Importance of CH/π hydrogen bonds in recognition of the core motif in proline-recognition domains: an ab initio fragment molecular orbital study

We examined CH/π hydrogen bonds in protein/ligand complexes involving at least one proline residue using the ab initio fragment molecular orbital (FMO) method and the program CHPI. FMO calculations were carried out at the Hartree–Fock (HF)/6‐31G*, HF/6‐31G**, second‐order Møller–Plesset perturbation (MP2)/6‐31G*, and MP2/6‐31G** levels for three Src homology 3 (SH3) domains and five proline‐recognition domains (PRDs) complexed with their corresponding ligand peptides. PRDs use a conserved set of aromatic residues to recognize proline‐rich sequences of specific ligands. Many CH/π hydrogen bonds were identified in these complexes. CH/π hydrogen bonds occurred, in particular, in the central part of the proline‐rich motifs. Our results suggest that CH/π hydrogen bonds are important in the recognition of SH3 and PRDs by their ligand peptides and play a vital role in the signal transduction system. Combined use of the FMO method and CHPI analysis is a valuable tool for the study of protein/protein and protein/ligand interactions and may be useful in rational drug design. © 2011 Wiley Periodicals, Inc. J Comput Chem 2011     

Synthesis and evaluation of α-helix mimetics based on a trans-fused polycyclic ether: sequence-selective binding to aspartate pairs in α-helical peptides

Inspired by the topological similarity between ladder-like cyclic ether skeletons and α-helical peptides, a trans-fused 6/6/6/6 tetracyclic ether containing two hydroxyl groups separated by a distance of 4.8 Å was designed as a scaffold for a nonpeptidic α-helix mimetic. Two alkyl guanidinium groups were attached to the hydroxyl groups to develop a synthetic receptor for the specific recognition of i + 4 spaced aspartate pairs on the surface of an α-helical peptide. A circular dichroism (CD) titration showed that this mode of molecular recognition stabilizes α-helical structures of peptides containing i + 4 spaced aspartate pairs.     

G4 Sensing Pyridyl-Thiazole Polyamide Represses c-KIT Expression in Leukemia Cells

Specific sensing and functional tuning of nucleic acid secondary structures remain less explored to date. Herein, we report a thiazole polyamide TPW that binds specifically to c-KIT1 G-quadruplex (G4) with sub-micromolar affinity and ∼1 : 1 stoichiometry and represses c-KIT proto-oncogene expression. TPW shows up to 10-fold increase in fluorescence upon binding with c-KIT1 G4, but shows weak or no quantifiable binding to other G4s and ds26 DNA. TPW can increase the number of G4-specific antibody (BG4) foci and mark G4 structures in cancer cells. Cell-based assays reveal that TPW can efficiently repress c-KIT expression in leukemia cells via a G4-dependent process. Thus, the polyamide can serve as a promising probe for G-quadruplex recognition with the ability to specifically alter c-KIT oncogene expression.     

Liposome fusion with orthogonal coiled coil peptides as fusogens: the efficacy of roleplaying peptides

Biological membrane fusion is a highly specific and coordinated process as a multitude of vesicular fusion events proceed simultaneously in a complex environment with minimal off-target delivery. In this study, we develop a liposomal fusion model system with specific recognition using lipidated derivatives of a set of four de novo designed heterodimeric coiled coil (CC) peptide pairs. Content mixing was only obtained between liposomes functionalized with complementary peptides, demonstrating both fusogenic activity of CC peptides and the specificity of this model system. The diverse peptide fusogens revealed important relationships between the fusogenic efficacy and the peptide characteristics. The fusion efficiency increased from 20% to 70% as affinity between complementary peptides decreased, (from K_F ≈ 10⁸ to 10⁴ M⁻¹), and fusion efficiency also increased due to more pronounced asymmetric role-playing of membrane interacting ‘K’ peptides and homodimer-forming ‘E’ peptides. Furthermore, a new and highly fusogenic CC pair (E₃/P1_K) was discovered, providing an orthogonal peptide triad with the fusogenic CC pairs P2_E/P2_K and P3_E/P3_K. This E₃/P1_k pair was revealed, via molecular dynamics simulations, to have a shifted heptad repeat that can accommodate mismatched asparagine residues. These results will have broad implications not only for the fundamental understanding of CC design and how asparagine residues can be accommodated within the hydrophobic core, but also for drug delivery systems by revealing the necessary interplay of efficient peptide fusogens and enabling the targeted delivery of different carrier vesicles at various peptide-functionalized locations.

