Decoding the Role of Water Dynamics in Ligand–Protein Unbinding: CRF₁R as a Test Case

The residence time of a ligand–protein complex is a crucial aspect in determining biological effect in vivo. Despite its importance, the prediction of ligand <i>k</i>_off still remains challenging for modern computational chemistry. We have developed aMetaD, a fast and generally applicable computational protocol to predict ligand–protein unbinding events using a molecular dynamics (MD) method based on adiabatic-bias MD and metadynamics. This physics-based, fully flexible, and pose-dependent ligand scoring function evaluates the maximum energy (RTscore) required to move the ligand from the bound-state energy basin to the next. Unbinding trajectories are automatically analyzed and translated into atomic solvation factor (SF) values representing the water dynamics during the unbinding event. This novel computational protocol was initially tested on two M₃ muscarinic receptor and two adenosine A_2A receptor antagonists and then evaluated on a test set of 12 CRF₁R ligands. The resulting RTscores were used successfully to classify ligands with different residence times. Additionally, the SF analysis was used to detect key differences in the degree of accessibility to water molecules during the predicted ligand unbinding events. The protocol provides actionable working hypotheses that are applicable in a drug discovery program for the rational optimization of ligand binding kinetics

Click Modification in the <i>N</i>⁶ Region of A₃ Adenosine Receptor-Selective Carbocyclic Nucleosides for Dendrimeric Tethering that Preserves Pharmacophore Recognition

Adenosine derivatives were modified with alkynyl groups on <i>N</i>⁶ substituents for linkage to carriers using Cu­(I)-catalyzed click chemistry. Two parallel series, both containing a rigid North-methanocarba (bicyclo[3.1.0]­hexane) ring system in place of ribose, behaved as A₃ adenosine receptor (AR) agonists: (5′-methyluronamides) or partial agonists (4′-truncated). Terminal alkynyl groups on a chain at the 3 position of a <i>N</i>⁶-benzyl group or simply through a <i>N</i>⁶-propargyl group were coupled to azido derivatives, which included both small molecules and G4 (fourth-generation) multivalent poly­(amidoamine) (PAMAM) dendrimers, to form 1,2,3-triazolyl linkers. The small molecular triazoles probed the tolerance in A₃AR binding of distal, sterically bulky groups such as 1-adamantyl. Terminal 4-fluoro-3-nitrophenyl groups anticipated nucleophilic substitution for chain extension and ¹⁸F radiolabeling. <i>N</i>⁶-(4-Fluoro-3-nitrophenyl)-triazolylmethyl derivative <b>32</b> displayed a <i>K</i>_i of 9.1 nM at A₃AR with ∼1000-fold subtype selectivity. Multivalent conjugates additionally containing click-linked water-solubilizing polyethylene glycol groups potently activated A₃AR in the 5′-methyluronamide, but not 4′ truncated series. <i>N</i>⁶-Benzyl nucleoside conjugate <b>43</b> (apparent <i>K</i>_i 24 nM) maintained binding affinity of the monomer better than a <i>N</i>⁶-triazolylmethyl derivative. Thus, the <i>N</i>⁶ region of 5′-methyluronamide derivatives, as modeled in receptor docking, is suitable for functionalization and tethering by click chemistry to achieve high A₃AR agonist affinity and enhanced selectivity

Control of Quality and Safety of Products as Fundamental Function of the State Concerning Defence of Consumers' Rights

У статті досліджуються питання щодо закріпленого ст. 14 Закону України “Про захист прав споживачів” права споживача на те, щоб продукція за звичайних умов її використання, зберігання і транспортування була безпечною для його життя, здоров’я, навколишнього природного середовища, а також не завдавала шкоди його майну. Дане дослідження проводиться з погляду на конституційний обов’язок держави захищати права споживачів та здійснювати контроль за якістю і безпечністю продукції.In the article carry out a research the consumers' right that products at the ordinary terms of its use, storage and transporting, were safe for their life, health, natural environment, and also did not harm his property. This research is conducted from the point of view to the constitutional duty of the state to protect rights for users and carry out control of quality and safety of products

Structure-Guided Design of A₃ Adenosine Receptor-Selective Nucleosides: Combination of 2-Arylethynyl and Bicyclo[3.1.0]hexane Substitutions

