Galectin-4 N-Terminal Domain: Binding Preferences Toward A and B Antigens With Different Peripheral Core Presentations.

The tandem-repeat Galectin-4 (Gal-4) contains two different domains covalently linked through a short flexible peptide. Both domains have been shown to bind preferentially to A and B histo blood group antigens with different affinities, although the binding details are not yet available. The biological relevance of these associations is unknown, although it could be related to its attributed role in pathogen recognition. The presentation of A and B histo blood group antigens in terms of peripheral core structures differs among tissues and from that of the antigen-mimicking structures produced by pathogens. Herein, the binding of the N-terminal domain of Gal-4 toward a group of differently presented A and B oligosaccharide antigens in solution has been studied through a combination of NMR, isothermal titration calorimetry (ITC), and molecular modeling. The data presented in this paper allow the identification of the specific effects that subtle chemical modifications within this antigenic family have in the binding to the N-terminal domain of Gal-4 in terms of affinity and intermolecular interactions, providing a structural-based rationale for the observed trend in the binding preferences.

Targeting the CRD F-face of Human Galectin-3 and Allosterically Modulating Glycan Binding by Angiostatic PTX008 and a Structurally Optimized Derivative.

Calix[4]arene PTX008 is an angiostatic agent that inhibits tumor growth in mice by binding to galectin-1, a ß-galactoside-binding lectin. To assess the affinity profile of PTX008 for galectins, we used 15 N,1 H HSQC NMR spectroscopy to show that PTX008 also binds to galectin-3 (Gal-3), albeit more weakly. We identified the contact site for PTX008 on the F-face of the Gal-3 carbohydrate recognition domain. STD NMR revealed that the hydrophobic phenyl ring crown of the calixarene is the binding epitope. With this information, we performed molecular modeling of the complex to assist in improving the rather low affinity of PTX008 for Gal-3. By removing the N-dimethyl alkyl chain amide groups, we produced PTX013 whose reduced alkyl chain length and polar character led to an approximately eightfold stronger binding than PTX008. PTX013 also binds Gal-1 more strongly than PTX008, whereas neither interacts strongly, if at all, with Gal-7. In addition, PTX013, like PTX008, is an allosteric inhibitor of galectin binding to the canonical ligand lactose. This study broadens the scope for galectin targeting by calixarene-based compounds and opens the perspective for selective galectin blocking.

Amino Acid-Based Synthesis and Glycosidase Inhibition of Cyclopropane-Containing Iminosugars.

Synthesis of four iminosugars fused to a cyclopropane ring is described using l-serine as the chiral pool. The key steps are large-scale preparation of an α,ß-unsaturated piperidinone followed by completely stereoselective sulfur ylide cyclopropanation. Stereochemistry of compounds has been studied by nuclear Overhauser effect spectroscopy (NOESY) experiments and 1H homonuclear decoupling to measure constant couplings. The activity of these compounds against different glycosidases has been evaluated. Although inhibition activity was low (compound 8a presents a (K i) of 1.18 mM against ß-galactosidase from Escherichia coli), interestingly, we found that compounds 8a and 8b increase the activity of neuraminidase from Vibrio cholerae up to 100%.

Molecular Recognition in C-Type Lectins: The Cases of DC-SIGN, Langerin, MGL, and L-Sectin.

Carbohydrates play a pivotal role in intercellular communication processes. In particular, glycan antigens are key for sustaining homeostasis, helping leukocytes to distinguish damaged tissues and invading pathogens from healthy tissues. From a structural perspective, this cross-talk is fairly complex, and multiple membrane proteins guide these recognition processes, including lectins and Toll-like receptors. Since the beginning of this century, lectins have become potential targets for therapeutics for controlling and/or avoiding the progression of pathologies derived from an incorrect immune outcome, including infectious processes, cancer, or autoimmune diseases. Therefore, a detailed knowledge of these receptors is mandatory for the development of specific treatments. In this review, we summarize the current knowledge about four key C-type lectins whose importance has been steadily growing in recent years, focusing in particular on how glycan recognition takes place at the molecular level, but also looking at recent progresses in the quest for therapeutics.

