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Background: PHI-501 has been developed as a novel inhibitor of NRAS mutated acute myeloid leukemia. Big data and artificial intelligence (AI)-based drug discovery platform and cell-based investigation identified PHI-501 repurposable against melanoma as a novel pan-RAF inhibitor. BRAF activated by the common V600E and other mutations, as well as by upstream NRAS mutation are frequent in malignant melanoma. Regardless of the therapeutic effect of class I BRAF inhibitors such as vemurafenib, dabrafenib and encorafenib, melanoma patients with BRAF non-V600/RAS mutations remain limited response. In this study, we evaluated PHI-501 to potently inhibit the tumor cell growth and overcome the limited response in melanoma. Methods: The growth inhibitory activity of PHI-501 was determined using CellTiter-Glo luminescent assay in a panel of cancer cell lines. The effect of PHI-501 on anchorage independent growth and cell proliferation compared to vemurafenib or belvarafenib (pan-RAF inhibitor) were tested in A375 (BRAFV600E), C8161 (BRAFG464E) and SK-MEL-2 (NRASQ61R) melanoma cell lines by soft agar assay, western blot and FACS analysis. To measure cell motility in a controlled environment, cells were cultured in monolayer followed by a wound-healing assay in a treatment with 0.1 µM of each compound for 12 hours. Anti-tumor activities of PHI-501 were evaluated in A375 or C8161 xenograft mouse in vivo model. Results: PHI-501 showed 3- to 119-fold more effective growth inhibition than other RAF inhibitors, vemurafenib and belvarafenib, in SK-MEL-2 (GI50: 0.15 µM for PHI-501 vs 7.13 µM and 0.37 µM, respectively) and C8161 (GI50: 0.33 µM for PHI-501 vs 39.56 µM and 2.10 µM, respectively) melanoma cell lines, and did not show a potential to paradoxical activation. PHI-501 induced melanoma cell apoptosis more efficiently than other RAF inhibitors, as shown in increased G0/G1-arrested and annexin V-positive cell population and abundant cleavage of PARP1 after drug treatment. In the cell wound-healing assay, PHI-501 exhibited promising antimetastatic potential via inhibiting migration of all tested melanoma cells by up to 70%, unlike other RAF inhibitors. Anti-tumor activity of PHI-501 was dose-proportional in A375 xenograft model. Tumor growth was significantly reduced in C8161 xenograft mice treated with PHI-501 (TGI: 75.0%), compared with vehicle and vemurafenib (TGI: 12.3%). Conclusion: PHI-501, a novel pan-RAF inhibitor, has potent oral anti-tumor activity. Melanoma cells harboring BRAF non-V600/NRAS or BRAF common V600E mutations exhibited significantly reduced proliferation, increased apoptosis and inhibited migration upon treatment with PHI-501. The results of this study suggest that PHI-501 has a potential to overcome the limited response in the treatment of melanoma, and warrant evaluation of PHI-501 as a single agent to treat both BRAF-and NRAS-mutated metastatic melanoma. Citation Format: Namkyeong Kim, Jung Hee Park, Ky-Youb Nam, June H-J Han, Kyu-Tae Kim, Sandip Sengupta, Jeong Hyeok Yoon, Taebo Sim. PHI-501, a novel and potent pan-RAF inhibitor in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1627.

Background: PHI-501 is a newly developed, highly effective, and orally accessible dual inhibitor for pan-RAF and discoidin domain receptor (DDR) that is a collagen-activated receptor tyrosine kinase. Mutations of the BRAF and NRAS are the most common genetic alterations in melanoma and consequently lead to activation of mitogen activated protein kinase (MAPK) signaling. The major drawback of melanoma treatment is the innate and acquired drug resistance to MAPK inhibitors. In this study, we investigated the inhibitory effects of a novel compound, PHI-501 on resistance to known BRAF and MEK inhibitors in melanoma. Methods: After compounds treatment, the growth-inhibiting activity was assessed using the CCK test. Inhibition of RAF/DDR1 pathway signaling was evaluated by western blotting. Using annexin-V/propidium iodide-stained cells and flow cytometry, apoptosis induction was assessed. To quantify cell mobility, monolayer cells were subjected to a wound-healing assay. Long-term therapy with dabrafenib (BRAF inhibitor), cobimetinib or trametinib (MEK inhibitor) led to the establishment of the drug-resistant SK-MEL-3 (BRAF V600E) and SK-MEL-30 (NRAS Q61K) melanoma cell line. Results: PHI-501 demonstrated potent growth inhibition (GI50 Conclusion: Melanoma cells resistant to RAF or MEK-targeted treatments or harboring NRAS mutation exhibited strong antiproliferative activity when treated with PHI-501, a highly potent pan-RAF/DDR dual inhibitor. The results of this study suggest that PHI-501, as a single agent, has the potential to overcome the restricted response in the treatment of melanoma. Citation Format: Sue Min Kim, Jung Hee Park, Ky-Youb Nam, June H-J Han, Kyu-Tae Kim, Jeong Hyeok Yoon, Sandip Sengupta, Taebo Sim, Sang Joon Shin. PHI-501, a novel pan-RAF/DDRs dual kinase inhibitor, overcomes BRAF or MEK inhibitor resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 411.

More than 80% of colorectal cancer patients have adenomatous polyposis coli (APC) mutations, which induce abnormal WNT/β-catenin activation. Tankyrase (TNKS) mediates the release of active β-catenin, which occurs regardless of the ligand that translocates into the nucleus by AXIN degradation via the ubiquitin-proteasome pathway. Therefore, TNKS inhibition has emerged as an attractive strategy for cancer therapy. In this study, we identified pyridine derivatives by evaluating in vitro TNKS enzyme activity and investigated N-([1,2,4]triazolo[4,3-a]pyridin-3-yl)-1-(2-cyanophenyl)piperidine-4-carboxamide (TI-12403) as a novel TNKS inhibitor. TI-12403 stabilized AXIN2, reduced active β-catenin, and downregulated β-catenin target genes in COLO320DM and DLD-1 cells. The antitumor activities of TI-12403 were confirmed by the viability of the colorectal cancer cells and its lack of visible toxicity in DLD-1 xenograft mouse model. In addition, combined 5-FU and TI-12403 treatment synergistically inhibited proliferation to a greater extent than that in a single drug treatment. Our observations suggest that TI-12403, a novel selective TNKS1 inhibitor, may be a suitable compound for anticancer drug development.

Radiotherapy is one of the most common treatments for cancer, but radioresistance and injury to normal tissue are considered major obstacles to successful radiotherapy. Thus, there is an urgent need to develop radiosensitizers to improve the therapeutic outcomes of radiotherapy in cancer patients. Our previous efforts to identify novel radiosensitizers, using high-throughput screening targeting p53 and Nrf2 revealed a promising N-phenylpyrimidin-2-amine (PPA) lead compound. In the present study, 17 derivatives of this lead compound were examined, and it was found that 4-(4-fluorophenyl)-N-(4-nitrophenyl)-6-phenylpyrimidin-2-amine (PPA5), 4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)-3-methoxy-N-methyl -benzamide (PPA13), 4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA14), 4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA15), and 4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)-N-methylbenzamide (PPA17) inhibited cell viability by more than 50%, with a marked increase in the proportion of cells arrested at the G2/M phase of cell cycle. Among these compounds, PPA15 markedly increased the sub-G1 cell population and increased the levels of cyclin B1 and the phosphorylation levels of cyclin-dependent kinase (CDK) 1. Combined treatment with radiation and PPA14 or PPA15 significantly decreased clonogenic survival. An in vitro kinase assay revealed that PPA15 inhibited multiple CDKs involved in cell cycle regulation. Compared with drug or radiation treatment alone, combined treatment with PPA15 and radiation resulted in the suppression of A549 tumor growth in mice by 59.5% and 52.7%, respectively. Treatment with PPA15 alone directly inhibited tumor growth by 25.7%. These findings suggest that the novel pan CDK inhibitor, PPA15, may be a promising treatment to improve the effectiveness of radiotherapy for the treatment of cancer. SIGNIFICANCE STATEMENT: Several inhibitors of CDK have been successfully evaluated in combination with other chemotherapeutics in clinical trials, but negative side effects have partially restricted their clinical use. In this study, we identified a novel pan-CDK inhibitor to increase radiosensitivity, and we hope this work will encourage the development of promising small-molecule radiosensitizers.

