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.

1 The sea urchin egg homogenate is an ideal model to characterize Ca 2+ -release mechanisms because of its reliability and high signal-to-noise-ratio. Apart from the InsP 3 -and ryanodine-sensitive Ca 2+ -release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist. 2 The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca 2+ -releasing agent, NAADP, compared to inositol trisphosphate (InsP 3 ) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors. 3 NAADP-induced Ca 2+ -release was blocked by L-type Ca 2+ -channel blockers and by Bay K 8644, while InsP 3 -and cyclic ADPR-induced Ca 2+ -release were insensitive to these agents. L-type Ca 2+ -channel blockers did not displace [ 32 P]-NAADP binding, suggesting that their binding site was di erent. Moreover, stopped-¯ow kinetic studies revealed that these agents blocked NAADP in a all-or-none fashion. 4 Similarly, a number of K + -channel antagonists blocked NAADP-induced Ca 2+ -release selectively over InsP 3 -and cyclic ADPR-induced Ca 2+ -release. Radioligand studies showed that these agents were not competitive antagonists. 5 As has been shown for InsP 3 and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca 2+ -release mechanisms. 6 The presence of K + was not essential for NAADP-induced Ca 2+ -release, since substitution of K + with other monovalent cations in the experimental media did not signi®cantly alter Ca 2+ release by NAADP. On the contrary, cyclic ADPR and InsP 3 -sensitive mechanisms were a ected profoundly, although to a di erent extent depending on the monovalent cation which substituted for K + . Similarly, modi®cations of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly a ected NAADPinduced Ca 2+ -release. While the alkaline condition permitted InsP 3 and cyclic ADPR-induced Ca 2+ -release, the acidic condition completely hampered both Ca 2+ -release mechanisms. 7 The present results characterize pharmacologically and biochemically the novel Ca 2+ -release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.

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

Exploiting oxidative stress has recently emerged as a plausible strategy for treatment of human cancer, and antioxidant defenses are implicated in resistance to chemotherapy and radiotherapy. Targeted suppression of antioxidant defenses could thus broadly improve therapeutic outcomes. Here, we identify the AMPK-related kinase NUAK1 as a key component of the antioxidant stress response pathway and reveal a specific requirement for this role of NUAK1 in colorectal cancer. We show that NUAK1 is activated by oxidative stress and that this activation is required to facilitate nuclear import of the antioxidant master regulator NRF2: Activation of NUAK1 coordinates PP1β inhibition with AKT activation in order to suppress GSK3β-dependent inhibition of NRF2 nuclear import. Deletion of NUAK1 suppresses formation of colorectal tumors, whereas acute depletion of NUAK1 induces regression of preexisting autochthonous tumors. Importantly, elevated expression of NUAK1 in human colorectal cancer is associated with more aggressive disease and reduced overall survival. This work identifies NUAK1 as a key facilitator of the adaptive antioxidant response that is associated with aggressive disease and worse outcome in human colorectal cancer. Our data suggest that transient NUAK1 inhibition may provide a safe and effective means for treatment of human colorectal cancer via disruption of intrinsic antioxidant defenses. .

MRCKα and MRCKβ (myotonic dystrophy kinase-related Cdc42-binding kinases) belong to a subfamily of Rho GTPase activated serine/threonine kinases within the AGC-family that regulate the actomyosin cytoskeleton. Reflecting their roles in myosin light chain (MLC) phosphorylation, MRCKα and MRCKβ influence cell shape and motility. We report further evidence for MRCKα and MRCKβ contributions to the invasion of cancer cells in 3-dimensional matrix invasion assays. In particular, our results indicate that the combined inhibition of MRCKα and MRCKβ together with inhibition of ROCK kinases results in significantly greater effects on reducing cancer cell invasion than blocking either MRCK or ROCK kinases alone. To probe the kinase ligand pocket, we screened 159 kinase inhibitors in an in vitro MRCKβ kinase assay and found 11 compounds that inhibited enzyme activity >80% at 3 µM. Further analysis of three hits, Y-27632, Fasudil and TPCA-1, revealed low micromolar IC50 values for MRCKα and MRCKβ. We also describe the crystal structure of MRCKβ in complex with inhibitors Fasudil and TPCA-1 bound to the active site of the kinase. These high-resolution structures reveal a highly conserved AGC kinase fold in a typical dimeric arrangement. The kinase domain is in an active conformation with a fully-ordered and correctly positioned αC helix and catalytic residues in a conformation competent for catalysis. Together, these results provide further validation for MRCK involvement in regulation of cancer cell invasion and present a valuable starting point for future structure-based drug discovery efforts.

Polo-like kinases (Plks) have several functions in mitotic progression and are upregulated in many tumor types. Small-molecule Plk inhibitors would be valuable as tools for studying Plk biology and for developing antitumor agents. Guided by homology modeling of the Plk1 kinase domain, we have discovered a chemical series that shows potent and selective Plk1 inhibition. The effects of one such optimized benzthiazole N-oxide, cyclapolin 1 (1), on purified centrosomes indicate that Plks are required to generate MPM2 epitopes, recruit gamma-tubulin and enable nucleation of microtubules. The compound can also promote loss of centrosome integrity and microtubule nucleating ability apparently through increased accessibility of protein phosphatases. We show that treatment of living S2 cells with cyclapolin 1 leads to collapsed spindles, in contrast to the metaphase-arrested bipolar spindles observed after RNAi. This different response to protein depletion and protein inhibition may have significance in the development of antitumor agents.

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.

Cancer cells acquire characteristics of deregulated growth, survival and increased metastatic potential. Genetic mutations that provide a selective advantage by promoting these characteristics have been termed 'drivers,' whereas mutations that do not contribute to disease initiation/progression are termed 'passengers.' The advent of high-throughput methodologies has facilitated large-scale screening of cancer genomes and the subsequent identification of novel somatic mutations. Although this approach has generated valuable results, the data remain incomplete until the functional consequences of these mutations are determined to differentiate potential drivers from passengers. ROCK1 is an essential effector kinase downstream of Rho GTPases, an important pathway involved in cell migration. The Cancer Genome Project identified three nonsynonymous mutations in the ROCK1 gene. We now show that these somatic ROCK1 mutations lead to elevated kinase activity and drive actin cytoskeleton rearrangements that promote increased motility and decreased adhesion, characteristics of cancer progression. Mapping of the kinase-interacting regions of the carboxy terminus combined with structural modeling provides an insight into how these mutations likely affect the regulation of ROCK1. Consistent with the frequency of ROCK1 mutations in human cancer, these results support the conclusion that there is selective pressure for the ROCK1 gene to acquire 'driver' mutations that result in kinase activation.