Transcript Chapter 2 Drug screening & discovery (design)

Chapter 2 Drug screening & discovery (design)
口服药物的共性：
•Lipinski归纳的“类药5规则”(Rule of Five)，概括了类药的最低标准，即分子
量在500以下；氢键的给体不超过5个；氢键的接受体不超过10个；计算的分配系
数(正辛醇-水系统)clogP值不超过5。上述原则只限于化合物经被动扩散机理的
吸收。
•化合物的柔性不宜过强。否则会存在许多种构象(RB <= 5)
•化合物不得含有重金属和反应活性基团。
基于结构的设计
• 在受体结构信息已知的情况下，可根据结合部位的三维结构信息，用分子对
接方法，对互补性好、评分高的化合物，可预计有较强的亲和力。若不知受
体的三维结构，可根据药效团特征筛选虚拟库，并以不同程度的限制条件，
“滤除”与药效团无相似性的分子。
知识产权的预测
•
化合物具备自主的知识产权和专利保护(IP)的前景，是开发决策的重要指
标，筛选虚拟库和组合库时要剔除已被其它专利覆盖或有可能侵权的化合
物。所以，完备的化合物检索查新系统可确保化合物结构的新颖性。
(emolecules.com)
先导化合物发现
1. 天然活性物质
2. 高通量筛选
3. 基于配体或底物的分子设计
4. 基于药物的副作用
5. 基于代谢作用
6. 幸运Serendipity
7. 基于片段的药物发现
1.天然生物活性物质作为先导物
1、天然活性物质直接作药物：利血平，万古霉素（糖肽类抗生素）
CH3
CH3
CH3
O
N
N
H H
O
O
O
CH3
H
Reserpine：肾上腺素能神经元阻断性
抗高血压药，提取于萝芙木属多种植物
H
O
O
O
O
O
CH3
CH3
2、天然活性物质作先导物，适当分子变换：紫杉醇，长春碱
H3C
O
H3C
O
O
OH
CH3
CH3
O
HN
O
H3C
H
O
O
O
OH
CH3
NH
O
H3C
CH3
H
O
H
O
HO
OH
H3C
CH3
O
CH3
OH
O
HO
CH3
O
O
CH3
Paclitaxel: 促进微管蛋白聚合，阻止其在
有丝分裂的功能，治疗卵巢癌、乳腺癌、
恶性黑色素瘤。
O
O
HO
O
O
O
O
CH3
docetaxel:水溶性增加，活性〉紫杉醇，
无交叉耐药
3、改造天然产物
HO H C
3
S
acety l CoA
O
CH3
HO
CoA
OH O
HMG CoA reductase
cholesterol
NADPH
Km=10mM
O
HO
OH
LDL-C: 引起动脉粥状样硬化、心肌梗死
桔青霉Penicillium citrinum提纯得到Lovastatin
HO
O
HO
O
OH
OH
O
O
O
H
O
H3C
H
CH3
H3C
Lovastatin
H
O
H3C
H
CH3
H
CH3
H3C
Ki = 1nM
H
CH3
statins
O
F
NH
F
H3C
N
OH
OH
O
CH3
OH
OH
N
CH3
OH
OH
O
Atorvastatin
(Lipitor, best sale)
H3C
Fluvastatin
Pitavastatin(Nisvastatin)
• 青蒿素
O
黄花蒿
Artemisia annula
O
O
生物利用度较低
复发率高
O
O
青蒿素
Artemisinin
O
蒿甲醚
Artemether
(Novartis PCT)
O
O
O
OCH3
• 喜树碱
OH
HO
喜树(中国）
Camptotheca acuminata
O
N
O
N
O
水溶性较差，毒性大
羟基喜树碱
Hydroxycamptothecin
N(CH3)2
OH
HO
拓扑替康
Topotecan （GSK 2007）
作用于DNA topoisomerase I （化疗药物）
治疗SCLC，卵巢癌，结肠癌
O
N
O
N
O
• 局麻药
O
N
南美洲古柯
Erythroxylum coca Lam
O
O
可卡因
Cocaine
H
O
O
N
O
普鲁卡因
Procaine （1905）
H 2N
