Microarray Analysis of Oligosaccharide‐Mediated Multivalent Carbohydrate–Protein Interactions and Their Heterogeneity

Carbohydrate–protein interactions (CPIs) are involved in a wide range of biological phenomena. Hence, the characterization and presentation of carbohydrate epitopes that closely mimic the natural environment is one of the long‐term goals of glycosciences. Inspired by the multivalency, heterogeneity and nature of carbohydrate ligand‐mediated interactions, we constructed a combinatorial library of mannose and galactose homo‐ and hetero‐glycodendrons to study CPIs. Microarray analysis of these glycodendrons with a wide range of biologically important plant and animal lectins revealed that oligosaccharide structures and heterogeneity interact with each other to alter binding preferences.     

Anti‐HIV‐1 Peptide Derivatives Based on the HIV‐1 Co‐receptor CXCR4

The human immunodeficiency virus type 1 (HIV‐1) uses CD4 and the co‐receptor CCR5 or CXCR4 in the process of cell entry. The negatively charged extracellular domains of CXCR4 (CXCR4‐ED) interact with positive charges on the V3 loop of gp120, facilitating binding via electrostatic interactions. The presence of highly conserved positively charged residues in the V3 loop suggests that CXCR4‐ED‐derived inhibitors might be broadly effective inhibitors. Synthetic peptide derivatives were evaluated for anti‐HIV‐1 activity. The 39‐mer extracellular N‐terminal region (NT) was divided into three fragments with 10‐mer overlapping sites ( N1 – N3 ), and these linear peptides were synthesized. Peptide N1 contains Met 1–Asp 20 and shows significant anti‐HIV‐1 activity. Extracellular loops 1 and 2 (ECL1 and 2) were mimicked by cyclic peptides C1 and C2 , which were synthesized by chemoselective cyclization. Cyclic peptides C1 and C2 show higher anti‐HIV‐1 activity than their linear peptide counterparts, L1 and L2 . The cytotoxicities of C1 and C2 are lower than those of L1 and L2 . These results indicate that Met 1–Asp 20 segments of the NT and cyclic peptides of ECL1 and ECL2 are potent anti‐HIV‐1 drug candidates.     

Helix‐Grafted Pleckstrin Homology Domains Suppress HIV‐1 Infection of CD4‐Positive Cells

The size, functional group diversity and three‐dimensional structure of proteins often allow these biomolecules to bind disease‐relevant structures that challenge or evade small‐molecule discovery. Additionally, folded proteins are often much more stable in biologically relevant environments compared to their peptide counterparts. We recently showed that helix‐grafted display—extensive resurfacing and elongation of an existing solvent‐exposed helix in a pleckstrin homology (PH) domain—led to a new protein that binds a surrogate of HIV‐1 gp41, a validated target for inhibition of HIV‐1 entry. Expanding on this work, we prepared a number of human‐derived helix‐grafted‐display PH domains of varied helix length and measured properties relevant to therapeutic and basic research applications. In particular, we showed that some of these new reagents expressed well as recombinant proteins in Escherichia coli, were relatively stable in human serum, bound a mimic of pre‐fusogenic HIV‐1 gp41 in vitro and in complex biological environments, and significantly lowered the incidence of HIV‐1 infection of CD4‐positive cells.     

Non‐natural Peptide Triazole Antagonists of HIV‐1 Envelope gp120

We investigated the derivation of non‐natural peptide triazole dual receptor site antagonists of HIV‐1 Env gp120 to establish a pathway for developing peptidomimetic antiviral agents. Previously we found that the peptide triazole HNG‐156 [R‐I‐N‐N‐I‐X‐W‐S‐E‐A‐M‐M‐CONH₂, in which X=ferrocenyltriazole‐Pro (FtP)] has nanomolar binding affinity to gp120, inhibits gp120 binding to CD4 and the co‐receptor surrogate mAb 17b, and has potent antiviral activity in cell infection assays. Furthermore, truncated variants of HNG‐156, typified by UM‐24 (Cit‐N‐N‐I‐X‐W‐S‐CONH₂) and containing the critical central stereospecific ^LX‐^LW cluster, retain the functional characteristics of the parent peptide triazole. In the current work, we examined the possibility of replacing natural with unnatural residue components in UM‐24 to the greatest extent possible. The analogue with the critical “hot spot” residue Trp 6 replaced with L ‐3‐benzothienylalanine (Bta) (KR‐41), as well as a completely non‐natural analogue containing D ‐amino acid substitutions outside the central cluster (KR‐42, ^DCit‐^DN‐^DN‐^DI‐X‐Bta‐^DS‐CONH₂), retained the dual receptor site antagonism/antiviral activity signature. The results define differential functional roles of subdomains within the peptide triazole and provide a structural basis for the design of metabolically stable peptidomimetic inhibitors of HIV‐1 Env gp120.     

