The Tumor-Selective Cytotoxic Agent β-Lapachone is a Potent Inhibitor of IDO1

β-lapachone is a naturally occurring 1,2-naphthoquinone-based compound that has been advanced into clinical trials based on its tumor-selective cytotoxic properties. Previously, we focused on the related 1,4-naphthoquinone pharmacophore as a basic core structure for developing a series of potent indoleamine 2,3-dioxygenase 1 (IDO1) enzyme inhibitors. In this study, we identified IDO1 inhibitory activity as a previously unrecognized attribute of the clinical candidate β-lapachone. Enzyme kinetics-based analysis of β-lapachone indicated an uncompetitive mode of inhibition, while computational modeling predicted binding within the IDO1 active site consistent with other naphthoquinone derivatives. Inhibition of IDO1 has previously been shown to breach the pathogenic tolerization that constrains the immune system from being able to mount an effective anti-tumor response. Thus, the finding that β-lapachone has IDO1 inhibitory activity adds a new dimension to its potential utility as an anti-cancer agent distinct from its cytotoxic properties, and suggests that a synergistic benefit can be achieved from its combined cytotoxic and immunologic effects

Spontaneous Rearrangement of the β20/β21 Strands in Simulations of Unliganded HIV‑1 Glycoprotein, gp120

Binding of the viral spike drives cell entry and infection by HIV-1 to the cellular CD4 and chemokine receptors with associated conformational change of the viral glycoprotein envelope, gp120. Crystal structures of the CD4–gp120–antibody ternary complex reveal a large internal gp120 cavity formed by three domainsthe inner domain, outer domain, and bridging sheet domainand are capped by CD4 residue Phe43. Several structures of gp120 envelope in complex with various antibodies indicated that the bridging sheet adopts varied conformations. Here, we examine bridging sheet dynamics using a crystal structure of gp120 bound to the F105 antibody exhibiting an open bridging sheet conformation and with an added V3 loop. The two strands of the bridging sheet β2/β3 and β20/β21 are dissociated from each other and are directed away from the inner and outer domains. Analysis of molecular dynamics (MD) trajectories indicates that the β2/β3 and β20/β21 strands rapidly rearrange to interact with the V3 loop and the inner and outer domains, respectively. Residue N425 on β20 leads the conformational rearrangement of the β20/β21 strands by interacting with W112 on the inner domain and F382 on the outer domain. An accompanying shift is observed in the inner domain as helix α1 exhibits a loss in helicity and pivots away from helix α5. The two simulations provide a framework for understanding the conformational diversity of the bridging sheet and the propensity of the β20/β21 strand to refold between the inner and outer domains of gp120, in the absence of a bound ligand

Spontaneous Rearrangement of the β20/β21 Strands in Simulations of Unliganded HIV‑1 Glycoprotein, gp120

Binding of the viral spike drives cell entry and infection by HIV-1 to the cellular CD4 and chemokine receptors with associated conformational change of the viral glycoprotein envelope, gp120. Crystal structures of the CD4–gp120–antibody ternary complex reveal a large internal gp120 cavity formed by three domainsthe inner domain, outer domain, and bridging sheet domainand are capped by CD4 residue Phe43. Several structures of gp120 envelope in complex with various antibodies indicated that the bridging sheet adopts varied conformations. Here, we examine bridging sheet dynamics using a crystal structure of gp120 bound to the F105 antibody exhibiting an open bridging sheet conformation and with an added V3 loop. The two strands of the bridging sheet β2/β3 and β20/β21 are dissociated from each other and are directed away from the inner and outer domains. Analysis of molecular dynamics (MD) trajectories indicates that the β2/β3 and β20/β21 strands rapidly rearrange to interact with the V3 loop and the inner and outer domains, respectively. Residue N425 on β20 leads the conformational rearrangement of the β20/β21 strands by interacting with W112 on the inner domain and F382 on the outer domain. An accompanying shift is observed in the inner domain as helix α1 exhibits a loss in helicity and pivots away from helix α5. The two simulations provide a framework for understanding the conformational diversity of the bridging sheet and the propensity of the β20/β21 strand to refold between the inner and outer domains of gp120, in the absence of a bound ligand