We developed a liposomal fusion model system with specific recognition using a set of heterodimeric coiled coil peptide pairs. This study unravels important structure–fusogenic efficacy relationships of peptide fusogens.     

Influence of hydrogen bonds between sidechains and cooperativity on self‐assembly of the cyclopeptides

The self‐assembly of four cyclic D,L‐octapeptides, [‐(D‐Ala‐Gln)₄‐], [‐(D‐Val‐Gln)₄‐], [‐(D‐Leu‐Gln)₄‐], and [‐(D‐Phe‐Gln)₄‐], was investigated on the theory level in detail. Based on these cyclic peptides, which contain L‐Gln residues and possess C₄ symmetry, a series of oligomers were constructed according to different stacking modes as well as interaction patterns. We employed the semiempirical molecular orbital method AM1 to optimize the structures of all the oligomers, some of which were further studied using density functional method B3PW91/6‐31G to calculate the interaction energies. The studies indicate that when these cyclopeptides aggregate to form oligomers, or even nanotubes, four more hydrogen bonds could form between the sidechains of L‐Gln residues in addition to eight hydrogen bonds formed between the backbones of adjacent two cyclic peptides, a result that would clearly affect the self‐assembling process of cyclic peptides. The main effects can be summarized as follows. First, the dimers of these cyclic peptides with C₄ symmetry are more stable than those with D₄ symmetry due to their additional H‐bonds between Gln sidechains. Second, for the self‐assembly of the cyclopeptides, there is a competition between parallel and antiparallel stacking modes in lower oligomers such as dimers. However, with an increasing degree of oligomerization, energetically there is an increased possibility for the cyclic peptides to take the parallel stacking mode in assembly. Finally, the synergetic effect of weak interactions is the fundamental driving force for cyclic peptides to form stable nanotubes. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

Effects of hydration on scale factors for ab initio force constants

Experimentally measured vibrational frequencies from the polar groups of peptides in aqueous solutions do not agree with frequencies calculated from scaled quantum mechanical force fields (SQMFF) using differential scale factors developed for molecules in the vapor phase. Measured stretching frequencies for carbonyl groups are more than 50 wavenumbers lower than the calculated values. On the other hand, frequencies for non-polar groups calculated using these scale factors are relatively accurate. Our goal is to develop a SQMFF that yields accurate calculated frequencies for peptides in aqueous solutions. To this end, we have calculated scale factors for ab initio force constants for formic acid, acetic acid, and acetone using a least squares fit of calculated and experimental frequencies. We compare these scale factors with changes observed in the ab initio force constants calculated for these molecules at various states of hydration. These force constants are calculated using fully optimized geometries for these hydrated molecules using the 4-31G basis. We present a comparison of the experimental and calculated frequencies, along with their potential energy distributions, for both vapor and aqueous phases. The results indicate that scale factors can simulate the effects of solvation on molecular force constants to yield accurate scaled ab initio force fields.     

Molecular modeling of class I and II alleles of the major histocompatibility complex in <Emphasis Type="Italic">Salmo salar</Emphasis>

