The role of water in computational and experimental derivation of binding thermodynamics in SH2 domains

We have studied the role of bound interface water molecules on the prediction of the thermodynamics of SH2 domain binding to tyrosyl phosphopeptides using a method based on accessible surface area buried upon association. We studied three phosphopeptide ligands, which have been shown by Lubman and Waksman (J Mol Biol;328:655, 2003) and Davidson et al. (JACS;124:205, 2002) to have similar binding free energies but very different thermodynamic signatures. The thermodynamic model is semiempirical and applies to the crystal structure of the SH2 domain-bound forms. We explored all possible combinations of bound interfacial waters. We show that the model does not predict the binding thermodynamics of either ligand. However, we identified the empirical formula describing the heat capacity change as the source of the problem. Indeed, systematic exploration of heat capacity change values between 0 and -300 cal/mol deg results in a sharp distribution of the number of ligand/SH2/water-subset structures that provide binding thermodynamics similar to experimental values. The heat capacity change values at which the distributions peak are different for each peptide. This prompted us to experimentally determine the heat capacity change for each of the peptides and we found them to coincide with the values of the peaks. The implications of such findings are discussed.     

Identification and specificity studies of small-molecule ligands for SH3 protein domains

The Src Homology 3 (SH3) domains are small protein-protein interaction domains that bind proline-rich sequences and mediate a wide range of cell-signaling and other important biological processes. Since deregulated signaling pathways form the basis of many human diseases, the SH3 domains have been attractive targets for novel therapeutics. High-affinity ligands for SH3 domains have been designed; however, these have all been peptide-based and no examples of entirely nonpeptide SH3 ligands have previously been reported. Using the mouse Tec Kinase SH3 domain as a model system for structure-based ligand design, we have identified several simple heterocyclic compounds that selectively bind to the Tec SH3 domain. Using a combination of nuclear magnetic resonance chemical shift perturbation, structure-activity relationships, and site-directed mutagenesis, the binding of these compounds at the proline-rich peptide-binding site has been characterized. The most potent of these, 2-aminoquinoline, bound with Kd = 125 microM and was able to compete for binding with a proline-rich peptide. Synthesis of 6-substituted-2-aminoquinolines resulted in ligands with up to 6-fold improved affinity over 2-aminoquinoline and enhanced specificity for the Tec SH3 domain. Therefore, 2-aminoquinolines may potentially be useful for the development of high affinity small molecule ligands for SH3 domains.     

Surface plasmon resonance thermodynamic and kinetic analysis as a strategic tool in drug design. Distinct ways for phosphopeptides to plug into Src- and Grb2 SH2 domains

Thermodynamic and kinetic studies of biomolecular interactions give insight into specificity of molecular recognition processes and advance rational drug design. Binding of phosphotyrosine (pY)-containing peptides to Src- and Grb2-SH2 domains was investigated using a surface plasmon resonance (SPR)-based method. This SPR assay yielded thermodynamic binding constants in solution, and the kinetic information contained in the SPR signal allowed kinetic analysis, which demonstrated distinct ways for pY ligands to interact with the SH2 domains. The results for binding to Src SH2 were consistent with sequestration of water molecules in the interface of the pYEEI peptide/Src SH2 complex. The results for a pYVNV peptide binding to Grb2 SH2 suggested a conformational change for Grb2 SH2 upon binding, which is not observed for Src SH2. Binding of a cyclic construct, allowing the pYVNV sequence in the bound conformation, did not have the expected entropy advantage. The results suggest an alternative binding mode for this construct, with the hydrophobic ring-closing part interacting with the protein. In all cases, except for full-length Grb2 protein, the affinity for the immobilized peptide at the SPR sensor and in solution was identical. This study demonstrates that SPR thermodynamic and kinetic analysis is a useful strategic tool in drug design.     

Hierarchy of simulation models in predicting structure and energetics of the Src SH2 domain binding to tyrosyl phosphopeptides.

