Flavopiridol Research Articles

Flavopiridol inhibits oocyte maturation, reduces oocyte quality and blocks cumulus cell function

Flavopiridol (FP) is a plant-derived flavonoidis and used to treat cancers, fungal infections and inflammation-related diseases. However, it is not clear whether it has side effects on the female reproductive system. In this study, we aimed to investigate the toxic effects and potential underlying mechanisms of FP on oocyte maturation and cumulus cell expansion in mice. Cumulus-oocyte complexes (COCs) were cultured in vitro with FP of gradient concentration (50–1000 nM), according to the plasma concentration of FP in the clinical trial. The maturation rate and cumulus expansion index of oocytes were counted and studied by immunofluorescence staining, qRT-PCR, oocyte chromosome preparation and so on. The results showed that the FP-exposed COCs inhibited the oocyte maturation and cumulus cell expansion, leading to cell apoptosis in a dose dependent way. Oocytes exposed to 500 nM FP showed abnormalities in the spindle structure and chromosome arrangement, ultimately leading to the oocyte maturation arrest and aneuploidy. This may be due to the excessive oxidative stress caused by mitochondrial membrane potential damage and mislocalization. Therefore, when FP is used for cancer treatment, its side effects on the female reproductive system should be seriously considered.

Targeted inhibition of MASTL kinase activity induces apoptosis in breast cancer

Microtubule-associated serine/threonine kinase-like (MASTL) (or Greatwall kinase (GWL)) is an important cell cycle regulating kinase that regulates the G2-M transition. Uncontrolled MASTL activity is implicated in breast cancer progression. To date, very few inhibitors have been reported against this protein. Here, structure-based computational modeling indicates that the natural product flavopiridol (FLV) binds strongly to MASTL and these results are validated using molecular dynamics simulation studies. An in vitro kinase assay reveals an EC50 (effective concentration) value of FLV to be 82.1 nM and a better IC50 compared to the positive reference compound, staurosporine. FLV is found to inhibit MASTL kinase activity, arresting the cell growth in the G1 phase and inducing apoptosis in breast cancer cells. Consistent with these results differential gene expression obtained using RNA sequencing studies, and validated by RT PCR and immunoblot analysis, indicate that MASTL inhibition induces cell cycle arrest and apoptotic-related genes. Furthermore, metastasis- and inflammation-related genes are downregulated. Thus, the deregulation of MASTL signaling pathways on targeted inhibition of its kinase activity is revealed. This study lays a strong foundation for investigating FLV as a lead compound in breast cancer therapeutics.

Analytical Quality-by-Design-Based Systematic Optimization of RP-HPLC Method Conditions to Analyze Simultaneously Chloroquine Phosphate and Flavopiridol in Stress-Induced Combined Drug Solution and Pharmaceutical Emulsions.

A design of experiments (DoE)-driven RP-HPLC method conditions was employed to analyze simultaneously chloroquine (CQ) phosphate and flavopiridol (FLAP) in emulsions and solution. After subjecting the various critical method attributes to preliminary risk assessment and screening by Pareto-chart-based fractional factorial design, the 17 runs were produced in Box-Behnken design for optimization. Analysis of variance, lack of fit, prediction equations, 3D response surface plots and contour plots were used to evaluate the critical analytical attributes such as retention time, tailing factor and theoretical plate count. The optimized RP-HPLC method conditions include 262nm as detection wavelength, 37°C temperature for column, 20-μl injection volume, 1-ml/min flow rate and mobile phase mixture [70:30 ratio of 0.4% triethylamine in methanol&sodium phosphate buffer (11mM, pH3.0)]. The studied validation parameters were found within the ICH-prescribed limits. Exposing the combined drug solution at oxidative stress condition resulted to diminish the FLAP recovery value (53.39 ± 0.86) and arrival of an extra chromatographic peak. However, the % drug entrapment efficiency values of 96.22 ± 2.47 and 85.86 ± 3.66, respectively, were noticed for CQ phosphate and FLAP in emulsions. Thus, DoE-driven approach could be helpful for systematically optimizing RP-HPLC method conditions.

