DRUG DISCOVERY

1
??????????????

• ????????????
• ????????
• ??

2
??

• ???????
• ???????????????????
• ????????????
• ??????????????????????
• ??????????????????????????????????????
• ????????????????????????

3
?????????
?????
???? DRUG DISCOVERY
1.?????????? 2.????????????
???????
1. ????? 2. ????
???? DRUG DEVELOPMENT
4
??????????????????
???? ???? ????
????? ??
????
??
?????
????
????
????? ????? ????????? ????
????? ????? ???? ????
5
?????(HTS)

• ???????????????????????????????,??????????????????
  ?,???????10000??????????????

6
The Evolution of HTS
7
?????

• ??----????????
• ??----???????????
• ??----???????????????
• ??----?????

8
??????????
?????
????
9
??????????
HTS
????
????
????
???
10
????????????

• ????????,????????????????????????

11
??????????

• ?????????????????????????????????,????????????????
  ??????????????????????????????????????

12
Current Drug Targets

• Membrane Receptors 45
• Enzymes 28
• Hormones and Factors 11
• Ion Channels 5
• DNA 2
• Nuclear Receptors 2
• Unknown 7
• N 483 J. Drews, Science (2000) 2871962

13
????

• Expression phenotype ?
• Mutant ?
• Antisense treatment ?
• Antibody treatment ?
• RNAi ?
• Knockout / knockin ?
• Inhibitor treatment ?

14
?? PCR?RT-PCR???cDNA???EST??????????RNA????????
???????????,????????
??? ???????? ?????? ??????
????????????
?????????????????????
???
??
E412nm 13,600 M-1cm-1
15
????

• ??????
• ?????????????????????????????????????
• ???????
• ???????????????(?????????)???????????????
• ?????????????????????????????????

16
??????
17
????(Photometry)
18
????????MetAP??
19
????????MetAP??
20
????(Fluorescence Intensity)
7-amino-4-methylcoumarin
rhodamine 110
resorufin
21
Fluorogenic Peptide Substrate
22
????(Fluorescence Polarization)
23
FP
Theoretical FP Behavior of a Series of
Fluorescent Dyes As a Function of MW
24
????
25
IMAP
26
Protein-DNA Binding FP HTS
27
Ser/Th Kinase Competitive FP Assay
28
???????(FRET)

• ??????????????????????????????????????????????????
  ????????????FRET??????????????????(10100?)?????
  ???????????????????????????????????

29
Fluorescence Resonance Energy Transfer (FRET)
Donor
Acceptor
30
???????

• ???????,????????????????,?????????????????????????
  ??????(tetramethylrhodamine)????50???????????55
  ?,EDANS?DABCYL???33?,EDANS???????46??FRET?????????
  ?????????

31
Fluorogenic (Quenched) Substrate
32
FRET

• Example of use of FRET in a protease assay
• Matayoshi et al., Science 247 954 (1990)

33
???????????(Time Resolved FRET)
34
LANCE Assay for Kinase
35
LANCE Assay for Transferase and Nuclear receptor
36
LANCE Assay for Protease and Helicase
37
AlphaScreen
38
AlphaScreen for PIP3
39
AlphaScreen for cAMP
40
BRET
41
???????(Scintillation Proximity Assay, SPA)
42
FlashPlate - SPA without Beads
Bind Receptor Or Target to Plate
Radiolabeled Tracer and Sample added
Only bound Tracer is Measured
43
????????????
44
????????????
45
?????????
46
???????????
47
?????

• ????????????????????????????
• ??????(?????pH???)
• ???????????
• ????
• ???????
• ???????????
• ????????,?????????????????????????????????????????
  ?,???????,?DNA????????

48
??????
49
??????(FLIPR)

• ??????????Fluo-3?????,????????????FLIPR???,???96??
  384???????????,???????????1?????????????????

50
FLIPR functional screening assay
L
?
PLC
GTP
GDP
inositol phosphates
Fluorescent dye
Ca2

fluorescent signal
51
????

• ?????????????????,?????????,???????????
• ????????,???????????????????????????(luciferase)??
  ????????(secreted alakaline phosphatase,
  SEAP)?????????(chloramiphenicol
  acetyltransferase, CAT)??-?????(?-galactosidase)??
  -???????????(GFP)?