Structure and energetics of the Src Src Homology 2 (SH2) domain binding with the recognition phosphopeptide pYEEI and its mutants are studied by a hierarchical computational approach. The proposed structure prediction strategy includes equilibrium sampling of the peptide conformational space by simulated tempering dynamics with the simplified, knowledge-based energy function, followed by structural clustering of the resulting conformations and binding free energy evaluation of a single representative from each cluster, a cluster center. This protocol is robust in rapid screening of low-energy conformations and recovers the crystal structure of the pYEEI peptide. Thermodynamics of the peptide-SH2 domain binding is analyzed by computing the average energy contributions over conformations from the clusters, structurally similar to the predicted peptide bound structure. Using this approach, the binding thermodynamics for a panel of studied peptides is predicted in a better agreement with the experiment than previously suggested models. However, the overall correlation between computed and experimental binding affinity remains rather modest. The results of this study show that small differences in binding free energies between the Ala and Gly mutants of the pYEEI peptide are considerably more difficult to predict than the structure of the bound peptides, indicating that accurate computational prediction of binding affinities still remains a major methodological and technical challenge.     

SAR and X-ray. A new approach combining fragment-based screening and rational drug design: application to the discovery of nanomolar inhibitors of Src SH2

(pp60)Src is a protein involved in signal transduction and is mainly expressed in neurones, platelets, and osteoclasts. Its precise biological role was recently discovered with the KO experiments by Soriano that gave rise to no other apparent phenotype than osteopetrosis, a disease resulting in excedent bone formation. The SH2 domain of the Src family specifically recognizes a sequence of tetrapeptide featuring a phosphotyrosine and a lipophilic aminoacid at the +1 and +3 positions. Recently we engaged in the search for SH2 ligands via modular peptidomimicry of this tetrapetide. This gave rise to several families of nanomolar inhibitors; the best one incorporated a caprolactam scaffold, a biphenyl moiety, and a phosphotyrosine. However, these inhibitors still incorporated the phosphate group that confers good binding affinity to the protein. Phosphates have undesirable features for drug candidates, namely, high rate of hydrolysis of the phosphate group by phosphatases and high charge content precluding cell penetration. Therefore, while searching for optimal non-peptide ligands for Src SH2, we looked for phosphate replacements. For this, we have designed an SAR by fragment crystallography approach. The start of this work resulted from two experimental observations. First, the fact that phenyl phosphate itself displayed detectable binding affinity for Src SH2 permitted us to perform a screening of small aromatic compounds as phenyl phosphate surrogates. Second, the obtention of large Src SH2 crystals displaying a channel large enough for soaking purposes allowed structure determination of over 40 of these small aromatic compounds bound in the phosphotyrosine binding pocket. This search and the way it gave rise to low nanomolar range Src SH2 inhibitors devoid of phosphate groups will be the subject of the present paper.     

Allosteric Binding Site and Activation Mechanism of Class C G-Protein Coupled Receptors: Metabotropic Glutamate Receptor Family

Metabotropic glutamate receptors (mGluR) are mainly expressed in the central nervous system (CNS) and contain eight receptor subtypes, named mGluR1 to mGluR8. The crystal structures of mGluR1 and mGluR5 that are bound with the negative allosteric modulator (NAM) were reported recently. These structures provide a basic model for all class C of G-protein coupled receptors (GPCRs) and may aid in the design of new allosteric modulators for the treatment of CNS disorders. However, these structures are only combined with NAMs in the previous reports. The conformations that are bound with positive allosteric modulator (PAM) or agonist of mGluR1/5 remain unknown. Moreover, the structural information of the other six mGluRs and the comparisons of the mGluRs family have not been explored in terms of their binding pockets, the binding modes of different compounds, and important binding residues. With these crystal structures as the starting point, we built 3D structural models for six mGluRs by using homology modeling and molecular dynamics (MD) simulations. We systematically compared their allosteric binding sites/pockets, the important residues, and the selective residues by using a series of comparable dockings with both the NAM and the PAM. Our results show that several residues played important roles for the receptors’ selectivity. The observations of detailed interactions between compounds and their correspondent receptors are congruent with the specificity and potency of derivatives or compounds bioassayed in vitro. We then carried out 100 ns MD simulations of mGluR5 (residue 26-832, formed by Venus Flytrap domain, a so-called cysteine-rich domain, and 7 trans-membrane domains) bound with antagonist/NAM and with agonist/PAM. Our results show that both the NAM and the PAM seemed stable in class C GPCRs during the MD. However, the movements of “ionic lock,” of trans-membrane domains, and of some activation-related residues in 7 trans-membrane domains of mGluR5 were congruent with the findings in class A GPCRs. Finally, we selected nine representative bound structures to perform 30 ns MD simulations for validating the stabilities of interactions, respectively. All these bound structures kept stable during the MD simulations, indicating that the binding poses in this present work are reasonable. We provided new insight into better understanding of the structural and functional roles of the mGluRs family and facilitated the future structure-based design of novel ligands of mGluRs family with therapeutic potential.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-015-9742-8) contains supplementary material, which is available to authorized users.KEY WORDS: allosteric, class C of G protein-coupled receptors, homology model, metabotropic glutamate receptor family, molecular dynamics simulation     

