Potential off-target effects of beta-blockers on gut hormone receptors: In silico study including GUT-DOCK-A web service for small-molecule docking.

The prolonged use of many currently available drugs results in the severe side effect of the disruption of glucose metabolism leading to type 2 diabetes mellitus (T2DM. Gut hormone receptors including glucagon receptor (GCGR) and the incretin hormone receptors: glucagon-like peptide 1 receptor (GLP1R) and gastric inhibitory polypeptide receptor (GIPR) are important drug targets for the treatment of T2DM, as they play roles in the regulation of glucose and insulin levels and of food intake. In this study, we hypothesized that we could compensate for the negative influences of specific drugs on glucose metabolism by the positive incretin effect enhanced by the off-target interactions with incretin GPCR receptors. As a test case, we chose to examine beta-blockers because beta-adrenergic receptors and incretin receptors are expressed in a similar location, making off-target interactions possible. The binding affinity of drugs for incretin receptors was approximated by using two docking scoring functions of Autodock VINA (GUT-DOCK) and Glide (Schrodinger) and juxtaposing these values with the medical information on drug-induced T2DM. We observed that beta-blockers with the highest theoretical binding affinities for gut hormone receptors were reported as the least harmful to glucose homeostasis in clinical trials. Notably, a recently discovered beta-blocker compound 15 ([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide was among the top-scoring drugs, potentially supporting its use in the treatment of hypertension in diabetic patients. Our recently developed web service GUT-DOCK (gut-dock.miningmembrane.com) allows for the execution of similar studies for any drug-like molecule. Specifically, users can compute the binding affinities for various class B GPCRs, gut hormone receptors, VIPR1 and PAC1R.

Drug-induced diabetes type 2: In silico study involving class B GPCRs.

A disturbance of glucose homeostasis leading to type 2 diabetes mellitus (T2DM) is one of the severe side effects that may occur during a prolonged use of many drugs currently available on the market. In this manuscript we describe the most common cases of drug-induced T2DM, discuss available pharmacotherapies and propose new ones. Among various pharmacotherapies of T2DM, incretin therapies have recently focused attention due to the newly determined crystal structure of incretin hormone receptor GLP1R. Incretin hormone receptors: GLP1R and GIPR together with the glucagon receptor GCGR regulate food intake and insulin and glucose secretion. Our study showed that incretin hormone receptors, named also gut hormone receptors as they are expressed in the gastrointestinal tract, could potentially act as unintended targets (off-targets) for orally administrated drugs. Such off-target interactions, depending on their effect on the receptor (stimulation or inhibition), could be beneficial, like in the case of incretin mimetics, or unwanted if they cause, e.g., decreased insulin secretion. In this in silico study we examined which well-known pharmaceuticals could potentially interact with gut hormone receptors in the off-target way. We observed that drugs with the strongest binding affinity for gut hormone receptors were also reported in the medical information resources as the least disturbing the glucose homeostasis among all drugs in their class. We suggested that those strongly binding molecules could potentially stimulate GIPR and GLP1R and/or inhibit GCGR which could lead to increased insulin secretion and decreased hepatic glucose production. Such positive effect on the glucose homeostasis could compensate for other, adverse effects of pharmacotherapy which lead to drug-induced T2DM. In addition, we also described several top hits as potential substitutes of peptidic incretin mimetics which were discovered in the drug repositioning screen using gut hormone receptors structures against the ZINC15 compounds subset.

How to design potent and selective DYRK1B inhibitors? Molecular modeling study.

DYRK1B protein kinase is an emerging anticancer target due to its overexpression in a variety of cancers and its role in cancer chemoresistance through maintaining cancer cells in the G0 (quiescent) state. Consequently, there is a growing interest in the development of potent and selective DYRK1B inhibitors for anticancer therapy. One of the major off-targets is another protein kinase, GSK3ß, which phosphorylates an important regulator of cell cycle progression on the same residue as DYRK1B and is involved in multiple signaling pathways. In the current work, we performed a detailed comparative structural analysis of DYRK1B and GSK3ß ATP-binding sites and identified key regions responsible for selectivity. As the crystal structure of DYRK1B has never been reported, we built and optimized a homology model by comparative modeling and metadynamics simulations. Calculation of interaction energies between docked ligands in the ATP-binding sites of both kinases allowed us to pinpoint key residues responsible for potency and selectivity. Specifically, the role of the gatekeeper residues in DYRK1B and GSK3ß is discussed in detail, and two other residues are identified as key to selectivity of DYRK1B inhibition versus GSK3ß. The analysis presented in this work was used to support the design of potent and selective azaindole-quinoline-based DYRK1B inhibitors and can facilitate development of more selective inhibitors for DYRK kinases.