DNMT3A Mutation-Induced CDK1 Overexpression Promotes Leukemogenesis by Modulating the Interaction between EZH2 and DNMT3A.

DNMT3A mutations are frequently identified in acute myeloid leukemia (AML) and indicate poor prognosis. Previously, we found that the hotspot mutation DNMT3A R882H could upregulate CDK1 and induce AML in conditional knock-in mice. However, the mechanism by which CDK1 is involved in leukemogenesis of DNMT3A mutation-related AML, and whether CDK1 could be a therapeutic target, remains unclear. In this study, using fluorescence resonance energy transfer and immunoprecipitation analysis, we discovered that increased CDK1 could compete with EZH2 to bind to the PHD-like motif of DNMT3A, which may disturb the protein interaction between EZH2 and DNMT3A. Knockdown of CDK1 in OCI-AML3 cells with DNMT3A mutation markedly inhibited proliferation and induced apoptosis. CDK1 selective inhibitor CGP74514A (CGP) and the pan-CDK inhibitor flavopiridol (FLA) arrested OCI-AML3 cells in the G2/M phase, and induced cell apoptosis. CGP significantly increased CD163-positive cells. Moreover, the combined application of CDK1 inhibitor and traditional chemotherapy drugs synergistically inhibited proliferation and induced apoptosis of OCI-AML3 cells. In conclusion, this study highlights CDK1 overexpression as a pathogenic factor and a potential therapeutic target for DNMT3A mutation-related AML.

On the inhibition of cytochrome P450 3A4 by structurally diversified flavonoids

Cytochrome P450 3A4 (CYP3A4) is the most versatile enzyme involved in drug metabolism. The time-dependent inhibition of CYP3A4 by acacetin, apigenin, chrysin, and pinocembrin was experimentally detected, but not entirely elaborated so far. Thus, a two-level QM/MM (Quantum Mechanics/Molecular Mechanics) model is developed to yield insights into the receptor-flavonoid recognition at the molecular scale. Active site residues and the flavonoid are modelled using SCC-DFTB-D (QM level), while the rest of the complex is treated using AMBER force field (MM level). QM/MM binding free energies are well correlated with experimental data, indicating the largest inhibitory effect of chrysin on enzyme activity at a submicromolar concentration. Consequently, quercetin (QUE) and flavopiridol (FLP) are observed as representative examples of structurally different flavonoids. The inhibition parameters for QUE and FLP are evaluated using the well-calibrated QM/MM strategy, thereby aiding to quantitatively conceive the functional behavior of the whole family of flavonoids. A kinetic threshold for further assessment of the drug-drug interactions underlying the time-dependent inhibition of CYP3A4 by flavonoids is explored. Communicated by Ramaswamy H. Sarma

Cyclin-Dependent Kinase 9 Inhibition Suppresses Necroptosis and Pyroptosis in the Progress of Endotoxemia.

The host innate immune response stands at the first line of defense against the outburst of pathogen invasion and their byproduct release. The balanced and coordinated expression of genes in normal immune responses is compromised in the progress of endotoxemia with exacerbated inflammation and massive cell death. In the present study, we identified cyclin-dependent kinase 9 (CDK9), the functional subunit of the positive transcription elongation factor b, as a master regulator of inflammatory gene transcription in the process of promoter-proximal pausing to productive elongation. Therapeutic pharmacological inhibition of CDK9 by flavopiridol (FVD) rescued mice from death in experimental models of fatal endotoxemia. In addition to alleviation of the cytokine storm in the circulation system following lethal endotoxin injection, FVD treatment significantly dampened the onset of inflammation in the livers and lungs and reduced the necroptosis and pyroptosis in livers. Moreover, CDK9 inhibition reduced inflammatory cytokine release and decreased cell death in the pro-inflammatory pyroptotic and necroptotic cell death pathway in monocytes in responses to lipopolysaccharide. In conclusion, CDK9 inhibition may affect the progress of endotoxemia by dampening inflammation and cell death including necroptosis and pyroptosis.

