A proposed bioactive form of peptide T and the design of peptidomimetics

Summary Peptide T is a non-natural octapeptide of sequence Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, taken from the sequence of the protein gp 120 of HIV. The peptide has been shown to bind competitively to the CD4 receptors of the helper/inducer lymphocytes T. The peptide is presently used for the treatment of AIDS-associated dementia and has been proven useful for the treatment of psoriasis. Using molecular modeling procedures, we studied the conformational profile of this peptide as well as those of several active and inactive analogs. The analysis of these results gave rise to the proposal of a bioactive conformation of the peptide, which can be described as a pseudo β-turn structure, involving the last four residues at the C-terminus of the peptide. The secondary structure is stabilized by a hydrogen bond between the hydroxyl hydrogen of the side chain of Thr⁵ and the carbonyl oxygen of Tyr⁷. From the bioactive form and different structure-activity relationship studies, a pharmacophore was proposed. This hypothesis was used to search on several 3D data bases. One of the hits obtained was the natural compound amigdalin, which was tested and exhibited moderate activity.     

A proposed bioactive form of peptide T and the design of peptidomimetics

Peptide T is a non-natural octapeptide of sequence Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, taken from the sequence of the protein gp120 of HIV. The peptide has been shown to bind competitively to the CD4 receptors of the helper/inducer lymphocytes T. The peptide is presently used for the treatment of AIDS-associated dementia and has been proven useful for the treatment of psoriasis. Using molecular modeling procedures, we studied the conformational profile of this peptide as well as those of several active and inactive analogs. The analysis of these results gave rise to the proposal of a bioactive conformation of the peptide, which can be described as a pseudo -turn structure, involving the last four residues at the C-terminus of the peptide. The secondary structure is stabilized by a hydrogen bond between the hydroxyl hydrogen of the side chain of Thr⁵ and the carbonyl oxygen of Tyr⁷. From the bioactive form and different structure–activity relationship studies, a pharmacophore was proposed. This hypothesis was used to search on several 3D data bases. One of the hits obtained was the natural compound amigdalin, which was tested and exhibited moderate activity.     

CD4-derived synthetic peptide blocks the binding of HIV-1 GP120 to CD4-bearing cells and prevents HIV-1 infection

The T cell surface glycoprotein CD4 plays an important role in mediating cellular immunity and serves as the receptor for human immunodeficiency virus. In order to identify primary sequences within the CD4 molecule that may be involved in the binding of the HIV-I envelope, we synthesized various peptides corresponding to the V1, V2, V3, and V4 domains of CD4. We tested the ability of these peptides to block the binding of purified HIV-I gp120 to CD4+ human lymphoblastic leukemia cells (CEM) using fluorescence-activated cell sorting. One of these peptides, corresponding to CD4 amino acids (74-95), when preincubated with gp120, blocked its subsequent binding to CEM cells by 80%. A truncated form of this peptide (81-95), was found to be as efficient as the longer peptide (74-95) in inhibiting the binding of gp120 to CEM cells. The same peptide did not block the binding of OKT4A or Leu3A anti-CD4 monoclonal antibodies, which were previously shown to block HIV-I binding to CD4. The peptides were also tested for their ability to block HIV-I infection of a T cell line in vitro. Only CD4 peptide (74-95) and the shorter fragment (81-95) succeeded in protecting T cells against infection with different HIV-I strains. All the other peptides examined had no effect on gp120 binding to CEM cells and did not block syncytia formation. Goat polyclonal antibodies against the CD4 peptide (74-95) gave modest interference of gp120 binding to CEM cells. These data suggest that the CD4 region (74-95) participates in the CD4-mediated binding and/or internalization of HIV-I virion.     

Effects of anti-gp120 monoclonal antibodies on CD4 receptor binding by the env protein of human immunodeficiency virus type 1.

