chapter 2 drug screening & discovery (design)
DESCRIPTION
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 (RBTRANSCRIPT
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)
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
先导化合物发现
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）
NH
N
O OO
O
OO
O OO
CH3
CH3
CH3
CH3
CH3
CH3
H
HH
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
2 、天然活性物质作先导物，适当分子变换：紫杉醇，长春碱
NH
O
O
OO
O OO
O
O
OHO
O
CH3
OH
OH
CH3
CH3
CH3
CH3
CH3
HO
ONHO
O
OH
O
CH3
CH3
CH3
CH3
OH O OH
CH3CH3
CH3
OH
OO
H
H
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。
docetaxel: 水溶性增加，活性〉紫杉醇，无交叉耐药
1.天然生物活性物质作为先导物
3 、改造天然产物
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
acetyl CoA OOOH
OH CH3 SCoA HMG CoA reductase
NADPH
O OH
OH
OH
CH3
cholesterol
O
OO
OOH
CH3
CH3
CH3
CH3
H
H HO
OHO
OOH
CH3
CH3
CH3
CH3
H
H H
OH
Lovastatin Ki = 1nM
Km=10M
N
O
F
OH
OH
CH3
CH3
O
NH
OH
N
CH3
CH3
OH
F
OH
OH
O
statins
Atorvastatin
(Lipitor, best sale)
Fluvastatin Pitavastatin(Nisvastatin)
• 青蒿素
青蒿素Artemisinin
黄花蒿Artemisia annula
蒿甲醚Artemether
(Novartis PCT)
O
O
O
O
O
O
O
O
O
OCH3
生物利用度较低复发率高
• 喜树碱
羟基喜树碱Hydroxycamptothecin
喜树 (中国）Camptotheca acuminata
拓扑替康Topotecan （ GSK 2007）
NN
O
O
OOH
HO
NN
O
O
OOH
HO
N(CH3)2
水溶性较差，毒性大
作用于 DNA topoisomerase I （化疗药物）治疗 SCLC ，卵巢癌，结肠癌
南美洲古柯Erythroxylum coca Lam
• 局麻药
可卡因Cocaine
普鲁卡因Procaine （ 1905）
N
O
O
O
OH
H2N
O
O
N
NH
O
N
CH3
CH3
CH3
CH3
Lidocaine（ 1943 Synthesis;
1949 on market)
• 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
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
Cons:Low hit rate: 0.001%Low druggabilityHigh false positive rate
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
KS AT KR ACP KS AT KR ACP TE KS AT ACP KS AT DH ER KR ACPAT ACP KS AT KR ACP KS AT KR ACP
SO
SO
HO
SO
HO
HO
SO
HO
HO
O
SO
HO
O
HO
SO
HO
HO
HO
O
SO
HO
HO
O
HO
HO
13
13
13
13
13
1313
13
9
9
9
9
9
9
15
5
5
¹¦ Äܵ¥Ôª1¹¦ Äܵ¥Ôª2
¹¦ Äܵ¥Ôª3¹¦ Äܵ¥Ôª4
¹¦ Äܵ¥Ôª5¹¦ Äܵ¥Ôª6
DEBS1 DEBS2 DEBS3
ºì ùËØA6-Deoxyerythronolide B
O
O CH3
HON(CH3)2OH
H3CO
O
CH3H3C
H3C
O
CH3
O
O
CH3
CH3
OH
OCH3CH3
OH
HO
H3C
OH
H3CO
O
CH3H3C
H3C
O
CH3
OH
OH
CH3
HO
H3C
9
1 3
6
12
9
125
56
3
岩白菜内酯的生物催化组合库
O
OOH
HO
CH3O
OH
OH
OH
O
O
OOH
HO
CH3O
OH
OH
OH
OOHO
OOH
HO
CH3O
OH
OH
OH
OCl
O
OHOH
OH
HOO
O
OOH
HO
CH3O
OH
OH
O
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O
