Parallel synthesis and biological evaluation of 5,6,7,8-tetrahydrobenzothieno[2,3-d]pyrimidin-4(3H)-one cytotoxic agents selective for p21-deficient cells

A novel series of inhibitors of cancer cell proliferation, selective against p21 cell cycle checkpoint-disrupted cells vs. cells with intact p21 checkpoint, were identified by high-throughput screening. Optimization of both ends of the lead molecule to improve potency, using parallel synthesis and iterative design, is described. The 2-(1,4-dibenzodioxane)-substituted derivative 14 was identified as a highly selective and potent agent displaying an IC50 of 91 nM in the p21-deficient cell line.     

Inhibition of tumor cell proliferation by thieno[2,3-d]pyrimidin-4(1H)-one-based analogs

On the basis of a screening lead from an assay using a pair of p21 isogenic cell lines (p21-proficient cells and p21-deficient cells) to identify chemoselective agents, a series of novel thieno[2,3-d]pyrimidin-4(1H)-one-based analogs was prepared. Some analogs inhibited the growth of human colon tumor cells.     

Cytodifferentiation potentiates aFGF-induced p21(ras)/Erk signaling pathway in rat cultured astrocytes.

MBP kinase detection assay revealed that acidic FGF (aFGF) augmented MBP kinase activity in a dose-dependent manner in astrocytes (AC). The molar potency of this action of aFGF in dibutyryl cyclic AMP (DBcAMP)-treated AC was significantly higher than that in quiescent AC. Consistently, the molar potency of accumulation of p21(ras)-GTP by aFGF was significantly higher in DBcAMP-treated AC than in quiescent AC. However, binding study showed that B(max) and K(D) for [(125)I]aFGF in DBcAMP-treated AC were quite similar to those in quiescent AC. Furthermore, the expression levels of Grb2, SOS, and p21(ras) were not changed by treatment of AC with DBcAMP. These results suggest that cytodifferentiation potentiates the p21(ras)/Erk signaling pathway in AC in response to aFGF without changing the expression levels of signaling molecules mediating from the FGF receptor to p21(ras).     

Successive phosphorylation of p27(KIP1) protein at serine-10 and C terminus crucially controls its potency to inactivate Cdk2

During the G(1)-S transition, the activity of Cdk2 is regulated by its association with p27(KIP1), which in rodent fibroblasts undergoes phosphorylation mainly at serine 10, threonine 187, and C-terminal threonine 197 by KIS, Cdk2, and Pim or ROCK, respectively. Recently Cdc6 the AAA+ ATPase, identified initially to assemble pre-replicative complexes on origins of replication and later to activate p21(CIP1)-inactivated Cdk2, was found also to activate p27-bound Cdk2 but only after the bound p27 is C-terminally phosphorylated. On the other hand, the biological significance of the serine 10 phosphorylation remains elusive aside from its involvement in the stability of p27 itself. We report here that serine 10 phosphorylation is required for efficient C-terminal phosphorylation of its own by PIM and ROCK kinases and critically controls the potency of p27 as a Cdk2 inhibitor. In vitro, PIM1 and active ROCK1 efficiently phosphorylated free as well as Cdk2-bound p27 but only when the p27 was phosphorylated at Ser-10 in advance. Consistently, a Ser-10 nonphosphorylatable mutant p27 protein was not phosphorylated at the C terminus in vivo. Furthermore, when double-phosphorylated, free p27 was no longer a potent inhibitor of Cdk2, and Cdk2-bound p27 could be removed by Cdc6 to reactivate the Cdk2. Thus, phosphorylation at these two sites crucially controls the potency of this CDK inhibitor in two distinct modes.     

Imidazo-pyrazine derivatives as potent CXCR3 antagonists

A general way of improving the potency of CXCR3 antagonists with fused hetero-bicyclic cores was identified. Optimization efforts led to the discovery of a series of imidazo-pyrazine derivatives with improved pharmacokinetic properties in addition to increased potency. The efficacy of the lead compound 21 is evaluated in a mouse lung inflammation model.     

1-Phenyl-5-pyrazolyl ureas: potent and selective p38 kinase inhibitors

Inhibitors of the MAP kinase p38 are potentially useful for the treatment of arthritis and osteoporosis. Several 2,3-dichlorophenyl ureas were identified as small-molecule inhibitors of p38 by a combinatorial chemistry effort. Optimization for cellular potency led to the discovery of a new class of potent and selective p38 kinase inhibitors, exemplified by the 1-phenyl-5-pyrazolyl urea 7 (IC50 = 13 nM).     

