Fragment-based lead discovery is becoming an increasingly popular strategy for drug discovery. Fragment screening identifies weakly binding compounds that require optimization to become high-affinity leads. As design of leads from fragments is challenging, reliable computational methods to guide optimization would be invaluable. We evaluated using molecular dynamics simulations and the free energy perturbation method (MD/FEP) in fragment optimization for the A2A adenosine receptor, a pharmaceutically relevant G protein-coupled receptor. Optimization of fragments exploring two binding site subpockets was probed by calculating relative binding affinities for 23 adenine derivatives, resulting in strong agreement with experimental data (R2 = 0.78). The predictive power of MD/FEP was significantly better than that of an empirical scoring function. We also demonstrated the potential of the MD/FEP to assess multiple binding modes and to tailor the thermodynamic profile of ligands during optimization. Finally, MD/FEP was applied prospectively to optimize three nonpurine fragments, and predictions for 12 compounds were evaluated experimentally. The direction of the change in binding affinity was correctly predicted in a majority of the cases, and agreement with experiment could be improved with rigorous parameter derivation. The results suggest that MD/FEP will become a powerful tool in structure-driven optimization of fragments to lead candidates.

Adenosine derivatives developed to activate adenosine receptors (ARs) revealed µM activity at serotonin 5HT2B and 5HT2C receptors (5HTRs). We explored the SAR at 5HT2Rs and modeled receptor interactions in order to optimize affinity and simultaneously reduce AR affinity. Depending on N6 substitution, small 5′-alkylamide modification maintained 5HT2BR affinity, which was enhanced upon ribose substitution with rigid bicyclo[3.1.0]hexane (North (N)-methanocarba), e.g. N6-dicyclopropylmethyl 4′-CH2OH derivative 14 (Ki 11 nM). 5′-Methylamide 23 was 170-fold selective as antagonist for 5HT2BR vs. 5HT2CR. 5′-Methyl 25 and ethyl 26 esters potently antagonized 5HT2Rs with moderate selectivity in comparison to ARs; related 6-N,N-dimethylamino analogue 30 was 5HT2R-selective. 5′ position flexibility of substitution was indicated in 5HT2BR docking. Both 5′-ester and 5′-amide derivatives displayed in vivo t1/2 of 3–4 h. Thus, we used GPCR modeling to repurpose nucleoside scaffolds in favor of binding at nonpurine receptors, as novel 5HT2R antagonists, with potential for cardioprotection, liver protection or CNS activity.

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.