DW71177: A novel [1,2,4]triazolo[4,3-a]quinoxaline-based potent and BD1-Selective BET inhibitor for the treatment of acute myeloid leukemia.

The bromodomain and extraterminal domain (BET) family proteins recognize acetyl-lysine (Kac) at the histone tail through two tandem bromodomains, i.e., BD1 and BD2, to regulate gene expression. BET proteins are attractive therapeutic targets in cancer due to their involvement in oncogenic transcriptional activation, and bromodomains have defined Kac-binding pockets. Here, we present DW-71177, a potent BET inhibitor that selectively interacts with BD1 and exhibits strong antileukemic activity. X-ray crystallography, isothermal titration calorimetry, and molecular dynamic studies have revealed the robust and specific binding of DW-71177 to the Kac-binding pocket of BD1. DW-71177 effectively inhibits oncogenes comparable to the pan-BET inhibitor OTX-015, but with a milder impact on housekeeping genes. It efficiently blocks cancer-associated transcriptional changes by targeting genes that are highly enriched with BRD4 and histone acetylation marks, suggesting that BD1-selective targeting could be an effective and safe therapeutic strategy against leukemia.

Synthesis and evaluation of tirbanibulin derivatives: a detailed exploration of the structure-activity relationship for anticancer activity.

Tirbanibulin, an FDA-approved microtubule-targeting agent (MTA) introduced in 2020, represents a pioneering treatment for precancerous actinic keratosis. Despite its failure to gain approval as an anticancer agent due to insufficient efficacy, there remains potential value in extending its application into malignancy treatment through tirbanibulin-based derivatives. Tirbanibulin possesses a distinctive dual mechanism of action involving microtubule and Src inhibition, distinguishing it from other MTAs. In spite of its unique profile, exploration of tirbanibulin's structure-activity relationship (SAR) and the development of its derivatives are significantly limited in the current literature. This study addresses this gap by synthesizing various tirbanibulin derivatives and exploring their SAR through modifications in the core amide motif and the eastern benzylamine part. Our results underscore the critical role of the pyridinyl acetamide core structure for optimal cellular potency, with favorable tolerance observed for modifications at the para position of the benzylamine moiety. Particularly noteworthy is the analogue modified with p-fluorine benzylamine, which exhibited favorable in vivo PK profiles. These findings provide crucial insights into the potential advancement of tirbanibulin-based compounds as promising anticancer agents.

A novel IRAK4/PIM1 inhibitor ameliorates rheumatoid arthritis and lymphoid malignancy by blocking the TLR/MYD88-mediated NF-κB pathway.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a pivotal enzyme in the Toll-like receptor (TLR)/MYD88 dependent signaling pathway, which is highly activated in rheumatoid arthritis tissues and activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL). Inflammatory responses followed by IRAK4 activation promote B-cell proliferation and aggressiveness of lymphoma. Moreover, proviral integration site for Moloney murine leukemia virus 1 (PIM1) functions as an anti-apoptotic kinase in propagation of ABC-DLBCL with ibrutinib resistance. We developed a dual IRAK4/PIM1 inhibitor KIC-0101 that potently suppresses the NF-κB pathway and proinflammatory cytokine induction in vitro and in vivo. In rheumatoid arthritis mouse models, treatment with KIC-0101 significantly ameliorated cartilage damage and inflammation. KIC-0101 inhibited the nuclear translocation of NF-κB and activation of JAK/STAT pathway in ABC-DLBCLs. In addition, KIC-0101 exhibited an anti-tumor effect on ibrutinib-resistant cells by synergistic dual suppression of TLR/MYD88-mediated NF-κB pathway and PIM1 kinase. Our results suggest that KIC-0101 is a promising drug candidate for autoimmune diseases and ibrutinib-resistant B-cell lymphomas.

Novel allosteric glutaminase 1 inhibitors with macrocyclic structure activity relationship analysis.

Glutamine-addicted cancer metabolism is recently recognized as novel cancer target especially for KRAS and KEAP1 co-occurring mutations. Selective glutaminase1 (GLS1) inhibition was reported using BPTES which has novel mode of allosteric inhibition. However, BPTES is a highly hydrophobic and symmetric molecule with very poor solubility which results in suboptimal pharmacokinetic parameters and hinders its further development. As an ongoing effort to identify more drug-like GLS1 inhibitors via systematic structure - activity relationship (SAR) analysis of BPTES analogs, we disclose our novel macrocycles for GLS1 inhibition with conclusive SAR analysis on the core, core linker, and wing linker, respectively. Selected molecules resulted in reduction in intracellular glutamate levels in LR (LDK378-resistant) cells which is consistent to cell viability result. Finally, compounds 13 selectively reduced the growth of A549 and H460 cells which have co-occurring mutations including KRAS and KEAP1.

