scholarly journals Cryo-EM structures reveal distinct mechanisms of inhibition of the human multidrug transporter ABCB1

2020 ◽  
Vol 117 (42) ◽  
pp. 26245-26253 ◽  
Author(s):  
Kamil Nosol ◽  
Ksenija Romane ◽  
Rossitza N. Irobalieva ◽  
Amer Alam ◽  
Julia Kowal ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Drug Disposition ◽  
Structural Data ◽  
Binding Pocket ◽  
Drug Binding ◽  
Structural Basis ◽  
Multidrug Transporter ◽  
Fab Fragment ◽  
Extrusion Mechanism ◽  
Inhibitory Antibodies

ABCB1 detoxifies cells by exporting diverse xenobiotic compounds, thereby limiting drug disposition and contributing to multidrug resistance in cancer cells. Multiple small-molecule inhibitors and inhibitory antibodies have been developed for therapeutic applications, but the structural basis of their activity is insufficiently understood. We determined cryo-EM structures of nanodisc-reconstituted, human ABCB1 in complex with the Fab fragment of the inhibitory, monoclonal antibody MRK16 and bound to a substrate (the antitumor drug vincristine) or to the potent inhibitors elacridar, tariquidar, or zosuquidar. We found that inhibitors bound in pairs, with one molecule lodged in the central drug-binding pocket and a second extending into a phenylalanine-rich cavity that we termed the “access tunnel.” This finding explains how inhibitors can act as substrates at low concentration, but interfere with the early steps of the peristaltic extrusion mechanism at higher concentration. Our structural data will also help the development of more potent and selective ABCB1 inhibitors.

scholarly journals Structural basis for subtype-specific inhibition of the P2X7 receptor

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Akira Karasawa ◽  
Toshimitsu Kawate
Keyword(s):  
P2x7 Receptor ◽  
Hydrophobic Interactions ◽  
Binding Pocket ◽  
Drug Binding ◽  
Structural Basis ◽  
Atp Binding ◽  
Allosteric Site ◽  
Channel Opening ◽  
A Site ◽  
Novel Drug

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

scholarly journals Structural Basis of Enantioselective Inhibition of Cyclooxygenase-1 by S-α-Substituted Indomethacin Ethanolamides

2007 ◽  
Vol 282 (38) ◽  
pp. 28096-28105 ◽  
Author(s):  
Christine A. Harman ◽  
Melissa V. Turman ◽  
Kevin R. Kozak ◽  
Lawrence J. Marnett ◽  
William L. Smith ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Drug Binding ◽  
Structural Basis ◽  
Cyclooxygenase 1 ◽  
Cox 2 ◽  
The Impact ◽  
First Time ◽  
Side Pocket ◽  
Cox 1 ◽  
Equal Efficacy

The modification of the nonselective nonsteroidal anti-inflammatory drug, indomethacin, by amidation presents a promising strategy for designing novel cyclooxygenase (COX)-2-selective inhibitors. A series of α-substituted indomethacin ethanolamides, which exist as R/S-enantiomeric pairs, provides a means to study the impact of stereochemistry on COX inhibition. Comparative studies revealed that the R- and S-enantiomers of the α-substituted analogs inhibit COX-2 with almost equal efficacy, whereas COX-1 is selectively inhibited by the S-enantiomers. Mutagenesis studies have not been able to identify residues that manifest the enantioselectivity in COX-1. In an effort to understand the structural impact of chirality on COX-1 selectivity, the crystal structures of ovine COX-1 in complexes with an enantiomeric pair of these indomethacin ethanolamides were determined at resolutions between 2.75 and 2.85Å. These structures reveal unique, enantiomer-selective interactions within the COX-1 side pocket region that stabilize drug binding and account for the chiral selectivity observed with the (S)-α-substituted indomethacin ethanolamides. Kinetic analysis of binding demonstrates that both inhibitors bind quickly utilizing a two-step mechanism. However, the second binding step is readily reversible for the R-enantiomer, whereas for the S-enantiomer, it is not. These studies establish for the first time the structural and kinetic basis of high affinity binding of a neutral inhibitor to COX-1 and demonstrate that the side pocket of COX-1, previously thought to be sterically inaccessible, can serve as a binding pocket for inhibitor association.

