Cyclobutanes represent an underutilised structural feature in medicinal chemistry, partially due to difficulties in forming them in an easy and controlled manner. Herein is described their application to a drug discovery project and development of the enamine [2+2] cyclisation; a straightforward synthesis of functionalised cyclobutanes. A library of 30 cyclobutane based integrin antagonists have been designed and synthesised to explore the SAR around the hit dual β3 integrin antagonist ICT9055. Several of which were shown to be highly potent antagonists inhibiting cancer cell adhesion, migration and invasion while remaining non-toxic. ICT9072 had comparable β3 activity to hit compound ICT9055 but also had activity against αvβ5 and therefore showed greater inhibition of migration of DLD-1 cells. This showed the ability to modify this scaffold for multi integrin antagonism and potential benefit of this. Synthetic studies towards the marine natural product providencin has led to the development of a previously unknown intramolecular enamine [2+2] cyclisation which has been shown to proceed in a diastereoselective manner. This reaction has been applied to the synthesis of a highly functionalised enatiopure cyclobutene suitable for inclusion into the total synthesis. A model furyl cyclobutane has also been synthesised to exemplify the route from the enantiopure cyclobutene through to the furyl cyclobutane fragment of providencin. / Yorkshire Cancer Research

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23806号 / 医博第4852号 / 新制||医||1058(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 森信 暁雄, 教授 上野 英樹, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第24009号 / エネ博第445号 / 新制||エネ||84(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー応用科学専攻 / (主査)教授 馬渕 守, 教授 土井 俊哉, 教授 濵 孝之 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

Macrophage integrin aDb2 promotes macrophage retention and accumulation within inflamed tissue, a key event in development of chronic inflammation. Recently, the P5 peptide was identified as a specific inhibitor for integrin aDb2 interaction with 2-(ω-carboxyethyl) pyrole (CEP), a ligand at inflammatory sites. This thesis aims to identify integrin aD I-domain amino acids involved in binding P5 peptide and likewise to CEP. We propose that non-conserved, basic amino acids of the integrin aDb2 I-domain are responsible for binding to P5 peptide and likewise to CEP. Eight amino acids were analyzed by generating six mutant aD I-domains: K180[A], R189[Q], K205[L], HHK223-225[NIT], K233[A], and K246[A]. Mutagenic constructs were created using PCR site-directed mutagenesis, then transformed into E.coli BL21 cells for IPTG-induced protein expression. Of the 6 mutant I-domains analyzed, amino acid K246 was critical in binding to P5 peptide and CEP through ForteBio Protein-Protein Assay, as well as to CEP by cell adhesion assay.

