Palmitic Acid Analogs Exhibit Nanomolar Binding Affinity for the HIV-1 CD4 Receptor and Nanomolar Inhibition of gp120-to-CD4 Fusion

Background

We recently reported that palmitic acid (PA) is a novel and efficient CD4 fusion inhibitor to HIV-1 entry and infection. In the present report, based on in silico modeling of the novel CD4 pocket that binds PA, we describe discovery of highly potent PA analogs with increased CD4 receptor binding affinities (K_d) and gp120-to-CD4 inhibition constants (K_i). The PA analogs were selected to satisfy Lipinski''s rule of drug-likeness, increased solubility, and to avoid potential cytotoxicity.

Principal Findings

PA analog 2-bromopalmitate (2-BP) was most efficacious with K_d ∼74 nM and K_i ∼122 nM, ascorbyl palmitate (6-AP) exhibited slightly higher K_d ∼140 nM and K_i ∼354 nM, and sucrose palmitate (SP) was least efficacious binding to CD4 with K_d ∼364 nM and inhibiting gp120-to-CD4 binding with K_i ∼1486 nM. Importantly, PA and its analogs specifically bound to the CD4 receptor with the one to one stoichiometry.

Significance

Considering observed differences between K_i and K_d values indicates clear and rational direction for improving inhibition efficacy to HIV-1 entry and infection. Taken together this report introduces a novel class of natural small molecules fusion inhibitors with nanomolar efficacy of CD4 receptor binding and inhibition of HIV-1 entry.     

Molecular interaction between HIV-1 major envelope glycoprotein and dextran sulfate

We investigated at the molecular level the interaction between, HIV-1 recombinant gp160 (rgp160) and low-molecular-weight dextran sulfate. We demonstrate the occurrence of a specific interaction between rgp160 and sulfated dextran beads, which is saturable, pH-dependent and inhibitable by soluble dextran sulfate but not by soluble dextran. This specific interaction has a low affinity, with an estimated K_d in the 10^?4 M range. In addition, the binding of rgp160 to soluble recombinant CD4 (sT4) can only be inhibited by the preincubation of rgp160, but not of sT4, with dextran sulfate. Taken together, these results demonstrate the occurrence of a low affinity, but specific interaction between dextran sulfate and rgp160. This may account, at least in part, for the anti-HIV-1 activity of dextran sulfate.     

Hormone activity of a synthetic decapeptide with the adrenocorticotropin-like sequence of human immunoglobulin G1

The antiproliferative and immunosuppressivein vitro effects ofimmunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11–20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10⁻¹¹−10⁻⁷ M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strainSalmonella typhimurium 415. By using a¹²⁵I-labeled “addressing” fragment of ACTH {[¹²⁵I]ACTH (13–24)}, we showed that MT-4 cells express specific receptors for ACTH (K _d 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [¹²⁵I]ACTH-(13–24) to these receptors withK _i1 of 0.38 andK _i2 of 0.34 nM, respectively. Specific receptors for ACTH (K _d 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to compete with labeled ACTH-(13–24) for binding to these receptors (K _i=1.8 nM), and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.     

Involvement of the second extracellular loop and transmembrane residues of CCR5 in inhibitor binding and HIV-1 fusion: insights into the mechanism of allosteric inhibition

C-C chemokine receptor 5 (CCR5), a member of G-protein-coupled receptors, serves as a coreceptor for human immunodeficiency virus type 1 (HIV-1). In the present study, we examined the interactions between CCR5 and novel CCR5 inhibitors containing the spirodiketopiperazine scaffolds AK530 and AK317, both of which were lodged in the hydrophobic cavity located between the upper transmembrane domain and the second extracellular loop (ECL2) of CCR5. Although substantial differences existed between the two inhibitors—AK530 had 10-fold-greater CCR5-binding affinity (K_d = 1.4 nM) than AK317 (16.7 nM)—their antiviral potencies were virtually identical (IC₅₀ = 2.1 nM and 1.5 nM, respectively). Molecular dynamics simulations for unbound CCR5 showed hydrogen bond interactions among transmembrane residues Y108, E283, and Y251, which were crucial for HIV-1-gp120/sCD4 complex binding and HIV-1 fusion. Indeed, AK530 and AK317, when bound to CCR5, disrupted these interhelix hydrogen bond interactions, a salient molecular mechanism enabling allosteric inhibition. Mutagenesis and structural analysis showed that ECL2 consists of a part of the hydrophobic cavity for both inhibitors, although AK317 is more tightly engaged with ECL2 than AK530, explaining their similar anti-HIV-1 potencies despite the difference in K_d values. We also found that amino acid residues in the β-hairpin structural motif of ECL2 are critical for HIV-1-elicited fusion and binding of the spirodiketopiperazine-based inhibitors to CCR5. The direct ECL2-engaging property of the inhibitors likely produces an ECL2 conformation, which HIV-1 gp120 cannot bind to, but also prohibits HIV-1 from utilizing the “inhibitor-bound” CCR5 for cellular entry—a mechanism of HIV-1's resistance to CCR5 inhibitors. The data should not only help delineate the dynamics of CCR5 following inhibitor binding but also aid in designing CCR5 inhibitors that are more potent against HIV-1 and prevent or delay the emergence of resistant HIV-1 variants.     