Sequential Alvocidib Followed by Cytarabine + Mitoxantrone in AML

Sequential Alvocidib Followed by Cytarabine + Mitoxantrone in AML

Role of microRNA-92b in the pathophysiology of acute transverse myelitis in children

Objective To explore microRNAs that play key regulatory roles in the pathophysiology of acute transverse myelitis in children, and to find therapeutic targets. Methods Twelve patients with acute transverse myelitis were enrolled as ATM group and three children with normal cerebrospinal fluid as the control group.MicroRNA in cerebrospinal fluid of children with acute transverse myelitis was detected by using microarray4.0 chip.Bioinformatics was used to demonstrate microRNA, which plays a key regulatory role, and to predict target genes.Real-time quantitative polymerase chain reaction (qPCR) technique was adopted for in biology and technology duplication.Enzyme-linked immunosorbent assay (ELISA) and Western blot technique were used to detect the expression of key miRNA target protein. The key candidate microRNA was inhibited/overexpressed in dorsal root ganglion neurons, and the function was verified in vitro.Flavopiridol was used to inhibit the activity of CDKs to verify that miR-92b worked through p57-CDKs-GAP-43 pathway. Results The characteristic elevation of miR-92b in cerebrospinal fluid samples of acute transverse myelitis was significant. Bioinformatics analysis showed that p57 was the target gene of miR-92b.The expression of miR-92b was contrary to the p57 protein.In vitro experiments showed that the length of axons in miR-92b mimics group was significantly shorter than that in the blank group.The axons of neurons in antimiR-92b group were obviously prolonged. In the miR-92b mimics+ Flavopiridol groups, the axons of neurons were still significantly prolonged compared with that in the blank group.Western blot showed that p57 and GAP-43 protein expression in miR-92b mimics group was lower than that in blank group.The expression of p57 and GAP-43 protein in antimiR-92b group was significantly higher than that in blank group.But in miR-92b mimics+ Flavopiridol group, the expression of p57 was lower compared with that in blank group, and the expression of GAP-43 protein was higher compared with that in blank group. Conclusions Up-regulation of miR-92b in children with acute transverse myelitis leads to a down-regulation of p57.The activity of CDKs is enhanced, which inhibits the expression of GAP-43 protein and the regeneration of axons in spinal cord injury region. MiR-92b is one of the key targets in the treatment of children with acute transverse myelitis. Key words: Acute transverse myelitis; Child; Cerebrospinal fluid; MicroRNA; Therapeutic target

Preferential Binding of Flavonoids with Bovine Serum Albumin: In-Silico and Spectroscopic Insight into Cytotoxic Competence

Flavonoids are polyphenolic secondary metabolites with numerous pharmacological advantages including their competence as anticancer molecules. Flavopiridol (FLAP) and riviciclib (RCB) are semi-synthetic analogs of a natural flavonoid rohitukine, isolated from the stem bark of an Indian medicinal plant, Dysoxylum binectariferum. These are cyclin-dependent kinase inhibitors (CDKIs), however, their specificity for biomolecules and mechanism of inhibition inside cell is not well deciphered. Considering the significance these inhibitors in cancer therapeutics, herein, we explored the molecular interaction mechanism between FLAP/RCB and bovine serum albumin (BSA) using various spectroscopic and molecular docking methods. Surface enhanced Raman, circular dichroism and absorption spectroscopy have been employed to understand the structural-conformational aspects and other binding parameters between FLAP/RCB and BSA. Vibrational spectroscopic studies reveal that interacts with the amino acid residues of BSA without interrupting with the microenvironment of around the residue Trp213. Further, the FLAP-BSA interactions are stabilized by hydrophobic and electrostatic forces, whereas, hydrophobic interactions are observed for binding between RCB and BSA. Comparative experimental results illustrate that the primary binding sites for FLAP and RCB on BSA are sites II and III. Conformationally, there is reduction in alpha-helical content of BSA, when complexed with FLAP/RCB indicating partial unfolding of BSA and increase in the turns and random coil structures of protein. The binding constant (Ka) estimated for the complexation is of the order of 105 and 104 for FLAP/RCB-BSA complexes respectively, specifying moderate to strong binding affinity. Further, molecular docking simulations have been performed, which are in corroboration with spectroscopic outcomes. Hence, the findings concluded here, paves a way in delineating the action mechanism of flavonoids that may lead to rational designing of flavonoid based new therapeutics and recognizing BSA as molecular target.

