Search

High affinity phenyl-piperidine P2Y(14)R antagonist 1 (PPTN) was modified with piperidine bridging moieties to probe receptor affinity and hydrophobicity. Various 2-azanorbornane, nortropane, isonortropane, isoquinuclidine, and ring-opened cyclopentylamino derivatives preserved human P2Y(14)R affinity (fluorescence binding assay), and their pharmacophoric overlay was compared. Enantiomeric 2-azabicyclo[2.2.1]hept-5-en-3-one precursors assured stereochemically unambiguous, diverse products. Pure (S,S,S) 2-azanorbornane enantiomer 15 (MRS4738) displayed higher affinity than 1 (3-fold higher affinity than enantiomer 16) and in vivo antihyperallodynic and antiasthmatic activity. Its double prodrug 143 (MRS4815) dramatically reduced lung inflammation in a mouse asthma model. Related lactams 21−24 and dicarboxylate 42 displayed intermediate affinity and enhanced aqueous solubility. Isoquinuclidine 34 (IC(50) 15.6 nM) and isonortropanol 30 (IC(50) 21.3 nM) had lower lipophilicity than 1. In general, rigidified piperidine derivatives did not lower lipophilicity dramatically, except those rings with multiple polar groups. P2Y(14)R molecular modeling based on a P2Y(12)R structure showed stable and persistent key interactions for compound 15.

Molecular docking is one of the most widely used computational approaches in the field of rational drug design, thanks to its favorable balance between the rapidity of execution and the accuracy of provided results. Although very efficient in exploring the conformational degrees of freedom available to the ligand, docking programs can sometimes suffer from inaccurate scoring and ranking of generated poses. To address this issue, several post-docking filters and refinement protocols have been proposed throughout the years, including pharmacophore models and molecular dynamics simulations. In this work, we present the first application of Thermal Titration Molecular Dynamics (TTMD), a recently developed method for the qualitative estimation of protein-ligand unbinding kinetics, to the refinement of docking results. TTMD evaluates the conservation of the native binding mode throughout a series of molecular dynamics simulations performed at progressively increasing temperatures with a scoring function based on protein-ligand interaction fingerprints. The protocol was successfully applied to retrieve the native-like binding pose among a set of decoy poses of drug-like ligands generated on four different pharmaceutically relevant biological targets, including casein kinase 1δ, casein kinase 2, pyruvate dehydrogenase kinase 2, and SARS-CoV-2 main protease.

Nucleoside diphosphates and triphosphates impact nearly every aspect of biochemistry, however, the use of such compounds as tools or medicinal leads for nucleotide-dependent enzymes and receptors is hampered due to rapid metabolism. Meanwhile, a successful strategy to address the instability of the monophosphate moiety in oligonucleotide therapeutics has been accomplished by their isosteric replacement with phosphorothioates. On the contrary, no practical methods exist to rapidly and controllably access stereopure di- and triphosphate thioisosteres of both natural and unnatural nucleosides. Here we show how a modular, reagent-based platform can enable the stereocontrolled and scalable synthesis of a library of such molecules. Such thioisosteric replacements can have profound effects on the potency and stability of lead candidates, as evidenced by data demonstrating that ligand-receptor interactions can be dramatically influenced by P-stereochemistry.

We previously reported 1

A known zwitterionic, heterocyclic P2Y14R antagonist 3a was substituted with diverse groups on the central phenyl and terminal piperidine moieties, following a computational selection process. The most potent analogues contained an uncharged piperidine bioisostere, prescreened in silico, while an aza-scan (central phenyl ring) reduced P2Y14R affinity. Piperidine amide 11, 3-aminopropynyl 19, and 5-(hydroxymethyl)isoxazol-3-yl) 29 congeners in the triazole series maintained moderate receptor affinity. Adaption of 5-(hydroxymethyl)isoxazol-3-yl gave the most potent naphthalene-containing (32; MRS4654; IC50, 15 nM) and less active phenylamide-containing (33) scaffolds. Thus, a zwitterion was nonessential for receptor binding, and molecular docking and dynamics probed the hydroxymethylisoxazole interaction with extracellular loops. Also, amidomethyl ester prodrugs were explored to reversibly block the conserved carboxylate group to provide neutral analogues, which were cleavable by liver esterase, and in vivo efficacy demonstrated. We have, in stages, converted zwitterionic antagonists into neutral molecules designed to produce potent P2Y14R antagonists for in vivo application.

