Arpromidine-related acylguanidines: synthesis and structure-activity relationships of a new class of guanidine-type histamine H2 receptor agonists with reduced basicity

There has been increasing evidence that histamine receptor stimulation might be an interesting aspect for the development of future drugs as well. Arpromidine and related imidazolylpropylguanidines are the most potent H2R agonists on the isolated guinea pig right atrium. The strongly basic guanidino group is essential for the agonistic activity but it is also responsible for very low oral bioavailability, non-H2R-mediated effects and lack of penetration across the blood-brain barrier. Therefore, the objective of this work was to design, synthesize and characterize histamine H2 receptor agonists, structurally related to arpromidine, but with lower basicity in order to achieve more favourable pharmacokinetic properties, in particular, oral bioavailability and penetration across the blood-brain barrier. The first part of this project was to develop a general synthetic route for the synthesis of NG-acylated imidazolylpropylguanidines. In Chapter 3, the development of a very convenient and straight forward synthetic route is described for the synthesis of NG-acylated guanidines, from the commercially available starting material, urocanic acid and guanidine, followed by coupling of acids. Then the synthesised compounds were pharmacologically tested on isolated guinea pig tissues (ileum: H1R, H3R; right atrium: H2R), on human H1R expressing cells (U373MG) and on membrane preparations of guinea pig and human histamine H2 or H4 receptor expressing Sf9 cells. The basicity of the acylguanidines is by 4-5 orders of magnitude lower than that of the corresponding guanidines. Acyl and alkyl guanidines are about equipotent as, for instance, demonstrated for the diphenylpropyl versus the diphenylpropanoyl and for impromidine versus the oxo-substituted analogue. Surprisingly, compounds with only one phenyl ring are equally or even more potent than the diaryl analogues. On the isolated guinea pig right atrium the most active H2R agonist, the 3-phenylbutanoyl substituted guanidine was about 30 times more potent than the natural ligand. However, the acylated imidazolylpropylguanidines are superior with respect to pharmacokinetic properties. A very important finding is that the compounds are absorbed from the gastrointestinal tract and are capable of penetrating through the blood-brain barrier. Centrally active H2R agonists will be useful pharmacological tools to study the role histamine H2 receptors in the brain. In Chapter 4, the bioisosteric replacement of the imidazolyl moiety in NG-acylated guanidine-type histamine H2 receptor agonists by a 2-amino-4-methylthiazol-5-yl group resulted in about the same H2R agonistic potency on the isolated guinea pig right atrium as well as in GTPase assays. Interestingly, in the GTPase assay on the human H2R the thiazolyl analogue with cyclohexylbutanoyl substituent was favoured compared to the corresponding phenylbutanoylguanidine. This may be interpreted a hint that a certain degree of selectivity for the human H2R may be achieved by structural variation of both the acyl and the heterocyclylpropyl group. Moreover, the aminothiazoles proved to be devoid of H3R antagonistic activity. Thus, the selectivity for H2R versus H3R can be considerably improved. In Chapter 5, the stereoselective preparation of enantiomers of several chiral new H2R agonists is described. The building block (R and S)-3-phenylbutanoic acid was synthesized from the achiral precursor, methyl (E)-but-2-enoate, via asymmetric conjugate addition of phenylboronic acid by using a catalytic amount of rhodium catalyst and chiral binap ligand, followed by hydrolysis of the methyl ester. The corresponding chiral pyridyl acids with high ee (95-99 %), were prepared by the lipase catalysed enantioselective acetylation of racemic 1-(2-pyridyl)ethanol, followed by SN2 displacement with sodium salt of dimethyl malonate, and finally the decarboxylation of saponified product. Interestingly, the preferred stereoisomers were conversely configured in case of the imidazolyl compound (R > S) and its aminothiazolyl analogue (S > R), respectively. This information is of particular interest with respect to the further refinement of receptor models, as the binding mode of imidazoles and aminothiazoles appears to be different. Meanwhile imidazolylpropylguanidines and corresponding acylated analogues were found to be useful building blocks beyond the preparation of H2R agonists, for instance for the synthesis of ligands preferentially binding to other histamine receptor subtypes, to neuropeptide Y Y1 or Y4 receptors. Thus, there is increasing evidence that in terms of medicinal chemistry the imidazolylpropylguanidine moiety and the acylated analogues may be considered �privileged structures�. An extremely promising perspective results from very recent studies: surprisingly, some of the acylguanidines proved to be rather potent as either agonists or inverse agonists at the recently discovered H4R

