target validation rget - rensselaer polytechnic institutewentlm3/example module dd.pdf · • entry...

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

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Target Validation - Suitability of Molecular Targetsfor Small Molecule Drug Discovery

Druggability

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Sample Module

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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


Sample Module

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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


Sample Module

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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)


Sample Module

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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)


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HIV-1 Replication in CD4+ Target Cells (Macrophages and Helper T Lymphocytes)

Uncoating

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Proviral DNA

Transcription

Viral mRNA

Protease

TAT, REV

Translation

protein

gp120

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N

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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)

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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

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CCR5 orCXCR4

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Sample Module

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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

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Resistant to infectionDelayed progression

Heterozygous (~ 18%)Wild type CCR5 (~ 80%)

Susceptible to Infection


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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.

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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

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Sample Module

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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)

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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.

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17-25 ntAntisense DNA

+

mRNA bindingto ribosome

RNaseH-inducedcleavage

Hybridization

DNA-RNA DuplexmRNA


Sample Module

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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.

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Sample Module

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Gene Silencing by siRNAs

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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

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OH

OH

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-2O3PO OH

-2O3PO OH

-2O3PO OH

-2O3PO OH


Sample Module

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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.


Sample Mod

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Consider binding of a growth factor to extracellulardomain of a Growth Factor Receptor (e.g., EGFR, VEGFR, FGFR):

GF

Cell membrane

P

P P

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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


Sample Module

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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


Sample Module

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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


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The mechanism of action for Eylea is slightly different other anti-VEGF agents in that its soluble receptor binds to VEGF.

Eylea


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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


Sample Module

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Affymetrix GeneChip™ DNA Microarray

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1.28 cm 90 m

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Fluorescent labeled target oligo

ArrayProbe

Fluorescence image ofhybridized probe array


Sample Modul

SSam

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Genomic DNA

RNA(e.g. viral or mRNA)

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Scan

GeneChip™ Assay

1. PCR

1. Reverse transcription2. PCR gives cDNA

3. Fragmentation4. Labeling

2. Fragmentation3. Labeling *

Hybridization

*

Oligo probe


Sample Moduleeeeeee

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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)


Sample Module

aG - C

b

G - C - C

- C - G - T

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CG -

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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



Mutation(G → C)


Sample Module

a

G - C - C

bas

C - C - G

C - G - T

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C -G -

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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



Mutation(G → A)


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

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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/)


Sample Module

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Steroyl-CoA Oleoyl-CoA

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MK-8245 (currently in clinical trials)


Sample Module

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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

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Phosphoramidite Method of Solid-Phase Oligonucleotide Synthesis

GE OligoProcess oligonucleotide synthesizer system

5'-G-C-A-3'

O

O

P

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O

OHO

O

P

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O

O

OH

N

N

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3'

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Sample ModuleGE Oli

syn

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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

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Toolbox for Phosphoramidite Method of Solid-Phase Oligonucleotide Synthesis

O

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N

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O

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NC

O

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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


Sample Module

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Phosphoramidite Method of Solid-Phase Oligonucleotide Synthesis


Sample Module

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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

3'

b1O

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O

3'

b1

additionalcycles using:BB4 BBx

1. I2, pyr, H2O2. Deprotection (TCA)3. Deprotection (NH4OH)4. Purification

H1. Deprotection (TCA)2. "activated BB3"

5'O

P

OH

DMTrOb3

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5'-G-C-A-3'

O

O

P

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O

OHO

O

P

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O

O

OH

N

N

NH2

OC

5'

3'

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NN

N

O

AN

NN

N

NH2

NH2


Sample Module

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Synthesis of GeneChip™ Microarrays

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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

P

G

P

G

P

O

P

O

P

O

P

G

P

G

P

T

P

O

P

T

P

T

G

T

G

A

T

T

T

C

A

G

T

G

C

A

G

P

G

P

O

P

O

P

O

P

Photolithographic Synthesis (Affymetrix)


Sample Module

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Photolithographic Mask

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Sample Module

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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


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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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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.

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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.


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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


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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


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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

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http://www.incyte.com/science/proteomics/index.htmlDrug Discovery (CHEM-4330/6330) Rensselaer Polytechnic Institute 44 Module - Target Validation

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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


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target validation rget - rensselaer polytechnic institutewentlm3/example module dd.pdf · • entry...

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