Adenosine receptor (AR) ligands are being developed for metabolic, cardiovascular, neurological, and inflammatory diseases and cancer. The ease of drug discovery is contingent on the availability of pharmacological tools. Fluorescent antagonist ligands for the human A

[Image: see text] The A(3) adenosine receptor (A(3)AR) is a target for pain, ischemia, and inflammatory disease therapy. Among the ligand tools available are selective agonists and antagonists, including radioligands, but most high-affinity non-nucleoside antagonists are limited in selectivity to primate species. We have explored the structure–activity relationship of a previously reported A(3)AR antagonist DPTN 9 (N-[4-(3,5-dimethylphenyl)-5-(4-pyridyl)-1,3-thiazol-2-yl]nicotinamide) for radiolabeling, including 3-halo derivatives (3-iodo, MRS7907), and characterized 9 as a high -affinity radioligand [(3)H]MRS7799. A(3)AR K(d) values were (nM): 0.55 (human), 3.74 (mouse), and 2.80 (rat). An extended methyl acrylate (MRS8074, 19) maintained higher affinity (18.9 nM) than a 3-((5-chlorothiophen-2-yl)ethynyl) derivative 20. Compound 9 had an excellent brain distribution in rats (brain/plasma ratio ∼1). Receptor docking predicted its orthosteric site binding by engaging residues that were previously found to be essential for AR binding. Thus the new radioligand promises to be a useful species-general antagonist tracer for receptor characterization and drug discovery.

A side-by-side pharmacological comparison of ribose and (N)-methanocarba (bicyclo[3.1.0]hexane) nucleosides as A3AR agonists indicated that the bicyclic pseudoribose ring constraint provided higher affinity/selectivity at human and mouse A3AR. The mean affinity enhancement for 5 pairs of 5'-methylamides was 11-fold at hA3AR and 42-fold at mA3AR. Novel C2-(5-fluorothien-2-ylethynyl) substitution enhanced affinity in the methanocarba but not ribose series, with highly hA3AR-selective 16 (MRS7334) displaying Ki 280 pM and favorable pharmacokinetics and off-target activity profile. Molecular dynamics comparison of 16 and its corresponding riboside 8 suggested a qualitative entropic advantage of 16 in hA3AR binding. The 5-F substitution tended to increase hA3AR affinity (cf. 5-Cl) for methanocarba but not ribose derivatives. A representative methanocarba agonist 4 was shown to interact potently exclusively with A3AR, among 240 GPCRs and 466 kinases. Thus, despite added synthetic difficulty, the (N)-methanocarba modification has distinct advantages for A3AR agonists, which have translational potential for chronic disease treatment.

We recently reported N(4)-substituted 3-methylcytidine-5′-α,β-methylenediphosphates as CD73 inhibitors, potentially useful in cancer immunotherapy. We now expand the structure–activity relationship of pyrimidine nucleotides as human CD73 inhibitors. 4-Chloro (MRS4598 16; K(i) = 0.673 nM) and 4-iodo (MRS4620 18; K(i) = 0.436 nM) substitution of the N(4)-benzyloxy group decreased K(i) by ~20-fold. Primary alkylamine derivatives coupled through a p-amido group with a varying methylene chain length (24 and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. X-ray structures of hCD73 with two inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between the C-terminal and N-terminal domains) as N(4)-benzyl groups in adenine inhibitors. Molecular dynamics identified stabilizing interactions and predicted conformational diversity. Thus, by N(4)-benzyloxy substitution, we have greatly enhanced the inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in tumor tissues in situ.

