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One of many modules taught in the course, Drug Discovery (CHEM-4330 and CHEM-6330) at Rensselaer Polytechnic Institute by Mark P. Wentland
Target Validation
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 1 Module - Target Validation
mple Module
ught in 30) at R
S
rget
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Target Validation - Suitability of Molecular Targetsfor Small Molecule Drug Discovery
Druggability
Link
to d
isea
se
low high
high
Most
Least
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 2 Module - Target Validation
Sample Module
ample Module
ample Module
MM
Screening Small Structure- Link toMolecular target (MT) (HTS) molecules Selectivity based design disease
Enzymes + + + +
GPCRs + +
Ion channels + − +
RNA + + + +
DNA + + − + +
Protein-protein interactions − − +
Biogenic amine transporters + + − +
Nuclear hormone receptors + + + +- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
+ MT is generally amenable to this property/technologyMT is somewhat amenable to this property/technology
− MT is not very amenable to this property/technology
Druggability of Molecular Targets for Small Molecule Drug Discovery
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 3 Module - Target Validation
Sample Module
mp
+ e M+M
− duul- - -
Target Discovery and Validation
A validated drug target is a protein or oligonucleotide that when modulated by a drug in abiological system, the desired therapeutic effect is realized.
• To some researchers, a target is well validated only when a small molecule shows efficacy in humans at tolerated doses.
• TV becomes a circular argument akin to the "chicken or the egg", namely what should comefirst, the target or the lead molecule?
- Target HTS lead drug: Biological/genetic approaches (a.k.a. reverse genetics)
- HTS target lead drug: Chemical approaches (a.k.a. forward genetics)
• Goal is to be as close to human as possible for most meaningful target validation, BUT technology tends to put you farthest away from the human.
• "Merck Benediction"biw50a
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 4 Module - Target Validation
Sample Module
rget es
ment akcule?
Biological/
al approac
eaning
Target Discovery and Validation• Biological/genetic approaches (a.k.a. reverse genetics; TV comes before HTS)
- Core technologies: functional genomics, bioinformatics, transgenic mice, antisense & siRNA oligonucleotides, and DNA microarrays
- Advantages cost and speed
- Disadvantages factors are reductionism, biological redundancy, poly-pharmacology
• Chemical approaches (a.k.a. forward genetics or chemical genomics; TV comes after HTS)
- Core technologies: HTS, bioinformatics, cell microarrays, and proteomics
- Advantages novel MTs & potential to overcome reductionism
- Disadvantages cost and speed
• Biological system can be:
- Human
- Animal
- Cell- or tissue-based
- Cell-free (e.g., standard enzyme inhibition assays)
- In silico (e.g., bioinformatics and virtual screening)
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 5 Module - Target Validation
Sample Module
and sp
tors are
forward
ormatics,
l to overc
Target Discovery and Validation in Humans • Entry of "Pioneer" vs. "Fast-follower" blockbuster drugs
(Cohen, F. J.; et. al, NRDD 2006, 5, 285-294. and Booth, B.; Zemmel, R. NRDD 2003, 2, 838-841):
- First in Class ("pioneer"): 28 % (e.g., Viagra)
- Fast-followers: 13 % (2-15 years late; e.g., Levitra)
• Both V and L take effect in about 30 minutes. Levitra's effect last forabout 5 hours; Viagra, about 4 hours.
- Differentiators: 31% (5-15 years late; e.g., Cialis)
• Cialis works a bit faster (within about 15 minutes), and the effectslast much longer - up to 36 hours.
- Late-comers: 28% (>15 years late; e.g., Allegra)
• Human genetics: CCR5 Antagonists as inhibitors of HIV entry
• Decode Population Genetics Approach (http://www.decode.com)
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 6 Module - Target Validation
("pio
: 13 %
ke effecra, abou
5 years la
hin abours.
