Attractive Target For Cancer Chemotherapy Research Articles

An "In Schizo" Evaluation System to Screen for Human Kinesin-5 Inhibitors.

Kinesin-5 motor proteins are essential for mitotic spindle formation and maintenance, ensuring accurate chromosome segregation. Human kinesin-5 is highly expressed in various cancer cells but not in nonproliferative tissues; therefore, it is expected to be an attractive target for cancer chemotherapy, with fewer adverse side effects. Many inhibitors have been developed and subjected to clinical trials; however, they have not yet been commercially distributed because of their poor efficacy and frequent drug resistance. Establishing in vivo assay systems to easily monitor inhibitory activity is necessary and valuable to develop more effective inhibitors. Here, we report a procedure to evaluate the inhibitory activity against human kinesin-5 using a fission yeast-based system called "in schizo". Our approach could further be used to screen for inhibitors against kinesin-5 and other human cancer-related targets.

Discovery and Characterization of Peptide Inhibitors for Calcium and Integrin Binding Protein 1.

Calcium and integrin binding protein 1 (CIB1) is an EF-hand-containing, small intracellular protein that has recently been implicated in cancer cell survival and proliferation. In particular, CIB1 depletion significantly impairs tumor growth in triple-negative breast cancer (TNBC). Thus, CIB1 is a potentially attractive target for cancer chemotherapy that has yet to be validated by a chemical probe. To produce a probe molecule to the CIB1 helix 10 (H10) pocket and demonstrate that it is a viable target for molecular intervention, we employed random peptide phage display to screen and select CIB1-binding peptides. The top peptide sequence selected, UNC10245092, was produced synthetically, and binding to CIB1 was confirmed by isothermal titration calorimetry (ITC) and a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Both assays showed that the peptide bound to CIB1 with low nanomolar affinity. CIB1 was cocrystallized with UNC10245092, and the 2.1 Å resolution structure revealed that the peptide binds as an α-helix in the H10 pocket, displacing the CIB1 C-terminal H10 helix and causing conformational changes in H7 and H8. UNC10245092 was further derivatized with a C-terminal Tat-derived cell penetrating peptide (CPP) to demonstrate its effects on TNBC cells in culture, which are consistent with results of CIB1 depletion. These studies provide a first-in-class chemical tool for CIB1 inhibition in cell culture and validate the CIB1 H10 pocket for future probe and drug discovery efforts.

The Novel Nature Microtubule Inhibitor Ivalin Induces G2/M Arrest and Apoptosis in Human Hepatocellular Carcinoma SMMC-7721 Cells In Vitro.

Background and Objectives: Microtubules are an attractive target for cancer chemotherapy. Previously, we reported that Ivalin exhibited excellent anti-migration and anti-invasion activities in human breast cancer cells. Here, we examined the microtubule inhibition effect of Ivalin in human hepatocellular carcinoma SMMC-7721 cells. Materials and Methods: We used the 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate the cell proliferation effect of Ivalin and flow cytometry analysis to detect the apoptotic and cell cycle arrest effects of Ivalin. Immunofluorescence staining was used to measure the effect of Ivalin on the cytoskeleton network, and Western blotting was used to detect the expression levels of Bax, Bcl-2, Cdc2, phosphor-Cdc2, Cdc25A, Cyclin B1, and tubulin. Results: Ivalin induced cell cycle G2/M arrest and subsequent triggered apoptosis in human hepatocellular carcinoma SMMC-7721 cells. Furthermore, microtubules were shown to be involved in Ivalin-meditated apoptosis. In this connection, Ivalin treatment suppressed cellular microtubule network formation by regulating microtubule depolymerization. Moreover, Western blotting revealed Cdc25A and Cyclin B1 were upregulated in Ivalin-meditated cell cycle arrest. Subsequently, the induction of Bax (a proapoptotic protein) and reduction of Bcl-2 (an anti-apoptotic protein) expression were observed in Ivalin-treated SMMC-7721 cells. Conclusion: Ivalin induced microtubule depolymerization, then blocked cells in mitotic phase, and eventually resulted in apoptosis in SMMC-7721 cells. Collectively, these data indicate that Ivalin, acting as a novel inhibitor of microtubules, could be considered as a promising lead in anticancer drug development.

