Total chemical synthesis by native chemical ligation of the all-D immunoglobulin-like domain 2 of Axl

Axl is a tyrosine kinases receptor playing crucial role in several cellular responses. The deregulation of Axl signaling has been associated to many high impact diseases ranging from cancer to multiple sclerosis. We report the successful procedure for the chemical synthesis of the Ig2 domain of Axl, one of the key extracellular regions of the receptor involved in ligand binding. The protein was synthesized in its d-enantiomeric form (D-Axl-2), opening the way to the selection of D-peptides selectively targeting Axl receptor through the mirror-image phage display peptide library screening approach.     

Circulating IgSF Proteins Inhibit Adhesion of Antibody Targeted Microspheres to Endothelial Inflammatory Ligands

Proposed methods for detecting circulatory system disease include targeting ultrasound contrast agents to inflammatory markers on vascular endothelial cells. For antibody-based therapies, soluble forms of the targeted adhesion proteins of the immunoglobulin superfamily (IgSF) reduce adhesion yet were left unaccounted in prior reports. Microspheres labeled simply with a maximum level of antibodies can reduce the diagnostic sensitivity by adhering to proteins expressed normally at a low level, while sparsely coated particles may be rendered ineffective by circulating soluble forms of the targeted proteins. A new microdevice technique is applied to simultaneously measure the adhesion profile to a series of IgSF-protein-coated surfaces. In this investigation, we quantify the in vitro binding characteristics of 5-μm microspheres to oriented intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) protein-coated surfaces in the presence of human serum at physiological concentrations. Defined regions of a slide were coated with recombinant chimeric Fc-human ICAM-1 and VCAM-1 in variable ratios but constant total concentration. Monoclonal human anti-ICAM-1 or anti-VCAM-1 antibodies in competition with non-binding mouse anti-rabbit antibodies coat the microsphere surface at a constant surface density with variable yet controlled surface activities. Using multiple slide surface IgSF protein and microsphere antibody concentrations, an adhesion profile was developed for the microspheres with and without IgSF proteins from human serum, which demonstrated that exposure to serum reduced microsphere binding, on average, more than 50% compared to the no-serum condition.. The serum effects were limited to antibodies on the microsphere, since binding inhibition was reversed after rinsing serum from the system and fresh antibody-coated microspheres were introduced. This analysis quantifies the binding effects of soluble IgSF proteins from human serum on antibody-based targeted ultrasound detection and drug delivery methods.     

Predicting the effects of amino acid replacements in peptide hormones on their binding affinities for class B GPCRs and application to the design of secretin receptor antagonists

Computational prediction of the effects of residue changes on peptide-protein binding affinities, followed by experimental testing of the top predicted binders, is an efficient strategy for the rational structure-based design of peptide inhibitors. In this study we apply this approach to the discovery of competitive antagonists for the secretin receptor, the prototypical member of class B G protein-coupled receptors (GPCRs). Proteins in this family are involved in peptide hormone-stimulated signaling and are implicated in several human diseases, making them potential therapeutic targets. We first validated our computational method by predicting changes in the binding affinities of several peptides to their cognate class B GPCRs due to alanine replacement and compared the results with previously published experimental values. Overall, the results showed a significant correlation between the predicted and experimental ΔΔG values. Next, we identified candidate inhibitors by applying this method to a homology model of the secretin receptor bound to an N-terminal truncated secretin peptide. Predictions were made for single residue replacements to each of the other nineteen naturally occurring amino acids at peptide residues within the segment binding the receptor N-terminal domain. Amino acid replacements predicted to most enhance receptor binding were then experimentally tested by competition-binding assays. We found two residue changes that improved binding affinities by almost one log unit. Furthermore, a peptide combining both of these favorable modifications resulted in an almost two log unit improvement in binding affinity, demonstrating the approximately additive effect of these changes on binding. In order to further investigate possible physical effects of these residue changes on receptor binding affinity, molecular dynamics simulations were performed on representatives of the successful peptide analogues (namely A17I, G25R, and A17I/G25R) in bound and unbound forms. These simulations suggested that a combination of the α-helical propensity of the unbound peptide and specific interactions between the peptide and the receptor extracellular domain contribute to their higher binding affinities.     