52
The Two-Hybrid System

• Two hybrid proteins are generated with
  transcription factor domains
• Both fusions are expressed in a yeast cell that
  carries a reporter gene whose expression is under
  the control of binding sites for the DNA-binding
  domain

Activation Domain
Prey Protein
Bait Protein
Binding Domain
Reporter Gene
53
The Two-Hybrid System

• Interaction of bait and prey proteins localizes
  the activation domain to the reporter gene, thus
  activating transcription.
• Since the reporter gene typically codes for a
  survival factor, yeast colonies will grow only
  when an interaction occurs.

Activation Domain
Prey Protein
Reporter mRNA
Bait Protein
Reporter mRNA
Reporter mRNA
Reporter mRNA
Binding Domain
Reporter mRNA
Reporter Gene
54
Quantum dots

• Invitrogen ???Qdot??
• Quantum dots ?????????????????,???????????????????
  ????
• ?????????,?????????
• ????,???????,???????????????,???????????????,?????
  ?????
• ???????,?????????????

Qdot???
55
High Content Screening
56
High Content Screening

• Cellomics
• ArrayScan
• KineticScan
• Molecular Devices
• Discovery-1

Visualize and analyze cell populations Identify
sub-cellular protein localization Perform
multi-parameter analysis
57
Translocation Assay

• Live cell assays to detect translocation of
  signaling proteins
• primary screening
• lead profiling
• Pathway screening for functional effect, rather
  than specific mode of action (e.g. in vitro
  binding, catalytic activity).
• Modulation of protein translocation is an
  alternative to inhibition of catalytic activity
  protein translocation modulators.

58
Protein Translocation is Essential for
Intracellular Signaling
The PKB/Akt Pathway
59
Greater Selectivity with Translocation Modulators

• Active Site inhibitors often lack specificity
  and selectivity ? unwanted side effects.
• Many signalling proteins with very different
  functions share similar active sites - e.g.
  Protein Kinase C family.

X
X
X
ATP and Substrate binding sites
Other binding sites could be targets for
specific, selective inhibitors.
PKC catalytic inhibitors all act on ATP-binding
site
60
GFP Fusion Proteins for Detecting Translocations

• Make stable, transfected cell lines.
• Quantify intracellular translocations of target
  in high throughput.

61
Membrane to Cytoplasm Translocation
Example Pathway Therapeutic area
PLCd Phosphoinositide Many
MARCKS PKC Cancer, Diabetes
Membrane Receptors Receptor internalization Many
Target PLCd-PH domain Host cell type
CHO-hIR Stimulation ATP Timescale 5-20 sec
62
Cytoplasm to Nucleus Translocation
Example Pathway Therapeutic area
p65 NFkB TNF etc. Inflammation
ERK1 MAPK Cancer
p38 Stress Inflammation
JNK cJun Cancer
b-catenin Wnt Cancer
Nuclear receptors RXR, LXR, estrogen etc. Many
TFs Transcription Many
Target Erk1 Host cell type HeLa Stimulation
FCS / hEGF Timescale 10 min
63
Vesicle to Plasma Membrane Translocation
Example Pathway Therapeutic area
GLUT4 Insulin Diabetes
Receptor channel recycling Endosomal Many
A human GLUT4 assay in Xcellsyz Ltd.
conditionally immortalized human fat and muscle
cell lines is under development.
Target GLUT4 Host cell type
CHO-hIR Stimulation Insulin Timescale 20 min
64
Cytoplasm to Membrane Translocation
Example Pathway Therapeutic area
PKCa Phosphoinositide Cancer
PKCb Phosphoinositide Diabetic complications
PKCe Phosphoinositide Cardiovascular
Akt/PKB Growth factor Cancer, Diabetes
Arno PI3-K Many
b-Arrestin GPCRs Many
Assay read on IN Cell Analyzer with the Membrane
Spot Analysis Module (MSPO).
Target Akt1 / PKBa Host cell type
CHO-hIR Stimulation IGF-1 / insulin Timescale
5 min
65
???????????

