Design and Synthesis of High‐Affinity Dimeric Inhibitors Targeting the Interactions between Gephyrin and Inhibitory Neurotransmitter Receptors

Gephyrin is the central scaffolding protein for inhibitory neurotransmitter receptors in the brain. Here we describe the development of dimeric peptides that inhibit the interaction between gephyrin and these receptors, a process which is fundamental to numerous synaptic functions and diseases of the brain. We first identified receptor‐derived minimal gephyrin‐binding peptides that displayed exclusive binding towards native gephyrin from brain lysates. We then designed and synthesized a series of dimeric ligands, which led to a remarkable 1220‐fold enhancement of the gephyrin affinity (K_D=6.8 nM ). In X‐ray crystal structures we visualized the simultaneous dimer‐to‐dimer binding in atomic detail, revealing compound‐specific binding modes. Thus, we defined the molecular basis of the affinity‐enhancing effect of multivalent gephyrin inhibitors and provide conceptually novel compounds with therapeutic potential, which will allow further elucidation of the gephyrin–receptor interplay.     

Affinity Maturation of Macrocyclic Peptide Modulators of Lys48-Linked Diubiquitin by a Twofold Strategy

Messenger RNA display of peptides containing non-proteinogenic amino acids, referred to as RaPID system, has become one of the leading methods to express libraries consisting of more than trillion-members of macrocyclic peptides, which allows for discovering de novo bioactive ligands. Ideal macrocyclic peptides should have dissociation constants (K_D) as low as single-digit values in the nanomolar range towards a specific target of interest. Here, a twofold strategy to discover optimized macrocyclic peptides within this affinity regime is described. First, benzyl thioether cyclized peptide libraries were explored to identify tight binding hits. To obtain more insights into critical sequence information, sequence alignment was applied to guide rational mutagenesis for the improvement of their binding affinity. Using this twofold strategy, benzyl thioether macrocyclic peptide binders against Lys48-linked ubiquitin dimer (K48-Ub2) were successfully obtained that display K_D values in the range 0.3–1.2 nm , which indicate binding two orders of magnitude stronger than those of macrocyclic peptides recently reported. Most importantly, this macrocyclic peptide also showed an improved cellular inhibition of the K48-Ub2 recognition by deubiquitinating enzymes and the 26S proteasome, resulting in the promotion of apoptosis in cancer cells.     

1H‐NMR relaxometric studies of interaction between apoptosis specific MRI paramagnetic contrast agents and micellar models of apoptotic cells

¹H‐NMR was previously used to analyze the interaction between peptides (E3 and R826) selected by phage display to target apoptotic cells and phospholipidic models of these cells. In order to avoid the use of apoptotic cells and to obtain a fast evaluation of the efficiency of the potential MRI contrast agents obtained by grafting these peptides and their scramble analogs on a paramagnetic gadolinium complex, their proton relaxometric behavior was investigated in the presence of micelles mimicking healthy and apoptotic cells. Their preferential interaction with 1,2‐dipalmitoyl‐sn‐glycero‐3‐phospho‐l ‐serine micelles mimicking apoptotic cells as compared with 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine micelles modeling healthy cells was shown by nuclear magnetic relaxation dispersion profiles and the enhancement of the transverse proton relaxation rates at 60 MHz. The association constant values confirm the stronger interaction of the selected conjugated peptides (K_a Gd‐PMN‐E3(gadolinium 2,2′,2′′,2′′′‐[((4‐carboxy)pyridine‐2,6‐diyl)bis(methylenenitrilo)]‐tetrakis acetate) grafted with E3 peptide): 2.43 10⁴ m ^?1; K_a Gd‐DTPA‐R826(gadolinium ((1‐p‐isothiocyanatobenzyl)‐diethylenetriaminepentaacetate) grafted with R826 peptide): 2.91 10⁴ m ^?1) as compared with their conjugated scrambles (K_a Gd‐PMN‐E3sc(gadolinium 2,2′,2′′,2′′′‐[((4‐carboxy)pyridine‐2,6‐diyl)bis(methylenenitrilo)]‐tetrakis acetate) grafted with E3 scramble peptide): 0.18 10⁴ m ^?1; K_a Gd‐DTPA‐R826sc(gadolinium ((1‐p‐isothiocyanatobenzyl)‐diethylenetriaminepentaacetate) grafted with R826 scramble peptide): 0.32 10⁴ m ^?1) even if the conjugation of E3 and R826 seems to decrease their interaction. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of binding constants between the antibiotic ristocetin A and D-Ala-D-Ala terminus peptides by affinity capillary electrophoresis