Neurotoxicological Profiling of Paraquat in Zebrafish Model.

Paraquat is a polar herbicide protecting plant products against invasive species, it requires careful manipulation and restricted usage because of its harmful potentials. Exposure to paraquat triggers oxidative damage in dopaminergic neurons and subsequently causes a behavioral defect in vivo. Thereby, persistent exposure to paraquat is known to increase Parkinson's disease risk by dysregulating dopaminergic systems in humans. Therefore, most studies have focused on the dopaminergic systems to elucidate the neurotoxicological mechanism of paraquat poisoning, and more comprehensive neurochemistry including histaminergic, serotonergic, cholinergic, and GABAergic systems has remained unclear. Therefore, in this study, we investigated the toxicological potential of paraquat poisoning using a variety of approaches such as toxicokinetic profiles, behavioral effects, neural activity, and broad-spectrum neurochemistry in zebrafish larvae after short-term exposure to paraquat and we performed the molecular modeling approach. Our results showed that paraquat was slowly absorbed in the brain of zebrafish after oral administration of paraquat. In addition, paraquat toxicity resulted in behavioral impairments, namely, reduced motor activity and led to abnormal neural activities in zebrafish larvae. This locomotor deficit came with a dysregulation of dopamine synthesis induced by the inhibition of tyrosine hydroxylase activity, which was also indirectly confirmed by molecular modeling studies. Furthermore, short-term exposure to paraquat also caused simultaneous dysregulation of other neurochemistry including cholinergic and serotonergic systems in zebrafish larvae. The present study suggests that this neurotoxicological profiling could be a useful tool for understanding the brain neurochemistry of neurotoxic agents that might be a potential risk to human and environmental health.

Discovery and Characterization of a Novel MASTL Inhibitor MKI-2 Targeting MASTL-PP2A in Breast Cancer Cells and Oocytes.

Although microtubule-associated serine/threonine kinase-like (MASTL) is a promising target for selective anticancer treatment, MASTL inhibitors with nano range potency and antitumor efficacy have not been reported. Here, we report a novel potent and selective MASTL inhibitor MASTL kinase inhibitor-2 (MKI-2) identified in silico through a drug discovery program. Our data showed that MKI-2 inhibited recombinant MASTL activity and cellular MASTL activity with IC50 values of 37.44 nM and 142.7 nM, respectively, in breast cancer cells. In addition, MKI-2 inhibited MASTL kinase rather than other AGC kinases, such as ROCK1, AKT1, PKACα, and p70S6K. Furthermore, MKI-2 exerted various antitumor activities by inducing mitotic catastrophe resulting from the modulation of the MASTL-PP2A axis in breast cancer cells. The MKI-2 treatment showed phenocopies with MASTL-null oocyte in mouse oocytes, which were used as a model to validate MKI-2 activity. Therefore, our study provided a new potent and selective MASTL inhibitor MKI-2 targeting the oncogenic MAST-PP2A axis in breast cancer cells.

Rotational Variance-Based Data Augmentation in 3D Graph Convolutional Network.

This work proposes the data augmentation by molecular rotation, with consideration that the protein-ligand binding events are rotation-variant. As a proof-of-concept, known active (i. e., 1-labeled) ligands to human ß-secretase 1 (BACE-1) are rotated for the generation of 0-labeled data, and the rotation-dependent prediction accuracy of 3D graph convolutional network (3DGCN) is investigated after data augmentation. The data augmentation makes the orientation-recognizing ability of 3DGCN improved significantly in the classification task for BACE-1/ligand binding. Furthermore, the data-augmented 3DGCN has a capability for predicting active ligands from a candidate dataset, via improved performance of orientation recognition, which would be applied to virtual drug screening and discovery.

Novel QSAR Models for Molecular Initiating Event Modeling in Two Intersecting Adverse Outcome Pathways Based Pulmonary Fibrosis Prediction for Biocidal Mixtures.