Molecular cloning of smg p21B and identification of smg p21 purified from bovine brain and human platelets as smg p21B

We have previously purified smg p21 from bovine brain membranes and isolated its cDNA from a bovine brain cDNA library. In the present studies, we have performed extensive screening of the bovine brain cDNA library with the cloned smg p21 cDNA as a probe and isolated another cDNA encoding a protein highly homologous to smg p21. The proteins encoded by the previously and newly isolated cDNAs are designated as smg p21A and -B, respectively. Since the partial amino acid sequences determined previously from the smg p21 purified from bovine brain were identical with the common amino acid sequences between smg p21A and -B, we have further sequenced smg p21 and identified it as smg p21B. We have also further sequenced the smg p21 purified from human platelet membranes and identified it as smg p21B. Amino acid sequence analysis indicates that smg p21A is identical with the rap1A and Krev-1 proteins and smg p21B is identical with the rap1B protein.     

Synthesis and SAR of p38alpha MAP kinase inhibitors based on heterobicyclic scaffolds

The synthesis and structure-activity relationships (SAR) of p38alpha MAP kinase inhibitors based on heterobicyclic scaffolds are described. This effort led to the identification of compound (21) as a potent inhibitor of p38alpha MAP kinase with good cellular potency toward the inhibition of TNF-alpha production. X-ray co-crystallography of an oxalamide analog (24) bound to unphosphorylated p38alpha is also disclosed.     

p21RNAi的pSUPER载体的构建及功能鉴定

目的：构建抑制p21基因表达的pSUPERRNAi载体（pSUPER—p21）并鉴定其功能。方法：化学合成一对编码短发夹RNA序列的、靶向大鼠p21基因的寡核苷酸链,各60个碱基,退火,克隆到经BglⅡ、HindⅢ酶切的pSUPER质粒上,构建重组RNAi质粒（pSUPER-p21）。通过双酶切鉴定及测序分析验证构建效果。将正确构建的质粒转染大鼠原代培养皮质神经元,western blotting检测经红藻氨酸处理的神经元中p21蛋白表达。结果：pSUPER-p21载体经双酶切鉴定及测序分析,结果表明60个碱基成功插入到预计位点,并且序列完全一致。Western blotting结果证实pSUPER-p21载体可特异性抑制红藻氨酸诱导的原代培养皮质神经元中p21蛋白表达的上调。结论：靶向p21的pSUPERRNAi载体构建成功,该载体可特异性抑制p21基因表达。     

Transforming ras proteins accelerate hormone-induced maturation and stimulate cyclic AMP phosphodiesterase in Xenopus oocytes.

Transforming Harvey (Ha) ras oncogene products accelerated the time course of Xenopus oocyte maturation induced by insulin, insulinlike growth factor 1, or progesterone. The transforming constructs, [Val-12]Ha p21 and [Val-12, Thr-59]Ha p21, displayed equal potency and efficacy in their abilities to accelerate the growth peptide-induced response. Normal Ha p21 was only 60% as powerful and one-fifth as potent as the mutants containing valine in the 12 position. In contrast, two nontransforming constructs, [Val-12, Ala-35, Leu-36, Thr-59]Ha p21 and [Val-12, Thr-59]Ha(term-174) p21, had no effect on the time course of hormone-induced maturation. Effects of the transforming ras proteins on hormone-induced maturation correlated with their abilities to stimulate in vivo phosphodiesterase activity measured after microinjection of 200 microM cyclic [3H] AMP. When p21 injection followed 90 min of insulin treatment, there was no increase in phosphodiesterase activity over that measured after hormone treatment or p21 injection alone, but additive effects of p21 and insulin on enzyme activity were observed during the first 90 min of insulin treatment. Even though normal Ha p21 and transforming [Val-12, Thr-59]Ha p21 stimulated oocyte phosphodiesterase to equal levels when coinjected with substrate at the initiation of the in vivo assay, the transforming protein elicited a more sustained stimulation of enzyme activity. These results suggest that stimulation of a cyclic AMP phosphodiesterase activity associated with insulin-induced maturation is involved in the growth-promoting actions of ras oncogene products in Xenopus oocytes.     

O-Isopropylidenation of carbohydrates catalyzed by vanadyl triflate

Vanadyl triflate has been identified as a mild and efficient catalyst for the chemoselective O-isopropylidenation of functionalized carbohydrates with acetone and acetone equivalents. The current protocol is compatible with a diverse array of protecting groups and the products can be readily isolated by simple aqueous wash.     

Biochemical and biological properties of the human N-ras p21 protein.

We characterized the normal (Gly-12) and two mutant (Asp-12 and Val-12) forms of human N-ras proteins produced by Escherichia coli. No significant differences were found between normal and mutant p21 proteins in their affinities for GTP or GDP. Examination of GTPase activities revealed significant differences between the mutant p21s: the Val-12 mutant retained 12% of wild-type GTPase activity, whereas the Asp-12 mutant retained 43%. Both mutant proteins, however, were equally potent in causing morphological transformation and increased cell motility after their microinjection into quiescent NIH 3T3 cells. This lack of correlation between transforming potency and GTPase activity or guanine nucleotide binding suggests that position 12 mutations affect other aspects of p21 function.     