Following our study of 4′-truncated (N)-methanocarba-adenosine derivatives that displayed unusually high mouse (m) A(3)AR affinity, we incorporated dopamine-related N(6) substituents in the full agonist 5′-methylamide series. N(6)-(2-(4-Hydroxy-3-methoxy-phenyl)ethyl) derivative MRS7618 11 displayed K(i) (nM) 0.563 at hA(3)AR (~20,000-fold selective) and 1.54 at mA(3)AR. 2-Alkyl ethers maintained A(3) affinity, but with less selectivity than 2-alkynes. Parallel functional assays of G protein-dependent and β-arrestin 2 (βarr2)-dependent pathways indicate these are full agonists but not biased. Through use of computational modeling, we hypothesized that phenyl OH/OMe groups interact with polar residues, particularly Gln261, on the mA(3)AR extracellular loops as the basis for the affinity enhancement. Although the pharmacokinetics indicated facile clearance of parent O-methyl catechol nucleosides 21 and 31, prolonged mA(3)AR activation in vivo was observed in a hypothermia model, suggested potential formation of active metabolites through demethylation. Selected analogues induced mouse hypothermia following i.p. injection, indicative of peripheral A(3)AR agonism in vivo.

(N)-Methanocarba ([3.1.0]bicyclohexyl) adenosines and corresponding ribosides were synthesized to identify novel A1 adenosine receptor (A1AR) agonists for CNS or peripheral applications. Human and mouse AR binding was determined to assess the constrained ring system’s A1AR compatibility. N6-Dicyclobutylmethyl ribose agonist (9, MRS7469, >2000-fold selective for A1AR) and known truncated N6-dicyclopropylmethyl methanocarba 7 (MRS5474) were drug-like. The pure diastereoisomer of known riboside 4 displayed high hA1AR selectivity. Methanocarba modification reduced A1AR selectivity of N6-dicyclopropylmethyl and endo-norbornyladenosines but increased ribavirin selectivity. Most analogues tested (ip) were inactive or weak in inducing mouse hypothermia, despite mA1AR full agonism and variable mA3AR efficacy, but strong hypothermia by 9 depended on A1AR, which reflects CNS activity (determined using A1AR or A3AR null mice). Conserved hA1AR interactions were preserved in modeling of 9 and methanocarba equivalent 24...

A known zwitterionic, heterocyclic P2Y(14)R antagonist 3a was substituted with diverse groups on the central phenyl and terminal piperidine moieties, following a computational selection process. The most potent analogues contained an uncharged piperidine bioisostere, prescreened in silico, while an aza-scan (central phenyl ring) reduced P2Y(14)R affinity. Piperidine amide 11, 3-aminopropynyl 19, and 5-(hydroxymethyl)isoxazol-3-yl) 29 congeners in the triazole series maintained moderate receptor affinity. Adaption of 5-(hydroxymethyl)isoxazol-3-yl gave the most potent naphthalene-containing (32; MRS4654; IC(50), 15 nM) and less active phenylamide-containing (33) scaffolds. Thus, a zwitterion was nonessential for receptor binding, and molecular docking and dynamics probed the hydroxymethylisoxazole interaction with extracellular loops. Also, amidomethyl ester prodrugs were explored to reversibly block the conserved carboxylate group to provide neutral analogues, which were cleavable by liver esterase, and in vivo efficacy demonstrated. We have, in stages, converted zwitterionic antagonists into neutral molecules designed to produce potent P2Y(14)R antagonists for in vivo application.