Intervenolin suppresses gastric cancer cell growth through the induction of TSP-1 secretion from fibroblast-like stromal cells.

Fibroblast-like stromal cells modulate the growth of cancer cells, both positively and negatively. Growth modulation is achieved through the secretion of regulatory factors as well as by proteins within the extracellular matrix. Those cellular interactions present attractive targets for cancer chemotherapy. It was demonsrated a novel natural compound, intervenolin (ITV), inhibited the in vitro growth of human gastric cancer cells when co-cultured with stromal cells. Importantly, the inhibition was enhanced by the presence of stromal cells. The present study reported a mechanism of ITV action. Human gastric fibroblast-like stromal cells (Hs738) were treated with ITV. The resultant conditioned medium (ITV CM) inhibited the growth of human gastric cancer cells and suppressed the level of c-Myc protein. This result suggested that ITV negatively modulated cancer cell growth by upregulating the secretion of factors originating from stromal cells in the co-culture system. To better understand the mechanism, ITV CM was subjected to proteomic analysis. The data revealed that one of the candidate regulators was thrombospondin-1 (TSP-1). Recombinant human TSP-1 protein inhibited the growth of gastric cancer cells. Moreover, the growth-inhibitory activities of ITV CM as well as that of recombinant TSP-1 were blocked by neutralizing antibody targeting TSP-1. These results suggested that ITV inhibited the growth of gastric cancer cells through its modulation of stromal cell function.

Abstract B205: Intervenolin suppresses gastric cancer cell growth via stromal secreted factors

Abstract Background: Tumor-stromal cell interactions are attractive targets for cancer chemotherapy because fibroblast-like stromal cells modulate cancer cells both positively and negatively through secreted factors as well as proteins of the extracellular matrix. We have focused on the negative regulation of cancer cells by secreted factors of stromal cells as it offers a novel strategy for discovering new cancer therapeutics. We used a coculture system to screen small molecules from natural sources such as microbial culture media, seeking molecules that modulated tumor-stromal cell interactions. In a recent study, we found a novel compound, intervenolin (ITV), from the culture medium of Nocardia sp. ML96-86F2. ITV inhibited the in vitro growth of human gastric cancer cells. Importantly, inhibition was greater when the cancer cells were cocultured with stromal cells. Here we report the mechanism of ITV action. Materials and Method: Human gastric cancer MKN-7 and MKN-74 cells were cultured with human gastric stromal Hs738 cells in the presence of ITV. The growth of MKN-7 and MKN-74 cells cultured in only CM taken from Hs738 cells treated with ITV (ITV CM) was also inhibited more strongly than it was cultured alone. We prepared serum-free CM from Hs738 cells to determine the anticancer factors secreted by ITV-treated HS738 cells using LC-MS/MS analysis. Results: Conditioned medium (CM) of ITV-treated Hs738 cells inhibited the growth of MKN-7 or MKN-74 cells and suppressed c-MYC protein level in MKN-74 cells. This result suggested that ITV induced the negative modulation of cancer cells through secreted factors from stromal cells. Proteome analysis of ITV-treated Hs738 CM revealed that one of the candidates could be thrombospondin-1 (TSP-1). As a result, recombinant TSP-1 inhibited the growth of gastric cancer cells and downregulated c-MYC. Conclusions: ITV inhibits gastric cancer cell growth through inducing TSP-1 secretion from Hs738 cells in coculture system in vitro. No conflict of interest. Citation Format: Junjiro Yoshida, Hikaru Abe, Takumi Watanabe, Manabu Kawada. Intervenolin suppresses gastric cancer cell growth via stromal secreted factors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B205.

From Natural Products to Designer Drugs: Development and Molecular Mechanisms Action of Novel Anti-Microtubule Breast Cancer Therapeutics.

