(R,R')-4'-methoxy-1-naphthylfenoterol targets GPR55-mediated ligand internalization and impairs cancer cell motility
Biochem Pharmacol. 2014 Feb 15;87(4):547-61. doi: 10.1016/j.bcp.2013.11.020. Epub 2013 Dec 16
Paul RK, Wnorowski A, Gonzalez-Mariscal I, Nayak SK, Pajak K, Moaddel R, Indig FE, Bernier M, Wainer IW
(R,R')-4'-Methoxy-1-naphthylfenoterol (MNF) promotes growth inhibition and apoptosis of human HepG2 hepatocarcinoma cells via cannabinoid receptor (CBR) activation. The synthetic CB1R inverse agonist, AM251, has been shown to block the anti-mitogenic effect of MNF in these cells; however, AM251 is also an agonist of the recently deorphanized, lipid-sensing receptor, GPR55, whose upregulation contributes to carcinogenesis. Here, we investigated the role of MNF in GPR55 signaling in human HepG2 and PANC-1 cancer cell lines in culture by focusing first on internalization of the fluorescent ligand Tocrifluor 1117 (T1117). Initial results indicated that cell pretreatment with GPR55 agonists, including the atypical cannabinoid O-1602 and l-α-lysophosphatidylinositol, dose-dependently reduced the rate of cellular T1117 uptake, a process that was sensitive to MNF inhibition. GPR55 internalization and signaling mediated by O-1602 was blocked by MNF in GPR55-expressing HEK293 cells. Pretreatment of HepG2 and PANC-1 cells with MNF significantly abrogated the induction of ERK1/2 phosphorylation in response to AM251 and O-1602. Moreover, MNF exerted a coordinated negative regulation of AM251 and O-1602 inducible processes, including changes in cellular morphology and cell migration using scratch wound healing assay. This study shows for the first time that MNF impairs GPR55-mediated signaling and, therefore, may have therapeutic potential in the management of cancer.
Isothermal titration calorimetry in membrane protein research
J Pharm Biomed Anal. 2014 Jan;87:313-25. doi: 10.1016/j.jpba.2013.09.003. Epub 2013 Sep 18
Draczkowski P, Matosiuk D, Jozwiak K
Isothermal titration calorimetry is a versatile method to characterize energetics of intermolecular reactions and in particular interactions between drug molecules and their macromolecular targets. The assay is widely used in medicinal chemistry to quantification and characterization of molecular mechanisms of these interactions. The article reviews applications of the method to study ligand interactions with various classes of embedded membrane proteins. These proteins constitute very important fraction of macromolecular targets for medicinal compounds used in current therapies and recently emerged for detailed structural, functional and biochemical characterization of their interactions with drug molecules.
Comparative molecular field analysis of fenoterol derivatives interacting with an agonist-stabilized form of the beta2-adrenergic receptor
Bioorg Med Chem. 2014 Jan 1;22(1):234-46. doi: 10.1016/j.bmc.2013.11.030. Epub 2013 Nov 23
Plazinska A, Pajak K, Rutkowska E, Jimenez L, Kozocas J, Koolpe G, Tanga M, Toll L, Wainer IW, Jozwiak K
The β₂-adrenergic receptor (β₂-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 β₂-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 β₂-AR conformation probed by [(3)H]-(R,R')-4'-methoxyfenoterol. The CoMFA model of the agonist-stabilized β₂-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the β₂-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.
Molecular interactions between mecamylamine enantiomers and the transmembrane domain of the human alpha4beta2 nicotinic receptor
Biochemistry. 2014 Feb 11;53(5):908-18. doi: 10.1021/bi400969x. Epub 2014 Jan 30
Bondarenko V, Targowska-Duda KM, Jozwiak K, Tang P, Arias HR
To characterize the binding sites of mecamylamine enantiomers on the transmembrane domain (TMD) of human (h) (α4)3(β2)2 and (α4)2(β2)3 nicotinic acetylcholine receptors (AChRs), we used nuclear magnetic resonance (NMR), molecular docking, and radioligand binding approaches. The interactions of (S)-(+)- and (R)-(-)-mecamylamine with several residues, determined by high-resolution NMR, within the hα4β2-TMD indicate different modes of binding at several luminal (L) and nonluminal (NL) sites. In general, the residues sensitive to each mecamylamine enantiomer are similar at both receptor stoichiometries. However, some differences were observed. The molecular docking experiments were crucial for delineating the location and orientation of each enantiomer in its binding site. In the (α4)2(β2)3-TMD, (S)-(+)-mecamylamine interacts with the L1 (i.e., between positions -3' and -5') and L2 (i.e., between positions 16' and 20') sites, whereas the β2-intersubunit (i.e., cytoplasmic end of two β2-TMDs) and α4/β2-intersubunit (i.e., cytoplasmic end of α4-TM1 and β2-TM3) sites are shared by both enantiomers. In the (α4)3(β2)2-TMD, both enantiomers bind with different orientations to the L1' (closer to ring 2') and α4-intrasubunit (i.e., at the cytoplasmic ends of α4-TM1 and α4-TM2) sites, but only (R)-(-)-mecamylamine interacts with the L2' (i.e., closer to ring 20') and α4-TM3-intrasubunit sites. Our findings are important because they provide, for the first time, a structural understanding of the allosteric modulation elicited by mecamylamine enantiomers at each hα4β2 stoichiometry. This advancement could be beneficial for the development of novel therapies for the treatment of several neurological disorders.
