 3-(benzo[d]oxazol-2-yl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine was identified as a novel hit by high throughput screening campaign. Structural modification of parent benzoxazole scaffolds by introducing substituents on phenyl displayed potent inhibitory activities toward GRK-2 and -5.

Novel bis-ortho-alkoxy-para-piperazinesubstituted-2,4-dianilinopyrimidines (KRCA-0008) as potent and selective ALK inhibitors for anticancer treatment.

The synthesis of bis-ortho-alkoxy-para-piperazinesubstituted-2,4-dianilinopyrimidines is described and their structure-activity-relationship to anaplastic lymphoma kinase (ALK) is presented. KRCA-0008 is selective and potent to ALK and Ack1, and displays drug-like properties without hERG liability. KRCA-0008 demonstrates in vivo efficacy comparable to Crizotinib in xenograft mice model.

Discovery of pan-IAP degraders via a CRBN recruiting mechanism.

Inhibitors of apoptosis proteins (IAPs), defined by the presence of baculovirus IAP repeat (BIR) protein domain, are critical regulators of cell survival and cell death processes. Cellular IAP 1/2 (cIAP1/2) and X-linked IAPs (XIAPs) regulate the innate immune signaling pathway through their E3 ubiquitin ligase activity. Peptidomimetics or small-molecule IAP antagonists have been developed to treat various diseases, such as cancer, infection, and inflammation. In this study, we synthesized and characterized IAP-cereblon (CRBN) heterodimerizing proteolysis-targeting chimera (PROTAC), which induces the degradation of cIAP1/2 and XIAP but not CRBN. We demonstrated that this PROTAC inhibits tumor necrosis factor alpha (TNFα)-induced innate immune response and cancer cell migration and invasion, leading to apoptotic cell death. Our study is the first to demonstrate that both cIAPs and XIAP are degradable when applied to the PROTAC strategy.

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.

Design and synthesis of 3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)-1H-quinolin-2-ones as VEGFR-2 kinase inhibitors.

A series of 3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)-1H-quinolin-2-ones have been identified as a new class of VEGFR-2 kinase inhibitors. A variety of (4,5,6,7-tetrahydro-imidazo[5,4-c]pyridin-2-yl)-acetic acid ethyl esters were synthesized, and their VEGFR-2 inhibitory activity was evaluated. Described herein are the preparation of the series and the effects of the compounds on VEGFR-2 kinase activity.

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.

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.

Augmentation of the antitumor effects of PARP inhibitors in triple-negative breast cancer via degradation by hydrophobic tagging modulation.

Triple-negative breast cancer (TNBC) has an aggressive phenotype and poor prognosis due to the lack of specific targeted treatments. The development of an effective therapeutic strategy with a novel mechanism is essential for TNBC management. Olaparib, a PARP inhibitor, has been approved for the treatment of breast or ovarian cancer patients with breast cancer gene 1/2 (BRCA1/2) mutations. Here, we report the development of a small molecule targeting PARP1 based on the hydrophobic tagging (HyT) method. Targeted protein misfolding and consequent degradation are caused by HyT. Hydrophobic-tagged olaparib induces the proteasome-dependent degradation of PARP1 and shows enhanced antitumor effects compared to olaparib in TNBC cells. In addition, hydrophobic-tagged olaparib causes ER stress-related unfolded protein response (UPR), autophagy, and apoptosis. These results point towards encouraging prospects for chemically modifying approved drugs that not only exhibit superior effects compared to those of the original drugs by triggering novel mechanisms but also provide great feasibility in the translational scenario.