g., All
ibitors
HIV-1 Replication in CD4+ Target Cells (Macrophages and Helper T Lymphocytes)
Uncoating
CD4
Assembly& budding
Proviral DNA
Transcription
Viral mRNA
Protease
TAT, REV
Translation
protein
gp120
HN
N
O
O
CH3
OHO
N3
Reverse transcriptase
Integrase
Attachment;fusion; entry
Zidovudine (AZT, 1987)RetrovirTM (GSK)
Saquinavir (1995) FortovaseTM (Roche)
Enfuvirtide (2003) FuzeonTM (Trimeris/Roche)36-amino acid synthetic peptide binds to gp41
Maraviroc (2007) SelzentryTM (Pfizer)
HN
NH
N
OH
O
OCONH2
PhN
HNO
H
H
Review: Frankel, A. D.; Young, J. A. T."HIV-1: Fifteen Proteins and an RNA."Annu. Rev. Biochem. 1998, 67, 1-25
Human Immunodeficiency Virus 1
gp41
gp120CD4
CCR5 orCXCR4
HN
O Ph
N N NN
H3C
i-Pr
FF
Raltegravir (2007) IsentressTM (Merck)
N
NHN
Me MeO
NN
OMe H
N
O
FOHMeO
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 7 Module - Target Validation
Sample Module
ample Module
ampleampleeee Me MMMe Me MMMe MMMe MMMeeeeeeee MMMMMMMMMMMMMMMMMMMMMMMMMMe MMe MMMe MMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM
VmRNA
MMe ModeeleleProt
ule
e Me MMee
Sa
oMoMM
Pr
(2007) Merck)
mmm
Hppplplmpmpmp Fpmmm
CCR5 Antagonists as HIV-1 Entry Inhibitors
• CCR5 is part of the chemokine GPCR receptor family - Binds 5-10 kDa proteins: MIP-1α, MIP-1β, RANTES - Over 15 identified and cloned - Directs migration of immune cells - Applicable to RA, atherosclerosis, transplants, HIV
• CCR5 antagonists as inhibitors of HIV-1 entry - Target validation: • Not a "pure" viral target • Homozygous individuals that lack a 32 bp deletion in the gene which expresses
CCR5 are resistant to R5 (M-tropic) HIV-1 infection
- Initial attempts to identify small molecule antagonists were disappointing
Recent reviews: Horuk, R. Nat. Rev. Drug Disc. 2009, 8, 23; Palani, A.; et al. J. Med. Chem. 2006, 49, 2851.
NH2
HO2C
out
in
CCR5 32 (< 2%)
Resistant to infectionDelayed progression
Heterozygous (~ 18%)Wild type CCR5 (~ 80%)
Susceptible to Infection
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 8 Module - Target Validation
Sample Module
f immutheroscle
bitors of H
a 32 bp deV-1 infec
ulul
sistant t
18%)
Module
Target Discovery & Validation - deCODE Population Genetics Approach
• Icelanders have very high compliance and accuracy in genealogy.
• The population has a rare resource in human genetics, namely three sets of data - genetic, disease and genealogical.
• deCODE has created a huge genealogy database of Icelanders and clusters patients into large extended families by disease state.
• High-throughput genotyping capabilities conduct effective genome- and population-wide linkage scans for disease-linked genes.
• Proprietary datamining software enable deCODE to identify small genetic regions shared by related patients.
• Detailed analysis of these regions allows deCODE to isolate key disease genes and then study their function and interaction with other genes within the pathogenesis of disease.
• deCODE's approach allows for a hypothesis-free discovery process and with focus on inherited components of human disease. This provides deCODE with population-validated drug targets and diagnostic markers.
biw50k
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 9 Module - Target Validation
Sample Module
esoal
e geneaease sta
lities cond.
eCODE to
to isolatewithin t
ry procesCODE w
deCODE Population Genetics Approach to Cardiovascular Diseases
• Genome-wide scan of 713 patients enlisted from a registry of 8000 Icelandic myocardial infarction victims under age 75 during 1981-2000 (296 Icelandic families) revealed:
- 4 SNP haplotype* in the gene encoding 5-lipoxygenase activating protein (FLAP)
• This gene confers a 2-fold greater risk of MI and stroke.
• Neutrophils from MI patients produce more LTB4 (proinflammatory agent) than controls
• deCODE has licensed a FLAP inhibitor from Bayer; DG031 has been in over 1000 asthma patients where Bayer found low efficacy but few side effects observed.
• In Phase II clinical trials, LTB4 and other biomarkers significantly reduced; PIII will evaluate for efficacy in preventing MI
Reference: Helgadottir, A. et al. Nature Genetics, 2004, 36, 233-239.*Haplotype - group of several SNPs linked physically on a single chromosome
biw50m
CO2H CO2H
OOH OH
CO2H
OH
Arachidonic acid 5-HPETE
FLAP
5-LO
LTB4
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 10 Module - Target Validation
Sample Module
gene
and strok
4 (proin
DG031effects obs
cantly r
ammmpmpamammplplpmpmmm
l5-HPETE
Target Identification and Validation using Mice
Transgenic Mice (i.e., reverse genetics):
• Many genotypic & phenotypic similarities exist between diseased mice and humans
• Mouse genome has been sequenced and is well-understood
• Transgenic mouse technology (gene deletions, additions and substitutions) is a mature and prospering field (Dunn, D. et. al, DDT 2005, 10, 757-767)
• Transgenic knockouts are most widely used since most therapeutics work by inhibition of some biochemical process.