Microtubule-targeted drugs (MTDs) have been on the forefront of breast cancer chemotherapy. Classic MTDs, such as paclitaxel and their semisynthetic derivatives, have achieved considerable success in the clinical management of breast neoplasms. In order to improve the specificity and to reduce undesirable, dose-limiting toxicities of these drugs, a plethora of novel compounds are being synthesized and investigated in laboratories worldwide. Due to their crucial roles during cell division, and to the fact that the suppression of their innate 'dynamic instability' can arrest cell cycle progression, microtubules formed an attractive target for cancer chemotherapy. Kadcyla (ado-trastuzumab emtansine), Halaven (eribulin mesylate), and Ixempra (Ixabepilone) are three relatively-novel, microtubule-targeting antibreast cancer drugs. Kadcyla was developed by conjugating a very potent derivative of the natural product maytansine to trastuzumab, a HER2-targeted monoclonal antibody. Kadcyla is a double-edged weapon, that is, it prevents receptor dimerization to inhibit cell proliferation, and then it enters inside the target tumour cell by receptor-mediated endocytosis and ensures death of the cell. Halaven (eribulin mesylate), created by simplifying the structure of the marine sponge-derived molecule Halichondrin B, works primarily by suppressing the growth rates of microtubules and thereby inducing cell cycle arrest and cell death. Ixabepilone, the semisynthetic analogue of epothilone B, suppresses the shortening rates of dynamic microtubules resulting in cell cycle inhibition and cell death. In order to improve the efficacy and reduce drug-induced side effects, novel therapeutic strategies, including liposome-mediated drug delivery, are being investigated.

Mechanism of microtubule stabilization by taccalonolide AJ

As a major component of the cytoskeleton, microtubules consist of αβ-tubulin heterodimers and have been recognized as attractive targets for cancer chemotherapy. Microtubule-stabilizing agents (MSAs) promote polymerization of tubulin and stabilize the polymer, preventing depolymerization. The molecular mechanisms by which MSAs stabilize microtubules remain elusive. Here we report a 2.05 Å crystal structure of tubulin complexed with taccalonolide AJ, a newly identified taxane-site MSA. Taccalonolide AJ covalently binds to β-tubulin D226. On AJ binding, the M-loop undergoes a conformational shift to facilitate tubulin polymerization. In this tubulin–AJ complex, the E-site of tubulin is occupied by GTP rather than GDP. Biochemical analyses confirm that AJ inhibits the hydrolysis of the E-site GTP. Thus, we propose that the β-tubulin E-site is locked into a GTP-preferred status by AJ binding. Our results provide experimental evidence for the connection between MSA binding and tubulin nucleotide state, and will help design new MSAs to overcome taxane resistance.

Chemotherapy Drug Response to the L858R-induced Conformational Change of EGFR Activation Loop in Lung Cancer.

Oncogenic L858R mutation of human epidermal growth factor receptor (EGFR) confers constitutive activation to the kinase and is frequently observed in the pathological process of metastatic lung cancer. Selective inhibition of EGFRL858R mutant over wild-type EGFR (EGFRWT ) has been established as an attractive target for cancer chemotherapy. Here, we performed long-term molecular dynamics (MD) simulations to reconstruct the complete dynamics trajectory of L858R-induced conformational change in EGFR activation loop (A-loop). It was found that the mutation considerably destabilizes A-loop in Src-like inactive conformation and promotes the loop conversion to DFG-in active form. Electrostatic force is primarily responsible for the conversion and stabilization upon the mutation. Binding free energy analysis revealed that Gefitinib exhibits strong selectivity for mutant over wild-type kinases. The A-loop conformation, but not L858R mutation, directly determines inhibitor affinity; the mutation can indirectly influence inhibitor binding via regulation of A-loop conformation. Subsequently, chemical similarity searching was carried out with the structural sketch of Gefitinib against a large library of drug/lead-like compounds, from which two hits were identified to have high selectivity for EGFRL858R over EGFRW ; they can potently inhibit the kinase mutant with IC50 values at nanomolar level. The selectivity is primarily originated from hydrogen bond interactions of inhibitor ligands with mutant but not with wild type due to the A-loop conformational difference.