A Discovery Strategy for Selective Inhibitors of c‐Src in Complex with the Focal Adhesion Kinase SH3/SH2‐binding Region

The c‐Src tyrosine kinase co‐operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c‐Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c‐Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3‐SH2 docking sequence, we screened a kinase‐biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c‐Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH‐4‐124‐2, with nanomolar inhibitory potency and fivefold selectivity for c‐Src when bound to the phospho‐focal adhesion kinase peptide. Molecular docking studies indicate that WH‐4‐124‐2 may preferentially inhibit the ‘DFG‐out’ conformation of the kinase active site. These findings suggest that interaction of c‐Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition.     

Structure-based design of compounds inhibiting Grb2-SH2 mediated protein-protein interactions in signal transduction pathways

Receptor protein tyrosine kinases are usually activated upon binding their growth factors, or other suitable ligands, to their extracellular domains. These activated receptors initiate cytoplasmic signalling cascades which, when aberrant, can result in different disease states, such as oncogenic transformation. Many receptor protein tyrosine kinases use Src homology 2 domains (SH2) to couple growth factor activation with intracellular signalling pathways to mediate cell control and other biological events. The characterization of the components involved in these signal transduction pathways has resulted in the identification of new attractive targets for therapeutic intervention. Such is the case for the protein-protein interactions involving the SH2 domain of growth factor receptor bound protein 2 (Grb2). Agents that specifically disrupt Grb2-SH2 binding interactions involved in aberrant signalling could potentially shut down these oncogenic pathways and thus block human malignancies. This paper reviews the structural characteristics of the Grb2-SH2 domain and the approaches which have been used to identify antagonists of the Grb2-SH2 domain. Examples have been selected from our own research to illustrate how the unique structural features of the ligand-bound Grb2-SH2 have been exploited to design potent and selective Grb2-SH2 antagonists.     

Conformationally constrained peptide analogues of pTyr-Glu-Glu-Ile as inhibitors of the Src SH2 domain binding

A series of conformationally constrained peptides were designed and synthesized as the Src SH2 domain ligands based on a tetrapeptide sequence pTyr-Glu-Glu-Ile (pYEEI). In general, the constrained peptides such as compounds 6, 7, and 11 (IC(50) = 1.1-1.5 microM) showed higher binding affinities to the Src SH2 domain relative to the corresponding linear peptides 8a, 9a, and 13a, respectively (IC(50) > 100 microM), and pYEEI (IC(50) = 6.5 microM), as evaluated by a fluorescence polarization assay. Molecular modeling studies revealed that in constrained peptides, the isoleucine side chain penetrates very deeply into the hydrophobic binding pocket (P + 3 site) of the Src SH2 domain. These constrained peptides can serve as novel templates for the design of small and nonpeptidic inhibitors of the Src SH2 domain.     