scholarly journals Structural basis for ligand modulation of the CCR2 conformational landscape

2018 ◽  
Author(s):  
Bryn C. Taylor ◽  
Christopher T. Lee ◽  
Rommie E. Amaro
Keyword(s):  
Chemokine Receptor ◽  
Chemokine Receptors ◽  
Drug Targets ◽  
Control Cell ◽  
Binding Pocket ◽  
Drug Binding ◽  
Structural Basis ◽  
Cc Chemokine ◽  
Wide Range ◽  
Dynamics Simulations

AbstractCC Chemokine Receptor 2 (CCR2) is a part of the chemokine receptor family, an important class of therapeutic targets. These class A G-protein coupled receptors (GPCRs) are involved in mammalian signaling pathways and control cell migration toward endogenous CC chemokine ligands. Chemokine receptors and their associated ligands are involved in a wide range of diseases and thus have become important drug targets. Of particular interest is CCR2, which has been implicated in cancer, autoimmunity driven type-1 diabetes, diabetic nephropathy, multiple sclerosis, asthma, atherosclerosis, neuropathic pain, and rheumatoid arthritis. Although promising, CCR2 antagonists have been largely unsuccessful to date. Here, we investigate the effect of an orthosteric and an allosteric antagonist on CCR2 dynamics by coupling long timescale molecular dynamics simulations with Markov-state model theory. We find that the antagonists shift CCR2 into several stable inactive conformations that are distinct from the crystal structure conformation, and that they disrupt a continuous internal water and sodium ion pathway preventing transitions to an active-like state. Several of these stable conformations contain a putative drug binding pocket that may be amenable to targeting with another small molecule antagonist. In the absence of antagonists, the apo dynamics reveal intermediate conformations along the activation pathway that provide insight into the basal dynamics of CCR2, and may also be useful for future drug design.

Structural basis for the drug extrusion mechanism by a MATE multidrug transporter

Nature ◽  
2013 ◽  
Vol 496 (7444) ◽  
pp. 247-251 ◽  
Author(s):  
Yoshiki Tanaka ◽  
Christopher J. Hipolito ◽  
Andrés D. Maturana ◽  
Koichi Ito ◽  
Teruo Kuroda ◽  
...  
Keyword(s):  
Structural Basis ◽  
Multidrug Transporter ◽  
Extrusion Mechanism

scholarly journals Structural basis for ligand modulation of the CCR2 conformational landscape

2019 ◽  
Vol 116 (17) ◽  
pp. 8131-8136 ◽  
Author(s):  
Bryn C. Taylor ◽  
Christopher T. Lee ◽  
Rommie E. Amaro
Keyword(s):  
Chemokine Receptor ◽  
Chemokine Receptors ◽  
Drug Targets ◽  
Control Cell ◽  
Binding Pocket ◽  
Drug Binding ◽  
Structural Basis ◽  
Allosteric Site ◽  
Cc Chemokine ◽  
Wide Range

CC chemokine receptor 2 (CCR2) is a part of the chemokine receptor family, an important class of therapeutic targets. These class A G-protein coupled receptors (GPCRs) are involved in mammalian signaling pathways and control cell migration toward endogenous CC chemokine ligands, named for the adjacent cysteine motif on their N terminus. Chemokine receptors and their associated ligands are involved in a wide range of diseases and thus have become important drug targets. CCR2, in particular, promotes the metastasis of cancer cells and is also implicated in autoimmunity-driven type-1 diabetes, diabetic nephropathy, multiple sclerosis, asthma, atherosclerosis, neuropathic pain, and rheumatoid arthritis. Although promising, CCR2 antagonists have been largely unsuccessful to date. Here, we investigate the effect of an orthosteric and an allosteric antagonist on CCR2 dynamics by coupling long-timescale molecular dynamics simulations with Markov-state model theory. We find that the antagonists shift CCR2 into several stable inactive conformations that are distinct from the crystal structure conformation and disrupt a continuous internal water and sodium ion pathway, preventing transitions to an active-like state. Several metastable conformations present a cryptic drug-binding pocket near the allosteric site that may be amenable to targeting with small molecules. Without antagonists, the apo dynamics reveal intermediate conformations along the activation pathway that provide insight into the basal dynamics of CCR2 and may also be useful for future drug design.