Summary Binding constants between the antibiotic ristocetin A (Rist A) and D-Ala-D-Ala terminus peptides were determined using affinity capillary electrophoresis (ACE). In these experiments two techniques are used to obtain binding constants. In the first, a plug of Rist A and non-interacting standards are injected and electrophoresed. Analysis of the change in the relative migration time ratio (RMTR) of Rist, relative to the non-interacting standards, as a function of the concentration of peptide, yields a value for the binding constant (Kb). In the second, samples of peptide and standards are injected and electrophoresed in increasing concentrations of Rist A in the running buffer. Analysis using theRMTR yields aK _b. The findings described here demonstrate the advantage of using ACE for estimating binding parameters between antibiotics and ligands.     

Encapsulation of AGE‐Breaker Alagebrium by Cucurbit[7]uril Improved the Stability of Both Its Carbonyl α‐Hydrogen and Thiazolium C2‐Hydrogen

As determined by both ¹H NMR and UV/Vis spectroscopic titration, ESI‐MS, isothermal titration calorimetry, and DFT molecular modeling, advanced glycation end products (AGE) breaker alagebrium (ALA) formed 1:1 guest–host inclusion complexes with cucurbit[7]uril (CB[7]), with a binding affinity, K_a, in the order of magnitude of 10⁵ m ^?1, thermodynamically driven by both enthalpy (ΔH=?6.79 kcal mol^?1) and entropy (TΔS=1.21 kcal mol^?1). For the first time, a dramatic inhibition of keto–enol tautomerism of the carbonyl α‐hydrogen of ALA has been observed, as evidenced by over an order of magnitude decrease of both the first step rate constant, k₁, and the second step rate constant, k₂, during hydrogen/deuterium exchange in D₂O. Meanwhile, as expected, the reactivity of C2‐hydrogen was also inhibited significantly, with an upshift of 2.09 pK_a units. This discovery will not only provide an emerging host molecule to modulate keto–enol tautomerism, but also potentially lead to a novel supramolecular formulation of AGE‐breaker ALA for improved stability and therapeutic efficacy.     

Peptide‐binding specificity of the prosurfactant protein C Brichos domain analyzed by electrospray ionization mass spectrometry

The C‐terminal domain of lung surfactant protein C (CTC) precursor (proSP‐C) is involved in folding of the transmembrane segment of proSP‐C. CTC includes a Brichos domain with homologs in cancer‐ and dementia‐associated proteins. Mutations in the Brichos domain cause misfolding of proSP‐C and hence amyloid fibril formation in interstitial lung disease. Electrospray ionization mass spectrometry (ESI‐MS) with collision‐induced dissociation (CID) experiments was applied to study non‐covalent interactions between human recombinant CTC or its Brichos domain, and SP‐C analogs, homotripeptides and peptides designed to model amyloid fibril formation. The results show that the Brichos domain contains the peptide‐binding function of CTC. In titration experiments, apparent dissociation constants (K_D) were in the micromolar range where triple‐valine showed the lowest K_D and triple‐tyrosine the highest. Non‐hydrophobic peptides failed to form complexes with Brichos. CID revealed that complexes with aromatic peptide ligands are more stable in the gas phase than complexes with non‐aromatic ligands. The Brichos domain was also shown to bind fibril‐forming peptides containing aromatic/hydrophobic residues. Copyright © 2009 John Wiley & Sons, Ltd.     

Chemically Triggered C–ON Bond Homolysis in Alkoxyamines. Part 2: DFT Investigation and Application of the pH Effect on NMP

In recent work, a 15‐fold increase in the C–ON bond homolysis rate constant k_d of 4‐pyridylethyl‐SG1‐based alkoxyamine was observed upon protonation of the pyridyl moiety in organic solvent. In this report, the pH dependence of k_d (pK_a = 4.7) is investigated in D₂O/CD₃OD (v/v 1:1). A 64‐fold increase in k_d is observed at acidic pH. Calculations show that the increase in k_d upon protonation is due to both an increase in the stabilization of the protonated 4‐pyridylethyl radical and an increase of the destabilization of the starting materials through an increase in the polarity of the alkyl fragment. This new alkoxyamine is applied to NMP of styrene and sodium styrene sulfonate.     

