A broad activity screen in support of a chemogenomic map for kinase signalling research and drug discovery.

Despite the development of a number of efficacious kinase inhibitors, the strategies for rational design of these compounds have been limited by target promiscuity. In an effort to better understand the nature of kinase inhibition across the kinome, especially as it relates to off-target effects, we screened a well-defined collection of kinase inhibitors using biochemical assays for inhibitory activity against 234 active human kinases and kinase complexes, representing all branches of the kinome tree. For our study we employed 158 small molecules initially identified in the literature as potent and specific inhibitors of kinases important as therapeutic targets and/or signal transduction regulators. Hierarchical clustering of these benchmark kinase inhibitors on the basis of their kinome activity profiles illustrates how they relate to chemical structure similarities and provides new insights into inhibitor specificity and potential applications for probing new targets. Using this broad dataset, we provide a framework for assessing polypharmacology. We not only discover likely off-target inhibitor activities and recommend specific inhibitors for existing targets, but also identify potential new uses for known small molecules.

Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress.

In late mitosis and early G1, replication origins are licensed for subsequent use by loading complexes of the minichromosome maintenance proteins 2-7 (Mcm2-7). The number of Mcm2-7 complexes loaded onto DNA greatly exceeds the number of replication origins used during S phase, but the function of the excess Mcm2-7 is unknown. Using Xenopus laevis egg extracts, we show that these excess Mcm2-7 complexes license additional dormant origins that do not fire during unperturbed S phases because of suppression by a caffeine-sensitive checkpoint pathway. Use of these additional origins can allow complete genome replication in the presence of replication inhibitors. These results suggest that metazoan replication origins are actually comprised of several candidate origins, most of which normally remain dormant unless cells experience replicative stress. Consistent with this model, using Caenorhabditis elegans, we show that partial RNAi-based knockdown of MCMs that has no observable effect under normal conditions causes lethality upon treatment with low, otherwise nontoxic, levels of the replication inhibitor hydroxyurea.

Nuclear ERM (ezrin, radixin, moesin) proteins: regulation by cell density and nuclear import.

The highly conserved ERM (ezrin-radixin-moesin) family of proteins function as molecular linkers between the actin cytoskeleton and transmembrane receptors. We now provide unequivocal evidence that full-length endogenous ezrin and moesin also localise to the nucleus in two independent mammalian cell lines. All three ERM family members can localise to the nucleus upon exogenous expression of their GFP-tagged counterparts, suggesting a common family trend. Furthermore, Dmoesin, the Drosophila ERM homologue, is present in the nucleus of an insect cell line and can localise to the nucleus when exogenously expressed in MDCK cells. The nuclear localisation of endogenous ezrin and moesin is regulated by cell density and is resistant to detergent extraction, suggesting tight association with nuclear structures. Furthermore, phosphorylation in the actin-binding domain is not a prerequisite for nuclear localisation. We have identified a specific nuclear localisation sequence, which is conserved and functional in all ERM family members, implying specific regulated nuclear import. Although the precise nuclear function of the ERM proteins is unknown, these data provide further evidence that an increasing number of cytoskeletal components directly link the plasma membrane with nuclear events.