OOH
HO
CH3O
OCOR1
OCOR2
OCOR3
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
NHNH2
OH
NH
NH2
O
NH2
O
NH
NH
NH2
CH3
S
O
NH
NH
NH2
CH3
OS
O
NH
NH
N
CH3
O
CH3
CH3
Activity(5-HT1) = 2 (vs. 5-HT)
Activity(5-HT2) = 1/25
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
Structural basis for molecular recognition at serotonin receptors. (2013) Science 340: 610-614
5-HT3 受体阻断剂
ONH
NH2
Cl
O
N CH3
CH3
CH3
O
NH
O
CH3
Cl
NH2
NNH N CH3
Cl
NH2
O
CH3
O
N
NN
O
CH3
CH3
N
N
NHO
CH3
NCH3
N
ON
H
MetoclopramideBlock dopamine & 5-HT3
止吐，胃动力药
CleboprideDopamine blocker胃动力不变
5-HT3 blocker> clebopride 300 folds无 dopamine 拮抗
Setrons: 治疗肿瘤化疗引起的恶心呕吐
granisetron palonosetronOndansetron
血管紧张素转化酶（ angiotensin-converting enzyme, ACE)抑制剂
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
ACE
（ angiotensin II: 强效收缩血管）
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
Angiotensin I Bradykinin: 血管舒张剂
ACE
Arg-Pro-Pro-Gly-Phe-Ser-Pro
Hypothetical active site of carboxypeptidase A
NH CH
R
C
O
NH CH C
CH2 O
O
Zn++
S1
S1'
O
C
O
CH2CH C
CH2 O
O
substrate
(R)-2-benzylsuccinic acid
Hypothetical binding of inhibitors to ACE
Zn++
S1 S1'
N CH
O
CCHNH
R2
O
O
CC
O
CH
R3
NH
HS2'
peptide inhibitor
C CH2 C
O
O
R2
CH C
O
CHNO
O
S CH2 C
O
O
R2
CH C
O
CHN
carboxyalkylproline
mercaptoalkylproline
N
O
O
OH
SH
CH3
NNH
OO
O
OH
OCH3
CH3
N
NH
O
OO
OH
OH
CH3
captopril enalapril Enalaprilat(enalapril 的代谢活性组分）
ACE小分子拟肽抑制剂
ACE的功能 羧肽酶 A 的作用模式 肽类抑制剂的结合模式
羧烷基脯氨酸卡托普利依那普利等
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，开始优化
²¼ÂíÁ¢°· Burimamide ¿Ú·þÎÞЧ
Ñ¡ÔñÐÔÞ׿¹ ¼Á
²¿ ·Ö¼¤¶¯¼Á
HN N
HN
S
NHCH3
HN N
HN
S
NH2
HN N
NH
NH2
S
+HN N
S NH2
NH
HN N
S NH2
NH
HN N
HN NH2
NH
HN N
NH2
¼×ÁòßäÌØ Metiamide 20%favoring form
favoring form
favoring form40%
3%80%
20%
6.80
6.25
7.25
5.90
6.80
cbapKa
H
R
S
HN
S
NHCH3
HN
S
NHCH3
NH2
HN N
R
N NH
R
+HN NH
R
·¨ ĪÌ涡 Famotidine1986
À×ÄáÌ涡 Ranitidine1983
Î÷ßäÌ涡 Cimetidine1976
S
NSO2NH2
NH2
S
NN
NH2
H2N
S
HN
CHNO2
NHCH3
O(CH3)2N
HN N
S
HN
NCN
NHCH3H3C
¼×ÁòßäÌØMetiamideÉöËðÉËºÍ Á£Ï¸ °ûȱ·¦ Ö¢HN N
S
HN
S
NHCH3H3C
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-Gly-
Asp （ 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。
H2N NH
NH
NH
NH
O
O
HN
NH
O
O
COOH
H2N
NH
HN
O
N
O
O COOHRGD
Sibrofiban
Roxifiban
H2N
NH
NO
NH
O
HN
OCH3
O
O
O CH3
4 、 SOSA磺胺治疗细菌感染时，发现利尿作用 .