X-linked inhibitor of apoptosis protein (XIAP) is an important regulator of cancer cell survival whose BIR3 domain (XIAP-BIR3) recognizes the Smac N-terminal tetrapeptide sequence (AVPI), making it an attractive protein-protein interaction (PPI) target for cancer therapies. We used the fragment molecular orbital (FMO) method to study the binding modes and affinities between XIAP-BIR3 and a series of its inhibitors (1–8) that mimic the AVPI binding motif; the inhibitors had common interactions with key residues in a hot spot region of XIAP-BIR3 (P1–P4 subpockets) with increased binding affinity mainly attributed to specific interactions with the P1 and P4 subpockets. Based on the structural information from FMO results, we proposed a novel XIAP natural product inhibitor, neoeriocitrin 10, which was derived from our preciously reported XIAP-BIR3 inhibitor 9, can be used as a highly potent candidate for XIAP-BIR3 inhibition. We also performed pair interaction energy decomposition analysis to investigate the binding energies between specific binding residues and individual ligands, showing that the novel natural product neoeriocitrin 10 had a higher binding affinity than epicatechin gallate 9. Molecular docking and dynamics simulations were performed to explore the mode of binding between 10 and XIAP-BIR3, demonstrating that 10 binds more strongly to the P1 and P4 pockets than 9. Overall, we present a novel natural product, neoeriocitrin 10, and demonstrate that the FMO method can be used to identify hot spots in PPIs and design new compounds for XIAP inhibition., Graphical Abstract Unlabelled Image

Purpose: Development of the first-in-class inhibitor of NRAS mutated acute myeloid leukemia. PHI-501 has potent activity against receptor tyrosine kinases and intracellular kinases related to STAT, AKT, and ERK signaling. Description: Preclinical testing of PHI-501 for efficacy against NRAS mutations both in vitro and in vivo xenografts. Summary: Mutations in the RAS genes (including KRAS, NRAS, and HRAS) are discovered in about 30% of all tumors. KRAS is the most frequently mutated gene in cancers found in pancreatic, colon, and lung, while NRAS mutations around the hot spots at codons 12, 13, and 61 are more common in AML (10.3%). PHI-501 is a novel small molecule inhibitor intended for the treatment of AML in patients expressing the N-RAS activating mutation. PHI-501 presented higher selectivity within a panel of 355 protein kinases. PHI-501 exerts their anti-proliferative effects by inducing apoptosis and G0-G1 arrest and significantly induce apoptotic markers (cleaved PARP and caspase 3) in both Ba/F3-NRAS-G12D and OCI-AML3 cells. The results of Western blot analysis showed that PHI-501 attenuate phosphorylation of p70S6K1, AKT, and p38 in OCI-AML3 After 21 days of the treatment in subcutaneous OCI-AML3 human AML xenograft model, PHI-501 showed significant inhibition of tumor growth (TGI) in a dose-dependent manner. Compared to the vehicle group, the 40 mg/kg group demonstrated a 74.0% lower average tumor volume (P Conclusion: The preclinical evaluation of PHI-501, a novel N-RAS inhibitor, showed clear evidence of anticancer activity for AML and improved efficacy in both in vitro and in vivo models. Consequently, PHI-501 is a new potent multi-kinase inhibitor with characteristics that warrant entry into human trials for the treatment of AML in patients expressing the N-RAS activating mutation. Citation Format: Ky-Youb Nam, Jung Hee Park, Kyung Ah Kim, Inje Shin, June H-J Han, Kyu-Tae Kim, Jeong Hyeok Yoon, Hanna Cho, Seung-Hye Choi, Sandip Sengupta, Taebo Sim. PHI-501, a potent and novel inhibitor of NRAS mutated acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5495.

The prevalence of a novel β-coronavirus (SARS-CoV-2) was declared as a public health emergency of international concern on 30 January 2020 and a global pandemic on 11 March 2020 by WHO. The spike glycoprotein of SARS-CoV-2 is regarded as a key target for the development of vaccines and therapeutic antibodies. In order to develop anti-viral therapeutics for SARS-CoV-2, it is crucial to find amino acid pairs that strongly attract each other at the interface of the spike glycoprotein and the human angiotensin-converting enzyme 2 (hACE2) complex. In order to find hot spot residues, the strongly attracting amino acid pairs at the protein–protein interaction (PPI) interface, we introduce a reliable inter-residue interaction energy calculation method, FMO-DFTB3/D/PCM/3D-SPIEs. In addition to the SARS-CoV-2 spike glycoprotein/hACE2 complex, the hot spot residues of SARS-CoV-1 spike glycoprotein/hACE2 complex, SARS-CoV-1 spike glycoprotein/antibody complex, and HCoV-NL63 spike glycoprotein/hACE2 complex were obtained using the same FMO method. Following this, a 3D-SPIEs-based interaction map was constructed with hot spot residues for the hACE2/SARS-CoV-1 spike glycoprotein, hACE2/HCoV-NL63 spike glycoprotein, and hACE2/SARS-CoV-2 spike glycoprotein complexes. Finally, the three 3D-SPIEs-based interaction maps were combined and analyzed to find the consensus hot spots among the three complexes. As a result of the analysis, two hot spots were identified between hACE2 and the three spike proteins. In particular, E37, K353, G354, and D355 of the hACE2 receptor strongly interact with the spike proteins of coronaviruses. The 3D-SPIEs-based map would provide valuable information to develop anti-viral therapeutics that inhibit PPIs between the spike protein of SARS-CoV-2 and hACE2.

The p53 tumor suppressor plays critical roles in cell cycle regulation and apoptotic cell death, with its activation capable of sensitizing cancer cells to radiotherapy or chemotherapy. To identify small molecules that induce apoptosis via increased p53 transcriptional activity, we used a novel in-house library containing 96 small-molecule compounds. Using a cell-based screening method with a p53-responsive luciferase-reporter assay system involving benzoxazole derivatives, we found that AU14022 administration significantly increased p53 transcriptional activity in a concentration-dependent manner. Treatment with AU14022 increased p53 protein expression, p53 Ser15 phosphorylation, p53-mediated expression of downstream target genes, and apoptosis in p53-wild-type HCT116 human colon cancer cells, but not in p53-knockout HCT116 cells. Additionally, p53-wild-type HCT116 cells treated with AU14022 exhibited mitochondrial dysfunction, including modulated expression of B-cell lymphoma-2 family proteins and cytochrome c release. Combination treatment with AU14022 and ionizing radiation (IR) synergistically induced apoptosis as compared with IR or AU14022 treatment alone, with further investigation demonstrating that cell cycle progression was significantly arrested at the G2/M phase following AU14022 treatment. Furthermore, in a mouse p53-wild-type HCT116 colon cancer xenograft model, combined treatment with AU14022 and IR inhibited tumor growth more effectively than radiation alone. Therefore, AU14022 treatment induced apoptosis through p53-mediated cell cycle arrest involving mitochondrial dysfunction, leading to enhanced radiosensitivity in colon cancer cells. These results provide a basis for further assessments of AU14022 as a promising anticancer agent.