• For 100 best-selling drugs, knockout phenotypes correlate well with drug efficacy. Will they model the next 100? (Zambrowicz, B. P.; Sands, A. T. NRDD 2003, 2, 38-51).
• Utility in target identification/validation: "Role for Stearoyl-CoA Desaturase-1 in Leptin-Mediated Weight Loss" (Friedman, J. M.; et. al, Science, 2002, 297, 240)
Mouse Mutagenesis with ENU (i.e., forward genetics)
• N-ethyl-N-nitrosourea (ENU) induces point mutations at a high rate permitting efficient systematic phenotype-based surveys of gene function.
• ENU can induce a series of alleles leading to loss-of-function and gain-of-function phenotypes. (see The Jackson Laboratories at www.jax.org)
biw51
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 11 Module - Target Validation
Sample Module
een s
echnolognn, D. et.
most wid
ut phen, B. P San
Role for SScience
)
at a high
Target Validation in Cells - Antisense Oligonucleotides
• Antisense oligonucleotides (AOs) are biochemical tools that efficiently define gene function in cells, tissues, and in vivo by regulating gene expression levels.
- AO technology is a mature and well-understood science - Amenable to 96-well format - AOs as therapeutics: • Vitravene® and Kynamro® are the only two marketed AOs • Others in the clinic for cancer and other diseases with unmet need
Melnikova, I. "RNA-based therapies" Nature Reviews Drug Discovery 2007, 6, 863-864.
biw52
17-25 ntAntisense DNA
+
mRNA bindingto ribosome
RNaseH-inducedcleavage
Hybridization
DNA-RNA DuplexmRNA
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 12 Module - Target Validation
Sample Module
rma
mro® arncer and
dodee
oduodu
HybridizaMMpl
Target Validation in Cells and In Vivo - siRNAs
• RNA interference (RNAi) ancient gene silencing method that uses small interfering RNA (siRNAs) to promote degradation of target mRNA.- Underpinnings: Studies in plants (1996) and mammalian cells (2001)- Science Magazine's "Breakthrough of the Year" in 2002 (12/20/02; p. 2296)- Target Validation - excellent tool to silence expression of a specific gene:
• siRNAs can knockdown target gene expression by > 90%• High specificity in target mRNA recognition• Thousands of mRNAs can be silenced by only a few siRNAs suggesting
amplification or catalytic activity
• The future is bright- Therapeutics - several are in clinical trials- RNomics - the hunt for novel RNA genes- siRNA arrays as high-throughput target validation tools
Reviews: Dorsett, Y.; Tuschl, T. NRDD 2004, 3, 318-329.Zamore, P. D.; Aronin, N. Nature Medicine, 2003, 9, 266-267.
biw52c
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 13 Module - Target Validation
Sample Module
"Breakxcellent
n target gRNA rec
n be sile
ools
Gene Silencing by siRNAs
biw52e
dsRNA (e.g., hairpin construct)
RISC formation
Target mRNA
RISC(RNA-induced
silencing complex)
Nuclease-induced cleavage
Synthetic dsRNA3'5'
siRNA (21-23 nt)HO OPO3
-2
Cellular kinaseDicer
(RNase III-like enzyme)
Binding to ribosome
RISC regenerated+
Degraded mRNA
HO
HO
OH
OH
-2O3PO OH
-2O3PO OH
-2O3PO OH
-2O3PO OH
-2O3PO OH
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 14 Module - Target Validation
Sample Module
MoRNA
nduce
Sme
ee MMooododddddddududududuuu
pleleleeeepl-2O3PO
dddddudududud
Age-related Macular Degeneration• AMD is the leading cause of vision loss and blindness in Americans
aged 65 and older.
• Wet Macular Degeneration: a more advanced and damaging form of the eye disease characterized by new blood vessels growth (neovascularization) beneath the retina and leak blood and fluid. This leakage causes permanent damage to light-sensitive retinal cells, which die off and create blind spots in central vision.
