Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2.

Covalently acting inhibitors constitute a large and growing fraction of approved small-molecule therapeutics as well as useful tools for a variety of in vitro and in vivo applications. Here, we aimed to develop a covalent antagonist of CC chemokine receptor 2 (CCR2), a class A GPCR that has been pursued as a therapeutic target in inflammation and immuno-oncology. Based on a known intracellularly binding CCR2 antagonist, several covalent derivatives were synthesized and characterized by radioligand binding and functional assays. These studies revealed compound 14 as an intracellular covalent ligand for CCR2. In silico modeling followed by site-directed mutagenesis confirmed that 14 forms a covalent bond with one of three proximal cysteine residues, which can be engaged interchangeably. To our knowledge, compound 14 represents the first covalent ligand reported for CCR2. Due to its unique properties, it may represent a promising tool for ongoing and future studies of CCR2 pharmacology.

Kinetics of human cannabinoid 1 (CB1) receptor antagonists: Structure-kinetics relationships (SKR) and implications for insurmountable antagonism.

While equilibrium binding affinities and in vitro functional antagonism of CB1 receptor antagonists have been studied in detail, little is known on the kinetics of their receptor interaction. In this study, we therefore conducted kinetic assays for nine 1-(4,5-diarylthiophene-2-carbonyl)-4-phenylpiperidine-4-carboxamide derivatives and included the CB1 antagonist rimonabant as a comparison. For this we newly developed a dual-point competition association assay with [3H]CP55940 as the radioligand. This assay yielded Kinetic Rate Index (KRI) values from which structure-kinetics relationships (SKR) of hCB1 receptor antagonists could be established. The fast dissociating antagonist 6 had a similar receptor residence time (RT) as rimonabant, i.e. 19 and 14 min, respectively, while the slowest dissociating antagonist (9) had a very long RT of 2222 min, i.e. pseudo-irreversible dissociation kinetics. In functional assays, 9 displayed insurmountable antagonism, while the effects of the shortest RT antagonist 6 and rimonabant were surmountable. Taken together, this study shows that hCB1 receptor antagonists can have very divergent RTs, which are not correlated to their equilibrium affinities. Furthermore, their RTs appear to define their mode of functional antagonism, i.e. surmountable vs. insurmountable. Finally, based on the recently resolved hCB1 receptor crystal structure, we propose that the differences in RT can be explained by a different binding mode of antagonist 9 from short RT antagonists that is able to displace unfavorable water molecules. Taken together, these findings are of importance for future design and evaluation of potent and safe hCB1 receptor antagonists.

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

We report on the synthesis and biological evaluation of a series of 1,2-diarylimidazol-4-carboxamide derivatives developed as CB1 receptor antagonists. These were evaluated in a radioligand displacement binding assay, a [35S]GTPγS binding assay, and in a competition association assay that enables the relatively fast kinetic screening of multiple compounds. The compounds show high affinities and a diverse range of kinetic profiles at the CB1 receptor and their structure-kinetic relationships (SKRs) were established. Using the recently resolved hCB1 receptor crystal structures, we also performed a modeling study that sheds light on the crucial interactions for both the affinity and dissociation kinetics of this family of ligands. We provide evidence that, next to affinity, additional knowledge of binding kinetics is useful for selecting new hCB1 receptor antagonists in the early phases of drug discovery.

Synthesis and evaluation of N-substituted 2-amino-4,5-diarylpyrimidines as selective adenosine A1 receptor antagonists.

We report the synthesis and biological evaluation of new 2-amino-4,5-diarylpyrimidines as selective antagonists at the adenosine A1 receptor. The scaffold they are based upon is a deaza variation of a previously reported collection of 3-amino-5,6-diaryl-1,2,4-triazines, members of which had a subnanomolar affinity but limited selectivity over the A2A subtype. Initially, similar structure-affinity relationships at the 5-aryl ring were established, and then emphasis was put on increasing selectivity at the hA1AR by introducing substituents on the N2-position, all the while maintaining a nanomolar affinity. Compound 3z, bearing a trans 4-hydroxycyclohexyl substituent, was identified as a potent (Ki(hA1AR) = 7.7 nM) and selective (Ki(hA2AAR) = 1389 nM) antagonist at the human adenosine A1 receptor. Computational docking was effected at the A1 and A2A subtypes, rationalizing the effect of the 4-hydroxycyclohexyl substituent on selectivity, in relation with the nature of the substituent on the 5-position of the pyrimidine.

A covalent antagonist for the human adenosine A2A receptor.

The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine.

5'-Substituted Amiloride Derivatives as Allosteric Modulators Binding in the Sodium Ion Pocket of the Adenosine A2A Receptor.