Flavopiridol Strongly Sensitizes Canine Lymphoma Cells to TRAIL-induced Apoptosis.

Targeting the extrinsic apoptotic pathway is an interesting option for anticancer therapy. A protein which such ability is Apo2 ligand, also known as TNF-related apoptosis-inducing ligand (TRAIL). The aim of this study was to examine the possibility of sensitizing resistant CLBL-1 canine lymphoma cells to TRAIL-induced apoptosis by using flavopiridol (FVP) a cyclin-dependent kinase inhibitor (CDKs). The CLBL-1 (canine B-cell lymphoma cell line) was used in the study. The effect of FVP and TRAIL treatment on apoptosis induction was assessed by flow cytometry and western blot. Although canine lymphoma cells were resistant to TRAIL-induced apoptosis, combination of this death ligand with FVP was able to overcome TRAIL resistance of CLBL-1 lymphoma cells. Our results demonstrated that although canine lymphoma cells were resistant to TRAIL-induced apoptosis, combination of this death ligand with FVP was able to overcome TRAIL resistance of CLBL-1 lymphoma cell line. Although further investigation is required to deepen the knowledge of TRAIL as an antitumor agent in canine cancers, our results open the door to future use of TRAIL-based treatment strategies in veterinary oncology.

Autophagy is essential for flavopiridol‑induced cytotoxicity against MCF‑7 breast cancer cells.

Flavopiridol (FP) exerts antitumoral effects by triggering tumor cell cycle arrest and cytotoxicity in human breast cancer cell lines. The potent antitumor activity of FP is through its inhibition of cyclin‑dependent kinases; however, this may not be the only mechanism of action. The present study aimed to investigate whether FP is able to induce autophagy and to examine the effects of autophagy on cell death in FP‑treated MCF‑7 human breast cancer cells. MCF‑7 cells were treated with either FP alone or FP in combination with chloroquine (CQ). Expression levels of autophagy‑related protein LC3B‑II and p62/sequestosome 1 (SQSTM1) were used to monitor autophagic flux. MCF‑7 cells were transfected with autophagy‑related 5 (ATG5) small interfering (si)RNA to block autophagy. Cell viability and cell cycle status were determined. Following incubation with FP, MCF‑7 cells exhibited significantly higher autophagy compared with untreated control cells, and the level of autophagy is comparable with cells under rapamycin induction, which was verified by immunodetection of LC3B‑II and p62/SQSTM1 expression and inhibition by CQ. The addition of CQ treatment or ATG5‑siRNA transfection against autophagy components attenuated the cytotoxic effects of FP treatment of MCF‑7 cells. Furthermore, this autophagy inhibition did not impair the FP‑induced cell cycle arrest. These results revealed that autophagy may be involved in FP‑induced MCF‑7 cell death and autophagy inhibition enhanced the tumor cell pro‑survival ability. It is possibly that potential autophagy regulatory drugs may be used as a chemotherapy adjuvant.

