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 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 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 Identification of a novel post-hydrolytic state in CFTR gating

2012 ◽  
Vol 139 (5) ◽  
pp. 359-370 ◽  
Author(s):  
Kang-Yang Jih ◽  
Yoshiro Sohma ◽  
Min Li ◽  
Tzyh-Chang Hwang
Keyword(s):  
Free Energy ◽  
Chloride Channel ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Fast Response ◽  
Structural Basis ◽  
Atp Binding ◽  
Closed State ◽  
Binding Domains ◽  
Abc Protein

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, ubiquitous proteins found in all kingdoms of life, catalyze substrates translocation across biological membranes using the free energy of ATP hydrolysis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of this superfamily in that it functions as an ATP-gated chloride channel. Despite difference in function, recent studies suggest that the CFTR chloride channel and the exporter members of the ABC protein family may share an evolutionary origin. Although ABC exporters harness the free energy of ATP hydrolysis to fuel a transport cycle, for CFTR, ATP-induced dimerization of its nucleotide-binding domains (NBDs) and subsequent hydrolysis-triggered dimer separation are proposed to be coupled, respectively, to the opening and closing of the gate in its transmembrane domains. In this study, by using nonhydrolyzable ATP analogues, such as pyrophosphate or adenylyl-imidodiphosphate as baits, we captured a short-lived state (state X), which distinguishes itself from the previously identified long-lived C2 closed state by its fast response to these nonhydrolyzable ligands. As state X is caught during the decay phase of channel closing upon washout of the ligand ATP but before the channel sojourns to the C2 closed state, it likely emerges after the bound ATP in the catalysis-competent site has been hydrolyzed and the hydrolytic products have been released. Thus, this newly identified post-hydrolytic state may share a similar conformation of NBDs as the C2 closed state (i.e., a partially separated NBD and a vacated ATP-binding pocket). The significance of this novel state in understanding the structural basis of CFTR gating is discussed.

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.

scholarly journals Identification and Biological Evaluation of CK2 Allosteric Fragments through Structure-Based Virtual Screening

Molecules ◽  
2020 ◽  
Vol 25 (1) ◽  
pp. 237 ◽  
Author(s):  
Chunqiong Li ◽  
Xuewen Zhang ◽  
Na Zhang ◽  
Yue Zhou ◽  
Guohui Sun ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Interaction Mechanism ◽  
Pharmacophore Model ◽  
Binding Pocket ◽  
Biological Evaluation ◽  
Atp Binding ◽  
Allosteric Site ◽  
Allosteric Inhibitors ◽  
Competitive Inhibitors ◽  
Reduced Toxicity

Casein kinase II (CK2) is considered as an attractive cancer therapeutic target, and recent efforts have been made to develop its ATP-competitive inhibitors. However, achieving selectivity with respect to related kinases remains challenging due to the highly conserved ATP-binding pocket of kinases. Allosteric inhibitors, by targeting the much more diversified allosteric site relative to the highly conserved ATP-binding pocket, might be a promising strategy with the enhanced selectivity and reduced toxicity than ATP-competitive inhibitors. The previous studies have highlighted the traditional serendipitousity of discovering allosteric inhibitors owing to the complicate allosteric modulation. In this current study, we identified the novel allosteric inhibitors of CK2α by combing structure-based virtual screening and biological evaluation methods. The structure-based pharmacophore model was built based on the crystal structure of CK2α-compound 15 complex. The ChemBridge fragment library was searched by evaluating the fit values of these molecules with the optimized pharmacophore model, as well as the binding affinity of the CK2α-ligand complexes predicted by Alloscore web server. Six hits forming the holistic interaction mechanism with the αD pocket were retained after pharmacophore- and Alloscore-based screening for biological test. Compound 3 was found to be the most potent non-ATP competitive CK2α inhibitor (IC50 = 13.0 μM) with the anti-proliferative activity on A549 cancer cells (IC50 = 23.1 μM). Our results provide new clues for further development of CK2 allosteric inhibitors as anti-cancer hits.

scholarly journals Pathway and mechanism of drug binding to chemokine receptors revealed by accelerated molecular simulations

2020 ◽  
Vol 12 (13) ◽  
pp. 1213-1225 ◽  
Author(s):  
Shristi Pawnikar ◽  
Yinglong Miao
Keyword(s):  
Molecular Dynamics ◽  
Binding Site ◽  
Chemokine Receptors ◽  
Cancer Metastasis ◽  
Drug Binding ◽  
Host Cells ◽  
Allosteric Site ◽  
Accelerated Molecular Dynamics ◽  
Hiv Entry ◽  
Novel Drug

Background: Chemokine GPCRs play key roles in biology and medicine. Particularly, CXCR4 promotes cancer metastasis and facilitate HIV entry into host cells. Plerixafor (PLX) is a CXCR4 drug, but the pathway and binding site of PLX in CXCR4 remain unknown. Results & methodology: We have performed molecular docking and all-atom simulations using Gaussian accelerated molecular dynamics (GaMD), which are consistent with previous mutation experiments, suggesting that PLX binds to the orthosteric site of CXCR4 as an antagonist. The GaMD simulations further revealed an intermediate allosteric binding site at the extracellular mouth of CXCR4. Conclusion: The newly identified allosteric site can be targeted for novel drug design targeting CXCR4 and other chemokine receptors.