Developing peptide-based multivalent antagonists of proliferating cell nuclear antigen and a fluorescence-based PCNA binding assay

Proliferating cell nuclear antigen (PCNA) is a critical player in cell proliferation. It interacts with a myriad of cellular proteins in genomic DNA replication and cell cycle control. This makes PCNA an attractive target for developing antiproliferative therapeutics. Indeed, the binding of a human tumor suppressor protein, p21, to PCNA contributes to its antiproliferative effect in cells. In this work, we report a fluorescence polarization-based binding assay for determining the affinity between the p21 peptide and human PCNA. To improve the potency of the p21-based PCNA antagonist, we exploited the homotrimeric structure of PCNA and developed multivalent peptide-based PCNA antagonists. The di- and trivalent p21-based antagonists bind to PCNA with low nanomolar dissociation constant. Moreover, we show that the multivalent PCNA antagonists inhibited PCNA-dependent DNA synthesis in a human cell extract with improved avidity when compared with the monovalent p21 peptide. The fluorescence polarization assay holds promise for the discovery of potent small-molecule PCNA inhibitors given its ready adaptability to a high-throughput screening format.     

Cdk2-dependent phosphorylation of p21 regulates the role of Cdk2 in cisplatin cytotoxicity

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. We found that an 18-kDa protein identified by mass spectrometry as p21(WAF1/Cip1) was phosphorylated by Cdk2 starting 12 h after cisplatin exposure. The analysis showed it was phosphorylated at serine 78, a site not previously identified. The adenoviral transduction of p21 before cisplatin exposure protects from cytotoxicity by inhibiting Cdk2. Although cisplatin causes induction of endogenous p21, the protection is inefficient. We hypothesized that phosphorylation of p21 at serine 78 could affect its role as a Cdk inhibitor, and thereby lessen its ability to protect from cisplatin cytotoxicity. To investigate the effect of serine 78 phosphorylation on p21 activity, we replaced serine 78 with aspartic acid, creating the phosphomimic p21(S78D). Mutant p21(S78D) was an inefficient inhibitor of Cdk2 and was inefficient at protecting TKPTS cells from cisplatin-induced cell death. We conclude that phosphorylation of p21 by Cdk2 limits the effectiveness of p21 to inhibit Cdk2, which is the mechanism for continued cisplatin cytotoxicity even after the induction of a protective protein.     

Optimization of small molecule agonists of the thrombopoietin (Tpo) receptor derived from a benzo[a]carbazole hit scaffold

The lead optimization of a novel series of benzo[a]carbazole-based small molecule agonists of the thrombopoietin (Tpo) receptor is reported. The chemical instability of the dihydro-benzo[a]carbazole lead 2 was successfully addressed in the design and evaluation of compounds which also demonstrated improved potency compared to 2. Members of the scaffold have been identified which are full agonists that demonstrate cellular functional potency <50 nM. Analog 21 demonstrates equivalent efficacy in the human megakaryocyte differentiation (CFU-mega) assay compared to Eltrombopag.     

Novel 1,4-benzodiazepine-2,5-diones as Hdm2 antagonists with improved cellular activity

The disruption of the p53-Hdm2 protein-protein interaction induces cell growth arrest and apoptosis. We have identified the 1,4-benzodiazepine-2,5-dione scaffold as a suitable template for inhibiting this interaction by binding to the Hdm2 protein. Several compounds have been made with improved potency, solubility, and cell-based activities.     

Activation of a human c-K-ras oncogene

The human lung carcinomas PR310 and PR371 contain activated c-K-ras oncogenes. The oncogene of PR371 was found to present a mutation at codon 12 of the first coding exon which substitutes cysteine for glycine in the encoded p21 protein. We report here that the transforming gene of PR310 tumor contains a mutation in the second coding exon. An A----T transversion at codon 61 results in the incorporation of histidine instead of glutamine in the c-K-ras gene product. By constructing c-K-ras/c-H-ras chimeric genes we show that this point mutation is sufficient to confer transforming potential to ras genes, and that a hybrid ras gene coding for a protein mutant at both codons 12 and 61 is also capable of transforming NIH3T3 cells. The relative transforming potency of p21 proteins encoded by ras genes mutant at codons 12, 61 or both has been analyzed. Our studies also show that the coding exons of ras genes, including the fourth, can be interchanged and the chimeric p21 ras proteins retain their oncogenic ability in normal rodent established cell lines.