Discovery, Development, and SAR of Aminothiazoles as LIMK Inhibitors with Cellular Anti-Invasive Properties.

As part of a program to develop a small molecule inhibitor of LIMK, a series of aminothiazole inhibitors were discovered by high throughput screening. Scaffold hopping and subsequent SAR directed development led to a series of low nanomolar inhibitors of LIMK1 and LIMK2 that also inhibited the direct biomarker p-cofilin in cells and inhibited the invasion of MDA MB-231-luc cells in a matrigel inverse invasion assay.

Interaction of yeast Rvs167 and Pho85 cyclin-dependent kinase complexes may link the cell cycle to the actin cytoskeleton.

BACKGROUND: . PHO85 encodes the catalytic subunit of a cyclin-dependent kinase (Cdk) in budding yeast and functions in phosphate and glycogen metabolism. Pho85 associated with the G1 cyclins Pcl1 and Pcl2 is also required for cell cycle progression in the absence of the Cdc28 cyclins Cln1 and Cln2. Loss of Pcl1, Pcl2 and related Pho85 cyclins results in budding defects, suggesting that Pcl-Pho85 complexes function in cell morphogenesis early in the cell cycle; their precise role is not clear, however. RESULTS: . To identify targets for Pcl-Pho85 kinases, we performed yeast two-hybrid interaction screens using Pcl2 and the related cyclin Pcl9. We identified RVS167, a gene involved in endocytosis, organization of the actin cytoskeleton, and cell survival after starvation. Like rvs167Delta mutants, pho85 mutants or strains deleted for the Pcl1,2-type Pho85 cyclins showed abnormal cell morphology on starvation, sensitivity to salt, random budding in diploids, and defects in endocytosis and in the actin cytoskeleton. Overexpression of Rvs167 in wild-type cells caused morphological abnormalities and growth arrest at high temperatures; these phenotypes were exacerbated by deleting PHO85. Rvs167 has a Src homology 3 (SH3) domain and five potential Pho85 phosphorylation sites; recombinant Rvs167 was phosphorylated by the Pcl2-Pho85 kinase in vitro. Maximal phosphorylation of Rvs167 in vivo required Pho85 and the Pcl1,2-type cyclins. CONCLUSIONS: . Rvs167 interacts with Pho85 cyclins and is implicated as a target of Pho85 kinases in vivo. Our results identify a connection between Cdks and the actin cytoskeleton; interaction of Rvs167 and Pcl-Pho85 Cdks might contribute to actin cytoskeleton regulation in response to stresses such as starvation.

A role for the Pcl9-Pho85 cyclin-cdk complex at the M/G1 boundary in Saccharomyces cerevisiae.

PHO85 is a cyclin-dependent kinase (CDK) with roles in phosphate and glycogen metabolism and cell cycle progression. As a CDK, Pho85 is activated by association with Pho85 cyclins (Pcls), of which 10 are known. PCL1, PCL2 and PCL9 are the only members of the Pho85 cyclin family that are expressed in a cell cycle-regulated pattern. We found that PCL9 is expressed in late M/early G1 phase of the cell cycle and is activated by the transcription factor, Swi5. This pattern of regulation is different from PCL1 and PCL2, which are expressed later in G1 phase and are regulated primarily by the transcription factor SBF. Co-immunoprecipitation experiments using in vitro translated proteins showed that Pcl9 and Pho85 form a complex. Furthermore, immunoprecipitated Pcl9 complexes from yeast lysates were capable of phosphorylating the exogenous substrate Pho4. The Pcl9-associated kinase activity was dependent on PHO85, showing that Pcl9 and Pho85 form a functionally active kinase complex in vivo. Deletion of PCL9 in diploid cells caused random, rather than bipolar, budding in 18% of cells. In contrast, deletion of PCL2, the closest relative of PCL9, had no effect on the budding pattern. Deleting more members of the PCL1,2 subfamily (which includes PCL9) increased the percentage of random budding in the cell population. When all members of the PCL1,2 subfamily were deleted, 73% of cells budded randomly, a value similar to that obtained when the CDK partner PHO85 was deleted. Our results show that PCL9 and PHO85 form a functional kinase complex and suggest a role for Pho85 CDKs at the M/G1 boundary.

Expression of the dystrophin isoform Dp71 in differentiating human fetal myogenic cultures.

The dystrophin gene defective in Duchenne muscular dystrophy (DMD) is extreme in size and complexity with several promoters which direct expression of different isoforms in different tissues. In contrast with adult skeletal muscle which expresses 427 kDa dystrophin, fetal muscle tissue expresses the 71 kDa ubiquitous isoform Dp71 as well as 427 kDa muscle dystrophin. To examine Dp71 expression in fetal muscle further, we have monitored its expression pattern in differentiating myogenic cultures of human fetal muscle origin. The presence of transcripts initiated from the Dp71 promoter was demonstrated by quantitative RT-PCR. The level of transcript expressed from the Dp71 promoter did not change significantly during myogenic differentiation, consistent with the housekeeping nature of the promoter. Measurements to determine the stability of the Dp71 mRNA indicated that it has a half-life of -20 h and, therefore, is somewhat more stable than the larger 14 kb muscle dystrophin mRNA (t1/2 = 16 h). In contrast with the constant level of Dp71 transcript during myogenic differentiation, the level of Dp71 protein increased significantly, perhaps due to changes in translation efficiency or protein stability. These results demonstrate expression and posttranscriptional upregulation of Dp71 in human fetal myogenic cultures.

Stability of the human dystrophin transcript in muscle.

The human dystrophin gene has 79 exons spanning >2300 kb making it the largest known gene. In previous studies we showed that approximately 16 h are required to transcribe the gene in myogenic cultures [Tennyson, C.N., Klamut, H.J. and Worton, R.G. (1995) Nature Genet. 9, 184-190]. To estimate the half-life of the dystrophin mRNA, the decay of the transcript was monitored by quantitative RT-PCR in cultured human fetal myotubes following exposure to actinomycin D. Results from this analysis indicated that the half-life of the dystrophin mRNA is 15.6 +/- 2.8 h in these cultures. Transcript accumulation profiles were predicted using a mathematical model which incorporated the measured half-life. The modeled accumulation profiles were consistent with observed profiles supporting the half-life measured using actinomycin D. The kinetic model was then used to predict the relative amount of nascent and mature dystrophin transcript at steady state. Measurements by quantitative RT-PCR indicated that in adult skeletal muscle tissue the concentration of mature dystrophin mRNA is 5-10 molecules per nucleus, demonstrating, as expected, that it is a low abundance transcript. Furthermore the ratio of nascent to mature dystrophin transcript indicated that dystrophin synthesis may not be at steady state in the adult skeletal muscle we tested. Alternatively, the kinetics of transcript production in skeletal muscle tissue may be different from those observed in cultured fetal myogenic cells.

The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced.

The largest known gene is the human dystrophin gene, which has 79 exons spanning at least 2,300 kilobases (kb). Transcript accumulation was monitored from four regions of the gene following induction of expression in muscle cell cultures. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) results indicate that approximately 12 h are required for transcription of 1,770 kb (at an average elongation rate of 2.4 kb min-1), extrapolating to a transcription time of 16 h for the complete gene. Accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5' end before transcription is complete providing strong evidence for cotranscriptional splicing. The rate of transcript accumulation was reduced at the 3' end of the gene relative to the 5' end, perhaps due to premature termination of transcription complexes.