Κατεύθυνση Σχεδιασμός και Ανάπτυξη νέων Φαρμακευτικών Ενώσεων - Φαρμακευτική Χημεία
Library of the School of Science

2019-09-17

2019

Stamatis Dimitrios

Κολοκούρης Αντώνιος, Αναπληρωτής Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ

Μελέτη της Ορθοστερικής Περιοχής Πρόσδεσης Αγωνιστών του Υποδοχέα Αδενοσίνης A3 με Υπολογιστικές Προσομοιώσεις και Πειράματα Μεταλλαξιγένεσης

Greek

Structural Characterization of Agonist Binding to A3 Adenosine Receptor Through Biomolecular Simulations and Mutagenesis Experiments

Adenosine A3 receptor (A3R), which is activated by adenosine, is over-expressed in various tumor cells and it is a promising drug target against cancer cell proliferation and other conditions including asthma, rheumatoid arthritis and ischemic injury. Currently there is no experimental structure of A3R and in this work the orthosteric binding site of A3R in complex with two agonists, the non-selective 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-D-ribofuranuronamide (NECA, (2)) and the selective 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide (IB-MECA, (3)) was studied. Molecular dynamics (MD) simulations of the wild-type (WT) A3R in complex with NECA (2) or IB-MECA (3) were performed to identify the residues important for agonist binding in the orthosteric site. Mutagenesis and functional assays were performed for the measurement of the activities (pIC50) of NECA (2) and IB-MECA (3) against the mutated forms of A3R, in order to further develop our theoretical observations. The application of Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) binding free energy calculations was able to distinguish mutations that reduce or negate NECA (2) or IB-MECA (3) activity from those that maintained or increased activity. The calculated ΔGeff values for both NECA (2) and IB-MECA (3) displayed good correlations with experimental activities (r=-0.76 and -0.69 respectively). The combined computational and experimental results suggested that agonist binding and receptor activation is realized through direct interactions with residues of the orthosteric area, such as (a) π-π interactions with F1685.29, (b) van der Waals interactions with L2466.51 and I2687.39, and (c) hydrogen bond interactions with T943.36, N2506.55 S2717.42 and H2727.43. Mutating these residues to alanine negated agonist activity. In general, mutation of the directly interacting residues V1695.30, W1855.46 and L2647.35 (with heavy atoms within 3,5 Å from the agonists) to alanine maintained agonist activity, although the first one increased the potency of IB-MECA (3) significantly. Moreover, although V1695.30 is considered to be a selectivity filter for A3R binders, when this residue was mutated to glutamic acid, the activity of IB-MECA (3) against A3R increased. Indirect interactions including residues L903.32 and M1775.38 were critical for both agonists binding. M1745.35 was important only for NECA (2), while I2536.58 was unimportant for the binding of both agonists. The study aimed at (a) highlighting features of the still-unsolved A3R that are important for IB-MECA (3)and NECA (2) binding, (b) assessing the accuracy of the MM-GBSA method for the calculation of binding free energies, and (c) combining the computational and experimental results to make conclusions about the design of effective ligands of A3R.

Science

adenosine receptor A3, Α3R, ΙΒ-MECA, NECA, molecular dynamics, mutagenesis, functional assays, MM-GBSA, orthosteric binding site

Yes

2

Yes

117

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https://pergamos.lib.uoa.gr/uoa/dl/object/2880823