Nucleoside derivatives are well represented as pharmaceuticals due to their druglike physicochemical properties, and some nucleoside drugs are designed to act on receptors. The purinergic signaling pathways for extracellular nucleosides and nucleotides, consisting of adenosine receptors, P2Y/P2X receptors for nucleotides, and enzymes such as adenosine (ribo)kinase, have been extensively studied. A general modification, i.e. a constrained, bicyclic ring system (bicyclo[3.1.0]hexane, also called methanocarba) substituted in place of a furanose ring, can increase nucleoside/nucleotide potency and/or selectivity at purinergic and antiviral targets and in interactions at diverse and unconventional targets. Compared to other common drug discovery scaffolds containing planar rings, methanocarba nucleosides display greater sp3 character (i.e. more favorable as drug-like molecules) and can manifest as sterically-constrained North (N) or South (S) conformations. Initially weak, off-target interactions of (N)-methanocarba adenosine derivatives were detected as leads that were structurally optimized to enhance activity and selectivity toward target proteins that normally do not recognize nucleosides. By this approach, novel modulators for 5HT(2) serotonin and κ-opioid receptors, dopamine (DAT) and ATP-binding cassette (ABC) transporters were found, and previously undetected antiviral activities were revealed. Thus, through methanocarba nucleoside synthesis, structure–activity relationships, and multi-target pharmacology, a robust purinergic receptor scaffold has been repurposed to satisfy the pharmacophoric requirements of various GPCRs, enzymes and transporters.

Since 2007, Metalloproteases (MMPs) have been considered potential targets for treating osteoarthritis (OA), for which the primary pathogenic event is the extensive degeneration of articular cartilage. MMP3 is an enzyme critical for these degenerative changes. However, problems of selectivity, low bioavailability and poor metabolic profile during clinical trials of MMPs inhibitors (MMPIs) led to limited beneficial effect and thus did not justify further pursuit of the clinical studies. In a previous work, a new alkyl derivative of hyaluronic acid (HA), HYADD4®, previously approved as intra-articular treatment for knee OA, was studied in vitro and in vivo as MMP3I. Molecular simulation studies confirmed the interaction between the alkyl side chain of this HA derivative and the additional S1′ pocket of MMP3. However, the high MW and the polar HA backbone of HYADD4® imply a high desolvation energy cost, which can potentially decrease its inhibitory potency. In this study, a new class of MMP3Is based on a small peptide backbone (CGV) chemically derivatized with an alkyl chain was developed through interactive cycles of design, synthesis and screening, accompanied by computational evaluation and optimization. Two MMP3Is, e(I) and l(II), were selected because of their effective inhibitory activity (3.2 and 10.2 µM, respectively) and water solubility. Both MMPIs showed a broad range of inhibitory effects against almost all the MMPs tested. In an in vitro model of inflammatory OA, e(I) was the most effective MMPI: at the concentration of 93 µM, it reversed inflammatory outcomes. Moreover, because of its amphiphilic structure, the e(I) MMPI promoted stable micellar formulation at concentrations higher than 0.2 mg/mL in water. The findings were confirmed by TEM and Nile red staining analysis. Based on these results, the e(I) MMPI can be considered a good candidate for the intra-articular treatment of OA, and the micellar formulation of this peptide in an aqueous buffer can potentially increase the bioavailability and, thus, the efficacy of the MMPIs.

The purinergic signaling system includes membrane-bound receptors for extracellular purines and pyrimidines, and enzymes/transporters that regulate receptor activation by endogenous agonists. Receptors include: adenosine (A(1), A(2A), A(2B) and A(3)) and P2Y (P2Y(1) P2Y(2), P2Y(4), P2Y(6), P2Y(11), P2Y(12), P2Y(13), and P2Y(14)) receptors (all GPCRs), as well as P2X receptors (ion channels). Receptor activation, especially accompanying physiological stress or damage, creates a temporal sequence of signaling to counteract this stress and either mobilize (P2Rs) or suppress (ARs) immune responses. Thus, modulation of this large signaling family has broad potential for treating chronic diseases. Experimentally determined structures represent each of the three receptor families. We focus on selective purinergic agonists (A(1), A(3)), antagonists (A(3), P2Y(14)), and allosteric modulators (P2Y(1), A(3)). Examples of applying structure-based design, including the rational modification of known ligands, are presented for antithrombotic P2Y(1)R antagonists and anti-inflammatory P2Y(14)R antagonists and A(3)AR agonists. A(3)AR agonists are a potential, nonaddictive treatment for chronic neuropathic pain.