Background: The FMS-like tyrosine kinase 3 (FLT3) is mutated in approximately 30% of acute myeloid leukemia (AML) patients either by internal tandem duplication (ITD) or by point mutations in the tyrosine kinase domain (TKD). PHI-101 is a Type I FLT3 inhibitor developed to overcome resistance in AML. PHI-101 showed potent cellular activity in vitro and in vivo for both FLT3 ITD mutations and a wide variety of FLT3 point mutations in TKD. In preclinical studies utilizing primary human FLT3-ITD leukemia cells, PHI-101 showed increased anti-leukemic activity compared to gilteritinib based on increased survival in mice transplanted with these samples. The Phase 1a and Phase 1b global clinical trial with PHI-101 (NCT04842370) is currently underway to assess the overall safety and efficacy of PHI-101 in refractory and relapsed AML. Study Design and Methods: The overall design of the AML clinical trial consists of two parts, phase 1a, the dose-escalation, and phase 1b, the dose-expansion trial utilizing oral PHI-101 tablets. Up to 5 dose levels are planned for phase 1a and a single subject enrolled per dose level until the occurrence of at least one subject with more than Grade 2 toxicity according to CTCAE5.0 criteria during the 28-day evaluation period. The dose escalation will then convert to a standard 3+3 scheme, with 3 to 6 subjects per dose level cohort over the 28-day cycle. If there are no dose-limiting toxicities (DLT), dose escalation to the next higher dose levels will proceed upon the recommendation of the Safety Monitoring Committee. Subjects with FLT3 mutations or FLT3 wild-type will be enrolled in the dose-escalation cohort, and blood samples are collected for the primary endpoint pharmacokinetics (PK) and for the exploratory pharmacodynamic (PD) endpoint evaluation, including plasma inhibitory assay and biomarker analysis. Results: The Phase 1a clinical trial of PHI-101 was initiated in June 2020 at a daily dose of 40mg for level 1. As of July 31, 2021, 8 patients with relapsed or refractory AML have been enrolled in the trial, all of whom had prior anti-leukemic treatments with intensive chemotherapy, hypomethylating agents and/or other FLT3 inhibitors. To date, 5 enrolled patients were available for safety assessment at three dose levels and have not experienced any dose-limiting toxicities (DLTs) from repeating doses of the 28-day DLT-assessment window. The leukemic blasts in marrow or peripheral blood were dramatically reduced by up to 98% with one cycle (28 days). The pharmacokinetic data of plasma PHI-101 varied in a dose-proportional manner and peak plasma concentrations (Cmax) were reached between 4 and 6 hours after once-daily dosing. The pharmacodynamic evaluation by plasma inhibitory assay showed that plasma from patients receiving at daily doses of PHI-101 inhibited more than 90% of the phosphorylation of the FLT3-ITD receptor. PHI-101 was well tolerated with these dose levels. This study is currently recruiting FLT3 mutation or wild-type AML patients at multiple sites in Korea and Australia. Conclusion: PHI-101 is a next-generation FLT3 inhibitor that showed a potent anti-leukemic activity and improved efficacy in primary AML samples harboring FLT3/ITD and FLT3/TKD mutations in preclinical studies. The current analysis of this trial indicated that PHI-101 is a very effective FLT3 inhibitor for both refractory and relapsed AML patients, including those that have relapsed on other FLT3 TKI. The assessments of safety, tolerability, and PK of PHI-101 to determine the recommended dose for expansion are ongoing in the Phase 1a clinical trial. Disclosures Lee: Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board; AbbVie: Honoraria, Other: Advisory board; Korean Society of Hematology: Membership on an entity's Board of Directors or advisory committees. Im: Pharos iBio Co., Ltd.: Current Employment. Nam: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Kim: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Han: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Yoon: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Small: Pharos iBio Co., Ltd.: Consultancy, Other: Scientific Advisory Board. The arrangement has been reviewed and approved by Johns Hopkins University in accordance with its conflict-of-interest policies, Research Funding; InSilico Medicine: Other: Scientific Advisory Board.

Purpose: Development of the potent and selective checkpoint kinase 2 (CHK2) inhibitor to overcome limiting clinical utility of poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Experimental Procedure: Preclinical evaluation of PHI-101 for cellular and molecular potency in ovarian and breast cancer cell lines and patient derived primary cells; it includes biochemical binding assay, cellular assays, animal efficacy studies, combination study, signaling pathway effect examination, and cell cycle analysis. Summary: CHK2 is a serine/threonine kinase and a cell cycle checkpoint regulator involved in the ATM-mediated DNA repair upon replication blocks and DNA damage. It has been proposed that Chk2 functions as a barrier to tumorigenesis by maintaining genomic stability, and this DNA damage induction is thought to prevent or delay genetic instability and tumorigenesis. The inhibition of CHK2 is a promising approach to achieve synthetic lethality of cancer cells when combined with PARP1 inhibitors. Solid cancer indications of PHI-101 was identified by the Chemiverse Network module which is an AI and Big data-based in-house drug discovery platform. Biochemical kinase assays for PHI-101 showed stronger affinity to CHK2 over CHK1 more than 5-fold. PHI-101 treatment of ovarian and breast cancer cells for 72 hrs elicit a synergistic lethal response in combination with PARP1 inhibitor Olaparib regardless of functional BRCA and P53 in the cells. PHI-101 also potentiates a countermeasure to dose-limiting toxicity triggered by genotoxic agents such as cisplatin and topotecan. The present results from in vivo and in vitro preclinical testing do demonstrate that PHI-101 is a highly potent inhibitor of CHK2 and may exert mono- and combinational therapeutic activity in ovarian and breast cancer model. Conclusion: The preclinical evaluation of PHI-101, a novel CHK2 inhibitor, showed clear evidence of anticancer activity for refractory ovarian and breast cancer cells and improved efficacy in both in vitro and in vivo models. Consequently, PHI-101 is currently under investigation in Phase 1 clinical trials for relapsed or refractory ovarian cancer patients. Citation Format: June H-J Han, Kyu-Tae Kim, Jeejin Im, Sojung Park, Min Kyung Choi, Inki Kim, Ky-Youb Nam, JeongHyeok Yoon. PHI-101, a potent and novel inhibitor of CHK2 in ovarian and breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1461.

Aims Proprotein convertase subtilisin/kexin type-9 (PCSK9), a molecular determinant of low-density lipoprotein (LDL) receptor (LDLR) fate, has emerged as a promising therapeutic target for atherosclerotic cardiovascular diseases. However, the precise mechanism by which PCSK9 regulates the internalization and lysosomal degradation of LDLR is unknown. Recently, we identified adenylyl cyclase-associated protein 1 (CAP1) as a receptor for human resistin whose globular C-terminus is structurally similar to the C-terminal cysteine-rich domain (CRD) of PCSK9. Herein, we investigated the role of CAP1 in PCSK9-mediated lysosomal degradation of LDLR and plasma LDL cholesterol (LDL-C) levels. Methods and results The direct binding between PCSK9 and CAP1 was confirmed by immunoprecipitation assay, far-western blot, biomolecular fluorescence complementation, and surface plasmon resonance assay. Fine mapping revealed that the CRD of PCSK9 binds with the Src homology 3 binding domain (SH3BD) of CAP1. Two loss-of-function polymorphisms found in human PCSK9 (S668R and G670E in CRD) were attributed to a defective interaction with CAP1. siRNA against CAP1 reduced the PCSK9-mediated degradation of LDLR in vitro. We generated CAP1 knock-out mice and found that the viable heterozygous CAP1 knock-out mice had higher protein levels of LDLR and lower LDL-C levels in the liver and plasma, respectively, than the control mice. Mechanistic analysis revealed that PCSK9-induced endocytosis and lysosomal degradation of LDLR were mediated by caveolin but not by clathrin, and they were dependent on binding between CAP1 and caveolin-1. Conclusion We identified CAP1 as a new binding partner of PCSK9 and a key mediator of caveolae-dependent endocytosis and lysosomal degradation of LDLR.

