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Chemical Biology 1 –
Pharmacology
10-17-14
Methods for studying protein
function – Loss of Function
• 1. Gene knockouts
• 2. Conditional knockouts
pre-translational
• 3. RNAi
• 4. Pharmacology (use of small molecules
to turn off protein function)
Pharmacology
• Advantages
1. Fast time scale
2. Only perturbs targeted sub-domains
3. graded dose response - tunability
4. Most drugs are small molecules
• Disadvantage
– Unlike genetic methods it is difficult to identify
ligands that are highly selective for a target.
Weiss WA, Taylor SS, Shokat KM. “Recognizing and exploiting differences between
RNAi and small-molecule inhibitors.” Nat Chem Biol. 2007 Dec;3(12):739-44.
Time Scale and Specificity
Small molecules are subdomain
specific
Example: PAK1 Kinase
Small molecules affect only one domain, while pre-translational methods
remove the entire protein from the cell.
Tunability
Allows the amount of inhibition/activity that is necessary
Reverse Chemical Genetics
(Pharmacology)
1. Identify a protein target of interest
– Develop an activity assay (enzymes) or a binding
assay (protein-ligand interactions) to screen
compounds
2. Test biased or unbiased panels of compounds against
protein target of interest
3. Optimize your initial lead compound by making analogs
(SAR) and by using any additional biochemical/structural
information. In parallel, screen optimized analogs
against other targets (selectivity)
Major challenges
• Druggability
– Many proteins do not appear to make
favorable interactions with drug-like small
molecules
Molecular Weight <900 Da
Kd < 1 mM (∆G < -8.4 kcal/mol)
No more than one or two fixed charges
– Estimated that only ~10% of all proteins are
druggable
Hopkins and Groom, Nat Reviews Drug Disc, 2002
Major challenges
• Selectivity
– Finding selective agonists and antagonists is
very challenging
– Knowing which other proteins to
counterscreen is difficult (easier for
mechanism-based or enzyme family-directed
ligands)
In some cases, chemistry and genetics can be used to circumvent these
problems.
Knight ZA, Shokat KM. “Chemical genetics: where genetics and pharmacology
meet. Cell. 2007 Feb 9;128(3):425-30.”
Koh JT. “Engineering selectivity and discrimination into ligand-receptor interfaces.”
Chem Biol. 2002 Jan;9(1):17-23. Review.
Identification of small molecule
inhibitors
2 classes
– 1. Enzyme Inhibitors
• Many effective strategies for identifying enzyme
inhibitors.
– 2. Protein-Protein Interaction Inhibitors
• Difficult to identify potent inhibitors of proteinprotein interactions.
Methods for discovering enzyme
inhibitors
• High throughput screening (parallel
synthesis and combinatorial chemistry)
• Mechanism-based (incorporate a
functionality that is unique for an enzyme
enzyme class (For example, proteases)
• Privileged scaffolds (kinases,
phosphodiesterases)
• Transition state analogs
Turk B.Targeting
proteases: successes,
failures and future
prospects.
Nat Rev Drug Discov.
2006 Sep;5(9):785-99.
Aspartyl Protease Inhibitors
HIV Protease Inhibitors
INHIBITORS OF
HIV-1 PROTEASE:
A “Major Success
of StructureAssisted Drug
Design” Alexander
Wlodawer, Jiri
Vondrasek. Annual
Review of
Biophysics and
Biomolecular
Structure. Volume
27, Page 249-284,
1998
HIV Protease Inhibitors
HIV Protease Inhibitors Resistance
More protease inhibitors
• Ketones (serine and cysteine proteases)
• Phosphonic and hydroxamic acids (metalloproteases)
transition state analog
hydroxamic acids
chelate
the active site
zinc
Protein Kinases
The human genome encodes
538 Protein kinases (483 are
catalytically active)
Kinase Inhibitors
Almost all inhibitors that have been developed bind in the ATP pocket
Synthesis of kinase inhibitors
Library
Synthesis
10,000
HN
N
N
HO
HN
N
N
Olomucine
Cdc2/CyB: 1µM
Cdk2/CyA: 1µM
N
H
R
HN
N
N
R'
N
H
Cl
Library
Screening
10,000
N
N
N
N
R"
HO
N
H
N
N
Hit
Cdc2/CyB: 340 pM
Cdk2/CyA: 340 pM
Gray et. al. Science (1998) 281, 533-538.
Approved kinase inhibitors
28 small molecule kinase inhibitors are now in the clinic
Gleevec (Imatinib) was the first clinically approved kinase
inhibitor (2003)
Protein-Protein Interaction (PPI)
Inhibitors
• Identification of potent PPI inhibitors is
very challenging. In general, standard
screening strategies don’t work.
• Conversion of Peptides/Proteins to Small
Molecules
• Innovative new strategies are needed
– for example, SAR by NMR
Conversion of Peptides/Proteins to Small Molecules
“SAR” by NMR Abbott Laboratories (Stephen Fesik)
Fragment based approach
- library of small compounds (several thousands)
- build up larger ligands
- n fragments may yield n2 compounds
NMR: 15N-HSQC of target protein (2D NMR)
Requirements
3D structure of target protein (NMR or other)
large quantities of 15N-labeled protein (> 100 mg)
NMR assignments of backbone N and HN atoms
size of protein <40 kDa
solubility: protein and ligands
Principle
start with known protein structure and 15N assignments
15N-HSQC of protein
15N-HSQC of protein plus ligand: identify shifted peaks
map these on protein surface: binding site
Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.;Fesik, S. W. Science 1996, 274, 1531.
“SAR” by NMR
“SAR” by NMR
1. Screen 100-5000 low molecular
weight (150 -300 MW) ligands to identify
weak binders. HSQC perturbations
identigy the site of binding
2. Screen for a second site of binding in
the presence of the first ligand
3. Use structural information to design
a linkage between the two identified ligands
∆G(linked ligand) = ∆G(fragment 1) +
∆G(fragment 2) + ∆G(linker) +
∆G(cooperativity)
∆G(linker) usually positive (entropic cost)
∆G(cooperativity) is a non-additive effect
Application: Bcl-xL/BH3
Proteins
First Site Ligands
First Site Ligands
Second Site Ligands
Linked Inhibitor
(Bcl-xL-ABT-737)
NO2
H
N S
O O
O
NH
S
N
N
N
Cl
Nature 2005 Jun 2;Vol. 435(7042):p. 677-81.
J. Med Chem. 2006 Jan 26;Vol. 49(2):p. 656-63.
J Med Chem. 2006 Feb 9;Vol. 49(3):p. 1165-81