In silico identification and biological evaluation of a selective MAP4K4 inhibitor against pancreatic cancer.

Inhibiting a specific target in cancer cells and reducing unwanted side effects has become a promising strategy in pancreatic cancer treatment. MAP4K4 is associated with pancreatic cancer development and correlates with poor clinical outcomes. By phosphorylating MKK4, proteins associated with cell apoptosis and survival are translated. Therefore, inhibiting MAP4K4 activity in pancreatic tumours is a new therapeutic strategy. Herein, we performed a structure-based virtual screening to identify MAP4K4 inhibitors and discovered the compound F389-0746 with a potent inhibition (IC50 120.7 nM). The results of kinase profiling revealed that F389-0746 was highly selective to MAP4K4 and less likely to cause side effects. Results of in vitro experiments showed that F389-0746 significantly suppressed cancer cell growth and viability. Results of in vivo experiments showed that F389-0746 displayed comparable tumour growth inhibition with the group treated with gemcitabine. These findings suggest that F389-0746 has promising potential to be further developed as a novel pancreatic cancer treatment.

Identification of a dual TAOK1 and MAP4K5 inhibitor using a structure-based virtual screening approach.

The STE20 kinase family is a complex signalling cascade that regulates cytoskeletal organisation and modulates the stress response. This signalling cascade includes various kinase mediators, such as TAOK1 and MAP4K5. The dysregulation of the STE20 kinase pathway is linked with cancer malignancy. A small-molecule inhibitor targeting the STE20 kinase pathway has therapeutic potential. In this study, a structure-based virtual screening (SBVS) approach was used to identify potential dual TAOK1 and MAP4K5 inhibitors. Enzymatic assays confirmed three potential dual inhibitors (>50% inhibition) from our virtual screening, and analysis of the TAOK1 and MAP4K5 binding sites indicated common interactions for dual inhibition. Compound 1 revealed potent inhibition of colorectal and lung cancer cell lines. Furthermore, compound 1 arrested cancer cells in the G0/G1 phase, which suggests the induction of apoptosis. Altogether, we show that the STE20 signalling mediators TAOK1 and MAP4K5 are promising targets for drug research.

CXCL12/CXCR4 Signaling Contributes to the Pathogenesis of Opioid Tolerance: A Translational Study.

BACKGROUND: Long-term opioid therapy for chronic pain may lead to analgesic tolerance, especially when administered intrathecally, thus preventing adequate pain relief. Discovering drug targets to treat opioid tolerance using a mechanism-based approach targeting opioid-induced neuroinflammation provides new therapeutic opportunities. In this study, we provide translational evidence that CXCL12/CXCR4 signaling contributes to the pathogenesis of opioid tolerance. METHODS: The CXCL12 levels in the cerebrospinal fluid of opioid-tolerant patients were compared with those of opioid-naive subjects. For further investigation, a rodent translational study was designed using 2 clinically relevant opioid delivery paradigms: daily intraperitoneal morphine injections and continuous intrathecal morphine infusion. We measured rats' tail flick responses and calculated the percentage of maximum possible effects (%MPE) to demonstrate opioid acute antinociception and the development of analgesic tolerance. The effects of exogenous CXCL12, CXCL12 neutralizing antibody, and receptor antagonist AMD3100 were investigated by intrathecal administration. Data were presented as mean ± SEM. RESULTS: CXCL12 was significantly upregulated in the cerebrospinal fluid of opioid-tolerant patients for 892 ± 34 pg/mL (n = 27) versus 755 ± 33 pg/mL (n = 10) in naive control subjects (P = .03). Furthermore, after 2 and 5 days of intrathecal morphine infusion, rat lumbar spinal cord dorsal horn CXCL12 messenger RNA levels were significantly upregulated by 3.2 ± 0.7 (P = .016) and 3.4 ± 0.3 (P = .003) fold, respectively. Results from the daily intraperitoneal morphine injection experiments revealed that administering an intrathecal infusion of CXCL12 for 24 hours before the first morphine injection did not decrease antinociception efficacy on day 1 but accelerated tolerance after day 2 (%MPE 49.5% vs 88.1%, P = .0003). In the intrathecal morphine coinfusion experiments, CXCL12 accelerated tolerance development (%MPE 9.4% vs 43.4% on day 1, P < .0001), whereas coadministration with CXCL12 neutralizing antibody attenuated tolerance (72.5% vs 43.4% on day 1, P < .0001; 47.6% vs 17.5% on day 2, P < .0001). Coadministration of receptor antagonist AMD 3100 can persistently preserve morphine analgesic effects throughout the study period (27.9% ± 4.1% vs 0.9% ± 1.6% on day 5, P = .03). CONCLUSIONS: The CXCL12/CXCR4 pathway contributes to the pathogenesis of opioid tolerance. Our study indicates that intervening with CXCL12/CXCR4 signaling has therapeutic potential for opioid tolerance.