Lidocaine
（1943 Synthesis;
1949 on market)
CH3
NH
N
O
CH3
CH3
CH3
• 动物毒素
–
–
–
–
蛇毒Bungarotoxin，N2受体拮抗剂肌松药
蛇毒Batroxobin，溶血栓酶抗栓药
鱼毒Tetrodotoxin，钠通道阻断剂心血管药物
蜂毒Apamin，钙通道阻断剂和钾通道开放剂心血管药物
2、高通量筛选（HTS）
多靶标对化合物库的筛选（目前的主要筛选方法）
立体筛选方法的灵敏性、微量化、自动化
大容量多样性化合物库
Cons:
Low hit rate: 0.001%
Low druggability
High false positive rate
组合化学
Combinatorial chemistry
•
•
同时制备含众多分子的化合物库
–
以代数级数增加构建块的数目，库容量则以几何级数增加
与高通量筛选（high-throughput screening, HTS）技术结合，可极大地加
快先导物发现和优化的速度
平行合成和混分合成
固相合成和液相合成
小分子组合合成
计算机辅助设计及虚拟库合成
组合生物合成
Combinatorial biosynthesis
• 基本原理
基因变异（混合、匹配、交换、突变等）
基因克隆
多种变异的酶系
多种非天然的天然物质
聚酮合酶催化合成红霉素
DEBS2
DEBS1
¹¦ Äܵ¥Ôª1
¹¦ Äܵ¥Ôª2
AT ACP KS AT KR ACP KS AT KR ACP
S
S
O
HO
O
9
HO
13
HO
¹¦ Äܵ¥Ôª5
¹¦ Äܵ¥Ôª4
KS AT ACP KS AT DH ER KR ACP
S
O
13
¹¦ Äܵ¥Ôª3
DEBS3
S
O
13
HO
HO
KS AT KR ACP KS AT KR ACP TE
S
S
O
O
O
5
9
O
5
9
O
HO
HO
HO
1
HO
HO
5
O
9
9
HO
13
S
O
HO
9
13
¹¦ Äܵ¥Ôª6
13
HO
13
HO
O
H3C
H3C
HO
H3C
CH3
9
OH 6
5
H3C
O
12
O
OH
CH3
HO
O
3
O
O
CH3
ºì ùËØA
O
H3C
N(CH3)2
O
CH3
OCH3
CH3
OH
CH3
H3C
HO
H3C
CH3
9
OH 6
5
H3C
O
CH3
12
1
OH
3
O
OH
CH3
6-Deoxyerythronolide B
13
岩白菜内酯的生物催化组合库
OH
OH
OH
O
CH3O
OH
O
HO
O
ÑҰײËÄÚõ¥
Ö¬·¾Ã¸
õ£»¯
ÌÇÜÕ»¯
ÌÇÜÕø
OH
OH
OH
O
OCOR2
O
OH
CH3O
OCOR3
OH
OH
OH
OH
HO
OH
O
CH3O
O
OH
O
HO
Cl
Ê×ÂÖÑÜÉúÎï 20¸ö
õ£»¯£º
õ¥£¬õ£°·
̼Ëáõ¥
°±¼×Ëáõ¥
ÌÇÜÕ»¯£º
ÌÇÜÕ
°±»ùÌÇÜÕ
ÌÇËáÜÕ
OH
O
O
HO
O
OH
OH
O
CH3O
CH3O
O
OH
OH
O
O
HO
ÂÈ´ú
HO
O
OCOR1
OH
±¹ý Ñõ»¯Ã¸
Ñõ»¯
Ñõ»¯»¹ Ô-£º
ôÇ»ù»¯
Ñõ»¯
ÍѼ׻ù»¯
µÚ¶þÂÖÑÜÉúÎï ¹² 600¸ö
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ÂÈ´ú
äå´ú
µâ´ú
O
Impact of high-throughput screening in biomedical research
Macarron, R. NATURE REVIEWS DRUG DISCOVERY 10: 188-195 (2011)
3、基于配体或底物的分子设计
•
酶反应过程：酶抑制剂
– 酶结构
– 底物、过渡态、产物结构
– ACEI、COX-2、GABA-T、MAO抑制剂等
•
抗代谢物：酶抑制剂，致死合成
•
与受体作用过程：激动剂或拮抗剂
– 受体结构
– 配体结构
– 肾上腺素能药物、胆碱能药物、甾体药物等
5-hydroxytryptamine (5-HT)：神经递质，低水平--〉偏头痛