We describe the structure-based design and synthesis of N-(2,6-difluorophenyl)-3-(9H-purin-6-yl)pyridine-2-amine derivatives as a selective pan-RAF kinase inhibitor. The synthesized compounds showed highly potent and specific inhibition of the BRAFV600E mutant cell line. Among them, N-(3-((3-(9H-purin-6-yl)pyridine-2-yl)amino)-2,4-difluorophenyl)furan-3-sulfonamide (4b) exhibited the most potent inhibitory activities against protein kinase enzymes BRAFV600E, BRAFWT, and CRAF (IC50 of 2, 2, and 1 nM, respectively) and a mutant cell line bearing a BRAFV600E mutation, A375P (GI50 of 7 nM).

Rab GTPases regulate various types of intracellular membrane trafficking in all eukaryotes. Since Rab27a and its multiple effectors are involved in exocytosis of lysosome-related organelles and play a major role in malignancy, compounds targeting Rab27a could be likely used to inhibit invasive growth and tumor metastasis. Thus, we designed and synthesized several compounds based on the previously reported Rab27a-targeting synthetic compounds identified by virtual screening, and investigated their anti-metastatic effects in MDA-MB231 and A375 cells. Among the synthesized compounds, (E)-N-(3-chlorophenyl)-6-(2-(3,4-dihydroxy benzylidene)hydrazinyl)pyridine-3-sulfonamide (3d) and (E)-N-benzyl-6-(2-(3,4-dihydroxy benzylidene)hydrazinyl)-N-methylpyridine-3-sulfonamide (3f) significantly inhibited the invasiveness of both tumor cell lines. Compounds 3d and 3f also decreased the levels of signature extracellular matrix marker proteins (fibronectin, collagen, and α-smooth muscle actin) and representative mesenchymal cell markers (N-cadherin and vimentin). Taken together, our results suggest that novel sulfonamide analogs have anti-metastatic activity in breast and melanoma cancer cell lines and may be used as therapeutic agents to treat malignant cancer.

Background: The zoonotic transmission of highly pathogenic avian influenza viruses and the global pandemic of H1N1 influenza in 2009 signified the need for a wider coverage of therapeutic options for the control of influenza. Methods: An in-house compound library was screened using a cytopathic effect inhibition assay. Selected hits were then tested in vivo and used as a core skeleton for derivative synthesis. Results: The hit compound (BMD-2601505) was effective [50% effective concentration (EC50) of 60-70 μM] in reducing the death rate of cells infected with human influenza A and B viruses as well as avian influenza A virus. Furthermore, BMD-2601505 reduced the weight loss and increased the survival after lethal infection. The compound was further modified to enhance its antiviral potency. Results show that one derivative with bromobenzene moiety was most effective (EC50 of 22-37 μM) against the influenza viruses tested. Conclusion: We identified a small benzamide compound exhibiting antiviral activity against influenza viruses. The results warrant further evaluation of antiviral activities against drug-resistant influenza isolates.

Background: FMS-like tyrosine kinase 3 (FLT3), a member of the class III receptor tyrosine kinase family, plays a pivotal role in regulating cell growth and differentiation of hematopoietic cells. FLT3 is mutated in approximately 30% of AML patients either by internal tandem duplication (ITD) within the juxtamembrane portion or by point-mutations in the kinase domain (TKD). Thus, FLT3 inhibitors including quizartinib (AC220), midostaurin (PKC412) and gilteritinib (ASP2215) have been developed and undergone clinical testing for the treatment of AML. However, patients frequently relapse due to intrinsic and extrinsic resistance to these FLT3 inhibitors in spite of the initial clinical efficacy. We have developed a potent third-generation FLT3 inhibitor, PHI-101, capable of overcoming some of these mechanisms of resistance. Methods: Biochemical kinase assays for PHI-101 have been performed on 9 different FLT3 mutants and wild type FLT3. Cellular potencies of PHI-101 have also been assessed using various patient-derived AML cells as well as MV4-11, MOLM14 and BaF3 cell lines transformed with human FLT3 mutants including single mutations [FLT3(ITD), FLT3(D835Y)], double or triple mutations [FLT3(ITD/D835Y), FLT3(ITD/F691L), FLT3(ITD/F691L/D835Y)]. In order to evaluate in vivo efficacies of oral administration of PHI-101, xenograft mouse models and in vivo bioluminescence imaging have been utilized. Results: PHI-101 possessed excellent enzymatic potencies against FLT3 potential resistant mutants such as ITD/D835V and the gatekeeper ITD/F691L mutation, as well as against FLT3 single activating mutants that include ITD, D835V, D835H, and D835Y. PHI-101 inhibited the phosphorylation of FLT3 and downstream STAT5 and ERK1/2 more effectively than quizartinib and gilteritinib at equivalent doses. Moreover, oral administration of PHI-101 induced tumor regression in the xenograft mouse models developed by injection of BaF3 cells transformed with FLT3-ITD, FLT3-TKD or FLT3-ITD/TKD mutants in a dose-dependent fashion with no appreciable toxicities. In luciferase-bearing blood circulating mouse models with FLT3-double mutants (ITD/D835Y and ITD/F691L) or triple mutants (ITD/D835Y/F691L), oral administration (30 mpk, QD) of PHI-101 diminished more than 89% of bioluminescent intensity and reduced leukemic burden. PHI-101 also showed increased efficacy in extending the lifespan of xenograft mice compared to quizartinib. PHI-101 strongly suppressed proliferation and induced apoptosis in primary AML samples harboring FLT3/ITD and FLT3/TKD mutations with little effect on wild-type FLT3 samples. Conclusion: PHI-101, an orally bioavailable novel small molecule, is a potent third-generation FLT3 inhibitor able to overcome resistance to several resistance mutations based on in vitro and in vivo experiments. PHI-101 possesses excellent in vitro and in vivo activities against not only FLT3 single activating mutations (ITD or TKD mutants) but also FLT3 double (ITD/D835Y or ITD/F691L) and triple (ITD/D835Y/F691L) resistant mutations with no pronounced toxicities. Preclinical evaluation of PHI-101 showed clear evidence of antileukemic activity and improved efficacy in both in vitro and in vivo models. PHI-101 is currently under investigation in first-in-human clinical trials with relapsed or refractory AML patients. Disclosures Nam: Pharos I&BT Co., Ltd.: Current equity holder in private company. Im:Pharos I&BT Co., Ltd.: Current Employment. Han:Pharos I&BT Co., Ltd.: Current equity holder in private company. Kim:Pharos I&BT Co., Ltd.: Current Employment. Yoon:Pharos I&BT Co., Ltd.: Current equity holder in private company. Cho:Pharos I&BT Co., Ltd.: Research Funding. Choi:Pharos I&BT Co., Ltd.: Research Funding. Young:Pharos I&BT Co., Ltd.: Research Funding. Nguyen:Pharos I&BT Co., Ltd.: Research Funding. Zhu:Pharos I&BT Co., Ltd.: Research Funding. Li:Pharos I&BT Co., Ltd.: Research Funding. Small:Pharos I&BT Co., Ltd.: Consultancy, Research Funding. Sim:Pharos I&BT Co., Ltd.: Research Funding.

