HIV-1 gp120 V3 loop for structure-based drug design

HIV-1 cell entry is mediated by sequential interactions of the envelope protein gp120 with the receptor CD4 and a coreceptor, usually CCR5 or CXCR4, depending on the individual virion. Considerable efforts on exploiting the HIV coreceptors as drug targets have led to the new class of coreceptor antagonists. While these antiretroviral drugs aim at preventing virus/coreceptor interaction by binding to host proteins, neutralizing antibodies directed against the coreceptor-binding sites on gp120 have attracted attention as possible vaccine candidates. However, both approaches are complicated by the multiple protective mechanisms of gp120 which allow for rapid escape from selective pressures exerted by drugs or antibodies. Thus, advances in rational drug and vaccine design rely heavily on improved insights into the relation between genotype and phenotype, the evolution of coreceptor usage, and, ultimately the structural biology of coreceptor usage and inhibition. The third variable (V3) loop of gp120, crucially involved in all these aspects, will be a major focus of this review.     

Synergistic Effect by Combining a gp120-Binding Protein and a gp41-Binding Antibody to Inactivate HIV-1 Virions and Inhibit HIV-1 Infection

Acquired immune deficiency syndrome (AIDS) has prevailed over the last 30 years. Although highly active antiretroviral therapy (HAART) has decreased mortality and efficiently controlled the progression of disease, no vaccine or curative drugs have been approved until now. A viral inactivator is expected to inactivate cell-free virions in the absence of target cells. Previously, we identified a gp120-binding protein, mD1.22, which can inactivate laboratory-adapted HIV-1. In this study, we have found that the gp41 N-terminal heptad repeat (NHR)-binding antibody D5 single-chain variable fragment (scFv) alone cannot inactivate HIV-1 at the high concentration tested. However, D5 scFv in the combination could enhance inactivation activity of mD1.22 against divergent HIV-1 strains, including HIV-1 laboratory-adapted strains, primary HIV-1 isolates, T20- and AZT-resistant strains, and LRA-reactivated virions. Combining mD1.22 and D5 scFv exhibited synergistic effect on inhibition of infection by divergent HIV-1 strains. These results suggest good potential to develop the strategy of combining a gp120-binding protein and a gp41-binding antibody for the treatment of HIV-1 infection.     

Solid-state NMR yields structural constraints on the V3 loop from HIV-1 Gp120 bound to the 447-52D antibody Fv fragment

Solid-state NMR measurements were performed on the complex of an 18-residue peptide derived from the V3 loop sequence of the gp120 envelope glycoprotein of the HIV-1 MN strain with Fv fragments of the human anti-gp120 monoclonal antibody 447-52D in a frozen glycerol/water solution. The peptide was uniformly (15)N- and (13)C-labeled in a 7-residue segment containing the conserved GPGR motif in the epitope. (15)N and (13)C NMR chemical shift assignments for the labeled segment were obtained from two-dimensional (13)C-(13)C and (15)N-(13)C magic-angle spinning NMR spectra. Reductions in (13)C NMR line widths and changes in chemical shifts upon complex formation indicate the adoption of a well-defined, antibody-dependent structure. Intramolecular (13)C-(13)C distances in the complex, which constrain the peptide backbone and side chain conformations in the GPGR motif, were determined from an analysis of rotational resonance (RR) data. Structural constraints from chemical shifts and RR measurements are in good agreement with recent solution NMR and crystallographic studies of this system, although differences regarding structural ordering of certain peptide side chains are noted. These experiments explore and help delineate the utility of solid state NMR techniques as structural probes of peptide/protein complexes in general, potentially including membrane-associated hormone/receptor complexes.     