5-HT receptor isomers: 5-HT1, 5-HT2, 5-HT3
NH2
OH
NH2
H3C
O
O
NH2
NH
NH
NH2
N
H
Activity(5-HT1) = 2 (vs. 5-HT)
N
H
Activity(5-HT2) = 1/25
H3C
O
H3C
NH S
O
CH3
NH S
NH2
O
N
O
CH3
N
H
N
H
Sumatriptan （GSK，2009 expired)：
Activity(5-HT1) = ¼
对5-HT2, 5-HT3，多巴胺和肾上腺能受体无作用
5-HT3受体阻断剂
NH
H3C
O
N
H3C
Cl
CH3
O
O
O
CH3
O
H2N
NH
Cl
H2N
N
NH2
N CH3
NH
O
Cl
H3C
Clebopride
Dopamine blocker
胃动力不变
Metoclopramide
Block dopamine & 5-HT3
止吐，胃动力药
O
N
CH3
N
N
5-HT3 blocker
> clebopride 300 folds
无dopamine 拮抗
CH3
O
CH3
N
N
N
N
CH3
NH
N
H
O
Ondansetron
granisetron
Setrons: 治疗肿瘤化疗引起的恶心呕吐
palonosetron
血管紧张素转化酶（angiotensin-converting enzyme, ACE)抑制剂
Angiotensin I
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu
ACE
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
（angiotensin II: 强效收缩血管）
Bradykinin: 血管舒张剂
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
ACE
Arg-Pro-Pro-Gly-Phe-Ser-Pro
Hypothetical active site of carboxypeptidase A
S1'
S1
Zn
NH
++
R
O
CH
C
CH2
NH
CH
O
C
substrate
O
CH2
O
C
O
CH2CH
O
(R)-2-benzylsuccinic acid
C
O
Hypothetical binding of inhibitors to ACE
S1
Zn
NH
S2'
S1'
R3
O
CH
C
H
++
NH
R2
O
CH
C
O
N CH
peptide inhibitor
C
O
O
O
C
R2
CH2 CH
O
C
O
N CH
carboxyalkylproline
C
O
R2
S
CH2 CH
O
C
O
N CH
mercaptoalkylproline
C
O
ACE小分子拟肽抑制剂
H3C
CH3
HS
O
OH
NH
O
O
CH3
O
O
N
OH
O
O
captopril
enalapril
ACE的功能
羧肽酶A的作用模式
依那普利等
卡托普利
OH
O
NH
N
CH3 O
N
OH
Enalaprilat
(enalapril的代谢活性组分）
肽类抑制剂的结合模式
羧烷基脯氨酸
Crystal Structure of the Human Angiotensin-Converting Enzyme-Lisinopril
Complex (2003) Nature 421: 551
H2受体拮抗剂类抗溃疡药
•
选定靶点－组胺H2受体 (确立研发目标－抑制胃酸分泌药物)
•
建立动物筛选模型－麻醉兔灌胃
•
从H2受体天然激动剂－组胺入手，以其为先导结构，保留咪唑环，改变侧链，开
NH2
始优化
HN
N
S
H
N
HN
NH2
N
HN
NH
² ·¿ ¼Ö¶ ¼¯¤
S
Á
N
NH
H
N
NH2
+
ÁS
Ñ Ô¡ Ð ÞÔñ¿×¼¹
NH
S
HN
NH2
NH2
N
N
NH2
H
N
HN
HN
N
N
H
HN
N
NHCH3
S
² ¼Áí °¢ Burimamide
·
