The activity of cyclin-dependent kinase 2 (CDK2) is essential for progression of cells from G 1 to the S phase of the mammalian cell cycle. CVT-313 is a potent CDK2 inhibitor, which was identified from a purine analog library with an IC 50 of 0.5 M in vitro. Inhibition was competitive with respect to ATP (K i ‫؍‬ 95 nM), and selective CVT-313 had no effect on other, nonrelated ATP-dependent serine/threonine kinases. When added to CDK1 or CDK4, a 8.5-and 430-fold higher concentration of CVT-313 was required for half-maximal inhibition of the enzyme activity. In cells exposed to CVT-313, hyperphosphorylation of the retinoblastoma gene product was inhibited, and progression through the cell cycle was arrested at the G 1 /S boundary. The growth of mouse, rat, and human cells in culture was also inhibited by CVT-313 with the IC 50 for growth arrest ranging from 1.25 to 20 M. To evaluate the effects of CVT-313 in vivo, we tested this agent in a rat carotid artery model of restenosis. A brief intraluminal exposure of CVT-313 to a denuded rat carotid artery resulted in more than 80% inhibition of neointima formation. These observations suggest that CVT-313 is a promising candidate for evaluation in other disease models related to aberrant cell proliferation.Cell cycle progression in mammalian cells is regulated by a family of cyclin-dependent protein kinases (CDKs), 1 that include CDK1, CDK2, CDK3, CDK4, and CDK6 (1). CDK2 is a serine/threonine kinase whose activity is essential for the G 1 to S transition during cell division. A number of proteins have been shown to be substrates for CDK2 phosphorylation, and among them are the retinoblastoma gene product (Rb) and other related pocket proteins, members of the E2F transcription factor family, cyclin E, and members of the CDK inhibitory proteins. It is also postulated that CDK2 phosphorylates and regulates proteins involved in DNA replication (2, 3). Two lines of evidence suggest that CDK2 activity is essential for cell proliferation; microinjection of antibodies directed against CDK2 blocks the progression of human diploid fibroblasts into S phase (4, 5), and overexpression of a dominant negative mutant of CDK2 in human osteosarcoma cells has a similar effect (6). The crucial role of CDK2 in controlling cell cycle progression suggests that CDK2 is an attractive target for treatment of aberrant cell proliferation.Smooth muscle cell proliferation is largely responsible for restenosis following angioplasty (7). A recent study has shown that CDK2 is activated very early after endothelial denudation in the rat carotid artery model of restenosis (8); moreover, antisense oligonucleotides directed against CDK2 were shown to be effective in reducing neointima formation in this model (9, 10). Arguably, the restenosis model can be used as a "proof of principle" for developing CDK2 inhibitors as drug candidates for the treatment of diseases related to aberrant cell proliferation. Olomoucine is a purine analog of ATP and is a specific inhibitor of CDK1 and CDK2 (11). Its potency, ho...

The D-type cyclins promote progression through the G 1 phase of the cell cycle and may provide a link between growth factors and the cell cycle machinery. We determined the nucleotide sequence of the 5-flanking region of the human cyclin D2 and cyclin D3 genes and identified the transcription start sites. Analysis of the upstream sequences required for transcription of the cyclin D2 and cyclin D3 genes in continuously dividing cells revealed marked differences in their regulatory elements. In the cyclin D2 gene positive elements were localized between positions ؊306 and ؊114 relative to the ATG codon at ؉1. Additional positive elements were localized between ؊444 and ؊345, whereas sequences that reduced transcription were identified between nucleotides ؊1624 and ؊892. In the cyclin D3 gene all of the positive elements required for maximal transcription were localized between nucleotides ؊366 and ؊167, and no negative elements were found. The activities of a reporter gene linked to the upstream regulatory sequences of the cyclin D2 gene but not the cyclin D3 gene were induced when starved cells were serum stimulated. This suggests that although the abundance of both the cyclin D2 and cyclin D3 mRNAs is increased by serum stimulation, only the cyclin D2 gene is up-regulated at the transcriptional level. Sequences between nucleotides ؊306 and ؊1624 of the cyclin D2 gene were necessary for serum inducibility.