Interactions of HIV-1 proteins gp120 and Nef with cellular partners define a novel allosteric paradigm

During the course of infection, a subset of HIV-1 proteins interacts with multiple cellular partners, sometimes in a hierarchical or sequential way. These proteins include those associated with the initial infection event, with the preparation of the cell for the replicative cycle of the virus and with the exit of new virions from the infected cell. It appears that the interactions of viral proteins with multiple cellular partners are mediated by the occurrence of ligand-induced conformational changes that direct the binding of these proteins to subsequent partners. Two of the most studied HIV-1 proteins that are known to interact with different cellular partners are gp120 and Nef. Here we discuss the interactions of these two proteins with their cellular partners and present new results indicating that the conformational changes undergone by these proteins define a novel allosteric paradigm. In the traditional view, conformational changes are thought to occur between well defined structural conformations of a protein. In gp120 and Nef, those changes involve conformations characterized by the presence of large regions devoid of stable secondary or tertiary structure. Those unstructured regions contain the binding determinants for subsequent partners and only become functionally competent by ligand-induced structuring or un-structuring of those regions. By switching binding epitopes between structured and unstructured conformations the binding affinity can be modulated by several orders of magnitude, thus effectively precluding binding against unwanted partners. A better understanding of these interactions would lead to improved strategies for inhibitor design against these viral targets.     

Discovery of novel polyamine analogs with anti-protozoal activity by computer guided drug repositioning

Chagas disease is a parasitic infection caused by the protozoa Trypanosoma cruzi that affects about 6 million people in Latin America. Despite its sanitary importance, there are currently only two drugs available for treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects and limited efficacy in the chronic stage of the disease. Polyamines are ubiquitous to all living organisms where they participate in multiple basic functions such as biosynthesis of nucleic acids and proteins, proliferation and cell differentiation. T. cruzi is auxotroph for polyamines, which are taken up from the extracellular medium by efficient transporters and, to a large extent, incorporated into trypanothione (bis-glutathionylspermidine), the major redox cosubstrate of trypanosomatids. From a 268-compound database containing polyamine analogs with and without inhibitory effect on T. cruzi we have inferred classificatory models that were later applied in a virtual screening campaign to identify anti-trypanosomal compounds among drugs already used for other therapeutic indications (i.e. computer-guided drug repositioning) compiled in the DrugBank and Sweetlead databases. Five of the candidates identified with this strategy were evaluated in cellular models from different pathogenic trypanosomatids (T. cruzi wt, T. cruzi PAT12, T. brucei and Leishmania infantum), and in vitro models of aminoacid/polyamine transport assays and trypanothione synthetase inhibition assay. Triclabendazole, sertaconazole and paroxetine displayed inhibitory effects on the proliferation of T. cruzi (epimastigotes) and the uptake of putrescine by the parasite. They also interfered with the uptake of others aminoacids and the proliferation of infective T. brucei and L. infantum (promastigotes). Trypanothione synthetase was ruled out as molecular target for the anti-parasitic activity of these compounds.     

Cytokine antibody arrays: a promising tool to identify molecular targets for drug discovery

Cytokines play important roles in normal cell functions and changes in cytokines have been implicated in many diseases. Recent efforts have focused on developing cytokine antibody arrays. These arrays allow investigators to simultaneously detect multiple cytokines in qualitative and quantitative ways. Cytokine antibody array systems feature high sensitivity, specificity and throughput. This novel technology opens up an expanding spectrum of applications in drug discovery, including target discovery, target validation, screening for lead compounds, compound optimization and clinical trials.     

Use of the quartz crystal microbalance to monitor ligand-induced conformational rearrangements in HIV-1 envelope protein gp120

We evaluated the potential of a quartz crystal microbalance with dissipation monitoring (QCM-D) to provide a sensitive, label-free method for detecting the conformational rearrangement of glycoprotein gp120 upon binding to different ligands. This glycoprotein is normally found on the envelope of the HIV-1 virus and is involved in viral entry into host cells. It was immobilized on the surface of the sensing element of the QCM-D and was exposed to individual solutions of several different small-molecule inhibitors as well as to a solution of a soluble form of the host cell receptor to which gp120 binds. Instrument responses to ligand-triggered changes were in qualitative agreement with conformational changes as suggested by other biophysical methods.

