H/D-Exchange Advantages Facilitate the Drug Discovery Process
• Ligand binding site identification
• Binding site and pharmacology correlations (e.g. agonist vs. antagonist)
• Enabling “fragment assembly” strategies in the absence of ligand-bound X-ray structures
• Ability to optimize selective LXR agonists, and predict in vivo effects
Drug binding and H/D-Exchange
Amide hydrogen/deuterium exchange, coupled with proteolysis and liquid chromatography-mass spectrometry (H/D-Ex), is gaining widespread use for the analysis of protein structure dynamics1,2, protein–protein interactions3,4, and protein–ligand interactions5-10.
In example below, the ligand binding domain of a nuclear receptor (NR), peroxisome proliferator-activated receptor gamma (PPAR-gamma), was exposed to various ligands and analyzed by H/D-Ex. NRs are a superfamily of transcriptional regulators that modulate biological processes as diverse as cellular differentiation and metabolism. 5-10 Their flexibility in regard to gene regulation has been attributed to their conformational flexibility. Many studies suggest that different types of ligands induce changes in conformation and dynamics to the ligand-binding domain (LBD) of PPAR-gamma. These changes consequently alter the expression profile of certain genes relating to lipid and glucose homeostasis. In fact, PPAR-gamma’s role in lipid and glucose homeostasis has made it a common target of strategies seeking to restore insulin sensitivity in patients with type II diabetes.
In this case, H/D-Exchange was used to detect differences (perturbations) in the protein dynamics of apo and drug-bound PPAR-gamma LBD. Three different classes of drugs were surveyed: agonists, partial agonists and antagonists. The value of this technology is that H/D-Ex can be used to bind newly identified ligands (or drug-leads) into groups that elicit similar conformation or dynamic responses upon complex formation.
The rate of amide hydrogen exchange is dependent upon local fluctuations in protein structure.5 For this reason the rate of H/D-Ex is a good indicator of regional flexibility within a protein. As illustrated above in panels A-E, ligand binding in all cases reduced the exchange rate and, by so doing, stabilized structural regions closely corresponding to the crystallographically determined rosiglitazone binding site (panel E). Since ligand binding typically involves the formation of an intermolecular network involving hydrogen bonds and/or electrostatic interactions with the protein, complex formation is accompanied by stabilization. Protein stabilization is readily detectable by H/D-Ex.
In general, full agonists GW1929 and rosiglitazone (panels C-D) stabilized PPAR-gamma LBD to a greater extent than antagonist GW9662, and partial agonist nTZDpa (panels A-B). The exchange of amide hydrogens in Helix 11 and 12 (circled region) was largely impeded upon binding agonist. Both of these helices are highly dynamic in apo PPAR-gamma LBD. Upon binding antagonist or partial agonist, helices 11 and 12 appear to be as dynamic as in the unbound form. The stabilization of helix 12 in the presence of agonist supports earlier studies showing that the maintenance of the helical conformation of this helix is critical for gene activation. These published results demonstrate the ability to predict the activities of various drugs by H/D-Exchange technology.
On the ability to optimize selective LXR agonists and predict in vivo effects as presented at the Keystone Nuclear Receptor Conference, March 21-26, 2010 by Peter Akerblad, PhD, Associate Principal Scientist/Team Leader Bioscience at AstraZeneca, Sweden. Read Dr. Akerblad’s poster titled “Linking Liver X Receptor Modulation to Effects In Vivo.”
1. Hamuro Y, Coales SJ, Southern MR, Nemeth-Cawley JF, Stranz DD, Griffin PR. Rapid analysis of protein structure and dynamics by hydrogen/deuterium exchange mass spectrometry. J Biomol Tech. 2003;14:171-182.
2. Hamuro Y, Molnar KS, Coales SJ, Yang BO, Simorellis A, Pochapsky TC. Hydrogen-deuterium exchange spectrometry for investigation of backbone dynamics of oxidized and reduced cytochrome P450cam. J Inorg Biochem. 2008;102:364.
3. Horn JR, Kraybill B, Petro EJ, Coales SJ, Morrow JA, Hamuro Y, et al. The role of protein dynamics in increasing the binding affinity of an engineered protein-protein interaction established by H/D exchange mass spectrometry. Biochemistry. 2006;45: 8488-8498.
4. Coales SJ, Tuske SJ, Tomasso JC, Hamuro Y. Epitope mapping by amide hydrogen/deuterium exchange coupled with proteolysis and liquid chromatography mass spectrometry. Rapid Commun Mass Sp. 2009;23(5):639-647.
5. Hamuro Y, Coales SJ, Morrow JA, Molnar KS, Tuske SJ, Southern MR, et al. Hydrogen/deuterium-exchange (H/D-Ex) of PPARgamma LBD in the presence of various modulators. Protein Sci. 2006;15:1883-1892.
6. Hamuro Y, Coales SJ, Morrow JA, Molnar KS, Tuske SJ, Southern MR, et al. Hydrogen/deuterium-exchange (H/D-Ex) of PPARγ LBD in the presence of various modulators. Protein Sci. 2006;15:1-10.
7. Luo L, Carson JD, Molnar KS, Tuske SJ, Coales SJ, Hamuro Y, et al. Conformation dependent ligand regulation of ATP hydrolysis by human KSP: Activation of basal hydrolysis and inhibition of microtubule-stimulated hydrolysis by a single, small molecule modulator. J Am Chem Soc. 2008;130:7584-7591.
8. Kornhaber GJ, Tropak MB, Maegawa G, Tuske SJ, Coales SJ, Mahuran DJ, et al. Isofagomine induced stabilization of glucocerebrosidase. ChemBioChem. 2008;9(16):2643-2649.
9. Tropak MB, Kornhaber GJ, Rigat B, Maegawa G, Buttner J, Blanchard J, et al. Identification of pharmacological chaperones for gaucher disease and characterization of their effects on b-Glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry. ChemBioChem. 2008;9(16):2650-2662.
10. Chandra V, Huang P, Hamuro Y, Raghuram S, Wang Y, Burris TP, et al. Structural organization of the intact PPARγ-RXRα nuclear receptor complex on DNA. Nature. 2008;456(7220):350-356.