purvalanol-a and Neoplasms

Members of the short-chain dehydrogenase/reductase (SDR) and aldo-keto reductase (AKR) superfamilies mediate the reduction of anthracyclines to their less potent C-13 alcohol metabolites. This reductive metabolism has been recognized as one of the most important factors that trigger anthracycline resistance in cancer cells. In our study, two purine analogues, purvalanol A and roscovitine, were identified as effective inhibitors of aldo-keto reductase 1C3 (AKR1C3), an enzyme that is overexpressed in many cancer types and is also a key player in tumour cell resistance to anthracyclines. Purvalanol A and roscovitine potently inhibited human recombinant AKR1C3 (Ki = 5.5 μM and 1.4 μM, respectively) and displayed similar activity in experiments with intact cells. Ligand-protein docking calculations suggested that both inhibitors occupied a part of the cofactor-binding site. Furthermore, we demonstrated that the combination of daunorubicin with purvalanol A or roscovitine exhibited a synergistic effect in AKR1C3 overexpressing cells. Based on these findings, it is possible to presume that purvalanol A and roscovitine may have the potential to enhance the therapeutic effectiveness and safety of anthracyclines via inhibition of AKR1C3.

Topics: Aldo-Keto Reductase Family 1 Member C3; Anthracyclines; Antineoplastic Agents; Cloning, Molecular; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; HCT116 Cells; Hep G2 Cells; Humans; Neoplasms; Purines; Roscovitine

Adaptive therapy (AT) aims to control tumour burden by maintaining therapy-sensitive cells to exploit their competition with resistant cells. This relies on the assumption that resistant cells have impaired cellular fitness. Here, using a model of resistance to a pharmacological cyclin-dependent kinase inhibitor (CDKi), we show that this assumption is valid when competition between cells is spatially structured. We generate CDKi-resistant cancer cells and find that they have reduced proliferative fitness and stably rewired cell cycle control pathways. Low-dose CDKi outperforms high-dose CDKi in controlling tumour burden and resistance in tumour spheroids, but not in monolayer culture. Mathematical modelling indicates that tumour spatial structure amplifies the fitness penalty of resistant cells, and identifies their relative fitness as a critical determinant of the clinical benefit of AT. Our results justify further investigation of AT with kinase inhibitors.

Topics: Animals; Antineoplastic Agents; Cell Culture Techniques; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinases; Drug Resistance, Neoplasm; Female; Humans; Mice; Mice, Nude; Models, Biological; Neoplasms; Piperazines; Protein Kinase Inhibitors; Purines; Pyridines; RNA, Small Interfering; Spheroids, Cellular; Tumor Burden; Xenograft Model Antitumor Assays

Studies into the genetic origins of tumor cell chemoactivity pose significant challenges to bioinformatic mining efforts. Connections between measures of gene expression and chemoactivity have the potential to identify clinical biomarkers of compound response, cellular pathways important to efficacy and potential toxicities; all vital to anticancer drug development. An investigation has been conducted that jointly explores tumor-cell constitutive NCI60 gene expression profiles and small-molecule NCI60 growth inhibition chemoactivity profiles, viewed from novel applications of self-organizing maps (SOMs) and pathway-centric analyses of gene expressions, to identify subsets of over- and under-expressed pathway genes that discriminate chemo-sensitive and chemo-insensitive tumor cell types. Linear Discriminant Analysis (LDA) is used to quantify the accuracy of discriminating genes to predict tumor cell chemoactivity. LDA results find 15% higher prediction accuracies, using ∼30% fewer genes, for pathway-derived discriminating genes when compared to genes derived using conventional gene expression-chemoactivity correlations. The proposed pathway-centric data mining procedure was used to derive discriminating genes for ten well-known compounds. Discriminating genes were further evaluated using gene set enrichment analysis (GSEA) to reveal a cellular genetic landscape, comprised of small numbers of key over and under expressed on- and off-target pathway genes, as important for a compound's tumor cell chemoactivity. Literature-based validations are provided as support for chemo-important pathways derived from this procedure. Qualitatively similar results are found when using gene expression measurements derived from different microarray platforms. The data used in this analysis is available at http://pubchem.ncbi.nlm.nih.gov/andhttp://www.ncbi.nlm.nih.gov/projects/geo (GPL96, GSE32474).

