Polo C.-H. Lam

Refinement of Glucagon-like Peptide 1 Docking to Its Intact Receptor Using Mid-region Photolabile Probes and Molecular Modeling

The glucagon-like peptide 1 (GLP1) receptor is an important drug target within the B family of G protein-coupled receptors. Its natural agonist ligand, GLP1, has incretin-like actions and the receptor is a recognized target for management of type 2 diabetes mellitus. Despite recent solution of the structure of the amino terminus of the GLP1 receptor and several close family members, the molecular basis for GLP1 binding to and activation of the intact receptor remains unclear. We previously demonstrated molecular approximations between amino- and carboxyl-terminal residues of GLP1 and its receptor. In this work, we study spatial approximations with the mid-region of this peptide to gain insights into the orientation of the intact receptor and the ligand-receptor complex. We have prepared two new photolabile probes incorporating a p-benzoyl-l-phenylalanine into positions 16 and 20 of GLP1(7–36). Both probes bound to the GLP1 receptor specifically and with high affinity. These were each fully efficacious agonists, stimulating cAMP accumulation in receptor-bearing CHO cells in a concentration-dependent manner. Each probe specifically labeled a single receptor site. Protease cleavage and radiochemical sequencing identified receptor residue Leu141 above transmembrane segment one as its site of labeling for the position 16 probe, whereas the position 20 probe labeled receptor residue Trp297 within the second extracellular loop. Establishing ligand residue approximation with this loop region is unique among family members and may help to orient the receptor amino-terminal domain relative to its helical bundle region.

Functional Importance of a Structurally Distinct Homodimeric Complex of the Family B G Protein-Coupled Secretin Receptor

Oligomerization of G protein-coupled receptors has been described, but its structural basis and functional importance have been inconsistent. Here, we demonstrate that the agonist occupied wild-type secretin receptor is predominantly in a guanine nucleotide-sensitive high-affinity state and exhibits negative cooperativity, whereas the monomeric receptor is primarily in a guanine nucleotide-insensitive lower affinity state. We previously demonstrated constitutive homodimerization of this receptor through the lipid-exposed face of transmembrane (TM) IV. We now use cysteine-scanning mutagenesis of 14 TM IV residues, bioluminescence resonance energy transfer (BRET), and functional analysis to map spatial approximations and functional importance of specific residues in this complex. All, except for three helix-facing mutants, trafficked to the cell surface, where secretin was shown to bind and elicit cAMP production. Cells expressing complementary-tagged receptors were treated with cuprous phenanthroline to establish disulfide bonds between spatially approximated cysteines. BRET was measured as an indication of receptor oligomerization and was repeated after competitive disruption of oligomers with TM IV peptide to distinguish covalent from noncovalent associations. Although all constructs generated a significant BRET signal, this was disrupted by peptide in all except for single-site mutants replacing five residues with cysteine. Of these, covalent stabilization of receptor homodimers through positions of Gly243, Ile247, and Ala250 resulted in a GTP-sensitive high-affinity state of the receptor, whereas the same procedure with Ala246 and Phe240 mutants resulted in a GTP-insensitive lower affinity state. We propose the existence of a functionally important, structurally specific high-affinity dimeric state of the secretin receptor, which may be typical of family B G protein-coupled receptors.

Molecular approximations between residues 21 and 23 of secretin and its receptor : Development of a model for peptide docking with the amino terminus of the secretin receptor

The structurally unique amino-terminal domain of class II G protein-coupled receptors is critically important for ligand binding and receptor activation. Understanding the precise role it plays requires detailed insights into the molecular basis of its ligand interactions and the conformation of the ligand-receptor complex. In this work, we used two high-affinity, full-agonist, secretin-like photolabile probes having sites for covalent attachment in positions 21 and 23 and used sequential proteolysis and sequencing of the labeled region of the receptor to identify two new spatial approximation constraints. The position 21 probe labeled receptor residue Arg15, whereas the position 23 probe labeled receptor residue Arg21. A homology model of the amino-terminal domain of the secretin receptor was developed using the NMR structure of the analogous domain of the corticotropin-releasing factor receptor. This was attached to a homology model of the secretin receptor transmembrane bundle, with the two domains oriented relative to each other based on continuity of the peptide backbone and by imposing a distance restraint recently identified between the amino-terminal WDN sequence and the region of the helical bundle above transmembrane segment six. Secretin was docked to this model using seven sets of spatial approximation constraints identified in previous photoaffinity labeling studies. This model was found to fully accommodate all existing constraints, as well as the two new approximations identified in this work.