Various heteroaryl and bicyclo-aliphatic analogues of zwitterionic biaryl P2Y(14) receptor (P2Y(14)R) antagonists were synthesized, and affinity was measured in P2Y(14)R-expressing Chinese hamster ovary cells by flow cytometry. Given this series’ low water solubility, various polyethylene glycol derivatives of the distally binding piperidin-4-yl moiety of moderate affinity were synthesized. Rotation of previously identified 1,2,3-triazole attached to the central m-benzoic acid core (25) provided moderate affinity but not indole and benzimidazole substitution of the aryl-triazole. The corresponding P2Y(14)R region is predicted by homology modeling as a deep, sterically limited hydrophobic pocket, with the outward pointing piperidine moiety being the most flexible. Bicyclic-substituted piperidine ring derivatives of naphthalene antagonist 1, e.g., quinuclidine 17 (MRS4608, IC(50) ≈ 20 nM at hP2Y(14)R/mP2Y(14)R), or of triazole 2, preserved affinity. Potent antagonists 1, 7a, 17, and 23 (10 mg/kg) protected in an ovalbumin/Aspergillus mouse asthma model, and PEG conjugate 12 reduced chronic pain. Thus, we expanded P2Y(14)R antagonist structure–activity relationship, introducing diverse physical–chemical properties.

Dopamine-derived N(6)-substituents, compared to N(6)-(2-phenylethyl), in truncated (N)-methanocarba (bicyclo[3.1.0]hexyl) adenosines favored high A(3) adenosine receptor (AR) affinity/selectivity, e.g. C2-phenylethynyl analogue 15 (MRS7591, K(i) 10.9/17.8 nM, at human/mouse A(3)AR). 15 was a partial agonist in vitro (hA(3)AR, cAMP inhibition, 31% E(max); mA(3)AR, [(35)S]GTP-γ-S binding, 16% E(max)) and in vivo, and also antagonized hA(3)AR in vitro. Distal H-bonding substitutions of the N(6)-(2-phenylethyl) moiety particularly enhanced mA(3)AR affinity by polar interactions with the extracellular loops, predicted using docking and molecular dynamics simulation with newly constructed mA(3)AR and hA(3)AR homology models. These hybrid models were based on an inactive antagonist-bound hA(1)AR structure for the upper part of TM2 and an agonist-bound hA(2A)AR structure for the remaining TM portions. These species-independent A(3)AR-selective nucleosides are low efficacy partial agonists and novel, nuanced modulators of the A(3)AR, a drug target of growing interest.

Dopamine-derived N6-substituents, compared to N6-(2-phenylethyl), in truncated (N)-methanocarba (bicyclo[3.1.0]hexyl) adenosines favored high A3 adenosine receptor (AR) affinity/selectivity, e.g., ...

Following our study of 4′-truncated (N)-methanocarba-adenosine derivatives that displayed unusually high mouse (m) A3AR affinity, we incorporated dopamine-related N6 substituents in the full agonist 5′-methylamide series. N6-(2-(4-Hydroxy-3-methoxy-phenyl)ethyl) derivative MRS7618 11 displayed Ki (nM) 0.563 at hA3AR (∼20,000-fold selective) and 1.54 at mA3AR. 2-Alkyl ethers maintained A3 affinity, but with less selectivity than 2-alkynes. Parallel functional assays of G protein-dependent and β-arrestin 2 (βarr2)-dependent pathways indicate these are full agonists but not biased. Through use of computational modeling, we hypothesized that phenyl OH/OMe groups interact with polar residues, particularly Gln261, on the mA3AR extracellular loops as the basis for the affinity enhancement. Although the pharmacokinetics indicated facile clearance of parent O-methyl catechol nucleosides 21 and 31, prolonged mA3AR activation in vivo was observed in a hypothermia model, suggested potential formation of active metabolites through demethylation. Selected analogues induced mouse hypothermia following i.p. injection, indicative of peripheral A3AR agonism in vivo.

