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Biomedical subjects

W C Shakespeare

Publications and source records attributed to W C Shakespeare.

12 recordsLinked to original sources
PubMed2006

Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases.

Vascular endothelial growth factor (VEGF) is the predominant pro-angiogenic cytokine in human malignancy, and its expression correlates with disease recurrence and poor outcomes in patients with colorectal cancer. Recently, expression of vascular endothelial growth factor receptors (VEGFRs) has been observed on tumours of epithelial origin, including those arising in the colon, but the molecular mechanisms governing potential VEGF-driven biologic functioning in these tumours are not well characterised. In this report, we investigated the role of Src family kinases (SFKs) in VEGF-mediated signalling in human colorectal carcinoma (CRC) cell lines. Vascular endothelial growth factor specifically activated SFKs in HT29 and KM12L4 CRC cell lines. Further, VEGF stimulation resulted in enhanced cellular migration, which was effectively blocked by pharmacologic inhibition of VEGFR-1 or Src kinase. Correspondingly, migration studies using siRNA clones with reduced Src expression confirmed the requirement for Src in VEGF-induced migration in these cells. Furthermore, VEGF treatment enhanced VEGFR-1/SFK complex formation and increased tyrosine phosphorylation of focal adhesion kinase, p130 cas and paxillin. Finally, we demonstrate that VEGF-induced migration is not due, at least in part, to VEGF acting as a mitogen. These results suggest that VEGFR-1 promotes migration of tumour cells through a Src-dependent pathway linked to activation of focal adhesion components that regulate this process.

Adenocarcinoma↗
PubMed2005

Protein phosphorylation and signal transduction modulation: chemistry perspectives for small-molecule drug discovery.

Protein phosphorylation has been exploited by Nature in profound ways to control various aspects of cell proliferation, differentiation, metabolism, survival, motility and gene transcription. Cellular signal transduction pathways involve protein kinases, protein phosphatases, and phosphoprotein-interacting domain (e.g., SH2, PTB, WW, FHA, 14-3-3) containing cellular proteins to provide multidimensional, dynamic and reversible regulation of many biological activities. Knowledge of cellular signal transduction pathways has led to the identification of promising therapeutic targets amongst these superfamilies of enzymes and adapter proteins which have been linked to various cancers as well as inflammatory, immune, metabolic and bone diseases. This review focuses on protein kinase, protein phosphatase and phosphoprotein-interacting cellular protein therapeutic targets with an emphasis on small-molecule drug discovery from a chemistry perspective. Noteworthy studies related to molecular genetics, signal transduction pathways, structural biology, and drug design for several of these therapeutic targets are highlighted. Some exemplary proof-of-concept lead compounds, clinical candidates and/or breakthrough medicines are further detailed to illustrate achievements as well as challenges in the generation, optimization and development of small-molecule inhibitors of protein kinases, protein phosphatases or phosphoprotein-interacting domain containing cellular proteins.

Drug Design↗
PubMed2001

A novel phosphotyrosine mimetic 4'-carboxymethyloxy-3'-phosphonophenylalanine (Cpp): exploitation in the design of nonpeptide inhibitors of pp60(Src) SH2 domain.

The novel phosphotyrosine (pTyr) mimetic 4'-carboxymethyloxy-3'-phosphonophenylalanine (Cpp) has been designed and incorporated into a series of nonpeptide inhibitors of the SH2 domain of pp60(c-Src) (Src) tyrosine kinase. A 2.2 A X-ray crystal structure of 1a bound to a mutant form of Lck SH2 domain provides insight regarding the structure-activity relationships and supports the design concept of this new pTyr mimetic.

Animals↗
PubMed2001

SH2 domain inhibition: a problem solved?

The past two years have witnessed a number of significant advances in the design of SH2 inhibitors of both Src and Grb2. For Src, several non-peptide templates have been developed with high affinity, and one case, in the context of bone-binding phosphotyrosine bioisostere, has yielded an in vivo active antiresorptive agent. Similarly, high-affinity Grb2 SH2 inhibitors with novel phosphotyrosine replacements have now been reported that demonstrate, for the first time, cellular activities consistent with an anticancer agent.