• ?????(Chemical diversity)
• ???(Drug-like)

66
Lipinski??

• MW lt 500
• ClogP lt 5
• H bond donors lt 5
• H bond acceptors lt 10

67
Properties of an Average Drug
68
Undesirable Compounds

• Unstable
• Reactive
• Michael acceptor
• Toxic
• Liquid
• General metal chelator
• Polyaromatic
• metallocompound

69
??????

• ?????????
• ?????
• ????????????????????
• ??????
• ?????????????,??????????
• ??????????????

70
?????????

• ?????????
• ???????????????????????
• ????????,??????????,????
• ??????SAR??,???????,????????????????

71
????

• ????????????????,?????????????(?????????????????)?
  ?????????????

72
????

• ??
• ??????,???????
• ???????---100,000 compounds (and more!) in a year
  with a few chemists and automation
• SAR???
• Drug-like?

73
????????

• ????????????????????????????????
• ????---??????????????---??????,???????????????????
  ?????????????????????????????????
• ???????????,???????????????????????,??????????????
  ?????????????????????????????

74
??????(Nature Product Pool)

• ??????????????????????,???????????????????????????
  ???????????????????????????,?????????????????????
  ?????

75
????????????????

• ????,????????????????????????,????????????????????
  
• ???20?????????????????????
• ?????????????????????????????

76
????????????????????

• ????????????????(?????????)?????????????,Haslam???
  ?
• 1)????????????????????
• 2)????????????????,?????????
• 3)????????????????,??????????????
• 4)???????????????,?????????,?????????????????????
  
• 5)??????????????????????????,??????????,?????????
  
• 6)???????????????????????,??????????????

77
???20?????????????????????

• 20??70???????????????????????????????
• ????????????????????,??????50000??????,???????????
  ?????????
• ??????????????????????????

78
?????????

• ?????????
• ??????????
• ????????

79
??????????????????????

• ??????????
• ???????
• ???????????
• ???????????
• ??????
• ?????????????
• ??????

80
???????????????

• 61 of 877 small molecule drugs introduced
  worldwide during the period 1981-2002 can be
  traced to, or were inspired by, natural products.

81
???? (Lovastatin)(HMG-CoA???)
Pravastatin
Fluvastatin
CompactinR1R2H Mevinolin R1CH3,R2H Simvasti
n R1CH3,R2CH3
Cerivastin
Atorvastin
82
????A(Cyclosporine A)
??Trichoderma polysporum??
????????????????
83
???
???(1, Paclitaxel, Taxol)????????Taxus
brevifolia?????????????????? ?????(2, Docetaxel,
Taxotere)??????????????????????????? 2001?????????
???20?????,???????????10?????????????????,????????
???????
84
????????
(A)
Camptothecin R1R2R3H Topotecan
R1H,R2CH2N(CH3)2,R3OH Irinotecan
R1CH2CH3,R2H,R3(A) 9-Aminocamptothecin
R1R3H,R2NH2 9-Nitrocamptothecin R1R3H,R2NO2
?????????DNA?????? (DNA toposomerase? ????
85
?????
Buprenorphone,???Morphine 25-50?,????????
Morphine,RH Codeine,RCH3
Pentazocine,???????30,??????
86
?????????
??????????????????????
??????? (diversity-oriented synthesis)
87
Galanthamine ???????????????????????-???????Secram
ine A ???
2527 compounds were synthesized
Shair M D and Kirchhausen T. Nature chemical
biology, 2006, 2 39-46
88
Compound Storage

• Solid
• Long-term storage
• DMSO solution
• Long-term storage (10 year?)
• Working solutions

89
Compound plates
80 Compounds
88 Compounds

• Mother Plates
• DMSO solutions from vendors
• Daughter Plates
• DMSO solutions
• 1 mg/ml
• Assay Plates
• Aqueous solutions
• 25 µg/mL compound
• 2.5 DMSO
• 20 µL

352 Compounds
90
Sample management technology

• ?????????????????????????????????????????????????
• ?????????????????????,??
• ????????
• ???????60,000,000???
• ?????????-80? ?
• ?????????
• ???????,??????,??,Cherry picking?
• ??
• ????????,?????????
• ??
• ???,???,??,Barcode.

91
Sample management technology

• ??Tecan??????????????

Click to view the video
REMP Large-size storeTM (LSS)
92
??????

• ????????,??????,????????