Blocking interaction of viral gp120 and CD4-expressing T cells by single-stranded DNA aptamers

To investigate the potential clinical application of aptamers to prevention of HIV infection, single-stranded DNA (ssDNA) aptamers specific for CD4 were developed using the systematic evolution of ligands by exponential enrichment approach and next generation sequencing. In contrast to RNA-based aptamers, the developed ssDNA aptamers were stable in human serum up to 12 h. Cell binding assays revealed that the aptamers specifically targeted CD4-expressing cells with high binding affinity (K_d = 1.59 nM), a concentration within the range required for therapeutic application. Importantly, the aptamers selectively bound CD4 on human cells and disrupted the interaction of viral gp120 to CD4 receptors, which is a prerequisite step of HIV-1 infection. Functional studies showed that the aptamer polymers significantly blocked binding of viral gp120 to CD4-expressing cells by up to 70% inhibition. These findings provide a new approach to prevent HIV-1 transmission using oligonucleotide aptamers.     

Binding of a thromboxane A2/prostaglandin H2 agonist [H]U46619 to washed human platelets

The binding characteristics of [³H]U46619 to washed human platelets were studied. [³H]U46619 binding to washed human platelets was saturable and displaceable. Kinetic studies yielded a K_d of 11 ± 4 nM (n=4). Scatchard analysis of equilibrium binding studies revealed one class of high affinity binding sites with a K_d of 20 ± 7nM and a B_max of 9.1 ± 2.3 fmole/10⁷ platelets (550 ± 141 binding sites per platelet) (n=4). A number of compounds that act as either agonists or antagonists of the TXA₂/PGH₂ receptor were tested for their ability to inhibit the binding of [³H]U46619 to washed human platelets. The K_ds of the agonists and antagonists were similar to their potencies to induce or inhibit platelet aggregation. These data provide some evidence that [³H]U46619 binds to the putative human platelet TXA₂/PGH₂ receptor.     

Vitellogenesis in the cockroach Nauphoeta cinerea: Separation of two classes of ovarian binding sites and calcium effects on binding and uptake

Two classes of vitellogenin binding sites with K_d-values of 7.3 nM and 290 nM were observed in follicle-membrane preparations of the cockroach Nauphoeta cinerea using a membrane-binding assay at pH 8. Separation of follicle cells and basal laminae from oocyte membranes prior to binding studies showed that the fraction consisting of follicle cells and basal laminae (FC/BL) contained high-affinity binding sites for vitellogenin (K_d=16.6 nM), whereas loweraffinity binding sites (K_d=200 nM) were found in the oocyte membrane fraction. The concentration of Ca²⁺ had a distinct effect on vitellogenin binding and uptake: maximal binding to the oocyte membrane fraction was observed at 0.3 mM Ca²⁺ and to the FC/BL fraction at 10 mM, whereas uptake of vitellogenin by oocytes in vitro was highest at 4 mM Ca²⁺. The calcium ionophore A23187 decreased vitellogenin uptake. This effect of A23187 could be counteracted by the calcium chelator Quin2. A hypothetical model for the uptake of vitellogenin into follicles of Nauphoeta cinerea is suggested.     

The evolution of HIV-1 and the origin of AIDS

The major cause of acquired immune deficiency syndrome (AIDS) is human immunodeficiency virus type 1 (HIV-1). We have been using evolutionary comparisons to trace (i) the origin(s) of HIV-1 and (ii) the origin(s) of AIDS. The closest relatives of HIV-1 are simian immunodeficiency viruses (SIVs) infecting wild-living chimpanzees (Pan troglodytes troglodytes) and gorillas (Gorilla gorilla gorilla) in west central Africa. Phylogenetic analyses have revealed the origins of HIV-1: chimpanzees were the original hosts of this clade of viruses; four lineages of HIV-1 have arisen by independent cross-species transmissions to humans and one or two of those transmissions may have been via gorillas. However, SIVs are primarily monkey viruses: more than 40 species of African monkeys are infected with their own, species-specific, SIV and in at least some host species, the infection seems non-pathogenic. Chimpanzees acquired from monkeys two distinct forms of SIVs that recombined to produce a virus with a unique genome structure. We have found that SIV infection causes CD4⁺ T-cell depletion and increases mortality in wild chimpanzees, and so the origin of AIDS is more ancient than the origin of HIV-1. Tracing the genetic changes that occurred as monkey viruses adapted to infect first chimpanzees and then humans may provide insights into the causes of the pathogenicity of these viruses.     