scholarly journals The structural basis for monoclonal antibody 5D2 binding to the tryptophan-rich loop of lipoprotein lipase

2020 ◽  
Vol 61 (10) ◽  
pp. 1347-1359 ◽  
Author(s):  
John G. Luz ◽  
Anne P. Beigneux ◽  
DeeAnn K. Asamoto ◽  
Cuiwen He ◽  
Wenxin Song ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Resonance Raman Spectroscopy ◽  
Structural Basis ◽  
Fab Fragment ◽  
X Ray ◽  
X Ray Crystallography ◽  
Uv Resonance Raman Spectroscopy ◽  
Proline Substitution ◽  
Lipoprotein Binding ◽  
Α Helix

For three decades, the LPL–specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and UV resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short α-helix, with two Trps projecting into the antigen recognition site. A proline substitution in the α-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.

scholarly journals Structural basis of a potent human monoclonal antibody against Zika virus targeting a quaternary epitope

2019 ◽  
Vol 116 (5) ◽  
pp. 1591-1596 ◽  
Author(s):  
Feng Long ◽  
Michael Doyle ◽  
Estefania Fernandez ◽  
Andrew S. Miller ◽  
Thomas Klose ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Zika Virus ◽  
Neutralizing Antibodies ◽  
Human Monoclonal Antibody ◽  
Structural Basis ◽  
E Proteins ◽  
Fab Fragment ◽  
Cryo Electron Microscopy ◽  
E Protein ◽  
Mouse Model Of Infection

Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus in the Flaviviridae family. In contrast to most other insect-transmitted flaviviruses, ZIKV also can be transmitted sexually and from mother to fetus in humans. During recent outbreaks, ZIKV infections have been linked to microcephaly, congenital disease, and Guillain-Barré syndrome. Neutralizing antibodies have potential as therapeutic agents. We report here a 4-Å-resolution cryo-electron microscopy structure of the ZIKV virion in complex with Fab fragments of the potently neutralizing human monoclonal antibody ZIKV-195. The footprint of the ZIKV-195 Fab fragment expands across two adjacent envelope (E) protein protomers. ZIKV neutralization by this antibody is presumably accomplished by cross-linking the E proteins, which likely prevents formation of E protein trimers required for fusion of the viral and cellular membranes. A single dose of ZIKV-195 administered 5 days after virus inoculation showed marked protection against lethality in a stringent mouse model of infection.

scholarly journals Transmembrane Helix 12 of the Staphylococcus aureus Multidrug Transporter QacA Lines the Bivalent Cationic Drug Binding Pocket

2007 ◽  
Vol 189 (24) ◽  
pp. 9131-9134 ◽  
Author(s):  
Karl A. Hassan ◽  
Ronald A. Skurray ◽  
Melissa H. Brown
Keyword(s):  
Staphylococcus Aureus ◽  
Transmembrane Helix ◽  
Binding Pocket ◽  
Drug Binding ◽  
Cation Binding ◽  
Multidrug Transporter ◽  
Transmembrane Segment ◽  
Binding Region ◽  
Bivalent Cation ◽  
Helix 12

ABSTRACT An acidic residue in transmembrane segment (TMS) 10 is important for recognition of bivalent cationic substrates by the QacA multidrug transporter. Remarkably, an acidic residue in TMS 12 compensated for the absence of such a residue in TMS 10, suggesting that TMS 12 is a component of the bivalent cation-binding region.

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