ABSTRACT
Human immunodeficiency virus-1 (HIV-1), the causative agent of AIDS, impacts millions of people. Entry into target cells is mediated by the HIV-1 envelope (Env) glycoprotein interacting with host receptor CD4, which triggers conformational changes allowing binding to a coreceptor and subsequent membrane fusion. Small molecule or peptide CD4-mimetic drugs mimic CD4's Phe43 interaction with Env by inserting into the conserved Phe43 pocket on Env subunit gp120. Here, we present single-particle cryo-EM structures of CD4-mimetics BNM-III-170 and M48U1 bound to a BG505 native-like Env trimer plus the CD4-induced antibody 17b at 3.7 Å and 3.9 Å resolution, respectively. CD4-mimetic-bound BG505 exhibits canonical CD4-induced conformational changes including trimer opening, formation of the 4-stranded gp120 bridging sheet, displacement of the V1V2 loop, and formation of a compact and elongated gp41 HR1C helical bundle. We conclude that CD4-induced structural changes on both gp120 and gp41 Env subunits are induced by binding to the gp120 Phe43 pocket.
Subject(s)
CD4 Antigens/chemistry , Guanidines/chemistry , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp41/chemistry , HIV-1/chemistry , Indenes/chemistry , Receptors, Virus/chemistry , Animals , Binding Sites , Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , CD4 Antigens/antagonists & inhibitors , CD4 Antigens/genetics , CD4 Antigens/metabolism , CHO Cells , Cloning, Molecular , Cricetulus , Cryoelectron Microscopy , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Guanidines/metabolism , HEK293 Cells , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/metabolism , HIV-1/metabolism , Humans , Indenes/metabolism , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/genetics , Receptors, Virus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolismABSTRACT
The HIV-1 envelope glycoproteins (Env) undergo conformational changes upon interaction of the gp120 exterior glycoprotein with the CD4 receptor. The gp120 inner domain topological layers facilitate the transition of Env to the CD4-bound conformation. CD4 engages gp120 by introducing its phenylalanine 43 (Phe43) in a cavity ("the Phe43 cavity") located at the interface between the inner and outer gp120 domains. Small CD4-mimetic compounds (CD4mc) can bind within the Phe43 cavity and trigger conformational changes similar to those induced by CD4. Crystal structures of CD4mc in complex with a modified CRF01_AE gp120 core revealed the importance of these gp120 inner domain layers in stabilizing the Phe43 cavity and shaping the CD4 binding site. Our studies reveal a complex interplay between the gp120 inner domain and the Phe43 cavity and generate useful information for the development of more-potent CD4mc.IMPORTANCE The Phe43 cavity of HIV-1 envelope glycoproteins (Env) is an attractive druggable target. New promising compounds, including small CD4 mimetics (CD4mc), were shown to insert deeply into this cavity. Here, we identify a new network of residues that helps to shape this highly conserved CD4 binding pocket and characterize the structural determinants responsible for Env sensitivity to small CD4 mimetics.
Subject(s)
CD4 Antigens/chemistry , HIV Envelope Protein gp120/chemistry , Phenylalanine/chemistry , Animals , Binding Sites , Biomimetics , CD4-Positive T-Lymphocytes/virology , Cell Line , Crystallization , Dogs , HEK293 Cells , HIV-1 , Humans , Protein Binding , Protein Domains , ThymocytesABSTRACT
The HIV-1 envelope glycoprotein (Env) (gp120-gp41)3 is the target for neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC). HIV-1 Env is flexible, sampling different conformational states. Before engaging CD4, Env adopts a closed conformation (State 1) that is largely antibody resistant. CD4 binding induces an intermediate state (State 2), followed by an open conformation (State 3) that is susceptible to engagement by antibodies that recognize otherwise occluded epitopes. We investigate conformational changes in Env that induce ADCC in the presence of a small-molecule CD4-mimetic compound (CD4mc). We uncover an asymmetric Env conformation (State 2A) recognized by antibodies targeting the conserved gp120 inner domain and mediating ADCC. Sera from HIV+ individuals contain these antibodies, which can stabilize Env State 2A in combination with CD4mc. Additionally, triggering State 2A on HIV-infected primary CD4+ T cells exposes epitopes that induce ADCC. Strategies that induce this Env conformation may represent approaches to fight HIV-1 infection.
