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Background Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are frequently found in various cancer types. IDH1 mutants produce 2-hydroxyglutarate (2-HG), an oncometabolite, from alpha-ketoglutarate (α-KG). This 2-HG plays a key role in tumorigenesis via inhibition of α-KG dependent enzymes. For this reason, IDH1 mutant could be an ideal target for the treatment of cancer. Materials and methods To find a new IDH1 inhibitor, 8,364 compounds were obtained from Korea Chemical Bank. Using high-throughput screening (HTS) of a chemical library, we unveiled a compound that could inhibit the IDH1 mutant. Results According to the enzyme assay, our compound (KRC-09) effectively inhibited the activity of IDH1 R132H mutant. In addition, KRC-09 decreased the concentration of intracellular 2-HG in the U-87 MG cell line harboring IDH1 R132H. Conclusion In this article, we present a novel chemical scaffold that suppresses the activity of an IDH1 mutant.

Simple Summary c-Met is known to be overexpressed in gastric cancers. Here, we developed anti-c-Met CAR T cell and measured its anti-tumor efficacy in vitro and in vivo. Our anti c-Met CAR T cells have shown selective killing of c-Met overexpressed gastric cancer cells. Based on our results, we suggest that anti-c-Met CAR T cell therapy could be effective for gastric cancer patients. Abstract Chimeric antigen receptor (CAR) technology has been highlighted in recent years as a new therapeutic approach for cancer treatment. Although the impressive efficacy of CAR-based T cell adoptive immunotherapy has been observed in hematologic cancers, limited effect has been reported on solid tumors. Approximately 20% of gastric cancer (GC) patients exhibit a high expression of c-Met. We have generated an anti c-Met CAR construct that is composed of a single-chain variable fragment (scFv) of c-Met antibody and signaling domains consisting of CD28 and CD3ζ. To test the CAR construct, we used two cell lines: the Jurkat and KHYG-1 cell lines. These are convenient cell lines, compared to primary T cells, to culture and to test CAR constructs. We transduced CAR constructs into Jurkat cells by electroporation. c-Met CAR Jurkat cells secreted interleukin-2 (IL-2) only when incubated with c-Met positive GC cells. To confirm the lytic function of CAR, the CAR construct was transduced into KHYG-1, a NK/T cell line, using lentiviral particles. c-Met CAR KHYG-1 showed cytotoxic effect on c-Met positive GC cells, while c-Met negative GC cell lines were not eradicated by c-Met CAR KHYG-1. Based on these data, we created c-Met CAR T cells from primary T cells, which showed high IL-2 and IFN-γ secretion when incubated with the c-Met positive cancer cell line. In an in vivo xenograft assay with NSG bearing MKN-45, a c-Met positive GC cell line, c-Met CAR T cells effectively inhibited the tumor growth of MKN-45. Our results show that the c-Met CAR T cell therapy can be effective on GC.

Immunomodulatory drugs (IMiDs) exert anti-myeloma activity by binding to the protein cereblon (CRBN) and subsequently degrading IKZF1/3. Recently, their ability to recruit E3 ubiquitin ligase has been used in the proteolysis targeting chimera (PROTAC) technology. Herein, we design and synthesize a novel IMiD analog TD-106 that induces the degradation of IKZF1/3 and inhibits the proliferation of multiple myeloma cells in vitro as well as in vivo. Moreover, we demonstrate that TD-428, which comprises TD-106 linked to a BET inhibitor, JQ1 efficiently induce BET protein degradation in the prostate cancer cell line 22Rv1. Consequently, cell proliferation is inhibited due to suppressed C-MYC transcription. These results, therefore, firmly suggest that the newly synthesized IMiD analog, TD-106, is a novel CRBN modulator that can be used for targeted protein degradation.

Molecular glue degraders, such as lenalidomide and pomalidomide, bind to cereblon (CRBN) E3 ligase and subsequently recruit neosubstrate proteins, Ikaros (IKZF1) and Aiolos (IKZF3), for the ubiquitination-proteasomal degradation process. In this study, we explored structure-activity relationship analysis for novel GSPT1 degraders utilizing a benzotriazinone scaffold previously discovered as a novel CRBN binder. In particular, we focused on the position of the ureido group on the benzotriazinone scaffold, substituent effect on the phenylureido group, and methyl substitution on the benzylic position of benzotriazinone. As a result, we identified 34f (TD-522), which exhibits strong anti-proliferative effects in both KG-1 (EC

