beta-Lactoglobulin (beta-LG) is the major whey protein of ruminant species and is also present in the milks of many, but not all, other species. Its amino-acid sequence and 3-dimensional structure show that it is a lipocalin, a widely diverse family, most of which bind small hydrophobic ligands and thus may act as specific transporters, as does serum retinol binding protein. Bovine beta-LG binds a wide range of ligands, but this may not be the reason for its presence in milk. In reviewing the structure and physicochemical properties of the protein, we present the structures of the ligands cholesterol (at a resolution of 2.0A, R = 0.221; Rfree = 0.295) and vitamin D2 (at a resolution of 2.4A, R = 0.212; Rfree = 0.297) each bound to the central binding cavity of bovine beta-LG at pH 7.3. Neither ligand is fully visible in the electron density maps, and the less well-ordered regions are the polar end groups at the mouth of the binding site. In a separate experiment, a mercury ion was bound to the free Cys121 (at a resolution of 2.2A, R = 0.218; Rfree = 0.288) in a way that transmitted a small structural change through Asp137 via Arg148 to the dimer interface. It is not clear if the known dissociation that arises from the reaction of beta-LG with HgCl2 results from this perturbation. In reviewing the structural studies that reveal the ligand binding sites for long-chain fatty acids, retinoids, and steroids, only the central location, common to all lipocalins so far examined, is occupied under the conditions used. We find that there is no crystallographic evidence of another ligand binding site in our crystals grown in approximately 1.3 M citrate, although low ionic strength studies in solution indicate the possible presence of at least one other low affinity site. The apparent ability of the binding site to accommodate a wide range of ligands may point to a possible physiological function. However, by considering the lipocalin family in general, and the species distribution of beta-LG in particular, some speculation as to the physiological function can be made. beta-Lactoglobulin has been reported as being implicated, inter alia, in hydrophobic ligand transport and uptake, enzyme regulation, and the neonatal acquisition of passive immunity. However, these functions do not appear to be consistent between species. Sequence comparisons among members of the lipocalin family reveal that glycodelin, found in the human endometrium during early pregnancy, is the most closely related to beta-LG. Although the function of glycodelin is also unknown, it appears to have effects on the immune system and/or to be involved in differentiation. It is proposed that beta-LG, over-expressed in the lactating mammary gland of many, but not all, species, is primarily an important source of amino acids for the offspring of those animals that produce it, but that this function arose by gene duplication from the physiologically essential glycodelin. The other functions that have been associated with beta-LG in the neonate are, therefore, fortu...

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In a routine screening of our small-molecule compound collection we recently identified 4-arylazo-3,5-diamino-1H-pyrazoles as a novel group of ATP antagonists with moderate potency against CDK2-cyclin E. A preliminary SAR study based on 35 analogues suggests ways in which the pharmacophore could be further optimized, for example, via substitutions in the 4-aryl ring. Enzyme kinetics studies with the lead compound and X-ray crystallography of an inhibitor-CDK2 complex demonstrated that its mode of inhibition is competitive. Functional kinase assays confirmed the selectivity toward CDKs, with a preference for CDK9-cyclin T1. The most potent inhibitor, 4-[(3,5-diamino-1H-pyrazol-4-yl)diazenyl]phenol 31b (CAN508), reduced the frequency of S-phase cells of the cancer cell line HT-29 in antiproliferation assays. Further observed cellular effects included decreased phosphorylation of the retinoblastoma protein and the C-terminal domain of RNA polymerase II, inhibition of mRNA synthesis, and induction of the tumor suppressor protein p53, all of which are consistent with inhibition of CDK9.

The interactions between the tumor suppressor protein p21 WAF1 and the cyclin-dependent kinase (CDK) complexes and with proliferating cell nuclear antigen (PCNA) regulate and coordinate the processes of cell-cycle progression and DNA replication. We present the x-ray crystal structure of PCNA complexed with a 16-mer peptide related to p21 that binds with a Kd of 100 nM. Two additional crystal structures of native PCNA provide previously undescribed structures of uncomplexed human PCNA and show that significant changes on ligand binding include rigidification of a number of flexible regions on the surface of PCNA. In the competitive binding experiments described here, we show that a 20-mer sequence from p21 can be associated simultaneously with PCNA and CDK͞cyclin complexes. A structural model for this quaternary complex is presented in which the C-terminal sequence of p21 acts like double-sided tape and docks to both the PCNA and cyclin molecules. The quaternary complex shows little direct interaction between PCNA and cyclin, giving p21 the role of an adaptor molecule. Taken together, the biochemical and structural results delineate a druggable inhibitor site on the surface of PCNA that may be exploited in the design of peptidomimetics, which will act independently of cyclin-groove inhibitors.p21 ͉ x-ray ͉ structure ͉ protein ͉ interaction P roliferating cell nuclear antigen (PCNA) is an essential auxiliary protein for the processes of both DNA replication and repair. It stimulates the activity of DNA polymerase ␦ and increases its processivity by acting as a clamp platform that slides along the DNA template (1-3). Apart from polymerase ␦, PCNA associates with a host of other proteins, either involved directly in DNA replication and repair or in the regulation of these processes (4). The presence of a common PCNA-binding motif in such proteins suggests that regulation may depend largely on PCNA partner proteins competing with one another for access to PCNA.Deregulation of PCNA expression is a hallmark of many proliferative diseases, and in the clinic PCNA serves as a general proliferative marker, especially in the prognosis of tumor development (5). In fact, PCNA expression levels are directly related to the malignancy of various tumors, and antisense oligonucleotidemediated suppression of PCNA expression was demonstrated to selectively inhibit gastric cancer cell proliferation in vitro and in vivo (6). Antisense strategies targeting PCNA mRNA also have shown promise in models of other proliferative diseases, including glomerular nephritis (7) and rheumatoid arthritis (8). The fact that PCNA is required absolutely for cell proliferation indicates that pharmacological modulation of PCNA function should not be able to be circumvented by compensatory pathways. Furthermore, the ablation of PCNA expression or function in cells under proliferative stimuli appears to constitute an apoptotic trigger (5), suggesting that effective elimination of hyperproliferative cells should be possible in a therapeutic setting.The tum...

The Open University's repository of research publications and other research outputs Discovery and characterization of 2-Anilino-4-(Thiazol-5-yl)Pyrimidine transcriptional CDK inhibitors as anticancer agents

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The cyclin-dependent kinases (CDKs) have been characterized in complex with a variety of inhibitors, but the majority of structures solved are in the inactive form. We have solved the structures of six inhibitors in both the monomeric CDK2 and binary CDK2/cyclinA complexes and demonstrate that significant differences in ligand binding occur depending on the activation state. The binding mode of two ligands in particular varies substantially in active and inactive CDK2. Furthermore, energetic analysis of CDK2/cyclin/inhibitors demonstrates that a good correlation exists between the in vitro potency and the calculated energies of interaction, but no such relationship exists for CDK2/inhibitor structures. These results confirm that monomeric CDK2 ligand complexes do not fully reflect active conformations, revealing significant implications for inhibitor design while also suggesting that the monomeric CDK2 conformation can be selectively inhibited.