Biological Evaluation of Multivalent Lewis X–MGL‐1 Interactions

Myeloid C‐type lectin receptors (CLRs) expressed by antigen‐presenting cells are pattern‐recognition receptors involved in the recognition of pathogens as well as of self‐antigens. The interaction of carbohydrate ligands with a CLR can trigger immune responses. Although several CLR ligands are known, there is limited insight into CLR targeting by carbohydrate ligands. The weak affinity of lectin–carbohydrate interactions often renders multivalent carbohydrate presentation necessary. Here, we have analyzed the impact of multivalent presentation of the trisaccharide Lewis X (Le^X) epitope on its interaction with the CLR macrophage galactose‐type lectin‐1 (MGL‐1). Glycan arrays, including N‐glycan structures with terminal Le^X, were prepared by enzymatic extension of immobilized synthetic core structures with two recombinant glycosyltransferases. Incubation of arrays with an MGL‐1‐hFc fusion protein showed up to tenfold increased binding to multiantennary N‐glycans displaying Le^X structures, compared to monovalent Le^X trisaccharide. Multivalent presentation of Le^X on the model antigen ovalbumin (OVA) led to increased cytokine production in a dendritic cell /T cell coculture system. Furthermore, immunization of mice with Le^X‐OVA conjugates modulated cytokine production and the humoral response, compared to OVA alone. This study provides insights into how multivalent carbohydrate–lectin interactions can be exploited to modulate immune responses.     

The Multivalent Effect in Glycosidase Inhibition: Probing the Influence of Valency,Peripheral Ligand Structure,and Topology with Cyclodextrin‐Based Iminosugar Click Clusters

In view of recent reports of a strong multivalent effect in glycosidase inhibition, a library of β‐CD‐based multivalent iminosugars has been efficiently synthesized by way of Cu^I‐catalyzed azide–alkyne cycloaddition (CuAAC). In combination with the first application of isothermal titration calorimetry (ITC) experiments to the study of multivalent iminosugar–enzyme interactions, the inhibition properties of these click clusters were evaluated on a panel of glycosidases. The structural parameters that were varied include valency, peripheral ligand structure, and topology. The inhibition results obtained with the iminosugar clusters further highlight the importance of multivalency in the inhibition of α‐mannosidase. Generally, the evaluated multivalent iminosugars displayed comparable thermodynamic signatures of binding towards α‐mannosidase (Jack bean): that is, large negative enthalpies of complexation coupled with small entropies of either sign. In addition, the enthalpy–entropy compensation observed in all tested cases may be attributed to a common mechanism of dissociation for the enzyme–multivalent iminosugar interactions. The measured binding stoichiometries indicated that each iminosugar cluster interacts with no more than one protein molecule.     

Tautomerism and Magnesium Chelation of HIV‐1 Integrase Inhibitors: A Theoretical Study

The tautomerism and corresponding transition states of four authentic HIV‐1 integrase (IN) inhibitor prototype structures, α,γ‐diketo acid, α,γ‐diketotriazole, dihydroxypyrimidine carboxamide and 4‐quinolone‐3‐carboxylic acid, were investigated at the B3LYP/6‐311++G(d,p) level in vacuum and in aqueous solvent models. To study the possible chelating modes of these tautomers with two magnesium ions—a process important for inhibition—we modeled an assembly of three formic acids, four water molecules and two Mg²⁺ ions as a template mimicking the binding site of IN. The DFT calculation results show that deprotonated enolized or phenolic hydroxy groups of specific tautomers in water lead to the most stable complexes, with the two magnesium ions separated by a distance of approximately 3.70 to 3.74 Å, and with each magnesium ion at the center of an octahedron. The drug candidate GS‐9137 (Gilead), based on the 4‐quinolone‐3‐carboxylic acid scaffold, and its analogues form similar but different chelating modes. When one water molecule in the complex is replaced by a methanol molecule, which mimics the terminal 3′‐OH of viral DNA, a good chelating complex is retained. This supports the hypothesis that, in the binding site of IN after 3′‐processing, the terminal 3′‐OH of viral DNA interacts with one Mg²⁺ by chelation.     