Spontaneous Rearrangement of the β20/β21 Strands in Simulations of Unliganded HIV‑1 Glycoprotein, gp120

Binding of the viral spike drives cell entry and infection by HIV-1 to the cellular CD4 and chemokine receptors with associated conformational change of the viral glycoprotein envelope, gp120. Crystal structures of the CD4–gp120–antibody ternary complex reveal a large internal gp120 cavity formed by three domainsthe inner domain, outer domain, and bridging sheet domainand are capped by CD4 residue Phe43. Several structures of gp120 envelope in complex with various antibodies indicated that the bridging sheet adopts varied conformations. Here, we examine bridging sheet dynamics using a crystal structure of gp120 bound to the F105 antibody exhibiting an open bridging sheet conformation and with an added V3 loop. The two strands of the bridging sheet β2/β3 and β20/β21 are dissociated from each other and are directed away from the inner and outer domains. Analysis of molecular dynamics (MD) trajectories indicates that the β2/β3 and β20/β21 strands rapidly rearrange to interact with the V3 loop and the inner and outer domains, respectively. Residue N425 on β20 leads the conformational rearrangement of the β20/β21 strands by interacting with W112 on the inner domain and F382 on the outer domain. An accompanying shift is observed in the inner domain as helix α1 exhibits a loss in helicity and pivots away from helix α5. The two simulations provide a framework for understanding the conformational diversity of the bridging sheet and the propensity of the β20/β21 strand to refold between the inner and outer domains of gp120, in the absence of a bound ligand

Localized Changes in the gp120 Envelope Glycoprotein Confer Resistance to Human Immunodeficiency Virus Entry Inhibitors BMS-806 and #155

BMS-806 and the related compound, #155, are novel inhibitors of human immunodeficiency virus type 1 (HIV-1) entry that bind the gp120 exterior envelope glycoprotein. BMS-806 and #155 block conformational changes in the HIV-1 envelope glycoproteins that are induced by binding to the host cell receptor, CD4. We tested a panel of HIV-1 envelope glycoprotein mutants and identified several that were resistant to the antiviral effects of BMS-806 and #155. In the CD4-bound conformation of gp120, the amino acid residues implicated in BMS-806 and #155 resistance line the “phenylalanine 43 cavity” and a water-filled channel that extends from this cavity to the inner domain. Structural considerations suggest a model in which BMS-806 and #155 bind gp120 prior to receptor binding and, upon CD4 binding, are accommodated in the Phe-43 cavity and adjacent channel. The integrity of the nearby V1/V2 variable loops and N-linked carbohydrates on the V1/V2 stem indirectly influences sensitivity to the drugs. A putative binding site for BMS-806 and #155 between the gp120 receptor-binding regions and the inner domain, which is thought to interact with the gp41 transmembrane envelope glycoprotein, helps to explain the mode of action of these drugs

Small-Molecule CD4 Mimics Interact with a Highly Conserved Pocket on HIV-1 gp120

Human immunodeficiency virus (HIV-1) interaction with the primary receptor, CD4, induces conformational changes in the viral envelope glycoproteins that allow binding to the CCR5 second receptor and virus entry into the host cell. The small molecule NBD-556 mimics CD4 by binding the gp120 exterior envelope glycoprotein, moderately inhibiting virus entry into CD4-expressing target cells, and enhancing CCR5 binding and virus entry into CCR5-expressing cells lacking CD4. Studies of NBD-556 analogues and gp120 mutants suggest that: 1) NBD-556 binds within the Phe 43 cavity, a highly conserved, functionally important pocket formed as gp120 assumes the CD4- bound conformation; 2) the NBD-556 phenyl ring projects into the Phe 43 cavity; 3) enhancement of CD4-independent infection by NBD-556 requires the induction of conformational changes in gp120; and 4) increased affinity of NBD-556 analogues for gp120 improves antiviral potency during infection of CD4-expressing cells

Structure-Based Design, Synthesis and Validation of CD4-Mimetic Small Molecule Inhibitors of HIV-1 Entry: Conversion of a Viral Entry Agonist to an Antagonist