Since bromodomain containing 4 (brd4) has been considered as a prominent cancer target, numerous attempts have been made to develop potent brd4 bromodomain inhibitors. The present study provided a novel chemical scaffold which inhibited brd4 activity. Mid-throughput screening against brd4 bromodomain was performed using alpha-screen and homogeneous time-resolved fluorescence assays. Furthermore, cell cytotoxicity and xenograft assays were performed to examine if the compound was effective both in vitro and in vivo. As a result, it was revealed that compounds having naphthalene-1,4-dione scaffold inhibited the binding of bromodomain to acetylated histone. The compounds with naphthalene-1,4-dione had cytotoxic effects against the Ty82 cell line, a NUT midline carcinoma cell line, whose proliferation is dependent on brd4 activity. A10, one of the compounds with naphthalene-1,4-dione scaffold, also exhibited tumor growth inhibition effects in the xenograft assay. In addition, the compounds exhibited cytotoxic effects against gastric cancer cell lines which were resistant to I-BET-762, a BET bromodomain inhibitor. In conclusion, the novel scaffold to suppress brd4 activity was effective against cancer cells both in vitro and in vivo.

Proteolysis-targeting chimera (PROTAC)-mediated protein degradation is a rapidly emerging therapeutic intervention that induces the degradation of targeted proteins. Herein, we report the design and biological evaluation of a series of androgen receptor (AR) PROTAC degraders for the treatment of metastatic castration-resistant prostate cancer. Predominantly, instead of thalidomide, we utilized the TD-106 scaffold, a novel cereblon (CRBN) binder that was identified in our previous study. Our results suggest that the linker position in the TD-106 CRBN binder is critical for the efficiency of AR degradation. The compounds attached to the 6-position of TD-106 promoted better degradation of AR than those at the 5- and 7-positions. Among the synthesized AR PROTACs, the representative degrader 33c (TD-802) effectively induced AR protein degradation, with a degradation concentration 50% of 12.5 nM and a maximum degradation of 93% in LNCaP prostate cancer cells. Additionally, most AR PROTAC degraders, including TD-802, displayed good liver microsomal stability and in vivo pharmacokinetic properties. Finally, we showed that TD-802 effectively inhibited tumor growth in an in vivo xenograft study.

Bromodomain-containing protein 4 (Brd4) is known to play a key role in tumorigenesis. It binds acetylated histones to regulate the expression of numerous genes. Because of the importance of brd4 in tumorigenesis, much research has been undertaken to develop brd4 inhibitors with therapeutic potential. As a result, various scaffolds for bromodomain inhibitors have been identified. To discover new scaffolds, we performed mid-throughput screening using two different enzyme assays, alpha-screen and ELISA. We found a novel bromodomain inhibitor with a unique scaffold, aristoyagonine. This natural compound showed inhibitory activity in vitro and tumor growth inhibition in a Ty82-xenograft mouse model. In addition, we tested Brd4 inhibitors in gastric cancer cell lines, and found that aristoyagonine exerted cytotoxicity not only in I-BET-762-sensitive cancer cells, but also in I-BET-762-resistant cancer cells. This is the first paper to describe a natural compound as a Brd4 bromodomain inhibitor.

Gastric cancer is a malignancy that has a high mortality rate. Although progress has been made in the treatment of gastric cancer, many patients experience cancer recurrence and metastasis. Folate receptor 1 (FOLR1) is overexpressed on the cell surface in over one-third of gastric cancer patients, but rarely is expressed in normal tissue. This makes FOLR1 a potential target for chimeric antigen receptor (CAR) T cell immunotherapy, although the function of FOLR1 has not been elucidated. CAR are engineered fusion receptor composed of an antigen recognition region and signaling domains. T cells expressing CAR have specific activation and cytotoxic effects against cancer cells containing the target antigen. In this study, we generated a CAR that targets FOLR1 composed of a single-chain variable fragment (scFv) of FOLR1 antibody and signaling domains consisting of CD28 and CD3ζ. Both FOLR1-CAR KHYG-1, a natural killer cell line, and FOLR1-CAR T cells recognized FOLR1-positive gastric cancer cells in a MHC-independent manner and induced secretion of various cytokines and caused cell death. Conclusively, this is the first study to demonstrate that CAR KHYG-1/T cells targeting FOLR1 are effective against FOLR1-positive gastric cancer cells.

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.

