Andrew Orry

Virtual Ligand Screening of the p300/CBP Histone Acetyltransferase: Identification of a Selective Small Molecule Inhibitor

The histone acetyltransferase (HAT) p300/CBP is a transcriptional coactivator implicated in many gene regulatory pathways and protein acetylation events. Although p300 inhibitors have been reported, a potent, selective, and readily available active-site-directed small molecule inhibitor is not yet known. Here we use a structure-based, in silico screening approach to identify a commercially available pyrazolone-containing small molecule p300 HAT inhibitor, C646. C646 is a competitive p300 inhibitor with a K(i) of 400 nM and is selective versus other acetyltransferases. Studies on site-directed p300 HAT mutants and synthetic modifications of C646 confirm the importance of predicted interactions in conferring potency. Inhibition of histone acetylation and cell growth by C646 in cells validate its utility as a pharmacologic probe and suggest that p300/CBP HAT is a worthy anticancer target.

Structure‐based identification of binding sites, native ligands and potential inhibitors for G‐protein coupled receptors

G‐protein coupled receptors (GPCRs) are the largest family of cell‐surface receptors involved in signal transmission. Drugs associated with GPCRs represent more than one fourth of the 100 top‐selling drugs and are the targets of more than half of the current therapeutic agents on the market. Our methodology based on the internal coordinate mechanics (ICM) program can accurately identify the ligand‐binding pocket in the currently available crystal structures of seven transmembrane (7TM) proteins [bacteriorhodopsin (BR) and bovine rhodopsin (bRho)]. The binding geometry of the ligand can be accurately predicted by ICM flexible docking with and without the loop regions, a useful finding for GPCR docking because the transmembrane regions are easier to model. We also demonstrate that the native ligand can be identified by flexible docking and scoring in 1.5% and 0.2% (for bRho and BR, respectively) of the best scoring compounds from two different types of compound database. The same procedure can be applied to the database of available chemicals to identify specific GPCR binders. Finally, we demonstrate that even if the sidechain positions in the bRho binding pocket are entirely wrong, their correct conformation can be fully restored with high accuracy (0.28 Å) through the ICM global optimization with and without the ligand present. These binding site adjustments are critical for flexible docking of new ligands to known structures or for docking to GPCR homology models. The ICM docking method has the potential to be used to “de‐orphanize” orphan GPCRs (oGPCRs) and to identify antagonists–agonists for GPCRs if an accurate model (experimentally and computationally validated) of the structure has been constructed or when future crystal structures are determined. Proteins 2003;51:423–433.

Structure-based development of target-specific compound libraries

The success or failure of a small-molecule drug discovery project ultimately lies in the choice of the scaffolds to be screened -- chosen from among the many millions of available compounds. Therefore, the methods used to design compound screening libraries are key for the development of new drugs that target a wide range of diseases. Currently, there is a trend towards the construction of receptor-structure-based focused libraries. Recent advances in high-throughput computational docking, NMR and crystallography have facilitated the development of these libraries. A structure-based target-specific library can save time and money by reducing the number of compounds to be experimentally tested, also improving the drug discovery success rate by identifying more-potent and specific binders.

The Challenge of Considering Receptor Flexibility in Ligand Docking and Virtual Screening

Computational ligand docking and screening is widely employed throughout the pharmaceutical industry to speed up the drug discovery process and identify drug candidates from very large pools of virtual compound libraries. When a ligand interacts with a receptor a number of structural changes within the ligand binding site might occur. It is therefore critical for these methods to accurately predict, or otherwise take into account the receptor flexibility upon ligand binding. This flexibility within the binding pocket explains why a diverse range of ligand sizes and shapes can sometimes bind to the same receptor pocket. This observation supersedes the notion that ligand-receptor interaction is a purely lock and key mechanism. The capability to correctly predict molecular interactions is critical for computer-aided molecular design technology. In this review, we discuss biological cases of receptor flexibility upon ligand binding that can range from large-scale movement of loops to single gate-keeper amino acid movements. In addition, we provide further evidence that rigid receptor docking alone will more than likely fail in the drug-discovery process. We then discuss computational methods, which have been developed to mimic flexibility within the binding pocket and predict ligand-receptor interactions. Early flexible receptor docking methods used soft-potential docking and rotamer libraries. More recently methods have focused on constructing an ensemble of structures generated by a variety of means including X-ray crystallography, NMR, Monte Carlo sampling, Normal Modes-based methods and Molecular Dynamics. It is evident that methods that ignore receptor flexibility can result in poorly docked solutions and therefore the challenge is to develop computational methods, which can accurately and efficiently predict this phenomenon.

Molecular basis of secretin docking to its intact receptor using multiple photolabile probes distributed throughout the pharmacophore.