Integrin-linked kinase as a novel molecular switch of the IL-6-NF-κB signaling loop in breast cancer.

Substantial evidence has clearly demonstrated the role of the IL-6-NF-κB signaling loop in promoting aggressive phenotypes in breast cancer. However, the exact mechanism by which this inflammatory loop is regulated remains to be defined. Here, we report that integrin-linked kinase (ILK) acts as a molecular switch for this feedback loop. Specifically, we show that IL-6 induces ILK expression via E2F1 upregulation, which, in turn, activates NF-κB signaling to facilitate IL-6 production. shRNA-mediated knockdown or pharmacological inhibition of ILK disrupted this IL-6-NF-κB signaling loop, and blocked IL-6-induced cancer stem cells in vitro and estrogen-independent tumor growth in vivo Together, these findings establish ILK as an intermediary effector of the IL-6-NF-κB feedback loop and a promising therapeutic target for breast cancer.

In silico identification of a novel Cdc2-like kinase 2 (CLK2) inhibitor in triple negative breast cancer.

Dysregulation of RNA splicing processes is intricately linked to tumorigenesis in various cancers, especially breast cancer. Cdc2-like kinase 2 (CLK2), an oncogenic RNA-splicing kinase pivotal in breast cancer, plays a significant role, particularly in the context of triple-negative breast cancer (TNBC), a subtype marked by substantial medical challenges due to its low survival rates. In this study, we employed a structure-based virtual screening (SBVS) method to identify potential CLK2 inhibitors with novel chemical structures for treating TNBC. Compound 670551 emerged as a novel CLK2 inhibitor with a 50% inhibitory concentration (IC50) value of 619.7 nM. Importantly, Compound 670551 exhibited high selectivity for CLK2 over other protein kinases. Functionally, this compound significantly reduced the survival and proliferation of TNBC cells. Results from a cell-based assay demonstrated that this inhibitor led to a decrease in RNA splicing proteins, such as SRSF4 and SRSF6, resulting in cell apoptosis. In summary, we identified a novel CLK2 inhibitor as a promising potential treatment for TNBC therapy.

Identification of selective dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitors and their effects on tau and microtubule.

The overexpression of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), commonly observed in neurodegenerative diseases like Alzheimer's disease (AD) and Down syndrome (DS), can induce the formation of neurofibrillary tangles (NFTs) and amyloid plaques. Hence, designing a selective DYRK1A inhibitor would result in a promising small molecule for treating neurodegenerative diseases. Developing selective inhibitors for DYRK1A has been a difficult challenge due to the highly preserved ATP-binding site of protein kinases. In this study, we employed a structure-based virtual screening (SBVS) campaign targeting DYRK1A from a database containing 1.6 million compounds. Enzymatic assays were utilized to verify inhibitory properties, confirming that Y020-3945 and Y020-3957 showed inhibitory activity towards DYRK1A. In particular, the compounds exhibited high selectivity for DYRK1A over a panel of 120 kinases, reduced the phosphorylation of tau, and reversed the tubulin polymerization for microtubule stability. Additionally, treatment with the compounds significantly reduced the secretion of inflammatory cytokines IL-6 and TNF-α activated by DYRK1A-assisted NFTs and Aß oligomers. These identified inhibitors possess promising therapeutic potential for conditions associated with DYRK1A in neurodegenerative diseases. The results showed that Y020-3945 and Y020-3957 demonstrated structural novelty compared to known DYRK1A inhibitors, making them a valuable addition to developing potential treatments for neurodegenerative diseases.