Amoxicillin Inactivation by Thiol-Catalyzed Cyclization Reduces Protein Haptenation and Antibacterial Potency.

Serum and cellular proteins are targets for the formation of adducts with the ß-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that several thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine, and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. Increased diketopiperazine conversion is also observed with human serum albumin but not with L-cysteine, which mainly forms the amoxicilloyl amide. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other ß-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions.

Complete oxidation of hydroxymethylfurfural to furandicarboxylic acid by aryl-alcohol oxidase.

BACKGROUND: 5-Hydroxymethylfurfural (HMF) is a highly valuable platform chemical that can be obtained from plant biomass carbohydrates. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a renewable substitute for the petroleum-based terephthalic acid in polymer production. RESULTS: Aryl-alcohol oxidase (AAO) from the white-rot fungus Pleurotus eryngii is able to oxidize HMF and its derivative 2,5-diformylfuran (DFF) producing formylfurancarboxylic acid (FFCA) thanks to its activity on benzylic alcohols and hydrated aldehydes. Here, we report the ability of AAO to produce FDCA from FFCA, opening up the possibility of full oxidation of HMF by this model enzyme. During HMF reactions, an inhibitory effect of the H2O2 produced in the first two oxidation steps was found to be the cause of the lack of AAO activity on FFCA. In situ monitoring of the whole reaction by 1H-NMR confirmed the absence of any unstable dead-end products, undetected in the HPLC analyses, that could be responsible for the incomplete conversion. The deleterious effect of H2O2 was confirmed by successful HMF conversion into FDCA when the AAO reaction was carried out in the presence of catalase. On the other hand, no H2O2 formation was detected during the slow FFCA conversion by AAO in the absence of catalase, in contrast to typical oxidase reaction with HMF and DFF, suggesting an alternative mechanism as reported in some reactions of related flavo-oxidases. Moreover, several active-site AAO variants that yield nearly complete conversion in shorter reaction times than the wild-type enzyme have been identified. CONCLUSIONS: The use of catalase to remove H2O2 from the reaction mixture leads to 99% conversion of HMF into FDCA by AAO and several improved variants, although the mechanism of peroxide inhibition of the AAO action on the aldehyde group of FFCA is not fully understood.

Unraveling Sugar Binding Modes to DC-SIGN by Employing Fluorinated Carbohydrates.

A fluorine nuclear magnetic resonance (19F-NMR)-based method is employed to assess the binding preferences and interaction details of a library of synthetic fluorinated monosaccharides towards dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN), a lectin of biomedical interest, which is involved in different viral infections, including HIV and Ebola, and is able to recognize a variety of self- and non-self-glycans. The strategy employed allows not only screening of a mixture of compounds, but also obtaining valuable information on the specific sugar-protein interactions. The analysis of the data demonstrates that monosaccharides Fuc, Man, Glc, and Gal are able to bind DC-SIGN, although with decreasing affinity. Moreover, a new binding mode between Man moieties and DC-SIGN, which might have biological implications, is also detected for the first time. The combination of the 19F with standard proton saturation transfer difference (1H-STD-NMR) data, assisted by molecular dynamics (MD) simulations, permits us to successfully define this new binding epitope, where Man coordinates a Ca2+ ion of the lectin carbohydrate recognition domain (CRD) through the axial OH-2 and equatorial OH-3 groups, thus mimicking the Fuc/DC-SIGN binding architecture.

Insights into real-time chemical processes in a calcium sensor protein-directed dynamic library.

Dynamic combinatorial chemistry (DCC) has proven its potential in drug discovery speeding the identification of modulators of biological targets. However, the exchange chemistries typically take place under specific reaction conditions, with limited tools capable of operating under physiological parameters. Here we report a catalyzed protein-directed DCC working at low temperatures that allows the calcium sensor NCS-1 to find the best ligands in situ. Ultrafast NMR identifies the reaction intermediates of the acylhydrazone exchange, tracing the molecular assemblies and getting a real-time insight into the essence of DCC processes at physiological pH. Additionally, NMR, X-ray crystallography and computational methods are employed to elucidate structural and mechanistic aspects of the molecular recognition event. The DCC approach leads us to the identification of a compound stabilizing the NCS-1/Ric8a complex and whose therapeutic potential is proven in a Drosophila model of disease with synaptic alterations.

