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.
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.
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.
We report here the development and optimization of a process synthesis for the human immunodeficiency virus-1 entry inhibitor BNM-III-170 bis-trifluoroacetate salt (1). The synthesis features a ...dynamic-kinetic resolution to establish the initial stereogenicity. By taking advantage of significant sequence modifications of our first-generation synthesis, in conjunction 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.
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.
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.
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.
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•HIV Env exists in several conformations, including an asymmetric State 2A•State 2A is difficult to trigger and is susceptible to non-neutralizing antibody attack•Sera from HIV+ individuals contain antibodies that can stabilize Env State 2A•Conditions triggering State 2A expose epitopes that induce ADCC
HIV-1 envelope glycoproteins (Env) are very flexible and exist in at least three different conformational states. Alsahafi et al. characterize a fourth Env state that is efficiently recognized by antibodies capable of mediating potent antibody-dependent cellular toxicity.
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 kinase 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 bryostatin 1.
Bryostatin brightness: To investigate the cellular distribution of tumor‐promoting versus non‐tumor‐promoting bryostatin (Bryo) analogues, we synthesized fluorescent variants of two bryostatin derivatives that had previously shown either phorbol ester‐like or bryostatin 1‐like biological activity in U937 leukemia cells. The compounds had different biology despite their similar subcellular localization.
Bryostatin 1 is a 20-member macrolactone natural product that exhibits a variety of extremely beneficial biological activities. Since it was first isolated in 1969 and characterized in 1982, ...bryostatin has garnered interest for its highly oxygenated complex structure and also for its potent effect on several diseases. Bryostatin 1 has been the focus of clinical trials for cancer, Alzheimer’s disease, stroke, and the human immunodeficiency virus. The biological activity is traced back to bryostatin 1’s nanomolar binding affinity for and ability to maintain activation of a family of isozymes, protein kinase C (PKC).Current work in this field of research focuses on the construction of structurally simplified bryostatin analogues that can mimic the biological effects, since the natural abundance of bryostatin 1 cannot support the necessary medical testing required. A new direction in bryostatin analogue design involves the removal of one of the ABC pyran rings at the core of the structure and replacing it with an aromatic phenyl or pyridine ring. Replacement of the C-ring pyran with a tetra-substituted aryl ring has helped reduce the bryostatin 1 natural C-ring synthesis of 18 steps down to a much more practical 11 steps. The major pharmacophores present in bryostatin 1 are mirrored in these aromatic analogues in a new way, therefore binding studies and biological analysis will be required to assess if they are acceptable for further analogue development.To further understand the mechanism of exogenous ligands PMA and bryostatin 1 on the translocation of PKC and downstream effects, a group of fluorescently labeled compounds have been synthesized. Fluorescently labeled Merle 44 and Merle 45 were synthesized to mimic two classes of analogues that tend to have opposite effects on the living system. Merle 45 has a very similar biological profile to bryostatin 1 through various cell assays. To complete the fluorescence study, a phorbol with the same BODIPY tag as Merle 44 and 45 was synthesized. Future biological studies will shed light on the rate of uptake of phorbol versus bryostatin analogues, along with the localization of PKC within the cell and translocation pathways. Förster resonance energy transfer (FRET) studies will also be performed to analyze the protein/ligand interactions, while monitoring the resulting PKC conformational change. These results will give more insight about how the high-affinity binding of these two structurally different compound classes to PKC can result in such vastly different downstream biological outcome.
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.
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-H⋯O hydrogen bonding.
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.
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