Enhancement of placenta growth factor expression by oncostatin M in human rheumatoid arthritis synovial fibroblasts.

Oncostatin M (OSM) belongs to IL-6 subfamily and is mostly produced by T lymphocytes. High levels of OSM are detected in the pannus of rheumatoid arthritis (RA) patients and it may arouse the inflammation responses in joints and eventually leads to bone erosion. Placenta growth factor (PLGF) is an angiogenic factor and highly homologous with vascular endothelial growth factor (VEGF). It has been recently reported that PLGF is highly expressed in synovial tissue and enhances the production of proinflammatory cytokines including TNF-α and IL-6. Here, we demonstrated that OSM increased mRNA and protein levels of PLGF in a time- and concentration-dependent manner in RA synovial fibroblasts. Inhibitors of JAK3 and PI3K antagonized OSM-induced production of PLGF. OSM enhanced the phosphorylation of Tyr705-STAT3, Ser727-STAT3, Ser473-Akt, and increased the nuclear translocation of phosphorylated STAT3 time-dependently. Transfection of dominant negative Akt or application of PI3K inhibitorLY294002 significantly inhibited p-Tyr705-STAT3, p-Ser727-STAT3, and PLGF expression, indicating that Akt is involved in JAK3/STAT3/PLGF signaling cascade. To further examine whether STAT3 binds to the promoter region of PLGF, Chip assay was used and it was found that OSM could bind with PLGF promoter, which was inhibited by JAK3 and PI3K inhibitors. Accumulation of PLGF in the pannus may contribute to the inflammation, angiogenesis and joints destruction in RA patients. These findings demonstrated the important role of OSM in the pathology network of RA and provided novel therapeutic drug targets for RA treatment.

Urolithin A exhibits a neuroprotective effect against Alzheimer's disease by inhibiting DYRK1A activity.

Alzheimer's disease (AD) is a devastating neurodegenerative disease with more than 50 million people suffer from it. Unfortunately, none of the currently available drugs is able to improve cognitive impairment in AD patients. Urolithin A (UA) is a metabolite obtained from ellagic acid and ellagitannin through the intestinal flora, and it has antioxidant and anti-inflammatory properties. Previous reports found that UA had neuroprotective effects in an AD animal model, but the detailed mechanism still needs to be elucidated. In this study, we performed kinase-profiling to show that dual-specific tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is the main target of UA. Studies showed that the level of DYRK1A in AD patients' brains was higher than that of healthy people, and it was closely related to the occurrence and progression of AD. Our results revealed that UA significantly reduced the activity of DYRK1A, which led to de-phosphorylation of tau and further stabilized microtubule polymerization. UA also provided neuroprotective effects by inhibiting the production of inflammatory cytokines caused by Aß. We further showed that UA significantly improved memory impairment in an AD-like mouse model. In summary, our results indicate that UA is a DYRK1A inhibitor that may provide therapeutic advantages for AD patients.

Identification and analysis of a selective DYRK1A inhibitor.

The dysregulation of DYRK1A is implicated in many diseases such as cancer, diabetes, and neurodegenerative diseases. Alzheimer's disease is one of the most common neurodegenerative disease and has elevated interest in DYRK1A research. Overexpression of DYRK1A has been linked to the formation of tau aggregates. Currently, an effective therapeutic treatment that targets DYRK1A is lacking. A specific small-molecule inhibitor would further our understanding of the physiological role of DYRK1A in neurodegenerative diseases and could be presented as a possible therapeutic option. In this study, we identified pharmacological interactions within the DYRK1A active site and performed a structure-based virtual screening approach to identify a selective small-molecule inhibitor. Several compounds were selected in silico for enzymatic and cellular assays, yielding a novel inhibitor. A structure-activity relationship analysis was performed to identify areas of interactions for the compounds selected in this study. When tested in vitro, reduction of DYRK1A dependent phosphorylation of tau was observed for active compounds. The active compounds also improved tau turbidity, suggesting that these compounds could alleviate aberrant tau aggregation. Testing the active compound against a panel of kinases across the kinome revealed greater selectivity towards DYRK1A. Our study demonstrates a serviceable protocol that identified a novel and selective DYRK1A inhibitor with potential for further study in tau-related pathologies.

