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Dimer型構造を起点とした低分子PD-1/PD-L1結合阻害剤の設計,合成および構造活性相関研究

川下, 誠司 筑波大学 DOI:10.15068/0002008166

2023.09.04

概要

Dimer 型構造を起点とした低分子 PD-1/PD-L1 結合阻害剤の
設計,合成および構造活性相関研究

2023年1月
川下

誠司

Dimer 型構造を起点とした低分子 PD-1/PD-L1 結合阻害剤の
設計,合成および構造活性相関研究

筑波大学大学院
理工情報生命学術院
生命地球科学研究群
生命産業科学学位プログラム
博士(生物工学)学位論文

川下誠司

目次
序論 ................................................................ 1
第1章

研究の背景.......................................................................................................................1

第1節

がん免疫療法と免疫チェックポイント阻害薬 ............................................................................... 1

第2節

PD-1/PD-L1 経路 ................................................................................................................................. 3

第3節

現行の抗体医療における問題点 ....................................................................................................... 4

第4節

低分子治療薬開発の意義 ................................................................................................................... 5

第5節

PD-1/PD-L1 複合体の X 線結晶構造 ................................................................................................. 6

第6節

中分子および低分子の PD-1/PD-L1 阻害化合物 ............................................................................. 7

第7節

ビフェニル構造を有する低分子 PD-1/PD-L1 阻害剤 ..................................................................... 8

第8節

「タンパク質-タンパク質相互作用を安定化する分子」の報告例 ............................................ 9

第9節

ビフェニル構造を有する化合物の評価結果 ................................................................................. 10

第10節 分子の対称性に着目したリガンド設計 ...................................................................................... 11

第2章

研究目的 ........................................................................................................................ 13

本論 ............................................................... 15
第1章

複合体結晶構造の C2 対称性に着目した,Dimer 型リガンドの設計,合成
および評価結果............................................................................................................. 15

第1節

緒言 .................................................................................................................................................... 15

第2節

Dimer 型化合物の設計と計算化学的手法による考察 .................................................................. 15

第3節

Dimer 型化合物の合成 ..................................................................................................................... 20

第4節

評価方法 ............................................................................................................................................ 23

第5節

Dimer 型化合物の評価 ..................................................................................................................... 26

第6節

化合物の作用様式の確認 ................................................................................................................. 30

第2章
第1節

Dimer 型化合物の構造活性相関 ................................................................................. 31
緒言 .................................................................................................................................................... 31

i

第2節

ビフェニル環(A 環)上の置換基(R1,R2)の変換.................................................................. 32

第3節

ジアルコキシフェニル環(B 環)上への置換基(R3,R4)の導入 .......................................... 41

第4節

リンカー部位の変換 ......................................................................................................................... 47

第5節

タンパク質近傍の水の排除を企図した,B 環上への置換基(R5)の導入 .............................. 52

第6節

分子内の電子反発によるコンホメーション規制を企図した,B 環内への窒素原子の導入... 58

第7節

細胞機能評価..................................................................................................................................... 62

第8節

Dimer 型化合物の作用様式の確認 .................................................................................................. 63

第3章

高活性かつ分子量が低減した骨格の取得 .................................................................65

第1節

緒言 .................................................................................................................................................... 65

第2節

低分子化を指向した化合物の設計 ................................................................................................. 65

第3節

化合物の合成..................................................................................................................................... 67

第4節

低分子化を指向した化合物の評価結果 ......................................................................................... 69

小括

........................................................................................................................................74

結論 ............................................................... 76
Experimental Section .................................................. 82
参考文献 .......................................................... 145
公表論文リスト
謝辞

ii

Abbreviations

aAPCs: artificial antigen presenting cells
AMPA receptor: α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor
APCs: antigen presenting cells
BET: bromodomain and extra-terminal domain
Boc: tert-butoxycarbonylBRD4: bromodomain-containing protein 4
CD: cluster of differentiation
CPME: cyclopentyl methyl ether
CTLA-4: cytotoxic T lymphocyte antigen-4
DCs: dendritic cells
Dess-Martin periodinane: 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-on
DIAD: diisopropyl azodicarboxylate
DIPEA: N,N-diisopropylethylamine
DMF: dimethylformamide
DMSO: dimethyl sulfoxide
EC50: half maximal effective concentration
GluR: glutamate receptor
HAC: heavy atom count
HRMS: high resolution mass spectrometry
ICOS: inducible costimulatory molecule
IFN-γ: interferon gamma
irAEs: immune-related adverse events