S
S
NNNH
O
ONH2
O
CH3
S
S
O
O
OO
NH2
NH2
Cl
Cl
SO
ONH
N
S
NH2
O
OCl
SSNH
O O
NH
O
O
NH2
Cl
Acetazolamide口服利尿 Dichlorphenamide chlorthiazide
hydrochlorthiazide
SNH
S
NH
OOO
O
NH2
Cl
SNH
S
NH
O OO
O
NH2
F
F
F
cyclothiazide长效利尿药
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
SNH
SN
O
O
NH2
CH3S
NHO
OO
NH
NH2
CH3
sulfasomizole Carbutamide, 更强的降糖作用，治疗糖尿病
O
N
NHNH2
NH
NH
O
N
CH3
CH3NH
NH2
NH2CH
NCH3
CH3
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
Isoniazid无抗抑郁
iproniazidphenelzine
tranylcypromine selegiline
抗抑郁药
O
CH3
N
NH
O
NH N
N N N
NNH
O
NH2
O
O
OH
O
OH
P
O
N
NHN
N
CH3
CH3
O
O
CH3
S
O
O
N
NCH3
Zaprinast
抗过敏药扩张血管、降血压抑制 PDE5 ：治疗心绞痛
cGMP GMPPDE5
cGMP ：扩张血管，增加血流量
sildenafil
cGMPNO第二信使增强勃起功能 (ED)
NN
CH3
S
O
O
O
CH3
N
NH
N
N
O
CH3
CH3
N
NHS
N
NH
N
N
CH3
CH3
O
CH3
CH3
O
O
O
N
NNH
O
CH3
O
O
O
H
H
模拟创新：
• 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
5、基于代谢作用
°ÙÀ˶àÏ¢ Prontosil
H2N N N SO2NH2
NH2
»Ç°· Sulfonilamide
H2N SO2NH2
»Çõ£°· ÀàÒ©Îï
• 抗疟药环氯胍
ÂÈëÒ Proguanil
Cl NHCNHCNHCH(CH3)2
NH NH
»· ÂÈëÒ Cycloguanil
Cl N NH2
N
N
H2N
H3CCH3
ÒÒ°· à×ठPyrimethamine
Cl NH2
N
N
H2N
C2H5
保泰松的代谢活化
NN
O O
CH2CH2CH2CH3
±£Ì©ËÉ Phenylbutazone
NN
O O
CH2CH2CH2CH3
OH
ôDz¼×Ú Oxyphenbutazone
´ÙÄòËáÅÅй
NN
O O
CH2CH2CHCH3
OH
NN
O O
CH2CH2S
O
»ÇßÁͪ Sulfinpyrazone£¨ ¿¹ Í´ ·ç Ò©£©
O
O
O
O
CH3O
CH3O
Îåζ×Ó±ûËØ Schizandrine C
O
CH3O
CH3O
O
O
O
COOCH3
COOCH3
O
O
CH3O
CH3O
O
O
COOCH3
COOCH3
OCH3
OCH3
O
O
O
O
Áª±½Ë«õ¥ Bifendate
6、幸运
结构解析错误
Penicillin
N
SNH
O
O
OCl
OH
Cl
CH3
CH3
OCH3H H
N+
N
O-
NH
Cl
CH3
Chlordiazepoxide
(Roche)
NH3
Cl
Pt ClNH3
cisplatin
NH3
NH3
O
O
O
O
Pt
carboplatin
NH2
NH2
OO
OO
Pt
NH2
NH2
Pt
H
H
O
O O
CH3
oxaliplatin lobaplatin
More good luck:
Target
LibrarySynthesis
Full screeningLipinski's RO5
Fragment grow, link and merge
Fragments:Fragment-based Screening
High ThroughputScreening
102 ~104 RO3 compounds
(~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)
7 、 Fragment based lead discovery
Fragment Library
Target
X-ray / NMR Structures
b ca
NMR CompetitiveBinding Experiment
ON
NH
N
O
O
NH
N
NH
O
OH
O
NHN
NNH
NH
ONH
N
NH
NNH
OH
O
N
NH
NNH
NH
N
NH
O
O
NH2
O
NH
NH
Evolution
Validation Biacore
HSQC
*Hubbard et al (2007), Curr Topics Med Chem, 7, 1568
Biacore
Fragment Based Lead Discovery
Clinical candidates of fragment-derived compounds
Compound Company Status Target Therapy areas
Vemurafenib Plexxikon/Roche
Phase IV B-RAF Melonoma (first FBDD drug on market, FDA approved in 2011)
ABT-263 Abbott Genentech
Phase II Bcl-xL Small-cell lung cancer, CML, Lymphoma, Hematological neoplasm Cancer
ABT-869 Abbott Phase II VEGF and PDGF receptor tyrosine kinase family members
Non-small-cell lungcancer, 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, gamma
Inflammatory disease
Cardiovascular disease
Non-insulin dependent
diabetes
LY-517717 Lilly Tularik Phase II Factor Xa Thrombosis
NVP-AUY-922
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
Ref: Expert Opin. Drug Discov. (2009) 4:1125
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.
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 methodsScreening
methodLibrary
sizePrimary
screeningProtein amount
Pros & Cons
Crystallography
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 • 3 weeks screening• Cross validation with NMR
HCS 5 – 30K Y • 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
NH
NOH
OH O
O
NO
O
NHOH
OHN
OOH
OH CO2Me
OOH
OH
O
O
O
HH
O
Cl
OH
OH Me
Known Ligands
Detailed DesignNVP-AUY922Phase I/II
Merging
Virtual Screen
Linking
NH
OS
O O
NH
S
N
ON
N
Cl
SO
O
F
F
F
ABT-263
Phase II
OHO
F
OH
B
A
Growing
NH
NNH
N
AT9283
Phase IINH
NNH
NNHO
NH
NNH
NNH
NH
ON
O
Fragment 1 M 0.07 M 0.003 M
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。”