¿ ·ÚÎþÐÞ §
N
HN
R
pKa
H
6.80
NH2
5.90
H
N
R
R
R
HN + NH
a
b
NH
N
c
20%
80%
3%
NHCH3
7.25
40%
S
H
N
NHCH3
S
6.25
favoring form
6.80
favoring form
S
¼×ÁòßäÌØ Metiamide
20%
favoring form
H
N
H3C
NHCH3
S
N
HN
S
H
N
H3C
NHCH3
S
N
HN
Î÷ßäÌ涡 Cimetidine
1976
NCN
H
N
(CH3)2N
¼×ÁòßäÌØMetiamide
ÉöËðÉËºÍ Á£Ï¸ °ûȱ·¦ Ö¢
S
O
NHCH3
CHNO2
NH2
H 2N
N
NSO2NH2
S
N
S
NH2
À×ÄáÌ涡 Ranitidine
1983
·¨ ĪÌ涡 Famotidine
1986
IIb/IIIa糖蛋白受体拮抗剂
•
•
•
•
血栓形成的关键步骤是纤维蛋白原与血小板IIb/IIIa受体结合。
被IIb/IIIa受体识别和相互作用的主要区段是纤维蛋白原的三肽片断Arg-GlyAsp（RGD）。
蛇毒或水蛭素中含有RGD的线形或环状肽，是阻断IIb/IIIa受体活化从而抑制血
小板聚集的药效团。
含有或模拟RGD结构的肽或拟肽可作为纤维蛋白原的拮抗剂，是创制抗血栓
药物的一个新途径。
O
NH
NH
H2N
N
H
NH
O
H
N
O
H2N
O
N
H
N
H
O
H
N
COOH
N
NH
RGD
O
O
H2N
O
N
O
O
N
H
Sibrofiban
OCH3
HN
Roxifiban
COOH
O
O
CH3
4、SOSA
磺胺治疗细菌感染时，发现利尿作用.
O
NH2
N
NH
O
N
S
O
O
S
H2N
N
S
CH3
O
S
O
O
NH2
Cl
O
S
O
S
NH2
O
Dichlorphenamide
O
O
O
Cl
cyclothiazide
长效利尿药
N
H
NH2
S
S
S
NH
F
F
N
H
hydrochlorthiazide
O
O
O
F
O
O
NH
NH2 Cl
O
NH
S
Cl
chlorthiazide
S
O
O
O
S
Cl
Acetazolamide
口服利尿
H2N
H
N
O
N
H
bendroflumethiazide
长效利尿药
Sulfasomizole治疗伤寒病，胰岛素释放急性和持久性低血糖
H2N
O
S
N
CH3
S
O
NH
H3C
O
NH
S
O
NH
O
NH2
sulfasomizole
Carbutamide, 更强的降糖作用，治疗糖尿病
Iproniazid治疗结核病，情绪提高 抑制单胺氧化酶MAO靶标抑郁型精神病
CH3
N
N
HN
NH
CH3
NH2
NH
O
NH
NH2
Isoniazid
无抗抑郁
O
phenelzine
iproniazid
CH3
NH2
CH
抗抑郁药
tranylcypromine
N
H3C
selegiline
Zaprinast
CH3
O
NH
O
NH N
H3C
N
N
O
sildenafil
N
N
N
NH2
N
N
N
S
OH
O
P
O
cGMP：扩张血
管，增加血流量
O
5
CH3
CH3
OH
cGMPNO第二信使
增强勃起功能(ED)
抗过敏药扩张血管、降血压抑制PDE5：
治疗心绞痛
PDE
cGMP
N
O
O
O
N
HN
O
O
N
H
CH3
GMP
模拟创新：
H3C
O
N
CH3
O
NH
N
O
O
H3C
S
O
N
N
N
N
H
N
H
O
CH3
N
N
O
H
N
S
HN
N
CH3
O
N
N
H
O
CH3
CH3
O
O
CH3
O
CH3
5、基于代谢作用
•
•
代谢活化
– 活性代谢物作为先导物
– 前药设计
代谢失活
– 软药设计
H2N
N
N
SO2NH2
NH2
°ÙÀ˶àÏ¢ Prontosil
H2N
SO2NH2
»Ç°· Sulfonilamide
»Çõ£°· ÀàÒ©
Îï
• 抗疟药环氯胍
H 2N
NH NH
Cl
N
Cl
NHCNHCNHCH(CH3)2
N
NH 2
N
H 3C