Metabolic reprogramming is associated with flavopiridol resistance in prostate cancer DU145 cells

Flavopiridol (FP) is a pan-cyclin dependent kinase inhibitor, which shows strong efficacy in inducing cancer cell apoptosis. Although FP is potent against most cancer cells in vitro, unfortunately it proved less efficacious in clinical trials in various aggressive cancers. To date, the molecular mechanisms of the FP resistance are mostly unknown. Here, we report that a small fraction human prostate cancer DU145 cells can survive long-term FP treatment and emerge as FP-resistant cells (DU145FP). These DU145FP cells show accumulated mitochondrial lesions with stronger glycolytic features, and they proliferate in slow-cycling and behave highly migratory with strong anti-apoptotic potential. In addition, the cells are less sensitive to cisplatin and docetaxel-induced apoptotic pressure, and over-express multiple stem cell associated biomarkers. Our studies collectively uncover for the first time that FP-resistant prostate cancer cells show metabolic remodeling, and the metabolic plasticity might be required for the FP resistance-associated cancer cell stemness up-regulation.

Effect of flavopiridol on the proliferation, invasiveness and apoptosis of human prostatic cancer cell line LNCaP

Objective To investigate the effect of flavopiridol on the proliferation, invasiveness and apoptosis of human prostate cancer cell line LNCaP, and to explore the possibility of its application in clinical treatment. Methods MTT assay was used to detect cell proliferation, cell invasion in vitro was detected by Transwell assay, and flow cytometer was used to observe apoptosis. Results Flavopiridol inhibited the growth of LNCaP cells in a concentration-dependent and time-dependent way (P < 0.05), and reduced the ability of invasion capacity. After treated by 10 nmol/L flavopiridol for 24 h, the apoptosis rate was increased significantly to (7.5±0.9) % compared with the control group [(5.3±0.5) %] (P < 0.05). Conclusion Flavopiridol can inhibit proliferation of LNCaP cells and induce apoptosis, which may be applicable for the treatment of prostate cancer. Key words: Prostatic neoplasms; Flavopiridol; Proliferation; Apoptosis; Invasiveness

Improved Anti-Tumor Activity of Novel Highly Bioactive Liposome-Bound TRAIL in Breast Cancer Cells.

Apo2-ligand/TRAIL, a member of the TNF cytokine superfamily capable of inducing apoptosis on tumor cells while sparing normal cells, is a promising anti-tumor agent. However, about 50% of human cancer are TRAIL resistant. Consequently, future TRAIL-based therapies will require the use of novel highly bioactive forms of TRAIL and/or the addition of sensitizing agents to TRAIL-induced apoptosis. Recently, we demonstrated that artificial lipid nanoparticles coated with bioactive TRAIL (LUV-TRAIL) greatly improved TRAIL activity and were able to induce apoptosis in chemoresistant hematological tumor cells. In this study, we have tested LUV-TRAIL-pro-apoptotic potential in human breast cancer. Comparative analysis of cytotoxicity induced by sTRAIL and LUV-TRAIL was performed using several human breast tumor cell lines with different sensitivity to TRAIL. In vivo anti-tumor activity of LUV-TRAIL was also studied using a xeno-graft tumor model. LUV-TRAIL improved not only sTRAIL in vitro cytotoxicity in all breast tumor cell lines tested but also showed more anti-tumor activity than sTRAIL in an in vivo xeno-graft tumor model. On the other hand, the concomitant treatment of LUV-TRAIL with the sensitizing agent flavopiridol (FVP) induced a higher level of cell death in TRAIL-resistant cell lines. TRAIL-sensitization induced by FVP was mediated by DR5 up-regulation, and interestingly TRAIL-apoptotic signaling was completely shifted towards DR5 upon FVP treatment. LUV-TRAIL could especially take advantage of this DR5 up-regulation, while sTRAIL was not able. To date, no special attention had been paid to this aspect of FVP-induced TRAIL-sensitization, may be because sTRAIL used were not able to take advantage of this DR5 up-regulation. Hence, LUV-TRAIL could be a better choice than sTRAIL to be used in combination with anti-tumor drugs inducing DR5 over-expression, since LUV-TRAIL is especially effective activating this death-receptor.