Monoclonal antibodies (MAbs) to defined peptide epitopes on gp120 from human immunodeficiency virus type 1 were used to investigate the involvement of their epitopes in gp120 binding to the CD4 receptor. Recombinant vaccinia viruses were constructed that expressed either full-length gp120 (v-ED6), or a truncated gp120 lacking 44 amino acids at the carboxyl terminus (v-ED4). Binding of these glycoproteins to the CD4 receptor was detected directly with metabolically labeled gp120 or indirectly with the gp120 MAbs. Truncated gp120 from v-ED4 bound to CD4-positive cells less than 1/12 as well as gp120 from v-ED6, indicating that the C-terminal region of gp120, which is conserved in numerous isolates of human immunodeficiency virus type 1, is critical for CD4 binding. However, MAb 110-1, which recognizes a peptide contained in the region deleted from v-ED4 (amino acids 489 through 511), did not inhibit binding of gp120 to CD4. MAb 110-1 also reacted with gp120 bound to the CD4 receptor, indicating that the epitope for this antibody does not directly interact with CD4. A second MAb, 110-4, which recognizes a peptide epitope located between amino acids 303 and 323 and has potent viral neutralizing activity, also bound to gp120 on the CD4 receptor. Furthermore, pretreatment of gp120 with MAb 110-4 at concentrations approximately 1,000-fold higher than those required for complete virus neutralization inhibited subsequent CD4 binding by only about 65%. Taken together, these data suggest that neutralization mediated by antibody 110-4 does not result from binding of this MAb to the CD4-binding site of gp120.     

Inhibitors of HIV-1 attachment. Part 4: A study of the effect of piperazine substitution patterns on antiviral potency in the context of indole-based derivatives

4-Fluoro- and 4-methoxy-1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione (2 and 3, respectively) have been characterized as potent inhibitors of HIV-1 attachment that interfere with the interaction of viral gp120 with the host cell receptor CD4. As part of an effort to understand fundamental aspects of this pharmacophore, discovered originally using a high throughput cell-based screen, modification and substitution of the piperazine ring was examined in the context of compounds 6a–ah. The piperazine ring was shown to be a critical element of the HIV-1 attachment inhibiting pharmacophore, acting as a scaffold to deploy the indole glyoxamide and benzamide in a topographical relationship that complements the binding site on gp120.     

Beta-turn Phe in HIV-1 Env binding site of CD4 and CD4 mimetic miniprotein enhances Env binding affinity but is not required for activation of co-receptor/17b site

HIV-1 enters a host cell after an initial interaction between viral envelope glycoprotein gp120 and cell surface receptor CD4, followed by a second interaction between gp120 and a cell surface chemokine receptor. CD4 residue Phe43 makes a significant contribution to the high-affinity interaction between CD4 and env. We and others have used scorpion toxin scaffolds to display and examine CD4 epitopes used for gp120 recognition. These peptides, which have a beta-turn Phe that acts as a Phe43 surrogate, compete with CD4 for gp120 binding and enhance the binding of gp120 to 17b, an antibody that binds near the co-receptor-binding site. In the current study, a scyllatoxin-scaffolded peptide, identified via phage epitope randomization and lacking a beta-turn Phe (indeed, containing no aromatic residues), was shown to behave in a distinctly CD4-like manner. This peptide, denoted [20EGLV23]ST, not only competed with CD4 for gp120 binding, but also enhanced the binding of gp120 to 17b. Quantitatively, an [20EGLV23]ST-gp120 complex exhibited the same 17b binding on-rate as a complex of gp120 with [20AGSF23]ST, a scyllatoxin-based CD4 mimetic peptide containing a beta-turn Phe. In view of this result, we examined the role of Phe43 in CD4 itself by comparing F43V D1D2 sCD4 versus D1D2 sCD4. Like the peptides, a close similarity was observed for both Phe43 and Phe43-less D1D2 sCD4s in enhancing gp120 binding to 17b. Further, when examined for their ability to enhance binding of gp120 to CCR5+ cells, [20EGLV23]ST and [20AGSF23]ST were found to have the same efficacy, after correcting for the difference in their gp120 affinities. These results show that, although Phe43 is important in maintaining high affinity in gp120 ligands, the aromatic residue is not necessary for triggering the conformational isomerization in gp120 that results in formation or exposure of the binding sites for the 17b antibody and the CCR5 receptor.     

Discovery of small-molecule human immunodeficiency virus type 1 entry inhibitors that target the gp120-binding domain of CD4

The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4(+) cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions.     