The molecular basis of ligand binding and activation of family B G protein-coupled receptors is not yet clear due to the lack of insight into the structure of intact receptors. Although NMR and crystal structures of amino-terminal domains of several family members support consistency in general structural motifs that include a peptide-binding cleft, there are variations in the details of docking of the carboxyl terminus of peptide ligands within this cleft, and there is no information about siting of the amino terminus of these peptides. There are also no empirical data to orient the receptor amino terminus relative to the core helical bundle domain. Here, we prepared a series of five new probes, incorporating photolabile moieties into positions 2, 15, 20, 24, and 25 of full agonist secretin analogues. Each bound specifically to the receptor and covalently labeled single distinct receptor residues. Peptide mapping of labeled wild-type and mutant receptors identified that the position 15, 20, and 25 probes labeled residues within the distal amino terminus of the receptor, whereas the position 24 probe labeled the amino terminus adjacent to TM1. Of note, the position 2 probe labeled a residue within the first extracellular loop of the receptor, a region not previously labeled, providing an important new constraint for docking the amino-terminal region of secretin to its receptor core. These additional experimentally derived constraints help to refine our understanding of the structure of the secretin-intact receptor complex and provide new insights into understanding the molecular mechanism for activation of family B G protein-coupled receptors.

Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping

While it is evident that the carboxyl‐terminal region of natural peptide ligands bind to the amino‐terminal domain of class B GPCRs, how their biologically critical amino‐terminal regions dock to the receptor is unclear. We utilize cysteine trapping to systematically explore spatial approximations among residues in the first five positions of secretin and in every position within the receptor extracellular loops (ECLs). Only Cys2 and Cys5 secretin analogues exhibited full activity and retained moderate binding affinity (IC50: 92±4 and 83±1 nM, respectively). When these peptides probed 61 human secretin receptor cysteine‐replacement mutants, a broad network of receptor residues could form disulfide bonds consistent with a dynamic ligand‐receptor interface. Two distinct patterns of disulfide bond formation were observed: Cys2 predominantly labeled residues in the amino terminus of ECL2 and ECL3 (relative labeling intensity: Ser340, 94±7%; Pro341, 84±9%; Phe258, 73±5%; Trp274 62±8%), and Cys5 labeled those in the carboxyl terminus of ECL2 and ECL3 (Gln348, 100%; Ile347, 73±12%; Glu342, 59±10%; Phe351, 58±11%). These constraints were utilized in molecular modeling, providing improved understanding of the structure of the transmembrane bundle and interconnecting loops, the orientation between receptor domains, and the molecular basis of ligand docking. Key spatial approximations between peptide and receptor predicted by this model (H1‐W274, D3‐N268, G4‐F258) were supported by mutagenesis and residue‐residue complementation studies.—Dong, M., Xu, X., Ball, A. M., Makhoul, J. A., Lam, P. C.‐H., Pinon, D. I., Orry, A., Sexton, P. M., Abagyan, R., Miller, L. J. Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping. FASEB J. 26, 5092–5105 (2012). www.fasebj.org

Secretin Occupies a Single Protomer of the Homodimeric Secretin Receptor Complex INSIGHTS FROM PHOTOAFFINITY LABELING STUDIES USING DUAL SITES OF COVALENT ATTACHMENT

The secretin receptor, a prototypic family B G protein-coupled receptor, forms a constitutive homodimeric complex that is stable even in the presence of hormone. Recently, a model of this agonist-bound receptor was built based on high resolution structures reported for amino-terminal domains of other family members. Although this model provided the best solution for all extant data, including 10 photoaffinity labeling constraints, a new such constraint now obtained with a position 16 photolabile probe was inconsistent with this model. As the secretin receptor forms constitutive homodimers, we explored whether secretin might dock across both protomers of the complex, an observation that could also contribute to the negative cooperativity observed. To directly explore this, we prepared six secretin analogue probes that simultaneously incorporated two photolabile benzoylphenylalanines as sites of covalent attachment, in positions known to label distinct receptor subdomains. Each bifunctional probe was a full agonist that labeled the receptor specifically and saturably, with electrophoretic migration consistent with labeling a single protomer of the homodimeric secretin receptor. No band representing radiolabeled receptor dimer was observed with any bifunctional probe. The labeled monomeric receptor bands were cleaved with cyanogen bromide to demonstrate that both of the photolabile benzoylphenylalanines within a single probe had established covalent adducts with a single receptor in the complex. These data are consistent with a model of secretin occupying a single secretin receptor protomer within the homodimeric receptor complex. A new molecular model accommodating all constraints is now proposed.

Molecular basis for binding and subtype selectivity of 1,4-benzodiazepine antagonist ligands of the cholecystokinin receptor.