iii

KD: dissociation constant
LAG-3: lymphocyte activation gene-3
LE: ligand efficiency
mAbs: monoclonal antibodies
MD: molecular dynamics
MeI: iodomethane
MLR: mixed lymphocyte reaction
MOM: methoxymethylMW: molecular weight
NaBH(OAc)3: sodium triacetoxyborohydride
NBS: N-bromosuccinimide
NCS: N-chlorosuccinimide
NFAT: nuclear factor of activated T-cell
PBMCs: peripheral blood mononuclear cells
PCy3: tricyclohexylphosphine
PDB: protein data bank
PD-1: programmed cell death-1
Pd2(dba)3: tris(dibenzylideneacetone)dipalladium(0)
PD-L1: programmed cell death-ligand 1
Pd(OAc)2: palladium(II) acetate
Pd(PPh3)4: tetrakis(triphenylphosphine)palladium(0)
PPh3: triphenylphosphine
PPI: protein-protein interaction
PPTS: pyridinium p-toluenesulfonate

iv

PROTAC: proteolysis targeting chimera
PTSA: p-toluenesulfonic acid monohydrate
SAR: structure-activity-relationship
SD: standard deviation
SEC: size exclusion chromatography
SPhos Pd G1: (2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl)[2-(2aminoethylphenyl)]palladium(II) chloride - methyl-t-butyl ether adduct
SPR: surface plasmon resonance
TBAF: tetra-n-butylammonium fluoride
TBS: tert-butyldimethylsilylTCR: T cell receptor
THF: tetrahydrofuran
THP: tetrahydropyranylTIM-3: T-cell immunoglobulin and mucin protein-3
TLR: toll-like receptor
Vilsmeier reagent: (chloromethylene)dimethyliminium chloride
VISTA: V-domain Ig suppressor of T-cell activation
Xantphos: 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
XPhos Pd G2: chloro(2-dicylcohexylphosphino-2′,4′,6′-tri-i-propyl-1,1′-biphenyl)(2′-amino-1,1′biphenyl-2-yl) palladium(II)

v

序論

第1章 研究の背景
第1節

がん免疫療法と免疫チェックポイント阻害薬

近年,免疫系を利⽤して抗腫瘍免疫を回復させるがん免疫療法の登場により,がん治療の
分野で⼤きな進展が⾒られる。がん免疫療法の歴史は古く,1950 年代には免疫監視機構の
概念によりがん免疫療法の理論的基礎が確⽴された 1。悪性腫瘍の進⾏における免疫細胞の
重要性は,19 世紀初頭から認識されていたが,免疫チェックポイント阻害療法の登場によ
り免疫腫瘍学(immuno-oncology)に対する科学的関⼼が再び⾼まっており,近年では外科
⼿術,細胞毒性化学療法,放射線療法,標的療法に続くがん治療の「5 本⽬の柱」とも⾔わ
れるほどに急速に発展している 2。
がん免疫療法は,化学療法や放射線療法等の従来の治療法とは対照的に,免疫細胞やサイ
トカインを利⽤して宿主の防御システムを構築し,その抗腫瘍免疫作⽤によってがん細胞
の排除を⾏う治療法である

3, 4

。がん免疫療法には,免疫賦活剤療法,サイトカイン療法,

養⼦細胞移植(ATC: adoptive T cell transfer therapy),遺伝⼦導⼊により改変した T 細胞を投
与する CAR-T 細胞療法(chimeric antigen receptors T-cell therapy),がんワクチン等が含まれ
るが,それらの中で最も⼤きな成功を収めているのが免疫チェックポイント阻害療法であ
る。免疫チェックポイント阻害剤は,その有効性と⽐較的管理しやすい副作⽤のプロファイ
ルにより,⼀部の固形がん(転移性メラノーマ,⾮⼩細胞がん)においては治療の第⼀選択
となっている 5–8。
免疫チェックポイントは,免疫応答の過程において T 細胞受容体(TCR: T cell receptor)
の抗原認識を調節するための⼀種のシグナルである。免疫チェックポイントには共刺激免
疫チェックポイントおよび共抑制免疫チェックポイントの 2 種類が含まれている(Table 1)。
共刺激免疫チェックポイントは免疫の進⾏を刺激し亢進させる機能を担い,CD28,ICOS,
1