Knowledge of the 3D structure of the binding groove of major histocompatibility (MHC) molecules, which play a central role in the immune response, is crucial to shed light into the details of peptide recognition and polymorphism. This work reports molecular modeling studies aimed at providing 3D models for two class I and two class II MHC alleles from Salmo salar (Sasa), as the lack of experimental structures of fish MHC molecules represents a serious limitation to understand the specific preferences for peptide binding. The reliability of the structural models built up using bioinformatic tools was explored by means of molecular dynamics simulations of their complexes with representative peptides, and the energetics of the MHC-peptide interaction was determined by combining molecular mechanics interaction energies and implicit continuum solvation calculations. The structural models revealed the occurrence of notable differences in the nature of residues at specific positions in the binding groove not only between human and Sasa MHC proteins, but also between different Sasa alleles. Those differences lead to distinct trends in the structural features that mediate the binding of peptides to both class I and II MHC molecules, which are qualitatively reflected in the relative binding affinities. Overall, the structural models presented here are a valuable starting point to explore the interactions between MHC receptors and pathogen-specific interactions and to design vaccines against viral pathogens.     

Delineating Binding Modes of Gal/GalNAc and Structural Elements of the Molecular Recognition of Tumor‐Associated Mucin Glycopeptides by the Human Macrophage Galactose‐Type Lectin

The human macrophage galactose‐type lectin (MGL) is a key physiological receptor for the carcinoma‐associated Tn antigen (GalNAc‐α‐1‐O‐Ser/Thr) in mucins. NMR and modeling‐based data on the molecular recognition features of synthetic Tn‐bearing glycopeptides by MGL are presented. Cognate epitopes on the sugar and matching key amino acids involved in the interaction were identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non‐glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for MGL binding, as is Ca²⁺. NMR data were complemented with molecular dynamics simulations and Corcema‐ST to establish a 3D view on the molecular recognition process between Gal, GalNAc, and the Tn‐presenting glycopeptides and MGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH–π contacts involving exclusively the NHAc moiety.     

Stiff-Stilbene Ligands Target G-Quadruplex DNA and Exhibit Selective Anticancer and Antiparasitic Activity

G-quadruplex nucleic acid structures have long been studied as anticancer targets whilst their potential in antiparasitic therapy has only recently been recognized and barely explored. Herein, we report the synthesis, biophysical characterization, and in vitro screening of a series of stiff-stilbene G4 binding ligands featuring different electronics, side-chain chemistries, and molecular geometries. The ligands display selectivity for G4 DNA over duplex DNA and exhibit nanomolar toxicity against Trypasanoma brucei and HeLa cancer cells whilst remaining up to two orders of magnitude less toxic to non-tumoral mammalian cell line MRC-5. Our study demonstrates that stiff-stilbenes show exciting potential as the basis of selective anticancer and antiparasitic therapies. To achieve the most efficient G4 recognition the scaffold must possess the optimal electronics, substitution pattern and correct molecular configuration.     

Macrocyclization of bis-indole quinolines for selective stabilization of G-quadruplex DNA structures

The recognition of G-quadruplex (G4) DNA structures as important regulatory elements in biological mechanisms, and the connection between G4s and the evolvement of different diseases, has sparked interest in developing small organic molecules targeting G4s. However, such compounds often lack drug-like properties and selectivity. Here, we describe the design and synthesis of a novel class of macrocyclic bis-indole quinolines based on their non-macrocyclic lead compounds. The effects of the macrocyclization on the ability to interact with G4 DNA structures were investigated using biophysical assays and molecular dynamic simulations. Overall, this revealed compounds with potent abilities to interact with and stabilize G4 structures and a clear selectivity for both G4 DNA over dsDNA and for parallel/hybrid G4 topologies, which could be attributed to the macrocyclic structure. Moreover, we obtained knowledge about the structure–activity relationship of importance for the macrocyclic design and how structural modifications could be made to construct improved macrocyclic compounds. Thus, the macrocyclization of G4 ligands can serve as a basis for the optimization of research tools to study G4 biology and potential therapeutics targeting G4-related diseases.

Macrocyclization improves the selectivity, affinity, and ability to stabilize G4 DNA structures.     

