Discovery of (E)-5-(benzylideneamino)-1H-benzo[d]imidazol-2(3H)-one derivatives as inhibitors for PTK6.

A lead compound 1, which inhibits the catalytic activity of PTK6, was selected from a chemical library. Derivatives of compound 1 were synthesized and analyzed for inhibitory activity against PTK6 in vitro and at the cellular level. Selected compounds were analyzed for cytotoxicity in human foreskin fibroblasts using MTT assays and for selectivity towards PTK members in HEK 293 cells. Compounds 20 (in vitro IC50=0.12µM) and 21 (in vitro IC50=0.52µM) showed little cytotoxicity, excellent inhibition of PTK6 in vitro and at the cellular level, and selectivity for PTK6. Compounds 20 and 21 inhibited phosphorylation of specific PTK6 substrates in HEK293 cells. Thus, we have identified novel PTK6 inhibitors that may be used as treatments for PTK6-positive carcinomas, including breast cancer.

The presence of outer arm fucose residues on the N-glycans of tissue inhibitor of metalloproteinases-1 reduces its activity.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MMPs) by binding at a 1:1 stoichiometry. Here we have shown the involvement of N-glycosylation in the MMP inhibitory ability of TIMP-1. TIMP-1, purified from HEK 293 cells overexpressing TIMP-1 (293 TIMP-1), showed less binding and inhibitory abilities to MMPs than TIMP-1 purified from fibroblasts or SF9 insect cells infected with TIMP-1 baculovirus. Following deglycosylation of TIMP-1, all forms of TIMP-1 showed similar levels of MMP binding and inhibition, suggesting that glycosylation is involved in the regulation of these TIMP-1 activities. Analysis of the N-glycan structures showed that SF9 TIMP-1 has the simplest N-glycan structures, followed by fibroblast TIMP-1 and 293 TIMP-1, in order of increasing complexity in their N-glycan structures. Further analyses showed that cleavage of outer arm fucose residues from the N-glycans of 293 TIMP-1 or knockdown of both FUT4 and FUT7 (which encode for fucosyltransferases that add outer arm fucose residues to N-glycans) enhanced the MMP-binding and catalytic abilities of 293 TIMP-1, bringing them up to the levels of the other TIMP-1. These results demonstrate that the ability of TIMP-1 to inhibit MMPs is at least in part regulated by outer arm fucosylation of its N-glycans.

The associated pyrazolopyrimidines PP1 and PP2 inhibit protein tyrosine kinase 6 activity and suppress breast cancer cell proliferation.

Protein tyrosine kinase (PTK)6, also known as breast tumor kinase, is a non-receptor tyrosine kinase. It is closely associated with, but evolutionarily distinct from, the Src family members. PTK6 has a role in proliferation, migration and invasion in various cancers, and therefore has been suggested as a potentially valuable therapeutic target. In an attempt to develop PTK6 inhibitors, chemicals known to inhibit various kinases were screened for their ability to inhibit PTK6. Pyrazolopyrimidine (PP)1, PP2 and a lymphocyte-specific protein tyrosine kinase inhibitor strongly inhibited the catalytic activity of PTK6 in vitro. These chemicals suppressed the phosphorylation of PTK6 substrate proteins, including signal transducer and activator of transcription 3, in human embryonic kidney (HEK) 293 cells expressing hyperactive PTK6. They also expressed selectivity towards PTK6 over other PTK members in HEK 293 cells. PP1 and PP2 specifically inhibited the PTK6-dependent proliferation of human breast carcinoma T-47D cells. PP1 and PP2 were more selective for PTK6 than for Src family kinases, and may be useful for the treatment of PTK6-positive malignant diseases such as breast cancer.

Growth-stimulatory activity of TIMP-2 is mediated through c-Src activation followed by activation of FAK, PI3-kinase/AKT, and ERK1/2 independent of MMP inhibition in lung adenocarcinoma cells.