O-methylated flavonol as a multi-kinase inhibitor of leukemogenic kinases exhibits a potential treatment for acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease with poor overall survival characterized by various genetic changes. The continuous activation of oncogenic pathways leads to the development of drug resistance and limits current therapeutic efficacy. Therefore, a multi-targeting inhibitor may overcome drug resistance observed in AML treatment. Recently, groups of flavonoids, such as flavones and flavonols, have been shown to inhibit a variety of kinase activities, which provides potential opportunities for further anticancer applications. PURPOSE: In this study, we evaluated the anticancer effects of flavonoid compounds collected from our in-house library and investigated their potential anticancer mechanisms by targeting multiple kinases for inhibition in AML cells. METHODS: The cytotoxic effect of the compounds was detected by cell viability assays. The kinase inhibitory activity of the selected compound was detected by kinase-based and cell-based assays. The binding conformation and interactions were investigated by molecular docking analysis. Flow cytometry was used to evaluate the cell cycle distribution and cell apoptosis. The protein and gene expression were estimated by western blotting and qPCR, respectively. RESULTS: In this study, an O-methylated flavonol (compound 11) was found to possess remarkable cytotoxic activity against AML cells compared to treatment in other cancer cell lines. The compound was demonstrated to act against multiple kinases, which play critical roles in survival signaling in AML, including FLT3, MNK2, RSK, DYRK2 and JAK2 with IC50 values of 1 - 2 µM. Compared to our previous flavonoid compounds, which only showed inhibitions against MNKs or FLT3, compound 11 exhibited multiple kinase inhibitory abilities. Moreover, compound 11 showed effectiveness in inhibiting internal tandem duplications of FLT3 (FLT3-ITDs), which accounts for 25% of AML cases. The interactions between compound 11 and targeted kinases were investigated by molecular docking analysis. Mechanically, compound 11 caused dose-dependent accumulation of leukemic cells at the G0/G1 phase and followed by the cells undergoing apoptosis. CONCLUSION: O-methylated flavonol, compound 11, can target multiple kinases, which may provide potential opportunities for the development of novel therapeutics for drug-resistant AMLs. This work provides a good starting point for further compound optimization.

A novel dual HDAC and HSP90 inhibitor, MPT0G449, downregulates oncogenic pathways in human acute leukemia in vitro and in vivo.

Acute leukemia is a highly heterogeneous disease; therefore, combination therapy is commonly used for patient treatment. Drug-drug interaction is a major concern of combined therapy; hence, dual/multi-target inhibitors have become a dominant approach for cancer drug development. HDACs and HSP90 are involved in the activation of various oncogenic signaling pathways, including PI3K/AKT/mTOR, JAK/STAT, and RAF/MEK/ERK, which are also highly enriched in acute leukemia gene expression profiles. Therefore, we suggest that dual HDAC and HSP90 inhibitors could represent a novel therapeutic approach for acute leukemia. MPT0G449 is a dual effect inhibitor, and it showed cytotoxic effectiveness in acute leukemia cells. Molecular docking analysis indicated that MPT0G449 possessed dual HDAC and HSP90 inhibitory abilities. Furthermore, MPT0G449 induced G2 arrest and caspase-mediated cell apoptosis in acute leukemia cells. The oncogenic signaling molecules AKT, mTOR, STAT3, STAT5, MEK, and ERK were significantly downregulated after MPT0G449 treatment in HL-60 and MOLT-4 cells. In vivo xenograft models confirmed the antitumor activity and showed the upregulation of acetyl-histone H3 and HSP70, biomarkers of pan-HDAC and HSP90 inhibition, with MPT0G449 treatment. These findings suggest that the dual inhibition of HDAC and HSP90 can suppress the expression of oncogenic pathways in acute leukemia, and MPT0G449 represents a novel therapeutic for anticancer treatment.

Ring-opening of five-membered heterocycles conjugated 4-isopropylresorcinol scaffold-based benzamides as HSP90 inhibitors suppressing tumor growth in vitro and in vivo.

