Molecular Mechanisms in the Selectivity of Nonsteroidal Anti-Inflammatory Drugs

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) 1 and 2 with varying degrees of selectivity. A group of COX-2 selective inhibitorscoxibsbinds in a time-dependent manner through a three-step mechanism, utilizing a side pocket in the binding site. Coxibs have been extensively probed to identify the structural features regulating the slow tight-binding mechanism responsible for COX-2 selectivity. In this study, we further probe a structurally and kinetically diverse data set of COX inhibitors in COX-2 by molecular dynamics and free energy simulations. We find that the features regulating the high affinities associated with time-dependency in COX depend on the inhibitor kinetics. In particular, most time-dependent inhibitors share a common structural binding mechanism, involving an induced-fit rotation of the side-chain of Leu531 in the main binding pocket. The high affinities of two-step slow tight-binding inhibitors and some slow reversible inhibitors can thus be explained by the increased space in the main binding pocket after this rotation. Coxibs that belong to a separate class of slow tight-binding inhibitors benefit more from the displacement of the neighboring side-chain of Arg513, exclusive to the COX-2 side-pocket. This displacement further stabilizes the aforementioned rotation of Leu531 and can explain the selectivity of coxibs for COX-2

Probing the Time Dependency of Cyclooxygenase‑1 Inhibitors by Computer Simulations

Time-dependent inhibition of the cyclooxygenases (COX) by a range of nonsteroidal anti-inflammatory drugs has been described since the first experimental assays of COX were performed. Slow tight-binding inhibitors of COX-1 bind in a two-step mechanism in which the EI → EI* transition is slow and practically irreversible. Since then, various properties of the inhibitors have been proposed to cause or affect the time dependency. Conformational changes in the enzyme have also been proposed to cause the time dependency, but no particular structural feature has been identified. Here, we investigated a series of inhibitors of COX-1 that are either time-independent or time-dependent using a combination of molecular dynamics simulations, binding free energy calculations, and potential of mean force calculations. We find that the time-dependent inhibitors stabilize a conformational change in the enzyme mainly identified by the rotation of a leucine side chain adjacent to the binding pocket. The induced conformation has been previously shown to be essential for the high binding affinities of tight-binding inhibitors in COX-1. The results of this work show that the structural features of the enzyme involved in both time-dependent and tight-binding inhibition are identical and further identify a structural mechanism responsible for the transition between the two enzyme–inhibitor complexes characteristic of slow tight-binding COX-1 inhibitors

Origin of the Enigmatic Stepwise Tight-Binding Inhibition of Cyclooxygenase‑1

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain, fever, inflammation, and some types of cancers. Their mechanism of action is the inhibition of isoforms 1 and 2 of the enzyme cyclooxygenase (COX-1 and COX-2, respectively). However, both nonselective and selective NSAIDs may have side effects that include gastric intestinal bleeding, peptic ulcer formation, kidney problems, and occurrences of myocardial infarction. The search for selective high-affinity COX inhibitors resulted in a number of compounds characterized by a slow, tight-binding inhibition that occurs in a two-step manner. It has been suggested that the final, only very slowly reversible, tight-binding event is the result of conformational changes in the enzyme. However, the nature of these conformational changes has remained elusive. Here we explore the structural determinants of the tight-binding phenomenon in COX-1 with molecular dynamics and free energy simulations. The calculations reveal how different classes of inhibitors affect the equilibrium between two conformational substates of the enzyme in distinctly different ways. The class of tight-binding inhibitors is found to exclusively stabilize an otherwise unfavorable enzyme conformation and bind significantly stronger to this state than to that normally observed in crystal structures. By also computing free energies of binding to the two enzyme conformations for 16 different NSAIDs, we identify an induced-fit mechanism and the key structural features associated with high-affinity tight binding. These results may facilitate the rational development of new COX inhibitors with improved selectivity profiles

Toward an Optimal Docking and Free Energy Calculation Scheme in Ligand Design with Application to COX‑1 Inhibitors

Cyclooxygenase-1 (COX-1) is one of the main targets of most pain-relieving pharmaceuticals. Although the enzyme is well characterized, it is known to be a difficult target for automated molecular docking and scoring. We collected from the literature a structurally diverse set of 45 nonsteroidal anti-inflammatory drugs (NSAIDs) and COX-2-selective inhibitors (coxibs) with a wide range of binding affinities for COX-1. The binding of this data set to a homology model of human COX-1 was analyzed with different combinations of molecular docking algorithms, scoring functions, and the linear interaction energy (LIE) method for estimating binding affinities. It is found that the computational protocols for estimation of binding affinities are extremely sensitive to the initial orientations of the ligands in the binding pocket. To overcome this limitation, we propose a systematic exploration of docking poses using the LIE calculations as a postscoring function. This scheme yields predictions in excellent agreement with experiment, with a mean unsigned error of 0.9 kcal/mol for binding free energies and structures of high quality. A significant improvement of the results is also seen when averaging over experimental data from several independent measurements

Discovery of Potent and Highly Selective A_2B Adenosine Receptor Antagonist Chemotypes

