New insights into the stereochemical requirements of the bradykinin B2 receptor antagonists binding

Bradykinin (BK) is a member of the kinin family, released in response to inflammation, trauma, burns, shock, allergy and some cardiovascular diseases, provoking vasodilatation and increased vascular permeability among other effects. Their actions are mediated through at least two G-protein coupled receptors, B1 a receptor up-regulated during inflammation episodes or tissue trauma and B2 that is constitutively expressed in a variety of cell types. The goal of the present work is to carry out a structure–activity study of BK B2 antagonism, taking into account the stereochemical features of diverse non-peptide antagonists and the way these features translate into ligand anchoring points to complementary regions of the receptor, through the analysis of the respective ligand-receptor complex. For this purpose an atomistic model of the BK B2 receptor was built by homology modeling and subsequently refined embedded in a lipid bilayer by means of a 600 ns molecular dynamics trajectory. The average structure from the last hundred nanoseconds of the molecular dynamics trajectory was energy minimized and used as model of the receptor for docking studies. For this purpose, a set of compounds with antagonistic profile, covering maximal diversity were selected from the literature. Specifically, the set of compounds include Fasitibant, FR173657, Anatibant, WIN64338, Bradyzide, CHEMBL442294, and JSM10292. Molecules were docked into the BK B2 receptor model and the corresponding complexes analyzed to understand ligand-receptor interactions. The outcome of this study is summarized in a 3D pharmacophore that explains the observed structure–activity results and provides insight into the design of novel molecules with antagonistic profile. To prove the validity of the pharmacophore hypothesized a virtual screening process was also carried out. The pharmacophore was used as query to identify new hits using diverse databases of molecules. The results of this study revealed a set of new hits with structures not connected to the molecules used for pharmacophore development. A few of these structures were purchased and tested. The results of the binding studies show about a 33 % success rate with a correlation between the number of pharmacophore points fulfilled and their antagonistic potency. Some of these structures are disclosed in the present work.Peer ReviewedPostprint (author's final draft

Discovery of new selective antagonists of G-protein coupled receptors of therapeutic interest

