Structure–Affinity Relationships and Structure–Kinetics Relationships of Pyrido[2,1-f]purine-2,4-dione Derivatives as Human Adenosine A₃ Receptor Antagonists

Abstract

We expanded on a series of pyrido[2,1-f]purine-2,4-dione derivatives as human adenosine A3 receptor (hA3R) antagonists to determine their kinetic profiles and affinities. Many compounds showed high affinities and a diverse range of kinetic profiles. We found hA3R antagonists with very short residence time (RT) at the receptor (2.2 min for 5) and much longer RTs (e.g., 376 min for 27 or 391 min for 31). Two representative antagonists (5 and 27) were tested in [35S]GTPγS binding assays, and their RTs appeared correlated to their (in)surmountable antagonism. From a kon–koff–KD kinetic map, we divided the antagonists into three subgroups, providing a possible direction for the further development of hA3R antagonists. Additionally, we performed a computational modeling study that sheds light on the crucial receptor interactions, dictating the compounds’ binding kinetics. Knowledge of target binding kinetics appears useful for developing and triaging new hA3R antagonists in the early phase of drug discovery.

Structure–Affinity Relationships and Structure–Kinetic Relationships of 1,2-Diarylimidazol-4-carboxamide Derivatives as Human Cannabinoid 1 Receptor Antagonists

“… 20 − 22 In the research field of GPCRs, a number of structure–kinetic relationship (SKR) studies have been published and the results suggest that the strategic combination of SKR with classic structure–affinity relationships (SAR) can improve the resulting decision process. 23 − 26 By doing so, ligand–receptor interactions can be better understood, as together they not only comprise the equilibrium state of a ligand–receptor interaction but also its metastable intermediates and/or transition states. 27 The binding kinetics driven drug discovery approach for the hCB 1 receptor has been validated in some aspects already by its application in the development of allosteric modulators of the hCB 1 receptor.…”

Development of Covalent Ligands for G Protein-Coupled Receptors: A Case for the Human Adenosine A3 Receptor

“…To examine the interaction between receptor residues possibly involved in covalent binding, we docked 17b into a ligand optimized homology model on the basis of the A 2A receptor crystal structure (PDB: 4EIY(27)), as described previously. 7 As detailed in Figure 6, the core structure of compound 17b interacted with the TM3, TM6, and EL2 regions. Additionally, the carbonyl-oxygen at the C 4 -position participated in H-bond formation with residue N250 6.55 and the methoxyl moiety at the C 8 -position functioned as H-bond acceptor with Q167 EL2 .…”

“…6−8 Using compound 1 , a nanomolar probe from the previous series, as the starting point, we further designed and synthesized compounds based on a previously suggested binding mode of the pyrido[2,1- f ]purine-2,4-dione scaffold. 7 When examining the suggested binding mode of this scaffold, we noted that this scaffold inserted into the binding pocket with a receptor interaction between TM3, TM6, and EL2. Two key H-bonds include the carbonyl-oxygen at the C 4 -position with residue N250 6.55 and the methoxy substituent at the C 8 -position bonding to Q167 EL2 .…”

“…Here, we performed a competition association assay to characterize the binding kinetics of 17b and 19 following previously reported procedures from our research group. 7 Using the on- and off-rate constants from [ 3 H]PSB, the k on ( k 3 ) and k off ( k 4 ) values for 17b were determined using the equations from the (equilibrium) Motulsky and Mahan model. 22 17b had a much slower association rate ( k on = 3.48 ± 0.22 × 10 5 M –1 min –1 ) than the radioligand and a negligible dissociation rate ( k off = 1.38 ± 0.22 × 10 –12 min –1 ), yielding an almost infinite residence time (RT = 7.63 ± 1.19 × 10 11 min), indicative of irreversible receptor binding by 17b .…”

“…In contrast, pretreatment of the hA 3 AR with 19 , followed by 2-Cl-IB-MECA agonist exposure resulted in surmountable antagonism (Figure 5B), i.e., shifting dose–response curves to the right with no alteration of its maximum effect. The extent of the shifts was used to construct a Schild plot as previously described, 7 which would have a slope of unity if the interaction is competitive and the p A 2 -value corresponds to the p K i value of the antagonist. The slope for 19 was found to be 1.1 ± 0.1 and the compound’s p A 2 value was 5.9 ± 0.1, comparable with its p K i value (6.3 ± 0.03), suggesting that 19 competed with 2-Cl-IB-MECA for the same receptor binding site.…”