A series of ring-opened dihydroxybenzamides have been designed and synthesized as heat shock protein 90 inhibitors. One of derivatives, compound 6b ((N-ethyl-2,4-dihydroxy-5-isopropyl-N-(pyridin-3-yl)benzamide)) demonstrated remarkable antiproliferative activity against in human KRAS mutant A549 and EGFR T790 M mutant H1975 lung cancer cell lines with GI50 values of 0.07 and 0.05 µM, respectively. It is also active against in other cancer cell lines, such as colorectal HCT116 (GI50 = 0.09 µM), liver Hep3B (GI50 = 0.20 µM) and breast MDA-MB-231 (GI50 = 0.09 µM), and shows no evidence of toxicity in normal cell line. Compound 6b has an IC50 of 110.18 nM in HSP90α inhibitory activity, slightly better than reference compound 1 (17-AAG, IC50 = 141.62 nM) and achieves the degradation of multiple HSP90 client proteins in a dose- and time-dependent manner and downstream signaling of Akt in a concentration- and time-dependent manner in the human A549 lung cancer cell line. In the Boyden chamber assay, compound 6b can efficiently inhibit the migration of A549 cells when compared to the reference compound 1. It also induce significant activity through the apoptotic pathway. Treatment with 6b showed no vision toxicity (IC50 > 10 µM) on 661w photoreceptor cells as compared to AUY922 (3a) with a 0.04 µM values of IC50 and has no effect in hERG test. In a bidirectional Caco-2 permeability assay, compound 6b was classified as a highly permeable compound which is not a substrate of efflux transporters. In a pharmacokinetic study in rats, 6b showed an F = 17.8% of oral bioavailability. The effect of metabolic stability of compound 6b in human hepatocytes showed a T1/2 of 67.59 min. Compound 6b (50 mg/kg, po, daily) exhibits antitumor activity with a 72% TGD (tumor growth delay) in human A549 lung xenograft. The combination of 6b and afatinib, orally administered, showed tumor growth suppression with 67.5% of TGI in lung H1975 xenograft model. Thus compound 6b is a lead compound for further development of potential agents to treat lung cancer.

Retraction: Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability.

[This retracts the article DOI: 10.18632/oncotarget.6427.].

Anti-metastatic activity of MPT0G211, a novel HDAC6 inhibitor, in human breast cancer cells in vitro and in vivo.

Triple-negative breast cancer (TNBC) is associated with an increased risk of metastasis and a poor prognosis. The invasive ability of TNBC relies on actin reorganization and is regulated by histone deacetylase 6 (HDAC6). The present study aimed to examine the effect of MPT0G211, a novel HDAC6 inhibitor, on cell migration and microtubule association in both in vitro and in vivo models of TNBC. Here MPT0G211 more selectively and potently targeted and inhibited HDAC6, compared with tubastatin A, another selective HDAC6 inhibitor. In vitro, MPT0G211 decreased the migration of the TNBC cell line MDA-MB-231, particularly when administered together with paclitaxel, and increased heat shock protein 90 (Hsp90) acetylation, leading to the dissociation of Hsp90 from aurora-A and proteasomal degradation. Furthermore, MPT0G211 significantly disrupted F-actin polymerization by increasing cortactin acetylation and downregulating slingshot protein phosphatase 1 (SSH1) and active cofilin expression. In vivo, MPT0G211 treatment significantly ameliorated TNBC metastasis. In conclusion, our results demonstrate that MPT0G211 reduces TNBC cell motility by promoting cortactin acetylation and aurora-A degradation, and inhibiting the cofilin-F-actin pathway via HDAC6 activity attenuation. MPT0G211 therefore demonstrates therapeutic potential for invasive TNBC.

Combination treatment strategy for pancreatic cancer involving the novel HDAC inhibitor MPT0E028 with a MEK inhibitor beyond K-Ras status.