(<i>N</i>)-Methanocarba adenosine 5′-methyluronamides containing known A₃ AR (adenosine receptor)-enhancing modifications, i.e., 2-(arylethynyl)­adenine and <i>N</i>⁶-methyl or <i>N</i>⁶-(3-substituted-benzyl), were nanomolar full agonists of human (h) A₃AR and highly selective (<i>K</i>_i ∼0.6 nM, <i>N</i>⁶-methyl 2-(halophenylethynyl) analogues <b>13</b> and <b>14</b>). Combined 2-arylethynyl-<i>N</i>⁶-3-chlorobenzyl substitutions preserved A₃AR affinity/selectivity in the (<i>N</i>)-methanocarba series (e.g., 3,4-difluoro full agonist MRS5698 <b>31</b>, <i>K</i>_i 3 nM, human and mouse A₃) better than that for ribosides. Polyaromatic 2-ethynyl <i>N</i>⁶-3-chlorobenzyl analogues, such as potent linearly extended 2-<i>p</i>-biphenylethynyl MRS5679 <b>34</b> (<i>K</i>_i hA₃ 3.1 nM; A₁, A_2A, inactive) and fluorescent 1-pyrene adduct MRS5704 <b>35</b> (<i>K</i>_i hA₃ 68.3 nM), were conformationally rigid; receptor docking identified a large, mainly hydrophobic binding region. The vicinity of receptor-bound C2 groups was probed by homology modeling based on recent X-ray structure of an agonist-bound A_2AAR, with a predicted helical rearrangement requiring an agonist-specific outward displacement of TM2 resembling opsin. Thus, the X-ray structure of related A_2AAR is useful in guiding the design of new A₃AR agonists

Structural Sweet Spot for A₁ Adenosine Receptor Activation by Truncated (N)-Methanocarba Nucleosides: Receptor Docking and Potent Anticonvulsant Activity

A₁ adenosine receptor (AR) agonists display antiischemic and antiepileptic neuroprotective activity, but peripheral cardiovascular side effects impeded their development. SAR study of <i>N</i>⁶-cycloalkylmethyl 4′-truncated (N)-methanocarba-adenosines identified <b>10</b> (MRS5474, <i>N</i>⁶-dicyclopropylmethyl, <i>K</i>_i = 47.9 nM) as a moderately A₁AR-selective full agonist. Two stereochemically defined <i>N</i>⁶-methynyl group substituents displayed narrow SAR; groups larger than cyclobutyl greatly reduced AR affinity, and those larger or smaller than cyclopropyl reduced A₁AR selectivity. Nucleoside docking to A₁AR homology model characterized distinct hydrophobic cyclopropyl subpockets, the larger “A” forming contacts with Thr270 (7.35), Tyr271 (7.36), Ile274 (7.39), and carbon chains of glutamates (EL2) and the smaller subpocket “B” forming contacts between TM6 and TM7. <b>10</b> suppressed minimal clonic seizures (6 Hz mouse model) without typical rotarod impairment of A₁AR agonists. Truncated nucleosides, an appealing preclinical approach, have more druglike physicochemical properties than other A₁AR agonists. Thus, we identified highly restricted regions for substitution around <i>N</i>⁶ suitable for an A₁AR agonist with anticonvulsant activity

Structure-Guided Design of A₃ Adenosine Receptor-Selective Nucleosides: Combination of 2-Arylethynyl and Bicyclo[3.1.0]hexane Substitutions

(<i>N</i>)-Methanocarba adenosine 5′-methyluronamides containing known A₃ AR (adenosine receptor)-enhancing modifications, i.e., 2-(arylethynyl)­adenine and <i>N</i>⁶-methyl or <i>N</i>⁶-(3-substituted-benzyl), were nanomolar full agonists of human (h) A₃AR and highly selective (<i>K</i>_i ∼0.6 nM, <i>N</i>⁶-methyl 2-(halophenylethynyl) analogues <b>13</b> and <b>14</b>). Combined 2-arylethynyl-<i>N</i>⁶-3-chlorobenzyl substitutions preserved A₃AR affinity/selectivity in the (<i>N</i>)-methanocarba series (e.g., 3,4-difluoro full agonist MRS5698 <b>31</b>, <i>K</i>_i 3 nM, human and mouse A₃) better than that for ribosides. Polyaromatic 2-ethynyl <i>N</i>⁶-3-chlorobenzyl analogues, such as potent linearly extended 2-<i>p</i>-biphenylethynyl MRS5679 <b>34</b> (<i>K</i>_i hA₃ 3.1 nM; A₁, A_2A, inactive) and fluorescent 1-pyrene adduct MRS5704 <b>35</b> (<i>K</i>_i hA₃ 68.3 nM), were conformationally rigid; receptor docking identified a large, mainly hydrophobic binding region. The vicinity of receptor-bound C2 groups was probed by homology modeling based on recent X-ray structure of an agonist-bound A_2AAR, with a predicted helical rearrangement requiring an agonist-specific outward displacement of TM2 resembling opsin. Thus, the X-ray structure of related A_2AAR is useful in guiding the design of new A₃AR agonists