Fast, metadynamics-based method for prediction of the stereochemistry-dependent relative free energies of ligand-receptor interactions
J Comput Chem. 2014 Apr 30;35(11):876-82. doi: 10.1002/jcc.23563. Epub 2014 Feb 24
Plazinska A, Plazinski W, Jozwiak K
The computational approach applicable for the molecular dynamics (MD)-based techniques is proposed to predict the ligand-protein binding affinities dependent on the ligand stereochemistry. All possible stereoconfigurations are expressed in terms of one set of force-field parameters [stereoconfiguration-independent potential (SIP)], which allows for calculating all relative free energies by only single simulation. SIP can be used for studying diverse, stereoconfiguration-dependent phenomena by means of various computational techniques of enhanced sampling. The method has been successfully tested on the β2-adrenergic receptor (β2-AR) binding the four fenoterol stereoisomers by both metadynamics simulations and replica-exchange MD. Both the methods gave very similar results, fully confirming the presence of stereoselective effects in the fenoterol-β2-AR interactions. However, the metadynamics-based approach offered much better efficiency of sampling which allows for significant reduction of the unphysical region in SIP.
fRMSDchiral: a novel algorithm to represent differences between positions of stereoisomers in complex with dissymmetric binding site
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Apr 1;955-956:110-5. doi: 10.1016/j.jchromb.2014.02.025. Epub 2014 Feb 28
Urniaz RD, Rutkowska EE, Plazinska A, Jozwiak K
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.
Antidepressant activity in mice elicited by 3-furan-2-yl-N-p-tolyl-acrylamide, a positive allosteric modulator of the alpha7 nicotinic acetylcholine receptor
Neurosci Lett. 2014 May 21;569:126-30. doi: 10.1016/j.neulet.2014.03.067. Epub 2014 Apr 5
Targowska-Duda KM, Feuerbach D, Biala G, Jozwiak K, Arias HR
The objective of the current study is to determine whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator of α7 nicotinic receptors (AChRs), produces antidepressant-like behavior in mice, and reactivates desensitized α7 AChRs expressed in CH3-α7 cells. Mice from both sexes were injected (i.p.) with PAM-2 (1.0mg/kg) on a daily basis for three weeks. Forced swim tests (FSTs) were performed on Day 1 and Day 7 to determine the acute and subchronic effects of PAM-2, respectively, and on Days 14 and 21 to determine its chronic activity. To examine the residual effects after drug treatment, a withdrawal period of two more weeks was continued with FSTs performed on Day 28 and 35. Our results indicate that: (1) PAM-2 does not induce acute antidepressant effects in male or female mice, (2) PAM-2 induces antidepressant effects in mice from both sexes after one (subchronic) and two (chronic) weeks, whereas at the third week (chronic), the antidepressant effect is decreased in male and increased in female mice. Since PAM-2 does not influence the locomotor activity of mice, the observed antidepressant activity is not driven by nonspecific motor-stimulant actions, (3) the residual antidepressant effect mediated by PAM-2 after one week of treatment cessation is observed only in female mice, and finally the Ca(2+) influx results indicate that (4) PAM-2 can reactivate desensitized α7 AChRs. Our results clearly indicate that PAM-2 elicits antidepressant activity, probably by enhancing the activity of the endogenous neurotransmitter acetylcholine on α7 AChRs, without inducing receptor desensitization, and that this activity is gender-dependent. This is the first time that an antidepressant activity is described for an α7 PAM, supporting further studies as potential therapeutic medications for depressive states.
Tyrosine 308 is necessary for ligand-directed Gs protein-biased signaling of beta2-adrenoceptor
J Biol Chem. 2014 Jul 11;289(28):19351-63. doi: 10.1074/jbc.M114.558882. Epub 2014 May 15
Woo AY, Jozwiak K, Toll L, Tanga MJ, Kozocas JA, Jimenez L, Huang Y, Song Y, Plazinska A, Pajak K, Paul RK, Bernier M, Wainer IW, Xiao RP
Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β2-adrenoceptor (β2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of β2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most β2-AR agonists cause both Gs and Gi activation, (R,R')-fenoterol preferentially activates β2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β2-AR essential for Gs-biased signaling. Following stimulation with a β2-AR-Gs-biased agonist (R,R')-4'-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β2-AR. Interestingly, Y308F substitution on β2-AR enabled (R,R')-4'-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering β2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.