Theoretical studies on the interactions and interferences of HIV-1 glycoprotein gp120 and its coreceptor CCR5

The interaction between the HIV gp120 protein and coreceptor CCR5 or CXCR4 of the host cell is critical in mediating the HIV entry process. A model for the CCR5-gp120 complex has been developed. In the model, the N-terminus of CCR5 binds to three discontinuous domains of gp120, including the fourth conserved (C4) region, β19/β20 connecting loop, and V3 loop. The second extra-cellular loop (ECL2) of CCR5 also interacts with the crown part of the gp120 V3 loop. The bindings of the three CCR5 antagonists, maraviroc, aplaviroc, and vicriviroc, to the trans-membrane domain of CCR5 have been modeled. The bindings are found to affect the conformation of the ECL2 domain, which in turn drives the N-terminus of CCR5 to an altered state. Aplaviroc is more hydrophilic than maraviroc and vicriviroc, and its binding is more interfered by solvent, resulting in a quite different effect to the structure of CCR5 compared with those of the other two molecules. The above results are in accord with experimental observations and provide a structural basis for further design of CCR5 antagonists.     

Modification and structure-activity relationship of a small molecule HIV-1 inhibitor targeting the viral envelope glycoprotein gp120

This paper describes selected modification and structure-activity relationship of the small molecule HIV-1 inhibitor, 4-benzoyl-1-[(4-methoxy-1H-pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2-(R)-methylpiperazine (BMS-378806). The results revealed: i) that both the presence and configuration (R vs. S) of the 3-methyl group on the piperazine moiety are important for the antiviral activity, with the 3-(R)-methyl derivatives showing the highest activity; ii) that the electronegativity of the C-4 substituent on the indole or azaindole ring seems to be important for the activity, with a small, electron-donating group such as a fluoro or a methoxy group showing enhanced activity, while a nitro group diminishes the activity; iii) that the N-1 position of the indole ring is not eligible for modification without losing activity; and iv) that bulky groups around the C-4 position of the indole or azaindole ring diminish the activity, probably due to steric hindrance in the binding. We found that a synthetic bivalent compound with two BMS-378806 moieties being tethered by a spacer demonstrated about 5-fold enhanced activity in an nM range against HIV-1 infection than the corresponding monomeric inhibitor. But the polyacrylamide-based polyvalent compounds did not show inhibitory activity at up to 200 nM.     

Identification of ligands for RNA targets via structure-based virtual screening: HIV-1 TAR

Binding of the Tat protein to TAR RNA is necessary for viral replication of HIV-1. We screened the Available Chemicals Directory (ACD) to identify ligands to bind to a TAR RNA structure using a four-step docking procedure: rigid docking first, followed by three steps of flexible docking using a pseudobrownian Monte Carlo minimization in torsion angle space with progressively more detailed conformational sampling on a progressively smaller list of top-ranking compounds. To validate the procedure, we successfully docked ligands for five RNA complexes of known structure. For ranking ligands according to binding avidity, an empirical binding free energy function was developed which accounts, in particular, for solvation, isomerization free energy, and changes in conformational entropy. System-specific parameters for the function were derived on a training set of RNA/ligand complexes with known structure and affinity. To validate the free energy function, we screened the entire ACD for ligands for an RNA aptamer which binds l-arginine tightly. The native ligand ranked 17 out of ca. 153,000 compounds screened, i.e., the procedure is able to filter out >99.98% of the database and still retain the native ligand. Screening of the ACD for TAR ligands yielded a high rank for all known TAR ligands contained in the ACD and suggested several other potential TAR ligands. Eight of the highest ranking compounds not previously known to be ligands were assayed for inhibition of the Tat-TAR interaction, and two exhibited a CD₅₀ of ca. 1 M.     

Aptamer Laden Liquid Crystals Biosensing Platform for the Detection of HIV-1 Glycoprotein-120

We report a label-free and simple approach for the detection of glycoprotein-120 (gp-120) using an aptamer-based liquid crystals (LCs) biosensing platform. The LCs are supported on the surface of a modified glass slide with a suitable amount of B40t77 aptamer, allowing the LCs to be homeotropically aligned. A pronounced topological change was observed on the surface due to a specific interaction between B40t77 and gp-120, which led to the disruption of the homeotropic alignment of LCs. This results in a dark-to-bright transition observed under a polarized optical microscope. With the developed biosensing platform, it was possible to not only identify gp-120, but obtained results were analyzed quantitatively through image analysis. The detection limit of the proposed biosensing platform was investigated to be 0.2 µg/mL of gp-120. Regarding selectivity of the developed platform, no response could be detected when gp-120 was replaced by other proteins, such as bovine serum albumin (BSA), hepatitis A virus capsid protein 1 (Hep A VP1) and immunoglobulin G protein (IgG). Due to attributes such as label-free, high specificity and no need for instrumental read-out, the presented biosensing platform provides the potential to develop a working device for the quick detection of HIV-1 gp-120.     