BACKGROUND: Oncogenic K-Ras signaling highly relies on the canonical Ras/MEK/ERK pathway to contribute to pancreatic cancer progression. However, numerous efforts of MEK inhibitors have failed to provide an optimal antitumor effect for pancreatic cancer in practice. The aim of the present work was to develop a more efficacious therapeutic intervention for MEK inhibitors through combination with histone deacetylase (HDAC) inhibitor MPT0E028. METHODS: The effects of combined therapy on cell viability, apoptosis, protein, and RNA expressions were determined by MTT assay, flow cytometry, western blotting, and quantitative PCR analysis. The AsPC-1 xenograft was used to assess antitumor effects in vivo. RESULTS: The co-administration of MPT0E028 and MEK inhibitor yielded synergistic effects on cell viability suppression both in K-Ras mutated and wild-type pancreatic cancer cells and also markedly triggered cell apoptosis. Surprisingly, ERK and epidermal growth factor receptor (EGFR) were activated by the long-term and low-concentration treatment of MPT0E028 or another HDAC inhibitor alone. Whereas, the pharmacological attenuation of ERK signaling dramatically abolished the MPTE028-induced p-ERK and EGFR expression. Overexpression of HDAC4, HDAC6, and MEK, respectively, reversed the cell death induced by the combined treatment. Finally, the combined treatment decreased the tumor volume in an AsPC-1 xenograft model compared to each individual treatment alone. CONCLUSIONS: The synergistic anti-survival effect of the combination was suggested to occur via compensation of the MEK inhibitor for activated ERK. Our results indicate that this combination strategy could benefit patients with pancreatic cancer beyond K-Ras status.

The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells.

BACKGROUND: There are some limitations of standard chemotherapy for acute leukemia. Vincristine and doxorubicin are commonly used for acute leukemia, but they may induce serious side effects such as cardiomyopathy and neurotoxicity. Furthermore, chemotherapy resistance occurs more and more frequently. Therefore, effective treatment strategies are needed. Histone deacetylase 6 inhibition is considered as a potential therapeutic strategy for acute leukemia, since it is observed that HDAC6 is overexpressed in acute leukemia and regulates tumor survival. Combination therapy for cancer is used to minimize adverse drug effects, reduce drug dosage, enhance efficacy, and prevent drug resistance. In order to improve efficacy of chemotherapy agents of acute leukemia, this study will investigate the effects of combination MPT0G211, a novel histone deacetylase 6 inhibitor, with doxorubicin or vincristine on human acute leukemia cells. RESULTS: MPT0G211 combined with doxorubicin induces DNA damage response on human acute myeloid leukemia cells. MPT0G211 can additionally increase Ku70 acetylation and release BAX to mitochondria. Ectopic expression of HDAC6 successively reversed the apoptosis triggered by the combined treatment. Moreover, co-treatment of MPT0G211 and vincristine may alter microtubule dynamics, triggering acute lymphoblastic leukemia cells arrest in mitotic phase followed by induction of the apoptotic pathway. Finally, MPT0G211 plus doxorubicin or vincristine can significantly improve the tumor growth delay in a tumor xenograft model. CONCLUSIONS: Collectively, our data highlighted that MPT0G211 in combination with chemotherapy drugs has significant anticancer activity, suggesting a novel strategy for the treatment of acute leukemia.

Impairment of social behaviors in Arhgef10 knockout mice.

Background: Impaired social interaction is one of the essential features of autism spectrum disorder (ASD). Our previous copy number variation (CNV) study discovered a novel deleted region associated with ASD. One of the genes included in the deleted region is ARHGEF10. A missense mutation of ARHGEF10 has been reported to be one of the contributing factors in several diseases of the central nervous system. However, the relationship between the loss of ARHGEF10 and the clinical symptoms of ASD is unclear. Methods: We generated Arhgef10 knockout mice as a model of ASD and characterized the social behavior and the biochemical changes in the brains of the knockout mice. Results: Compared with their wild-type littermates, the Arhgef10-depleted mice showed social interaction impairment, hyperactivity, and decreased depression-like and anxiety-like behavior. Behavioral measures of learning in the Morris water maze were not affected by Arhgef10 deficiency. Moreover, neurotransmitters including serotonin, norepinephrine, and dopamine were significantly increased in different brain regions of the Arhgef10 knockout mice. In addition, monoamine oxidase A (MAO-A) decreased in several brain regions. Conclusions: These results suggest that ARHGEF10 is a candidate risk gene for ASD and that the Arhgef10 knockout model could be a tool for studying the mechanisms of neurotransmission in ASD. Trial registration: Animal studies were approved by the Institutional Animal Care and Use Committee of National Taiwan University (IACUC 20150023). Registered 1 August 2015.

