Vanadate trapping of nucleotide at the ATP-binding sites of human multidrug resistance P-glycoprotein exposes different residues to the drug-binding site

AbstractSulfonylureas are anti-diabetic medications that act by inhibiting pancreatic KATP channels composed of SUR1 and Kir6.2. The mechanism by which these drugs interact with and inhibit the channel has been extensively investigated, yet it remains unclear where the drug binding pocket resides. Here, we present a cryo-EM structure of the channel bound to a high-affinity sulfonylurea drug glibenclamide and ATP at 3.8Å resolution, which reveals in unprecedented details of the ATP and glibenclamide binding sites. Importantly, the structure shows for the first time that glibenclamide is lodged in the transmembrane bundle of the SUR1-ABC core connected to the first nucleotide binding domain near the inner leaflet of the lipid bilayer. Mutation of residues predicted to interact with glibenclamide in our model led to reduced sensitivity to glibenclamide. Our structure provides novel mechanistic insights of how sulfonylureas and ATP interact with the KATP channel complex to inhibit channel activity.

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COVID19 Approved Drug Repurposing: Pocket Similarity Approach

10.26434/chemrxiv.12722483 ◽

2020 ◽

Author(s):

Mohamed Fadlalla

Keyword(s):

Binding Site ◽

Binding Sites ◽

Drug Repurposing ◽

Drug Binding ◽

Drug Binding Site ◽

Main Protease ◽

Major Outbreak ◽

Target Binding ◽

Approved Drugs

<p>SARS CoV 2 has spread worldwide and caused a major outbreak of coronavirus disease 2019 (COVID-19). To date, no licensed drug or a vaccine is available against COVID19.</p><p>Starting from all of the resolved SARS CoV2 crystal structures, this study aims to find inhibitors for all of the SARS CoV2 proteins. To achieve this, I used PocketMatch to test the similarity of approved drugs binding sites against all of the binding sites found on SARS CoV 2 proteins. After that docking was used to confirm the results.</p><p>I found drugs that inhibit the main protease, Nsp12 and Nsp3. The discovered drugs are either in clinical trials (Sildenafil, Lopinavir, Ritonavir) or have in vitro antiviral activity (Nelfinavir, Indinavir, Amprenavir, depiqulinum , Gemcitabine, Raltitrexed, Aprepitant, montelukast, Ouabain, Raloxifene) whether against SARS CoV 2 or other viruses. In addition to this, further analysis of pockets revealed a steroidal pocket that might open the door to hypotheses on why the mortality of men is higher than women.</p><p>Many of the in silico repurposing studies test binding of the compound to the target using docking. The significance of this study adds to the similarity between the drug binding site and the target binding site. This takes into consideration the dynamic behaviour of the pocket after ligand binding.</p><div><br></div>

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Ultrastructural localization of drug binding sites

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081127 ◽

1978 ◽

Vol 36 (2) ◽

pp. 52-53

Author(s):

S.F. Hoff ◽

A.J. Macinnis

Keyword(s):

Binding Site ◽

Binding Sites ◽

Phosphotungstic Acid ◽

Ultrastructural Localization ◽

Drug Binding ◽

Electron Microscopic ◽

Electron Microscopic Autoradiography ◽

Drug Binding Site ◽

Drug Binding Sites ◽

Conventional Electron Microscopy

The need for a high resolution morphological method of identifying drug binding sites has been mentioned by several authors. Electron microscopic autoradiography may identify statisically an organelle as a drug binding site, but in practice has proven unreliable. This is primarily due to the tissue preparatory procedures used in conventional electron microscopy, which denature proteins and extract many proteins and lipid components from the tissue. As the drug binding sites are denatured and/or extracted, drugs are displaced from their “receptors.” Then the drugs are either extracted or attached to an anomolous binding site created by the preparatory procedure. Sjöstrand's minimal denaturation technique is based on a sound physical-chemical rationale and was modified slightly to accommodate the drug localization procedure used in this study.It was found after many experiments that phosphotungstic acid (PTA) at pH7 had the highest specificity for the drugs used in this study and the lowest affinity for biological structures.

