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.

Synthesis and evaluation of potent (iso)ellipticine-based inhibitors of MYLK4 accessed via expeditious synthesis from isoquinolin-5-ol.

The K2S2O8-mediated generation of p-iminoquinone contributed to the regioselective substitution of isoquinolin-5,8-dione. This hydroxyl group-guided substitution was also applied to selected heterocycles and addressed the regioselectivity issue of quinones. This study has provided an expeditious pathway from isoquinolin-5-ol (5) to ellipticine (1) and isoellipticine (2), which benefits the comprehensive comparison of their activity. Compounds 1 and 2 displayed marked MYLK4 inhibitory activity with IC50 values of 7.1 and 6.1 nM, respectively. In the cellular activity of AML cells (MV-4-11 and MOLM-13), compound 1 showed better AML activity than compound 2.

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.

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.

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.

Structure-based virtual screening and biological evaluation of novel small-molecule BTK inhibitors.

Bruton tyrosine kinase (BTK) is linked to multiple signalling pathways that regulate cellular survival, activation, and proliferation. A covalent BTK inhibitor has shown favourable outcomes for treating B cell malignant leukaemia. However, covalent inhibitors require a high reactive warhead that may contribute to unexpected toxicity, poor selectivity, or reduced effectiveness in solid tumours. Herein, we report the identification of a novel noncovalent BTK inhibitor. The binding interactions (i.e. interactions from known BTK inhibitors) for the BTK binding site were identified and incorporated into a structure-based virtual screening (SBVS). Top-rank compounds were selected and testing revealed a BTK inhibitor with >50% inhibition at 10 µM concentration. Examining analogues revealed further BTK inhibitors. When tested across solid tumour cell lines, one inhibitor showed favourable inhibitory activity, suggesting its potential for targeting BTK malignant tumours. This inhibitor could serve as a basis for developing an effective BTK inhibitor targeting solid cancers.

Discovery of a novel cyclin-dependent kinase 8 inhibitor with an oxindole core for anti-inflammatory treatment.

Chronic inflammation is an underlying cause in a number of diseases. Cyclin-dependent kinase 8 (CDK8) has been implicated as an inflammatory mediator, indicating its potential as an anti-inflammatory target. Herein, we performed structure-based virtual screening (SBVS) to identify novel CDK8 inhibitors. The pharmacological interactions for CDK8 were identified and incorporated into a SBVS protocol. Selected compounds were tested in enzymatic assays, and one compound was confirmed to be a CDK8 inhibitor with a 50% inhibitory concentration (IC50) value of 1684.4 nM. Comparing structural analogs identified a compound, F059-1017, with greater potency (IC50 558.1 nM). When tested in cell lines, the compounds displayed low cytotoxicity. Cellular assays revealed that the identified CDK8 inhibitors can reduce phosphorylation and expression of signaling mediators associated with inflammation. In addition, results of kinase profiling showed that compound F059-1017 is selective towards CDK8. These findings suggest that the new inhibitors have great potential as lead compounds for developing novel anti-inflammatory therapeutics.

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.

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.

A site-moiety map and virtual screening approach for discovery of novel 5-LOX inhibitors.

The immune system works in conjunction with inflammation. Excessive inflammation underlies various human diseases, such as asthma, diabetes and heart disease. Previous studies found that 5-lipoxygenase (5-LOX) plays a crucial role in metabolizing arachidonic acid into inflammatory mediators and is a potential therapeutic target. In this study, we performed an in silico approach to establish a site-moiety map (SiMMap) to screen for new 5-LOX inhibitors. The map is composed of several anchors that contain key residues, moiety preferences, and their interaction types (i.e., electrostatic (E), hydrogen-bonding (H), and van der Waals (V) interactions) within the catalytic site. In total, we identified one EH, one H, and five V anchors, within the 5-LOX catalytic site. Based on the SiMMap, three 5-LOX inhibitors (YS1, YS2, and YS3) were identified. An enzyme-based assay validated inhibitory activity of YS1, YS2, and YS3 against 5-LOX with an IC50 value of 2.7, 4.2, and 5.3 µM, respectively. All three inhibitors significantly decrease LPS-induced TNF-α and IL-6 production, which suggests its potential use an anti-inflammatory agent. In addition, the identified 5-LOX inhibitors contain a novel scaffold. The discovery of these inhibitors presents an opportunity for designing specific anti-inflammatory drugs.