CD137 等の分⼦が該当する。⼀⽅,共抑制免疫チェックポイントは免疫の進⾏を阻害する
機能を担い,PD-1, CTLA-4, VISTA 等の分⼦が含まれる。免疫系が病原体を攻撃していると
き,これらの免疫チェックポイント分⼦のはたらきにより正常組織を損傷から保護する。こ
の制御機構は,腫瘍形成時にはその抗腫瘍免疫反応に対してブレーキの役割を果たす。すな
わち,腫瘍細胞上に発現するこれらのチェックポイント分⼦のリガンドが結合すると,腫瘍
細胞に対する T 細胞の攻撃が抑制される。免疫チェックポイント療法は,共刺激免疫チェ
ックポイントに対するアゴニスト,または共抑制免疫チェックポイントに対する阻害剤を
⽤いることにより免疫系を機能させ,抗腫瘍免疫反応を再活性化する治療法である 9。この
うち現在までに⽶国⾷品医薬品局(FDA: Food and Drug Administration)から承認された免疫
チェックポイント阻害剤は,いずれも共抑制受容体あるいはそのリガンドへの結合を介し
て受容体―リガンド間の結合を阻害する抗体であり,抗 CTLA-4 抗体(Ipilimumab),抗 PD1 抗体(Nivolumab,Pembrolizumab,Cemiplimab,Dostarlimab)
,抗 PD-L1 抗体(Avelumab,
Durvalumab,Atezolizumab)および抗 LAG-3 抗体(Relatlimab)がある。

Table 1. T 細胞応答を制御する共刺激および共抑制免疫チェックポイント分子の例
受容体

リガンド

(T 細胞上に発現)

(APCs 上に発現)

CD28

CD80 / CD86

ICOS

B7RP1

CD137

CD137L

CTLA-4

CD80 / CD86

共抑制

PD-1

PD-L1 / PD-L2

チェックポイント

TIM-3

Galectin-9

LAG-3

MHC class II

共刺激
チェックポイント

2

免疫チェックポイントの中で,その機能が最もよく研究されているのが細胞傷害性 T リ
ンパ球関連抗原 4(CTLA-4: cytotoxic T lymphocyte antigen-4,CD152 としても知られる)と,
プログラム細胞死タンパク質1(PD-1: programmed cell death-1,CD279 としても知られる)
である。
CTLA-4 は T 細胞上に発現する共抑制チェックポイントである。マクロファージ,樹状細
胞(DCs: dendritic cells),B 細胞等の抗原提⽰細胞(APCs: antigen presenting cells)からのシ
グナルによって開始される T 細胞の活性化に伴ってその表⾯への発現が亢進し

10

,その発

現量は TCR(および CD28)による刺激の強度に依存する。CTLA-4 は CD28 と構造的に類
似しており,リガンドである CD80 や CD86 を CD28 と共有することで,T 細胞の持続的な
活性化と増殖を阻害して,細胞周期の停⽌を引き起こす 11。また CTLA-4 ⽋損マウスは致死
性の⾃⼰免疫疾患を発症しリンパ球増殖の表現型が⾒られることから,⽣体において強⼒
な免疫抑制機能をもつと考えられている

12

。この様な CTLA-4 の機能によって,TCR に対

するリガンドの濃度や親和性が⼤きく変化した場合にも T 細胞の活性化レベルが⼀定に保
たれる。
CTLA-4 の作⽤を阻害することでがんに対する T 細胞の治療効果を⾼めることができる
という考えのもと抗 CTLA-4 抗体の開発が実施され 13,2011 年に Ipilimumab が最初の免疫
チェックポイント阻害剤として FDA から承認された 14, 15。

第2節 PD-1/PD-L1 経路
PD-1 は T 細胞,B 細胞,NK 細胞等の表⾯に発現する共抑制チェックポイントであり,
その主な役割は末梢組織で抗原を認識したエフェクターT 細胞による組織内の炎症反応の
制御である。PD-1 は T 細胞の活性化により発現が誘導される 16。 ...

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bromodomain

inhibition

promotes

anti-tumor

immunity

by

suppressing

PD-L1

express ...

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