Bovine ␤-lactoglobulin (␤-Lg) has been studied extensively in both the isolated and the naturally occurring states. It is a commercially important whey protein of obvious nutritional value but, so far, one that has no clearly identified biological function.

Following the identification through virtual screening of 4-(2,4-dimethyl-thiazol-5-yl)pyrimidin-2-ylamines as moderately potent inhibitors of cyclin-dependent kinase-2 (CDK2), a CDK inhibitor analogue program was initiated. The first aims were to optimize potency and to evaluate the cellular mode of action of lead candidate molecules. Here the synthetic chemistry, the structure-guided design approach, and the structure-activity relationships (SARs) that led to the discovery of 2-anilino-4-(thiazol-5-yl)pyrimidine ATP-antagonistic CDK2 inhibitors, many with very low nM K(i)s against CDK2, are reported. Furthermore, X-ray crystal structures of four representative analogues from our chemical series in complex with CDK2 are presented, and these structures are used to rationalize the observed biochemical SARs. Finally results are reported that show, using the most potent CDK2 inhibitor compound from the current series, that the observed antiproliferative and proapoptotic effects are consistent with cellular CDK2 and CDK9 inhibition.

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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...