The effect of antagonists on the conformational exchange of the retinoid X receptor alpha ligand‐binding domain

The effect of retinoid X receptor (RXR) antagonists on the conformational exchange of the RXR ligand‐binding domain (LBD) remains poorly characterized. To address this question, we used nuclear magnetic resonance spectroscopy to compare the chemical shift perturbations induced by RXR antagonists and agonists on the RXRα LBD when partnered with itself as a homodimer and as the heterodimeric partner with the peroxisome proliferator‐activated receptor γ (PPARγ) LBD. Chemical shift mapping on the crystal structure showed that agonist binding abolished a line‐broadening effect caused by a conformational exchange on backbone amide signals for residues in helix H3 and other regions of either the homo‐ or hetero‐dimer, whereas binding of antagonists with similar binding affinities failed to do so. A lineshape analysis of a glucocorticoid receptor‐interacting protein 1 NR box 2 coactivator peptide showed that the antagonists enhanced peptide binding to the RXRα LBD homodimer, but to a lesser extent than that enhanced by the agonists. This was further supported by a lineshape analysis of the RXR C‐terminal residue, threonine 462 (T462) in the homodimer but not in the heterodimer. Contrary to the agonists, the antagonists failed to abolish a line‐broadening effect caused by a conformational exchange on the T462 signal corresponding to the RXRα LBD–antagonist–peptide ternary complex. These results suggest that the antagonists lack the ability of the agonists to shift the equilibrium of multiple RXRα LBD conformations in favor of a compact state, and that a PPARγ LBD‐agonist complex can prevent the antagonist from enhancing the RXRα LBD‐coactivator binding interaction. Copyright © 2009 John Wiley & Sons, Ltd.     

基于结构和药效团特征的人类腺苷受体拮抗剂选择性比较

腺苷受体是重要的治疗靶标,选择性腺苷受体拮抗剂具有广泛的临床应用前景.本文通过同源模建构建了腺苷A₁、A_2B和A₃受体的结构,采用LigandScout 3.12软件分别构建了腺苷受体四种亚型的拮抗剂药效团模型.然后利用Schrödinger程序中的Induced Fit Docking模块完成受体-拮抗剂结合模式的预测,并与药效团结果进行比对.结果发现,由于结合口袋部位的残基在家族间高度保守,模建得到的各个亚型受体的初始结构活性口袋部位极为相似,无法用于亚型选择性拮抗剂的识别.而腺苷受体四种亚型拮抗剂药效团的药效特征与空间排布都不同,并与以前突变实验信息相吻合.研究结果说明,结合口袋部位的优化是模建中的关键步骤,基于配体的药效团模型所包含的一系列药效特征元素如氢键受体、氢键供体、疏水基团、芳环中心,可以很好地表征受体结合部位氢键、疏水空腔的位置及其方向.本文研究结果可以为进一步的优化同源模建结果,寻找新型的人类腺苷受体选择性拮抗剂提供理论依据.     

Protein ligand design: from phage display to synthetic protein epitope mimetics in human antibody Fc-binding peptidomimetics

Phage display is a powerful method for selecting peptides with novel binding functions. Synthetic peptidomimetic chemistry is a powerful tool for creating structural diversity in ligands as a means to establish structure-activity relationships. Here we illustrate a method of bridging these two methodologies, by starting with a disulfide bridged phage display peptide which binds a human antibody Fc fragment (Delano et al. Science 2000, 287, 1279) and creating a backbone cyclic beta-hairpin peptidomimetic with 80-fold higher affinity for the Fc domain. The peptidomimetic is shown to adopt a well-defined beta-hairpin conformation in aqueous solution, with a bulge in one beta-strand, as seen in the crystal structure of the phage peptide bound to the Fc domain. The higher binding affinity of the peptidomimetic presumably reflects the effect of constraining the free ligand into the conformation required for binding, thus highlighting in this example the influence that ligand flexibility has on the binding energy. Since phage display peptides against a wide variety of different proteins are now accessible, this approach to synthetic ligand design might be applied to many other medicinally and biotechnologically interesting target proteins.     