Purpose: Develop a next generation FLT3 TKI to overcome previous resistance mechanisms. Description: Preclinical testing of PHI-101 for efficacy against FLT3 mutations both in vitro and in vivo in cell lines and primary FLT3 mutant leukemia samples. Summary: Mutations in the FLT3 gene are common in AML, occurring in approximately 1/3 of patients and result in poor prognosis for AML patients with FLT3 internal tandem duplication (ITD) mutations. Thus, it is a well-validated target for the development of tyrosine kinase inhibitors (TKI). A number of FLT3 TKI have been developed and the latest generation have proven increasingly successful at achieving thorough inhibition of FLT3 kinase activity in vivo, a necessary requirement for clinical efficacy against FLT3 mutant AML. Major reasons for the failure of some FLT3 TKI to achieve thorough inhibition include tight binding to alpha 1 acid glycoprotein (AGP) resulting in inadequate free levels of the drug and selection for resistance mutations within FLT3 itself. Resistance mutations within FLT3 have included those at D835 and F691 among others. We present here the initial preclinical data on a new FLT3 TKI, PHI-101, developed to overcome some of these mechanisms of resistance. PHI-101 is a potent small molecule inhibitor of both FLT3/ITD and FLT3/D835Y mutations with an IC50 of 5 nM and 11.1 nM, respectively, in cell-based assays. It is active against a number of FLT3/ITD resistance mutations including FLT3/ITD/A627T, FLT3/ITD/N676D, FLT3/ITD/F691L, with IC50s of 0.1nM, 2.3nM, 14.6nM, respectively. It is also active against FLT3 kinase domain (FLT3/KD) resistance mutations, with IC50s against FLT3/D835A and FLT3/840GS of 5.3nM and 9.6nM, respectively. Furthermore, it is active against the FLT3/ITD/KD and FLT3/ITD/F691 resistance mutations. We also demonstrate its effectiveness in inhibiting FLT3 activity by Western blot analysis. Importantly, when analyzed in the human plasma inhibitory assay (PIA), its IC50 shifts only 8.5-fold, unlike several other FLT3 TKI with shifts of >200-fold, rendering them unable to sufficiently inhibit FLT3 mutations in vivo. PHI-101 monotherapy induces dose-dependent regression of tumor growth measured with bioluminescence in FLT3-ITD mutant xenograft models. In addition, PHI-101 significantly inhibits proliferation and induces apoptosis in primary AML samples expressing FLT3/ITD and FLT3/KD mutations, but not wild-type FLT3. These effects suggest it is selective for primary AML with FLT3 mutations, comparable to that of Giltertinib, the newly-approved FLT3 TKI. PHI-101 will soon undergo first-in-human clinical trials in FLT3 mutant AML patients. Conclusion: PHI-101 is a new FLT3 TKI with characteristics that warrant entry into human trials. Citation Format: Ky-Youb Nam, Jeejin Im, JeongHyeok Yoon, David Young, Li Li, Ruiqi Zhu, Bao Nguyen, Donald Small. PHI-101, a next generation FLT3 inhibitor for acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4226.

Various adamantane sulfonamides showed potent inhibitory activity against 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). In continuation of our efforts to discover a more potent, selective and metabolically stable 11β-HSD1 inhibitor in mice as well as in humans, we optimized the adamantane sulfonamide using structure-based molecular modeling. Compound 3, which has alkyl side chains on the linker, demonstrated a potent inhibitory activity against human and mouse 11β-HSD1 (IC50 of 0.6 nM and 26 nM, respectively) and good physicochemical properties as a new anti-diabetes drug candidate.

Aims Elevated levels of glucocorticoid hormones cause glucose intolerance, visceral obesity, insulin resistance, hypertension, and dyslipidemia. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) represents as an attractive therapeutic target for treatment of metabolic syndrome and type 2 diabetes. This study investigated whether 11β-HSD1 inhibition by a novel selective inhibitor, ( 1S,3R,4S,5S,7S )-4-(1-((2-fluoro- N -methylphenylsulfonamido)-methyl) cyclopropanecarboxamido) adamantane-1-carboxamide (UI-1499) ameliorated metabolic abnormalities in diabetic mice. Main methods The in vitro activity of 11β-HSD1 was measured using the homogeneous time resolved fluorescence (HTRF) assay. Differentiated adipocytes were used to evaluate cellular 11β-HSD1 activity. To determine the inhibitory effects on 11β-HSD1 in tissues, we performed ex vivo studies using liver and epididymal fat isolated from C57BL/6J mice. KKAy mice were administered with UI-1499 to evaluate whether this compound ameliorated metabolic abnormalities in vivo in diabetic animals. Key findings UI-1499 had highly potent inhibitory activity in mouse, monkey and human 11β-HSD1, derived from liver microsomes. Oral administration of 45 mg/kg UI-1499 significantly inhibited 11β-HSD1 activity in C57BL/6J mouse liver and epididymal fat tissues. In KKAy mice, oral administration of UI-1499 (10 and 30 mg/kg for 3 weeks) lowered fasting blood glucose and HbA1c levels; these effects were comparable to those of pioglitazone. Further, UI-1499 treatment lowered plasma low-density lipoprotein (LDL) level in KKAy mice. Significance These results suggest that the 11β-HSD1 inhibitor, UI-1499, may serve as a novel drug candidate for the treatment of type 2 diabetes and metabolic syndrome.

E-mail: hkchoi45@jwu.ac.krReceived September 11, 2014, Accepted October 8, 2014Nuclear factor-erythroid 2-related factor 2 (Nrf2) regulates the expression of over 200 genes of antioxidant andphase II drug-metabolizing enzymes, and is highly expressed in non-small cell lung cancer (NSCLC). Ninederivatives of 4-(2-cyclohexylethoxy)aniline were designed. Our previous study demonstrated that IM3829increases radiosensitivity of several lung cancer cells in vitro and in vivo. Here, biological effects of IM3829derivatives ( 2a-2i) were evaluated. Compound 2g derivative effectively inhibits mRNA and protein expressionof Nrf2 and HO-1. In addition, we observed over two fold enhancement in IR-induced cell death, from2.90 ± 0.22 to 6.02 ± 0.87, in H1299 cancer cell-line. Among the nine derivatives, compound 2g derivativeexhibited the highest enhancement of radiosensitizing effect via inhibition of Nrf2 activity. Key Words : Radiosensitizer, Nrf2 inhibitor, Reactive oxygen species, Radiotherapy, Free radicalsIntroductionRadiotherapy is an effective treatment modality for solidtumors that can accompany surgery. The dosage of radio-therapy on the tumor is limited due to the side effects on thesurrounding normal tissues. On the other hand, fractionatedionizing radiation (IR) can lead to acquisition of radio-resistance cancer cells, which results in decreasing treatmentefficiency. Therefore, efforts have been made to developradiosensitizer or combination therapeutic agents with en-hanced efficacy of radiotherapy. Cancer cells harbor highly developed antioxidant systemsto maintain redox homeostasis that ensures cell survival withaberrant metabolism.

Focal adhesion kinase 1 (FAK1) is known to promote tumor progression and metastasis by controlling cell movement, invasion, survival and the epithelial-to-mesenchymal transition in the tumor microenvironment. As recent reports imply that FAK1 is highly associated with tumor cell development and malignancy, the inhibition of FAK1 activity could be an effective therapeutic approach for inhibiting the growth and metastasis of tumor cells. In this study, we aimed to determine the effect of a novel synthetic FAK1 inhibitor 2-[2-(2-methoxy-4-morpholin-4-yl-phenylamino)-5-trifluoromethyl-pyrimidin-4-ylamino]-N-methyl-benzamide, (MPAP) on lung cancer cells. MPAP suppressed cancer cell proliferation and the phosphorylation of FAK1. Combined treatment with MPAP and irradiation (IR) showed enhanced suppression of cancer cell proliferation in wild-type p53 cells and more intense suppression in p53-null cells. In addition, the combination treatment effectively induced G1 cell cycle arrest in a p53-independent manner. In an in vivo tumor xenograft mouse model, treatment with both MPAP and IR reduced tumor growth more than the treatment with IR or MPAP alone. Overall, these data demonstrate that the radiosensitizing effect of MPAP is mediated by the regulation of retinoblastoma protein (RB) phosphorylation in a p53-independent manner.