Novel Strategies for Model-Building of G Protein-Coupled Receptors

The G protein-coupled receptors constitute still the most densely populated proteinfamily encompassing numerous disease-relevant drug targets. Consequently, medicinal chemistry is expected to pursue targets from that protein family in that hits need to be generated and subsequently optimized towards viable clinical candidates for a variety of therapeutic areas. For the purpose of rationalizing structure-activity relationships within such optimization programs, structural information derived from the ligand's as well as the macromolecule's perspective is essential. While it is relatively straightforward to define pharmacophore hypotheses based on comparative modelling of structurally and biologically characterized low-molecular weight ligands, a deeper understanding of the molecular recognition event underlying, remains challenging, since the principally available amount of experimentally derived structural data on GPCRs is extremely scarse when compared to, e.g., soluble enzymes. In this context, the protein modelling methodologies introduced, developed, optimized, and applied in this thesis provide structural models that are capable of assisting in the development of structural hypotheses on ligand-receptor complexes. As such they provide a valuable structural framework not only for a more detailed insight into ligand-GPCR interaction, but also for guiding the design process towards next-generation compounds which should display enhanced affinity. The model building procedure developed in this thesis systematically follows a hierarchical approach, sequentially generating a 1D topology, followed by a 2D topology that is finally converted into a 3D topology. The determination of a 1D topology is based on a compartmentalization of the linear amino acid sequence of a GPCR of interest into the extracellular, intracellular, and transmembrane sequence stretches. The entire chapter 3 of this study elaborates on the strengths and weaknesses of applying automated prediction tools for the purpose of identifying the transmembrane sequence domains. Based on an once derived 1D topology, a type of in-plane projection structure for the seven transmembrane helices can be derived with the aide of calculated vectorial property moments, yielding the 2D topology. Thorough bioinformatics studies revealed that only a consensus approach based on a conceptual combination of different methods employing a carefully made selection of parameter sets gave reliable results, emphasizing the danger to fully automate a GPCR modelling procedure. Chapter 4 describes a procedure to further expand the 2D topological findings into 3D space, exemplified on the human CCK-B receptor protein. This particular GPCR was chosen as the receptor of interest, since an enormous experimentally derived and structurally relevant data-set was available. Within the computational refinement procedure of constructed GPCR models, major emphasis was laid on the explicit treatment of a non-isotropic solvent environment during molecular mechanics (i.e. energy minimization and molecular dynamics simulations) calculations. The majority of simulations was therefore carried out in a tri-phasic solvent box accounting for a central lipid environment, flanked by two aqueous compartments, mimicking the extracellular and cytoplasmic space. Chapter 5 introduces the reference compound set, comprising low-molecular weight compounds modulating CCK receptors, that was used for validation purposes of the generated models of the receptor protein. Chapter 6 describes how the generated model of the CCK-B receptor was subjected to intensive docking studies employing compound series introduced in chapter 5. It turned out that by applying the DRAGHOME methodology viable structural hypotheses on putative receptor-ligand complexes could be generated. Based on the methodology pursued in this thesis a detailed model of the receptor binding site could be devised that accounts for known structure-activity relationships as well as for results obtained by site-directed mutagenesis studies in a qualitative manner. The overall study presented in this thesis is primarily aimed to deliver a feasibility study on generating model structures of GPCRs by a conceptual combination of tailor-made bioinformatics techniques with the toolbox of protein modelling, exemplified on the human CCK-B receptor. The generated structures should be envisioned as models only, not necessarily providing a detailed image of reality. However, consistent models, when verified and refined against experimental data, deliver an extremely useful structural contextual platform on which different scientific disciplines such as medicinal chemistry, molecular biology, and biophysics can effectively communicate

Cílené mutace lidského histaminového H4 receptoru: Postavení Arg-341 za vzájemného působení H4R agonistů cyanoguanidinového typu