Molecular dynamics simulations for human CAR inverse agonists

Constitutive androstane receptor (CAR), along with pregnane x receptor (PXR), is an important metabolic sensor in the hepatocytes. Like all other nuclear receptors (NRs), CAR works in concert with coregulator proteins, coactivators, and corepressors which bind to the NRs. The main basis for the receptor to distinguish between coactivators and corepressors is the position of the C-terminal helix 12 (H12), which is determined by the bound NR ligand. CAR, having constitutive activity, can be repressed or further activated by its ligands. Crystal structure of human CAR bound to an agonist and a coactivator peptide is available, but no structural information on an inverse agonist-bound human CAR and a corepressor exists. In our previous molecular dynamics (MD) studies, no corepressor peptide was included. Therefore, probably due to the strong interactions which keep the relatively short H12 of CAR in the active position, the structural changes elicited by inverse agonists were very subtle, and H12 of CAR seemed to more or less retain its active conformation. Here, we have run a series of MD simulations to study the movement of H12 in the presence of both activating and repressing ligands as well as a corepressor peptide. The presence of the corepressor on the coregulator surface of CAR induced a clear shift of H12 of the inverse agonists-bound CAR. In general, H12 moved toward H10 and not away from the ligand binding domain, as seen in some other NRs. However, H12 of CAR is short enough that this movement seems to be adequate to accommodate the binding of the corepressor.     

Targeting metastable coiled-coil domains by computational design

Approximately 30% of eukaryotic genomes are predicted to encode partially unfolded proteins. Many of these unstructured domains contact multiple partners in short-lived interactions critical for cellular homeostasis. Understanding the functional implications of these transient binding events is a current challenge that could be addressed with designed peptide inhibitors. Most current protein design methodologies, however, target only structurally well-defined, stable structures. To address this limitation, we implemented a computational design strategy that alternates between a fixed backbone sequence search for binding specificity and structural optimization of the designed interfaces. We applied this method to create specific peptide inhibitors of the C-terminal metastable coiled-coil domain of the essential yeast septin Cdc12p. Specific binding of the designed sequences was demonstrated by circular dichroism and equilibrium ultracentrifugation. Our results validate computational methods to design specific peptide ligands to protein domains lacking intrinsic structural stability and set the stage for functional analysis of Cdc12p coiled coil function in vivo.     

Ligand-guided optimization of CXCR4 homology models for virtual screening using a multiple chemotype approach

CXCR4 is a G-protein coupled receptor for CXCL12 that plays an important role in human immunodeficiency virus infection, cancer growth and metastasization, immune cell trafficking and WHIM syndrome. In the absence of an X-ray crystal structure, theoretical modeling of the CXCR4 receptor remains an important tool for structure–function analysis and to guide the discovery of new antagonists with potential clinical use. In this study, the combination of experimental data and molecular modeling approaches allowed the development of optimized ligand-receptor models useful for elucidation of the molecular determinants of small molecule binding and functional antagonism. The ligand-guided homology modeling approach used in this study explicitly re-shaped the CXCR4 binding pocket in order to improve discrimination between known CXCR4 antagonists and random decoys. Refinement based on multiple test-sets with small compounds from single chemotypes provided the best early enrichment performance. These results provide an important tool for structure-based drug design and virtual ligand screening of new CXCR4 antagonists.     