Phosphoinositide, phosphopeptide and pyridone interactions of the Abl SH2 domain

Signaling proteins are localized and regulated by Src homology 2 domains which recognize phosphotyrosine-containing sequences. Recently, noncanonical ligands have been proposed for Src homology 2 domains including that of Abl and its breakpoint cluster region fusion, which causes chronic myelogenous leukemia. Here, the Abl Src homology 2 domain's binding sites and affinities for phosphotyrosine- and phosphoserine-containing motifs, phosphoinositides as well as a pyridone-based peptidomimetic inhibitor were determined using nuclear magnetic resonance spectroscopy in order to define their roles. The cognate Crk peptide ligand was bound with an affinity of 69 microM and, like the higher affinity peptidomimetic, engages the phosphotyrosine and +3 hydrophobic pockets while putative phosphoserine-containing breakpoint cluster region ligands are ruled out. Surprisingly, phosphatidylinositol 4, 5 bisphosphate interacts with an overlapping site through an electrostatic mechanism that does not appear to involve hydrophobic insertion into micelles. The conserved Arg36 residue in the FLVRES motif is required for both phosphotyrosine binding and for localization to phosphatidylinositol 4, 5 bisphosphate-containing liposomes, while Arg59 in the betaD strand is necessary for the phosphoinositide interaction. Thus the Src homology 2 domain of Abl, a myristoylated and membrane-localized protein, is able to interact directly with phosphoinositides through a multifunctional basic site that overlaps the phosphotyrosine pocket.     

Molecular recognition properties of the C-terminal Sh3 domain of the Cbl associated protein,Cap

Abstract: A phage-displayed combinatorial peptide library was used to define the specificity of one of the three Src homology 3 (SH3) domains in a novel cytoskeletal protein, named CAP, for Cbl Associated Protein. The C-terminal SH3 domain was used to affinity select peptides with the consensus, PXPPXRXSSL, from a library of X₆PXXPX₆ peptides. Peptide sequences resembling this consensus were identified in two signal transduction proteins, c-Cbl and son-on-sevenless (Sos), previously shown to interact with the C-terminal SH3 domain of CAP. Genetic fusion of 16 and 14 amino acid segments of c-Cbl and Sos, respectively, to bacterial alkaline phosphatase confirmed that these segments were potential ligand sites for the C-terminal SH3 domain of CAP. Alanine-scanning mutagenesis of the c-Cbl peptide ligand confirmed that most of the residues, which were conserved among the peptide ligands selected from the combinatorial peptide library, contributed to binding to the C-terminal SH3 domain of CAP.     

Identification of non-phosphate-containing small molecular weight inhibitors of the tyrosine kinase p56 Lck SH2 domain via in silico screening against the pY + 3 binding site

The protein p56 lymphoid T cell tyrosine kinase (Lck) is predominantly expressed in T lymphocytes where it plays a critical role in T-cell-mediated immune response. Lck participates in phosphotyrosine-dependent protein-protein interactions through its modular binding unit, the Src homology-2 (SH2) domain. Accordingly, virtual screening methods combined with experimental assays were used to identify small molecular weight nonpeptidic compounds that block Lck SH2 domain-dependent interactions. Virtual screening included scoring normalization procedures and postdocking structural clustering that is shown to facilitate the selection of active compounds. By targeting the well-defined hydrophobic binding pocket known to impart specificity on Lck-protein interactions (i.e., pY + 3 site), inhibitors of the Lck SH2 domain were discovered that omit the phosphotyrosine (pY) or related moieties. The 34 out of 196 computationally selected compounds were shown to inhibit Lck SH2 domain association with phosphorylated immunoreceptor tyrosine based activation motifs peptide. Twenty-four of the active compounds were further tested for their ability to modulate biological function. Thirteen of these compounds showed inhibitory activity in mixed lymphocyte culture assay. Fluorescence titration experiments on four of these active compounds further verified their binding to the SH2 domain. Because of their simple chemical structures, these small organic compounds have the potential to act as lead compounds for the development of novel immunosuppressant drugs.     