Bupropion-induced inhibition of alpha7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus
Eur J Pharmacol. 2014 Oct 5;740:103-11. doi: 10.1016/j.ejphar.2014.06.059. Epub 2014 Jul 9
Vázquez-Gómez E, Arias HR, Feuerbach D, Miranda-Morales M, Mihailescu S, Targowska-Duda KM, Jozwiak K, García-Colunga J
The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2+ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 µM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50=54 µM) and binding affinity (Ki=63 µM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation.
Homo- and hetero-oligomerization of beta2-adrenergic receptor in receptor trafficking, signaling pathways and receptor pharmacology
Cell Signal. 2014 Oct;26(10):2259-65. doi: 10.1016/j.cellsig.2014.06.016. Epub 2014 Jul 15
Wnorowski A, Jozwiak K
The β2-adrenergic receptor (β2AR) is the prototypic member of G protein-coupled receptors (GPCRs) involved in the production of physiological responses to adrenaline and noradrenaline. Research done in the past few years vastly demonstrated that β2AR can form homo- and hetero-oligomers. Despite the fact that currently this phenomenon is widely accepted, the spread and relevance of β2AR oligomerization are still a matter of debate. This review considers the progress achieved in the field of β2AR oligomerization with focus on the implications of the receptor-receptor interactions to β2AR trafficking, pharmacology and downstream signal transduction pathways.
Design, stereoselective synthesis, configurational stability and biological activity of 7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide
Bioorg Med Chem. 2014 Sep 1;22(17):4667-76. doi: 10.1016/j.bmc.2014.07.017. Epub 2014 Jul 18
Carrozzo MM, Battisti UM, Cannazza G, Puia G, Ravazzini F, Falchicchio A, Perrone S, Citti C, Jozwiak K, Braghiroli D, Parenti C, Troisi L
Chiral 5-arylbenzothiadiazine derivatives have recently attracted particular attention because they exhibit an interesting pharmacological activity as AMPA receptor (AMPAr) positive modulators. However, investigations on their configurational stability suggest a rapid enantiomerization in physiological conditions. In order to enhance configurational stability, preserving AMPAr activity, we have designed the novel compound (R,S)-7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide bearing a pyrrolo moiety coupled with the 5-(furan-3-yl) substituent on benzothiadiazine core. A stereoselective synthesis was projected to obtain single enantiomer of the latter compound. Absolute configuration was assigned by X-ray crystal structure. Patch clamp experiments evaluating the activity of single enantiomers as AMPAr positive allosteric modulator showed that R stereoisomer is the active component. Molecular modeling studies were performed to explain biological results. An on-column stopped-flow bidimensional recycling HPLC procedure was applied to obtain on a large scale the active enantiomer with enantiomeric enrichment starting from the racemic mixture of the compound.
The effect of moonlighting proteins on the adhesion and aggregation ability of Lactobacillus helveticus
Anaerobe. 2014 Oct 13;30C:161-168. doi: 10.1016/j.anaerobe.2014.10.002
Waśko A, Polak-Berecka M, Paduch R, Jóźwiak K
The goal of this study was to identify moonlighting proteins in Lactobacillus helveticus that play an important role in adhesion and aggregation. The label-free method was used for identification and analysis of expression of cellular proteins. The analysis revealed the presence of eight moonlighting proteins in the cell envelope of Lb. helveticus. The tested strains mainly differed with respect to the presence of S-layer proteins and the level of expression of moonlighting proteins in Lb. helveticus strain T159. These surface proteins give the cell a hydrophobic character and play a role in specific interactions with intestinal epithelium cells and with other bacteria. In Lb. helveticus T159, the S-layer associated with moonlighting proteins could act as adherence factors, which was evidenced by the high capability of adhesion, auto- and coaggregation. The hydrophobicity, adhesion and aggregation abilities provide biological activities in food products and they are regarded as an important criterion for probiotic selection.
Synthesis, in vitro and in vivo studies, and molecular modeling of N-alkylated dextromethorphan derivatives as non-competitive inhibitors of alpha3beta4 nicotinic acetylcholine receptor
Bioorg Med Chem. 2014 Dec 15;22(24):6846-56. doi: 10.1016/j.bmc.2014.10.036. Epub 2014 Oct 31
Jozwiak K, Targowska-Duda KM, Kaczor AA, Kozak J, Ligeza A, Szacon E, Wrobel TM, Budzynska B, Biala G, Fornal E, Poso A, Wainer IW, Matosiuk D
9 N-alkylated derivatives of dextromethorphan are synthesized and studied as non-competitive inhibitors of α3β4 nicotinic acetylcholine receptors (nAChRs). In vitro activity towards α3β4 nicotinic acetylcholine receptor is determined using a patch-clamp technique and is in the micromolar range. Homology modeling, molecular docking and molecular dynamics of ligand-receptor complexes in POPC membrane are used to find the mode of interactions of N-alkylated dextromethorphan derivatives with α3β4 nAChR. The compounds, similarly as dextromethorphan, interact with the middle portion of α3β4 nAChR ion channel. Finally, behavioral tests confirmed potential application of the studied compounds for the treatment of addiction.