Models, Molecular↗
PubMed2001

Bone-targeted Src SH2 inhibitors block Src cellular activity and osteoclast-mediated resorption.

Src, a nonreceptor tyrosine kinase, is an important regulator of osteoclast-mediated resorption. We have investigated whether compounds that bind to the Src SH2 domain inhibit Src activity in cells and decrease osteoclast-mediated resorption. Compounds were examined for binding to the Src SH2 domain in vitro using a fluorescence polarization binding assay. Experiments were carried out with compounds demonstrating in vitro binding activity (nmol/L range) to determine if they inhibit Src SH2 binding and Src function in cells, demonstrate blockade of Src signaling, and lack cellular toxicity. Cell-based assays included: (1) a mammalian two-hybrid assay; (2) morphological reversion and growth inhibition of cSrcY527F-transformed cells; and (3) inhibition of cortactin phosphorylation in csk-/- cells. The Src SH2 binding compounds inhibit Src activity in all three of these mechanism-based assays. The compounds described were synthesized to contain nonhydrolyzable phosphotyrosine mimics that bind to bone. These compounds were further tested and found to inhibit rabbit osteoclast-mediated resorption of dentine. These results indicate that compounds that bind to the Src SH2 domain can inhibit Src activity in cells and inhibit osteoclast-mediated resorption.

Actins↗
PubMed2000

A Src SH2 selective binding compound inhibits osteoclast-mediated resorption.

BACKGROUND: The observations that Src(-/-) mice develop osteopetrosis and Src family tyrosine kinase inhibitors decrease osteoclast-mediated resorption of bone have implicated Src in the regulation of osteoclast-resorptive activity. We have designed and synthesized a compound, AP22161, that binds selectively to the Src SH2 domain and demonstrated that it inhibits Src-dependent cellular activity and inhibits osteoclast-mediated resorption. RESULTS: AP22161 was designed to bind selectively to the Src SH2 domain by targeting a cysteine residue within the highly conserved phosphotyrosine-binding pocket. AP22161 was tested in vitro for binding to SH2 domains and was found to bind selectively and with high affinity to the Src SH2 domain. AP22161 was further tested in mechanism-based cellular assays and found to block Src SH2 binding to peptide ligands, inhibit Src-dependent cellular activity and diminish osteoclast resorptive activity. CONCLUSIONS: These results indicate that a compound that selectively inhibits Src SH2 binding can be used to inhibit osteoclast resorption. Furthermore, AP22161 has the potential to be further developed for treating osteoporosis.

Amino Acid Sequence↗
PubMed1999

An efficient synthesis of a 4'-phosphonodifluoromethyl-3'-formyl-phenylalanine containing SRC SH2 ligand.

A CuBr-mediated, regioselective cross-coupling between methyl 2,5-diiodobenzoate (4) and [(diethoxyphosphinyl)difluoromethyl]zinc bromide is reported. Palladium-catalyzed incorporation of an amino acid side chain, followed by subsequent modifications resulted in the rapid construction of 2. Compound 2 was designed to engage Cys188 of the Src SH2 domain, however, this was not observed spectroscopically.

Binding Sites↗
PubMed1998

Modulation of receptor and receptor subtype affinities using diastereomeric and enantiomeric monosaccharide scaffolds as a means to structural and biological diversity. A new route to ether synthesis.

We show that carbohydrates constitute an attractive source of readily available, stereochemically defined scaffolds for the facile attachment of side chains contained in genetically encoded and other amino acids. beta-D- and beta-L-glucose, L-mannose, and the 6-deoxy-6-N-analogue of beta-D-glucose have been employed to synthesize peptidomimetics that bind the SRIF receptors on AtT-20 mouse pituitary cells, five cloned human receptor subtypes (hSSTRs), and the NK-1 receptor. The affinity profile of various sugar-based ligands at the hSSTRs is compared with that of SRIF. Compound 19 bound hSSTR4 with a Ki of 100 nM. Subtle structural changes affect affinities. Evidence is presented that suggests that one compound (8) binds both the AtT-20 cell receptors and the five hSSTRs via a unique mode. The SARs of the glycosides at SRIF receptors differ markedly from those at the NK-1 receptor. For example a 4-benzyl substituent is important for SRIF receptor binding, but the 4-desbenzyl analogue 27 was highly potent (IC50 of 27 nM) at the NK-1 receptor. A new, nonbasic method for the synthesis of base-sensitive ethers from primary and secondary alcohols is also described.