The sodium ion site is an allosteric site conserved among many G protein-coupled receptors (GPCRs). Amiloride 1 and 5-(N,N-hexamethylene)amiloride 2 (HMA) supposedly bind in this sodium ion site and can influence orthosteric ligand binding. The availability of a high-resolution X-ray crystal structure of the human adenosine A2A receptor (hA2AAR), in which the allosteric sodium ion site was elucidated, makes it an appropriate model receptor for investigating the allosteric site. In this study, we report the synthesis and evaluation of novel 5'-substituted amiloride derivatives as hA2AAR allosteric antagonists. The potency of the amiloride derivatives was assessed by their ability to displace orthosteric radioligand [(3)H]4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol ([(3)H]ZM-241,385) from both the wild-type and sodium ion site W246A mutant hA2AAR. 4-Ethoxyphenethyl-substituted amiloride 12l was found to be more potent than both amiloride and HMA, and the shift in potency between the wild-type and mutated receptor confirmed its likely binding to the sodium ion site.

Predicting Binding Affinities for GPCR Ligands Using Free-Energy Perturbation.

The rapid growth of structural information for G-protein-coupled receptors (GPCRs) has led to a greater understanding of their structure, function, selectivity, and ligand binding. Although novel ligands have been identified using methods such as virtual screening, computationally driven lead optimization has been possible only in isolated cases because of challenges associated with predicting binding free energies for related compounds. Here, we provide a systematic characterization of the performance of free-energy perturbation (FEP) calculations to predict relative binding free energies of congeneric ligands binding to GPCR targets using a consistent protocol and no adjustable parameters. Using the FEP+ package, first we validated the protocol, which includes a full lipid bilayer and explicit solvent, by predicting the binding affinity for a total of 45 different ligands across four different GPCRs (adenosine A2AAR, ß1 adrenergic, CXCR4 chemokine, and δ opioid receptors). Comparison with experimental binding affinity measurements revealed a highly predictive ranking correlation (average spearman ρ = 0.55) and low root-mean-square error (0.80 kcal/mol). Next, we applied FEP+ in a prospective project, where we predicted the affinity of novel, potent adenosine A2A receptor (A2AR) antagonists. Four novel compounds were synthesized and tested in a radioligand displacement assay, yielding affinity values in the nanomolar range. The affinity of two out of the four novel ligands (plus three previously reported compounds) was correctly predicted (within 1 kcal/mol), including one compound with approximately a tenfold increase in affinity compared to the starting compound. Detailed analyses of the simulations underlying the predictions provided insights into the structural basis for the two cases where the affinity was overpredicted. Taken together, these results establish a protocol for systematically applying FEP+ to GPCRs and provide guidelines for identifying potent molecules in drug discovery lead optimization projects.

Structure-Affinity Relationships (SARs) and Structure-Kinetics Relationships (SKRs) of Kv11.1 Blockers.

Kv11.1 (hERG) blockers with comparable potencies but different binding kinetics might display divergent pro-arrhythmic risks. In the present study, we explored structure-kinetics relationships in four series of Kv11.1 blockers next to their structure-affinity relationships. We learned that despite dramatic differences in affinities and association rates, there were hardly any variations in the dissociation rate constants of these molecules with residence times (RTs) of a few minutes only. Hence, we synthesized 16 novel molecules, in particular in the pyridinium class of compounds, to further address this peculiar phenomenon. We found molecules with very short RTs (e.g., 0.34 min for 37) and much longer RTs (e.g., 105 min for 38). This enabled us to construct a k on-k off-KD kinetic map for all compounds and subsequently divide the map into four provisional quadrants, providing a possible framework for a further and more precise categorization of Kv11.1 blockers. Additionally, two representative compounds (21 and 38) were tested in patch clamp assays, and their RTs were linked to their functional IC50 values. Our findings strongly suggest the importance of the simultaneous study of ligand affinities and kinetic parameters, which may help to explain and predict Kv11.1-mediated cardiotoxicity.

Structure-kinetics relationships of Capadenoson derivatives as adenosine A1 receptor agonists.

We report the synthesis and biological evaluation of new derivatives of Capadenoson, a former drug candidate that was previously advanced to phase IIa clinical trials. 19 of the 20 ligands show an affinity below 100 nM at the human adenosine A1 receptor (hA1AR) and display a wide range of residence times at this target (from approx. 5 min (compound 10) up to 132 min (compound 5)). Structure-affinity and structure-kinetics relationships were established, and computational studies of a homology model of the hA1AR revealed crucial interactions for both the affinity and dissociation kinetics of this family of ligands. These results were also combined with global metrics (Ligand Efficiency, cLogP), showing the importance of binding kinetics as an additional way to better select a drug candidate amongst seemingly similar leads.

