Abstract
A human dopamine transporter (DAT) cryo-EM structure was recently reported, as stabilized by an allosteric inhibitor, i.e. rigid nucleoside MRS7292 1, a tropane orthosteric inhibitor and Zn(2+). We have synthesized multiple North (N)-methanocarba-adenosine analogues of 1, with N(6), C2 and 4' modifications to examine their effects on DAT radioligand binding (either enhancement or inhibition) and at the norepinephrine (NET) and serotonin (SERT) transporters (generally inhibition). Small N(6) groups provided the most pronounced DAT enhancement (≥500 % at 10 μM for N(6)-methyl 2 and N(6)-cyclopropyl 8 5'-ethyl esters, and N(6)-methyl-4'-cyanomethyl 44 analogues). Various N(6)-(ω-phenyl-alkyl) groups caused binding inhibition and compensated for the removal of stabilizing interactions shown in the 1-DAT structure, e.g. an N7 H-bond, by accessing a previously uncharacterized DAT distal binding region. The optimal chain length was five methylenes for bitopic N(6)-(ω-phenyl-alkyl) derivatives, e.g. 16 and 31, having favored 5'-ethyl ester (but not 4'-cyanomethyl) and 2-(arylethynyl) groups. Surprisingly, the previously noted reduced DAT activity with a 2-iodo group, could be compensated by N(6)-(ω-phenyl-alkyl) groups. N(6)-(6-Phenylhexyl)-2-iodo compounds, 53 (5'-hydroxy, K(i) 0.89 μM, pan-inhibitor) and 54 (4'-cyanomethyl, K(i) 0.40 μM), inhibited DAT binding. Chiral, branched N(6) groups displayed binding stereoselectivity. Key nucleosides were docked to outward-facing hDAT cryo-EM structures. Molecular dynamics simulation predicted π-π interactions of the N(6)-(ω-phenyl-alkyl) substituent and aromatic side chains of F208(EL2) and H375(EL4) (aromatic conserved at NET) to define a preferred binding mode for the extended chain. Thus, we characterized the transporter SAR of this series, either enhancement or inhibition of orthosteric radioligand binding, and transport inhibition, and with DAT structural predictions.