A novel and highly stereoselective route for the synthesis of non-racemic 3-substituted isoindolin-1-one targets

A new, versatile and highly stereoselective approach for the synthesis of non-racemic 3-substituted isoindolin-1-ones is described from a readily available chiral template. The potential of this new protocol is demonstrated through the synthesis of an enantiomerically enriched 3-alkyl N-H isoindolin-1-one target with an e.e. of 98%.     

The application of perfluorooctanoate to investigate trimerization of the human immunodeficiency virus-1 gp41 ectodomain by electrophoresis

The transmembrane glycoprotein gp41 of human immunodeficiency virus has been proposed to form trimer-of-hairpin during virus-cell membrane fusion. To investigate its oligomerization propensity under soluble and membrane-mimic conditions, sodium salt of perfluorooctanoate (PFO) was applied. A recombinant gp41 ectodomain devoid of disulfide linkage was overexpressed in Escherichia coli and characterized by MS and circular dichroism spectropolarimetry in PFO solution in comparison to SDS. The helical content of this ectodomain in PFO is higher than that in SDS. Notably, PFO employed in PAGE clearly conduced to the formation of trimer under the optimized condition as visualized in the gel. In addition, the proteins expressed from the two mutants in the heptad repeat (HR) domains of gp41, I62P, and N126K, were also examined by the PFO-PAGE analysis for functional ramification of molecular organization. Remarkably, the I62P mutation completely abolished the gp41 trimer formation, whereas the N126K mutation resulted in a more stable trimer. The data suggested that PFO-PAGE analysis is appropriate for evaluating the effect of mutations on the trimerization of gp41 and other fusion proteins which may be implicated in the alteration of their fusogenicity.     

Inhibitor docking screened by the modified SAFE_p scoring function: application to cyclic urea HIV-1 PR inhibitors

Our laboratory has in the past developed a method for the prediction of ligand binding free energies to proteins, referred to as SAFE_p (Solvent free energy predictor). Previously, we have applied this protocol for the prediction of the binding free energy of peptidic and cyclic urea HIV-1 PR inhibitors, whose X-ray structures bound to enzyme are known. In this work, we present the first account of a docking simulation, where the ligand conformations were screened and inhibitor ranking was predicted on the basis of a modified SAFE_p approach, for a set of cyclic urea-HIV-1 PR complexes whose structures are not known. We show that the optimal dielectric constant for docking is rather high, in line with the values needed to reproduce some protein residue properties, like pKa's. Our protocol is able to reproduce most of the observed binding ranking, even in the case that the components of the equation are not fitted to experimental data. Partition of the binding free energy into pocket and residue contributions sheds light into the importance of the inhibitor's fragments and on the prediction of "hot spots" for resistance mutations.     

Gold Manno‐Glyconanoparticles: Multivalent Systems to Block HIV‐1 gp120 Binding to the Lectin DC‐SIGN