ÂÈë ÒProguanil
CH 3
»· ÂÈë ÒCycloguanil
H2 N
N
Cl
NH2
N
C 2H 5
ÒÒ
°· à×ठPyrimethamine
保泰松的代谢活化
OH
N
N
N
N
O
O
O
O
CH2CH2CH2CH3
CH2CH2CH2CH3
ôDz¼×Ú Oxyphenbutazone
±£Ì©ËÉ Phenylbutazone
N
N
N
N
O
O
O
CH2CH2CHCH3
OH
´ÙÄòËáÅÅй
O
CH2CH2S
O
»ÇßÁͪ Sulfinpyrazone
£¨ ¿¹ Í´ ·ç Ò ©
£©
6、幸运
O
结构解析错误
O
CH3O
O
CH3O
O
CH3O
O
O
O
O
O
CH3O
O
Îå ζ×Ó±ûËØ Schizandrine C
CH3O
COOCH3
CH3O
COOCH3
OCH3
O
O
COOCH3
O
COOCH3
O
OCH3
O
O
Áª±½Ë«õ¥ Bifendate
More good luck:
H3C
O
NH CH3
O
H
N
H
CH3
S
NH
Cl
N
O
CH3
O
Cl
Cl
+
N
-
O
HO
Penicillin
Chlordiazepoxide
(Roche)
O
Cl
H3N
Pt
O
H3N
Cl
NH3
Pt
H3N
O
O
H
NH2 O
O
carboplatin
N
H2
oxaliplatin
CH3
Pt
Pt
O
cisplatin
H2
N
O
O
H
NH2 O
lobaplatin
O
7、Fragment based lead discovery
Fragment-based
Screening
Fragments:
102 ~104 RO3 compounds
Fragment grow, link
and merge
Target
Library
Synthesis
High Throughput
Screening
Full screening
Lipinski's RO5
(~106 compounds,
~$$2M/Screening)
Small is better (sample bigger chemical space, higher hit rate, higher Ligand
Efficiency, less false positive results, more intuitive to medicinal chemists)
Fragment Based Lead Discovery
Target
a bc
Biacore
Validation
X-ray / NMR Structures
HSQC
NMR Competitive
Binding Experiment
O
H2N
O
H
N
NH
O
NH
N
N
N
NH
NH
HN
Biacore
O
H
N
N
H
HO
N
NH
O
N
NH
N
N
N
NH
NH
H
N
O
HN
O
OH
N
Fragment Library
*Hubbard et al (2007), Curr Topics Med Chem, 7, 1568
O
N
NH
O
O
Evolution
Clinical candidates of fragment-derived compounds
Compound
Company
Status
Target
Therapy areas
Vemurafenib
Plexxikon/R
oche
Phase IV
B-RAF
Melonoma (first FBDD drug
on market, FDA approved in
2011)
ABT-263
Abbott
Genentech
Phase II
Bcl-xL
ABT-869
Abbott
Phase II
VEGF and PDGF
receptor tyrosine
kinase family
members
Small-cell lung cancer, CML,
Lymphoma, Hematological
neoplasm Cancer
Non-small-cell lung
cancer, etc.