Topics: Algorithms; Antineoplastic Agents; Camptothecin; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cluster Analysis; Colchicine; Computational Biology; Cyclin-Dependent Kinases; Dasatinib; Databases, Genetic; Fluorouracil; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Neoplasms; Oligonucleotide Array Sequence Analysis; Paclitaxel; Pimozide; Purines; Pyrimidines; Terfenadine; Thiazoles; Tubulin Modulators; Verapamil

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain holds promise for the treatment of neurological diseases and has yielded new insight into brain cancer. However, the complete repertoire of signaling pathways that governs the proliferation and self-renewal of NSCs, which we refer to as the 'ground state', remains largely uncharacterized. Although the candidate gene approach has uncovered vital pathways in NSC biology, so far only a few highly studied pathways have been investigated. Based on the intimate relationship between NSC self-renewal and neurosphere proliferation, we undertook a chemical genetic screen for inhibitors of neurosphere proliferation in order to probe the operational circuitry of the NSC. The screen recovered small molecules known to affect neurotransmission pathways previously thought to operate primarily in the mature central nervous system; these compounds also had potent inhibitory effects on cultures enriched for brain cancer stem cells. These results suggest that clinically approved neuromodulators may remodel the mature central nervous system and find application in the treatment of brain cancer.

Topics: Animals; Cell Survival; Cells, Cultured; Mice; Molecular Structure; Neoplasms; Neurons; Pharmaceutical Preparations; Sensitivity and Specificity; Stem Cells

To clarify the relationship between CDC2 kinase activity and radiation-induced apoptosis, we examined whether the cyclin-dependent kinase (CDK) inhibitor purvalanol A enhanced radiation-induced apoptosis in gastric tumor cells. MKN45 cells exposed to 20 Gy of X rays increased the CDC2 kinase activity and the expression of regulatory proteins (phospho-CDC2 and cyclin B1) of the G2/M phase, followed by activation of the G2/M checkpoint, whereas the treatment of X-irradiated MKN45 cells with 20 microM purvalanol A suppressed the increase in the CDC2 kinase activity and expression of the G2/M-phase regulatory proteins and reduced the fraction of the cells in the G2/M phase in the cell cycle. Furthermore, this treatment resulted in not only a significant increase in radiation-induced apoptosis but also the loss of clonogenicity in both MKN45 (p53-wild) and MKN28 (p53-mutated) cells. The expression of anti-apoptosis proteins, inhibitor of apoptosis protein (IAP) family members (survivin and XIAP) and BCL2 family members (Bcl-X(L) and Bcl-2), in purvalanol A-treated cells with and without X rays was significantly lower than for cells exposed to X rays alone. These results suggest that the inhibition of radiation-induced CDC2 kinase activity by purvalanol A induces apoptosis through the enhancement of active fragments of caspase 3.

Topics: Apoptosis; CDC2 Protein Kinase; Cell Cycle Proteins; Cell Division; Cell Line; G2 Phase; Humans; Neoplasms; Purines; Up-Regulation; X-Rays