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.].

Isoindoline scaffold-based dual inhibitors of HDAC6 and HSP90 suppressing the growth of lung cancer in vitro and in vivo.

This study reports the synthesis of a series of 2-aroylisoindoline hydroxamic acids employing N-benzyl, long alkyl chain and acrylamide units as diverse linkers. In-vitro studies led to the identification of N-benzyl linker-bearing compound (10) and long chain linker-containing compound (17) as dual selective HDAC6/HSP90 inhibitors. Compound 17 displays potent inhibition of HDAC6 isoform (IC50 = 4.3 nM) and HSP90a inhibition (IC50 = 46.8 nM) along with substantial cell growth inhibitory effects with GI50 = 0.76 µM (lung A549) and GI50 = 0.52 µM (lung EGFR resistant H1975). Compound 10 displays potent antiproliferative activity against lung A549 (GI50 = 0.37 µM) and lung H1975 cell lines (GI50 = 0.13 µM) mediated through selective HDAC6 inhibition (IC50 = 33.3 nM) and HSP90 inhibition (IC50 = 66 nM). In addition, compound 17 also modulated the expression of signatory biomarkers associated with HDAC6 and HSP90 inhibition. In the in vivo efficacy evaluation in human H1975 xenografts, 17 induced slightly remarkable suppression of tumor growth both in monotherapy as well as the combination therapy with afatinib (20 mg/kg). Moreover, compound 17 could effectively reduce programmed death-ligand 1 (PD-L1) expression in IFN-γ treated lung H1975 cells in a dose dependent manner suggesting that dual inhibition of HDAC6 and HSP90 can modulate immunosuppressive ability of tumor area.

N-alkyl-hydroxybenzoyl anilide hydroxamates as dual inhibitors of HDAC and HSP90, downregulating IFN-γ induced PD-L1 expression.

Novel dual inhibitors of histone deacetylase (HDAC) and heat-shock protein 90 (HSP90) are synthesized and evaluated. These compounds are endowed with potent HDAC and HSP90 inhibitory activities with IC50 values in nanomolar range with Compound 20 (HDAC IC50 = 194 nM; HSP90α IC50 = 153 nM) and compound 26 (HDAC IC50 = 360 nM; HSP90α IC50 = 77 nM) displaying most potent HDAC and HSP90α inhibitory activities. Both of these compounds induce HSP70 expression and down regulate HSP90 client proteins which play important roles in the regulation of survival and invasiveness in cancer cells. In addition, compounds 20 and 26 induce acetylation of α-tubulin and histone H3. Significantly, compounds 20 and 26 could effectively reduce programmed death-ligand 1 (PD-L1) expression in IFN-γ treated lung H1975 cells in a dose dependent manner. These findings suggest that dual inhibition of HDAC and HSP90 that can modulate immunosuppressive ability of tumor area may provide a better therapeutic strategy for cancer treatment in the future.

Amide-tethered quinoline-resorcinol conjugates as a new class of HSP90 inhibitors suppressing the growth of prostate cancer cells.

The study is focused on the design and synthesis of amide tethered quinoline-resorcinol hybrid constructs as a new class of HSP90 inhibitor. In-vitro studies of the synthetic compounds led to the identification of compound 11, which possesses potent cell growth inhibitory effects against HCT116, Hep3B and PC-3 cell lines, exerted through HSP90 inhibition. Compound 11 triggers degradation of HSP90 client proteins along with concomitant induction of HSP70, demonstrates apoptosis inducing ability and causes G2M phase cell cycle arrest in PC-3 cells. Molecular modeling was used to dock compound 11 into the HSP90 active site and key interactions with the amino acid residues of the HSP90 chaperone protein were determined.

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.

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.

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.