[Image: see text] A side-by-side pharmacological comparison of ribose and (N)-methanocarba (bicyclo[3.1.0]hexane) nucleosides as A(3)AR agonists indicated that the bicyclic pseudoribose ring constraint provided higher affinity/selectivity at human and mouse A(3)AR. The mean affinity enhancement for 5 pairs of 5′-methylamides was 11-fold at hA(3)AR and 42-fold at mA(3)AR. Novel C2-(5-fluorothien-2-ylethynyl) substitution enhanced affinity in the methanocarba but not ribose series, with highly hA(3)AR-selective 16 (MRS7334) displaying K(i) 280 pM and favorable pharmacokinetics and off-target activity profile. Molecular dynamics comparison of 16 and its corresponding riboside 8 suggested a qualitative entropic advantage of 16 in hA(3)AR binding. The 5-F substitution tended to increase hA(3)AR affinity (cf. 5-Cl) for methanocarba but not ribose derivatives. A representative methanocarba agonist 4 was shown to interact potently exclusively with A(3)AR, among 240 GPCRs and 466 kinases. Thus, despite added synthetic difficulty, the (N)-methanocarba modification has distinct advantages for A(3)AR agonists, which have translational potential for chronic disease treatment.

Unquestionably, water appears to be an active player in the noncovalent protein-ligand binding process, as it can either bridge interactions between protein and ligand or can be replaced by the bound ligand. Accordingly, in the last decade, alternative computational methodologies have been sought with the aim of predicting the position and thermodynamic profile of water molecules (i.e., hydration sites) in the binding site using either the ligand-bound or ligand-free protein conformation. Herein, we present an alternative approach, named AquaMMapS, that provides a three-dimensional sampling of putative hydration sites. Interestingly, AquaMMapS can post-inspect molecular dynamics (MD) trajectories obtained from different MD engines using indifferently crystallographic or docking-driven structures as a starting point. Moreover, AquaMMapS is naturally integrated into supervised molecular dynamics (SuMD) simulations, presenting the possibility to inspect hydration sites during the ligand-protein association process. Finally, a penalty scoring method, named AquaMMapScoring(AMS), was developed to evaluate the number and nature of the water molecules displaced by a ligand approaching its binding site during the binding event, guiding a medicinal chemist to explore the most suitable regions of a ligand that can be decorated either with or without interfering with the interaction networks mediated by water molecules with specific recognition regions of the protein.

A small-molecule combinatorial library of 24 compounds with 2-aminoimidazole and 2-aminoimidazolyl-thiazole derivatives was synthesized using a 2-chloro trityl resin. The generated compound library was tested against all the human adenosine receptors subtypes. The 2-aminoimidazole derivatives (6a-6l) showed weak to moderate affinity towards the human adenosine receptors. Further modification to 2-aminoimidazolyl-thiazole derivatives (12a-12l) resulted in an improvement of affinity at adenosine A1, A2A and A3 receptor subtypes. Compound 12b was the most potent and selective non-xanthine human adenosine A3 receptor antagonist of this series. A receptor-based modeling study was performed to explore the possible binding mode of these novel 2-aminoimidazole and 2-aminoimidazolyl-thiazole derivatives into human adenosine A1, A2A and A3 receptor subtypes.

A novel series of variously substituted N-[3-(9H-carbazol-9-yl)-2-hydroxypropyl]-arylsulfonamides has been synthesized and assayed for β-Secretase (BACE1) inhibitory activity. BACE1 is a widely recognized drug target for the prevention and treatment of Alzheimer's Disease (AD). The introduction of benzyl substituents on the nitrogen atom of the arylsulfonamide moiety has so far led to the best results, with three derivatives showing IC50 values ranging from 1.6 to 1.9 μM. Therefore, a significant improvement over the previously reported series of N-carboxamides (displaying IC50’s ≥ 2.5 μM) has been achieved, thus suggesting an active role of the sulfonamido-portion in the inhibition process. Preliminary molecular modeling studies have been carried out to rationalize the observed structure-activity relationships.