Differences in the mannose oligomer specificities of the closely related lectins from Galanthus nivalis and Zea mays strongly determine their eventual anti-HIV activity

Background

In a recent report, the carbohydrate-binding specificities of the plant lectins Galanthus nivalis (GNA) and the closely related lectin from Zea mays (GNA_maize) were determined by glycan array analysis and indicated that GNA_maize recognizes complex-type N-glycans whereas GNA has specificity towards high-mannose-type glycans. Both lectins are tetrameric proteins sharing 64% sequence similarity.

Results

GNA_maize appeared to be ~20- to 100-fold less inhibitory than GNA against HIV infection, syncytia formation between persistently HIV-1-infected HuT-78 cells and uninfected CD4⁺ T-lymphocyte SupT1 cells, HIV-1 capture by DC-SIGN and subsequent transmission of DC-SIGN-captured virions to uninfected CD4⁺ T-lymphocyte cells. In contrast to GNA, which preferentially selects for virus strains with deleted high-mannose-type glycans on gp120, prolonged exposure of HIV-1 to dose-escalating concentrations of GNA_maize selected for mutant virus strains in which one complex-type glycan of gp120 was deleted. Surface Plasmon Resonance (SPR) analysis revealed that GNA and GNA_maize interact with HIV III_B gp120 with affinity constants (K_D) of 0.33 nM and 34 nM, respectively. Whereas immobilized GNA specifically binds mannose oligomers, GNA_maize selectively binds complex-type GlcNAcβ1,2Man oligomers. Also, epitope mapping experiments revealed that GNA and the mannose-specific mAb 2G12 can independently bind from GNA_maize to gp120, whereas GNA_maize cannot efficiently bind to gp120 that contained prebound PHA-E (GlcNAcβ1,2man specific) or SNA (NeuAcα2,6X specific).

Conclusion

The markedly reduced anti-HIV activity of GNA_maize compared to GNA can be explained by the profound shift in glycan recognition and the disappearance of carbohydrate-binding sites in GNA_maize that have high affinity for mannose oligomers. These findings underscore the need for mannose oligomer recognition of therapeutics to be endowed with anti-HIV activity and that mannose, but not complex-type glycan binding of chemotherapeutics to gp120, may result in a pronounced neutralizing activity against the virus.     

Characterization of Recombinant Integrase of Human Immunodeficiency Virus Type 1 (Isolate Bru)

Integration of the human immunodeficiency virus type 1 (HIV-1) DNA into the human genome requires the virusencoded integrase protein. The recombinant integrase protein of HIV-1 (isolate Bru) was prepared by constructing a plasmid based on pET-15b encoding the integrase gene. Integrase of HIV-1 was purified using a bacterial expression system (Escherichia coli). The main kinetic parameters of HIV-1 integrase (K _m = (3.7 ± 0.2)·10^–10 M, k _cat = (1.2 ± 0.3)·10^–7 sec^–1) were determined using an oligonucleotide duplex constructed on the basis of the U5-terminal sequence of proviral HIV-1 DNA as the substrate. Inhibition of integrase by aurintricarbonic acid ([I]₅₀ = 6.3 ± 0.4 M) and dependence of integrase activity on Mg²⁺ and Mn²⁺ concentration were studied.     

Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule

The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K_d = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K_d, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K_d = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.     

An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus

Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5′ untranslated region that contains all residues necessary for efficient RNA packaging (Ψ^WT; residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Ψ^WT]₂) binding 12 ± 2 NC molecules with high affinity (K_d = 17 ± 7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Ψ^M), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (∼ 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.     

Characterization of binding of [3H]PD 128907, A selective Dopamine D3 receptor agonist ligand,to CHO-K1 cells

PD 128907 [4a R, 10 b R-(+)-trans- 3, 4, 4a, 10 b - tetrahydro - 4- n-propy12 H,5H-[1] benzopyrano[4,3-b]1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (K_i, 1 nM) versus human D₂ receptors (K_i, 1183 nM) and a 10000-fold selectivity versus human D₄ receptors (K_i, 7000 nM) using [³H]spiperone as the radioligand in CHO-K1-cells. Studies with [³H]PD 128907, showed saturable, high affinity binding to human D₃ receptors expressed in CHO-K1 cells (CHO-K1-D₃) with an equilibrium dissociation constant (K_d) of 0.99 nM and a binding density (B_max) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D₂ receptors (CHO-K1-D₂). The rank order of potency for inhibition of [³H]PD 128907 binding with reference DA agents was consistent with reported values for D₃ receptors. These results indicate that [³H]PD 128907 is a new, highly selective D₃ receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DA D₃ receptors.     

