No abstract

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.

TFIIIC2 is a general factor essential for transcription of 5S RNA, tRNA, and VA RNA genes by mammalian RNA polymerase III and consists of two forms designated THIIC2a and TFIIIC2b. TFIIIC2a and TFIIIC2b share common subunits of 220, 102, 90, and 63 kD but differ with respect to transcription activity and the presence of a presumptive 110-kD subunit in the active form (TFIIIC2a). Because both forms can bind the promoter directly, a selective role for the 110-kD subunit in the regulation of RNA polymerase III activity has been suggested. To investigate this possibility, we have cloned and expressed a cDNA encoding the l l0-kD subunit (TFIIICB). Immunoprecipitation studies with anti-TFIIICI3 antibodies have confirmed that TFIIIC[3 is a bona fide subunit present only in TFIIIC2a, that TFIIIC2a and the general factor TFIIIC1 are associated in unfractionated extracts, and that previously undetected polypeptides (potential TFIIIC1 subunits) can be isolated in association with TFIIIC2a. Previous studies have shown that increases in RNA polymerase III activity during infection of cells by adenovirus (with concomitant E1A expression) or during cell growth at high serum concentration results from an increased activity in the TFIIIC fraction. Studies with antibodies to TFIIICB have shown that this is strongly correlated with a selective increase in the cellular concentration of the TFIIIC~ l l0-kD subunit and a concomitant rise in the ratio of the active-to-inactive forms of TFIIIC2.

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast Schizosaccharomyces pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of Cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of Cdc2. In this report, Cdc25 is shown to accumulate to a very high level in cells arrested in S. This shows that mechanisms which modulate the abundance of Cdc25 are unconnected to the DNA replication checkpoint. Using a Cdc2/cyclin B activation assay, we found that Cdc25 activity increased ϳ10-fold during transit through M phase. Cdc25 was activated by phosphorylations that were dependent on Cdc2 activity in vivo. Cdc25 activation was suppressed in cells arrested in G 1 and S. However, Cdc25 was more highly modified and appeared to be somewhat more active in S than in G 1 . This finding might be connected to the fact that progression from G 1 to S increases the likelihood that constitutive Cdc25 overproduction will cause inappropriate mitosis.

Human transcription factor IIIC (hTFIIIC) is a multisubunit complex that directly recognizes promoter elements and recruits TFIIIB and RNA polymerase III. Here we describe the cDNA cloning and characterization of the 90-kDa subunit (hTFIIIC90) that is present within a DNA-binding subcomplex (TFIIIC2) of TFIIIC. hTFIIIC90 has no specific homology to any of the known yeast TFIIIC subunits. Immunodepletion and immunoprecipitation studies indicate that hTFIIIC90 is a bona fide subunit of TFIIIC2 and absolutely required for RNA polymerase III transcription. hTFIIIC90 shows interactions with the hTFIIIC220, hT-FIIIC110, and hTFIIIC63 subunits of TFIIIC, the hTFIIIB90 subunit of TFIIIB, and the human RPC39 (hRPC39) and hRPC62 subunits of an initiation-specific subcomplex of RNA polymerase III. These interactions may facilitate both TFIIIB and RNA polymerase III recruitment to the preinitiation complex by TFIIIC. We show that hTFIIIC90 has an intrinsic histone acetyltransferase activity with a substrate specificity for histone H3.RNA polymerase III transcribes genes encoding small structural RNAs that include 5S RNA, tRNA, adenovirus-associated (VA) RNA, and the U6 and 7SK RNAs. Together with RNA polymerase III, transcription factor IIIC (TFIIIC) and TFIIIB suffice for transcription of tRNA, VA RNA, and yeast U6 RNA genes, whereas expression of the 5S gene is additionally dependent on TFIIIA (reviewed in references 14, 19, 49, and 50). Mammalian U6 and 7SK genes require PTF (SNAPc/ PBP), TFIIIC1, and an alternative form of TFIIIB to direct transcription by RNA polymerase III (reviewed in reference 38).In the simplest cases, preinitiation complex assembly on class III genes involves direct promoter recognition by TFIIIC (A and B boxes in tRNA, VA RNA, and yeast U6 RNA genes) and TFIIIB and RNA polymerase III recruitment through interactions with TFIIIC (reviewed in references 18, 46, and 47). Consistent with conservation of the assembly pathway from yeast to human, there is a corresponding conservation in structure and function of RNA polymerase III, TFIIIB, and a subset of TFIIIC subunits (reviewed in references 18, 46, and 47).TFIIIC has been most extensively characterized in yeast, where it is composed of six polypeptides (138, 131, 95, 91, 60, and 55 kDa) (reviewed in references 1 and 28) and binds to both A and B boxes, whereas human TFIIIC (hTFIIIC) contains at least nine subunits and can be resolved into a fivesubunit (220-, 110-, 102-, 90-, and 63-kDa) complex (TFIIIC2) that binds weakly to the B box and a less well-characterized complex (TFIIIC1) that stabilizes the binding of TFIIIC2 to the A and B boxes (21,44,47,53,54). Whereas the two largest hTFIIIC subunits are not conserved (24, 27, 34), the 102-kDa subunit (hTFIIIC102) and the hTFIIIC63 subunit are conserved in structure and in their interactions with TFIIIB and RNA polymerase III subunits (18). Thus, interactions of hTFIIIC102 (homologue of yeast TFIIIC131 [yTFIIIC131]) with hTFIIIB90 (homologue of yTFIIIB70) and of hTFIIIB90 with human RPC39 (hRPC39) (...