We investigated the inhibitory effects of hesperidin on melanogenesis. To find melanosome transport inhibitor from natural products, we collected the structural information of natural products from Korea Food and Drug Administration (KFDA) and performed pharmacophore-based in silico screening for Rab27A and melanophilin (MLPH). Hesperidin did not inhibit melanin production in B16F10 murine melanoma cells stimulated with α-melanocyte stimulating hormone (α-MSH), and also did not affect the catalytic activity of tyrosinase. But, hesperidin inhibited melanosome transport in melanocyte and showed skin lightening effect in pigmented reconstructed epidermis model. Therefore, we suggest that hesperidin is a useful inhibitor of melanosome transport and it might be applied to whitening agent.

By environmental stresses, cells can initiate a signaling pathway in which eukaryotic translation initiation factor 2-alpha (eIF2-α) is involved to regulate the response. Phosphorylation of eIF2-α results in the reduction of overall protein neogenesis, which allows cells to conserve resources and to reprogram energy usage for effective stress control. To investigate the role of eIF2-α in cell stress responses, we conducted a viability-based compound screen under endoplasmic reticulum (ER) stress condition, and identified 1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate (AMC-01) and its derivatives as eIF2-α-inactivating chemical. Molecular characterization of this signaling pathway revealed that AMC-01 induced inactivation of eIF2-α by phosphorylating serine residue 51 in a dose- and time-dependent manner, while the negative control compounds did not affect eIF2-α phosphorylation. In contrast with ER stress induction by thapsigargin, phosphorylation of eIF2-α persisted for the duration of incubation with AMC-01. By pathway analysis, AMC-01 clearly induced the activation of protein kinase RNA-activated (PKR) kinase and nuclear factor-κB (NF-κB), whereas it did not modulate the activity of PERK or heme-regulated inhibitor (HRI). Finally, we could detect a lower protein translation rate in cells incubated with AMC-01, establishing AMC-01 as a potent chemical probe that can regulate eIF2-α activity. We suggest from these data that AMC-01 and its derivative compounds can be used as chemical probes in future studies of the role of eIF2-α in protein synthesis-related cell physiology.

With the introduction of bioisosteres of the guanidinium group together with scaffold hopping, 35 zanamivir analogs with C-4-modification were synthesized, and their inhibitory activities against both group-1 and group-2 neuraminidase (H5N1 and H3N2) were determined. Compound D26 exerts the most potency, with IC(50) values of 0.58 and 2.72 μM against N2 and N1, respectively. Further preliminary anti-avian influenza virus (AIV, H5N1) activities against infected MDCK cells were evaluated, and D5 exerts ∼58% protective against AIV infection, which was comparable to zanamivir (∼67%). In a rat pharmacokinetic study, compound D5 showed an increased plasma half-life (t(1/2)) compared to zanamivir following either intravenous or oral administration. This study may represent a new start point for the future development of improved anti-AIV agents.

E-mail: gyoonhee@yonsei.ac.krReceived September 26, 2011, Accepted March 12, 2012Key Words : Histone deacetylase (HDAC), Quantitative structure-activity relationship (QSAR), Lactam basedhistone deacetylase inhibitor Histone acetyltransferases (HAT) and histone deacetylases(HDAC) regulate the chromatin structure related to tran-scriptional activity; epigenetic control.

E-mail: kyn@youai.co.krReceived October 31, 2011, Accepted December 21, 2011P-gp (P-glycoprotein) is a member of the ATP binding cassette (ABC) family of transporters. It transportsmany kinds of anticancer drugs out of the cell. It plays a major role as a cause of multidrug resistance (MDR).MDR function may be a cause of the failure of chemotherapy in cancer and influence pharmacokineticproperties of many drugs. Hence classification of candidate drugs as substrates or nonsubstrate of the P-gp isimportant in drug development. Therefore to identify whether a compound is a P-gp substrate or not, in silicomethod is promising. Recursive Partitioning (RP) method was explored for prediction of P-gp substrate. A setof 261 compounds, including 146 substrates and 115 nonsubstrates of P-gp, was used to training and validation.Using molecular descriptors that we can interpret their own meaning, we have established two models forprediction of P-gp substrates. In the first model, we chose only 6 descriptors which have simple physicalmeaning. In the training set, the overall predictability of our model is 78.95%. In case of test set, overallpredictability is 69.23%. Second model with 2D and 3D descriptors shows a little better predictability (overallpredictability of training set is 79.29%, test set is 79.37%), the second model with 2D and 3D descriptors showsbetter discriminating power than first model with only 2D descriptors. This approach will be used to reduce thenumber of compounds required to be run in the P-gp efflux assay.Key Words : ADME prediction, P-Glycoprotein, Recursive partitioning, 2D Descriptors, 3D DescriptorsIntroductionAbsorption, distribution, metabolism, excretion, and toxi-city (ADMET) properties are very important in the drugdiscovery.

The NF-κB system of transcription factors plays a crucial role in inflammatory diseases, making it an important drug target. We combined quantitative structure activity relationships for predicting the activity of new compounds and quantitative dynamic models for the NF-κB network with intracellular concentration models. GFA-MLR QSAR analysis was employed to determine the optimal QSAR equation. To validate the predictability of the IKKβ QSAR model for an external set of inhibitors, a set of ordinary differential equations and mass action kinetics were used for modeling the NF-κB dynamic system. The reaction parameters were obtained from previously reported research. In the IKKb QSAR model, good cross-validated q 2 (0.782) and conventional r 2 (0.808) values demonstrated the correlation between the descriptors and each of their activities and reliably predicted the IKKβ activities. Using a developed simulation model of the NF-κB signaling pathway, we demonstrated differences in IκB mRNA expression between normal and different inhibitory states. When the inhibition efficiency increased, inhibitor 1 (PS-1145) led to long-term oscillations. The combined computational modeling and NF-κB dynamic simulations can be used to understand the inhibition mechanisms and thereby result in the design of mechanism-based inhibitors.

The original red isomers and l-phenylalanine derivative isomers of monascus pigment, which have a side chain of either C5H11 or C7H15, were produced via Monascus fermentation, and then purified. The degradation patterns of the pigments relating to incubation conditions were investigated. For all tested compounds, a total of seven major fragments were formed during incubation, five in presence of sunlight irradiation and two with no sunlight. The red isomers R5 and R7 showed the same pattern whereas the L-Phe derivative isomers P5 and P7 exhibited another pattern. All of the pigments appeared to be highly sensitive to sunlight irradiation considering that with sunlight, diverse fragments were produced and the side chains C5H11 and C7H15 were degraded. The fragments are thought to be formed via degradation and combination reactions of pigments based on a comparison of the fragment molecular masses. Theoretically stable isomers of an original red pigment and some amino acid derivatives were selected based on a comparison of their Hartree conformational energies, which were estimated using geometric optimization based on ab initio molecular orbital calculations. The greater stability of amino acid derivatives was confirmed based on maximum UV-absorption wavelengths and isomer population rates.