Enhancement of human immunodeficiency virus type 1 infectivity by replacing the region including Env derived from defective particles with an ability to form particle-mediated syncytia in CD4+T cells

The infection and subsequent replication rates of human immunodeficiency virus type 1 (HIV-1) affect the pathogenicity. The initial stage of HIV-1 infection is largely regulated by viral envelope sequence. We previously reported that the defective doughnut-shaped particles produced from a persistently infected cell clone, named L-2, obtained from human CD4+ T-cell line MT-4 that was persistently infected with HIV-1 LAI strain, efficiently form particle-mediated syncytia with uninfected human CD4+ T-cell line, MOLT-4. Here, we prepared a molecular clone (pL2) containing the L-2 provirus to characterize the viral genetic region contributing to this activity to form particle-mediated syncytia. Several recombinants were constructed with pNL4-3 by replacing the pL2-derived region including full-length env. Characterization of the particles obtained by transfection with these recombinant clones confirmed that pL2-derived env carried the particle-mediated syncytia formation activity. It is noteworthy that the pL2-derived env region could also contribute to enhancement of infectivity in CD4+ T-cell lines as well as primary peripheral blood mononuclear cells (PBMCs). Thus, the HIV-1 particle-mediated syncytium formation activity could also contribute to the enhancement of HIV-1 infectivity.     

Muscarinic receptor sites in human foetal brain

The specific binding of the muscarinic cholinergic ligand N-methylscopolamine to human foetal brain has been measured. A level of binding of 64 pmol/g protein was found with a dissociation constant, K_d of 0.27 nM. Values of 0.17 nM min^?1 and 0.048 min^?1 for the association rate constant, K_on, and the dissociation rate constant K_off respectively, were obtained. The pharmacological properties of the binding site were found to be very similar to those reported for muscarinic receptors from adult mammalian brain except that the binding of pirenzepine and the nicotinic antagonists d-tubocurarine and decamethonium shows differences from that seen in adult brain.     

[3H]Vasopressin binding to rat hippocampal synaptic plasma membrane: Kinetic and pharmacological characterization

Characterization of specific vasopressin binding sites to rat hippocampal membranes has been assayed using tritiated lysine-vasopressin labelled on the tyrosyl residue. At 30°C specific [³H]vasopressin binding was saturable. The estimated equilibrium dissociation constant was 7.1 nM, the mean maximal binding capacity was 78 fmol/mg protein. Arginine-vasopressin has a high affinity (K_d = 2.8 nM) and dDAVP has a low affinity (K_d = 249 nM) for hippocampal synaptic membranes. (OH)AVP and Phe²Orn⁸VT are at least as active as AVP in inhibiting [³H]vasopressin binding. Adenylate cyclase was activated by VIP and inhibited by PIA, but not affected by lysine-vasopressin.     

Isolation and Characterization of a Replication-Competent Molecular Clone of an HIV-1 Circulating Recombinant Form (CRF33_01B)

A growing number of emerging HIV-1 recombinants classified as circulating recombinant forms (CRFs) have been identified in Southeast Asia in recent years, establishing a molecular diversity of increasing complexity in the region. Here, we constructed a replication-competent HIV-1 clone for CRF33_01B (designated p05MYKL045.1), a newly identified recombinant comprised of CRF01_AE and subtype B. p05MYKL045.1 was reconstituted by cloning of the near full-length HIV-1 sequence from a newly-diagnosed individual presumably infected heterosexually in Kuala Lumpur, Malaysia. The chimeric clone, which contains the 5′ LTR (long terminal repeat) region of p93JP-NH1 (a previously isolated CRF01_AE infectious clone), showed robust viral replication in the human peripheral blood mononuclear cells. This clone demonstrated robust viral propagation and profound syncytium formation in CD4⁺, CXCR4-expressing human glioma NP-2 cells, indicating that p05MYKL045.1 is a CXCR4-using virus. Viral propagation, however, was not detected in various human T cell lines including MT-2, M8166, Sup-T1, H9, Jurkat, Molt-4 and PM1. p05MYKL045.1 appears to proliferate only in restricted host range, suggesting that unknown viral and/or cellular host factors may play a role in viral infectivity and replication in human T cell lines. Availability of a CRF33_01B molecular clone will be useful in facilitating the development of vaccine candidates that match the HIV-1 strains circulating in Southeast Asia.     

Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.