称号及び氏名

博士（理学）RADWAN SAID RADWAN MOHAMMAD EL-HAGGAR

学位授与の日付

平成 22 年 3 月 31 日論

「Molecular Design of Small Organic Molecules as Mimics for
Biologically Active Peptides with α-Helical Conformation（α -ヘリックス構造をもつ生理活性ペプチドに関する低分子ミミック化合物の分子設計）」

文

名論文審査委員

主査
副査
副査
副査

藤井徳富
多田
神川郁雄
哲俊治
憲

論文要旨
Chapter 1: Introduction
In the fields of medicine, biotechnology and pharmacology, Drug Discovery is
the process by which drugs are discovered and/or designed.
The first step in designing new ligands is to discover a lead compound which isa chemical compound that has pharmacological or biological activity and whose
chemical structure is used as a starting point for chemical modifications in order toimprove potency, selectivity, and/or pharmacokinetic parameters. Despite theadvances in technology and understanding of biological systems, development of new
strategies for the design and synthesis of small organic compounds that targeting theinterfaces of protein-protein interactions is still challenging. Thus, the development of
new strategies for the rapid design and synthesis of such ligands (lead compounds)
is essential to analyze and control the biological processes of each protein.
Therefore, the aim of this work is to use the directed evolution and the structure
based drug design for development of a new strategy for the design of small organic
compounds that target the protein-protein interactions using Granulocyte-Colony
Stimulation Factor (G-CSF) receptor as a model protein. G-CSF is one of the important
cytokines that stimulates the bone marrow to release granulocytes and stem cells into theblood. The interaction of G-CSF with its receptor stimulates the survival, proliferation,differentiation, and function of neutrophil precursors and mature neutrophils. So ligands,
which can inhibit the interaction between G-CSF and its receptor, would be interested ascytotoxic agents for treatment of cancer.
Chapter 2: Molecular Design and Synthesis of the Target Ligands
In this work, a new strategy for the ligand design has been developed which uses
conformationally constrained peptide libraries. Screening of the libraries provides rigidly
folding peptides that have high binding affinity to the target proteins. In addition, therigid structure of the selected peptide provides the information about the spatial
orientation of the pharmacophore which facilitate the structure based design of the-1-peptidomimetics.
Our group has examined the directed evolution of peptides in a phage-displayed
library and has successfully synthesized a de novo designed helix-loop-helix peptide
consisting of 35 amino acids. Previously, screening of the peptide library has isolated apeptide, named P8-2KA, which can specifically bind to G-CSF receptor.
2-1. Design of tri-Substituted Ligands
The structure modeling and alanine scanning have shown that amino acids leu28, lys29
and glu32or Arg35 are essential for the receptor binding and the biological activity andbased on information of the pharmacophore several small molecule peptidomimetics
have been designed (Figure 1).

Figure 1: Structure based design from active peptide to peptidomimetic scaffold. a) Design basedon amino acids Leu28, Lys29 and Glu32. b) Design based on amino acids Leu28, Lys29 and Arg35.

2-2. Synthesis and Evaluation of tri-Substituted Ligands
Literature survey revealed that the biphenyl framework could act as an effective
scaffold for the design of small ligands for G-CSF receptor. Therefore, I designed andsynthesized tri-substituted biphenyl ligands I-VI with three residues, which simulate theside chains of the effective amino acids of P8-2KA, two of them are at 3- and 5- positions
and the third residue at variable position of the other benzene ring at 2`-, 3`- or 4`
positions.
H2 NO

O

CompoundI
IIIII

OPosition2`2`3`-

ROH
NH2
OH

CompoundIVVVI

Position3`4`4`-

R
NH2OH
NH2R

After synthesis and confirmation of the structures using the different spectral analyses,
the binding affinity toward the G-CSF receptor using the SPR Biacore technology has
been evaluated and pleasantly, compound IV could bind to G-CSF receptor with a Kd
around 110 μM and this result was supported by PyMol Modeling studies (Figure 2).
b)

RU70a)H2 N

60OO50Resp. Diff.

40

3020

NH2

100Kd-10400

450500Time550

IV=110 O

600s

Figure 2: a) SPR sensorgram of the IV. b) Superimposed model of compound IV and P8-2KA.

