京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13109号 / 論医博第2127号 / 新制||医||1022(附属図書館) / (主査)教授 今中 雄一, 教授 川上 浩司, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>c-Myc is one of the most important oncogenes. G-quadruplex DNA secondary structure formed in the proximal promoter region of c-Myc functions as a transcription silencer and is targetable by small molecules. Therefore, the c-Myc promoter G-quadruplex (MycG4) is an attractive anticancer drug target. Protein recognition of MycG4 is essential for its transcriptional regulating. Nucleolin was discovered as a major MycG4 binding protein in 2009. It shows a remarkably higher binding affinity for MycG4 over its known substrate NRE_RNA and overexpression of nucleolin represses the activity of the c-Myc promoter. However, little is known about its molecular recognition of MycG4. Here, we use X-ray crystallography combined with other biochemical and biophysical methods to understand how nucleolin recognizes MycG4. Nucleolin is a 77 kD protein with a modular organization. The four RNA-binding domains (RBD) of nucleolin are the minimal domains for high affinity binding with MycG4. We show that nucleolin prefers the c-Myc parallel G-quadruplex with a 6-nt central loop (Myc161) that is the thermodynamically favored conformation. Using a custom G4 DNA microarray, we optimized the MycG4 sequence with over 10-fold increased binding affinity to nucleolin. Fabs are widely used tools to facilitate crystallization and we have discovered Fabs that specifically bind the nucleolin-MycG4 complex using a phage display screening. This approach enabled us to obtain crystals of the nucleolin-MycG4-Fab ternary complex diffracted at 2.6 Å and we determined the crystal structure. In the structure, the parallel MycG4 is very well-defined with two K⁺ between the three G-treads. The central 6-nt loop residue protrude from the G4-core and extensively recognized by the nucleolin. Only RBD1 and RBD2 of nucleolin are seen in the crystal structures and interact extensively with the 6-nt central loop and 5′-flanking of MycG4. The binding surface and area of the globular MycG4 by nucleolin is much more extensive than NRE_RNA and involves an extra binding site. Fab binds to both RBD1 and 3′-end of MycG4 to stabilize the complex. The well-defined partial RBD2-3 linker and a cavity close to the 1-nt T19 loop suggest that the missing RBD3 likely binds the 3^rd loop of MycG4. This structure is the first MycG4-protein complex structure. It will help understand MycG4 and nucleolin interactions and the development of MycG4 targeted cancer therapeutics. This structure also provides novel insights into how proteins recognize the globular G-quadruplexes, highlighting the potential of G-quadruplexes as a platform for multivalent interactions such as with multiple tandem RBDs.</p>

Ligand-targeted therapeutics are a rapidly growing class of anticancer agents. This class of therapeutics is typically bifunctional molecules that use a targeting moiety to selectively deliver potent, typically nonspecific, cytotoxic agents to cancer cells while sparing normal cells. The low-molecular-weight of ligant-targeted therapeutics allows for better tumor penetration, especially in the case of solid tumors where the size of antibodies is a limiting factor for effective treatment. Unfortunately, the poor pharmacokinetic profiles of many of these conjugates present a challenge, which limits their tremendous therapeutic potential. Dose-limiting toxicity is also observed due to the need for high doses and frequent administration. This dissertation describes our work to develop a fundamentally new approach for targeting cancer. Our approach could potentially reduce the toxicity and enhance the pharmacokinetic properties of targeted anticancer agents, which would decrease dosing frequency and improve the lives of cancer patients.

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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The structurally novel diterpenoid paclitaxel (Taxol®), originally isolated from <i>Taxus brevifolia</i>, is one of the most promising new anticancer drugs. Its structural complexity and unique biological activity have provided the impetus for a number of structure-activity relationship (SAR) studies for the last twenty years, with the aim of developing analogs with improved bioactivity. Because of the absence of information on the structure-activity relationship of the C-6 position and the ring C skeleton of paclitaxel, it was goal of this research to synthesize paclitaxel analogs modified in ring C in order to evaluate the effects of these modifications on biological activity and to reveal the chemistry of paclitaxel. The inactivity of the C-6 methylene group towards chemical modifications has been overcome by the formation of a double bond at the C- 6 and C-7 positions. Modification of the C-6 position has been achieved for the first time and over 20 new paclitaxel analogs modified at both the C-6 and C-7 positions have been synthesized. Biological evaluation of these compounds reveal that the C-6 and C-7 positions do not play significant roles in the biological activity of paclitaxel, although the two deoxygenated paclitaxel analogs, 7-deoxy-6α-hydroxypaclitaxel and 7,lO-dideoxy-6ahydroxypaclitaxel, were found to be more active than paclitaxel. Modification of the ring C skeleton has been accomplished for the first time, and several new C-<i>nor</i>-paclitaxel analogs have been synthesized. Biological evaluation showed that these C-<i>nor</i>-paclitaxel analogs were less active than paclitaxel, indicating that the ring C skeleton plays a crucial role in the biological activity of paclitaxel. Biological evaluation also showed that all oxetane ring-opened paditaxel analogs were essentially inactive. These results indicate that changes in the size and conformation of ring C and the attached oxetane ring make a significant contribution to the activity of paclitaxel. / Ph. D.

No / A novel series of 1,4-disubstituted chloroethylaminoanthraquinones, containing alkylating chloroethylamino functionalities as part of a rigid piperidinyl or pyrrolidinyl ring-system, have been prepared. The target compounds were prepared by ipso-displacement of halides of various anthraquinone chromophores by either hydroxylated or chlorinated piperidinyl- or pyrrolidinyl-alkylamino side chains. The chloroethylaminoanthraquinones were shown to alkylate guanine residues of linearized pBR322 (1¿20 ¿M), and two symmetrically 1,4-disubstituted anthraquinones (compounds 14 and 15) were shown to interstrand cross-link DNA in the low nM range. Several 1,4-disubstituted chloroethylaminoanthraquinones were potently cytotoxic (IC50 values: ¿40 nM) in human ovarian cancer A2780 cells. Two agents (compounds 18 and 19) exhibited mean GI50 values of 96 nM and 182 nM, respectively, in the NCI human tumor cell line panel. Derivatization of the potent DNA cross-linking agent 15 to an N-oxide resulted in loss of the DNA unwinding, DNA interstrand cross-linking and cytotoxic activity of the parent molecule.

No / Croton membranaceus is used by herbalists and traditional healers in Ghana for the management of various cancers, especially prostate cancers. A methanolic extract of the roots showed cytotoxic activities against two cancer cell lines, and bioassay-guided fractionation of this extract revealed that the cytotoxic activity resided mostly in the ethyl acetate fraction. Six compounds were isolated from this fraction, including a new furano-clerodane diterpenoid (1), for which the trivial name crotomembranafuran is suggested. This compound exhibited an IC50 value of 4.1 microgram/mL (10.6 microM) against human prostate (PC-3) cells, providing some support for the traditional use of C. membranaceus in the treatment of cancers