Molecular docking is a powerful tool in the field of computer-aided molecular design. In particular, it is the technique of choice for the prediction of a ligand pose within its target binding site. A multitude of docking methods is available nowadays, whose performance may vary depending on the data set. Therefore, some non-trivial choices should be made before starting a docking simulation. In the same framework, the selection of the target structure to use could be challenging, since the number of available experimental structures is increasing. Both issues have been explored within this work. The pose prediction of a pool of 36 compounds provided by D3R Grand Challenge 2 organizers was preceded by a pipeline to choose the best protein/docking-method couple for each blind ligand. An integrated benchmark approach including ligand shape comparison and cross-docking evaluations was implemented inside our DockBench software. The results are encouraging and show that bringing attention to the choice of the docking simulation fundamental components improves the results of the binding mode predictions.

New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A1 and/or A2A receptor subtypes. On the whole, the novel derivatives 1–24 shared scarce or no affinities for the off-target hA2B and hA3 ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (Ki = 150 nM) and the best selectivity for the hA2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA2A (Ki = 123 nM) and A1 AR affinity (Ki = 25 nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (Ki = 11 nM) and good selectivity for the hA1 AR. The 5-(N4-substituted-piperazin-1-yl) derivatives 15–24 bind the hA1 AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.

The ribose of protein-bound nucleosides and nucleotides displays preferred conformations (usually either North or South), which can be exploited to design enhanced analogues having chemically fixed conformations. We introduce a computational protocol for assembling data from the protein database (PDB) on the ribose and ribose-like conformation of small molecule ligands when complexed with purinergic signaling proteins (including receptors, enzymes and transporters, and related intracellular pathways). Some targets prefer exclusively North (adenosine and P2Y(1) receptors, CD73, adenosine kinase ATP/ADP-binding site, adenosine deaminase), others prefer South (P2Y(12) receptor, E-NTPDase2) or East (adenosine kinase substrates), while others (P2XRs) allow various conformations.

(N)-Methanocarba ([3.1.0]bicyclohexyl) adenosines and corresponding ribosides were synthesized to identify novel A(1) adenosine receptor (A(1)AR) agonists for CNS or peripheral applications. Human and mouse AR binding was determined to assess the constrained ring system’s A(1)AR compatibility. N(6)-Dicyclobutylmethyl ribose agonist (9, MRS7469, >2000-fold selective for A(1)AR) and known truncated N(6)-dicyclopropylmethyl methanocarba 7 (MRS5474) were drug-like. The pure diastereoisomer of known riboside 4 displayed high hA(1)AR selectivity. Methanocarba modification reduced A(1)AR selectivity of N(6)-dicyclopropylmethyl and endo-norbornyl-adenosines, but increased ribavirin selectivity. Most analogues tested (ip.) were inactive or weak in inducing mouse hypothermia, despite mA(1)AR full agonism and variable mA(3)AR efficacy, but strong hypothermia by 9 depended on A(1)AR, which reflects CNS activity (determined using A(1)AR or A(3)AR null mice). Conserved hA(1)AR interactions were preserved in modeling of 9 and methanocarba equivalent 24 (~400-fold A(1)AR-selective). Thus, we identified, and characterized in vivo, ribose and methanocarba nucleosides, including with A(1)AR-enhancing N(6)-dicyclobutylmethyl-adenine and 1,2,4-triazole-3-carboxamide (40, MRS7451) nucleobases.

Structure-based drug design (SBDD) has matured within the last two decades as a valuable tool for the optimization of low molecular weight lead compounds to highly potent drugs. The key step in SBDD requires knowledge of the three-dimensional structure of the target-ligand complex, which is usually determined by X-ray crystallography. In the absence of structural information for the complex, SBDD relies on the generation of plausible molecular docking models. However, molecular docking protocols suffer from inaccuracies in the description of the interaction energies between the ligand and the target molecule, and often fail in the prediction of the correct binding mode. In this context, the appropriate selection of the most accurate docking protocol is absolutely relevant for the final molecular docking result, even if addressing this point is absolutely not a trivial task. D3R Grand Challenge 2015 has represented a precious opportunity to test the performance of DockBench, an integrate informatics platform to automatically compare RMDS-based molecular docking performances of different docking/scoring methods. The overall performance resulted in the blind prediction are encouraging in particular for the pose prediction task, in which several complex were predicted with a sufficient accuracy for medicinal chemistry purposes.