Computational identification of novel entry inhibitor scaffolds mimicking primary receptor CD4 of HIV-1 gp120

Virtual screening of novel entry inhibitor scaffolds mimicking primary receptor CD4 of HIV-1 gp120 was carried out in conjunction with evaluation of their potential inhibitory activity by molecular modeling. To do this, pharmacophore models presenting different sets of the hotspots of cellular receptor CD4 for its interaction with gp120 were generated. These models were used as the templates for identification of CD4-mimetic candidates by the pepMMsMIMIC screening platform. Complexes of these candidates with gp120 were built by high-throughput ligand docking and their stability was estimated by molecular dynamics simulations and binding free energy calculations. As a result, five top hits that exhibited strong attachment to the two well-conserved hotspots of the gp120 CD4-binding site were selected for the final analysis. In analogy to CD4, the identified compounds make hydrogen bonds with Asp-368_gp120 and multiple van der Waals contacts with the gp120 residues that bind to Phe-43_CD4, resulting in destruction of the critical interactions of gp120 with Phe-43_CD4 and Arg-59_CD4. The complexes of the CD4-mimetic candidates with gp120 show relative conformational stability within the molecular dynamics simulations and expose the high percentage occupancies of intermolecular hydrogen bonds, in line with the data on the binding free energy calculations. In light of these findings, the identified compounds are considered as good scaffolds for the development of new functional antagonists of viral entry with broad HIV-1 neutralization.     

The Conformation and Orientation of a 27-Residue CCR5 Peptide in a Ternary Complex with HIV-1 gp120 and a CD4-Mimic Peptide

Interaction of CC chemokine receptor 5 (CCR5) with the human immunodeficiency virus type 1 (HIV-1) gp120/CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of two to four tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied both in its free form and in a ternary complex with deglycosylated gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27), which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by 2-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4, while the C-terminal segment of Nt-CCR5(1-27) points toward the target cell membrane, reflecting an Nt-CCR5 orientation that differs by 180° from that of a previous model. A gp120 site that could accommodate ^CCR5Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated ^CCR5Tyr14 with ^gp120Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion, furthering our understanding of CCR5 recognition by HIV-1.     

Introducing metallocene into a triazole peptide conjugate reduces its off-rate and enhances its affinity and antiviral potency for HIV-1 gp120

In this work, we identified a high affinity and potency metallocene-containing triazole peptide conjugate that suppresses the interactions of HIV-1 envelope gp120 at both its CD4 and co-receptor binding sites. The ferrocene-peptide conjugate, HNG-156, was formed by an on-resin copper-catalysed [2+3] cycloaddition reaction. Surface plasmon resonance interaction analysis revealed that, compared to a previously reported phenyl-containing triazole conjugate HNG-105 (105), peptide 156 had a higher direct binding affinity for several subtypes of HIV-1 gp120 due mainly to the decreased dissociation rate of the conjugate-gp120 complex. The ferrocene triazole conjugate bound to gp120 of both clade A (92UG037-08) and clade B (YU-2 and SF162) virus subtypes with nanomolar KD in direct binding and inhibited the binding of gp120 to soluble CD4 and to antibodies that bind to HIV-1YU-2 gp120 at both the CD4 binding site and CD4-induced binding sites. HNG-156 showed a close-to nanomolar IC50 for inhibiting cell infection by HIV-1BaL whole virus. The dual receptor site antagonist activity and potency of HNG-156 make it a promising viral envelope inhibitor lead for developing anti-HIV-1 treatments.     

Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor

The primary event in the infection of cells by HIV is the interaction between the viral envelope glycoprotein, gp120, and its cellular receptor, CD4. A recombinant form of gp120 was found to bind to a recombinant CD4 antigen with high affinity. Two gp120-specific murine monoclonal antibodies were able to block the interaction between gp120 and CD4. The gp120 epitope of one of these antibodies was isolated by immunoaffinity chromatography of acid-cleaved gp120 and shown to be contained within amino acids 397-439. Using in vitro mutagenesis, we have found that deletion of 12 amino acids from this region of gp120 leads to a complete loss of binding. In addition, a single amino acid substitution in this region results in significantly decreased binding, suggesting that sequences within this region are directly involved in the binding of gp120 to the CD4 receptor.     