-2-Unfortunately, all changes applied to optimize the activity of ligand IV resulted indisappearance of the activity except only when the amide side chain was changed toamine side chain, the resultant compound XIII could bind to the receptor but with nospecificity.
OO

H 2NOOH2 NOOH 2NOONH2NH2NH2VII

VIIIIXNo Binding

No Binding

No Binding

OO

H 2NO

O

H 2N

NH2XIOO

NH2

NH2O

XII

No BindingO

No Binding

H2 NOO

H 2N

NH2X

O

XIIIO

Binds with no specificity

No Binding

Although changing the biphenyl scaffold with the phenyl-pyrrol scaffold resulted ininactive compound, XIV, the N-benzyl aniline scaffold gives active compound XV with aKd around 370 μM.
H 2NOOH 2NNOOHNNH2OXIVH 2NXVO2-3. Design of tetra-Substituted Ligands
Motivated by the studies that showed the importance of amino acids Leu28, Lys29,
Glu32 and/or Arg35 to receptor binding and the biological activity, I decided to design andsynthesize peptidomimetics that contain four substituents simulating the side chains ofthese important amino acids (Figure 3). Thus compound XVI was designed by additionof a fourth substituent that simulates Glu32 to the active compound IV. As expected theactivity was increased more than five folds as the Kd was around 20 μM (Figure 3).
OO

H2 NNH2Kd = 20O μM
Kinetics:
O Spot=1-r
HO Fc=2RU
20XVIResponse

15
1050-5-20

020

4060
Time

80

100120140s

Figure 3: a) SPR sensorgram of the XVI. b) Superimposed model of compound XVI and P8-2KA.

-3-2-4. Synthesis and Evaluation of tetra-Substituted Ligands
Once more, all modifications applied to compound XVI for optimizing the activity
resulted in inactive compounds XVII-XXO

O

H2NO

O

H2N

NH2O

NH2OO

HOOOO

H2NOHOHO
H 2NOO

H2NOO

HOO

HOXVIIXVIIIXIXXXNo Binding

No Binding

No Binding

No Binding

In addition, changing the position of the four
substituents was interested and XXI was chosen
as next target, but it showed a weak binding
towards G-CSF receptor.

H2 NOOOOH

Weak binding
NH2XXI

Next, the addition of the fourth chain to the N-benzyl aniline scaffold afforded
compound XXII which binds to the receptor with Kd = 47 μM, however, modifications
applied to compound XXII gave inactive compounds XXIII-XXV.
OO

H2 NOHOOO

H2 NONHOH2 NXXIIOON

H2 NOHXXIIOO

H2 NO
NNH2Kd = 47 μM

OO

H2 NN

NH2OXINo Binding

No Binding

OOH
XXVO

No Binding

Chapter 3: Binding Affinity
The binding affinities of compounds I-XXVII were evaluated by SPR with BIAcore
S51 apparatus and, in conclusion, we were able to apply a novel methodology to designand synthesize small molecules binding to G-CSF receptor based on conformationallyconstrained helix-loop-helix peptide libraries. The biphenyl scaffold shows better
binding affinity than the N-benzyl aniline scaffold and, in the both scaffolds, thetetra-substituted ligands showed better binding affinity to the receptor than thetri-substituted ligands.

-4-審査結果の要旨
藤井研究室は，ファージ表層ディスプレイ技術と有機合成化学を組み合わせることにより，従来のとは異なった独創的な新しいリード化合物設計法を提案した。この方法は２つのステップから構成される。第１ステップでは，立体構造モチーフ（α-ヘリックス）をもつペプチドのファージ表層提示ライブラリーを構築し，標的タンパク質に作用するペプチドをスクリーニングする。このライブラリーから得られるペプチドは，強固な立体構造を持っているのでファーマコフォアとその空間配置を容易に決定することができる。そこで，第２ステップでは，ペプチドから得られた立体構造情報をもとに低分子リード化合物を設計する。申請者は，この第２ステップを担当し，顆粒球コロニ−刺激因子（G-CSF）受容体に結合するα−ヘリックス構造ペプチドから受容体結合性低分子化合物の創出に成功した。
G-CSF 受容体結合α−ヘリックスペプチドでは， i, i + 2, i + 4, i + 5 の位置の４つのアミノ酸が受容体結合に関与する。そこで，申請者は，これらのアミノ酸の空間配置を再現できる低分子化合物として，２つのビフェニル誘導体を設計した。これらの分子を土台として対応するアミノ酸の官能基を導入し，受容体結合低分子の設計に成功した。また，官能基変換による構造活性相関を検討し，設計の妥当性を証明した。
以上のように，申請者は，ファージ表層ディスプレイ技術と有機合成化学を組み合わせることにより，新しいリード化合物設計法を開発した。本法では，手間のかかる標的タンパク質の立体構造解析や高価な低分子ライブラリーを必要としないので，迅速かつ低コストのリード化合物探索が達成される。顕著な新規性と独創性があり，また，本人自身が分子設計，有機合成，受容体結合実験までのすべてを手がけており，申請者を博 士 学 位 に 値
す る 能 力 を も つ も の と 判 断 す る 。したがって，本学位論文審査委員会は、当該論文の審査ならびに最終試験の結果に基づき、申請者に対して博士（理学）の学位を授与することが適当であると結論した。

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