Subject(s)
Antibody-Dependent Cell Cytotoxicity , CD4-Positive T-Lymphocytes/virology , HIV Antibodies/immunology , HIV-1/immunology , env Gene Products, Human Immunodeficiency Virus/immunology , CD4 Antigens/metabolism , Cells, Cultured , Humans , Protein Binding , Protein Conformation , env Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
HIV-1 conceals epitopes of its envelope glycoproteins (Env) recognized by antibody (Ab)-dependent cellular cytotoxicity (ADCC)-mediating antibodies. These Abs, including anti-coreceptor binding site (CoRBS) and anti-cluster A antibodies, preferentially recognize Env in its "open" conformation. The binding of anti-CoRBS Abs has been shown to induce conformational changes that further open Env, allowing interaction of anti-cluster A antibodies. We explored the possibility that CoRBS Abs synergize with anti-cluster A Abs to engage Fc-gamma receptors to mediate ADCC. We found that binding of anti-CoRBS and anti-cluster A Abs to the same gp120 is required for interaction with soluble dimeric FcγRIIIa in enzyme-linked immunosorbent assays (ELISAs). We also found that Fc regions of both Abs are required to optimally engage FcγRIIIa and mediate robust ADCC. Taken together, our results indicate that these two families of Abs act together in a sequential and synergistic fashion to promote FcγRIIIa engagement and ADCC.IMPORTANCE The "open" CD4-bound conformation of HIV-1 envelope glycoproteins is the primary target of antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies present in HIV-positive (HIV+) sera, such as anti-coreceptor binding site and anti-cluster A antibodies. Here we report that the binding of these two families of antibodies is required to engage FcγRIIIa and mediate ADCC.
Subject(s)
Epitopes/immunology , HIV Antibodies/immunology , HIV Infections/prevention & control , HIV-1/immunology , Receptors, IgG/metabolism , T-Lymphocytes/immunology , env Gene Products, Human Immunodeficiency Virus/immunology , Antibody-Dependent Cell Cytotoxicity , Binding Sites , HIV Antibodies/metabolism , HIV Infections/immunology , HIV Infections/virology , Humans , Protein Binding , Receptors, IgG/immunology , Recombinant Proteins/immunologyABSTRACT
We report here the development and optimization of a process synthesis for the HIV-1 entry inhibitor BNM-III-170 bis-TFA salt (1). The synthesis features a dynamic-kinetic resolution (DKR) to establish the initial stereogenicity. By taking advantage of significant sequence modifications of our first generation synthesis, inconjunction with the low solubility of late-stage intermediates, the overall efficiency of the synthesis has been significantly improved, now to proceed in an overall yield of 9.64% for the 16-steps, requiring only a single chromatographic separation.
ABSTRACT
To investigate the cellular distribution of tumor-promoting vs. non-tumor-promoting bryostatin analogues, we synthesized fluorescently labeled variants of two bryostatin derivatives that have previously shown either phorbol ester-like or bryostatin-like biological activity in U937 leukemia cells. These new fluorescent analogues both displayed high affinity for protein kinaseâ C (PKC) binding and retained the basic properties of the parent unlabeled compounds in U937 assays. The fluorescent compounds showed similar patterns of intracellular distribution in cells, however; this argues against an existing hypothesis that various patterns of intracellular distribution are responsible for differences in biological activity. Upon further characterization, the fluorescent compounds revealed a slow rate of cellular uptake; correspondingly, they showed reduced activity for cellular responses that were only transient upon treatment with phorbol ester or bryostatinâ 1.
Subject(s)
Bryostatins/chemistry , Fluorescent Dyes/chemistry , Humans , Phorbol Esters/chemistry , Protein Binding , Protein Kinase C/metabolism , U937 CellsABSTRACT
The title compound, C(11)H(22)N(2)O(2), has one chiral center and packs in the monoclinic space group P2(1). The asymmetric unit has five crystallographically independent mol-ecules, four of which engage in inter-molecular N-Hâ¯O hydrogen bonding.
ABSTRACT
Two classes of gem-dimethyl 4-n-pentenyl glycosides (i.e., C2-series and C3-series) have been prepared and studied in both the glycosylation and hydrolysis manifolds utilizing NBS as the sole stoichiometric activator. These novel glycosylating agents, which are analogues of Fraser-Reid's 4-n-pentenyl glycosyl donors, show increased reactivity in side-by-side studies by virtue of the gem-dimethyl effect.
Subject(s)
Glycosides/chemical synthesis , Glycosides/chemistry , Glycosylation , Hydrolysis , Molecular Structure , StereoisomerismABSTRACT
The study examined differences in patient anxiety and patient satisfaction between patients who experienced surgery at a hospital-based ambulatory surgery center versus a freestanding ambulatory surgery center. Forty-seven participants completed the State Trait Anxiety Inventory and Press Ganey Ambulatory Surgery Survey. Patients at both types of facilities experienced moderate levels of preoperative anxiety and were highly satisfied with care received. No significant differences were found in preoperative anxiety, overall satisfaction with care, or overall satisfaction with nursing care. The site where the surgery was performed may not be a determining factor in patient anxiety or satisfaction levels.