Three novel families of A_2B adenosine receptor antagonists were identified in the context of the structural exploration of the 3,4-dihydropyrimidin-2­(1<i>H</i>)-one chemotype. The most appealing series contain imidazole, 1,2,4-triazole, or benzimidazole rings fused to the 2,3-positions of the parent diazinone core. The optimization process enabled identification of a highly potent (3.49 nM) A_2B ligand that exhibits complete selectivity toward A₁, A_2A, and A₃ receptors. The results of functional cAMP experiments confirmed the antagonistic behavior of representative ligands. The main SAR trends identified within the series were substantiated by a molecular modeling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human A_2A receptor

Enantiospecific Recognition at the A_2B Adenosine Receptor by Alkyl 2‑Cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylates

A novel family of structurally simple, potent, and selective nonxanthine A_2BAR ligands was identified, and its antagonistic behavior confirmed through functional experiments. The reported alkyl 2-cyanoimino-4-substituted-6-methyl-1,2,3,4-tetrahy-dropyrimidine-5-carboxylates (<b>16</b>) were designed by bioisosteric replacement of the carbonyl group at position 2 in a series of 3,4-dihydropyrimidin-2-ones. The scaffold (<b>16</b>) documented herein contains a chiral center at the heterocycle. Accordingly, the most attractive ligand of the series [(±)<b>16b</b>, <i>K</i>_i <b>=</b> 24.3 nM] was resolved into its two enantiomers by chiral HPLC, and the absolute configuration was established by circular dichroism. The biological evaluation of both enantiomers demonstrated enantiospecific recognition at A_2BAR, with the (<i>S</i>)-<b>16b</b> enantiomer retaining all the affinity (<i>K</i>_i <b>=</b> 15.1 nM), as predicted earlier by molecular modeling. This constitutes the first example of enantiospecific recognition at the A_2B adenosine receptor and opens new possibilities in ligand design for this receptor

Discovery of Potent and Highly Selective A_2B Adenosine Receptor Antagonist Chemotypes

Three novel families of A_2B adenosine receptor antagonists were identified in the context of the structural exploration of the 3,4-dihydropyrimidin-2­(1<i>H</i>)-one chemotype. The most appealing series contain imidazole, 1,2,4-triazole, or benzimidazole rings fused to the 2,3-positions of the parent diazinone core. The optimization process enabled identification of a highly potent (3.49 nM) A_2B ligand that exhibits complete selectivity toward A₁, A_2A, and A₃ receptors. The results of functional cAMP experiments confirmed the antagonistic behavior of representative ligands. The main SAR trends identified within the series were substantiated by a molecular modeling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human A_2A receptor

Effect of Nitrogen Atom Substitution in A₃ Adenosine Receptor Binding: <i>N</i>‑(4,6-Diarylpyridin-2-yl)acetamides as Potent and Selective Antagonists

We report the first family of 2-acetamidopyridines as potent and selective A₃ adenosine receptor (AR) antagonists. The computer-assisted design was focused on the bioisosteric replacement of the N1 atom by a CH group in a previous series of diarylpyrimidines. Some of the generated 2-acetamidopyridines elicit an antagonistic effect with excellent affinity (<i>K</i>_i < 10 nM) and outstanding selectivity profiles, providing an alternative and simpler chemical scaffold to the parent series of diarylpyrimidines. In addition, using molecular dynamics and free energy perturbation simulations, we elucidate the effect of the second nitrogen of the parent diarylpyrimidines, which is revealed as a stabilizer of a water network in the binding site. The discovery of 2,6-diaryl-2-acetamidopyridines represents a step forward in the search of chemically simple, potent, and selective antagonists for the hA₃AR, and exemplifies the benefits of a joint theoretical–experimental approach to identify novel hA₃AR antagonists through succinct and efficient synthetic methodologies

Discovery of 3,4-Dihydropyrimidin-2(1<i>H</i>)‑ones As a Novel Class of Potent and Selective A_2B Adenosine Receptor Antagonists

We describe the discovery and optimization of 3,4-dihydropyrimidin-2­(1<i>H</i>)-ones as a novel family of (nonxanthine) A_2B receptor antagonists that exhibit an unusually high selectivity profile. The Biginelli-based hit optimization process enabled a thoughtful exploration of the structure–activity and structure–selectivity relationships for this chemotype, enabling the identification of ligands that combine structural simplicity with excellent hA_2B AdoR affinity and remarkable selectivity profiles

Effect of Nitrogen Atom Substitution in A₃ Adenosine Receptor Binding: <i>N</i>‑(4,6-Diarylpyridin-2-yl)acetamides as Potent and Selective Antagonists

We report the first family of 2-acetamidopyridines as potent and selective A₃ adenosine receptor (AR) antagonists. The computer-assisted design was focused on the bioisosteric replacement of the N1 atom by a CH group in a previous series of diarylpyrimidines. Some of the generated 2-acetamidopyridines elicit an antagonistic effect with excellent affinity (<i>K</i>_i < 10 nM) and outstanding selectivity profiles, providing an alternative and simpler chemical scaffold to the parent series of diarylpyrimidines. In addition, using molecular dynamics and free energy perturbation simulations, we elucidate the effect of the second nitrogen of the parent diarylpyrimidines, which is revealed as a stabilizer of a water network in the binding site. The discovery of 2,6-diaryl-2-acetamidopyridines represents a step forward in the search of chemically simple, potent, and selective antagonists for the hA₃AR, and exemplifies the benefits of a joint theoretical–experimental approach to identify novel hA₃AR antagonists through succinct and efficient synthetic methodologies