Abstract 2623: Molecular mechanism of synergy between BET-protein bromodomain antagonist (BA) and pTEFb kinase inhibitor against human AML blast progenitor cells

Abstract We have previously reported that treatment with BET-protein bromodomain antagonist (BA) reduces the enhancer/promoter occupancy of BRD4 and induces the expression of HEXIM1, which sequesters and inhibits pTEFb (a complex of CDK9 and cyclin T). pTEFb phosphorylates serine 2 in the C-terminal heptad repeats in the stalled RNA pol II (RNAP2), causing its pause-release and the mRNA transcript elongation. While relatively sparing normal bone marrow progenitor cells, BA treatment attenuates the expression of cMYC, BCL2 and CDK4/6 and induces apoptosis of cultured (OCI-AML3, MOLM13 and MV4-11) and primary AML blast progenitor cells (BPCs), including those expressing FLT3-ITD. Because of its ability to inhibit CDK9 and pTEFb, we determined whether co-treatment with flavopiridol (FP), a known inhibitor of CDK9, would enhance the activity of BA against the cultured and primary AML BPCs. Our findings demonstrate that co-treatment with BA and FP synergistically induced apoptosis of the cultured and primary AML BPCs (CI of &amp;lt; 1.0). Combination of BA and FP, versus each drug alone, was associated with greater attenuation of the levels of p-RNAP2, cMYC, BCL-2 and CDK4/6 but greater induction of HEXIM1 and p21 levels. Notably, co-treatment with FP and BA markedly reduced the levels of the anti-apoptotic protein MCL1, which is known to be transcriptionally regulated by pTEFb-mediated phosphorylation of p-RNAP2. However, treatment with BA and FP did not affect the levels of cyclin T or CDK9. Co-treatment with BA and FP also synergistically induced apoptosis of MOLM13-TKIR cells (expressing FLT3-ITD) that exhibit &amp;gt; 100-fold in vitro resistance to FLT3 tyrosine kinase inhibitors. We next determined whether HEXIM1 levels regulate the activity of BA and FP in the AML BPCs. While shRNA-mediated stable knockdown of HEXIM1 significantly reduced the lethal activity of BA in OCI-AML3/HKD and MOLM13/HKD cells, tetracycline-inducible ectopic overexpression of HEXIM1 significantly enhanced BA-induced apoptosis of MOLM13 and OCI-AML3 cells. Notably, the significant improvement in the median survival of NOD/SCID mice treated with JQ1 (50 mg/kg/5 days per week/for 3 weeks) was not observed in the similarly treated mice engrafted with MOLM13/HKD cells (p &amp;lt; 0.01). The lethality induced by co-treatment with JQ1 and FP was significantly reduced in MOLM13/HKD versus MOLM13 cells; conversely, it was significantly increased in MOLM13 cells with ectopic overexpression of HEXIM1 (p &amp;lt; 0.01). These findings indicate that co-treatment with BA and FP exerts synergistic lethality, and BA-mediated induction of HEXIM1 is mechanistically involved in mediating the lethal effects of BA and FP against human AML BPCs. Citation Format: Santhana G. T. Devaraj, Bhavin Shah, Warren Fiskus, Baohua Sun, Saikat Saha, Sai Ravi Pingali, Swaminathan P. Iyer, Sunil Sharma, James E. Bradner, Kapil N. Bhalla. Molecular mechanism of synergy between BET-protein bromodomain antagonist (BA) and pTEFb kinase inhibitor against human AML blast progenitor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2623. doi:10.1158/1538-7445.AM2015-2623

Sequence-dependent binding of flavonoids to duplex DNA.