Tissue inhibitors of metalloproteinases (TIMPs) control extracellular matrix (ECM) homeostasis by inhibiting the activity of matrix metalloproteinases (MMPs), which are associated with ECM turnover. Recent studies have revealed that TIMPs are implicated in tumorigenesis in both MMP-dependent and MMP-independent manners. We examined a mechanism by which TIMP-2 stimulated lung adenocarcinoma cell proliferation, independent of MMP inhibition. The stimulation of growth by TIMP-2 in A549 cells required c-Src kinase activation. c-Src kinase activity, induced by TIMP-2, concomitantly increased FAK, phosphoinositide 3-kinase (PI3-kinase)/AKT, and ERK1/2 activation. Selective knockdown of integrin α3ß1, known as a TIMP-2 receptor, did not significantly change TIMP-2 growth promoting activity. Furthermore, we showed that high TIMP-2 expression in lung adenocarcinomas is associated with a worse prognosis from multiple cohorts, especially for stage I lung adenocarcinoma. Through integrated analysis of The Cancer Genome Atlas data, TIMP-2 expression was significantly associated with the alteration of driving genes, c-Src activation, and PI3-kinase/AKT pathway activation. Taken together, our results demonstrate that TIMP-2 stimulates lung adenocarcinoma cell proliferation through c-Src, FAK, PI3-kinase/AKT, and ERK1/2 pathway activation in an MMP-independent manner.

Molecular dissection of the interaction between the SH3 domain and the SH2-Kinase Linker region in PTK6.

PTK6 (also known as Brk) is an intracellular tyrosine kinase that contains SH3, SH2, and tyrosine kinase catalytic (Kinase) domains. The SH3 domain of PTK6 interacts with the N-terminal half of the linker (Linker) region between the SH2 and Kinase domains. Site-directed mutagenesis and surface plasmon resonance studies showed that a tryptophan residue (Trp44) in the SH3 domain and proline residues in the Linker region, in the order of Pro177, Pro175, and Pro179, contribute to the interaction. The three-dimensional modeled structure of the SH3-Linker complex was in agreement with the biochemical data. Disruption of the intramolecular interaction between the SH3 domain and the Linker region by mutation of Trp44, Pro175, Pro177, and Pro179 markedly increased the catalytic activity of PTK6 in HEK 293 cells. These results demonstrate that Trp44 in the SH3 domain and Pro177, Pro175, and Pro179 in the N-terminal half of the Linker region play important roles in the SH3-Linker interaction to maintain the protein in an inactive conformation along with the phosphorylated Tyr447-SH2 interaction.

An intramolecular interaction between SH2-kinase linker and kinase domain is essential for the catalytic activity of protein-tyrosine kinase-6.

Protein-tyrosine kinase-6 (PTK6, also known as Brk) is a non-receptor tyrosine kinase that contains SH3, SH2, and catalytic (Kinase) domains. We have identified an intramolecular interaction between the linker (Linker) region connecting the SH2 and Kinase domains and the Kinase domain. Residue Trp-184 within the Linker region is essential for the Linker-Kinase interaction but not for the Linker-SH3 interaction. A recombinant PTK6 Kinase domain connected to the Linker region had catalytic activity in terms of autophosphorylation, phosphorylation of a PTK6 substrate, BKS, and phosphorylation of an oligopeptide substrate, whereas the Kinase domain itself, or one connected to a Linker region containing a W184A substitution, did not. The introduction of the W184A mutation into PTK6 also abrogated autophosphorylation and phosphorylation of another PTK6 substrate, Sam68, as well as phosphorylation of intracellular proteins. It also abolished the ability of PTK6 to promote proliferation and prevent apoptosis of HEK 293 cells, as well as to permit anchorage-independent colony formation. Therefore, unlike Src family members, in which the Linker-Kinase interaction inhibits catalytic activity, in PTK6 this interaction has an essential positive role.

Solution structure and backbone dynamics of the non-receptor protein-tyrosine kinase-6 Src homology 2 domain.

Human protein-tyrosine kinase-6 (PTK6, also known as breast tumor kinase (Brk)) is a member of the non-receptor protein-tyrosine kinase family and is expressed in two-thirds of all breast tumors. To understand the structural basis of PTK6 function, we have determined the solution structure and backbone dynamics of the PTK6-Src homology 2 (SH2) domain using multidimensional NMR spectroscopy. The solution structure clearly indicates that the SH2 domain of human PTK6 contains a consensus alpha/beta-fold and a Tyr(P) peptide binding surface, which are common to other SH2 domains. However, two of the alpha-helices (alphaA and alphaB) are located on opposite faces of the central beta-sheet. In addition, the topological arrangement of a central four-stranded antiparallel beta-sheet (strands betaA, betaB, betaC, and betaD) differs from that of other Src family members. Backbone dynamics and Tyr(P) peptide titration experiments revealed that the putative ligand binding sites of the PTK6-SH2 domain undergo distinctive internal motions when compared with other regions of the protein. Surface plasmon resonance analysis showed that the Tyr(P) peptide had a dissociation constant of about 60 microm, which is substantially weaker binding than previously reported for Src family members. The solution structure together with data from the ligand binding mode of PTK6-SH2 provides insight into the molecular basis of the autoinhibitory role of PTK6.