A Novel Selective JAK2 Inhibitor Identified Using Pharmacological Interactions.

The JAK2/STAT signaling pathway mediates cytokine receptor signals that are involved in cell growth, survival and homeostasis. JAK2 is a member of the Janus kinase (JAK) family and aberrant JAK2/STAT is involved with various diseases, making the pathway a therapeutic target. The similarity between the ATP binding site of protein kinases has made development of specific inhibitors difficult. Current JAK2 inhibitors are not selective and produce unwanted side effects. It is thought that increasing selectivity of kinase inhibitors may reduce the side effects seen with current treatment options. Thus, there is a great need for a selective JAK inhibitor. In this study, we identified a JAK2 specific inhibitor. We first identified key pharmacological interactions in the JAK2 binding site by analyzing known JAK2 inhibitors. Then, we performed structure-based virtual screening and filtered compounds based on their pharmacological interactions and identified compound NSC13626 as a potential JAK2 inhibitor. Results of enzymatic assays revealed that against a panel of kinases, compound NSC13626 is a JAK2 inhibitor and has high selectivity toward the JAK2 and JAK3 isozymes. Our cellular assays revealed that compound NSC13626 inhibits colorectal cancer cell (CRC) growth by downregulating phosphorylation of STAT3 and arresting the cell cycle in the S phase. Thus, we believe that compound NSC13626 has potential to be further optimized as a selective JAK2 drug.

Inhibition of osteoporosis by the αvß3 integrin antagonist of rhodostomin variants.

Integrins are heterodimeric cell surface receptors that mediate cell-cell and cell-matrix interaction. The vitronectin and osteopontin receptor αvß3 integrin has increased expression levels and is implicated in the adhesion, activation, and migration of osteoclasts on the bone surface as well as osteoclast polarization. αvß3 integrin plays an important role in osteoclast differentiation and resorption. In addition, Arg-Gly-Asp (RGD)-containing peptides, small molecular inhibitors, and antibodies to αvß3 integrin have been shown to inhibit bone resorption in vitro and in vivo. Here we examined the effects of a disintegrin HSA-ARLDDL a genetically modified mutant of rhodostomin conjugated with human serum albumin, which is highly selective of αvß3, on RANKL-induced osteoclastogenesis and ovariectomy (OVX)-induced osteoporosis. In RANKL-induced osteoclastogenesis, HSA-ARLDDL significantly inhibited osteoclast formation, and IC50 was at nM range. Post-treatment HSA-ARLDDL also inhibits osteoclast formation. Furthermore, weekly administration of HSA-ARLDDL significantly inhibits the increase in serum bone resorption marker levels and decrease in cancellous bone loss in tibia and femur induced by OVX. On the other hand, HSA-ARLDDL did not affect the differentiation and calcium deposition of osteoblasts. These results indicate that the highly selective and long-acting αvß3 integrin antagonists could be developed as effective drugs for postmenopausal osteoporosis.

Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability.

Here, we report a novel non-epigenetic function of histone deacetylase (HDAC) 8 in activating cancer stem cell (CSC)-like properties in breast cancer cells by enhancing the stability of Notch1 protein. The pan-HDAC inhibitors AR-42 and SAHA, and the class I HDAC inhibitor depsipeptide, suppressed mammosphere formation and other CSC markers by reducing Notch1 expression in MDA-MB-231 and SUM-159 cells. Interrogation of individual class I isoforms (HDAC1-3 and 8) using si/shRNA-mediated knockdown, ectopic expression and/or pharmacological inhibition revealed HDAC8 to be the primary mediator of this drug effect. This suppression of Notch1 in response to HDAC8 inhibition was abrogated by the proteasome inhibitor MG132 and siRNA-induced silencing of Fbwx7, indicating Notch1 suppression occurred through proteasomal degradation. However, co-immunoprecipitation analysis indicated that HDAC8 did not form complexes with Notch1 and HDAC inhibition had no effect on Notch1 acetylation. In a xenograft tumor model, the tumorigenicity of breast cancer cells was decreased by HDAC8 knockdown. These findings suggest the therapeutic potential of HDAC8 inhibition to suppress Notch1 signaling in breast cancer.