Approaches for Differentiation and Interconverting GPCR Agonists and Antagonists.

Predicting the functional preferences of the ligands was always a highly demanding task, much harder that predicting whether a ligand can bind to the receptor. This is because of significant similarities of agonists, antagonists and inverse agonists which are binding usually in the same binding site of the receptor and only small structural changes can push receptor toward a particular activation state. For G protein-coupled receptors, due to a large progress in crystallization techniques and also in receptor thermal stabilization, it was possible to obtain a large number of high-quality structures of complexes of these receptors with agonists and non-agonists. Additionally, the long-time-scale molecular dynamics simulations revealed how the activation processes of GPCRs can take place. Using both theoretical and experimental knowledge it was possible to employ many clever and sophisticated methods which can help to differentiate agonists and non-agonists, so one can interconvert them in search of the optimal drug.

Selected secondary metabolites in Echium vulgare L. populations from nonmetalliferous and metalliferous areas.

The aim of this study was to evaluate the effect of severe environmental conditions prevailing on metalliferous waste heaps and heavy metal-contaminated growth substrates on accumulation of selected secondary metabolites, antioxidant capacity, and heavy metal concentration in two metallicolous (MC, MZ) and one nonmetallicolous (NM) populations of Echium vulgare L. The shoots and the roots of the three studied populations were collected from their natural habitats. Additionally, the plants were cultivated on different growth substrates, i.e. a contaminated substrate obtained from the areas of growth of the MZ and MC populations and an uncontaminated one from the NM population site. Several compounds, i.e. allantoin, rutin, rosmarinic acid, chlorogenic acid, and 4-hydroxybenzoic acid were identified in the shoots. Moreover, rosmarinic acid, allantoin, and shikonin were measured in the roots. The adverse environmental conditions contributed to a ca. 10- and 4-fold increase in the concentration of allantoin in the roots and shoots, respectively, as well as a ca. 4-fold and ca. 3-fold increase in the level of 4-hydroxybenzoic acid and shikonin, respectively, in comparison with the plants from the uncontaminated site. Similarly, a great impact of the contaminated substrate on the compounds was demonstrated in the soil experiment. Regardless of the populations, even ca. 20-fold higher levels of allantoin and shikonin were observed in plants grown on the MC and MZ substrates. In contrast, the chlorogenic acid concentration was lower in plants collected from the metalliferous areas and in all populations cultivated on the contaminated substrates in comparison with plants from the uncontaminated soil. Unambiguous results were obtained in the case of rutin, i.e. decreased accumulation in both metallicolous populations from the natural environment and increased accumulation in plants grown on the contaminated substrates. The high concentrations of heavy metals in the substrates contributed to high HM concentrations in plant tissues. However, some differences were observed between the metallicolous and nonmetallicolous populations - the accumulation of metals was lower in the shoots and higher in the roots of the NM population, compared with the MZ and MC populations.

Comparison of Analytical Methods in Chemometric Fingerprinting of Metallicolous and Non-metallicolous Populations of Echium vulgare L.

INTRODUCTION: Adverse environmental conditions usually change plant biochemical pathways resulting in accumulation or decreased content of both primary and secondary metabolites. The chemometric fingerprinting analysis proves to be a useful tool to reveal phytochemical differentiation between plants inhabiting heavy metal-contaminated and uncontaminated areas. OBJECTIVE: Development and assessment of four analytical techniques - high performance capillary electrophoresis (HPCE), thin-layer chromatography (TLC), mass spectrometry (MS), and Fourier transform infrared (FTIR) spectroscopy in chemometric fingerprinting of metallicolous and non-metallicolous populations of Echium vulgare L. MATERIAL AND METHODS: Twenty-one crude methanol extracts of shoot samples representing three populations of Echium vulgare L., two originating from highly metal polluted areas and one from an unpolluted area, were investigated using four analytical methods: HPCE, TLC, MS, and FTIR spectroscopy. Data pre-processing (denoising, background subtracting, horizontal alignment) followed by principal component analysis (PCA), hierarchical clustering analysis (HCA), and phytochemical difference index (DI) calculations facilitated exploration of the differences and similarities between the populations. RESULTS: Clear phytochemical divergence between metallicolous and non-metallicolous populations of Echium vulgare was found. The suitability of the analytical techniques for revealing phytochemical markers and discrimination of individuals originating from different populations differed and in general increased in the order: TLC < MS = HPCE < FTIR. CONCLUSION: The chemometric methods applied were successful in discrimination between samples from polluted and unpolluted areas, showing a potential perspective for environmental quality control. Copyright © 2016 John Wiley & Sons, Ltd.