A Novel Redox-Sensing Histidine Kinase That Controls Carbon Catabolite Repression in Azoarcus sp. CIB.

We have identified and characterized the AccS multidomain sensor kinase that mediates the activation of the AccR master regulator involved in carbon catabolite repression (CCR) of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB. A truncated AccS protein that contains only the soluble C-terminal autokinase module (AccS') accounts for the succinate-dependent CCR control. In vitro assays with purified AccS' revealed its autophosphorylation, phosphotransfer from AccS'∼P to the Asp60 residue of AccR, and the phosphatase activity toward its phosphorylated response regulator, indicating that the equilibrium between the kinase and phosphatase activities of AccS' may control the phosphorylation state of the AccR transcriptional regulator. Oxidized quinones, e.g., ubiquinone 0 and menadione, switched the AccS' autokinase activity off, and three conserved Cys residues, which are not essential for catalysis, are involved in such inhibition. Thiol oxidation by quinones caused a change in the oligomeric state of the AccS' dimer resulting in the formation of an inactive monomer. This thiol-based redox switch is tuned by the cellular energy state, which can change depending on the carbon source that the cells are using. This work expands the functional diversity of redox-sensitive sensor kinases, showing that they can control new bacterial processes such as CCR of the anaerobic catabolism of aromatic compounds. The AccSR two-component system is conserved in the genomes of some betaproteobacteria, where it might play a more general role in controlling the global metabolic state according to carbon availability.IMPORTANCE Two-component signal transduction systems comprise a sensor histidine kinase and its cognate response regulator, and some have evolved to sense and convert redox signals into regulatory outputs that allow bacteria to adapt to the altered redox environment. The work presented here expands knowledge of the functional diversity of redox-sensing kinases to control carbon catabolite repression (CCR), a phenomenon that allows the selective assimilation of a preferred compound among a mixture of several carbon sources. The newly characterized AccS sensor kinase is responsible for the phosphorylation and activation of the AccR master regulator involved in CCR of the anaerobic degradation of aromatic compounds in the betaproteobacterium Azoarcus sp. CIB. AccS seems to have a thiol-based redox switch that is modulated by the redox state of the quinone pool. The AccSR system is conserved in several betaproteobacteria, where it might play a more general role controlling their global metabolic state.

Glycosylated Cell-Penetrating Peptides (GCPPs).

The cell membrane regulates the exchange of molecules and information with the external environment. However, this control barrier hinders the delivery of exogenous bioactive molecules that can be applied to correct cellular malfunctions. Therefore, the traffic of macromolecules across the cell membrane represents a great challenge for the development of the next generation of therapies and diagnostic methods. Cell-penetrating peptides are short peptide sequences capable of delivering a broad range of biomacromolecules across the cellular membrane. However, penetrating peptides still suffer from limitations, mainly related to their lack of specificity and potential toxicity. Glycosylation has emerged as a potential promising strategy for the biological improvement of synthetic materials. In this work we have developed a new convergent strategy for the synthesis of penetrating peptides functionalized with glycan residues by an oxime bond connection. The uptake efficiency and intracellular distribution of these glycopeptides have been systematically characterized by means of flow cytometry and confocal microscopy and in zebrafish animal models. The incorporation of these glycan residues into the peptide structure influenced the internalization efficiency and cellular toxicity of the resulting glycopeptide hybrids in the different cell lines tested. The results reported herein highlight the potential of the glycosylation of penetrating peptides to modulate their activity.

Differential recognition of Haemophilus influenzae whole bacterial cells and isolated lipooligosaccharides by galactose-specific lectins.