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 15 Module - Target Validation
Sample Mod
r.
generacharabeneat
nent dablind spo
Consider binding of a growth factor to extracellulardomain of a Growth Factor Receptor (e.g., EGFR, VEGFR, FGFR):
GF
Cell membrane
P
P P
P
GF
Ligand (GF) binding causes a conformational change in receptor leading to clustering of the cytoplasmic tyrosine kinase domains kinase activation via autophosphorylation
Substrate protein phosphorylation
and signal transduction
Binding of a Growth Factor to its Receptor
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 16 Module - Target Validation
Sample Module
am
o
plplModMod
ee Me Mpleplee
dd
n receptoon via a
Potential of siRNAs as Therapeutics: First Clinical Trials
• AMD (Age-Related Macular Degeneration) and DR (Diabetic Retinopathy)
- Central to the pathogenesis of AMD and DR is ocular neovascularization via binding of Vascular Endothelial Growth Factor (VEGF) to its receptor (VEGFR).
• Clinical trials initiated for two siRNAs for AMD via intraocular administration:
- Bevasiranib reduces production of VEGF (Phase III).
- Sirna-027 reduces production of VEGFR (Phase II).
- Both dropped due to efficacy issues
• Hurdles remain
- Challenges of in vivo siRNA delivery
- Safety concerns
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 17 Module - Target Validation
Sample Module
hogenEndoth
wo siRN
tion of VE
VEGF
KINGSTON, ON, June 18, 2012 /PRNewswire/ - At the center of the debate of how to potentially save the healthcare system millions of dollars are two drugs used to treat wet age-related macular degeneration (AMD), the leading cause of blindness in seniors. One, Lucentis, is FDA and Health Canada-approved and costs $1,700. The other, Avastin, is used off-label and costs one-tenth the price. Eye doctors currently use both drugs to treat AMD and some studies have shown that they are equally effective.
Avastin is a monoclonal antibody that binds directly to VEGF to form a protein complex which is incapable of further binding to the VEGF receptor.
Lucentis is a monoclonal antibody fragment (Fab) derived from the same parent mouse antibody as Avastin. It is much smaller than the parent molecule and has been affinity matured to provide stronger binding to VEGF-A
Avastin versus Lucentis for AMD
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 18 Module - Target Validation
mple Module
Sam
aculatis, is F
d off- atreat AM
The mechanism of action for Eylea is slightly different other anti-VEGF agents in that its soluble receptor binds to VEGF.
Eylea
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 19 Module - Target Validation
Sample Modulea is sligh
ptor binds
DNA Microarray Technology
GeneChip™ by Affymetrix (a.k.a. DNA probe arrays)
• Each array (1.28 cm2) contains 50-500K different probes complimentary to genetic information of interest
• Each probe cell (90 μm2) contains millions of copies of a specific 18- to 22-mer oligonucleotide
• When array is exposed to fluorescently-labeled oligo target, hybridization occurs only in those probes containing complimentary code
• Fluorescence is detected by an H-P GeneArray scanner and software prepares nucleotide and genotype report.
• Typical applications of DNA microarrays involve comparative genotyping and polymorphismanalysis for target identification/validation and diagnosis, e.g.:
- "Role for Stearoyl-CoA Desaturase-1 in Leptin-Mediated Weight Loss" - Jeffrey M. Friedman and coworkers, Science, 2002, 297, 240.
- Detect mutations in the tumor-suppressor gene p53.
