TY - JOUR
T1 - Unraveling binding mechanism and kinetics of macrocyclic Gαq protein inhibitors
AU - Voss, Jan H.
AU - Nagel, Jessica
AU - Rafehi, Muhammad
AU - Guixà-González, Ramon
AU - Malfacini, Davide
AU - Patt, Julian
AU - Kehraus, Stefan
AU - Inoue, Asuka
AU - König, Gabriele M.
AU - Kostenis, Evi
AU - Deupi, Xavier
AU - Namasivayam, Vigneshwaran
AU - Müller, Christa E.
N1 - Funding Information:
This work was supported by the Deutsche Forschungsgemeinschaft ( DFG ) ( FOR2372 , MU-1665/7-2 ) and by the Swiss National Science Foundation (SNF; grant number 192780 ) to XD.
Publisher Copyright:
© 2021
PY - 2021/11
Y1 - 2021/11
N2 - G proteins represent intracellular switches that transduce signals relayed from G protein-coupled receptors. The structurally related macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent, selective inhibitors of the Gαq protein family. We recently discovered that radiolabeled FR and YM display strongly divergent residence times, which translates into significantly longer antiasthmatic effects of FR. The present study is aimed at investigating the molecular basis for this observed disparity. Based on docking studies, we mutated amino acid residues of the Gαq protein predicted to interact with FR or YM, and recombinantly expressed the mutated Gαq proteins in cells in which the native Gαq proteins had been knocked out by CRISPR-Cas9. Both radioligands showed similar association kinetics, and their binding followed a conformational selection mechanism, which was rationalized by molecular dynamics simulation studies. Several mutations of amino acid residues near the putative binding site of the “lipophilic anchors” of FR, especially those predicted to interact with the isopropyl group present in FR but not in YM, led to dramatically accelerated dissociation kinetics. Our data indicate that the long residence time of FR depends on lipophilic interactions within its binding site. The observed structure-kinetic relationships point to a complex binding mechanism of FR, which likely involves snap-lock- or dowel-like conformational changes of either ligand or protein, or both. These experimental data will be useful for the design of compounds with a desired residence time, a parameter that has now been recognized to be of utmost importance in drug development.
AB - G proteins represent intracellular switches that transduce signals relayed from G protein-coupled receptors. The structurally related macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent, selective inhibitors of the Gαq protein family. We recently discovered that radiolabeled FR and YM display strongly divergent residence times, which translates into significantly longer antiasthmatic effects of FR. The present study is aimed at investigating the molecular basis for this observed disparity. Based on docking studies, we mutated amino acid residues of the Gαq protein predicted to interact with FR or YM, and recombinantly expressed the mutated Gαq proteins in cells in which the native Gαq proteins had been knocked out by CRISPR-Cas9. Both radioligands showed similar association kinetics, and their binding followed a conformational selection mechanism, which was rationalized by molecular dynamics simulation studies. Several mutations of amino acid residues near the putative binding site of the “lipophilic anchors” of FR, especially those predicted to interact with the isopropyl group present in FR but not in YM, led to dramatically accelerated dissociation kinetics. Our data indicate that the long residence time of FR depends on lipophilic interactions within its binding site. The observed structure-kinetic relationships point to a complex binding mechanism of FR, which likely involves snap-lock- or dowel-like conformational changes of either ligand or protein, or both. These experimental data will be useful for the design of compounds with a desired residence time, a parameter that has now been recognized to be of utmost importance in drug development.
KW - Conformational selection
KW - FR900359
KW - Gq protein
KW - Molecular dynamics simulation
KW - Residence time
KW - YM-254890
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U2 - 10.1016/j.phrs.2021.105880
DO - 10.1016/j.phrs.2021.105880
M3 - Article
C2 - 34506902
AN - SCOPUS:85115013455
VL - 173
JO - Pharmacological Research
JF - Pharmacological Research
SN - 1043-6618
M1 - 105880
ER -