Affinity-guided protein conjugation: the trilogy of covalent protein labeling,assembly and inhibition

Specific and dynamic biological interactions pave the blueprint of signal networks in cell. For example, a great variety of specific protein-ligand interactions define how intracellular signals flow. Taking advantage of the specificity of these interactions, we postulate an “affinity-guided covalent conjugation” strategy to lock binding ligands through covalent reactions between the ligand and the receptor protein. The presence of a nucleophile close to the ligand binding site of a protein is sine qua none of this reaction. Specific noncovalent interaction of a ligand derivative (which contains an electrophile at a designed position) to the ligand binding site of the protein brings the electrophile to the close proximity of the nucleophile. Subsequently, a conjugation reaction spontaneously takes place between the nucleophile and the electrophile, and leads to an intermolecular covalent linkage. This strategy was first showcased in coiled coil peptides which include a cysteine mutation at a selected position. The short peptide sequence was used for covalent labeling of cell surface receptors. The same strategy was then used to guide the design of a set of protein Lego bricks for covalent assembly of protein complexes of unnatural geometry. We finally made “reactive peptides” for natural adaptor proteins that play significant roles in signal transduction. The peptides were designed to react with a single domain of the multidomain adaptor protein, delivered into the cytosol of neurons, and re-directed the intracellular signal of neuronal migration. The trilogy of protein labeling, assembly, and inhibition of intracellular signals, all through a specific covalent bond, fully demonstrated the generality and versatility of “affinity-guided covalent conjugation” in various applications.     

Weak interaction between inhibition peptides and a soluble receptor of fusion protein in the liquid phase.

Fluorescence polarization analysis (FPA) of a liquid-phase method was carried out with a glycosylphosphatidylinositol (GPI) anchored membrane receptor bone marrow stromal cell antigen 1 (BST-1, CD157) as a model receptor for medical screening. A soluble receptor, BST1-Fc, was prepared by fusing the extracellular domain of BST-1 and the Fc region of human immunoglobulin G (IgG). The binding curves of BST1-Fc with a fluorescently labeled ligand peptide, or its three derivatives, were developed using ordinary FPA in the liquid phase. The obtained dissociation constants (Kd) were comparable with those reported as measured with SPR of a solid-phase method, except for one derivative peptide with Kd larger than 7000 nM. Competitive FPA was carried out, and it was demonstrated that a very weak interaction, which would be difficult to detect with SPR or other solid-phase methods, could be analyzed with both ordinary and competitive FPA.     

An improved method for the synthesis of cellulose membrane-bound peptides with free C termini is useful for PDZ domain binding studies

SPOT synthesis permits parallel synthesis and screening of thousands of cellulose membrane-bound peptides to study protein-protein interactions in a proteomic context. Recognition of C-terminal residues is one of the most common binding features of PDZ domains. Unfortunately, most solid support-bound peptide libraries lack a free C terminus due to C-terminal fixation on the solid support. To overcome this restriction, we developed a robust methodology based on our previous strategy for generating peptides with authentic C termini. To validate this improved method, we screened a human peptide library of 6223 C termini with the syntrophin PDZ domain. Furthermore, using the same library, new peptide ligands derived from membrane proteins and receptors were found for the ERBIN PDZ domain. Finally, we identified the protein kinase breakpoint cluster region, which is known as a negative regulator of cell proliferation and oncogenic transformation, as an ERBIN ligand.     

Structural Insights into Ligand—Receptor Interactions Involved in Biased Agonism of G-Protein Coupled Receptors

G protein-coupled receptors (GPCRs) are versatile signaling proteins that mediate complex cellular responses to hormones and neurotransmitters. Ligand directed signaling is observed when agonists, upon binding to the same receptor, trigger significantly different configuration of intracellular events. The current work reviews the structurally defined ligand – receptor interactions that can be related to specific molecular mechanisms of ligand directed signaling across different receptors belonging to class A of GPCRs. Recent advances in GPCR structural biology allow for mapping receptors’ binding sites with residues particularly important in recognition of ligands’ structural features that are responsible for biased signaling. Various studies show particular role of specific residues lining the extended ligand binding domains, biased agonists may alternatively affect their interhelical interactions and flexibility what can be translated into intracellular loop rearrangements. Studies on opioid and angiotensin receptors indicate importance of residues located deeper within the binding cavity and direct interactions with receptor residues linking the ortosteric ligand binding site with the intracellular transducer binding domain. Collection of results across different receptors may suggest elements of common molecular mechanisms which are responsible for passing alternative signals from biased agonists.     