Modeling of peptides containing D-amino acids: implications on cyclization

Cyclic peptides are therapeutically attractive due to their high bioavailability, potential selectivity, and scaffold novelty. Furthermore, the presence of D-residues induces conformational preferences not followed by peptides consisting of naturally abundant L-residues. Therefore, comprehending how amino acids induce turns in peptides, subsequently facilitating cyclization, is significant in peptide design. Here, we performed 20-ns explicit-solvent molecular dynamics simulations for three diastereomeric peptides with stereochemistries: LLLLL, LLLDL, and LDLDL. Experimentally LLLLL and LDLDL readily cyclize, whereas LLLDL cyclizes in low yield. Simulations at 310 K produced conformations with inter-terminal hydrogen bonds that correlated qualitatively with the experimental cyclization trend. Energies obtained for representative structures from quantum chemical (B3LYP/PCM/cc-pVTZ//HF/6-31G*) calculations predicted pseudo-cyclic and extended conformations as the most stable for LLLLL and LLLDL, respectively, in agreement with the experimental data. In contrast, the most stable conformer predicted for peptide LDLDL was not a pseudo-cyclic structure. Moreover, D-residues preferred the experimentally less populated α_L rotamers even when simulations were performed at a higher temperature and with strategically selected starting conformations. Energies calculated with molecular mechanics were consistent only with peptide LLLLL. Thus, the conformational preferences obtained for the all L-amino acid peptide were in agreement with the experimental observations. Moreover, refinement of the force field is expected to provide far-reaching conformational sampling of peptides containing D-residues to further develop force field-based conformational-searching methods. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Quantitative Detection of G‐Quadruplex DNA in Live Cells Based on Photon Counts and Complex Structure Discrimination

G‐quadruplex DNA show structural polymorphism, leading to challenges in the use of selective recognition probes for the accurate detection of G‐quadruplexes in vivo. Herein, we present a tripodal cationic fluorescent probe, NBTE , which showed distinguishable fluorescence lifetime responses between G‐quadruplexes and other DNA topologies, and fluorescence quantum yield (Φ_f) enhancement upon G‐quadruplex binding. We determined two NBTE ‐G‐quadruplex complex structures with high Φ_f values by NMR spectroscopy. The structures indicated NBTE interacted with G‐quadruplexes using three arms through π–π stacking, differing from that with duplex DNA using two arms, which rationalized the higher Φ_f values and lifetime response of NBTE upon G‐quadruplex binding. Based on photon counts of FLIM, we detected the percentage of G‐quadruplex DNA in live cells with NBTE and found G‐quadruplex DNA content in cancer cells is 4‐fold that in normal cells, suggesting the potential applications of this probe in cancer cell detection.     

NON-SPECIFIC AND SPECIFIC RECOGNITION MECHANISMS OF BACTERIAL AND MAMMALIAN CELL MEMBRANES

The recognition of bacteria by mammalian cells, or vice versa, involves specific, i.e. ligand-receptor interactions, and nonspecific, physicochemical factors, e.g. surface charge and hydrophobicity. Specific interactions can be of non-immunological character, viz. carbohydrate-specified, lectin-like cell-cell association, conveyed by bacterial adhesins, e.g. fimbriae or mammalian cell appendages for instance on macrophages. Other bacterial adhesins bind to receptor substances adsorbed onto the mammalian cells like serum proteins, or molecules being part of the histocompatibility antigen complex, e.g. β2-microglobulin. Immunological recognition comprises association between antibody or complement-coated (opsonized) particles with Fc- and C3b-receptors on phagocytic cells (polymorphonuclear leukocytes, macrophages, Kuppfer cells). On the other hand, these apparently specific interactions between ligands and receptors identified at the molecular level, also achieve general physicochemical alterations.

The present communication reviews experimental data on the dualistic character of the association between bacteria and animal cells, i.e. the interplay between specific and non-specific factors that promote or counteract cell-cell recognition.     