Ketamine Metabolites Enantioselectively Decrease Intracellular D-Serine Concentrations in PC-12 Cells.

D-Serine is an endogenous NMDA receptor co-agonist that activates synaptic NMDA receptors modulating neuronal networks in the cerebral cortex and plays a key role in long-term potentiation of synaptic transmission. D-serine is associated with NMDA receptor neurotoxicity and neurodegeneration and elevated D-serine concentrations have been associated with Alzheimer's and Parkinsons' diseases and amyotrophic lateral sclerosis. Previous studies have demonstrated that the ketamine metabolites (rac)-dehydronorketamine and (2S,6S)-hydroxynorketamine decrease intracellular D-serine concentrations in a concentration dependent manner in PC-12 cells. In the current study, PC-12 cells were incubated with a series of ketamine metabolites and the IC50 values associated with attenuated intracellular D-serine concentrations were determined. The results demonstrate that structural and stereochemical features of the studied compounds contribute to the magnitude of the inhibitory effect with (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine displaying the most potent inhibition with IC50 values of 0.18 ± 0.04 nM and 0.68 ± 0.09 nM. The data was utilized to construct a preliminary 3D-QSAR/pharmacophore model for use in the design of new and more efficient modulators of D-serine.

Computational methods for studying G protein-coupled receptors (GPCRs).

The functioning of GPCRs is classically described by the ternary complex model as the interplay of three basic components: a receptor, an agonist, and a G protein. According to this model, receptor activation results from an interaction with an agonist, which translates into the activation of a particular G protein in the intracellular compartment that, in turn, is able to initiate particular signaling cascades. Extensive studies on GPCRs have led to new findings which open unexplored and exciting possibilities for drug design and safer and more effective treatments with GPCR targeting drugs. These include discovery of novel signaling mechanisms such as ligand promiscuity resulting in multitarget ligands and signaling cross-talks, allosteric modulation, biased agonism, and formation of receptor homo- and heterodimers and oligomers which can be efficiently studied with computational methods. Computer-aided drug design techniques can reduce the cost of drug development by up to 50%. In particular structure- and ligand-based virtual screening techniques are a valuable tool for identifying new leads and have been shown to be especially efficient for GPCRs in comparison to water-soluble proteins. Modern computer-aided approaches can be helpful for the discovery of compounds with designed affinity profiles. Furthermore, homology modeling facilitated by a growing number of available templates as well as molecular docking supported by sophisticated techniques of molecular dynamics and quantitative structure-activity relationship models are an excellent source of information about drug-receptor interactions at the molecular level.

Reprint of: fRMSDchiral: a novel algorithm to represent differences between positions of stereoisomers in complex with dissymmetric binding site.

The ability of molecules to distinguish between optical isomers is crucial for living systems. The change of position of one enantiomer in respect to the position of the second enantiomer within an asymmetric binding site may be analyzed on different levels. Root Mean Square Deviation (RMSD) may be used for such analyses with low precision. Additional fragment level variants of RMSD allow for more precise definition of differences in location of the main molecular features responsible for recognition of stereoisomers by a selector. Three fRMSDchiral parameters appear to be very useful to precisely quantify the change in orientations of stereoisomers. Proposed calculation emerges as interesting assistance in interpretation of consequences of formation differential interaction(s) responsible for a chiral recognition process.

fRMSDchiral: a novel algorithm to represent differences between positions of stereoisomers in complex with dissymmetric binding site.

The ability of molecules to distinguish between optical isomers is crucial for living systems. The change of position of one enantiomer in respect to the position of the second enantiomer within an asymmetric binding site may be analyzed on different levels. Root Mean Square Deviation (RMSD) may be used for such analyses with low precision. Additional fragment level variants of RMSD allow for more precise definition of differences in location of the main molecular features responsible for recognition of stereoisomers by a selector. Three fRMSDchiral parameters appear to be very useful to precisely quantify the change in orientations of stereoisomers. Proposed calculation emerges as interesting assistance in interpretation of consequences of formation differential interaction(s) responsible for a chiral recognition process.