Bacterial surfaces are decorated with carbohydrate structures that may serve as ligands for host receptors. Based on their ability to recognize specific sugar epitopes, plant lectins are extensively used for bacteria typing. We previously observed that the galactose-specific agglutinins from Ricinus communis (RCA) and Viscum album (VAA) exhibited differential binding to nontypeable Haemophilus influenzae (NTHi) clinical isolates, their binding being distinctly affected by truncation of the lipooligosaccharide (LOS). Here, we examined their binding to the structurally similar LOS molecules isolated from strains NTHi375 and RdKW20, using microarray binding assays, saturation transfer difference NMR, and molecular dynamics simulations. RCA bound the LOSRdKW20 glycoform displaying terminal Galß(1,4)Glcß, whereas VAA recognized the Galα(1,4)Galß(1,4)Glcß epitope in LOSNTHi375 but not in LOSRdKW20, unveiling a different presentation. Binding assays to whole bacterial cells were consistent with LOSNTHi375 serving as ligand for VAA, and also suggested recognition of the glycoprotein HMW1. Regarding RCA, comparable binding to NTHi375 and RdKW20 cells was observed. Interestingly, an increase in LOSNTHi375 abundance or expression of HMW1 in RdKW20 impaired RCA binding. Overall, the results revealed that, besides the LOS, other carbohydrate structures on the bacterial surface serve as lectin ligands, and highlighted the impact of the specific display of cell surface components on lectin binding.

Structure and N-acetylglucosamine binding of the distal domain of mouse adenovirus 2 fibre.

Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenoviruses, it is a member of the genus Mastadenovirus, family Adenoviridae. The MAdV-2 genome has a single fibre gene that expresses a 787 residue-long protein. Through analogy to other adenovirus fibre proteins, it is expected that the carboxy-terminal virus-distal head domain of the fibre is responsible for binding to the host cell, although the natural receptor is unknown. The putative head domain has little sequence identity to adenovirus fibres of known structure. In this report, we present high-resolution crystal structures of the carboxy-terminal part of the MAdV-2 fibre. The structures reveal a domain with the typical adenovirus fibre head topology and a domain containing two triple ß-spiral repeats of the shaft domain. Through glycan microarray profiling, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and site-directed mutagenesis, we show that the fibre specifically binds to the monosaccharide N-acetylglucosamine (GlcNAc). The crystal structure of the complex reveals that GlcNAc binds between the AB and CD loops at the top of each of the three monomers of the MAdV-2 fibre head. However, infection competition assays show that soluble GlcNAc monosaccharide and natural GlcNAc-containing polymers do not inhibit infection by MAdV-2. Furthermore, site-directed mutation of the GlcNAc-binding residues does not prevent the inhibition of infection by soluble fibre protein. On the other hand, we show that the MAdV-2 fibre protein binds GlcNAc-containing mucin glycans, which suggests that the MAdV-2 fibre protein may play a role in viral mucin penetration in the mouse gut.

Avenues to Characterize the Interactions of Extended N-Glycans with Proteins by NMR Spectroscopy: The Influenza Hemagglutinin Case.

Long-chain multiantenna N-glycans are extremely complex molecules. Their inherent flexibility and the presence of repetitions of monosaccharide units in similar chemical environments hamper their full characterization by X-ray diffraction or standard NMR methods. Herein, the successful conformational and interaction analysis of a sialylated tetradecasaccharide N-glycan presenting two LacNAc repetitions at each arm is presented. This glycan has been identified as the receptor of the hemagglutinin protein of pathogenic influenza viruses. To accomplish this study, a N-glycan conjugated with a lanthanide binding tag has been synthesized, enabling analysis of the system by paramagnetic NMR. Under paramagnetic conditions, the NMR signals of each sugar unit in the glycan have been determined. Furthermore, a detailed binding epitope of the tetradecasaccharide N-glycan in the presence of HK/68 hemagglutinin is described.

Deciphering the Inhibition of the Neuronal Calcium Sensor 1 and the Guanine Exchange Factor Ric8a with a Small Phenothiazine Molecule for the Rational Generation of Therapeutic Synapse Function Regulators.