The whole family of structurally distinct flavonoids has been recognized as a valuable source of prospective anticancer agents. There is experimental evidence demonstrating that some flavonoids, like flavopiridol (FLP) and quercetin (QUE), bind to DNA influencing their key physiological function. FLP is involved in the combined mode of interaction (intercalation and minor groove binding), while QUE is viewed as a minor groove binder. From a physical standpoint, experimental and theoretical studies have not so far provided a sufficiently consistent picture of the nature of interaction with DNA. Herein the sequence-dependent binding of FLP and of QUE (two representative examples of the structurally different flavonoids) with duplex DNA, containing a variety of the sequences of eight nucleotides (I: GGGGCCCC, II: GGCCGGCC, III: AAAATTTT, IV: AAGCGCTT, V: GCGCGCGC) in the 5'-strand, is investigated using a sophisticated molecular dynamics (MD) approach. For various parts (helix, backbone, bases) of the DNA structure, the change of asymptotic (in terms of an infinite length of MD simulation) configurational entropy, being the thermodynamic consequence of DNA flexibility change due to ligand binding, is explored. As far as the sequence-dependent extent of DNA flexibility change upon QUE (or FLP) binding is concerned, for the entire double helix, increased flexibility is observed for I (or I ≈ II), while increased rigidity is found to be in the order of V > III > II > IV (or III > V > IV) for the rest of sequences. For the backbone, increased rigidity in the order of V > III > II > IV > I (or III > V > IV > I > II) is generally observed. For the nucleobases, increased flexibility is determined for I and II (I > II for both ligands), while increased rigidity in the order of V ≈ III > IV (or III > V > IV) is reported for the other sequences. Of the overall increased rigidity of the DNA structure upon ligand binding that is observed for the sequences III, IV, and V, about 50-70% comes from the sugar-phosphate backbone. Noteworthy is that the increased flexibility of the entire double helix and of the complete system of nucleobases upon ligand binding is only established for sequence I. The insights are further subtly substantiated by considering the configurational entropy contributions at the level of individual nucleobase pairs and of individual nucleo-base pair steps and by analyzing the sequence dependent estimates of intra-base pair entropy and inter-base pair entropy. The GGC triplet, which is part of the central tetramer (GGCC) of I, is concluded to be critical for binding of flavonoids, while the effect of the presence of ligand to the flexibility of nucleobases is localized through the intra-base pair motion of the intercalation site and its immediate vicinity. G-rich DNA sequences with consecutive Gs going before and/or after the critical GGC code (such as I: GGGGCCCC) are proposed to be uniquely specific for flavonoids. The configurational entropy contribution, as an upper bound of the true entropy contribution to the free energy in noncovalent binding, is demonstrated to influence the fundamental discrimination (intercalation vs groove binding) of DNA-flavonoid recognition modes. Some interesting implications for the structure-based design of optimal DNA binders are discussed.

Discovery of novel 5-fluoro-N2,N4-diphenylpyrimidine-2,4-diamines as potent inhibitors against CDK2 and CDK9.

Based on a 3D-QSAR pharmacophore derived from a diverse set of known cyclin-dependent kinase 9 (CDK9) inhibitors and a composite pharmacophore extracted from the complex structure of flavopiridol (FVP)-CDK9, thirty novel 5-fluoro-N2,N4-diphenylpyrimidine-2,4-diamine derivatives were designed and synthesized. Initial tests against four tumor cell lines with the sulforhodamine B (SRB) assay identified a series of potent compounds with GI50 values at lower micromolar or submicromolar level. Most of the highly cytotoxic compounds exhibited potent inhibitory activities against both CDK2/cyclin E1 and CDK9/cyclin T1. Notably, inhibitions against the two enzymes were generally correlated well with the cytotoxicity of these compounds. Appreciable inhibition was also observed for selected compounds in the anti-HIV-1 assay. Docking studies on compounds 6d and 9g provided conducive clues to further structural optimization.

