Chemical genetics approach to restoring p27Kip1 reveals novel compounds with antiproliferative activity in prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>The cyclin-dependent kinase (CDK) inhibitor p27^Kip1is downregulated in a majority of human cancers due to ectopic proteolysis by the ubiquitin-proteasome pathway. The expression of p27 is subject to multiple mechanisms of control involving several transcription factors, kinase pathways and at least three different ubiquitin ligases (SCF^SKP2, KPC, Pirh2), which regulate p27 transcription, translation, protein stability and subcellular localization. Using a chemical genetics approach, we have asked whether this control network can be modulated by small molecules such that p27 protein expression is restored in cancer cells.</p> <p>Results</p> <p>We developed a cell-based assay for measuring the levels of endogenous nuclear p27 in a high throughput screening format employing LNCaP prostate cancer cells engineered to overexpress SKP2. The assay platform was optimized to Z' factors of 0.48 - 0.6 and piloted by screening a total of 7368 chemical compounds. During the course of this work, we discovered two small molecules of previously unknown biological activity, SMIP001 and SMIP004, which increase the nuclear level of p27 at low micromolar concentrations. SMIPs (small molecule inhibitors of p27 depletion) also upregulate p21^Cip1, inhibit cellular CDK2 activity, induce G1 delay, inhibit colony formation in soft agar and exhibit preferential cytotoxicity in LNCaP cells relative to normal human fibroblasts. Unlike SMIP001, SMIP004 was found to downregulate SKP2 and to stabilize p27, although neither SMIP is a proteasome inhibitor. Whereas the screening endpoint - nuclear p27 - was robustly modulated by the compounds, SMIP-mediated cell cycle arrest and apoptosis were not strictly dependent on p27 and p21 - a finding that is explained by parallel inhibitory effects of SMIPs on positive cell cycle regulators, including cyclins E and A, and CDK4.</p> <p>Conclusions</p> <p>Our data provide proof-of-principle that the screening platform we developed, using endogenous nuclear p27 as an endpoint, presents an effective means of identifying bioactive molecules with cancer selective antiproliferative activity. This approach, when applied to larger and more diverse sets of compounds with refined drug-like properties, bears the potential of revealing both unknown cellular pathways globally impinging on p27 and novel leads for chemotherapeutics targeting a prominent molecular defect of human cancers.</p

Autosomal dominant hypercalciuria in a mouse model due to a mutation of the epithelial calcium channel, TRPV5

Contains fulltext : 118040.pdf (publisher's version ) (Open Access)Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1). Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5) and 6 (Trpv6) genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P). Compared to wild-type littermates, heterozygous (Trpv5(682P/+)) and homozygous (Trpv5(682P/682P)) mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and approximately 10% of males developed tubulointerstitial nephritis. Trpv5(682P/682P) mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D(3) concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5(682P/+) and Trpv5(682P/682P) mice consistent with a trafficking defect. In addition, Trpv5(682P/682P) mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D(28K), consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings indicate that the TRPV5 S682P mutant is functionally significant and study of HCALC1, a novel model for autosomal dominant hypercalciuria, may help further our understanding of renal calcium reabsorption and hypercalciuria