N‐Alkyl Ammonium Resorcinarene Salts as High‐Affinity Tetravalent Chloride Receptors

N‐Alkyl ammonium resorcinarene salts (NARYs, Y=triflate, picrate, nitrate, trifluoroacetates and NARBr) as tetravalent receptors, are shown to have a strong affinity for chlorides. The high affinity for chlorides was confirmed from a multitude of exchange experiments in solution (NMR and UV/Vis), gas phase (mass spectrometry), and solid‐state (X‐ray crystallography). A new tetra‐iodide resorcinarene salt (NARI) was isolated and fully characterized from exchange experiments in the solid‐state. Competition experiments with a known monovalent bis‐urea receptor ( 5 ) with strong affinity for chloride, reveals these receptors to have a much higher affinity for the first two chlorides, a similar affinity as 5 for the third chloride, and lower affinity for the fourth chloride. The receptors affinity toward chloride follows the trend K₁?K₂?K₃≈ 5 >K₄, with K_a=5011 m ^?1 for 5 in 9:1 CDCl₃/[D₆]DMSO.     

De Novo Design and Synthesis of a γ‐Turn Peptidomimetic Scaffold and Its Application as JNK3 Allosteric Ligand

As a way to develop a neuroprotective agent for the JNK3‐JIP1‐binding site, peptidomimetics of JIP‐1 as JNK3 allosteric regulators have been examined. The study consisted of in silico scaffold hopping, molecular docking, solution and solid‐phase peptide syntheses, and K_d measurements using surface plasmon resonance. As a peptidomimetic of JIP1, heptamer mimetic 16 (K_d=2.72 μm ) displayed a higher affinity than decamer JIP1 (K_d=23.6 μm ). The high affinity of 16 implies that the characteristic γ‐turn mimetic structure, “”Φ‐X‐Φ“ hydrophobic motif in 16 , increased its affinity toward the JIP‐site of JNK3.     

Template‐Assembled Synthetic G‐Quadruplex (TASQ): A Useful System for Investigating the Interactions of Ligands with Constrained Quadruplex Topologies

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular‐like G‐quadruplex motif 1 (parallel G‐quadruplex conformation), an intramolecular G‐quadruplex 2 , and a duplex DNA 3 have been designed and developed. The method is based on the concept of template‐assembled synthetic G‐quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G‐quadruplex conformation. Various known G‐quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a π‐stacking binding mode showed a higher binding affinity for intermolecular‐like G‐quadruplexes 1 , whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2 . In addition, the present method has also provided information about the selectivity of ligands for G‐quadruplex DNA over the duplex DNA. A numerical parameter, termed the G‐quadruplex binding mode index (G4‐BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G‐quadruplex 1 against intramolecular G‐quadruplex 2 . The G‐quadruplex binding mode index (G4‐BMI) of a ligand is defined as follows: G4‐BMI=K_D^intra/K_D^inter, where K_D^intra is the dissociation constant for intramolecular G‐quadruplex 2 and K_D^inter is the dissociation constant for intermolecular G‐quadruplex 1 . In summary, the present work has demonstrated that the use of parallel‐constrained quadruplex topology provides more precise information about the binding modes of ligands.     

Remarkably selective recognition of iodobenzene derivatives by a macrocyclic bis-Pt(II) metallohost

We designed and synthesized self‐assembled bis‐Pt^II dimer 1? 4 BF₄ with quino[8,7‐b][1,10]phenanthroline as an extended π‐face contact area, which acts as the first artificial receptor with high affinity toward iodinated aromatic compounds significantly based on noncovalent iodine ??? aromatic‐plane interactions in a “side‐on” fashion. Despite their structural similarity to a previously reported metallohost 2 ⁴⁺ that bears 2,2′:6′,2′′‐terpyridine units, a dramatic change in selectivity toward substituted benzene derivatives was observed for 1 ⁴⁺. ¹H NMR spectroscopic titration revealed a high affinity of 1 ⁴⁺ towards haloarenes, with exceptionally large association constants for 2‐iodophenol (K_a=16 000 M ^?1) and 1,2‐diiodobenzene (K_a=21 000 M ^?1), which are 93‐ and 140‐fold higher, respectively, than the values obtained for 2 ⁴⁺. In addition, 1 ⁴⁺ showed a remarkably high affinity and selectivity toward 2,6‐diiodophenol (K_a=35 000 M ^?1), which is an important substructure of the thyroid hormone T₄. X‐ray crystallography and theoretical calculations strongly suggest that “side‐on” iodine ??? aromatic‐plane interactions and π–π stacking contribute to the strong 1,2‐diiodobenzene and 2,6‐diiodophenol binding. The results obtained here give unique and valuable insight into the nature of halogen atom interactions in their “side‐on” region with an electropositive aromatic plane, which may provide useful guidance for designing artificial receptors for iodinated biomolecules.     

Synthetic Receptors for the High‐Affinity Recognition of O‐GlcNAc Derivatives

The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water‐soluble carbohydrate receptors (“synthetic lectins”). Both systems show outstanding affinities for derivatives of N‐acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside GlcNAc‐β‐OMe with K_a≈20 000 m ^?1, whereas the other one binds an O‐GlcNAcylated peptide with K_a≈70 000 m ^?1. These values substantially exceed those usually measured for GlcNAc‐binding lectins. Slow exchange on the NMR timescale enabled structural determinations for several complexes. As expected, the carbohydrate units are sandwiched between the pyrenes, with the alkoxy and NHAc groups emerging at the sides. The high affinity of the GlcNAcyl–peptide complex can be explained by extra‐cavity interactions, raising the possibility of a family of complementary receptors for O‐GlcNAc in different contexts.     

Organic Ligand Binding by a Hydrophobic Cavity in a Designed Tetrameric Coiled‐Coil Protein

The design and characterization of a hydrophobic cavity in de novo designed proteins provides a wide range of information about the functions of de novo proteins. We designed a de novo tetrameric coiled‐coil protein with a hydrophobic pocketlike cavity. Tetrameric coiled coils with hydrophobic cavities have previously been reported. By replacing one Leu residue at the a position with Ala, hydrophobic cavities that did not flatten out due to loose peptide chains were reliably created. To perform a detailed examination of the ligand‐binding characteristics of the cavities, we originally designed two other coiled‐coil proteins: AM2, with eight Ala substitutions at the adjacent a and d positions at the center of a bundled structure, and AM2W, with one Trp and seven Ala substitutions at the same positions. To increase the association of the helical peptides, each helical peptide was connected with flexible linkers, which resulted in a single peptide chain. These proteins exhibited CD spectra corresponding to superhelical structures, despite weakened hydrophobic packing. AM2W exhibited binding affinity for size‐complementary organic compounds. The dissociation constants, K_d, of AM2W were 220 nM for adamantane, 81 μM for 1‐adamantanol, and 294 μM for 1‐adamantaneacetic acid, as measured by fluorescence titration analyses. Although it was contrary to expectations, AM2 did not exhibit any binding affinity, probably due to structural defects around the designed hydrophobic cavity. Interestingly, AM2W exhibited incremental structure stability through ligand binding. Plugging of structural defects with organic ligands would be expected to facilitate protein folding.     

Cyclometalated iridium(III) complex of 6‐hydroxydipyrido[3,2‐a:2′,3′‐c]phenazine: synthesis,and acid–base and avid DNA binding properties

A new cyclometalated Ir(III) complex [Ir(ppy)₂(hdppz)]PF₆ (Hppy = 2‐phenylpyridine and hdppz = 6‐hydroxydipyrido[3,2‐a:2′,3′‐c]phenazine) was synthesized and characterized. The pH effects on the UV–vis absorption spectra were studied and ground‐state acid ionization constant pK_a values of the complex were derived. The calf thymus DNA (ct‐DNA) binding properties of the complex were investigated with UV‐vis absorption spectrophotometric titrations, DNA competitive binding with ethidium bromide, DNA melting experiments, viscosity measurements and density functional theory (DFT) calculations. The complex was demonstrated to act as a ct‐DNA intercalator with a large DNA binding constant value of (6.06 ± 0.32) × 10⁶ M ⁻¹ in 50 mM NaCl. The avid DNA binding affinity observed was rationalized by the DFT calculations. Copyright © 2011 John Wiley & Sons, Ltd.     

Ethyl Xanthate and Propyl Xanthate as Activators and Inhibitors of Mushroom Tyrosinase in Different Concentrations

The effect of ethyl xanthate(I) and propyl xanthate(II) on the kinetics of hydroxylation by mushroom tyrosinase (MT) has been investigated at 20°C in 10 mM phosphate buffer solution, pH 6.8. 4‐[(4‐Methyl‐phenyl)azo]‐phenol (MePAPh) was used as a synthetic substrate for the enzyme for cresolase reaction. The results show that ethyl xanthate and propyl xanthate can activate or inhibit the cresolase activity of mushroom tyrosinase depending on the concentration of these effectors. Both I and II act uncompetitive at relatively high concentrations (20‐50 μM). The inhibition constant (K_i) values for I and II are 13.8 and 11 μM, respectively. However, both I and II act as activators at relatively low concentrations (0‐11.5 μM). Activation of the enzyme in low concentrations of xanthates arises from increasing the affinity of binding for the substrate as well as increasing the enzyme catalytic constant. The activation constant (K_a) values for I and II are 1.88 and 2.68 μM, respectively. The enzyme has two distinct sites for both effectors. The first one is a high‐affinity activation site and the other is a low‐affinity inhibition site.     

Application of plug–plug technique to ACE experiments for discovery of peptides binding to a larger target protein: A model study of calmodulin‐binding fragments selected from a digested mixture of reduced BSA

To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a “plug–plug” technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan‐X, β‐endorphin, and oxytocin, as candidates for calmodulin‐binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug–plug technique, the ACE–MS screening methodology successfully selected calmodulin‐binding peptides from a random library with diverse constituents, such as protease digests of BSA. Three peptides with K_d values between 8–147 μM for calmodulin were obtained from a Glu‐C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE–MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein.     

Cucurbit[7]uril: A High‐Affinity Host for Encapsulation of Amino Saccharides and Supramolecular Stabilization of Their α‐Anomers in Water

Cucurbit[7]uril (CB[7]), an uncharged and water‐soluble macrocyclic host, binds protonated amino saccharides (D ‐glucosamine, D ‐galactosamine, D ‐mannosamine and 6‐amino‐6‐deoxy‐D ‐glucose) with excellent affinity (K_a=10³ to 10⁴ M ^?1). The host–guest complexation was confirmed by NMR spectroscopy, isothermal titration calorimetry (ITC), and MALDI‐TOF mass spectral analyses. NMR analyses revealed that the amino saccharides, except D ‐mannosamine, are bound as α‐anomers within the CB[7] cavity. ITC analyses reveal that CB[7] has excellent affinity for binding amino saccharides in water. The maximum affinity was observed for D ‐galactosamine hydrochloride (K_a=1.6×10⁴ M ^?1). Such a strong affinity for any saccharide in water using a synthetic receptor is unprecedented, as is the supramolecular stabilization of an α‐anomer by the host.     

Constrained Peptides with Target‐Adapted Cross‐Links as Inhibitors of a Pathogenic Protein–Protein Interaction

Bioactive conformations of peptides can be stabilized by macrocyclization, resulting in increased target affinity and activity. Such macrocyclic peptides proved useful as modulators of biological functions, in particular as inhibitors of protein–protein interactions (PPI). However, most peptide‐derived PPI inhibitors involve stabilized α‐helices, leaving a large number of secondary structures unaddressed. Herein, we present a rational approach towards stabilization of an irregular peptide structure, using hydrophobic cross‐links that replace residues crucially involved in target binding. The molecular basis of this interaction was elucidated by X‐ray crystallography and isothermal titration calorimetry. The resulting cross‐linked peptides inhibit the interaction between human adaptor protein 14‐3‐3 and virulence factor exoenzyme S. Taking into consideration that irregular peptide structures participate widely in PPIs, this approach provides access to novel peptide‐derived inhibitors.     

Simultaneous high-throughput determination of interaction kinetics for drugs and cyclodextrins by high performance affinity chromatography with mass spectrometry detection

The individual determination of the apparent dissociation rate constant (k_d,app) using high performance affinity chromatography (HPAC) is a tedious process requiring numerous separate tests and massive data fitting, unable to provide the apparent association rate constant (k_a) and equilibrium binding constant (K_a). In this study, a HPAC with mass spectrometry detection (HPAC-MS/MS) was employed to determine the drug-cyclodextrin (CD) interaction kinetics with low sample loading quantity (<10 ng per injection for single compound) and high-throughput yield as twenty drugs determined in one injection. The k_d,app measured by HPAC-MS/MS approach were 0.89 ± 0.07, 4.34 ± 0.01, 1.48 ± 0.01 and 7.77 ± 0.04 s⁻¹ for ketoprofen, trimethoprim, indapamide and acetaminophen, with k_d,app for acetaminophen consistent with that from the HPAC method with UV detector in our previous studies. For twenty drugs with diverse structures and chemical properties, good correlationship was found between k_d,app measured by single compound analysis method and high-throughput HPAC-MS/MS approach, with the correlation coefficient of 0.987 and the significance F less than 0.001. Comprehensive quantification of k_a,app, k_d,app and K_a values was further performed based on the measurement of k_d,app by peak profiling method and K_a by the peak fitting method. And the investigation of the drug-CD interaction kinetics under different conditions indicated that the column temperature and mobile phase composition significantly affected the determination of k_a,app, k_d,app and K_a while also dependent on the acidity and basicity of drugs. In summary, the high-throughput HPAC-MS/MS approach has been demonstrated high efficiency in determination of the drug-CD primary interaction kinetic parameter, especially, k_d,app, being proven as a novel tool in screening the right CD for the solubilization of the right drug.