- Determine resistance patterns in HIV-PR)- Pharmacogenomics (e.g., Roche's AmpliChip® CYP450)
See: http://www.affymetrix.com/products/index.htmlbiw56
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 20 Module - Target Validation
Sample Module
2) contain
uorescentary cod
-P GeneA
lve comand diagn
diated We
Affymetrix GeneChip™ DNA Microarray
biw57a
*
**
***
1.28 cm 90 m
Same 18-22 mers
Fluorescent labeled target oligo
ArrayProbe
Fluorescence image ofhybridized probe array
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 21 Module - Target Validation
Sample Modul
SSam
uleuleuleleululleleleuleuleululleleuuuleuleuuuuuuullulullulululllulullullllleleleleleuleuleleleuleleleleleleleldudududuuduluululdudududududuluuldudududududududududududududududududuuuuuuuuuuuluuuuluuuuuuuuuludulelelleleeelelelllelelllllleeeeeeeeeeeeeleleeleeeleleeeeeeeeeeee
Genomic DNA
RNA(e.g. viral or mRNA)
biw58
+
Scan
GeneChip™ Assay
1. PCR
1. Reverse transcription2. PCR gives cDNA
3. Fragmentation4. Labeling
2. Fragmentation3. Labeling *
Hybridization
*
Oligo probe
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 22 Module - Target Validation
Sample Moduleeeeeee
Samp
ntation
MMMe Me MMplele
biw62a
GeneChip™ p53 Probe Array
Wildtype reference sequence (antisense):
Complimentarysense probe set
Codon 2485' 3'
G - C - C - G - T - A - C - T - T - G - A - C - C - T - C - C - G - G
Wildtype at firstbase of codon 248
Interrogation of first base of codon 248
G - C - C - G - T - A - C - T - T - G - T - C - C - T - C - C - G - G
G - C - C - G - T - A - C - T - T - G - G - C - C - T - C - C - G - G
G - C - C - G - T - A - C - T - T - G - C - C - C - T - C - C - G - G
C - G - G - C - A - T - G - A - A - C - C - G - G - A - G - G - C - C - A - T T - G - G - G -
Mutation(C → G)
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 23 Module - Target Validation
Sample Module
aG - C
b
G - C - C
- C - G - T
- G - T -
CG -
Mbiw62b
GeneChip™ p53 Probe Array
Wildtype reference sequence (antisense):
Complimentarysense probe set
Codon 2485' 3'
G - C - C - G - T - A - C - T - T - G - G - A - C - T - C - C - G - G
Wildtype at middle base of codon 248
Interrogation of middle base of codon 248
G - C - C - G - T - A - C - T - T - G - G - T - C - T - C - C - G - G
G - C - C - G - T - A - C - T - T - G - G - G - C - T - C - C - G - G
G - C - C - G - T - A - C - T - T - G - G - C - C - T - C - C - G - G
C - G - G - C - A - T - G - A - A - C - C - G - G - A - G - G - C - C - A - T T - G - G - G -
Mutation(G → C)
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 24 Module - Target Validation
Sample Module
a
G - C - C
bas
C - C - G
C - G - T
- T - A - C
C -G -
biw62c
GeneChip™ p53 Probe Array
Wildtype reference sequence (antisense):
Complimentarysense probe set
Codon 2485' 3'
G - C - C - G - T - A - C - T - T - G - G - C - A - T - C - C - G - G
Wildtype at last base of codon 248
Interrogation of last base of codon 248
G - C - C - G - T - A - C - T - T - G - G - C - T - T - C - C - G - G
G - C - C - G - T - A - C - T - T - G - G - C - G - T - C - C - G - G
G - C - C - G - T - A - C - T - T - G - G - C - C - T - C - C - G - G
C - G - G - C - A - T - G - A - A - C - C - G - G - A - G - G - C - C - A - T T - G - G - G -
Mutation(G → A)
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 25 Module - Target Validation
Sample Module
a
G - C -
C - C - G
- C - G - T
- T - A - C
C- G -
Molecular Target Identification and Validation - Obesity and Leptin
• Leptin (16 kDa cytokine) promotes weight loss by suppressing appetite and stimulating metabolism
- ob/ob and db/db mutant mice are obese
- Leptin clinical trials: Successes in rodents did not translate to humans
biw56ex
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 26 Module - Target Validation
Samp
nt m
Succes
Module
Molecular Target Identification and Validation - Obesity and Leptin
• Leptin (16 kDa cytokine) promotes weight loss by suppressing appetite and stimulating metabolism
- ob/ob and db/db mutant mice are obese
- Leptin clinical trials: Successes in rodents did not translate to humans
• "Role for Stearoyl-CoA Desaturase-1 in Leptin-Mediated Weight Loss" - Jeffrey M. Friedman and coworkers, Science, 2002, 297, 240)
- Liver RNA from leptin-treated ob/ob mice subjected to microarray analysis
• Gene encoding SCD-1 (enzyme involved in fatty acid metabolism and insulin action) ranked highest among 15 gene clusters with distinct patterns of expression
• Leptin suppresses SCD-1 RNA levels and enzymatic activity• Transgenic mice lacking SCD-1 were lean and hypermetabolic• ob/ob mice lacking SCD-1 gene were less obese than controls
- 2002: SCD-1 potential target for obesity via inhibition
- 2004: $157M deal struck between Xenon Pharmaceuticals and Novartis to develop obesity drugs acting through SCD-1.