Improvement of Ligand Affinity and Thermodynamic Properties by NMR‐Based Evaluation of Local Dynamics and Surface Complementarity in the Receptor‐Bound State

The thermodynamic properties of a ligand in the bound state affect its binding specificity. Strict binding specificity can be achieved by introducing multiple spatially defined interactions, such as hydrogen bonds and van der Waals interactions, into the ligand–receptor interface. These introduced interactions are characterized by restricted local dynamics and improved surface complementarity in the bound state. In this study, we experimentally evaluated the local dynamics and the surface complementarity of weak‐affinity ligands in the receptor‐bound state by forbidden coherence transfer analysis in free‐bound exchange systems (Ex‐FCT), using the interaction between a ligand, a myocyte‐enhancer factor 2A (MEF2A) docking peptide, and a receptor, p38α, as a model system. The Ex‐FCT analyses successfully provided information for the rational design of a ligand with higher affinity and preferable thermodynamic properties for p38α.     

Thermodynamic basis for promiscuity and selectivity in protein-protein interactions: PDZ domains, a case study

Like other protein-protein interaction domains, PDZ domains are involved in many key cellular processes. These processes often require that specific multiprotein complexes be assembled, a task that PDZ domains accomplish by binding to specific peptide motifs in target proteins. However, a growing number of experimental studies show that PDZ domains (like other protein-protein interaction domains) can engage in a variety of interactions and bind distinct peptide motifs. Such promiscuity in ligand recognition raises intriguing questions about the molecular and thermodynamic mechanisms that can sustain it. To identify possible sources of promiscuity and selectivity underlying PDZ domain interactions, we performed molecular dynamics simulations of 20 to 25 ns on a set of 12 different PDZ domain complexes (for the proteins PSD-95, Syntenin, Erbin, GRIP, NHERF, Inad, Dishevelled, and Shank). The electrostatic, nonpolar, and configurational entropy binding contributions were evaluated using the MM/PBSA method combined with a quasi-harmonic analysis. The results revealed that PDZ domain interactions are characterized by overwhelmingly favorable nonpolar contributions and almost negligible electrostatic components, a mix that may readily sustain promiscuity. In addition, despite the structural similarity in fold and in recognition modes, the entropic and other dynamical aspects of binding were remarkably variable not only across PDZ domains but also for the same PDZ domain bound to distinct ligands. This variability suggests that entropic and dynamical components can play a role in determining selectivity either of PDZ domain interactions with peptide ligands or of PDZ domain complexes with downstream effectors.     

Rewiring cell adhesion

The adhesion of cells is mediated by the binding of several cell-surface receptors to ligands found in the extracellular matrix. These receptors often have overlapping specificities for the peptide ligands, making it difficult to understand the roles for discrete receptors in cell adhesion, migration, and differentiation as well as to direct the selective adhesion of cell types in tissue-engineering applications. To overcome these limitations, we developed a strategy to rewire the receptor-ligand interactions between a cell and substrate to ensure that adhesion is mediated by a single receptor with unique specificity. The strategy combines a genetic approach to engineer the cell surface with a chimeric integrin receptor having a unique ligand binding domain with a surface chemistry approach to prepare substrates that present ligands that are bound by the new binding domain. We show that Chinese hamster ovary cells that are engineered with a chimeric beta1 integrin adhere, signal, and even migrate on a synthetic matrix.     