Evaluation of Molecular Modeling of Agonist Binding in Light of the Crystallographic Structure of an Agonist-Bound A_2A Adenosine Receptor

Molecular modeling of agonist binding to the human A_2A adenosine receptor (AR) was assessed and extended in light of crystallographic structures. Heterocyclic adenine nitrogens of cocrystallized agonist overlaid corresponding positions of the heterocyclic base of a bound triazolotriazine antagonist, and ribose moiety was coordinated in a hydrophilic region, as previously predicted based on modeling using the inactive receptor. Automatic agonist docking of 20 known potent nucleoside agonists to agonist-bound A_2AAR crystallographic structures predicted new stabilizing protein interactions to provide a structural basis for previous empirical structure activity relationships consistent with previous mutagenesis results. We predicted binding of novel C2 terminal amino acid conjugates of A_2AAR agonist CGS21680 and used these models to interpret effects on binding affinity of newly synthesized agonists. d-Amino acid conjugates were generally more potent than l-stereoisomers and free terminal carboxylates more potent than corresponding methyl esters. Amino acid moieties were coordinated close to extracellular loops 2 and 3. Thus, molecular modeling is useful in probing ligand recognition and rational design of GPCR-targeting compounds with specific pharmacological profiles

Evaluation of Molecular Modeling of Agonist Binding in Light of the Crystallographic Structure of an Agonist-Bound A_2A Adenosine Receptor

Molecular modeling of agonist binding to the human A_2A adenosine receptor (AR) was assessed and extended in light of crystallographic structures. Heterocyclic adenine nitrogens of cocrystallized agonist overlaid corresponding positions of the heterocyclic base of a bound triazolotriazine antagonist, and ribose moiety was coordinated in a hydrophilic region, as previously predicted based on modeling using the inactive receptor. Automatic agonist docking of 20 known potent nucleoside agonists to agonist-bound A_2AAR crystallographic structures predicted new stabilizing protein interactions to provide a structural basis for previous empirical structure activity relationships consistent with previous mutagenesis results. We predicted binding of novel C2 terminal amino acid conjugates of A_2AAR agonist CGS21680 and used these models to interpret effects on binding affinity of newly synthesized agonists. d-Amino acid conjugates were generally more potent than l-stereoisomers and free terminal carboxylates more potent than corresponding methyl esters. Amino acid moieties were coordinated close to extracellular loops 2 and 3. Thus, molecular modeling is useful in probing ligand recognition and rational design of GPCR-targeting compounds with specific pharmacological profiles

Evaluation of Molecular Modeling of Agonist Binding in Light of the Crystallographic Structure of an Agonist-Bound A_2A Adenosine Receptor

Molecular modeling of agonist binding to the human A_2A adenosine receptor (AR) was assessed and extended in light of crystallographic structures. Heterocyclic adenine nitrogens of cocrystallized agonist overlaid corresponding positions of the heterocyclic base of a bound triazolotriazine antagonist, and ribose moiety was coordinated in a hydrophilic region, as previously predicted based on modeling using the inactive receptor. Automatic agonist docking of 20 known potent nucleoside agonists to agonist-bound A_2AAR crystallographic structures predicted new stabilizing protein interactions to provide a structural basis for previous empirical structure activity relationships consistent with previous mutagenesis results. We predicted binding of novel C2 terminal amino acid conjugates of A_2AAR agonist CGS21680 and used these models to interpret effects on binding affinity of newly synthesized agonists. d-Amino acid conjugates were generally more potent than l-stereoisomers and free terminal carboxylates more potent than corresponding methyl esters. Amino acid moieties were coordinated close to extracellular loops 2 and 3. Thus, molecular modeling is useful in probing ligand recognition and rational design of GPCR-targeting compounds with specific pharmacological profiles

Optimization of Adenosine 5′-Carboxamide Derivatives as Adenosine Receptor Agonists Using Structure-Based Ligand Design and Fragment Screening

Structures of G protein-coupled receptors (GPCRs) have a proven utility in the discovery of new antagonists and inverse agonists modulating signaling of this important family of clinical targets. Applicability of active-state GPCR structures to virtual screening and rational optimization of agonists, however, remains to be assessed. In this study of adenosine 5′ derivatives, we evaluated the performance of an agonist-bound A_2A adenosine receptor (AR) structure in retrieval of known agonists and then employed the structure to screen for new fragments optimally fitting the corresponding subpocket. Biochemical and functional assays demonstrate high affinity of new derivatives that include polar heterocycles. The binding models also explain modest selectivity gain for some substituents toward the closely related A₁AR subtype and the modified agonist efficacy of some of these ligands. The study suggests further applicability of in silico fragment screening to rational lead optimization in GPCRs