Comparative molecular field analysis of fenoterol derivatives interacting with an agonist-stabilized form of the ß2-adrenergic receptor.

The ß2-adrenergic receptor (ß2-AR) agonist [(3)H]-(R,R')-methoxyfenoterol was employed as the marker ligand in displacement studies measuring the binding affinities (Ki values) of the stereoisomers of a series of 4'-methoxyfenoterol analogs in which the length of the alkyl substituent at α' position was varied from 0 to 3 carbon atoms. The binding affinities of the compounds were additionally determined using the inverse agonist [(3)H]-CGP-12177 as the marker ligand and the ability of the compounds to stimulate cAMP accumulation, measured as EC50 values, were determined in HEK293 cells expressing the ß2-AR. The data indicate that the highest binding affinities and functional activities were produced by methyl and ethyl substituents at the α' position. The results also indicate that the Ki values obtained using [(3)H]-(R,R')-methoxyfenoterol as the marker ligand modeled the EC50 values obtained from cAMP stimulation better than the data obtained using [(3)H]-CGP-12177 as the marker ligand. The data from this study was combined with data from previous studies and processed using the Comparative Molecular Field Analysis approach to produce a CoMFA model reflecting the binding to the ß2-AR conformation probed by [(3)H]-(R,R')-4'-methoxyfenoterol. The CoMFA model of the agonist-stabilized ß2-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the ß2-AR is governed to a greater extend by steric effects than binding to the [(3)H]-CGP-12177-stabilized conformation(s) in which electrostatic interactions play a more predominate role.

Lipophilicity--methods of determination and its role in medicinal chemistry.

Lipophilicity is a physicochemical property of crucial importance in medicinal chemistry. On the molecular level it encodes information on the network of inter- and intramolecular forces affecting drug transport through lipid structures as well as drug's interactions with the target protein. In result, on the organism level, lipophilicity is an important factor defining pharmacokinetics and pharmacodynamics of a drug substance. Thus, it is a meaningful parameter that found innumerable applications in drug development, Quantitative Structure-Activity Relationships (QSARs) and Quantitative Structure-Pharmacokinetic Relationships (QSPkRs) analyses. This report reviews the importance of lipophilicity on each step of the presence of a medicinal substance in the organism and describes progress in experimental methods of its determination. It has been documented that the retention of a compound in reversed-phase liquid chromatography is governed by its lipophilicity and shows significant correlation with n-octanol/water partition coefficient. Hence, reversed phase chromatography may provide relevant information about the compounds' property. Elaboration of biomimetic stationary phases provides better insight into biological partition processes. Nowadays, there is an urgent need for both precise and quick procedures for quantification of molecular lipophilicity.

The stereoselective sulfate conjugation of 4'-methoxyfenoterol stereoisomers by sulfotransferase enzymes.

The presystemic sulfate conjugation of the stereoisomers of 4'-methoxyfenoterol, (R,R')-MF, (S,S')-MF, (R,S')-MF, and (S,R')-MF, was investigated using commercially available human intestinal S9 fractions, a mixture of sulfotransferase (SULT) enzymes. The results indicate that the sulfation was stereospecific and that an S-configuration at the ß-OH carbon of the MF molecule enhanced the maximal formation rates with (S,R')-MF (S,S')-MF (R,S')-MF ≈ (R,R')-MF, and competition studies demonstrated that (S,R')-MF is an effective inhibitor of (R,R')-MF sulfation (IC(50) = 60 µM). In addition, the results from a cDNA-expressed human SULT isoform screen indicated that SULT1A1, SULT1A3, and SULT1E1 can mediate the sulfation of all four MF stereoisomers. Previously published molecular models of SULT1A3 and SULT1A1 were used in docking simulations of the MF stereoisomers using Molegro Virtual Docker. The models of the MF-SULT1A3 and MF-SULT1A1 complexes indicate that each of the two chiral centers of MF molecule plays a role in the observed relative stabilities. The observed stereoselectivity is the result of multiple hydrogen bonding interactions and induced conformational changes within the substrate-enzyme complex. In conclusion, the results suggest that a formulation developed from a mixture of (R,R')-MF and (S,R')-MF may increase the oral bioavailability of (R,R')-MF.