Reversed-phase liquid chromatography as a tool in the determination of the hydrophilicity/hydrophobicity of amino acid side-chains at a ligand-receptor interface in the presence of different aqueous environments. I. Effect of varying receptor hydrophobicity

We have developed further a chromatographic model for studying the hydrophobic interactions which characterize the way a ligand binds to its receptor. This model is based on observing the retention behaviour of de novo designed model 18-residue amphipathic alpha-helical peptides (representing the hydrophobic binding domain of a ligand) on reversed-phase packings by varying hydrophobicity (representing a receptor protein with a hydrophobic binding pocket). Mutants of the "native" peptide ligand (which contains seven Leu residues in its non-polar face) were designed by replacing one residue in the center of the extremely non-polar face of the amphipathic alpha-helix. Through reversed-phase liquid chromatography of these peptides at pH 2.0 on cyano and C18 columns, we have demonstrated how an increase in receptor hydrophobicity (represented by an increase in column stationary phase hydrophobicity; cyano --> C18) significantly enhances hydrophilicity of polar amino acid side-chains at the ligand-receptor interface while moderately enhancing the hydrophobicity of non-polar side-chains. The addition of salt (100 mM sodium perchlorate) to the aqueous environment surrounding the binding site of receptor and ligand was also shown to have a profound effect on side-chain hydrophilicity/hydrophobicity in the binding interface. This effect was particularly dramatic for the positively charged side-chains Arg, Lys and His, whose significant enhancement of hydrophobicity in the presence of the cyano column contrasted with their increase in hydrophilicity in the presence of the considerably more hydrophobic C18 stationary phase. Our results have major implications to understanding the influence of hydrophobic and aqueous environment on hydrophilicity/hydrophobicity of amino acid side-chains and the role side-chains play in the folding and stability of proteins.     

Development of selective high affinity antagonists, agonists, and radioligands for the P2Y1 receptor

The P2Y(1) receptor is a member of the P2Y family of nucleotide-activated G protein-coupled receptors, and it is an important therapeutic target based on its broad tissue distribution and essential role in platelet aggregation. We have designed a set of highly selective and diverse pharmacological tools for studying the P2Y(1) receptor using a rational approach to ligand design. Based on the discovery that bisphosphate analogues of the P2Y(1) receptor agonist, ADP, are partial agonists/competitive antagonists of this receptor, an iterative approach was used to develop competitive antagonists with enhanced affinity and selectivity. Halogen substitutions of the 2-position of the adenine ring provided increased affinity while an N(6) methyl substitution eliminated partial agonist activity. Furthermore, various replacements of the ribose ring with symmetrically branched, phosphorylated acyclic structures revealed that the ribose is not necessary for recognition at the P2Y(1) receptor. Finally, replacement of the ribose ring with a five member methanocarba ring constrained in the Northern conformation conferred dramatic increases in affinity to both P2Y(1) receptor antagonists as well as agonists. These combined structural modifications have resulted in a series of selective high affinity antagonists of the P2Y(1) receptor, two broadly applicable radioligands, and a high affinity agonist capable of selectively activating the P2Y(1) receptor in human platelets. Complementary receptor modeling and computational ligand docking have provided a putative structural framework for the drug-receptor interactions. A similar rational approach is being applied to develop selective ligands for other subtypes of P2Y receptors.     

Multimerization of an Apoptogenic TRAIL‐Mimicking Peptide by Using Adamantane‐Based Dendrons

We have developed a straightforward strategy to multimerize an apoptogenic peptide that mimics the natural tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) by using adamantane‐based dendrons as multivalent scaffolds. The selective binding affinity of the ligands to TRAIL receptor 2 (TR2) was studied by surface plasmon resonance, thus demonstrating that the trimeric and hexameric forms of the peptide exert an increased affinity of about 1500‐ and 20 000‐fold, respectively, relative to the monomer. Moreover, only the trimeric and hexameric ligands were able to induce cell death in TR2 expressing cells (BJAB), thus confirming that a multivalent form of the peptide is necessary to trigger a substantial TR2‐dependent apoptotic response in vitro. These results provide interesting insight into the multivalency effect on biological ligand/receptor interactions for future therapeutic applications.