Cyclin-dependent kinase 2 is a serine/threonine protein kinase essential for progression of the mammalian cell cycle from G 1 to S phase. CDK2 mRNA has been shown to be induced by serum in several cultured cell types. Therefore, we set out to identify elements that regulate the transcription of the human CDK2 gene and to characterize its structure. This paper describes the cloning of a ϳ2.4-kilobase pair genomic DNA fragment from the upstream region of the human CDK2 gene. This fragment contains five transcription initiation sites within a 72-nucleotide stretch. A 200-base pair sub-fragment that confers 70% of maximal basal promoter activity was shown to contain two synergistically acting Sp1 sites. However, a much larger DNA fragment containing ϳ1.7 kilobase pairs of upstream sequence is required for induction of promoter activity following serum stimulation. The intron exon boundaries of seven exons in this gene were also identified, and this information will be useful for analyzing genomic abnormalities associated with CDK2.Cyclin-dependent kinases (CDKs) 1 are the catalytic subunits of a family of serine/threonine protein kinase complexes that are also composed of a cyclin regulatory subunit (1-3). Most members of the CDK family are involved in regulating the progression of the eukaryotic cell cycle at various stages throughout G 1 , S, G 2 , and M phases (4). Other CDKs are involved in regulation of other processes in the cell, including phosphate metabolism (5) and transcription (6, 7).CDK2 is a member of the CDK family whose activity is restricted to the G 1 /S phase of the cell cycle. Several experiments demonstrated that CDK2 is essential for the mammalian cell cycle progression; micro-injection of antibodies directed against CDK2 blocked the progression of human diploid fibroblasts into S phase (8, 9), and overexpression of a CDK2 dominant negative mutant in human osteosarcoma cells had a similar effect (10).CDK2 is subject to an elaborate series of post-translational modifications. Although it has no kinase activity itself, kinase activity is conferred by association of CDK2 with a regulatory subunit, cyclin A or cyclin E, and by phosphorylation of Thr-160. Conversely, CDK2 activity is repressed by phosphorylation of Thr-14 or Tyr-15. Another layer of complexity is added to the regulatory scheme by CDK inhibitory proteins that can bind to CDK2 and inhibit the activity of the cyclin-kinase complex (4).While much attention has been given to the post-translational regulation of CDK2, we and others have found that CDK2 is also regulated at the transcriptional level. HoriguchiYamada et al. (11) reported a 3-fold increase in CDK2 mRNA in HL60 cells following stimulation with the phorbol ester 12-otetradecanoyl 13-acetate. Other groups (12) had similar findings with serum-stimulated human keratinocytes and human lung fibroblasts. Tanguay et al. (13) found induction of CDK2 expression in primary B lymphocytes following anti-IgM stimulation. These data suggest that transcriptional regulation of CDK2 could ...

The phosphoinositide 3-kinases (PI3Ks) have been linked to an extraordinarily diversified group of cellular functions making these enzymes compelling targets for the treatment of disease. A large body of evidence has linked PI3Kγ to the modulation of autoimmune and inflammatory processes making it an intriguing target for drug discovery. Our high-throughput screening (HTS) campaign revealed two hits that were nominated for further optimization studies. The in vitro activity of the first HTS hit, designated as the sulfonylpiperazine scaffold, was optimized utilizing structure-based design. However, nonoptimal pharmacokinetic properties precluded this series from further studies. An overlay of the X-ray structures of the sulfonylpiperazine scaffold and the second HTS hit within their complexes with PI3Kγ revealed a high degree of overlap. This feature was utilized to design a series of hybrid analogues including advanced leads such as 31 with desirable potency, selectivity, and oral bioavailability.

Recurrent genetic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have been identified in multiple tumor types. The most frequent mutation, IDH1 R132H, is a gain-of-function mutation resulting in an enzyme-catalyzing conversion of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). A high-throughput assay quantifying consumption of NADPH by IDH1 R132H has been optimized and implemented to screen 3 million compounds in 1536-well formats. The primary high-throughput screening hits were further characterized by RapidFire-mass spectrometry measuring 2-HG directly. Multiple distinct chemotypes were identified with nanomolar potencies (6-300 nM). All inhibitors were found to be inactive against the wild-type IDH1 homodimers. An IDH1 heterodimer between wild-type and R132H mutant is capable of catalyzing conversion of α-KG to 2-HG and isocitrate to α-KG. Interestingly, one of the inhibitors, EXEL-9324, was found to inhibit both conversions by the IDH1 heterodimer. This indicates the R132H/WT heterodimer may adopt conformations distinct from that of the R132H/R132H homodimer. Further enzymatic studies support this conclusion as the heterodimer exhibited a significantly lower apparent Michaelis-Menten constant for α-KG (K(m)=110 µM) compared with the R132H homodimer (K(m)= 1200 µM). The enhanced apparent affinity for α-KG suggests R132H/WT heterodimeric IDH1 can produce 2-HG more efficiently at normal intracellular levels of α-KG (approximately 100 µM).