Molecular recognition is a crucial issue when aiming to interpret the mechanism of known active substances as well as to develop novel active candidates. Unfortunately, simulating the binding process is still a challenging task because it requires classical MD experiments in a long microsecond time scale that are affordable only with a high-level computational capacity. In order to overcome this limiting factor, we have recently implemented an alternative MD approach, named supervised molecular dynamics (SuMD), and successfully applied it to G protein-coupled receptors (GPCRs). SuMD enables the investigation of ligand-receptor binding events independently from the starting position, chemical structure of the ligand, and also from its receptor binding affinity. In this article, we present an extension of the SuMD application domain including different types of proteins in comparison with GPCRs. In particular, we have deeply analyzed the ligand-protein recognition pathways of six different case studies that we grouped into two different classes: globular and membrane proteins. Moreover, we introduce the SuMD-Analyzer tool that we have specifically implemented to help the user in the analysis of the SuMD trajectories. Finally, we emphasize the limit of the SuMD applicability domain as well as its strengths in analyzing the complexity of ligand-protein recognition pathways.

[Image: see text] Exploring at the molecular level, all possible ligand–protein approaching pathways and, consequently, identifying the energetically favorable binding sites is considered crucial to depict a clear picture of the whole scenario of ligand–protein binding. In fact, a ligand can recognize a protein in multiple binding sites, adopting multiple conformations in every single binding site and inducing protein modifications upon binding. In the present work, we would like to present how it is possible to couple a supervised molecular dynamics (SuMD) approach to explore, from an unbound state, the most energetically favorable recognition pathways of the ligand to its protein, with an enthalpic and entropic characterization of the most stable ligand–protein bound states, using the protein kinase CK2α as a prototype study. We identified two accessory binding pockets surrounding the ATP-binding site having a strong enthalpic contribution but a different configurational entropy contribution, suggesting that they play a different role.

Supervised MD (SuMD) is a computational method that enables the exploration of ligand-receptor recognition pathway in a reduced timescale. The performance speedup is due to the incorporation of a tabu-like supervision algorithm on the ligand-receptor approaching distance into a classic molecular dynamics (MD) simulation. SuMD enables the investigation of ligand-receptor binding events independently from the starting position, chemical structure of the ligand (small molecules or peptides), and also from its receptor-binding affinity. The application of SuMD highlights an appreciable capability of the technique to reproduce the crystallographic structures of several ligand-protein complexes and can provide high-quality protein-ligand models of for which yet experimental confirmation of binding mode is not available.

[1,2,4]Triazolo[1,5-c]pyrimidine is a promising platform to develop adenosine receptor antagonists. Here, we tried to investigate the effect of the substituent at the 8 position of [1,2,4]triazolo[1,5-c]pyrimidine derivatives on affinity and selectivity at the human A

Riluzole, approved by the US Food and Drug Administration (FDA) in 1995, is the most widespread oral treatment for the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). The drug, whose mechanism of action is still obscure, mitigates progression of the illness, but unfortunately with only limited improvements. Herein we report the first demonstration, using a combination of computational and in vitro studies, that riluzole is an ATP-competitive inhibitor of the protein kinase CK1 isoform δ, with an IC50 value of 16.1 μm. This allows us to rewrite its possible molecular mechanism of action in the treatment of ALS. The inhibition of CK1δ catalytic activity indeed links the two main pathological hallmarks of ALS: transactive response DNA-binding protein of 43 kDa (TDP-43) proteinopathy and glutamate excitotoxicity, exacerbated by the loss of expression of glial excitatory amino acid transporter-2 (EAAT2).