Affinity and kinetics study of anthranilic acids as HCA2 receptor agonists.

Structure-affinity relationship (SAR) and structure-kinetics relationship (SKR) studies were combined to investigate a series of biphenyl anthranilic acid agonists for the HCA2 receptor. In total, 27 compounds were synthesized and twelve of them showed higher affinity than nicotinic acid. Two compounds, 6g (IC50=75nM) and 6z (IC50=108nM) showed a longer residence time profile compared to nicotinic acid, exemplified by their kinetic rate index (KRI) values of 1.31 and 1.23, respectively. The SAR study resulted in the novel 2-F, 4-OH derivative (6x) with an IC50 value of 23nM as the highest affinity HCA2 agonist of the biphenyl series, although it showed a similar residence time as nicotinic acid. The SAR and SKR data suggest that an early compound selection based on binding kinetics is a promising addition to the lead optimization process.

Agonists for the adenosine A1 receptor with tunable residence time. A Case for nonribose 4-amino-6-aryl-5-cyano-2-thiopyrimidines.

We report the synthesis and evaluation of previously unreported 4-amino-6-aryl-5-cyano-2-thiopyrimidines as selective human adenosine A1 receptor (hA1AR) agonists with tunable binding kinetics, this without affecting their nanomolar affinity for the target receptor. They show a very diverse range of kinetic profiles (from 1 min (compound 52) to 1 h (compound 43)), and their structure-affinity relationships (SAR) and structure-kinetics relationships (SKR) were established. When put in perspective with the increasing importance of binding kinetics in drug discovery, these results bring new evidence of the consequences of affinity-only driven selection of drug candidates, that is, the potential elimination of slightly less active compounds that may display preferable binding kinetics.

Removal of human ether-à-go-go related gene (hERG) K+ channel affinity through rigidity: a case of clofilium analogues.

Cardiotoxicity is a side effect that plagues modern drug design and is very often due to the off-target blockade of the human ether-à-go-go related gene (hERG) potassium channel. To better understand the structural determinants of this blockade, we designed and synthesized a series of 40 derivatives of clofilium, a class III antiarrhythmic agent. These were evaluated in radioligand binding and patch-clamp assays to establish structure-affinity relationships (SAR) for this potassium channel. Efforts were especially focused on studying the influence of the structural rigidity and the nature of the linkers composing the clofilium scaffold. It was shown that introducing triple bonds and oxygen atoms in the n-butyl linker of the molecule greatly reduced affinity without significantly modifying the pKa of the essential basic nitrogen. These findings could prove useful in the first stages of drug discovery as a systematic way of reducing the risk of hERG K(+) channel blockade-induced cardiotoxicity.

Strategies to reduce HERG K+ channel blockade. Exploring heteroaromaticity and rigidity in novel pyridine analogues of dofetilide.

Drug-induced blockade of the human ether-a-go-go-related gene K(+) channel (hERG) represents one of the major antitarget concerns in pharmaceutical industry. SAR studies of this ion channel have shed light on the structural requirements for hERG interaction but most importantly may reveal drug design principles to reduce hERG affinity. In the present study, a novel library of neutral and positively charged heteroaromatic derivatives of the class III antiarrhythmic agent dofetilide was synthesized and assessed for hERG affinity in radioligand binding and manual patch clamp assays. Structural modifications of the pyridine moiety, side chain, and peripheral aromatic moieties were evaluated, thereby revealing approaches for reducing hERG binding affinity. In particular, we found that the extra rigidity imposed close to the positively charged pyridine moiety can be very efficient in decreasing hERG affinity.

Synthesis of (-)-swainsonine and (-)-8-epi-swainsonine by the addition of allenylmetals to chiral α,ß-alkoxy sulfinylimines.

The asymmetric synthesis of (-)-swainsonine and (-)-8-epi-swainsonine is reported through the addition of either the allenylzinc or the allenyl lithio cyanocuprate reagents derived from [3-(methoxymethoxy)prop-1-ynyl]trimethylsilane to enantiopure α,ß-dialkoxy N-tert-butanesulfinylimines derived from d-erythronolactone.

Rearrangement of 2,5-bis(silylated)-N-Boc pyrroles into the corresponding 2,4-species.

The rearrangement of 2,5-bis(silylated)-N-Boc pyrroles in their 2,4-isomers is shown to proceed under mild acidic conditions. A reasonable mechanism, based on literature as well as experiments, is proposed to rationalize this transformation.