) in TACE enzyme evaluated for discovery of potent TACE inhibitors. We have employed molecular dynamics simulations to compute the relative free energy of hydration and binding to TACE for gelastatin (1) and its hydroxamate analogue (2). The relative free energy difference is directly described in this article using the free energy perturbation approach as a means to accurately predict the TACE inhibitor of gelastatin analogues. The results show that th e good agreement between the experimental and theoretical relative free energies of binding, gelastatin hydroxamate (2) binds stronger to TACE by ‒3.37 kcal/mol. The desolvation energy costs significantly reduced binding affinity, hydroxamate group associated with high desolvation energy formed strong favorable interactions with TACE with more than compensated for the solvation costs and therefore led to an improvement in relative binding affinity.

G-protein coupled receptors (GPCRs) are involved in a wide variety of physiological processes and are known to be targets for nearly 50% of drugs. The various functions of GPCRs are affected by their cognate ligands which are mainly classified as agonists and antagonists. The purpose of this study is to develop a Bayesian classification model, that can predict a compound as either human GPCR agonist or antagonist. Total 6627 compounds experimentally determined as either GPCR agonists or antagonists covering all the classes of GPCRs were gathered to comprise the dataset. This model distinguishes GPCR agonists from GPCR antagonists by using chemical fingerprint, FCFP_6. The model revealed distinctive structural characteristics between agonistic and antagonistic compounds: in general, 1) GPCR agonists were flexible and had aliphatic amines, and 2) GPCR antagonists had planar groups and aromatic amines. This model showed very good discriminative ability in general, with pretty good discriminant statistics for the training set (accuracy: 90.1%) and a good predictive ability for the test set (accuracy: 89.2%). Also, receiver operating characteristic (ROC) plot showed the area under the curve (AUC) to be 0.957, and Matthew's Correlation Coefficient (MCC) value was 0.803. The quality of our model suggests that it could aid to classify the compounds as either GPCR agonists or antagonists, especially in the early stages of the drug discovery process.

Developing effective tools for predicting absorption, distribution, metabolism, excretion properties and toxicity (ADME/T) of new chemical entities in the early stage of drug design is one of the most important tasks in drug discovery and development today. As one of these attempts, support vector machines (SVM) has recently been exploited for the prediction of ADME/T related properties. However, two problems in SVM modeling, i.e. feature selection and parameters setting, are still far from solved. The two problems have been shown to be crucial to the efficiency and accuracy of SVM classification. In particular, the feature selection and optimal SVM parameters setting influence each other, which indicates that they should be dealt with simultaneously. In this account, we present an integrated practical solution, in which genetic-based algorithm (GA) is used for feature selection and grid search (GS) method for parameters optimization. hERG ion-channel inhibitor classification models of ADME/T related properties has been built for assessing and testing the proposed GA-GS-SVM. We generated 6 different models that are 3 different single models and 3 different ensemble models using training set - 1891 compounds and validated with external test set - 175 compounds. We compared single model with ensemble model to solve data imbalance problems. It was able to improve accuracy of prediction to use ensemble model.

A series of coumarin types MMP inhibitors were designed based on gelastatin hydroxamates (1) and evaluated for TACE, cellular TNF-α, and NO inhibitory activities. Among them, compounds 9b had potent inhibitory activities in enzymatic and cellular assays and good selectivity to MMP-2 and MMP-9. Further investigation of 9b will be carried out for its efficacy in RA animal model system.

We synthesized a series of delta-lactam-based HDAC inhibitors that were identified with various degrees of anti-inflammatory and cell growth inhibitory activities. Compounds possessing significant HDAC inhibitory activity exhibited both anti-inflammatory and cell growth inhibitory activities as well as significant tumor growth inhibition in the in vivo tumor xenograft experiments. Besides, these compounds demonstrated anti-inflammatory properties in vitro via suppression of the production of the proinflammatory cytokine TNF-alpha and nitric oxide by LPS-stimulated RAW264.7 cells.

RAS-driven tumors are often difficult to treat with conventional therapies and therefore, novel treatment strategies are necessary. The present study describes a promising targeted therapeutic strategy against non-small cell lung cancer (NSCLC) harboring KRAS mutations, which has intrinsic resistance to MEK inhibition. Results showed that intrinsic resistance to MEK inhibition occurred via high AKT expression by PI3K activation as a bypass pathway. The HSP90 inhibitor AUY922 suppressed PI3K-AKT-mTOR and RAF-MEK-ERK, and rendered cells sensitive to trametinib (GSK1120212). Synergy from the combination of the two drugs was observed in only sub-therapeutic concentrations of either drug. Dual inhibition of the HSP90 and MEK signaling pathways with sub-therapeutic doses may represent a potent therapeutic strategy to treat KRAS-mutant NSCLC with intrinsic resistance to MEK inhibition and to resolve the toxicity observed upon dual inhibition of AKT and MEK at therapeutic doses in clinical trials.

Triple-negative breast cancer (TNBC) is an aggressive cancer with a poor prognosis due to its epithelial‑to-mesenchymal transition (EMT) phenotype. Cancer patients often experience several detrimental effects of cancer treatment, such as chemoresistance, radioresistance and the maintenance of cancer stem cells due to EMT. Thus, EMT signaling is considered to be a valuable therapeutic target for cancer treatment, and its inhibition is being attempted as a new treatment option for TNBC patients. Previously, we showed that 3-(2-chlorobenzyl)-1,7-dimethyl-1H-imidazo[2,1-f]purine‑2,4(3H,8H)-dione (IM-412) inhibits transforming growth factor-β (TGF-β)-induced differentiation of human lung fibroblasts through both Smad-dependent and -independent pathways. In the present study, we examined the inhibitory effect of IM-412 on EMT pathways and invasiveness in TNBC cells since the TGF-β signaling pathway is a typical signaling pathway that functions in EMT. IM-412 not only potently suppressed the migration and invasion of MDA-MB-231 cells, but also lowered the expression of mesenchymal markers and EMT-activating transcription factors in these cells. IM-412 inhibited the activation of several signaling proteins, including Smad2/Smad3, p38MAPK, Akt and JNK, and it also attenuated the phosphorylation of FGFR1 and FGFR3. Collectively, our findings suggest that the synthetic compound IM-412 suppressed the EMT process in MDA-MB-231 cells and thereby effectively inhibited the migration and invasion of these cancer cells. Thus, IM-412 could serve as a novel therapeutic agent for malignant cancers.

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca2+) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca2+ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca2+ (iCa2+) levels for a prolonged period of time. Ca2+ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca2+ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca2+ supplementation to patient undergoing treatment for metastatic cancer.

The hydroxamic acid analogues (2) of the natural product gelastatins (1) were prepared by 1 step conversion reaction. The synthetic analogues (2) showed potent enzymatic inhibitory activities against MMP-2, MMP-9, and TACE IC50's of 6, 23, and 28 nM, respectively. In addition, 2 were able to inhibit TNF-alpha production effectively in mice as well as in a macrophage cell line, RAW 264.7. The protective effect of 2 also was examined on LPS-induced acute septic shock model. The mechanism of TNF-alpha inhibition was examined by RT-PCR and Western blot analyses. The relation of TACE and alpha-secretase was examined using cellular alpha-secretase assays on IMR-32 and SH-SY5Y cell lines. The docking mode of 2 with the catalytic domain of TACE was illustrated to analyze the binding mode for the further analogue design.

We report the synthesis of a series of monoanionic phosphotyrosyl (pTyr) mimetic-containing tripeptides based on 'Fmoc-Glu(OBn)-Xxx-Leu-amide' (where Xxx = pTyr mimetic) and their N-terminally modified derivatives. The inhibitory potencies of compounds were tested against YopH and human PTP1B enzymes. Several compounds exhibited noteworthy activity against both YopH and PTP1B. Among the N-terminally modified analogues, 5-methylindole derivative 30 was found to be the best moiety to replace base-labile Fmoc group. A mode of binding with YopH is proposed for tripeptides 21, 30, and 31.

