Chemical Biology 1 – Pharmacology

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. Advantages 1. 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 Dec;3(12):

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 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 Feb 9;128(3): ” Koh JT. “Engineering selectivity and discrimination into ligand-receptor interfaces.” Chem Biol Jan;9(1): 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 Sep;5(9):

Aspartyl Protease Inhibitors

HIV Protease Inhibitors INHIBITORS 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 , 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 Olomucine Cdc2/CyB: 1µM Cdk2/CyA: 1µM Library Synthesis 10,000 Library Screening 10,000 Hit Cdc2/CyB: 340 pM Cdk2/CyA: 340 pM Gray et. al. Science (1998) 281,

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

“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, Conversion of Peptides/Proteins to Small Molecules

“SAR” by NMR

1.Screen low molecular weight ( 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

Second Site Ligands

Linked Inhibitor

(Bcl-xL-ABT-737) Nature 2005 Jun 2;Vol. 435(7042):p J. Med Chem Jan 26;Vol. 49(2):p J Med Chem Feb 9;Vol. 49(3):p