93
???(Microplate)

• ???(Microplate)????????????????????????????????,??
  ??????????????????????????????????????????????????
  ????,???????????????????????????????
• ???????????,???????????,??????????????????????????
  ,96???384?????????????(Society of Biomolecular
  Screening task force "Standards" in Automation
  and instrumentation)??????????,?864?1536?3456,??96
  00?????????????????????????????????????,????????

94
Microplates

• ?????????,??????????????????,??
• ?????????
• ???????????
• ??????????

95
Microplates from Corning
96
Matrix of Wells

• Well Row Column Volume
• 96 8 12 300
• 384 16 24 100
• 1536 32 48 10

96
384
864
1536
97
Microplates

• ?????????

???? ??
?? ?????
??? ?????? ????????
???? ????,????
???? ??
????(PS) ???,????,??????
??? (PP) ??????,???????
UV???? ?????,??UV??
98
Microplates

• ???????????
• ???????????????????????????
• ?????
• ???????

???? 96? 384? 1536? 3456?
???? 75-250 mL 20-100 mL 2-10 mL 1mL
99
Microplates

• ??????????

?? ??
????????? Corning??NBSTM?????????
????????? ?????, ??ELISA, DELFIA
??????????? Corning???CelllBind?????????? ??????? ?????????????????
?????? ?DNA,??,?????????
100
?????

• ????,??????????????????????????,????????????
• ????(Stand-alone)???????????(Liquid
  Dispenser)?????
• ???(Workstation)???????(????????)???,
  ???????(Liquid Handling)????????????

101
History 1980s, Zymark

• ???????????Zymark?????BenchmateTM??,??????????????
  ???????????HPLC??????????????????????????????????,
  ?????????????????????,????????????

102

• ?????????????????Beckman Coulter???Biomek1000,????
  ??????????????????????(????)??????
• Biomek2000 ??????????????,???????????????????????
  ?????????????
• BiomekFX?????????,?????????????????,?????????????
  ???????

103
Biomek 2000 Automation Workstation
104
?????????
105
Beckman Biomek FX

• 96-channel pipetting head
• High-capacity for simultaneous liquid transfer
• Span-8 pipettor
• Flexible with individual movement and liquid
  sensing capability for hit picking and plate
  reformatting

106
????

• UV-visible Light Absorbance
• Fluorescence
• Fluorescence Intensity
• Time-resolved Fluorescence (TRF)
• Fluorescence Polarization (FP)
• Luminescence
• Radioactivity

107
Microplate Readers

• SpectraMax Plus 384
• UV absorbance
• Continuous 190 - 1000 nm,
• Reads 96 wells, 5 sec 384 wells,16 sec

• SpectraMax Gemini XS
• Luminescence, fluorescence and time-resolved
  fluorescence
• Continuous 250-850 nm
• Read 96 wells, 27 sec 384 wells, 2 min 30 sec

108
VICTOR2 V Multi-label Reader

• Filter-based instrument
• Five technologies
• Absorbance
• Luminescence
• Fluorescence
• Time-resolved fluorescence
• Fluorescence polarization

109
Microplate reader

• ?????????????,????,??????
• PerkinElmer???EnVision ??????36s??????1536???????
  ???
• ??????,????,????,??????,????,??????????

EnVision (PERKINELMER)
Safire² (TECAN)
SpectraMax M5 (Molecular Devices)
110
?????(Automated Robotic System)

• ??????????????????????????,??????????,????????????
  ??????????????????????????????,???????????????????
  ????????????

111
????????

• ???
• ???
• ???
• ????
• ????
• ???????????,???,??????????????

112
?????

• (?????) Articulated arm robots
• (??????? )Cylindrical coordinate robots
• ( ?????) Overhead gantry robots

113
????? ( Articulated arm robots ORCA Robot )
114
???????( Cylindrical coordinate robots SEICO
Robot )
115
( ?????) Overhead gantry robots
116
???(Gripper )
117
  ??????(Linear servo track)

• ????????????????,???????????????????,?????????????
  ?????,??????????????????????

118
???????(Typical peripheral devices)

• ??????? (Microplate storage unit)
• ?????? (Bar code scanner)
• ????(Liquid transfer)
• ???( incubator)

119
Microplate storage unit
carousel
stacker
120
ORCA System
121

• Thank You!