Towards a species-selective acetylcholinesterase inhibitor to control the mosquito vector of malaria, Anopheles gambiae

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.

Spatial Approximation between Secretin Residue Five and the Third Extracellular Loop of Its Receptor Provides New Insight into the Molecular Basis of Natural Agonist Binding

The amino terminus of class II G protein-coupled receptors plays an important role in ligand binding and receptor activation. Understanding of the conformation of the amino-terminal domain of these receptors has been substantially advanced with the solution of nuclear magnetic resonance and crystal structures of this region of receptors for corticotrophin-releasing factor, pituitary adenylate cyclase-activating polypeptide, and gastric inhibitory polypeptide. However, the orientation of the amino terminus relative to the receptor core and how the receptor gets activated upon ligand binding remain unclear. In this work, we have used photoaffinity labeling to identify a critical spatial approximation between residue five of secretin and a residue within the proposed third extracellular loop of the secretin receptor. This was achieved by purification, deglycosylation, cyanogen bromide cleavage, and sequencing of labeled wild-type and mutant secretin receptors. This constraint has been used to refine our evolving molecular model of secretin docked at the intact receptor, which for the first time includes refined helical bundle and loop regions and reflects a peptide-binding groove within the receptor amino terminus that directs the amino terminus of the peptide toward the receptor body. This model is fully consistent with the endogenous agonist mechanism for class II G protein-coupled receptor activation, where ligand binding promotes the interaction of a portion of the receptor amino terminus with the receptor body to activate it.

Triazole-linked reduced amide isosteres: an approach for the fragment-based drug discovery of anti-Alzheimer's BACE1 inhibitors.

In the course of a β-site APP-cleaving enzyme 1 (BACE1) inhibitor discovery project an in situ synthesis/screening protocol was employed to prepare 120 triazole-linked reduced amide isostere inhibitors. Among these compounds, four showed modest (single digit micromolar) BACE1 inhibition. Our ligand design was based on a potent reduced amide isostere 1, wherein the P(2) amide moiety was replaced with an anti-1,2,3-triazole unit. Unfortunately, this replacement resulted in a 1000-fold decrease in potency. Docking studies of triazole-linked reduced amide isostere A3Z10 and potent oxadiazole-linked tertiary carbinamine 2a with BACE1 suggests that the docking poses of A3Z10 and 2a in the active sites are quite similar, with one exception. In the docked structures the placement of the protonated amine that engages D228 differs considerably between 2a and A3Z10. This difference could account for the lower BACE1 inhibition potency of A3Z10 and related compounds relative to 2a.

Fluorescence resonance energy transfer analysis of secretin docking to its receptor: mapping distances between residues distributed throughout the ligand pharmacophore and distinct receptor residues.

Full structural characterization of G protein-coupled receptors has been limited to rhodopsin, with its uniquely stable structure and ability to be crystallized. For other members of this important superfamily, direct structural insights have been limited to NMR structures of soluble domains. Two members of the Class II family have recently had the structures of their isolated amino-terminal regions solved by NMR, yet it remains unclear how that domain is aligned with the heptahelical transmembrane bundle domain of those receptors. Indeed, three distinct orientations have been suggested for different members of this family. In the current work, we have utilized fluorescence resonance energy transfer to establish the distances between four residues distributed throughout fully biologically active, high affinity analogues of secretin and distinct residues in each of four extracellular regions of the intact secretin receptor. These 16 distance constraints were utilized along with nine photoaffinity labeling spatial approximation constraints to study the three proposed orientations of the peptide-binding amino terminus and helical bundle domains of this receptor. In the best model, the carboxyl terminus of secretin was found to bind in a groove above the β-hairpin region of the receptor amino terminus, with its amino-terminal end adjacent to the third extracellular loop and top of transmembrane segment VI. This refined model of the intact receptor was also fully consistent with the spatial approximation of the Trp48-Asp49-Asn50 endogenous agonist segment with the third extracellular loop region that it has been shown to photolabel. This provides strong evidence for the orientation of peptide-binding and signaling domains of a prototypic Class II G protein-coupled receptor.

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.