Animals↗
PubMed1998

A novel mechanism-based mammalian cell assay for the identification of SH2-domain-specific protein-protein inhibitors.

BACKGROUND: Many intracellular signal-transduction pathways are regulated by specific protein-protein interactions. These interactions are mediated by structural domains within signaling proteins that modulate a protein's cellular location, stability or activity. For example, Src-homology 2 (SH2) domains mediate protein-protein interactions through short contiguous amino acid motifs containing phosphotyrosine. As SH2 domains have been recognized as key regulatory molecules in a variety of cellular processes, they have become attractive drug targets. RESULTS: We have developed a novel mechanism-based cellular assay to monitor specific SH2-domain-dependent protein-protein interactions. The assay is based on a two-hybrid system adapted to function in mammalian cells where the SH2 domain ligand is phosphorylated, and binding to a specific SH2 domain can be induced and easily monitored. As examples, we have generated a series of mammalian cell lines that can be used to monitor SH2-domain-dependent activity of the signaling proteins ZAP-70 and Src. We are utilizing these cell lines to screen for immunosuppressive and anti-osteoclastic compounds, respectively, and demonstrate here the utility of this system for the identification of small-molecule, cell-permeant SH2 domain inhibitors. CONCLUSIONS: A mechanism-based mammalian cell assay has been developed to identify inhibitors of SH2-domain-dependent protein-protein interactions. Mechanism-based assays similar to that described here might have general use as screens for cell-permeant, nontoxic inhibitors of protein-protein interactions.

Animals↗
PubMed1996

ARL 17477, a potent and selective neuronal NOS inhibitor decreases infarct volume after transient middle cerebral artery occlusion in rats.

We tested the effects of administration of a selective neuronal nitric oxide synthase (nNOS) inhibitor, ARL 17477, on ischemic cell damage and regional cerebral blood flow (rCBF), in rats subjected to transient (2 h) middle cerebral artery (MCA) occlusion and 166 h of reperfusion (n = 48) and in rats without MCA occlusion (n = 25), respectively. Animals were administered ARL 17477 (i.v.): 10 mg/kg; 1 mg/kg; 3mg/kg; N-nitro-L-arginine (L-NA) 10 mg/kg L-NA 1 mg/kg; and Vehicle. Administration of ARL 17477 1 mg/kg, 3 mg/kg and 10 mg/kg reduced ischemic infarct volume by 53 (p < 0.05), 23, and 6.5%, respectively. L-NA 1 mg/kg and 10 mg/kg increased infarct volume by 2 and 15%, respectively (p > 0.05). Administration of ARL 17477 (10 mg/kg) significantly (p < 0.05) decreased rCBF by 27 +/- 5.3 and 24 +/- 14.08% and cortical NOS activity by 86 +/- 14.9 and 91 +/- 8.9% at 10 min or 3 h, respectively, and did not alter mean arterial blood pressure (MABP). L-NA (10 mg/kg) significantly reduced rCBF by 23 +/- 9.8% and NOS activity by 81 +/- 7% and significantly (p < 0.05) increased MABP. Treatment with 3 mg/kg and 1 mg/kg ARL 17477 reduced rCBF by only 2.4 +/- 4.5 and 0%, respectively, even when NOS activity was reduced by 63 +/- 13.4 and 45 +/- 15.7% at 3 h, respectively, (p < 0.05). The data demonstrate that ARL 17477 inhibits nNOS in the rat brain and causes a dose-dependent reduction in infarct volume after transient MCA occlusion.

Amidines↗