Common and differential recognition of structural features in synthetic peptides by the catalytic domain and the Src-Homology 2 (SH2) domain of pp60c-src

The relative efficiencies of the catalytic domain of the src-family kinase pp60^c-src in phosphorylating four peptide substrates including (i) src-optimal peptide (AEEEIYGEFEAKKKK), (ii) “-YEEI-peptide” (KKTHQEEEEPQYEEIPIYL), (iii) cdc2(6–20) (KVEKIGEGTY GVVYK), (iv) src-autophosphorylation site peptide (ADFGLARLIEDNEY TARG) and the relative efficiencies of its SH2 domain in binding the phosphorylated forms of these peptide substrates were compared. The results show that the src-optimal peptide, “-YEEI-peptide, ” cdc2(6–20) peptide were phosphorylated by the catalytic domain with high efficiency and that the phosphorylated form of all three peptides could bind the SH2 domain of the kinase, confirming the hypothesis proposed by Songyang and co-workers that the catalytic domain of pp60^c-src phosphorylates sites which are recognized by its own SH2 domain (Songyang et al. (1995) Nature 373, 536–539). The four peptides were phosphorylated by the kinase with relative efficiencies in the order of Src-optimal peptide > “-YEEI-peptide” > cdc2(6–20) ? src-autophosphorylation site peptide. However, the Tyr(P)-Src-optimal peptide and [pY]. ¹⁵cdc2(6–20) bound to the SH2 domain of the kinase with an affinity at least an order of magnitude lower than that of the tight-binding peptide, “-pYEEI-peptide.” Thus, our study suggests that the catalytic and SH2 domains of pp60^c-src recognize overlapping but not identical determinants in the local structure around the tyrosine phosphorylation site of the substrate peptides.     

Effects of halogenation on tyrosine phosphorylation and peptide binding to the SRC homology 2 domain of lymphocyte-specific protein tyrosine kinase

Phosphorylation of tyrosine residues by protein tyrosine kinases (PTK) and phosphotyrosine/Src homology 2 (SH2) domain interactions are crucial not only for signal transduction but also for regulation of PTK activity. Tyrosine residues also receive nitration and halogenation under oxidative conditions. It has been reported that nitration of tyrosine residue caused peptides to be a poor substrate for PTK and that nitrotyrosine residues could bind to SH2 domains as a phosphotyrosine mimic to activate Src family kinase. However, the effect of halogenation on tyrosine phosphorylation or SH2 domain binding is not well understood. We examined the phosphorylation of model peptides containing 3-halotyrosine or 3-nitrotyrosine using typical receptor tyrosine kinase, epidermal growth factor receptor (EGFR), and nonreceptor tyrosine kinase, lymphocyte-specific protein tyrosine kinase (Lck). The EGFR- and Lck-mediated phosphorylation was markedly inhibited by tyrosine halogenation. Iodination showed the strongest inhibition of the phosphorylation among four types of halogenation, and its inhibitory effect was stronger than that of nitration. We also examined the effect of iodination and nitration of tyrosine residues on binding to the SH2 domain of Lck, using a model peptide containing the phosphoTyr-Glu-Glu-Ile motif, which has a high affinity for the SH2 domain. The relative affinities of the modified peptides whose phosphotyrosine was substituted with unphosphorylated tyrosine, 3-nitrotyrosine, and 3-iodotyrosine, and of the model peptide were 0.024, 0.26, 1, and 16, respectively. These results suggest that tyrosine iodination may have an effect on the phosphorylation or binding to the SH2 domain similar to nitration. Tyrosine iodination possibly modulates signal transduction, with the potential impairment of cell function.     

Exploring kainate receptor pharmacology using molecular dynamics simulations

Ionotropic glutamate receptors (iGluRs) are enticing targets for pharmaceutical research; however, the search for selective ligands is a laborious experimental process. Here we introduce a purely computational procedure as an approach to evaluate ligand-iGluR pharmacology. The ligands are docked into the closed ligand-binding domain and during the molecular dynamics (MD) simulation the bi-lobed interface either opens (partial agonist/antagonist) or stays closed (agonist) according to the properties of the ligand. The procedure is tested with closely related set of analogs of the marine toxin dysiherbaine bound to GluK1 kainate receptor. The modeling is set against the abundant binding data and electrophysiological analyses to test reproducibility and predictive value of the procedure. The MD simulations produce detailed binding modes for analogs, which in turn are used to define structure-activity relationships. The simulations suggest correctly that majority of the analogs induce full domain closure (agonists) but also distinguish exceptions generated by partial agonists and antagonists. Moreover, we report ligand-induced opening of the GluK1 ligand-binding domain in free MD simulations. The strong correlation between in silico analysis and the experimental data imply that MD simulations can be utilized as a predictive tool for iGluR pharmacology and functional classification of ligands.     