Solvent polarity-dependent structural refolding: A CD and NMR study of a 15 residue peptide

A close association between the HIV surface protein gp120 and the CD4 T cell receptor initiates the viral multiplication cycle. A 15 amino acid peptide (LAV) within the CD4 binding domain of gp 120 has been shown to retain receptor binding ability. The structural behavior of the LAV peptide has been studied by CD and NMR methods in aqueous solution and upon addition of trifluoroethanol (TFE) to emulate the relatively apolar conditions at the membrane bound receptor. Previous work has shown that the LAV peptide folds into a β-pleated structure in more polar buffer/TFE mixtures, while a concerted structural change can be observed at a concentration of 60% TFE (v/v). This abrupt, cooperative refolding from a regular β-sheet to a helical secondary structure is known as “switch” behavior. Former CD experiments with LAV sequence variants have supported the assumption that four amino acids at the N-terminus (LPCR) are indispensable for the “switch.” The tetrad has a strong β-turn forming potential. The suggestion has been formulated that the tetrad can act as a nucleation site governing the refolding. The present NMR study of the LAV peptide in TFE gives evidence for a 3₁₀-helix suggesting that the tetrad adopts a type III β-turn and promotes the formation of a similar bend in the next overlapping tetrad until the sequence is restructured into a 3₁₀-helix at a critical polarity favoring intrachain hydrogen bonds. © 1995 Wiley-Liss, Inc.     

Mode of action for linear peptide inhibitors of HIV-1 gp120 interactions

The linear peptide 12p1 (RINNIPWSEAMM) was previously isolated from a phage display library and was found to inhibit interaction of HIV-1 gp120 with both CD4 and a CCR5 surrogate, mAb 17b [Ferrer, M., and Harrison, S. (1999) J. Virol. 73, 5795-5802]. In this work, we investigated the mechanism that leads to this dual inhibition of gp120 binding. We found that there is a direct interaction of 12p1 with gp120, which occurs with a binding stoichiometry of 1:1. The peptide inhibits binding of monomeric YU2 gp120 to both sCD4 and 17b at IC(50) values of 1.1 and 1.6 microM, respectively. The 12p1 peptide also inhibited the binding of these ligands to trimeric envelope glycoproteins, blocked the binding of gp120 to the native coreceptor CCR5, and specifically inhibited HIV-1 infection of target cells in vitro. Analyses of sCD4 saturation of monomeric gp120 in the presence or absence of a fixed concentration of peptide suggest that 12p1 suppression of CD4 binding to gp120 is due to allosteric inhibitory effects rather than competitive inhibition of CD4 binding. Using a panel of gp120 mutants that exhibit weakened inhibition by 12p1, the putative binding site of the peptide was mapped to a region immediately adjacent to, but distinguishable from, the CD4 binding footprint. In the case of the peptide, the effects of single-12p1 residue substitutions and various peptide truncations indicate that the side chain of Trp7 and other structural elements of 12p1 are critical for gp120 binding or efficient inhibition of binding of a ligand to gp120. Finally, 12p1 was unable to inhibit binding of sCD4 to a gp120 mutant that is believed to resemble the CD4-induced conformation of gp120. These results suggest that 12p1 preferentially binds gp120 prior to engagement of CD4; binding of the peptide to gp120 limits the interaction with ligands (CD4 and CCR5) that are generally crucial for viral entry. More importantly, these results indicate that 12p1 binds to a unique site that may prove to be a prototypic target for novel CD4-gp120 inhibitors.     

Correlation of beta-bend conformations of tetrapeptides with their activities in CD4-receptor binding assays

Conformational analysis, based on ECEPP (Empirical Conformational Energy Program for Peptides) using the chain build-up procedure, was applied to determine the low-energy conformations for a series of tetrapeptides. The tetrapeptides are components of larger peptides which have been found to bind to the CD4 receptor of monocytes. Several previous studies have implicated the tetrapeptide units investigated here as being critical to the biological activities of the full peptides. Five such tetrapeptides were studied: Ser-Ser-Asn-Tyr (from ribonuclease A), Thr-Thr-Asn-Tyr (from peptide T, known to block human immunodeficiency virus from attaching to CD4+ T cells), Thr-Ile-Asn-Tyr (from polio virus coat protein, which is less active than the other peptides in binding to CD4 receptors), Ser-Ser-Ala-Tyr (from the gp 120 coat protein of human immunodeficiency virus, a variant of the peptide T sequence, active in blocking viral attachment to CD4+ cells), and the tetrapeptide from an active synthetic pentapeptide, Asn-Thr-Lys-Tyr (from Asn-Thr-Lys-Tyr-Thr). Using a 7 kcal/mol cutoff, the low-energy conformations for each peptide were computed. Approximately 20,000 conformations were computed for each tetrapeptide. Residue probability profiles were determined for each tetrapeptide. All tetrapeptides except for the polio sequence showed flexibility in the sense that many low-energy conformations were possible. In previous studies, it was postulated that the critical tetrapeptide units would adopt conformations similar to the one observed in a segment of ribonuclease A, residues 22-25, a beta-bend, which is part of an octapeptide segment (residues 19-26) that is homologous to the sequence of peptide T.(ABSTRACT TRUNCATED AT 250 WORDS)     