Background: Allosteric ligands targeting cholecystokinin receptors are needed. Results: Stereochemically distinct iodinated 1,4-benzodiazepine antagonists of type 1 and 2 cholecystokinin receptors dock to analogous intramembranous pockets that have distinct shape and molecular determinants. Conclusion: The geometry of the binding pockets and specific residue interactions are unique for each receptor. Significance: The predictive power of these insights should be useful in the discovery of lead compounds and in their refinement. Allosteric binding pockets in peptide-binding G protein-coupled receptors create opportunities for the development of small molecule drugs with substantial benefits over orthosteric ligands. To gain insights into molecular determinants for this pocket within type 1 and 2 cholecystokinin receptors (CCK1R and CCK2R), we prepared a series of receptor constructs in which six distinct residues in TM2, -3, -6, and -7 were reversed. Two novel iodinated CCK1R- and CCK2R-selective 1,4-benzodiazepine antagonists, differing only in stereochemistry at C3, were used. When all six residues within CCK1R were mutated to corresponding CCK2R residues, benzodiazepine selectivity was reversed, yet peptide binding selectivity was unaffected. Detailed analysis, including observations of gain of function, demonstrated that residues 6.51, 6.52, and 7.39 were most important for binding the CCK1R-selective ligand, whereas residues 2.61 and 7.39 were most important for binding CCK2R-selective ligand, although the effect of substitution of residue 2.61 was likely indirect. Ligand-guided homology modeling was applied to wild type receptors and those reversing benzodiazepine binding selectivity. The models had high predictive power in enriching known receptor-selective ligands from related decoys, indicating a high degree of precision in pocket definition. The benzodiazepines docked in similar poses in both receptors, with C3 urea substituents pointing upward, whereas different stereochemistry at C3 directed the C5 phenyl rings and N1 methyl groups into opposite orientations. The geometry of the binding pockets and specific interactions predicted for ligand docking in these models provide a molecular framework for understanding ligand selectivity at these receptor subtypes. Furthermore, the strong predictive power of these models suggests their usefulness in the discovery of lead compounds and in drug development programs.

Kinesin-1 and mitochondrial motility control by discrimination of structurally equivalent but distinct subdomains in Ran-GTP-binding domains of Ran-binding protein 2

The pleckstrin homology (PH) domain is a versatile fold that mediates a variety of protein–protein and protein–phosphatidylinositol lipid interactions. The Ran-binding protein 2 (RanBP2) contains four interspersed Ran GTPase-binding domains (RBDn = 1–4) with close structural homology to the PH domain of Brutons tyrosine kinase. The RBD2, kinesin-binding domain (KBD) and RBD3 comprise a tripartite domain (R2KR3) of RanBP2 that causes the unfolding, microtubule binding and biphasic activation of kinesin-1, a crucial anterograde motor of mitochondrial motility. However, the interplay between Ran GTPase and R2KR3 of RanBP2 in kinesin-1 activation and mitochondrial motility is elusive. We use structure–function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260 000 mitochondrial-motility-related events to find mutually exclusive subdomains in RBD2 and RBD3 towards Ran GTPase binding, kinesin-1 activation and mitochondrial motility regulation. The RBD2 and RBD3 exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic kinetics of kinesin-1 activation or regulation of mitochondrial motility. Further, KBD alone and R2KR3 stimulate and suppress, respectively, multiple biophysical parameters of mitochondrial motility. The regulation of the bidirectional transport of mitochondria by either KBD or R2KR3 is highly coordinated, because their kinetic effects are accompanied always by changes in mitochondrial motile events of either transport polarity. These studies uncover novel roles in Ran GTPase-independent subdomains of RBD2 and RBD3, and KBD of RanBP2, that confer antagonizing and multi-modal mechanisms of kinesin-1 activation and regulation of mitochondrial motility. These findings open new venues towards the pharmacological harnessing of cooperative and competitive mechanisms regulating kinesins, RanBP2 or mitochondrial motility in disparate human disorders.

Molecular Basis for Benzodiazepine Agonist Action at the Type 1 Cholecystokinin Receptor

Background: Cholecystokinin receptor type 1 (CCK1R) stimulates satiety. Results: Binding and activity of a CCK1R agonist/CCK2R antagonist are studied at wild-type and chimeric receptors, and ligand-guided model refinement is utilized. Conclusion: The small molecule agonist docking site is distinct from the antagonist site, with benzodiazepines docked with consistent pose, including approximation with Leu7.39. Significance: The molecular model and determinants for small molecule agonist action should facilitate drug development. Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu7.39 that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a “trigger” for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu7.39, whereas the role of this residue was less clear for chemically distinct agonists.