Conspectus This Account provides an overview of a multidisciplinary consortium focused on structure-based strategies to devise small molecule antagonists of HIV-1 entry into human T-cells, which if successful would hold considerable promise for the development of prophylactic modalities to prevent HIV transmission and thereby alter the course of the AIDS pandemic. Entry of the human immunodeficiency virus (HIV) into target T-cells entails an interaction between CD4 on the host T-cell and gp120, a component of the trimeric envelope glycoprotein spike on the virion surface. The resultant interaction initiates a series of conformational changes within the envelope spike that permits binding to a chemokine receptor, formation of the gp41 fusion complex, and cell entry. A hydrophobic cavity at the CD4–gp120 interface, defined by X-ray crystallography, provided an initial site for small molecule antagonist design. This site however has evolved to facilitate viral entry. As such, the binding of prospective small molecule inhibitors within this gp120 cavity can inadvertently trigger an allosteric entry signal. Structural characterization of the CD4–gp120 interface, which provided the foundation for small molecule structure-based inhibitor design, will be presented first. An integrated approach combining biochemical, virological, structural, computational, and synthetic studies, along with a detailed analysis of ligand binding energetics, revealed that modestly active small molecule inhibitors of HIV entry can also promote viral entry into cells lacking the CD4 receptor protein; these competitive inhibitors were termed small molecule CD4 mimetics. Related congeners were subsequently identified with both improved binding affinity and more potent viral entry inhibition. Further assessment of the affinity-enhanced small molecule CD4 mimetics demonstrated that premature initiation of conformational change within the viral envelope spike, prior to cell encounter, can lead to irreversible deactivation of viral entry machinery. Related congeners, which bind the same gp120 site, possess different propensities to elicit the allosteric response that underlies the undesired enhancement of CD4-independent viral entry. Subsequently, key hotspots in the CD4–gp120 interface were categorized using mutagenesis and isothermal titration calorimetry according to the capacity to increase binding affinity without triggering the allosteric signal. This analysis, combined with cocrystal structures of small molecule viral entry agonists with gp120, led to the development of fully functional antagonists of HIV-1 entry. Additional structure-based design exploiting two hotspots followed by synthesis has now yielded low micromolar inhibitors of viral entry

A Model of Peptide Triazole Entry Inhibitor Binding to HIV‑1 gp120 and the Mechanism of Bridging Sheet Disruption

Peptide triazole (PT) entry inhibitors prevent HIV-1 infection by blocking the binding of viral gp120 to both the HIV-1 receptor and the coreceptor on target cells. Here, we used all-atom explicit solvent molecular dynamics (MD) to propose a model for the encounter complex of the peptide triazoles with gp120. Saturation transfer difference nuclear magnetic resonance (STD NMR) and single-site mutagenesis experiments were performed to test the simulation results. We found that docking of the peptide to a conserved patch of residues lining the “F43 pocket” of gp120 in a bridging sheet naïve gp120 conformation of the glycoprotein led to a stable complex. This pose prevents formation of the bridging sheet minidomain, which is required for receptor–coreceptor binding, providing a mechanistic basis for dual-site antagonism of this class of inhibitors. Burial of the peptide triazole at the gp120 inner domain–outer domain interface significantly contributed to complex stability and rationalizes the significant contribution of hydrophobic triazole groups to peptide potency. Both the simulation model and STD NMR experiments suggest that the I-X-W [where X is (2<i>S</i>,4<i>S</i>)-4-(4-phenyl-1<i>H</i>-1,2,3-triazol-1-yl)­pyrrolidine] tripartite hydrophobic motif in the peptide is the major contributor of contacts at the gp120–PT interface. Because the model predicts that the peptide Trp side chain hydrogen bonding with gp120 S375 contributes to the stability of the PT–gp120 complex, we tested this prediction through analysis of peptide binding to gp120 mutant S375A. The results showed that a peptide triazole KR21 inhibits S375A with 20-fold less potency than WT, consistent with predictions of the model. Overall, the PT–gp120 model provides a starting point for both the rational design of higher-affinity peptide triazoles and the development of structure-minimized entry inhibitors that can trap gp120 into an inactive conformation and prevent infection