A tyrosine-sulfated peptide based on the N terminus of CCR5 interacts with a CD4-enhanced epitope of the HIV-1 gp120 envelope glycoprotein and inhibits HIV-1 entry

The sequential association of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 with CD4 and a seven-transmembrane segment coreceptor such as CCR5 or CXCR4 initiates entry of the virus into its target cell. The N terminus of CCR5, which contains several sulfated tyrosines, plays a critical role in the CD4-dependent association of gp120 with CCR5 and in viral entry. Here we demonstrate that a tyrosine-sulfated peptide based on the N terminus of CCR5, but not its unsulfated analogue, inhibits infection of macrophages and peripheral blood mononuclear cells by CCR5-dependent, but not CXCR4-dependent, HIV-1 isolates. The sulfated peptide also inhibited the association of CCR5-expressing cells with gp120-soluble CD4 complexes and, less efficiently, with MIP-1alpha. Moreover, this peptide inhibited the precipitation of gp120 by 48d and 23e antibodies, which recognize CD4-inducible gp120 epitopes, but not by several other antibodies that recognize proximal epitopes. The ability of the sulfated peptide to block 48d association with gp120 was dependent in part on seven tropism-determining residues in the third variable (V3) and fourth conserved (C4) domains of gp120. These data underscore the important role of the N-terminal sulfate moieties of CCR5 in the entry of R5 HIV-1 isolates and localize a critical contact between gp120 and CCR5.     

Conformational study of fragments of envelope proteins (gp120: 254-274 and gp41: 519-541) of HIV-1 by NMR and MD simulations.

The envelope proteins, gp 120 and gp41 of HIV-1, play a crucial role in receptor (CD4+ lymphocytes) binding and membrane fusion. The fragment 254-274 of gp120 is conserved in all strains of HIV and, as a part of the full gp120 protein, behaves as 'immunosilent', but as an individual fragment it is 'immunoreactive'. When this fragment binds to its receptor, it activates the fusion domain of gp41 allowing viral entry into the host CD4+ cells. The conformation of fragment 254-274 of the gp120 domain and fragment 519-541 of the gp41 domain was studied by NMR and MD simulations. The studies were carried out in three varied media--water, DMSO-d6 and hexafluoroacetone (HFA). The fusogenic nature of the gp41 domain peptide was investigated by 31P NMR experiments with model bilayers prepared from dimyristoyl-L-alpha-phosphatidylcholine (DMPC). The solvent was seen to exert a major effect on the structure of the two peptides. Fragment (254-274) of gp120 in DMSO-d6 had a type I beta-turn around the tetrad Val9-Ser10-Thr11-Gln12 while in HFA a helical structure spanning the region Ile5 to Gln12 was seen with the remaining part of the peptide in a random coil structure. It is possible that the beta-turn may constitute an initiation site for the formation of the helix. In water at pH 4.5, the peptide adopted a beta-sheet. The NMR results for fragment 519-541 of gp41 are conclusive of a beta-sheet structure in DMSO-d6, a conformation which may help in insertion into the membrane, a notion also put forward by others. The 31P NMR studies of DMPC vesicles with this fragment show its fusogenic nature, promoting fusion of unilamellar vesicles to larger agglomerates like multilamellar ones.     

Expression and characterization of a single-chain polypeptide analogue of the human immunodeficiency virus type 1 gp120-CD4 receptor complex

The infection of CD4(+) host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further, the FLSC and TcSC analogues bound specifically to CCR5 (R5) and blocked R5 virus infection. Thus, these single-chain chimeric molecules represent the first generation of soluble recombinant proteins that mimic the gp120-CD4 complex intermediate that arises during HIV replication.     