The tyrosine kinase pp60src (Src) is the prototypical member of a family of proteins that participate in a broad array of cellular signal transduction processes, including cell growth, differentiation, survival, adhesion, and migration. Abnormal Src family kinase (SFK) signaling has been linked to several disease states, including osteoporosis and cancer metastases. Src has thus emerged as a molecular target for the discovery of small-molecule inhibitors that regulate Src kinase activity by binding to the ATP pocket within the catalytic domain. Here, we present crystal structures of the kinase domain of Src in complex with two purine-based inhibitors: AP23451, a small-molecule inhibitor designed to inhibit Src-dependent bone resorption, and AP23464, a small-molecule inhibitor designed to inhibit the Src-dependent metastatic spread of cancer. In each case, a trisubstituted purine template core was elaborated using structure-based drug design to yield a potent Src kinase inhibitor. These structures represent early examples of high affinity purine-based Src family kinase-inhibitor complexes, and they provide a detailed view of the specific protein-ligand interactions that lead to potent inhibition of Src. In particular, the 3-hydroxyphenethyl N9 substituent of AP23464 forms unique interactions with the protein that are critical to the picomolar affinity of this compound for Src. The comparison of these new structures with two relevant kinase-inhibitor complexes provides a structural basis for the observed kinase inhibitory selectivity. Further comparisons reveal a concerted induced-fit movement between the N- and C-terminal lobes of the kinase that correlates with the affinity of the ligand. Binding of the most potent inhibitor, AP23464, results in the largest induced-fit movement, which can be directly linked to interactions of the hydrophenethyl N9 substituent with a region at the interface between the two lobes. A less pronounced induced-fit movement is also observed in the Src-AP23451 complex. These new structures illustrate how the combination of structural, computational, and medicinal chemistry can be used to rationalize the process of developing high affinity, selective tyrosine kinase inhibitors as potential therapeutic agents.

Topics: Adenine; Adenosine Triphosphate; Bone and Bones; Carbon; Catalytic Domain; Cyclin-Dependent Kinase 2; Drug Design; Enzyme Inhibitors; Humans; Ligands; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Models, Molecular; Neoplasms; Nitrogen; Organophosphonates; Protein Conformation; Purines; Pyrimidines; src-Family Kinases; Structure-Activity Relationship; Substrate Specificity

Determination of pharmacokinetic properties in the intact animal remains a major bottleneck in drug discovery. Cassette dosing involves administration of a cocktail of drugs to individual animals. Here we describe the cassette dosing properties of a 107-membered library of 2,6,9-trisubstituted purine cyclin-dependent kinase 2 (CDK2) inhibitors. A three-step parallel synthesis approach produced compounds with purity ranging from 63% to 100%. Cassette dosing was validated by comparing the pharmacokinetic parameters obtained following i.v. administration of a mixture of olomoucine, R-roscovitine (CYC202), and bohemine, each at 16.6 mg/kg, with results for administration of single agents at 50 mg/kg. No significant difference was observed between the pharmacokinetic parameters of agents when dosed in combination compared with those of individual compounds. CYC202 showed the highest area under the curve (AUC) and the longest elimination half-life (t(1/2)). Further cassettes evaluated the library of trisubstituted purines with CYC202 and purvalanol A included as pharmacokinetic standards in a validated limited sampling strategy. The ratios of pharmacokinetic parameters to that of CYC202 [AUC, maximum concentration (C(max)), and t(1/2)] remained similar when compounds were tested in two different cassettes or as individual compounds. Following dosing of the same cassette on three different days, there was less than 20% variation in pharmacokinetic parameters between days. The structure-pharmacokinetics relationship showed that the favored purine substituents are benzylamine and veratrylamine at position 6, amino-2 propanol at position 2, and methylpropyl or hydroxyethyl at position 9. Without cassette dosing, this study would have used 3 times as many animals and would have taken 4 times longer, illustrating the power of this method in lead optimization.

Topics: Animals; Area Under Curve; CDC2-CDC28 Kinases; Cyclin-Dependent Kinase 2; Dose-Response Relationship, Drug; Drug Design; Drug Evaluation, Preclinical; Enzyme Inhibitors; Female; Free Radicals; Gene Library; Mice; Mice, Inbred BALB C; Models, Chemical; Neoplasms; Purines; Roscovitine; Structure-Activity Relationship; Time Factors