- 2011: From this deal, a small molecule SCD-1 inhibitor is believed to be in the clinic (http://www.xenon-pharma.com/)
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 27 Module - Target Validation
Sample Module
Succe
Desaturcience 2
ob/ob mic
involvesters with
nd enzyand hyperm
e than
tion
cals
ScoAO
SCD-1
N
F
Br
O N
NNN
NHO2C
ScoAO
Steroyl-CoA Oleoyl-CoA
O2, 2H+
+ 2H2O
MK-8245 (currently in clinical trials)
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 28 Module - Target Validation
Sample Module
SSSSS
MMMMMoMoMoooMMMo
Spotted Arrays vs. Photolithographic Synthesis (Affymetrix)Spotted microarrays- Probes (oligonucleotides or cDNA )are synthesized prior to deposition then "spotted" onto glass- Robotics commonly used (http://en.wikipedia.org/wiki/DNA_microarray)
- Approach utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface.
- The resulting "grid" of probes represents the nucleic acid profiles of the prepared probes and is ready to receive complementary cDNA targets oligos derived from experimental or clinical samples.
- Technique is used to produce "in-house" printed microarrays.- These arrays may be easily customized for each experiment
Pirrung, M. C.; “How to Make a DNA Chip” Angew. Chem. Int. Ed. 2002, 41, 1276Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 29 Module - Target Validation
Sample Module
obes an
s represery cDNA
ouse" printfor each ex
Phosphoramidite Method of Solid-Phase Oligonucleotide Synthesis
GE OligoProcess oligonucleotide synthesizer system
5'-G-C-A-3'
O
O
P
OO
O
OHO
O
P
OO
O
O
OH
N
N
NH2
OC
5'
3'
GNH
NN
N
O
AN
NN
N
NH2
NH2
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 30 Module - Target Validation
Sample ModuleGE Oli
syn
NH2
SPhosphoramidite Method of Solid-Phase Oligonucleotide Synthesis
Summary:- The cycle starts with the first building block (BB1) immobilized to a solid support via its 3' -OH group- All subsequent building block starting materials (BB2→BBx ) must be blocked/protected at the 5' -OH
[dimethoxytrityl (DMTr)] and amine groups (N-benzoyl or N-isobutyryl).- Coupling of the next nucleoside occurs at the 5' -OH group of the previous nucleotide. - The growing oligo stays bound to resin during the reaction series and is made in the 3‘ → 5' direction- Purification at each step is easily accomplished by washing the oligo-bound resin.- After deprotections, the final oligonucleotide has free OH groups at both its 3' and 5' ends.
Steps (somewhat abbreviated):1. Coupling reaction: BB2 is added (to BB1) in the form of an activated nucleoside phosphoramidite derivative
whose 5‘-OH bears a DMTr blocking group so it cannot polymerize with the phosphoramidite.2. Capping (with acetic anhydride/pyridine) to acetylate any unreacted 5'-OH of BB1 (prevents that unwanted
strand from growing again).3. Oxidation: To stabilized the newly introduces phosphite linkage, it is oxidized with iodine/N-methylimidazole to a
much more stable phosphate linkage. 4. Deprotection: Detritylation of the newly introduce BB at the 5' -OH group by trichloroacetic acid (TCA)5. Cycle repetition: Repetition of above mentioned steps until the desired full length oligonucleotide is made (each
cycle is approximately 99% efficient)6. Deblocking: Treatment of the oligo with NH4OH at 55oC for 5 hours amount of time removes:
- The cyanoethyl groups- The heterocyclic amine protecting groups- The oligo from the resin to give final oligo with free OH groups at both its 3' and 5' ends