scholarly journals Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site

2006 ◽  
Vol 401 (1) ◽  
pp. 39-47 ◽  
Author(s):  
Sheng Li ◽  
Yongcheng Lu ◽  
Baozhen Peng ◽  
Jianping Ding
Keyword(s):  
Enzymatic Activity ◽  
Binding Site ◽  
Active Site ◽  
Atp Binding ◽  
Allosteric Site ◽  
Nucleotide Synthesis ◽  
Structural Stabilization ◽  
Atp Binding Site ◽  
Phosphate Ions ◽  
A Site

PRPP (phosphoribosylpyrophosphate) is an important metabolite essential for nucleotide synthesis and PRS (PRPP synthetase) catalyses synthesis of PRPP from R5P (ribose 5-phosphate) and ATP. The enzymatic activity of PRS is regulated by phosphate ions, divalent metal cations and ADP. In the present study we report the crystal structures of recombinant human PRS1 in complexes with SO42− ions alone and with ATP, Cd2+ and SO42− ions respectively. The AMP moiety of ATP binds at the ATP-binding site, and a Cd2+ ion binds at the active site and in a position to interact with the β- and γ-phosphates of ATP. A SO42− ion, an analogue of the activator phosphate, was found to bind at both the R5P-binding site and the allosteric site defined previously. In addi-tion, an extra SO42− binds at a site at the dimer interface between the ATP-binding site and the allosteric site. Binding of this SO42− stabilizes the conformation of the flexible loop at the active site, leading to the formation of the active, open conformation which is essential for binding of ATP and initiation of the catalytic reaction. This is the first time that structural stabilization at the active site caused by binding of an activator has been observed. Structural and biochemical data show that mutations of some residues at this site influence the binding of SO42− and affect the enzymatic activity. The results in the present paper suggest that this new SO42−-binding site is a second allosteric site to regulate the enzymatic activity which might also exist in other eukaryotic PRSs (except plant PRSs of class II), but not in bacterial PRSs.

scholarly journals Recent Advances in the Discovery of CK2 Allosteric Inhibitors: From Traditional Screening to Structure-Based Design

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 870 ◽  
Author(s):  
Xiaolan Chen ◽  
Chunqiong Li ◽  
Dada Wang ◽  
Yu Chen ◽  
Na Zhang
Keyword(s):  
Protein Kinase Ck2 ◽  
Binding Pocket ◽  
Atp Binding ◽  
Binding Modes ◽  
Allosteric Site ◽  
Allosteric Inhibitors ◽  
Atp Binding Site ◽  
Recent Advances ◽  
Allosteric Sites ◽  
Kinase Ck2

Protein kinase (CK2) has emerged as an attractive cancer therapeutic target and recent efforts have been made to develop its inhibitors. However, the development of selective inhibitors remains challenging because of the highly conserved ATP-binding pocket (orthosteric site) of kinase family. As an alternative strategy, allosteric inhibitors, by targeting the much more diversified allosteric site relative to the conserved ATP-binding site, achieve better pharmacological advantages than orthosteric inhibitors. Traditional serendipitous screening and structure-based design are robust tools for the discovery of CK2 allosteric inhibitors. In this review, we summarize the recent advances in the identification of CK2 allosteric inhibitors. Firstly, we briefly present the CK2 allosteric sites. Then, the allosteric inhibitors targeting the well-elucidated allosteric sites (α/β interface, αD pocket and interface between the Glycine-rich loop and αC-helix) are highlighted in the discovery process and possible binding modes with the allosteric sites are described. This study is expected to provide valuable clues for the design of CK2 allosteric inhibitors.

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 of rifampin inactivation by rifampin phosphotransferase

2016 ◽  
Vol 113 (14) ◽  
pp. 3803-3808 ◽  
Author(s):  
Xiaofeng Qi ◽  
Wei Lin ◽  
Miaolian Ma ◽  
Chengyuan Wang ◽  
Yang He ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Conformational Changes ◽  
Bacterial Infections ◽  
Binding Pocket ◽  
Resistance Mechanisms ◽  
Binding Domain ◽  
Structural Basis ◽  
Atp Binding ◽  
Toggle Switch ◽  
The Cost

Rifampin (RIF) is a first-line drug used for the treatment of tuberculosis and other bacterial infections. Various RIF resistance mechanisms have been reported, and recently an RIF-inactivation enzyme, RIF phosphotransferase (RPH), was reported to phosphorylate RIF at its C21 hydroxyl at the cost of ATP. However, the underlying molecular mechanism remained unknown. Here, we solve the structures of RPH from Listeria monocytogenes (LmRPH) in different conformations. LmRPH comprises three domains: an ATP-binding domain (AD), an RIF-binding domain (RD), and a catalytic His-containing domain (HD). Structural analyses reveal that the C-terminal HD can swing between the AD and RD, like a toggle switch, to transfer phosphate. In addition to its catalytic role, the HD can bind to the AD and induce conformational changes that stabilize ATP binding, and the binding of the HD to the RD is required for the formation of the RIF-binding pocket. A line of hydrophobic residues forms the RIF-binding pocket and interacts with the 1-amino, 2-naphthol, 4-sulfonic acid and naphthol moieties of RIF. The R group of RIF points toward the outside of the pocket, explaining the low substrate selectivity of RPH. Four residues near the C21 hydroxyl of RIF, His825, Arg666, Lys670, and Gln337, were found to play essential roles in the phosphorylation of RIF; among these the His825 residue may function as the phosphate acceptor and donor. Our study reveals the molecular mechanism of RIF phosphorylation catalyzed by RPH and will guide the development of a new generation of rifamycins.

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