In order to characterize the hydrophobic parameters of N-acetyl amino acid amides in 1-octanol/water, a theoretical calculation was carried out using a solvation free energy density model. The hydrophobicity parameters of the molecules are obtained with the cons ideration of the solvation free energy over the solvent volume surrounding the solute, using a grid model. Our method can account for the solvent accessible surface area of the molecules according to conformational variations. Through a comparison of the hydrophobicity of our calculation and that of other experimental/theoretical works, the solvation free energy density model is proven to be a useful tool for the evaluation of the hydrophobicity of amino acids and peptides. In order to evaluate the solvation free energy density model as a method of calculating the activity of drugs using the hydrophobicity of its building blocks, the contracture of Bradykinin potentiating pentapeptide was also predicted from the hydrophobicity of each residue. The solvation free energy density model can be used to employ descriptors for the prediction of peptide activities in drug discovery, as well as to calculate the hydrophobicity of amino acids.

The three-dimensional solution structure obtained by NMR of the A35T mutant vnd/NK-2 homeodomain bound to the vnd/NK-2 consensus 16 bp DNA sequence was determined. This mutation to threonine from alanine in position 35 in helix II of the vnd/NK-2 homeodomain is associated with early embryonic lethality in Drosophila melanogaster. Although the unbound mutant protein is not structured, in the DNA-bound state it adopts the three-helix fold characteristic of all known homeodomains, but with alterations relative to the structure of the wild-type analogue. These structural modifications occur, and are accompanied by a 50-fold reduction in the DNA binding affinity, even though most of the protein-DNA interactions originally seen for the wild-type homeodomain are found likewise in the threonine analogue. Alterations include torsional angle changes in the loop between helix I and helix II, and in the turn between helix II and helix III, as well as in a distortion of the usual antiparallel orientation of helix I with respect to helix II. The alteration of the position of leucine 40 in the A35T mutant is proposed to explain the observed 1.27 ppm upfield shift of the corresponding amide proton resonance relative to the value observed for the wild-type analogue. A detailed comparison of the structures of the mutant A35T and wild-type vnd/NK-2 homeodomains bound to the cognate DNA is presented. The consequences of the structural alteration of the DNA-bound A35T mutant vnd/NK-2 protein may constitute the basis of the observed early embryonic lethality.

We have performed ab initio calculation on the active site of HIV-1 protease. The FEP method was used to determine the binding free energy of four different of protonated states of HIV-1 protease with inhibitor. The structure of the active site and hole structure was taken from the X-ray crystallographic coordinates of the C 2 symmetric inhibitor A74704 protease bound. The active site was modeled with the fragment molecules of binding pocket, acetic acid/ acetate anion (Asp25, Asp125), formamide (amide bond of Thr26/Gly27, Thr126/ Gly127), and methanol as inhibitor fragment. All possibly protonated states of the active site were considered, which were diprotonated state (0, 0), monoprotonated (-1, 0),(0, -1) and diunprotonated state (-1, -1). Once the binding energy ΔEbind, of each model was calculated, more probabilistic protonated states can be proposed from binding energy. From ab-initio results, the FEP simulations were performed for the three following mutations: I) Asp25 Asp125 → AspH25 Asp125, ii) Asp25 Asp125 → Asp25 AspH125, iii) AspH25 Asp125 → AspH25 AspH125. The free energy difference between the four states gives the information of the more realistic protonated state of active site aspartic acid. These results provide a theoretical prediction of the protonation state of the catalytic aspartic residues for A74707 complex, and may be useful for the evaluation of potential therapeutic targets.

A four-channel impedance measurement system, including a two-channel goniometer, to analyse human arm movement, was constructed. Impedances and joint angles were simultaneously measured for wrist and elbow movements. As the impedance changes resulting from wrist and elbow movements depended heavily on electrode placement, the optimum electrode configurations for those movements were determined by searching for high correlation coefficients, large impedance changes and minimum interferences in ten subjects (age: 29 +/- 6 years). The optimum electrode configurations showed very strong relationships between the wrist joint angle and forearm impedance (correlation coefficient= 0.95 +/- 0.04) and between the elbow joint angle and upper arm impedance (correlation coefficient= -0.97 +/- 0.03). Although the measured impedance changes of the wrist (1.1 +/-1.5 ohms) and elbow (-5.0 +/-2.9 ohms) varied between individuals, the reproducibilities of wrist and elbow impedance changes of five subjects were 5.8 +/- 1.8% and 4.6 +/- 1.4% for the optimum electrode pairs, respectively. It is proposed that this optimum electrode configuration would be useful for future studies involving the accurate measurement of arm movements by the impedance method.

This work is focused on analyzing ion-pair interactions and showing the effect of solvent induced inter-atomic attractions in various dielectric environments. To estimate the stability of ion-pairs, SCI-PCM ab initio MO calculations were carried out. We show that the solvent-induced attraction or ‘cavitation’ energy of the ion-pair interactions in solution that arises mainly from the stabilization of the water molecules by the generation of an electrostatic field. In fact, even the strong electrostatic interaction characteristic of ion-pair interactions in the gas phase cannot overcome the destabilization or reorganization of the water molecules around solute cavities that arise from cancellation of the electrostatic field. The solvent environment, possibly supplemented by some specific solvent molecules, may help place the solute molecule in a cavity whose surroundings are characterized by an infinite polarizable dielectric medium. This behavior suggests that hydrophobic residues at a protein surface could easily contact the side chains of other nearby residues through the solvent environment, instead of by direct intra-molecular interactions.

The behavior of terminally blocked amino acids, N -acetyl alanine amide (NAA), N -acetyl leucine amide (NAL), and N -acetyl glutatmine amide (NAQ) at the 1-octanol/water continuum interface was investigated by Monte Carlo simulation with the solvation free energy density (SFED) calculation method. All the model amino acids have free energy minimum near the interface. These amino acids can have various conformations such as α-helix, β-sheet, and C 7−eq , however the minimum energy conformations are obtained as α-helix for all the model amino acids at the interface region.

Background Polo-like kinase-1 (Plk1) plays a crucial role in cell proliferation and the inhibition of Plk1 has been considered as a potential target for specific inhibitory drugs in anti-cancer therapy. Several research groups have identified peptide-based inhibitors that target the polo-box domain (PBD) of Plk1 and bind to the protein with high affinity in in vitro assays. However, inadequate proteolytic resistance and cell permeability of the peptides hinder the development of these peptide-based inhibitors into novel therapeutic compounds. Methodology/Principal Findings In order to overcome the shortcomings of peptide-based inhibitors, we designed and synthesized small molecule inhibitors. Among these molecules, bg-34 exhibited a high binding affinity for Plk1-PBD and it could cross the cell membrane in its unmodified form. Furthermore, bg-34-dependent inhibition of Plk1-PBD was sufficient for inducing apoptosis in HeLa cells. Moreover, modeling studies performed on Plk1-PBD in complex with bg-34 revealed that bg-34 can interact effectively with Plk1-PBD. Conclusion/Significance We demonstrated that the molecule bg-34 is a potential drug candidate that exhibits anti-Plk1-PBD activity and possesses the favorable characteristics of high cell permeability and stability. We also determined that bg-34 induced apoptotic cell death by inhibiting Plk1-PBD in HeLa cells at the same concentration as PEGylated 4j peptide, which can inhibit Plk1-PBD activity 1000 times more effectively than bg-34 can in in vitro assays. This study may help to design and develop drug-like small molecule as Plk1-PBD inhibitor for better therapeutic activity.