The adverse outcome pathway (AOP) was introduced as an alternative method to avoid unnecessary animal tests. Under the AOP framework, an in silico methods, molecular initiating event (MIE) modeling is used based on the ligand-receptor interaction. Recently, the intersecting AOPs (AOP 347), including two MIEs, namely peroxisome proliferator-activated receptor-gamma (PPAR-γ) and toll-like receptor 4 (TLR4), associated with pulmonary fibrosis was proposed. Based on the AOP 347, this study developed two novel quantitative structure-activity relationship (QSAR) models for the two MIEs. The prediction performances of different MIE modeling methods (e.g., molecular dynamics, pharmacophore model, and QSAR) were compared and validated with in vitro test data. Results showed that the QSAR method had high accuracy compared with other modeling methods, and the QSAR method is suitable for the MIE modeling in the AOP 347. Therefore, the two QSAR models based on the AOP 347 can be powerful models to screen biocidal mixture related to pulmonary fibrosis.

Development and structure-activity relationship study of SHP2 inhibitor containing 3,4,6-trihydroxy-5-oxo-5H-benzo[7]annulene.

SHP2, a non-receptor protein tyrosine phosphatase encoded by PTPN11 gene, plays an important role in the cell growth and proliferation. Activating mutations of SHP2 have been reported as a cause of various human diseases such as solid tumors, leukemia, and Noonan syndrome. The discovery of SHP2 inhibitor can be a potent candidate for the treatment of cancers and SHP2 related human diseases. Several reports on a small molecule targeting SHP2 have published, however, there are limitations on the discovery of SHP2 phosphatase inhibitors due to the polar catalytic site environment. Allosteric inhibitor can be an alternative to catalytic site inhibitors. 3,4,6-Trihydroxy-5-oxo-5H-benzo[7]annulene 1 was obtained as an initial hit with a 0.097 µM of IC50 from high-throughput screening (HTS) study. After the structure-activity relationship (SAR) study, compound 1 still showed the most potent activity against SHP2. Moreover, compound 1 exerted good potency against SHP2 expressing 2D and 3D MDA-MB-468.

DITMD-induced mitotic defects and apoptosis in tumor cells by blocking the polo-box domain-dependent functions of polo-like kinase 1.

DITMD (1, 3- Dioxolo[4,5-g] isoquinolinium 5, 6, 7, 8- tetrahydro- 4- methoxy- 6, 6- dimethyl- 5- [2- oxo- 2- (2-pyridinyl)ethyl] - iodide) is a natural product-like compound with a hydrocotarnine moiety. The aim of this study was to investigate the anticancer effects of DITMD including mitotic arrest, apoptosis, radiosensitization, and to further explore its possible mechanism. DITMD (3-30 µM) induced an obvious cell cycle delay at G2/M transition and apoptosis in HeLa cells. In a validation study, DITMD caused chromosome alignment defects and accumulation of mitotic markers such as polo-like kinase 1, cyclin B1, and phospho-histone H3. DITMD pre-treatment for 11 h also significantly decreased the cells' survival after X-ray irradiation. In mechanism studies, DITMD inhibited the polo-box domain of polo-like kinase 1 but not the conserved kinase domain. Molecular modeling also suggests that DITMD binds at the phosphate group recognition site and inhibits the action on phospho-peptide ligands. In addition, DITMD was analyzed as a PLHSpT competitive inhibitor with an IC50 value of 2.1 µM and exhibited good selectivity against 105 distinct kinases. Taken together, these results indicate that DITMD induced chromosome alignment defects, apoptosis and radio-sensitization, and suggest that one mechanism underlying these anticancer effects involves inhibiting the polo-box domain-dependent functions of polo-like kinase 1.

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.

Identification of new pyrrole alkaloids from the fruits of Lycium chinense.

Three new minor pyrrole alkaloids, 3-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl]pentanedioic acid (1), (2R)-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl]-1-methoxy-1-oxobutanoic acid (2), and methyl (2R)-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]-4-methylpentanoate (3) were isolated from the fruits of Lycium chinense Miller (Solanaceae), along with the known compound, methyl (2R)-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]-3-(phenyl)propanoate (4). The structures of 1-4 were elucidated by analysis of their 1D- and 2D-NMR and HRMS data. The absolute configurations of 2-4, possessing a stereogenic center in each structure, were determined by comparison of their experimental electronic circular dichroism (ECD) with those of calculated ECD values.

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.

A novel aminothiazole KY-05009 with potential to inhibit Traf2- and Nck-interacting kinase (TNIK) attenuates TGF-ß1-mediated epithelial-to-mesenchymal transition in human lung adenocarcinoma A549 cells.