AT-7519
Astex
Phase II
CDK family
members
Multiple myeloma cancer
AT-9283
Astex
Phase II
Aurora kinase
family members
Flt3 tyrosine
kinase, Jak2
tyrosine kinase
Abl tyrosine
kinase
Hematological
neoplasm
Solid tumor
Compound
Company
Status
Target
Therapy areas
DG-051
deCODE
Phase II
Leukotriene A4
hydrolase
Myocardial infarction
PLX-204
Plexxikon
Phase II
PPAR alpha, delta, Inflammatory disease
gamma
Cardiovascular disease
Non-insulin dependent
diabetes
LY-517717
Lilly Tularik
Phase II
Factor Xa
Thrombosis
NVP-AUY922
Vernalis
Novartis
Phase II
ATPase Hsp 90
Cancer Solid tumor
ABT-518
Abbott
Phase I
Gelatinase
Metalloprotease-2
Metalloprotease-9
Solid tumor
IC-776
Lilly ICOS
Phase I
CD11a, ICAM
Inflammatory disease,
Psoriasis, Autoimmune
disease
AT-13387
Astex
Phase I
Hsp 90
Cancer
Compound
Company
Status
Target
Therapy areas
PLX-4032
Plexxikon
Roche
Phase I
Raf B protein
kinase
Melanoma Cancer
PLX-5568
Plexxikon
Roche
Phase I
Raf protein kinase Pain, Polycystic kidney
disease
SNS-314
Sunesis
Phase I
Aurora protein
kinase
Cancer solid tumor
AT-13148
Astex, ICR
CRT
AstraZeneca
Phase I
AKT protein
kinase
Cancer
SGX-393
Lilly (SGX)
IND for
Phase I
Abl tyrosine
kinase
Cancer
Weak binding between the target and a small molecule fragment is detected by
biophysical methods, e.g., NMR, SPR or cross-validated by these two techniques.
Ref: Expert Opin. Drug Discov. (2009) 4:1125
Fragment Selection
RO3:
110 < MW <250 ~ 300
cLogP < 2 ~ 3 (or cLogD < 2 ~ 3)
2 < N+O < 6
logS > -4.5
TPSA < 110
Maximize the shape and electrical diversities (Openbabel + Pipeline Pilot)
to cover the chemical space for prototypical compounds
High ligand efficiency
LE = RTln(KD)/HAC
LE improves from ~0.3 to ~0.5 during hits to leads (H2L)
Final:
1K~2K fragment library (Commercial library: Maybridge or
Chembridge)
Most used Fragments Screening methods
Screening
method
Library
size
Primary
screening
Protein
amount
Pros & Cons
Crystallograp
hy
1K
N
>10mg
• Low hit rate, low throughput
• Heavily involved in H2L, LO.
NMR
1K
Y
>10mg
• High hit rate (2~8%, indicate
druggability).
• Ligand based (STD & WaterLOGSY)
observation in cocktail
•Less false positive results
ITC
1 – 2K
N
~g
SPR
2 – 5K
Y
~mg
HCS
5 – 30K
Y
• 3 weeks screening
• Cross validation with NMR
• require bioassay development for
different targets (difficulty in
outsourcing)
• High false positive rate
>10 fragment derived compounds in clinical phase II from pharmaceutical
companies, e.g., Novartis, Lily, Abbott, Genentech, Astex, Vernalis, deCODE,
Plexxikon.
>20 fragment derived compounds in clinical phase I since 2005.
Hits to leads
Evolving Fragments - In Practice
Growing
O
N
NH
NH
N
O
N
H
N N
H
N N
H
N
N
H
0.07 mM
Fragment 1 mM
N
NH
N
H
N N
H
AT9283
Phase II
0.003 mM
F F
OH
O
Linking
O
H
N
O
O
O
S
F
O H
N
S
S
O
N
O
N
F
ABT-263
N
A
OH
B
Phase II
Cl
Merging
N
CO2Me
HO
H
N
O
O
O
HO
HO
O
Me
O
HH
HO
N
N
H
O
OH
HO
HO
O
Virtual Screen
OH
O
Cl
O
OH
HO
N
Known Ligands
Detailed Design
NVP-AUY922
Phase I/II
O
药物化学总论（2010，p609)
“在发现苗头和先导物的早期研究阶段，同时关注分子的物化性质和活性，以降低
新药研发链中各个环节上的风险率，已经成为业界之共识。基于片段的药物设计，
辅以配体效率的“监督”，已在国外许多药厂和大学蓬勃展开，大有超过高通量筛
选的势头。应用FBDD的13年来，已有一批候选药物进入临床试验阶段。这种研发
策略，涉及了分子药理学、结构生物学、分子模拟、化学合成和药物化学等多种学
科和技术，整合性很强。在我国，尚未发现采用该方法的研究报道。本节之目的在
于呼唤和催生FBDD在我国的实施。”