JUNB promotes the survival of Flavopiridol treated human breast cancer cells

Chemotherapy resistance is a major obstacle to achieving durable progression-free-survival in breast cancer patients. Identifying resistance mechanisms is crucial to the development of effective breast cancer therapies. Immediate early genes (IEGs) function in the initial cellular reprogramming response to alterations in the extracellular environment and IEGs have been implicated in cancer cell development and progression. The purpose of this study was to investigate the influence of kinase inhibitors on IEG expression in breast cancer cells. The results demonstrated that Flavopiridol (FP), a CDK9 inhibitor, effectively reduced gene expression. FP treatment, however, consistently produced a delayed induction of JUNB gene expression in multiple breast cancer cell lines. Similar results were obtained with Sorafenib, a multi-kinase inhibitor and U0126, a MEK1 inhibitor. Functional studies revealed that JUNB plays a pro-survival role in kinase inhibitor treated breast cancer cells. These results demonstrate a unique induction of JUNB in response to kinase inhibitor therapies that may be among the earliest events in the progression to treatment resistance.

Comparative drug screening in NUT midline carcinoma

Background:The NUT midline carcinoma (NMC) is a rare but fatal cancer for which systematic testing of therapy options has never been performed.Methods:On the basis of disease biology, we compared the efficacy of the CDK9 inhibitor flavopiridol (FP) with a panel of anticancer agents in NMC cell lines and mouse xenografts.Results:In vitro anthracyclines, topoisomerase inhibitors, and microtubule poisons were among the most cytotoxic drug classes for NMC cells, while efficacy of the bromodomain inhibitor JQ1 varied considerably between lines carrying different BRD4 (bromodomain-containing protein 4)–NUT (nuclear protein in testis) translocations. Efficacy of FP was comparable to vincristine and doxorubicin, drugs that have been previously used in NMC patients. All three compounds showed significantly better activity than etoposide and vorinostat, agents that have also been used in NMC patients. Statins and antimetabolites demonstrated intermediate single-agent efficacy. In vivo, vincristine significantly inhibited tumour growth in two different NMC xenografts. Flavopiridol in vivo was significantly effective in one of the two NMC xenograft lines, demonstrating the biological heterogeneity of this disease.Conclusions:These results demonstrate that FP may be of benefit to a subset of patients with NMC, and warrant a continued emphasis on microtubule inhibitors, anthracyclines, and topoisomerase inhibitors as effective drug classes in this disease.

Inhibition of cdk9 during Herpes Simplex Virus 1 Infection Impedes Viral Transcription

During herpes simplex virus 1 (HSV-1) infection there is a loss of the serine-2 phosphorylated form of RNA polymerase II (RNAP II) found in elongation complexes. This occurs in part because RNAP II undergoes ubiquitination and proteasomal degradation during times of highly active viral transcription, which may result from stalled elongating complexes. In addition, a viral protein, ICP22, was reported to trigger a loss of serine-2 RNAP II. These findings have led to some speculation that the serine-2 phosphorylated form of RNAP II may not be required for HSV-1 transcription, although this form is required for cellular transcription elongation and RNA processing. Cellular kinase cdk9 phosphorylates serine-2 in the C-terminal domain (CTD) of RNAP II. To determine if serine-2 phosphorylated RNAP II is required for HSV-1 transcription, we inhibited cdk9 during HSV-1 infection and measured viral gene expression. Inhibition was achieved by adding cdk9 inhibitors 5,6-dichlorobenzimidazone-1-β-D-ribofuranoside (DRB) or flavopiridol (FVP) or by expression of a dominant–negative cdk9 or HEXIM1, which in conjunction with 7SK snRNA inhibits cdk9 in complex with cyclin 1. Here we report that inhibition of cdk9 resulted in decreased viral yields and levels of late proteins, poor formation of viral transcription-replication compartments, reduced levels of poly(A)+ mRNA and decreased RNA synthesis as measured by uptake of 5-bromouridine into nascent RNA. Importantly, a global reduction in viral mRNAs was seen as determined by microarray analysis. We conclude that serine-2 phosphorylation of the CTD of RNAP II is required for HSV-1 transcription.