Scaffold Repurposing of Nucleosides (Adenosine Receptor Agonists): Enhanced Activity at the Human Dopamine and Norepinephrine Sodium Symporters

Abstract

We have repurposed (N)-methanocarba adenosine derivatives (A3 adenosine receptor (AR) agonists) to enhance radioligand binding allosterically at the human dopamine (DA) transporter (DAT) and inhibit DA uptake. We extended the structure-activity relationship of this series with small N6-alkyl substitution, 5′-esters, deaza modifications of adenine, and ribose restored in place of methanocarba. C2-(5-halothien-2-yl)-ethynyl 5′-methyl 9 (MRS7292) and 5′-ethyl 10 (MRS7232) esters enhanced binding at DAT (EC50 ∼35 nM) and at norepinephrine transporter (NET). 9 and 10 were selective for DAT compared to A3AR in the mouse, but not human. At DAT, binding of two structurally dissimilar radioligands was enhanced; NET binding of only one radioligand was enhanced; SERT radioligand binding was minimally affected. 10 was more potent than cocaine at inhibiting DA uptake (IC50 = 107 nM). Ribose analogues were weaker in DAT interaction than corresponding bicyclics. Thus, we enhanced the neurotransmitter transporters activity of rigid nucleosides while reducing A3AR affinity.

Polypharmacology of conformationally locked methanocarba nucleosides

“…MRS5980 and its congeners were demonstrated to have drug-like properties in ADMET tests, including oral bioavailability [34]. The high affinity (N)-methanocarba agonist MRS5980 ( 1 ) was twofold and 32-fold more-potent in binding to the human ( h ) and mouse ( m )A 3 AR, respectively, than the corresponding ribose derivative MRS7294 ( 2 ) [35]. Similarly, the N 6 -propyl equivalents of 1 and 2 (not shown) displayed even more pronounced difference; the binding affinity of the (N)-methanocarba analog was sixfold higher than the ribose equivalent at the h A 3 AR and 88-fold in affinity higher at the m A 3 AR [35].…”

“…The high affinity (N)-methanocarba agonist MRS5980 ( 1 ) was twofold and 32-fold more-potent in binding to the human ( h ) and mouse ( m )A 3 AR, respectively, than the corresponding ribose derivative MRS7294 ( 2 ) [35]. Similarly, the N 6 -propyl equivalents of 1 and 2 (not shown) displayed even more pronounced difference; the binding affinity of the (N)-methanocarba analog was sixfold higher than the ribose equivalent at the h A 3 AR and 88-fold in affinity higher at the m A 3 AR [35]. At the rat (r) A 3 AR, agonist affinity was better maintained with respect to h A 3 AR in the methanocarba series compared with the ribose series [7].…”

“…Structures of methanocarba nucleosides that interact with: (a) P2Y 1 R, (b) ENT1, (c) polymerases and kinases [35,45,62]. …”

Thermostabilization and purification of the human dopamine transporter (hDAT) in an inhibitor and allosteric ligand bound conformation

“…We discovered that the ligand binding activity of detergent extracted hDAT could be preserved if the extraction was performed in the presence of(N)-methanocarba nucleoside analogs(MRS compounds)(Fig S1b)(23, 24). The MRS compounds, which contain a rigidifying bicyclo[3.1.0]hexane ring system in place of ribose, were identified as allosteric ligands of hDAT during the off-target activity screening experiments.…”

“…hDAT is the major target of addictive psychostimulants such as cocaine, which bind to the active site and prevent the conformational transition of the transporter, thereby inhibiting the reuptake of dopamine(DA)(4, 21, 22). In addition to these potent inhibitors, certain(N)-methanocarba nucleoside analogs(MRS compounds, named after the Molecular Recognition Section of NIH) interact with hDAT and prevent transport(23, 24). The nucleoside analogues were initially derived from A 3 adenosine receptor(AR) agonists, and were subsequently structurally modified(Fig S1a) to enhance the interaction with DAT.…”