Transforming growth factor (TGF)-ß triggers the epithelial-to-mesenchymal transition (EMT) of cancer cells via well-orchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/ß-catenin. Since EMT-induced motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anti-cancer therapies. Traf2- and Nck-interacting kinase (TNIK) has recently been considered as a first-in-class anti-cancer target molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied. Here, using 5-(4-methylbenzamido)-2-(phenylamino)thiazole-4-carboxamide (KY-05009) with TNIK-inhibitory activity, its efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009 in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay (Ki, 100 nM). In A549 cells, KY-05009 significantly and strongly inhibited the TGF-ß-activated EMT through the attenuation of Smad and non-Smad signaling pathways, including the Wnt, NF-κB, FAK-Src-paxillin-related focal adhesion, and MAP kinases (ERK and JNK) signaling pathways. Continuing efforts to identify and validate potential therapeutic targets associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based disorders, such as cancer metastasis and fibrosis.

Biological evaluation of tanshindiols as EZH2 histone methyltransferase inhibitors.

EZH2 is the core subunit of Polycomb repressive complex 2 catalyzing the methylation of histone H3 lysine-27 and closely involved in tumorigenesis. To discover small molecule inhibitors for EZH2 methyltransferase activity, we performed an inhibitor screen with catalytically active EZH2 protein complex and identified tanshindiols as EZH2 inhibitors. Tanshindiol B and C potently inhibited the methyltransferase activity in in vitro enzymatic assay with IC50 values of 0.52µM and 0.55µM, respectively. Tanshindiol C exhibited growth inhibition of several cancer cells including Pfeiffer cell line, a diffuse large B cell lymphoma harboring EZH2 A677G activating mutation. Tanshindiol treatment in Pfeiffer cells significantly decreased the tri-methylated form of histone H3 lysine-27, a substrate of EZH2, as revealed by Western blot analysis and histone methylation ELISA. Based on enzyme kinetics and docking studies, we propose that tanshindiol-mediated inhibition of EZH2 activity is competitive for the substrate S-adenosylmethionine. Taken together, our findings strongly suggest that tanshindiols possess a unique anti-cancer activity whose mechanism involves the inhibition of EZH2 activity and would provide chemically valuable information for designing a new class of potent EZH2 inhibitors.

ALK inhibitors of bis-ortho-alkoxy-para-piperazinesubstituted-pyrimidines and -triazines for cancer treatment.

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.

A novel assessment of nefazodone-induced hERG inhibition by electrophysiological and stereochemical method.

Nefazodone was used widely as an antidepressant until it was withdrawn from the U.S. market in 2004 due to hepatotoxicity. We have investigated methods to predict various toxic effects of drug candidates to reduce the failure rate of drug discovery. An electrophysiological method was used to assess the cardiotoxicity of drug candidates. Small molecules, including withdrawn drugs, were evaluated using a patch-clamp method to establish a database of hERG inhibition. Nefazodone inhibited hERG channel activity in our system. However, nefazodone-induced hERG inhibition indicated only a theoretical risk of cardiotoxicity. Nefazodone inhibited the hERG channel in a concentration-dependent manner with an IC50 of 45.3nM in HEK-293 cells. Nefazodone accelerated both the recovery from inactivation and its onset. Nefazodone also accelerated steady-state inactivation, although it did not modify the voltage-dependent character. Alanine mutants of hERG S6 and pore region residues were used to identify the nefazodone-binding site on hERG. The hERG S6 point mutants Y652A and F656A largely abolished the inhibition by nefazodone. The pore region mutant S624A mildly reduced the inhibition by nefazodone but T623A had little effect. A docking study showed that the aromatic rings of nefazodone interact with Y652 and F656 via π-π interactions, while an amine interacted with the S624 residue in the pore region. In conclusion, Y652 and F656 in the S6 domain play critical roles in nefazodone binding.

Novel 2,4-dianilino-5-fluoropyrimidine derivatives possessing ALK inhibitory activities.

A new series of 2,4-dianilino-5-fluoropyrimidine derivatives were designed and synthesized and their anaplastic lymphoma kinase (ALK) inhibitory activities were evaluated by biochemical and cell-based assays in order to discover a new ALK inhibitor. Most compounds synthesized showed good inhibitory activities against ALK and good cytotoxic activities in H3122 cell line. The best compound 6f showed good activity against wild-type ALK along with crizotinib-resistant mutant ALK, and it showed 6 times better activity in cell-based assay than crizotinib. Some SAR studies were performed by the comparisons of the activities between 6 and the designed-synthesized compounds.

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.