An Octa-Urea [Pd2L4]4+ Cage that Selectively Binds to n-octyl-α-D-Mannoside

Designing compounds for the selective molecular recognition of carbohydrates is a challenging task for supramolecular chemists. Macrocyclic compounds that incorporate isophtalamide or bisurea spacers linking two aromatic moieties have proven effective for the selective recognition of all-equatorial carbohydrates. Here, we explore the molecular recognition properties of an octa-urea [Pd₂L₄]⁴⁺ cage complex ( 4 ). It was found that small anions like NO₃⁻ and BF₄⁻ bind inside 4 and inhibit binding of n-octyl glycosides. When the large non-coordinating anion ‘BAr^F’ was used, 4 showed excellent selectivity towards n-octyl-α-D-Mannoside with binding in the order of K_a≈16 M⁻¹ versus non-measurable affinities for other glycosides including n-octyl-β-D-Glucoside (in CH₃CN/H₂O 91 : 9).     

Molecular Recognition of the Hybrid‐2 Human Telomeric G‐Quadruplex by Epiberberine: Insights into Conversion of Telomeric G‐Quadruplex Structures

Human telomeres can form DNA G‐quadruplex (G4), an attractive target for anticancer drugs. Human telomeric G4s bear inherent structure polymorphism, challenging for understanding specific recognition by ligands or proteins. Protoberberines are medicinal natural‐products known to stabilize telomeric G4s and inhibit telomerase. Here we report epiberberine (EPI) specifically recognizes the hybrid‐2 telomeric G4 predominant in physiologically relevant K⁺ solution and converts other telomeric G4 forms to hybrid‐2, the first such example reported. Our NMR structure in K⁺ solution shows EPI binding induces extensive rearrangement of the previously disordered 5′‐flanking and loop segments to form an unprecedented four‐layer binding pocket specific to the hybrid‐2 telomeric G4; EPI recruits the (?1) adenine to form a “quasi‐triad” intercalated between the external tetrad and a T:T:A triad, capped by a T:T base pair. Our study provides structural basis for small‐molecule drug design targeting the human telomeric G4.     

Evaluation of Novel Third‐Strand Bases for the Recognition of a C⋅G Base Pair in the Parallel DNA Triple‐Helical Binding Motif

We describe the synthesis and the incorporation into oligonucleotides of the novel nucleoside building blocks 9, 10 , and 16 , carrying purine‐like double H‐bond‐acceptor bases. These base‐modified nucleosides were conceived to recognize selectively a cytosine⋅guanine (C⋅G) inversion site within a homopurine⋅homopyrimidine DNA duplex, when constituent of a DNA third strand designed to bind in the parallel binding motif. While building block 16 turned out to be incompatible with standard oligonucleotide‐synthesis conditions, UV/triplex melting experiments with third‐strand 15‐mers containing β‐D ‐nucleoside 6 (from 9 ) showed that recognition of the four natural Watson‐Crick base pairs follows the order G⋅C≈C⋅G>A⋅T>T⋅A. The recognition is sequence‐context sensitive, and G⋅C or C⋅G recognition does not involve protonated species of β‐D ‐nucleoside 6 . The data obtained fit (but do not prove) a structural model for C⋅G recognition via one conventional and one C−H⋅⋅⋅O H‐bond. The unexpected G⋅C recognition is best explained by third‐strand base intercalation. A comparison of the triplex binding properties of these new bases with those of 4‐deoxothymine (5‐methylpyrimidine‐2(1H)‐one, ^{4 H}T), previously shown to be C⋅G selective but energetically weak, is also described.     

The Thiophene “Sigma‐Hole” as a Concept for Preorganized,Specific Recognition of G⋅C Base Pairs in the DNA Minor Groove

In spite of its importance in cell function, targeting DNA is under‐represented in the design of small molecules. A barrier to progress in this area is the lack of a variety of modules that recognize G ? C base pairs (bp) in DNA sequences. To overcome this barrier, an entirely new design concept for modules that can bind to mixed G ? C and A ? T sequences of DNA is reported herein. Because of their successes in biological applications, minor‐groove‐binding heterocyclic cations were selected as the platform for design. Binding to A ? T sequences requires hydrogen‐bond donors whereas recognition of the G‐NH₂ requires an acceptor. The concept that we report herein uses pre‐organized N‐methylbenzimidazole (N‐MeBI) thiophene modules for selective binding with mixed bp DNA sequences. The interaction between the thiophene sigma hole (positive electrostatic potential) and the electron‐donor nitrogen of N‐MeBI preorganizes the conformation for accepting an hydrogen bond from G‐NH₂. The compound–DNA interactions were evaluated with a powerful array of biophysical methods and the results show that N‐MeBI‐thiophene monomer compounds can strongly and selectively recognize single G ? C bp sequences. Replacing the thiophene with other moieties significantly reduces binding affinity and specificity, as predicted by the design concept. These results show that the use of molecular features, such as sigma‐holes, can lead to new approaches for small molecules in biomolecular interactions.     