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Proximity of Bound Hoechst 33342 to the ATPase Catalytic Sites Places the Drug Binding Site of P-glycoprotein within the Cytoplasmic Membrane Leaflet†

Biochemistry ◽

10.1021/bi0120897 ◽

2002 ◽

Vol 41 (14) ◽

pp. 4744-4752 ◽

Cited By ~ 59

Author(s):

Qin Qu ◽

Frances J. Sharom

Keyword(s):

Binding Site ◽

Cytoplasmic Membrane ◽

Drug Binding ◽

Hoechst 33342 ◽

Drug Binding Site ◽

P Glycoprotein ◽

Catalytic Sites

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Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol

Biochemical Journal ◽

10.1042/bj20060632 ◽

2006 ◽

Vol 401 (2) ◽

pp. 597-605 ◽

Cited By ~ 79

Author(s):

Yasuhisa Kimura ◽

Noriyuki Kioka ◽

Hiroaki Kato ◽

Michinori Matsuo ◽

Kazumitsu Ueda

Keyword(s):

Atpase Activity ◽

Molecular Mass ◽

Binding Site ◽

Drug Binding ◽

Soluble Form ◽

Strongly Correlated ◽

Drug Binding Site ◽

Hydrophobic Compounds ◽

P Glycoprotein ◽

Human Mdr1

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug–MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on Km values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). Vmax values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.

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Computational Pharmacogenetics of P-Glycoprotein Mediated Antiepileptic Drug Resistance

The Open Bioinformatics Journal ◽

10.2174/1875036201811010197 ◽

2018 ◽

Vol 11 (1) ◽

pp. 197-207 ◽

Cited By ~ 2

Author(s):

Sindhu Varghese ◽

Ashok Palaniappan

Keyword(s):

Drug Resistance ◽

Treatment Failure ◽

Conformational Changes ◽

Binding Sites ◽

Docking Studies ◽

Drug Binding ◽

Experimental Investigations ◽

Binding Affinities ◽

Drug Binding Site ◽

P Glycoprotein

Background:The treatment of epilepsy using antiepileptogenic drugs is complicated by drug resistance, resulting in treatment failure in more than one-third of cases. Human P-glycoprotein (hPGP;MDR1) is a known epileptogenic mediator.Methods:Given that experimental investigations have suggested a role for pharmacogenetics in this treatment failure, it would be of interest to study hPGP polymorphisms that might contribute to the emergence of drug resistance. Changes in protein functional activity could result from mutations as well as altered abundance. Bioinformatics approaches were used to assess and rank the functional impact of 20 missenseMDR1polymorphisms and the top five were selected. The structures of the wildtype and variant hPGP were modelled based on the mouse PGP structure. Docking studies of the wildtype and variant hPGP with four standard anti-epileptic drugs were carried out.Results:Our results revealed that the drug binding site with respect to the wildtype protein was uniform. However, the variant hPGP proteins displayed a repertoire of binding sites with stronger binding affinities towards the drug.Conclusion:Our studies indicated that specific polymorphisms inMDR1could drive conformational changes of PGP structure, facilitating altered contacts with drug-substrates and thus modifying their bioavailability. This suggests thatMDR1polymorphisms could actively contribute to the emergence of pharmaco-resistance in antiepileptic therapy.

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Drug-binding properties of rat α-foetoprotein. Specificities of the phenylbutazone-binding and warfarin-binding sites

Biochemical Journal ◽

10.1042/bj2390451 ◽

1986 ◽

Vol 239 (2) ◽

pp. 451-458 ◽

Cited By ~ 8

Author(s):

F Hervé ◽

K M Rajkowski ◽

M T Martin ◽

P Dessen ◽

N Cittanova

Keyword(s):

Binding Site ◽

Binding Sites ◽

Drug Binding ◽

Foetal Development ◽

Drug Binding Site ◽

Varied Chemical ◽

Competitive Protein Binding ◽

Potential Risks ◽

Drug Binding Sites ◽

Binding Mechanisms

Rat alpha-foetoprotein (alpha-FP) strongly binds the drugs warfarin and phenylbutazone, as does albumin; however, the binding sites for the two drugs seemed to be different. This possibility and the specificity of this/these drug-binding site(s) of rat alpha-FP were investigated by competitive protein-binding experiments with a variety of drugs, representing different pharmacological groups, and biomolecules that are strongly bound by the foetal protein and that are suspected to play a specific role during foetal development. The binding mechanisms were further investigated by using comparisons between computer-derived theoretical displacement curves and experimental points in order to distinguish different possible binding models. The results indicate: that warfarin and phenylbutazone are bound at two distinct sites on rat alpha-FP and that a negative modulatory effect is exerted between the two sites; that the degree of specificity of these two drug-binding sites is different, since the warfarin-binding site appears to be specific for the binding of coumarinic and anthranilic drugs whereas that for phenylbutazone is able to bind substances of very varied chemical structure and is more hydrophobic; that the phenylbutazone-binding site is the site that binds oestrogens that thyroid hormones and, probably, fatty acids and bilirubin are bound at (an)other site(s) but exert negative modulatory effects on phenylbutazone binding. The nature of the different binding areas of rat alpha-FP is compared with that of those already proposed for albumin. The potential risks of toxicity of such interactions between drugs and/or biomolecules on foetal development are also discussed.

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