Pyrido[2,3-b][1,5]benzoxazepin-5(6H)-one derivatives as CDK8 inhibitors.

CDK8 is a cyclin-dependent kinase that forms part of the mediator complex, and modulates the transcriptional output from distinct transcription factors involved in oncogenic control. Overexpression of CDK8 has been observed in various cancers, representing a potential target for developing novel CDK8 inhibitors in cancer therapeutics. In the course of our investigations to discover new CDK8 inhibitors, we designed and synthesized tricyclic pyrido[2,3-b][1,5]benzoxazepin-5(6H)-one derivatives, by introduction of chemical complexity in the multi-kinase inhibitor Sorafenib taking into account the flexibility of the P-loop motif of CDK8 protein observed after analysis of structural information of co-crystallized CDK8 inhibitors. In vitro evaluation of the inhibitory activity of the prepared compounds against CDK8 led us to identify compound 2 as the most potent inhibitor of the series (IC50 = 8.25 nM). Co-crystal studies and the remarkable selectivity profile of compound 2 are presented. Compound 2 showed moderate reduction of phosphorylation of CDK8 substrate STAT1 in cells, in line with other reported Type II CDK8 inhibitors. We propose herein an alternative to find a potential therapeutic use for this chemical series.

Targeting RING domains of Mdm2-MdmX E3 complex activates apoptotic arm of the p53 pathway in leukemia/lymphoma cells.

Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2-p53 interface or MdmX ((MDM4), mouse double minute 4)-p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2-MdmX really interesting new gene (RING)-RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2-MdmX RING-RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2-MdmX RING domain inhibitors)) that specifically inhibit Mdm2-MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2-MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2-MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.

On the relevance of defining protein structures in cancer research

Are three-dimensional structures of proteins relevant in the study of cancer? The knowledge of the three-dimensional structure of a protein is crucial to gain a full understanding of its function, and structural determination has already shown its potential for guided drug design. The knowledge of the structures of proteins and their complexes with other biological macromolecules helps to elucidate functional networks and provide a better understanding of the functionally relevant behaviour of the molecular machinery of the cell. To study the cell, we must be able to work with proteins, to elucidate how they diffuse and move, to know their interacting partners, and to understand the changes induced by those interactions. Three-dimensional structures give us a picture of the protein and thereby the opportunity to introduce mutations that alter its affinity and specificity for other interactions helping us to understand the physico-chemical mechanisms that control their function. In turn these can lead to the development of novel therapies (AU)

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Family 7 cellobiohydrolases from Phanerochaete chrysosporium: crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 A resolution and homology models of the isozymes.

Cellobiohydrolase 58 (Cel7D) is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporium, constituting approximately 10 % of the total secreted protein in liquid culture on cellulose. The enzyme is classified into family 7 of the glycosyl hydrolases, together with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) from Trichoderma reesei. Like those enzymes, it catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration. The structure of the catalytic module (431 residues) of Cel7D was determined at 3.0 A resolution using the structure of Cel7A from T. reesei as a search model in molecular replacement, and ultimately refined at 1.32 A resolution. The core structure is a beta-sandwich composed of two large and mainly antiparallel beta-sheets packed onto each other. A long cellulose-binding groove is formed by loops on one face of the sandwich. The catalytic residues are conserved and the mechanism is expected to be the same as for other family members. The Phanerochaete Cel7D binding site is more open than that of the T. reesei cellobiohydrolase, as a result of deletions and other changes in the loop regions, which may explain observed differences in catalytic properties. The binding site is not, however, as open as the groove of the corresponding endoglucanase. A tyrosine residue at the entrance of the tunnel may be part of an additional subsite not present in the T. reesei cellobiohydrolase. The Cel7D structure was used to model the products of the five other family 7 genes found in P. chrysosporium. The results suggest that at least two of these will have differences in specificity and possibly catalytic mechanism, thus offering some explanation for the presence of Cel7 isozymes in this species, which are differentially expressed in response to various growth conditions.

Cellobiohydrolase 58 (P.c. Cel 7D) is complementary to the homologous CBH I (T.r. Cel 7A) in enantioseparations.

Cellobiohydrolase 58 (EC 3.2.1.91, P.c. Cel 7D) from Phanerochaete chrysosporium was immobilized on silica and the resulting material, CBH 58-silica, was then used as a chiral stationary phase (CSP) in liquid chromatographic separations of enantiomers. The enantioselectivities obtained on CBH 58-silica were compared with those on CBH I-silica (a phase based on a corresponding cellulase from Trichoderma reesei). CBH 58-silica displayed higher selectivity than CBH I-silica for the more hydrophilic compounds, such as atenolol and metoprolol, although great similarities in chiral separation of beta-adrenergic antagonists were found between the two phases. None of the acidic compounds tested could be resolved on the CBH 58 phase. Moreover, the solutes were retained more on the CBH 58 phase in general, indicating an improved application potential in bioanalysis. Addition of cellobiose or lactose, both of which are inhibitors of cellulases, to the mobile phase impaired the enantioselectivity, indicating an overlap of the enantioselective and catalytic sites. The chiral analytes also functioned as competitive inhibitors and their inhibition constants were determined.