Protein-protein interactions (PPIs) are known to play an essential role between the neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor Ric8a to regulate synapse function, emerging as a druggable interface for synaptopathies such as the fragile X syndrome (FXS). Recently, the phenothiazine FD44 has been identified as an inhibitor of this PPI, decreasing the abnormally high synapse number and enhancing associative learning in a FXS animal model. Here, we have integrated advanced experimental and computational studies to obtain important structural insights into Drosophila NCS-1/FD44 recognition to understand the basis of its affinity and specificity and generate improved PPI regulators. This has allowed the identification of a new small drug-like molecule, IGS-1.76, which efficiently inhibits the human NCS-1/Ric8a complex with improved binding potency. The crystal structure of the Drosophila NCS-1/IGS-1.76 complex demonstrates that the new inhibitor, although chemically different from FD44, shares the same mechanism of action and constitutes a new hit candidate for FXS.

Chameleon-like behavior of indolylpiperidines in complex with cholinesterases targets: Potent butyrylcholinesterase inhibitors.

Alzheimer's disease (AD) is the most common form of dementia worldwide with an increasing prevalence for the next years. The multifactorial nature of AD precludes the design of new drugs directed to a single target being probably one of the reasons for recent failures. Therefore, dual binding site acetylcholinesterase (AChE) inhibitors have been revealed as cognitive enhancers and ß-amyloid modulators offering an alternative in AD therapy field. Based on the dual ligands NP61 and donepezil, the present study reports the synthesis of a series of indolylpiperidines hybrids to optimize the NP61 structure preserving the indole nucleus, but replacing the tacrine moiety of NP61 by benzyl piperidine core found in donepezil. Surprisingly, this new family of indolylpiperidines derivatives showed very potent and selective hBuChE inhibition. Further studies of NMR and molecular dynamics have showed the capacity of these hybrid molecules to change their bioactive conformation depending on the binding site, being capable to inhibit with different shapes BuChE and residually AChE.

Breaking the Limits in Analyzing Carbohydrate Recognition by NMR Spectroscopy: Resolving Branch-Selective Interaction of a Tetra-Antennary N-Glycan with Lectins.

The biological recognition of complex-type N-glycans is part of many key physiological and pathological events. Despite their importance, the structural characterization of these events remains unsolved. The inherent flexibility of N-glycans hampers crystallization and the chemical equivalence of individual branches precludes their NMR characterization. By using a chemoenzymatically synthesized tetra-antennary N-glycan conjugated to a lanthanide binding tag, the NMR signals under paramagnetic conditions discriminated all four N-acetyl lactosamine antennae with unprecedented resolution. The NMR data revealed the conformation of the N-glycan and permitted for the first time the direct identification of individual branches involved in the recognition by two N-acetyllactosamine-binding lectins, Datura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA120).

From dual binding site acetylcholinesterase inhibitors to allosteric modulators: A new avenue for disease-modifying drugs in Alzheimer's disease.

The lack of an effective treatment for Alzheimer' disease (AD), an increasing prevalence and severe neurodegenerative pathology boost medicinal chemists to look for new drugs. Currently, only acethylcholinesterase (AChE) inhibitors and glutamate antagonist have been approved to the palliative treatment of AD. Although they have a short-term symptomatic benefits, their clinical use have revealed important non-cholinergic functions for AChE such its chaperone role in beta-amyloid toxicity. We propose here the design, synthesis and evaluation of non-toxic dual binding site AChEIs by hybridization of indanone and quinoline heterocyclic scaffolds. Unexpectely, we have found a potent allosteric modulator of AChE able to target cholinergic and non-cholinergic functions by fixing a specific AChE conformation, confirmed by STD-NMR and molecular modeling studies. Furthermore the promising biological data obtained on human neuroblastoma SH-SY5Y cell assays for the new allosteric hybrid 14, led us to propose it as a valuable pharmacological tool for the study of non-cholinergic functions of AChE, and as a new important lead for novel disease modifying agents against AD.