Connective Tissue Growth Factor (CTGF) is a matricellular protein that has been shown to mediate cell adhesion, and as a consequence, it regulates cell proliferation, migration, differentiation and gene transcription. Although previous in vivo and in vitro studies supported the anabolic role of CTGF in skeletogenesis, to date mechanisms of this effect remain unknown. So far, no specific receptor has been identified for CTGF, although previous studies have shown that integrins can serve as functional signaling receptors for CTGF. The CTGF-integrin interaction initiates intracellular signaling cascades that ultimately regulate cell cytoskeleton reorganization, gene transcription and cell function. To study the effect of CTGF on osteoblasts, we first conducted adhesion assays using the MC3T3-E1 osteoblastic cell line. We confirmed that osteoblasts adhere to rCTGF in a concentration-dependent manner and we showed this adhesion has characteristics of integrin mediated adhesions. Next, we used an array of blocking antibodies directed against the individual alpha and beta; integrin subunits that are known to be expressed in osteoblasts. Significant decreases in cell adhesion were observed upon treatment with anti-alpha-v or anti-beta1 blocking antibodies. Subsequent coimmunoprecipitation analyses demonstrated that CTGF interacts with alpha-v and beta1 integrins in osteoblasts. Furthermore, we showed that the specificity of this CTGF-integrin interaction occurs in the C-terminal domain (fourth module) of CTGF. The immunefluorescence staining of cells cultured on substrates of rCTGF, fibronectin (positive control) or BSA (negative control) demonstrated that osteoblast adhesion to rCTGF results in actin cytoskeleton reorganization, focal adhesion formation, enhanced cell spreading and Rac activation. These series of events are necessary for proper cell-matrix interaction and integrins' downstream signaling initiation. Next, through alkaline phosphatase (ALP) staining and activity assays, as well as Alizarin red staining, we demonstrated that osteoblast attachment to CTGF matrix enhances cell maturation, bone nodule formation and matrix mineralization. To investigate whether the effect of CTGF on osteoblast differentiation involves activation of specific signaling molecules, we performed Western blot and chromatin immunoprecipitation (ChIP) assays. Osteoblasts cultured on rCTGF expressed higher levels of both total and phosphorylated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) compared to the cells cultured on BSA. In addition, these osteoblasts showed an increase in runt-related transcription factor 2 (Runx2) binding to the osteocalcin gene promoter compared to the negative control. These experiments confirmed CTGF's effect on enhancing osteoblast differentiation through regulation of important signaling molecules. In another series of experiments, we used primary osteoblasts isolated from CTGF KO mice, their WT littermates, or WT cells infected to overexpress (OE) CTGF to study the effect of different levels of endogenous CTGF on osteoblast cytoskeleton reorganization and motility. Our assays showed enhanced cell adhesion, spreading and Rac expression in CTGF OE osteoblasts, while in CTGF KO osteoblasts, cell adhesion, spreading and Rac expression were significantly decreased. In contrast, CTGF OE osteoblasts that showed high adhesion and spreading, exhibited diminished cell motility and low levels of RhoA expression, while KO cells migrated quickly and expressed high levels of RhoA. Together, these experiments establish CTGF as an adhesion protein for osteoblasts; they demonstrate that the alpha-v beta1 integrin is a functional signaling receptor for CTGF; they confirm that osteoblast differentiation is enhanced when cultured on CTGF matrix through activation of regulatory signaling molecules; and finally, these experiments establish a role for CTGF in the regulation of small RhoGTPases expression, which in turn implies a significant role for CTGF in cell cytoskeleton reorganization and motility. / Cell Biology

Gliomas are the most common and difficult to treat tumors of the central nervous system. Current treatments often fail to slow progression of disease due to the high invasive nature of glioma leading to a high percentage of recurrence. Our previous studies have demonstrated that the levels of alpha; 9 beta; 1 integrin found on high grade glioma were significantly increased in comparison to normal brain tissue where the levels were negligible. We also found that interaction between alpha; 9 beta; 1 integrin and nerve growth factor (NGF) plays a major role in progression of experimental tumor. Another receptor for NGF the common neurotrophin receptor p75NTR is also overexpressed in high grade glioma. p75NTR forms a high affinity complex with the specific NGF receptor, TrkA leading to an increase in cell proliferation and survival. In the absence of an association, p75NTR is involved in transferring pro-apoptotic signals through the JNK pathway. We have found that the α 9 integrin subunit of α 9 β 1 forms a stable, cation independent complex with p75NTR on the cell membrane of glioma both in vitro using glioma derived immortalized cells lines and in vivo using glioma tissue. The co-expression of p75NTR with α 9 β 1 integrin led to optimization of integrin-dependent cellular activities such as cell survival, proliferation, and migration. Co-expression of p75NTR was also required for implanted glioma cells to migrate in a glioma-like perivascular manner away from the site of implantation as was seen in the in vivo quail chorioallantoic membrane assay. / Biology

No / We examined the influence of S-nitrosoglutathione (GSNO) on alpha(IIb)beta(3) integrin-mediated platelet adhesion to immobilised fibrinogen. GSNO induced a time- and concentration-dependent inhibition of platelet adhesion. Inhibition was cGMP-independent and associated with both reduced platelet spreading and protein tyrosine phosphorylation. To investigate the cGMP-independent effects of NO we evaluated integrin beta(3) phosphorylation. Adhesion to fibrinogen induced rapid phosphorylation of beta(3) on tyrosines 773 and 785, which was reduced by GSNO in a cGMP independent manner. Similar results were observed in suspended platelets indicating that NO-induced effects were independent of spreading-induced signalling. This is the first demonstration that NO directly regulates integrin beta(3) phosphorylation.