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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
• 3. RNAi
• 4. Pharmacology (use of small molecules to turn off protein function)
pre-translational
Pharmacology
• Disadvantage– Unlike genetic methods it is difficult to identify
ligands that are highly selective for a target.
• Advantages1. Fast time scale
2. Only perturbs targeted sub-domains
3. graded dose response - tunability
4. Most drugs are small molecules
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 methodsremove 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
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)
2. Test biased or unbiased panels of compounds against protein target of interest
Major challenges
• Druggability– Many proteins do not appear to make
favorable interactions with drug-like small molecules
Molecular Weight <900 Da
Kd < 1 M (∆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 protein-
protein 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 InhibitorsINHIBITORS OF HIV-1 PROTEASE: A “Major Success of Structure-Assisted 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 acidschelatethe active sitezinc
Protein Kinases
The human genome encodes538 Protein kinases (483 arecatalytically active)
Kinase Inhibitors
Almost all inhibitors that have been developed bind in the ATP pocket
Synthesis of kinase inhibitors
OlomucineCdc2/CyB: 1µMCdk2/CyA: 1µM
LibrarySynthesis
10,000
LibraryScreening
10,000
HitCdc2/CyB: 340 pMCdk2/CyA: 340 pM
Gray et. al. Science (1998) 281, 533-538.
Approved kinase inhibitors
28 small molecule kinase inhibitors are now in the clinicGleevec (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
“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)
Requirements3D structure of target protein (NMR or other)large quantities of 15N-labeled protein (> 100 mg)NMR assignments of backbone N and HN atomssize of protein <40 kDasolubility: protein and ligands
Principlestart with known protein structure and 15N assignments15N-HSQC of protein15N-HSQC of protein plus ligand: identify shifted peaksmap these on protein surface: binding site
Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.;Fesik, S. W. Science 1996, 274, 1531.
Conversion of Peptides/Proteins to Small Molecules
“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)
OHN
N
N
Cl
NO2
NH
S
N
SOO
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