Mite allergoids coupled to nonoxidized mannan from Saccharomyces cerevisae efficiently target canine dendritic cells for novel allergy immunotherapy in veterinary medicine.

We have recently reported that grass pollen allergoids conjugated with nonoxidized mannan of Saccharomyces cerevisae using glutaraldehyde results in a novel hypoallergenic mannan-allergen complex with improved properties for allergen vaccination. Using this approach, human dendritic cells show a better allergen uptake and cytokine profile production (higher IL-10/IL-4 ratio) for therapeutic purposes. Here we aim to address whether a similar approach can be extended to dogs using canine dendritic cells. Six healthy Spanish Greyhound dogs were used as blood donors to obtain canine dendritic cells (DC) derived from peripheral blood monocytes. Allergens from Dermatophagoides farinae mite were polymerized and conjugated with nonoxidized mannan. Nuclear magnetic resonance (NMR), gel electrophoresis (SDS-PAGE), immunoblotting and IgE-ELISA inhibition studies were conducted to evaluate the main characteristics of the allergoid obtained. Mannan-allergen conjugate and controls were assayed in vitro for canine DC uptake and production of IL-4 and IL-10. The results indicate that the conjugation of D. farinae allergens with nonoxidized mannan was feasible using glutaraldehyde. The resulting product was a polymerized structure showing a high molecular weight as detected by NMR and SDS-PAGE analysis. The mannan-allergen conjugate was hypoallergenic with a reduced reactivity with specific dog IgE. An increase in both allergen uptake and IL-10/IL-4 ratio was obtained when canine DCs were incubated with the mannan-allergen conjugate, as compared with the control allergen preparations (unmodified D. farinae allergens and oxidized mannan-allergen conjugate). We conclude that hypoallergenic D. farinae allergens coupled to nonoxidized mannan is a novel allergen preparation suitable for canine allergy immunotherapy targeting dendritic cells.

NMR and Molecular Recognition of N-Glycans: Remote Modifications of the Saccharide Chain Modulate Binding Features.

Glycans play a key role as recognition elements in the communication of cells and other organisms. Thus, the analysis of carbohydrate-protein interactions has gained significant importance. In particular, nuclear magnetic resonance (NMR) techniques are considered powerful tools to detect relevant features in the interaction between sugars and their natural receptors. Here, we present the results obtained in the study on the molecular recognition of different mannose-containing glycans by Pisum sativum agglutinin. NMR experiments supported by Corcema-ST analysis, isothermal titration calorimetry (ITC) experiments, and molecular dynamics (MD) protocols have been successfully applied to unmask important binding features and especially to determine how a remote branching substituent significantly alters the binding mode of the sugar entity. These results highlight the key influence of common structural modifications in natural glycans on molecular recognition processes and underscore their importance for the development of biomedical applications.

Fluoroacetamide Moieties as NMR Spectroscopy Probes for the Molecular Recognition of GlcNAc-Containing Sugars: Modulation of the CH-π Stacking Interactions by Different Fluorination Patterns.

We herein propose the use of fluoroacetamide and difluoroacetamide moieties as sensitive tags for the detection of sugar-protein interactions by simple 1 H and/or 19 F NMR spectroscopy methods. In this process, we have chosen the binding of N,N'-diacetyl chitobiose, a ubiquitous disaccharide fragment in glycoproteins, by wheat-germ agglutinin (WGA), a model lectin. By using saturation-transfer difference (STD)-NMR spectroscopy, we experimentally demonstrate that, under solution conditions, the molecule that contained the CHF2 CONH- moiety is the stronger aromatic binder, followed by the analogue with the CH2 FCONH- group and the natural molecule (with the CH3 CONH- fragment). In contrast, the molecule with the CF3 CONH- isoster displayed the weakest intermolecular interaction (one order of magnitude weaker). Because sugar-aromatic CH-π interactions are at the origin of these observations, these results further contribute to the characterization and exploration of these forces and offer an opportunity to use them to unravel complex recognition processes.