The Characteristic Structure of Anti‐HIV Actinohivin in Complex with Three HMTG D1 Chains of HIV‐gp120

The anti‐HIV lectin actinohivin (AH) specifically interacts with HMTG (high‐mannose‐type glycan), which is attached to the glycoprotein gp120 of HIV‐1 in a process in which the three branched mannotriose chains (D1, D2, and D3) of HMTG exhibit different binding affinities, it being estimated that that of D1 is the strongest, that of D3 is weaker, and that of D2 is undetectable. These properties have been ascribed to the stereochemical differences in linkages between the second and the third mannose residues of the three chains. In order to clarify the interaction geometry between AH and the major target D1, an X‐ray determination of the crystal structure of AH in complex with D1—which is α(1,2)mannotriose composed of three mannose (Man) residues linked together only by α(1,2) bonding—has been performed. In each of the three D1‐binding pockets of AH, two Man residues of D1 are accommodated at zones 1 and 2 in the pocket, in the same way as those found in the α(1,2)mannobiose‐bound AH crystals. However, an OMIT map shows poor densities at both ends of the two residues. This suggests the existence of positional disorder of D1 in the pocket: the two zones are each occupied by two Man residues in two different modes, with mode A involving the Man1 and Man2 residues and mode B the Man2 and Man3 residues. In each mode, D1 is stabilized by adopting a double‐bracket‐shaped conformation through C?H ??? O interactions. In mode B, however, the Man1 residue, which is the most sensitive residue to AH binding, protrudes wholly into the solvent region without contacts with AH. In mode A, in contrast, the Man3 residue interacts with the essential hydrophobic amino acid residues (Tyr and Leu conserved between the three pockets) of AH. Therefore, mode A is likely to be the one that occurs when whole HMTG is bound. In this mode, the two hydroxy groups (O3 and O4) of the Man2 residue are anchored in zone 2 by four hydrogen bonds with Asp, Asn, and Tyr residues of AH. In addition, it has been found that an isolated water molecule buried in the hydrophobic long loop bridges between Asp of AH and the hydroxy group of Man2 through hydrogen bonds. The most interesting feature is found in the interaction of the Man1 and Man3 residues with AH. All eight hydroxy groups of the two residues are completely exposed in the solvent region, whereas their hydrophobic parts make contacts with a Leu residue and two Tyr residues so that the shape of D1 and the surface of AH fit well over a wide area. These structural characteristics are potentially useful for development of AH to produce more effective antiretroviral drugs to suppress the infectious expansion of HIV/AIDS and to help expedite an end to the HIV/AIDS pandemic in the near future.     

Development and Characterization of Peptidic Fusion Inhibitors Derived from HIV‐1 gp41 with Partial D‐Amino Acid Substitutions

Interactions between C34 and N36 : Synthetic peptides with D ‐amino acid substitutions that mimic the human immunodeficiency virus (HIV) gp41 HR2 region may lead to new peptidic anti‐HIV‐1 drugs that retain potent antiviral activity while being more resistant to proteolytic degradation.

     

Covalent Inhibition of HIV‐1 Integrase by N‐Succinimidyl Peptides

We present a new approach for the covalent inhibition of HIV‐1 integrase (IN) by an LEDGF/p75‐derived peptide modified with an N‐terminal succinimide group. The covalent inhibition is mediated by direct binding of the succinimide to the amine group of a lysine residue in IN. The peptide serves as a specific recognition sequence for the target protein, while the succinimide serves as the binding moiety. The combination of a readily synthesizable peptide precursor with easy and efficient binding to the target protein makes this approach a promising new strategy for designing lead compounds.     

Induced‐Fit Docking Approach Provides Insight into the Binding Mode and Mechanism of Action of HIV‐1 Integrase Inhibitors

A three‐dimensional model of a complex between HIV‐1 integrase (IN), viral DNA, and metal ions that we recently built was used as a target for a docking method (induced‐fit docking, IFD) that accurately predicts ligand binding modes and concomitant structural changes in the receptor. Six different well‐known integrase strand transfer inhibitors (INSTIs): L‐708,906, L‐731,988, S‐1360, L‐870,810, raltegravir, and elvitegravir were thus used as ligands for our docking simulations. The obtained IFD results are consistent with the mechanism of action proposed for this class of IN inhibitors, that is, metal chelating/binding agents. This study affords new insight into the possible mechanism of inhibition and binding conformations for INSTIs. The impact on our hypothesis of specific mutations associated with IN inhibitor resistance was also evaluated. All these findings might have implications for integrase‐directed HIV‐1 drug discovery efforts.