Spatial Structure Model of the CD4 Receptor-Binding Site of the HIV Envelope Protein gp120

Abstract

In this study we have undertaken attempt to predict 3D structure of the CD4 receptor-binding site of the HIV envelope protein gp120. The structure of this site has been constructed by the analysis of low-energy conformers of peptide T, an HIV reproduction inhibitor with amino acid sequence corresponding to the fragment Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr of protein gp120, ensuring the interaction of virus with T4 lymphocytes. To do this, the following researches have been carried out: i) the spatial structure models of peptide T and similar fragment 4–11 of an analogues of vasoactive intestinal peptide have been modeled by the restrained molecular mechanics method developed earlier, ii) conformational parameters of these models have been compared to geometrical characteristics of homologous segments of unrelated proteins with known spatial structures. The following major conclusions have been made based on the comparative analysis: i) the conformation of C-terminal fragment Thr-Thr-Asn-Tyr-Thr of peptide T, responsible for the biological activity of the molecule, does not undergo the essential distortions while embedding into the peptide chains of unrelated proteins; ii) this conformation, that is realized in isolated molecule and includes two consecutive reverse turns of the polypeptide chain, adequately describes the main conformational features of an appropriate site of the HIV protein gp120; iii) the fragment Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr of protein gp120 accepts one of six spatial forms which are characteristic for peptide T.     

Use of a gp120 binding assay to dissect the requirements and kinetics of human immunodeficiency virus fusion events

Binding of the extracellular subunit of human immunodeficiency type 1 (HIV-1) envelope (Env) glycoprotein (gp120) to CD4 triggers the induction or exposure of a highly conserved coreceptor binding site in gp120 that helps mediate membrane fusion. Characterizing the structural features involved in gp120-coreceptor binding and the conditions under which binding occurs is important for understanding the fusion process, the evolution of pathogenic strains in vivo, the identification of novel anti-HIV compounds, and the development of HIV vaccines that utilize triggered structures of Env. Here we use the kinetics of interaction between CCR5 and gp120 to understand temporal and structural changes that occur during viral fusion. Using saturation binding and homologous competition analysis, we estimated the K(d) of interaction between CCR5 and gp120 from the macrophage tropic HIV-1 strain JRFL to be 4 nM. Unlike Env-mediated fusion, gp120 binding to CCR5 did not require divalent cations or elevated temperatures. Binding was not significantly affected by the pH of binding, G-protein coupling of CCR5, or partial gp120 deglycosylation. Oligomeric, uncleaved JRFL gp140 failed to bind CCR5 despite its ability to bind CD4 and monoclonal antibody 17b, suggesting that the uncleaved ectodomain of gp41 interferes with full exposure of the chemokine receptor binding site. Exposure of the chemokine receptor binding site on gp120 could be induced rapidly by CD4, but exposure of this site was lost upon CD4 dissociation from gp120, indicating that the conformational changes in gp120 induced by CD4 binding are fully reversible. The functional gp120-soluble CD4 complex was remarkably stable over time and temperature ranges, offering the possibility that complexes in which the highly conserved coreceptor binding site in gp120 is exposed can be used for vaccine development.     

Peptides from second extracellular loop of C-C chemokine receptor type 5 (CCR5) inhibit diverse strains of HIV-1

To initiate HIV entry, the HIV envelope protein gp120 must engage its primary receptor CD4 and a coreceptor CCR5 or CXCR4. In the absence of a high resolution structure of a gp120-coreceptor complex, biochemical studies of CCR5 have revealed the importance of its N terminus and second extracellular loop (ECL2) in binding gp120 and mediating viral entry. Using a panel of synthetic CCR5 ECL2-derived peptides, we show that the C-terminal portion of ECL2 (2C, comprising amino acids Cys-178 to Lys-191) inhibit HIV-1 entry of both CCR5- and CXCR4-using isolates at low micromolar concentrations. In functional viral assays, these peptides inhibited HIV-1 entry in a CD4-independent manner. Neutralization assays designed to measure the effects of CCR5 ECL2 peptides when combined with either with the small molecule CD4 mimetic NBD-556, soluble CD4, or the CCR5 N terminus showed additive inhibition for each, indicating that ECL2 binds gp120 at a site distinct from that of N terminus and acts independently of CD4. Using saturation transfer difference NMR, we determined the region of CCR5 ECL2 used for binding gp120, showed that it can bind to gp120 from both R5 and X4 isolates, and demonstrated that the peptide interacts with a CD4-gp120 complex in a similar manner as to gp120 alone. As the CCR5 N terminus-gp120 interactions are dependent on CD4 activation, our data suggest that gp120 has separate binding sites for the CCR5 N terminus and ECL2, the ECL2 binding site is present prior to CD4 engagement, and it is conserved across CCR5- and CXCR4-using strains. These peptides may serve as a starting point for the design of inhibitors with broad spectrum anti-HIV activity.