Univerzita Karlova v Praze Farmaceutická fakulta v Hradci Králové Katedra biochemických věd Kandidát: Kateřina Ládová Školitel: Prof. Dr. Armin Buschauer Doc. RNDr. Lenka Skálová, Ph.D. Název diplomové práce: Cílené mutace lidského histaminového H4 receptoru: Postavení Arg-341 za vzájemného působení H4R agonistů cyanoguanidinového typu Lidský histaminový H4 receptor (hH4R) byl objeven v roce 2000. Předpokládá se, že H4R zaujímá postavení v imunologických pochodech a může tak být potenciálním cílem vývoje nových léků v terapii zánětlivých onemocnění. Naklonování a exprese hH4Ru odstartovaly hledání selektivních agonistů a antagonistů. Nedávno byl v sérii agonistů cyanoguanidinového typu identifikován vysoce účinný a receptorově selektivní H4R agonista, UR-PI376, (2-cyano-1-[4-(1-H-imidazol-4-yl)butyl]-3-[(2-fenylthio)ethyl] guanidin), s jasnými preferencemi k hH4Ru před H4Rem myšovitých (mH4R). Dle molekulárních modelových studií, UR-PI376 tvoří dvě vodíkové vazby mezi cyanoguanidinovým zbytkem a argininem 341 hH4Ru, což vedlo k doměnce, že Arg-341 je důvodem, proč je UR-PI376 selektivnější k hH4Ru než k lidskému histaminovému H3 receptoru (hH3R) a proč upřednostňuje hH4R před mH4Rem. Abychom objasnili roli této aminokyseliny ve vzájemné interakci s cyanoguanidiny, vytvořili jsme tři mutanty:...Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Ládová Consultant: Prof. Dr. Armin Buschauer Doc. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Site-directed mutagenesis of the human histamine H4 receptor: The role of Arg-341 in the interaction with cyanoguanidine-type H4R agonists The human histamine H4 receptor (hH4R) was discovered in 2000. The H4R is supposed to be involved in immunological processes and is considered a potential drug target, e. g., for the treatment of inflammatory diseases. Cloning and expression of the hH4R inspired to the search for selective agonists and antagonists. Recently, UR-PI376 (2-cyano-1-[4-(1H- imidazol-4-yl)butyl]-3-[(2-phenylthio)ethyl]guanidine) was identified within a series of cyanoguanidines as a highly potent and subtype-selective H4R agonist with pronounced preference for the human over the murine H4R (mH4R). According to molecular modelling studies, the cyanoguanidine moiety of UR-PI376 forms charge-assisted hydrogen bonds with Arg-341 of the hH4R, suggesting this amino acid brings about selectivity for the hH4R over the hH3R and is the reason for the preference of UR-PI376 for hH4R over mH4R as well. To elucidate the role of this amino acid in the interaction with...Department of Biochemical SciencesKatedra biochemických vědFaculty of Pharmacy in Hradec KrálovéFarmaceutická fakulta v Hradci Králov

Novel Strategies for Model-Building of G Protein-Coupled Receptors

The G protein-coupled receptors constitute still the most densely populated proteinfamily encompassing numerous disease-relevant drug targets. Consequently, medicinal chemistry is expected to pursue targets from that protein family in that hits need to be generated and subsequently optimized towards viable clinical candidates for a variety of therapeutic areas. For the purpose of rationalizing structure-activity relationships within such optimization programs, structural information derived from the ligand's as well as the macromolecule's perspective is essential. While it is relatively straightforward to define pharmacophore hypotheses based on comparative modelling of structurally and biologically characterized low-molecular weight ligands, a deeper understanding of the molecular recognition event underlying, remains challenging, since the principally available amount of experimentally derived structural data on GPCRs is extremely scarse when compared to, e.g., soluble enzymes. In this context, the protein modelling methodologies introduced, developed, optimized, and applied in this thesis provide structural models that are capable of assisting in the development of structural hypotheses on ligand-receptor complexes. As such they provide a valuable structural framework not only for a more detailed insight into ligand-GPCR interaction, but also for guiding the design process towards next-generation compounds which should display enhanced affinity. The model building procedure developed in this thesis systematically follows a hierarchical approach, sequentially generating a 1D topology, followed by a 2D topology that is finally converted into a 3D topology. The determination of a 1D topology is based on a compartmentalization of the linear amino acid sequence of a GPCR of interest into the extracellular, intracellular, and transmembrane sequence stretches. The entire chapter 3 of this study elaborates on the strengths and weaknesses of applying automated prediction tools for the purpose of identifying the transmembrane sequence domains. Based on an once derived 1D topology, a type of in-plane projection structure for the seven transmembrane helices can be derived with the aide of calculated vectorial property moments, yielding the 2D topology. Thorough bioinformatics studies revealed that only a consensus approach based on a conceptual combination of different methods employing a carefully made selection of parameter sets gave reliable results, emphasizing the danger to fully automate a GPCR modelling procedure. Chapter 4 describes a procedure to further expand the 2D topological findings into 3D space, exemplified on the human CCK-B receptor protein. This particular GPCR was chosen as the receptor of interest, since an enormous experimentally derived and structurally relevant data-set was available. Within the computational refinement procedure of constructed GPCR models, major emphasis was laid on the explicit treatment of a non-isotropic solvent environment during molecular mechanics (i.e. energy minimization and molecular dynamics simulations) calculations. The majority of simulations was therefore carried out in a tri-phasic solvent box accounting for a central lipid environment, flanked by two aqueous compartments, mimicking the extracellular and cytoplasmic space. Chapter 5 introduces the reference compound set, comprising low-molecular weight compounds modulating CCK receptors, that was used for validation purposes of the generated models of the receptor protein. Chapter 6 describes how the generated model of the CCK-B receptor was subjected to intensive docking studies employing compound series introduced in chapter 5. It turned out that by applying the DRAGHOME methodology viable structural hypotheses on putative receptor-ligand complexes could be generated. Based on the methodology pursued in this thesis a detailed model of the receptor binding site could be devised that accounts for known structure-activity relationships as well as for results obtained by site-directed mutagenesis studies in a qualitative manner. The overall study presented in this thesis is primarily aimed to deliver a feasibility study on generating model structures of GPCRs by a conceptual combination of tailor-made bioinformatics techniques with the toolbox of protein modelling, exemplified on the human CCK-B receptor. The generated structures should be envisioned as models only, not necessarily providing a detailed image of reality. However, consistent models, when verified and refined against experimental data, deliver an extremely useful structural contextual platform on which different scientific disciplines such as medicinal chemistry, molecular biology, and biophysics can effectively communicate