7. Purification and desalting to remove ammonium ions) is achieved by PAGE, HPLC, or reverse phase HPLC.
http://www.atdbio.com/content/17/Solid-phase-oligonucleotide-synthesisDrug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 31 Module - Target Validation
Sample Module
occnd to res
sily accomonucleotid
in the formit cannoylate any un
e linkage
5' -OH groupesired f
amount o
Toolbox for Phosphoramidite Method of Solid-Phase Oligonucleotide Synthesis
O
HOO
N
NN
N
N
NH
O
OCH3
NH
NN
N
O
NHCOiPr
A GT
b1 4
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P
N(iPr)2
O
DMTrOO
b1 4
O
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NC
O
HOO
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N
N
NHCOPh
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NHCOPh
DNA Bases (b1 4):
R = resin; = resin + tether
BB1 BB2 BBx
(Dimethoxytrityl)
CH3O
CH3O
COPh =
O
CO-iPr = DMTr =O
CH3
CH3(Benzoyl) (iso-buturyl)
Ac2O = [CH3C(=O)]2OAcetic anhydride)N
pyr =
(Pyridine) Tetrazaole
N
NNH
N
TCA = Cl3CO2H(trichloroacetic aci)
NMI =
(N-methylimidazole)
NNCH3
Protecting groups/reagents
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 32 Module - Target Validation
Sample Module
N
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M
DNA B
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Phosphoramidite Method of Solid-Phase Oligonucleotide Synthesis
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 33 Module - Target Validation
Sample Module
mplmplmplmplmplmplmplmplmplmplmplmplmplmplmplmpmpmpmpmpmpmpmpmpmpmpmpmpmpmpmpmpmpl
Smmp
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H-Phosphonate Method of Solid-Phase Oligonucleotide Synthesis
O
P
O
O
DMTrOO
b1 4
BB2 BBx
H
BB1
"activated BB2"
(BB2 + Ac2O)
5'O
O
P
O
O
DMTrO
O
O
b2
3'
b1
O
O
P
OH
O
O
O
b2
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additionalcycles using:BB4 BBx
1. I2, pyr, H2O2. Deprotection (TCA)3. Deprotection (NH4OH)4. Purification
H1. Deprotection (TCA)2. "activated BB3"
5'O
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O
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OH
N
N
NH2
OC
5'
3'
GNH
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N
O
AN
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N
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NH2
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 34 Module - Target Validation
Sample Module
ppppppppppppppppp
34
MoMo
Synthesis of GeneChip™ Microarrays
biw59
Photo-protected substrates illuminated with light through
photolithographic mask
G - PO
P
O
P
O
P
O
P
O
P
OH OH O
P
O
P
O
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G
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Photolithographic Synthesis (Affymetrix)
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 35 Module - Target Validation
Sample Module
mpplmm
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Photolithographic Mask
biw60
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 36 Module - Target Validation
Sample Module
mplemplmple Modu
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Proteomics in Drug Discovery
Proteomics: An alternative/complimentary technique to genomics
• The study of the proteome (complete set of proteins encoded by the genome)
Underpinnings:
• Most drugs work on protein molecular targets
• mRNA levels usually don’t correlate with corresponding protein levels (contrast to genomics, where protein expression is inferred from mRNA levels)
• Post-transcriptional splicing of mRNA yields different proteins.
• Post-Translational Modification (PTM)
- Phosphorylation, glycosylation, farnesylation
- Formation of quaterny structures (e.g., heterodimers)
• Epigenetics (changes in gene expression that occur without a change in DNA sequence)
• Protein-protein interactions: Most diseases are not monogenic; therefore a protein-based approach is ideally suited to understand how proteins network in the majority.
See: http://www.nature.com/reviews/focus/proteomics
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 37 Module - Target Validation
Sample Module
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RNA yields
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Life Cycle of Proteins - The MT of Choice for Small Molecule Drug Discovery
biw75d
DNA - ~25K genes inhuman genome
mRNA - up to 100K transcripts in transcriptome
Transcription mRNA splicing Translation
Cleavage
pre-RNA
Pre-protein
Degradation
NH2
CO2H
PTM
PPI
CO2H
H2N
CO2H
OPO3-2
H2N
Proteins - up to 1M in proteome
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 38 Module - Target Validation
Sample M
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"Everything should be made as simple as possible, but not simpler."
Albert Einstein quote (from Cell, 2003, 112, 48):
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Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 39 Module - Target Validation
Sample Module
shoulde, but no
Systems Biology - A New "Golden Age" of Biology?
• Created to address critical issues in the DD process, namely:
- Reductionism: Traditional biology studies structure/function of proteins and genes in isolation.
- Biological redundancy: Biological systems have built-in backup systems to ensure that accidental changes (e.g., mutations) are not always harmful.
- Poly-pharmacology: Drugs often interact with multiple proteins and work via modulation of multiple pathways.
• Systems biology studies the complex interaction of biological macromolecules simultaneously to understand how they network and work together in, for example, signal transduction.
• Unit-operation iterative approach borrowed from engineers:
Review: Davidov, E. J. and coworkers, "Advancing drug discovery through systems biology." Drug Discovery Today, 2003, 8, 175-183.
biw75f
Manipulatebiological system
Measure multiplevariables simultaneously
Mathematicalmodeling
Data mining
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 40 Module - Target Validation
Sample Module
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Systems Biology• Protein Interaction Maps
- "A Systems Approach to Mapping DNA Damage Response Pathways"Workman, C. T.; et al, Science 2006, 312, 1054.