Structures of a diverse set of colchicine binding site inhibitors in complex with tubulin provide a rationale for drug discovery.

Microtubules are dynamic assemblies of αβ-tubulin heterodimers and have been recognized as highly attractive targets for cancer chemotherapy. A broad range of agents bind to tubulin and interfere with microtubule assembly. Despite having a long history of characterization, colchicine binding site inhibitors (CBSIs) have not yet reached the commercial phase as anti-cancer drugs to date. We determined the structures of tubulin complexed with a set of structurally diverse CBSIs (lexibulin, nocodazole, plinabulin and tivantinib), among which nocodazole and tivantinib are both binary-function inhibitors targeting cancer-related kinases and microtubules simultaneously. High resolution structures revealed the detailed interactions between these ligands and tubulin. Our results showed that the binding modes of the CBSIs were different from previous docking models, highlighting the importance of crystal structure information in structure-based drug design. A real structure-based pharmacophore was proposed to rationalize key common interactions of the CBSIs at the colchicine domain. Our studies provide a solid structural basis for developing new anti-cancer agents for the colchicine binding site. The atomic coordinates and structure factors for tubulin complexed with lexibulin, nocodazole, plinabulin and tivantinib have been deposited in the Protein Data Bank under accession codes 5CA0, 5CA1, 5C8Y and 5CB4, respectively.

Pgp efflux pump decreases the cytostatic effect of CENP-E inhibitor GSK923295

Human kinesin CENP-E is an attractive target for cancer chemotherapy. The allosteric CENP-E inhibitor GSK923295 was proposed as a promising anticancer compound with potent cytostatic effect. In our work, we have analyzed the influence of the Pgp efflux pump on the cytostatic effect of GSK923295. We have demonstrated that multidrug resistant MESSA Dx5 cells overexpressing Pgp are 70–80 times more resistant to GSK923295 than their parental counterpart MESSA cells. Addition of 20 µM verapamil restored the drug sensibility of MESSA Dx5 cells. Combinations of GSK923295 with verapamil showed nearly additive effects in MESSA and synergistic effects in MESSA Dx5 cells. Our results demonstrate that tumors possessing Pgp could be more resistant to GSK923295, and that overexpression of Pgp can decrease the therapeutic effect of this drug. Development of structural analogs of GSK923295 which would not be a substrate of the Pgp efflux pump or addition of Pgp pump inhibitors can significantly improve the cytostatic effect of this drug.

Targeting met mediated epithelial-mesenchymal transition in the treatment of breast cancer.

Mesenchymal epithelial transition factor receptor (Met) is a receptor tyrosine kinase that plays a critical role in promoting cancer cell malignant progression. Met is activated by its ligand hepatocyte growth factor (HGF). HGF-dependent Met activation plays an important role in stimulating epithelial-mesenchymal transition (EMT) in tumor cells, resulting in increased tumor cell proliferation, survival, motility, angiogenesis, invasion, and metastasis. The HGF/Met axis has thus attracted great interest as a potential target in the development of novel cancer therapies. In an effort to suppress tumor cell malignant progression, efforts have been made to develop agents capable of inhibiting inhibit Met-induced EMT, including specific Met tyrosine kinase inhibitors, HGF antagonists that interfere with HGF binding to Met, and antibodies that prevent Met activation and/or dimerization. Tocotrienols, a subgroup within the vitamin E family of compounds, display potent anticancer activity that results, at least in part, from inhibition of HGF-dependent Met activation and signaling. The present review will provide a brief summary of the increasing importance of the HGF/Met axis as an attractive target for cancer chemotherapy and the role of tocotrienols in suppressing Met activation, signaling and HGF-induced EMT in breast cancer cells. Evidence provided suggests that γ-tocotrienol therapy may afford significant benefit in the treatment of breast cancers characterized by Met dysregulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-014-0030-5) contains supplementary material, which is available to authorized users.

Abstract B53: Triterpene Fraction of Cucumis trigonus - A Novel Microtubule Inhibitor and Antitumor Agent, Induce Cell Death in Cancer Cells.

Abstract Taxol-based combination chemotherapy remains the predominant treatment for cancer. However, taxane related drug resistance and neurotoxicity have promoted us to develop substitute treatment strategies. Kinesis spindle protein which is crucial for bipolar spindal formation and duplicate chromosome separation in early phase of mitosis, has emerged as an attractive target for cancer chemotherapy. The Aim of study was to investigate the mechanism of inhibition of kinesis spindle protein that plays an essential role in establishing mitotic spindle bipolarity, by the novel small molecular inhibitor. Antiproliferative activity of triterpene fraction of cucumis trigonus(TF25CT) in cancer cell line was accessed by cell viability assay (MTT and SRB). TF25CT showed dose dependent inhibition of cell proliferation of cancer cells. The antiproliferative effect of TF25CT was also associated with induction of apoptosis as well as morphology changes and DNA fragment. The morphology of Apoptosis nuclei was quantified using Hoechst stating and PI fragmentation. The degree of DNA fragmentation was analyzed by using agarose gel electrophoresis. The observations indicate that induced apoptotic- liked in morphological changes such as cell shrinkage and blebbing. Suppression of microtubule dynamics is responsible for the ability of TF25CT to inhibit the mitotic progression and cell proliferation using fluorescent microscope, we examined the effect of TF25CT on dynamics of individual microtubule in two living human cancer cell line. Taken together the kinesis protein inhibitor represents an imported compound for further investigate as novel anticancer therapeutics. Citation Format: Viralkumar Laxmanbhai Patel, Ashish D. Wadhwani, Vijayan P. Triterpene fraction of Cucumis trigonus, a novel microtubule inhibitor and antitumor agent, induces cell death in cancer cells. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B53.

Structure–Activity Relationships of Carboline and Carbazole Derivatives as a Novel Class of ATP-Competitive Kinesin Spindle Protein Inhibitors

The kinesin spindle protein (KSP) is a mitotic kinesin involved in the establishment of a functional bipolar mitotic spindle during cell division. It is considered to be an attractive target for cancer chemotherapy with reduced side effects. Based on natural product scaffold-derived fused indole-based inhibitors and known biphenyl-type KSP inhibitors, various carboline and carbazole derivatives were synthesized and biologically evaluated. β-Carboline and lactam-fused carbazole derivatives exhibited remarkably potent KSP inhibitory activity and mitotic arrest in prometaphase with formation of an irregular monopolar spindle. The planar tri- and tetracyclic analogs inhibited KSP ATPase in an ATP-competitive manner just like biphenyl-type inhibitors.

Abstract 2986: Partial inhibition of Plk1 is cytotoxic despite normal spindle structure

Abstract Polo-like kinase 1 (Plk1) is a multifunctional mitotic kinase that has emerged as an attractive target for cancer chemotherapy. Frequently over-expressed in tumors, Plk1 is absent in the surrounding non-proliferating tissue. Furthermore, Plk1 depletion in tumor cell lines produces monopolar spindles and mitotic arrest that ultimately leads to cell death. However, it is unclear whether monopolar spindles mediate cytotoxicity at physiologic drug concentrations. We used a chemical genetic system to test the effect of partial inhibition of Plk1 in human cells. We discovered that partial inhibition of Plk1 produces a cytotoxic response in the absence of a prolonged mitotic delay. We hypothesized that partial inhibition disrupts mitosis via alternate mechanisms from the archetypal monopolar spindle-induced prometaphase arrest. We performed time-lapse microscopy experiments after exposure to low (0-1 μM) and high (10 μM) concentrations of the ATP analog, 3-MBPP1. As expected, 90% (53/60) of untreated cells successfully completed mitosis, whereas all cells (28/28) treated with 10 μM exhibited a monopolar-spindle induced prometaphase arrest. Surprisingly, 29-40% of cells treated with 3-MBPP1 exhibited a transient prometaphase delay, followed by impaired anaphase segregation and frequent cytokinesis failure. Indirect immunofluorescence demonstrates aberrations in chromosome and microtubule dynamics, which we are further characterizing. Partial inhibition of Plk1 leads to cytotoxicity by a spindle-independent mechanism. These results are important for developing Plk1-targeted therapies. First, they demonstrate that continuous dosing of an oral Plk1 inhibitor is likely to act by a different mechanism from intermittent high doses of an intravenous drug. Second, partial inhibition may provide a more physiologically relevant background to study function of kinase inhibitor activity in the treatment of cancer. Finally, identification of the responsible substrate may reveal the most sensitive biomarker for kinase inhibition in patient samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2986. doi:10.1158/1538-7445.AM2011-2986

A potent chemotherapeutic strategy in prostate cancer: S-(methoxytrityl)-L-cysteine, a novel Eg5 inhibitor

Docetaxel-based combination chemotherapy remains the predominant treatment for castration-resistant prostate cancer. However, taxane-related drug resistance and neurotoxicity have prompted us to develop substitute treatment strategies. Eg5 (kinesin spindle protein), which is crucial for bipolar spindle formation and duplicated chromosome separation during the early phase of mitosis, has emerged as an attractive target for cancer chemotherapy. The aim of this study was to investigate the anticancer efficacy of S-(methoxytrityl)-L-cysteine (S(MeO)TLC), a novel Eg5 inhibitor in prostate cancer. Eg5 expression was examined in human prostate cancer cell lines and tissue microarrays were constructed from clinical specimens. Antiproliferative activity of S(MeO)TLC in prostate cancer cells was assessed by a cell viability assay. The anticancer effect and inhibitory mechanism of S(MeO)TLC in prostate cancer cells was further explored by Hoechst staining, flow cytometry and immunofluorescence. In addition, the antitumor effect of S(MeO)TLC on subcutaneous xenograft models was assessed. Eg5 expression was identified in PC3, DU145 and LNCaP cells. More than half of prostate cancer clinical specimens displayed Eg5 expression. S(MeO)TLC exhibited more powerful anticancer activity in prostate cancer cells compared with the other four Eg5 inhibitors tested. S(MeO)TLC induced cell death after arresting dividing cells at mitosis with distinct monopolar spindle formation. S(MeO)TLC exhibited its significant inhibitory activity (P<0.05) on subcutaneous xenograft models also through induction of mitotic arrest. We conclude that Eg5 is a good target for prostate cancer chemotherapy, and S(MeO)TLC is a potent promising anticancer agent in prostate cancer.

Quinazoline-urea, new protein kinase inhibitors in treatment of prostate cancer

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2), two protein tyrosine kinases, are involved in pathological disorders and the progression of different types of carcinomas. Concomitant inhibition of both tyrosine kinase activities appears to be an attractive target for cancer chemotherapy. A series of new quinazoline derivatives substituted by amide, urea, or carbamic acid ester groups have been synthesized. The biological activities of these new compounds have been evaluated for their enzyme inhibition and antiproliferative activities.

The Protein Farnesyltransferase Regulates HDAC6 Activity in a Microtubule-dependent Manner

The cytoplasmic deacetylase HDAC6 is an important regulator of cellular pathways that include response to stress, protein folding, microtubule stability, and cell migration, thus representing an attractive target for cancer chemotherapy. However, little is known about its upstream regulation. Our previous work has implicated HDAC6 as a new protein target for the farnesyltransferase inhibitors (FTIs), although HDAC6 lacks a farnesylation motif. Here we show that the protein farnesyltransferase (FTase) and HDAC6 are present in a protein complex together with microtubules in vivo and in vitro. FTase binds microtubules directly via its alpha subunit, and this association requires the C terminus of tubulin. Treatment with an FTI removed FTase, but not HDAC6, from the protein complex, suggesting that the active form of FTase is bound to microtubules. Importantly, the removal of FTase from microtubules abrogated HDAC6 activity, as did a stable knockdown of the alpha subunit of FTase (FTalphaKD). Interestingly, the FTalphaKD cells showed increased sensitivity to the antiproliferative effects of Taxol and the FTI lonafarnib when used either as single agents or in combination as compared with parental cells. Altogether, these data suggest that FTase, via its tubulin-association, is a critical upstream regulator of HDAC6 activity and that FTase expression could help stratify cancer patients that would most benefit from this treatment.

Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to Diminish S-Adenosylmethionine Synthesis

We exploited the fact that leukemic cells utilize significantly higher levels of S-adenosylmethionine (SAMe) than normal lymphocytes and developed tools that selectively diminished their survival under physiologic conditions. Using RNA interference gene silencing technology, we modulated the kinetics of methionine adenosyltransferase-II (MAT-II), which catalyzes SAMe synthesis from ATP and l-Met. Specifically, we silenced the expression of the regulatory MAT-IIbeta subunit in Jurkat cells and accordingly shifted the K(m L-Met) of the enzyme 10-15-fold above the physiologic levels of l-Met, thereby reducing enzyme activity and SAMe pools, inducing excessive apoptosis and diminishing leukemic cell growth in vitro and in vivo. These effects were reversed at unphysiologically high l-Met (>50 microm), indicating that diminished leukemic cell growth at physiologic l-Met levels was a direct result of the increase in MAT-II K(m L-Met) due to MAT-IIbeta ablation and the consequent reduction in SAMe synthesis. In our NOD/Scid IL-2Rgamma(null) humanized mouse model of leukemia, control shRNA-transduced Jurkat cells exhibited heightened engraftment, whereas cells lacking MAT-IIbeta failed to engraft for up to 5 weeks post-transplant. These stark differences in malignant cell survival, effected by MAT-IIbeta ablation, suggest that it may be possible to use this approach to disadvantage leukemic cell survival in vivo with little to no harm to normal cells.

Dissecting the Determinants of Cyclin-Dependent Kinase 2 and Cyclin-Dependent Kinase 4 Inhibitor Selectivity

Cyclin dependent kinases are a key family of kinases involved in cell cycle regulation and are an attractive target for cancer chemotherapy. The roles of four residues of the cyclin-dependent kinase active site in inhibitor selectivity were investigated by producing cyclin-dependent kinase 2 mutants bearing equivalent cyclin-dependent kinase 4 residues, namely F82H, L83V, H84D, and K89T. Assay of the mutants with a cyclin-dependent kinase 4-selective bisanilinopyrimidine shows that the K89T mutation is primarily responsible for the selectivity of this compound. Use of the cyclin-dependent kinase 2-selective 6-cyclohexylmethoxy-2-(4'-sulfamoylanilino)purine (NU6102) shows that K89T has no role in the selectivity, while the remaining three mutations have a cumulative influence. The results indicate that certain residues that are not frequently considered in structure-aided kinase inhibitor design have an important role to play.

Identification of a quinoxaline derivative that is a potent telomerase inhibitor leading to cellular senescence of human cancer cells.

Telomere maintenance is essential for the continued proliferation of dividing cells, and is implicated in chromosome stability and cell immortalization. Telomerase activity allows cells to maintain their telomeric DNA and contributes to the indefinite replicative capacity of cancer cells. Telomerase is expressed in most cancer cells, but not in normal somatic cells, suggesting that telomerase is an attractive target for cancer chemotherapy. Here we screened a chemical library for inhibition of human telomerase, and identified 2,3,7-trichloro-5-nitroquinoxaline (TNQX) as a potent inhibitor. TNQX showed a potent inhibitory effect, with 50% inhibition at approximately 1.4 microM, and did not inhibit DNA and RNA polymerases, including retroviral reverse trancriptase. A series of enzyme kinetic experiments suggested that TNQX is a mixed-type non-competitive inhibitor, with an inhibitor-binding site distinct from the binding sites for the telomeric substrate (TS) primer and the dNTPs. Long-term cultivation of the MCF7 cell line with a drug concentration that did not cause acute cytotoxicity resulted in progressive telomere erosion followed by an increased incidence of chromosome abnormalities and induction of the senescence phenotype. The results presented here indicate that TNQX is a highly potent and selective anti-telomerase agent with good potential for further development as a promising anti-cancer agent.