G protein-coupled receptor allosterism and complexing

G protein-coupled receptors (GPCRs) represent the largest family of cell-surface receptors. These receptors are natural allosteric proteins because agonist-mediated signaling by GPCRs requires a conformational change in the receptor protein transmitted between two topographically distinct binding sites, one for the agonist and another for the G protein. It is now becoming increasingly recognized, however, that the agonist-bound GPCR can also form ternary complexes with other ligands or "accessory" proteins and display altered binding and/or signaling properties in relation to the binary agonist-receptor complex. Allosteric sites on GPCRs represent novel drug targets because allosteric modulators possess a number of theoretical advantages over classic orthosteric ligands, such as a ceiling level to the allosteric effect and a potential for greater GPCR subtype-selectivity. Because of the noncompetitive nature of allosteric phenomena, the detection and quantification of such effects often relies on a combination of equilibrium binding, nonequilibrium kinetic, and functional signaling assays. This review discusses the development and properties of allosteric receptor models for GPCRs and the detection and quantification of allosteric effects. Moreover, we provide an overview of the current knowledge regarding the location of possible allosteric sites on GPCRs and candidate endogenous allosteric modulators. Finally, we discuss the potential for allosteric effects arising from the formation of GPCR oligomers or GPCRs complexed with accessory cellular proteins. It is proposed that the study of allosteric phenomena will become of progressively greater import to the drug discovery process due to the advent of newer and more sensitive GPCR screening technologies

The structure and function of the human ghrelin receptor

The peptide hormone, ghrelin, exerts its physiological effects through a G-protein-coupled receptor called the ghrelin-R. The ghrelin-R displays a high degree of constitutive activity, signalling through the inositol phosphate pathway in the absence of bound agonist. TMs III and VI have been reported to be central to the activation of Family A GPCRs, with interactions between the two helices stabilising the ground state. During activation conformational rearrangements result in these interactions being broken, with new contacts forming and stabilising the active state. Investigation of the ghrelin-R constitutive activity gives an insight into the mechanisms involved in receptor activation. In this study the role of specific individual residues in the ghrelin-R has been investigated and the effect of disrupting or introducing intramolecular interactions was addressed. Site-directed mutagenesis and functional assays revealed that ghrelin-R constitutive activity can be increased and decreased with mutation of residues within the TM domains, specifically TMs III, VI and VII. The extracellular loops have been found to be involved in ligand binding and activation in a number of Family A GPCRs. The residues within ECL2 of the ghrelin-R were systematically mutated to alanine to determine their role. In particular, one residue, Asn196, was identified as being critical in ghrelin-R function and may be forming stabilising interactions which maintain ghrelin-R constitutive activity. The data presented in this thesis provide an insight into the structure and function of the ghrelin-R and the underlying molecular mechanisms of ghrelin-R constitutive activity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Translational Studies on Inflammation

Inflammation is known worldwide, from the bench to the bedside, but it is a hard theme to approach with one single point of view.In this sense, a selection of translational studies would support the medical-scientific community to better understand the complex network of the inflammatory process, its maintenance, and potential treatment targets. The eleven chapters that compose this book present interesting insights into inflammation and its mechanisms, merging classic background with innovative approaches. From the molecular basis to experimental models, the chapters selected for this book bring to readers at different academic levels updated and practical data on inflammation. Find out what drives interdisciplinary medical research on inflammation and enjoy this informative collection

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease