Identification of Novel Fragments Binding to the PDZ1-2 Domain of PSD-95

Inhibition of PSD-95 has emerged as a promising strategy for the treatment of ischemic stroke, as shown with peptide-based compounds that target the PDZ domains of PSD-95. In contrast, developing potent and drug-like small molecules against the PSD-95 PDZ domains has so far been unsuccessful. Here, we explore the druggability of the PSD-95 PDZ1-2 domain and use fragment screening to investigate if this protein is prone to binding small molecules. We screened 2500 fragments by fluorescence polarization (FP) and validated the hits by surface plasmon resonance (SPR), including an inhibition counter-test, and found four promising fragments. Three ligand efficient fragments were shown by ¹H,¹⁵N HSQC NMR to bind in the small hydrophobic P⁰ pockets of PDZ1-2, and one of them underwent structure-activity relationship (SAR) studies. Overall, we demonstrate that fragment screening can successfully be applied to PDZ1-2 of PSD-95 and disclose novel fragments that can serve as starting points for optimization towards small-molecule PDZ domain inhibitors.     

Discovery, structure-activity relationship studies, and crystal structure of nonpeptide inhibitors bound to the Shank3 PDZ domain

Shank is the central scaffolding protein of the postsynaptic density (PSD) protein complex found in cells of the central nervous system. Cellular studies indicate a prominent role of the protein in the organization of the PSD, in the development of neuronal morphology, in neuronal signaling, and in synaptic plasticity, thus linking Shank functions to the molecular basis of learning and memory. Mutations in the Shank gene have been found in several neuronal disorders including mental retardation, typical autism, and Asperger syndrome. Shank is linked to the PSD complex via its PDZ domain that binds to the C‐terminus of guanylate‐kinase‐associated protein (GKAP). Here, small‐molecule inhibitors of Shank3 PDZ domain are developed. A fluorescence polarization assay based on an identified high‐affinity peptide is established, and tetrahydroquinoline carboxylates are identified as inhibitors of this protein–protein interaction. Chemical synthesis via a hetero‐Diels–Alder strategy is employed for hit optimization, and structure–activity relationship studies are performed. Best hits possess K_i values in the 10 μM range, and binding to the PDZ domain is confirmed by ¹H,¹⁵N HSQC NMR experiments. One of the hits crystallizes with the Shank3 PDZ domain. The structure, analyzed at a resolution of 1.85 Å, reveals details of the binding mode. Finally, binding to PDZ domains of PSD‐95, syntrophin, and DVL3 was studied using ¹H,¹⁵N HSQC NMR spectroscopy.     

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein–protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling‐transduction complexes. In recent years, PDZ domains have emerged as novel and exciting drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C‐terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys/Arg residue in the ligand‐binding site of the second PDZ domain of PSD‐95, by employing a semisynthetic approach. We generated six semisynthetic PDZ domains comprising different proteogenic and nonproteogenic amino acids representing subtle changes of the conserved Lys/Arg residue. These were tested with four peptide interaction partners, representing the two different binding modes. The results highlight the role of a positively charged amino acid in the β1–β2 loop of PDZ domains, and show subtle differences for canonical and noncanonical interaction partners, thus providing additional insight into the mechanism of PDZ/ligand interaction.     

Improving Binding Affinity and Stability of Peptide Ligands by Substituting Glycines with D‐Amino Acids

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer‐related protease urokinase‐type plasminogen activator (uPA), we observed a glycine residue that has a positive ? dihedral angle when bound to the target. We hypothesized that replacing it with a D ‐amino acid, which favors positive ? angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D ‐serine in the bicyclic peptide indeed improved inhibitory activity (1.75‐fold) and stability (fourfold). X‐ray‐structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ? angles are found in many protein‐bound peptides. These results suggest that the glycine‐to‐D ‐amino acid mutagenesis strategy could be broadly applied.     

Improving the Binding Affinity of in‐Vitro‐Evolved Cyclic Peptides by Inserting Atoms into the Macrocycle Backbone

Cyclic peptides binding to targets of interest can be generated efficiently with powerful in vitro display techniques, such as phage display or mRNA display. The cyclic peptide libraries screened with these methods are generated by altering in a combinatorial fashion the amino acid sequence of the peptides, the number of amino acids in the macrocycle rings, and the cyclization chemistry. A structural element that cannot easily be varied in the cyclic peptides is the backbone, which is built from amino acids, each of which contributes three atoms to the macrocyclic ring structure. Here, we proposed to improve the affinity of a phage‐selected bicyclic peptide inhibitor of coagulation factor XII (FXII) by screening variants with one or two carbon atoms inserted into different positions of the backbone, and thus tapping into a structural space that was not sampled by phage display. Two mutants showed 4.7‐ and 2.5‐fold improved K_i values. The better one blocked FXII with a K_i of 1.5±0.1 nm and inhibited activation of the intrinsic coagulation pathway (EC_2x 1.7 μm) . The strategy of ring size variation by one or several atoms should be generally applicable for the affinity maturation of in‐vitro‐evolved cyclic peptides.     

Rigidified Clicked Dimeric Ligands for Studying the Dynamics of the PDZ1‐2 Supramodule of PSD‐95

PSD‐95 is a scaffolding protein of the MAGUK protein family, and engages in several vital protein–protein interactions in the brain with its PDZ domains. It has been suggested that PSD‐95 is composed of two supramodules, one of which is the PDZ1‐2 tandem domain. Here we have developed rigidified high‐affinity dimeric ligands that target the PDZ1‐2 supramodule, and established the biophysical parameters of the dynamic PDZ1‐2/ligand interactions. By employing ITC, protein NMR, and stopped‐flow kinetics this study provides a detailed insight into the overall conformational energetics of the interaction between dimeric ligands and tandem PDZ domains. Our findings expand our understanding of the dynamics of PSD‐95 with potential relevance to its biological role in interacting with multivalent receptor complexes and development of novel drugs.     

High‐Affinity Carbohydrate Binding by Trimeric Benzoboroxoles Measured on Carbohydrate Arrays

Carbohydrates are involved in a wide range of biological processes of pharmaceutical relevance. The selective recognition of carbohydrates is therefore of great interest in biology and medicine. In this study we present the synthesis of fluorescent multimeric benzoboroxoles and the analysis of multivalent binding processes to immobilized carbohydrate arrays by fluorescence spectroscopy. We observed high binding affinities of trimeric benzoboroxoles by determination of K_Dsurf values for their interaction with α‐Gal on glass chips. The observed K_Dsurf values were in the mid‐nM range (49 and 104 nM ) and are comparable to the K_Dsurf values for binding of natural lectins, such as that of ConA to immobilized α‐Man (79 nM ). The array technology was found to be an excellent tool for studying the binding processes of multivalent lectin mimetics with respect to profiling and quantitation.     

Asymmetric Dimerization of Disubstituted Ketenes Catalyzed by N‐Heterocyclic Carbenes

A series of chiral N‐heterocyclic carbenes (NHCs), derived from L ‐pyrogutamic acid, were found to be efficient catalysts for the asymmetric dimerization of alkylarylketenes to give the corresponding α‐quaternary β‐alkylidenyl‐β‐lactones in good yields with up to 97% ee. A chiral NHC with a proximal hydroxy group is superior in comparison with the corresponding NHC with its hydroxy group protected.     

Ligand‐Induced Dimerization of a Truncated Parallel MYC G‐Quadruplex

Binding of an indoloquinoline derivative with an aminoalkyl side chain to a truncated sequence from the MYC promoter region was studied through isothermal titration calorimetry (ITC). The targeted MYC3 sequence lacks 3′‐flanking nucleotides and forms a monomeric parallel quadruplex (G4) with a blunt‐ended 3′‐outer tetrad under the solution conditions employed. Analysis of ITC isotherms reveals multiple binding equilibria with the initial formation of a 1:2 ligand/quadruplex complex. Evaluation of electrophoretic mobilities as well as NMR spectral data confirm ligand‐induced dimerization of MYC3 quadruplexes with the ligand sandwiched between the two 3′‐outer tetrads. Additional ligand molecules in excess bind to the 5′‐outer tetrads of the sandwich complex. Such a ligand‐promoted G4 dimerization may be exploited for the controlled assembly or disassembly of G4 aggregates to expand on present quadruplex‐based technologies.     

Identifying Small‐Molecule Binding Sites for Epigenetic Proteins at Domain–Domain Interfaces

Epigenetics is a rapidly growing field in drug discovery. Of particular interest is the role of post‐translational modifications to histones and the proteins that read, write, and erase such modifications. The development of inhibitors for reader domains has focused on single domains. One of the major difficulties of designing inhibitors for reader domains is that, with the notable exception of bromodomains, they tend not to possess a well‐enclosed binding site amenable to small‐molecule inhibition. As many of the proteins in epigenetic regulation have multiple domains, there are opportunities for designing inhibitors that bind at a domain–domain interface which provide a more suitable interaction pocket. Examination of X‐ray structures of multiple domains involved in recognising and modifying post‐translational histone marks using the SiteMap algorithm identified potential binding sites at domain–domain interfaces. For the tandem plant homeodomain–bromodomain of SP100C, a potential inter‐domain site identified computationally was validated experimentally by the discovery of ligands by X‐ray crystallographic fragment screening.     

Heterotypic Sam–Sam Association between Odin‐Sam1 and Arap3‐Sam: Binding Affinity and Structural Insights

Arap3 is a phosphatidylinositol 3 kinase effector protein that plays a role as GTPase activator (GAP) for Arf6 and RhoA. Arap3 contains a sterile alpha motif (Sam) domain that has high sequence homology with the Sam domain of the EphA2‐receptor (EphA2‐Sam). Both Arap3‐Sam and EphA2‐Sam are able to associate with the Sam domain of the lipid phosphatase Ship2 (Ship2‐Sam). Recently, we reported a novel interaction between the first Sam domain of Odin (Odin‐Sam1), a protein belonging to the ANKS (ANKyrin repeat and Sam domain containing) family, and EphA2‐Sam. In our latest work, we applied NMR spectroscopy, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize the association between Arap3‐Sam and Odin‐Sam1. We show that these two Sam domains interact with low micromolar affinity. Moreover, by means of molecular docking techniques, supported by NMR data, we demonstrate that Odin‐Sam1 and Arap3‐Sam might bind with a topology that is common to several Sam‐Sam complexes. The revealed structural details form the basis for the design of potential peptide antagonists that could be used as chemical tools to investigate functional aspects related to heterotypic Arap3‐Sam associations.     

Conversion of Low‐Affinity Peptides to High‐Affinity Peptide Binders by Using a β‐Hairpin Scaffold‐Assisted Approach

Affinity maturation of protein‐targeting peptides is generally accomplished by homo‐ or heterodimerization of known peptides. However, applying a heterodimerization approach is difficult because it is not clear a priori what length or type of linker is required for cooperative binding to a target. Thus, an efficient and simple affinity maturation method for converting low‐affinity peptides into high‐affinity peptides would clearly be advantageous for advancing peptide‐based therapeutics. Here, we describe the development of a novel affinity maturation method based on a robust β‐hairpin scaffold and combinatorial phage‐display technology. With this strategy, we were able to increase the affinity of existing peptides by more than four orders of magnitude. Taken together, our data demonstrate that this scaffold‐assisted approach is highly efficient and effective in generating high‐affinity peptides from their low‐affinity counterparts.     

Phosphatidylethanolamine‐Binding Proteins,Including RKIP,Exhibit Affinity for Phosphodiesterase‐5 Inhibitors

Identifying protein “interactors” of drugs is of great importance to understand their mode of action and possible cross‐reactivity to off‐target protein binders. In this study, we profile proteins that bind to PF‐3717842, a high‐affinity phosphodiesterase‐5 (PDE5) inhibitor, by using a refined affinity pulldown approach with PF‐3717842 immobilized beads. By performing these pulldowns in rat testis tissue lysate, we strongly and specifically enriched for PDE5 and a few other PDEs. In addition to these expected affinity‐enriched proteins we also detect rodent‐specific phosphatidylethanolamine‐binding protein 2 (PEBP2), as a putative binder to the PDE5 inhibitor. By using recombinant forms of the related murine mPEBP2, mPEBP1 and human hPEBP1 (also known as Raf kinase inhibitor protein or RKIP) we confirm that they all can bind strongly to immobilized as well as soluble PF‐3717842. As the phosphatidylethanolamine‐binding proteins are involved in various important signal transduction pathways, the synthetic PDE5 inhibitor used here might form a platform to synthesize enhanced binders/inhibitors of the family of PEBP proteins. Our approach shows how chemical proteomics might be used to profile the biochemical space (interactome) of small molecule inhibitors.     

Ligand‐Biased and Probe‐Dependent Modulation of Chemokine Receptor CXCR3 Signaling by Negative Allosteric Modulators

Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target G protein‐coupled receptors (GPCRs). The CXC chemokine receptor 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer. Herein we report the discovery of an 8‐azaquinazolinone derivative (N‐{1‐[3‐(4‐ethoxyphenyl)‐4‐oxo‐3,4‐dihydropyrido[2,3‐d]pyrimidin‐2‐yl]ethyl}‐4‐(4‐fluorobutoxy)‐N‐[(1‐methylpiperidin‐4‐yl)methyl]butanamide, 1 b ) that can inhibit CXC chemokine 11 (CXCL11)‐dependent G protein activation over β‐arrestin recruitment with 187‐fold selectivity. This compound also demonstrates probe‐dependent activity, that is, it inhibits CXCL11‐ over CXCL10‐mediated G protein activation with 12‐fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.     

Electrochemical and Conformational Consequences of Copper (CuI and CuII) Binding to β‐Amyloid(1–40)

Copper‐induced structural rearrangements of Aβ40 structure and its redox properties are described in this study. Electrochemical and fluorescent methods are used to characterise the behaviour of Aβ–Cu species. The data suggest that time‐dependent folding of Aβ–Cu species may cause changes in the redox potentials.

     

Selective DNA‐Binding by Designed Bisbenzamidine‐Homeodomain Chimeras

We report the construction of conjugates between three variants of the helix 3 region of a Q50K engrailed homeodomain and bisbenzamidine minor‐groove DNA binders. The hybrid featuring the sequence of the native protein failed to bind to DNA; however, modifications that increased the α‐helical folding propensity of the peptide allowed specific DNA binding by a bipartite (major/minor groove) interaction.     

Glycopeptide Dendrimers with High Affinity for the Fucose‐Binding Lectin LecB from Pseudomonas aeruginosa

Dendritic antibacterial agents : Glycopeptide dendrimer biofilm inhibitors were synthesized combinatorially and optimized for binding to the fucose‐specific lectin LecB, which has high affinity of fucose. These dendritic ligands are potential antibacterial agents against Pseudomonas aeruginosa, an antibiotic resistant human pathogen.

     

Architectural Repertoire of Ligand‐Binding Pockets on Protein Surfaces

Knowledge of the three‐dimensional structure of ligand binding sites in proteins provides valuable information for computer‐assisted drug design. We present a method for the automated extraction and classification of ligand binding site topologies, in which protein surface cavities are represented as branched frameworks. The procedure employs a growing neural gas approach for pocket topology assignment and pocket framework generation. We assessed the structural diversity of 623 known ligand binding site topologies based on framework cluster analysis. At a resolution of 5 Å only 23 structurally distinct topology groups were formed; this suggests an overall limited structural diversity of ligand‐accommodating protein cavities. Higher resolution allowed for identification of protein‐family specific pocket features. Pocket frameworks highlight potentially preferred modes of ligand–receptor interactions and will help facilitate the identification of druggable subpockets suitable for ligand affinity and selectivity optimization.