- "A Protein Interaction Map of Drosophila melanogaster." Giot. L.; et al, Science 2003, 302, 1727
- Cellzome: www.cellzome.com
- Gene Network Sciences: www.gnsbiotech.com
- Protein interaction maps: www.hprd.org
• Database of human biological processes:
- www.reactome.org
Review: Blow, N. "Systems biology: Untangling the protein web" Nature, 2009, 460, 415-418.
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 41 Module - Target Validation
Sample Module
et al, S
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Proteomics in Drug Discovery - Objectives
• Bridge the gap between genome sequence and cellular behavior
• Identify phenotypic differences at the protein level (“where the action is”)
• Understand systems biology and how proteins network
• When used in a comparative manner:
- Identify new molecular targets and markers
- Validate molecular targets (e.g., lovastatin shown to affect proteins associated with cholesterol metabolism)
- Develop assays for side-effects (a.k.a. toxicoproteomics)biw76
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 42 Module - Target Validation
Sample Moduledifferen
ogy and
manner
ple :
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Proteomics in Drug Discovery
Core technologies:
• 2D-PAGE (by size and charge) coupled with mass spectroscopy (MS); silver and fluorescent dyes enable femtomole detection.
• Bioinformatics
• Yeast two-hybrid (Y2H) system to study protein-protein interactions
• Chip-based microarrays: ProteinChip™ chips have addressable sites for binding proteins from crude samples; MS of bound protein generates structural information
• Role of MS- MALDI-TOF-MS (Burnum, K. E.; et. al. Annu. Rev. Anal. Chem. 2008, 1, 698)- ESI-MS for high sensitivity- LC-IMS-TOF-MS/MS offers four degrees of separation- BIA-MS for protein interaction analysis- SELDI-MS for ProteinChip™ analysis
Limitations/shortcomings:
• Proteome very complex (up to 100-1000K proteins?)
• Higher throughput and sensitivity needed (e.g., no protein equivalent of PCR)biw77
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 43 Module - Target Validation
Sample Module
stem to
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nu. Re
paration
Protein Separation and Analysis:
Protein Characterization:
Proteomics - 2D Gel Platform
• Extract protein from gel• Treat with a succession of enzymes to convert protein to 5-10 AA peptides• Separate fragments by capillary electrophoresis• Analyze fragments by electrospray MS/MS to determine sequence and if glycosylated or phosphorylated• Bioinformatics and genomics now help match sequence information to known proteins/genes
...MSTLYASEID... ...ATGCCTAG...
biw78
Protein Protein sequence Gene sequence
Cellular proteins from diseased sample
Control sample
pHsize
2-D gelelectrophoresis
http://www.incyte.com/science/proteomics/index.htmlDrug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 44 Module - Target Validation
Sample Module
det or pho
Bioinforquence info
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Yeast Two-Hybrid System to Study Protein-Protein Interactions
biw78dLead reference: Uetz, P. and coworkers, Nature, 2000, 403, 623-627.
Bait Y
LexA DBDPrey X
B42 AD
LexA UAS
Bait Y
LexA DBDPrey X
B42 AD
Reporter gene
+
Activation of transcription
Invitrogen Dual Bait Hybrid HunterTM utilizes fusion protein technology: - DBD = DNA Binding Domain - AD = Activation Domain - UAS = Upstream Activating Sequences - When LexA gene turned on, the cell turns blue
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 45 Module - Target Validation
Sample Module
MoMo
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Chemical Genomics
• Underpinnings: The use of chemical tools (i.e., small molecules) to understand biology
• Target validation comes after ligand is identified via HTS (i.e., ligand use to validate MT)
- Considerable historical precedent (e.g., DNA gyrase, GABAA)
• NIH Roadmap for Medical Research (http://nihroadmap.nih.gov/):
- Emphasis on screening targets out of favor in Pharma (i.e., "undruggable" targets)
- uHTS format (1536 wells) to screen 100K compounds (initially) 1M (later) against genes, proteins, and cellular or organismic (yeast, zebra fish) phenotypes associated with any disease or any part of the genome.
- "Hit-to-probe" rather than hit-to-lead
- Probe molecules will be made available to all without encumbrances
- Informatics and technology development crucial to success of MLI
see: Terstappen, G. C.; et al, Nature Reviews Drug Discovery, 2007, 6, 891.biw79
Drug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 46 Module - Target Validation
Sample Module
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