Helix Formation and Folding in γ‐Peptides and Their Vinylogues

A complete overview of all possible periodic structures with characteristic H‐bonding patterns is provided for oligomers composed of γ‐amino acids (γ‐peptides) and their vinylogues by a systematic conformational search on hexamer model compounds employing ab initio MO theory at various levels of approximation (HF/6‐31G*, DFT/B3LYP/6‐31G*, SCRF/HF/6‐31G*, PCM//HF/6‐31G*). A wide variety of structures with definite backbone conformations and H‐bonds formed in forward and backward directions along the sequence was found in this class of foldamers. All formally conceivable H‐bonded pseudocycles between 7‐ and 24‐membered rings are predicted in the periodic hexamer structures, which are mostly helices. The backbone elongation in comparison to α‐ and β‐peptides allows several possibilities to realize identical H‐bonding patterns. In good agreement with experimental data, helical structures with 14‐ and 9‐membered pseudocycles are most stable. It is shown that the introduction of an (E)‐double bond into the backbone of the γ‐amino acid constituents, which leads to vinylogous γ‐amino acids, supports the folding into helices with larger H‐bonded pseudocycles in the resulting vinylogous γ‐peptides. Due to the considerable potential for secondary‐structure formation, γ‐peptides and their vinylogues might be useful tools in peptide and protein design and even in material sciences.     

CH/pi hydrogen bonds determine the selectivity of the Src homology 2 domain to tyrosine phosphotyrosyl peptides: an ab initio fragment molecular orbital study

The CH/pi hydrogen bond is a weak molecular force occurring between CH groups (soft acids) and pi-systems (soft bases), and has been recognized to be important in the interaction of proteins with their specific ligands. For instance, it is well known that Src homology-2 protein (SH2) recognizes its specific pTyr peptide in two key regions, pTyr-binding region and specificity-determining region, by the use of attractive molecular forces, including the CH/pi hydrogen bond. We hypothesized that the CH/pi hydrogen bond plays a key role in determining the selectivity of SH2 proteins, and studied this issue by the ab initio fragment molecular orbital (FMO) method. The FMO calculations were carried out, at the HF/6-31G* and MP2/6-31G* level, for SH2 domains of Src, Grb2, P85alpha(N), Syk, and SAP, in complex with corresponding pTyr peptides. CH/pi hydrogen bonds have in fact been found to be important in stabilizing the structure of the complexes. We conclude that the CH/pi hydrogen bond plays an indispensable role in the recognition of SH2 domains with their specific pTyr peptides, thus playing a vital role in the signal transduction system.     

Simultaneous Stabilization and Multimerization of a Peptide α‐Helix by Stapling Polymerization

Maintaining specific conformations of peptide ligands is crucial for improving the efficacy of biological interactions. Here, a one‐pot polymerization strategy for stabilizing the α‐helical conformation of peptides while simultaneously constructing multimeric ligands is presented. The new method, termed stapling polymerization, uses radical polymerization between acryloylated peptide side chains and vinylic monomers. Studies with model peptides indicate that i, i+7 crosslinking is effective for the helix stabilization, whereas i, i+4 crosslinking is not. The stapling polymerization results in the formation of peptide–polyacrylamide conjugates that include ≈3–16 peptides in a single conjugate. This stapling polymerization provides a simple but powerful methodology to fabricate multimeric α‐helices that can further be developed to modulate multivalent biomacromolecular interactions.