Bromodomain-containing protein 4 (BRD4) is known to regulate the expression of c-Myc to control the proliferation of cancer cells. Therefore, development of small-molecule inhibitors targeting the bromodomain has been widely studied. However, some clinical trials on BRD4 inhibitors have shown its drawbacks such as toxicity including the loss of organ weight. Here, we report the development of the novel and promising scaffold, 1H-indazol-4,7-dione, as a bromodomain inhibitor and synthesized derivatives for the inhibition of binding of bromodomain to acetylated histone peptide. Through this effort, we obtained 6-chloro-5-((2,6-difluorophenyl)amino)-1H-indazole-4,7-dione (5i), which showed a highly potent activity with a half-maximal inhibitory concentration (IC50) of 60 nM. The in vivo xenograft assay confirmed that the 1H-indazol-4,7-dione compound reduced the tumor size significantly. These results show that the 1H-indazol-4,7-dione scaffold is highly potent against bromodomain.

Syntheses of various bis-ortho-alkoxy-para-piperazineanilino-pyrimidines and -triazines of KRCA-0008 analogs are described and their structure-activity-relationship to anaplastic lymphoma kinase (ALK) is discussed. 5-trifluoromethyl-2,4-pyrimidine analog (2) seems to be most potent in both biochemical and cellular assay in this study, however it shows inferior mice xenograft activity to Crizotinib presumably due to its sub-optimal PK parameters. 4,6-disubstituted pyrimidine and 2,4-disubstituted triazine derivatives of KRCA-0008 are less potent or inactive to ALK wt., and this observation is explained with their molecular modeling compared to KRCA-0008.

The piperidine fragment in ceritinib was replaced with diverse aliphatic amines to improve inherent resistance issues of ceritinib. While most of the prepared compounds exhibit as similar in vitro activities as ceritinib, compound 10 shows encouraging activities against wild-type ALK as well as crizotinib-resistant mutants including extremely resistant G1202R mutant with an IC50 of 1.8 nM. Furthermore, pharmacokinetic profiles of 10 is apparently better than that of ceritinib. In murine xenograft studies, compound 10 turns out to be as active as ceritinib, suggesting that further optimization of 10 may lead to clinical candidates overcoming ALK mutant issues.

Ceritinib, an ALK inhibitor, was hurriedly approved by the US FDA last year, and demonstrates impressive results in EML4-ALK positive patients. To get a superior ALK inhibitor, we synthesized several ceritinib derivatives with minor modifications to the phenylpiperidine moiety. Biochemical and cellular assays demonstrated the improved activity of KRCA-386 over that of ceritinib. KRCA-386 has superior inhibitory activity against ALK mutants commonly found in crizotinib-resistant patients. Particularly, KRCA-386 has considerably greater activity than ceritinib against the G1202R mutant, one of the most challenging mutations to overcome. The cell cycle analysis indicates that ALK inhibitors induce G1/S arrest, resulting in apoptosis. The in vivo xenograft data also demonstrate that KRCA-386 is significantly better than ceritinib. KRCA-386 dosed at 25 mpk caused 105% tumor growth inhibition (TGI) compared to 72% TGI with ceritinib dosed at 25 mpk. ( n = 8, P = 0.010) The kinase profiling assay revealed that several kinases, which are known to be critical for tumor growth, are inhibited by KRCA-386, but not by ceritinib. We anticipate that this characteristic of KRCA-386 enhances its in vivo efficacy. In addition, KRCA-386 shows excellent blood brain barrier penetration compared to ceritinib. These results suggest that KRCA-386 could be useful for crizotinib-resistant patients with brain metastases.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2058-y) contains supplementary material, which is available to authorized users.

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor. Since differentiation can attenuate or halt the growth of tumor cells, an image-based phenotypic screening was performed to find out drugs inducing morphological differentiation of GBMs. Bexarotene, a selective retinoid X receptor agonist, showed strong inhibition of neurospheroidal colony formation and migration of cultured primary GBM cells. Bexarotene treatment reduced nestin expression, while significantly increasing glial fibrillary acidic protein (GFAP) expression. The effect of bexarotene on gene expression profile was compared with the activity of all-trans retinoic acid (ATRA), a well-known differentiation inducer. Both drugs largely altered the gene expression pattern into a tumor-ameliorating direction. These drugs increased the gene expression levels of Kruppel-like factor 9 (KLF9), regulator of G-protein signaling 4 (RGS4), growth differentiation factor 15 (GDF15), angiopoietin-like protein 4 (ANGPTL4), and lowered the level of chemokine receptor type 4 (CXCR4). However, transglutaminase 2 (TG2) induction, an adverse effect of ATRA, was much weaker in bexarotene treated primary GBM cells. Consistently, the TG2 enzymatic activity was negligibly affected by bexarotene treatment. It is important to control TG2 overexpression since its upregulation is correlated with tumor transformation and drug resistance. Bexarotene also showed in vivo tumoricidal effects in a GBM xenograft mouse model. Therefore, we suggest bexarotene as a more beneficial differentiation agent than ATRA for GBM.

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.