Macrocyclization in the design of Grb2 SH2 domain-binding ligands exhibiting high potency in whole-cell systems

While most SH2 domains bind phosphotyrosyl (pTyr) containing peptides in extended fashion, the growth factor receptor-bound protein 2 (Grb2) SH2 domain preferentially binds ligands in bend conformations. Accordingly, incorporation of bend-inducing functionality into synthetic ligands could potentially enhance their affinity for this SH2 domain. A macrocyclic tripeptide mimetic that contains a simplified pTyr surrogate lacking an alpha-nitrogen has recently been shown to exhibit high Grb2 SH2 domain-binding affinity in extracellular ELISA-based assays. However, the same compound is largely ineffective in whole-cell assays. It is known that acidic functionality originating from the alpha-nitrogen of pTyr residues or from the alpha-position of P0 pTyr mimetics not only increases binding affinity of peptides to Grb2 SH2 domains in extracellular assays but also enhances potency in cell-based systems. Such functionality is absent from the previously reported macrocycle. Therefore, the current study was undertaken to examine the effects of introducing carboxylic functionality at the pTyr mimetic alpha-position of macrocyclic ligands. It was found that such a modification not only enhanced Grb2 SH2 domain binding in extracellular assays but also conferred high efficacy in whole-cell systems. The most potent compound of the current study exhibited an IC(50) value of 0.002 microM in an extracellular ELISA-based assay, and in MDA-MB-453 cells, it both inhibited the association of Grb2 with p185(erbB-2) and exhibited antimitogenic effects with submicromolar IC50 values.     

Antagonists of the Src homology 2 (SH2) domains of Grb2, Src, Lck and ZAP-70

Src homology 2 (SH2) domains are protein modules that mediate intracellular protein-protein interactions in signal transduction pathways. The specific association of an SH2 domain with a phosphotyrosine-containing sequence of another protein induces a cascade of molecular interactions that effect a wide range of cellular processes. Alterations in these signaling pathways have been associated with the development and progression of a broad range of pathologies. Because of the regulatory role of SH2 domains in these signal transduction pathways, specific SH2 domains can be ideal targets for intervention with therapeutic agents in many different disease indications (e.g. cancer, osteoporosis, disorders of the immune and cardiovascular systems). Among the SH2 domains pursued as drug discovery targets in the last few years are those of Grb2, Src, Lck and ZAP-70. This review focuses on contributions in the design and synthesis of antagonists of these particular SH2 domains. Specific examples have been selected to illustrate how structure-based design approaches have been used to progress in this area of research.     

Development of a solid-phase binding assay and identification of nonpeptide ligands for the FynB Src homology 2 domain.

The nonreceptor tyrosine kinase FynB is known to be required in the induction of long-term potentiation (LTP), a cellular mechanism for learning and memory. Ligands of the FynB SH2 domain as a possible FynB activator are, thus, of great interest. In this study, a solid-phase ligand binding assay was established to meet the screening requirement of high-throughput and ease of use, and in an attempt to find the specific ligands for the FynB SH2 domain. This assay measures the competitive inhibition of the binding of the biotinylated phosphopeptide (GGSETDDY*AEIID), derived from a binding sequence in human focal adhesion kinase, to the SH2 domain of FynB precoated as a glutathione S-transferase fusion protein on a solid-phase. Using this high-throughput screening method for SH2 ligands, a modest size of chemical library was screened, and two non-peptide compounds, 4-acetamidobenzene sulfinic acid and 1-allylpyridinium 3-sulfonate, were identified by their strong binding affinity to the FynB SH2 domain. This result demonstrates the feasibility of the developed assay in high-throughput screening. Further studies on the molecular structures of the identified SH2-binding ligands will allow presentation of specific models for ligand-domain complexes for improving the ligands and will help to develop a potential lead compound for improving LTP.