The HIV envelope glycoprotein gp120 takes advantage of the high‐mannose clusters on its surface to target the C‐type lectin dendritic cell‐specific intracellular adhesion molecule‐3‐grabbing non‐integrin (DC‐SIGN) on dendritic cells. Mimicking the cluster presentation of oligomannosides on the virus surface is a strategy for designing carbohydrate‐based antiviral agents. Bio‐inspired by the cluster presentation of gp120, we have designed and prepared a small library of multivalent water‐soluble gold glyconanoparticles (manno‐GNPs) presenting truncated (oligo)mannosides of the high‐mannose undecasaccharide Man₉GlcNAc₂ and have tested them as inhibitors of DC‐SIGN binding to gp120. These glyconanoparticles are ligands for DC‐SIGN, which also interacts in the early steps of infection with a large number of pathogens through specific recognition of associated glycans. (Oligo)mannosides endowed with different spacers ending in thiol groups, which enable attachment of the glycoconjugates to the gold surface, have been prepared. manno‐GNPs with different spacers and variable density of mannose (oligo)saccharides have been obtained and characterized. Surface plasmon resonance (SPR) experiments with selected manno‐GNPs have been performed to study their inhibition potency towards DC‐SIGN binding to gp120. The tested manno‐GNPs completely inhibit the binding from the micro‐ to the nanomolar range, while the corresponding monovalent mannosides require millimolar concentrations. manno‐GNPs containing the disaccharide Manα1‐2Manα are the best inhibitors, showing more than 20 000‐fold increased activity (100 % inhibition at 115 nM ) compared to the corresponding monomeric disaccharide (100 % inhibition at 2.2 mM ). Furthermore, increasing the density of dimannoside on the gold platform from 50 to 100 % does not improve the level of inhibition.     

The potent anti-HIV protein cyanovirin-N contains two novel carbohydrate binding sites that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinity: implications for binding to the HIV envelope protein gp120.

Cyanovirin-N (CVN) is a monomeric 11 kDa cyanobacterial protein that potently inactivates diverse strains of human immunodeficiency virus (HIV) at the level of cell fusion by virtue of high affinity interactions with the surface envelope glycoprotein gp120. Several lines of evidence have suggested that CVN-gp120 interactions are in part mediated by N-linked complex carbohydrates present on gp120, but experimental evidence has been lacking. To this end we screened a comprehensive panel of carbohydrates which represent structurally the N-linked carbohydrates found on gp120 for their ability to inhibit the fusion-blocking activity of CVN in a quantitative HIV-1 envelope-mediated cell fusion assay. Our results show that CVN specifically recognizes with nanomolar affinity Man(9)GlcNAc(2) and the D1D3 isomer of Man(8)GlcNAc(2). Nonlinear least squares best fitting of titration data generated using the cell fusion assay show that CVN binds to gp120 with an equilibrium association constant (K(a)) of 2.4 (+/- 0.1) x 10(7) M(-1) and an apparent stoichiometry of 2 equiv of CVN per gp120, Man(8)GlcNAc(2) D1D3 acts as a divalent ligand (2 CVN:1 Man(8)) with a K(a) of 5.4 (+/- 0.5) x 10(7) M(-1), and Man(9)GlcNAc(2) functions as a trivalent ligand (3 CVN:1 Man(9)) with a K(a) of 1.3 (+/- 0.3) x 10(8) M(-1). Isothermal titration calorimetry experiments of CVN binding to Man(9)GlcNAc(2) at micromolar concentrations confirmed the nanomolar affinity (K(a) = 1.5 (+/- 0.9) x 10(8) M(-1)), and the fitted data indicated a stoichiometry equal to approximately one (1 Man(9):1 CVN). The 1:1 stoichiometry at micromolar concentrations suggested that CVN has not only a high affinity binding site-relevant to the studies at nM concentrations-but a lower affinity site as well that facilitates cross-linking of CVN-oligomannose at micromolar concentrations or higher. The specificity of CVN for Man(8) D1D3 and Man(9) over the D1D2 isomer of Man(8) indicated that the minimum structure required for high affinity binding comprises Manalpha1 --> 2Manalpha. By following the (1)H-(15)N correlation spectrum of (15)N-labeled CVN upon titration with this disaccharide, we unambiguously demonstrate that CVN recognizes and binds to the disaccharide Manalpha1 --> 2Manalpha via two distinct binding sites of differing affinities located on opposite ends of the protein. The high affinity site has a K(a) of 7.2 (+/- 4) x 10(6) M(-1) and the low affinity site a K(a) of 6.8 (+/- 4) x 10(5) M(-1) as determined by isothermal titration calorimetry. Mapped surfaces of the carbohydrate binding sites are presented, and implications for binding to gp120 are discussed.