GPCR are integral membrane receptor proteins that are characterized by heptahelical transmembrane domains connected by intracellular and extracellular loops. GPCRs are an attractive class of proteins for drug discovery, with more than 50% of all drugs regulating GPCR function, and some 30% of these drugs directly target GPCRs. Despite the number of GPCR crystal structures determined recently, they only represent a small fraction of total number of GPCRs known. Homology modelling has been the methodology used to fill the gap. However, the low sequence similarity between targets and templates hampers these studies. Aimed at overcoming these drawbacks template selection and the refinement process were studied in this work. Thus, several atomistic models of rat M3 muscarinic receptor were constructed from human M2 muscarinic receptor, human histamine 1 receptor and bovine rhodopsin receptor as templates. Moreover, in order to determine the effect of ligand in the simulation system, an extra model of M2 receptor was refined with NMS bound inside and an extra model refined without ligand. Results show the sampling time 500ns is adequate simulation time and molecular dynamics simulation of the protein embedded in a lipid bilayer as a refinement process improves on the homology models. Specifically, the refinement process can correct the length of the TM segment of the target receptor; the accuracy of the model greatly depends on the proximity of the template and the target in the phylogenetic tree and finally, the presence of a ligand produces a faster equilibration of the system. This methodology was used to study the pharmacological profile of bradykinin receptors B1 and B2. The B1 receptor was constructed using the chemokine CXC4 and bovine rhodopsin receptors as templates. Antagonists selected for the docking studies include Compound 11, Compound 12, Chroman28, SSR240612, NPV-SAA164 and PS020990. Analysis of the ligand-receptor complexes permitted the definition of a pharmacophore that describes the stereochemical requirements of antagonist binding. For the B2 receptor, a similar procedure was followed using the same template. In this case, the set of compounds used were Fasitibant, FR173657, Anatibant, WIN64338, Bradyzide, CHEMBL442294, and JSM10292. The outcome of this study is summarized in a 3D pharmacophore that explains the observed structure-activity results and provides insight into the design of novel molecules with antagonistic profile. To prove the validity of the pharmacophoric hypotheses, a virtual screening process was carried out. The results of the binding studies show about a 33% success rate with a correlation between the number of pharmacophore points fulfilled and their antagonistic potency. Some of these structures are disclosed in this thesis. Moreover, the B1R and B2R pharmacophores developed were compared and the observed differences permitted to explain the stereochemical requirements for receptor-selective ligands. The final study of this study was to establish a rational explanation for the role of zinc in preventing the dimerization of the serotonin 5-Hydroxytryptamine 1A receptor (5-HT1A) and Galanin receptor 1 (GALR1) involved in depression. Homology modeling was used to build atomistic models of these receptors using the crystallographic structures of 5-HT1B and κ– opioid receptor, respectively. First, prospective zinc binding sites were identified for the 5-HT1A using a molecular probe. Second, heterodimers of the two receptors were constructed with different interfaces: TM4 and TM5; TM6 and TM7; TM1 and TM2. Analysis of the 12 zinc-binding sites and the heterodimer interfaces suggests that there is a coincidence between zinc binding sites and heterodimerization interfaces providing a rational explanation for the role of zinc in the molecular processes associated with heterodimer preventionLos receptores acoplados a proteínas G (GPCRs) son proteínas de membrana que se caracterizan por dominios transmembrana heptahelicoidales conectados por lazos intracelulares y extracelulares. GPCRs son un atractivo grupo de proteínas para el descubrimiento de nuevos fármacos puesto que más del 50% de los medicamentos en el mercado que regulan su función y alrededor del 30% que tienen un GPCR como diana. A pesar del gran número de estructuras cristalográficas de GPCRs que se han determinado recientemente, estas solamente representan una pequeña fracción del número total de GPCRs. La homología de secuencia se utiliza de forma rutinaria para llenar el vacío, sin embargo, la baja identidad de secuencia entre miembros de esta familia obstaculiza estos estudios. Con el objetivo de superar estos inconvenientes, tanto el proceso de selección de la plantilla, como el proceso de refinamiento del modelo han sido estudiados en este trabajo. Se construyeron modelos atómicos del receptor muscarínico M3 de rata a partir del receptor humano M2 muscarínico, del de histamina humano 1 y de la rodopsina bovina como plantilla. Por otra parte, con el fin de determinar el efecto del ligando en el proceso de refinamiento, el receptor M2 fue refinado con el ligando NMS y además se construyó un modelo sin ligando. Los resultados muestran que un tiempo de muestreo 500ns es adecuado y que la dinámica molecular representa un proceso de refinamiento adecuado. Esta metodología se utilizó para estudiar el perfil farmacológico de los receptores de bradiquinina B1 y B2. El receptor B1 se construyó usando los receptores CXC4 de quimoquina y rodopsina bovina como plantillas. Los antagonistas seleccionados para los estudios de anclaje incluyen el Compuesto 11, el Compuesto 12, Chroman28, SSR240612, NVP-SAA164 y PS020990. El análisis de los complejos ligando-receptor permite la definición de un farmacóforo que describe los requisitos estereoquímicos de unión de antagonistas. Para el receptor B2, se siguió un procedimiento similar utilizando las mismas plantillas. En este caso, el conjunto de los compuestos utilizados fueron Fasitibant, FR173657, Anatibant, WIN64338, Bradyzide, CHEMBL442294 y JSM10292. El resultado de este estudio se resume en un farmacóforo 3D que explica los resultados estructura-actividad observados y ofrece información sobre el diseño de nuevas moléculas con el perfil antagonista. Para probar la validez de las hipótesis farmacofóricas, se llevó a cabo un proceso de cribado virtual. Los resultados de los estudios de unión muestran sobre una tasa de éxito del 33% con una correlación entre el número de puntos farmacóforicos cumplido y su potencia antagonista. Algunas de estas estructuras se describen en esta tesis. Por otra parte, los farmacóforos de B1R y B2R desarrollados se compararon y a través de las diferencias observadas explicar los requisitos estereoquımicos para que los ligandos sean selectivos. El estudio final de este trabajo fue el establecer una explicación racional para el papel del zinc en la prevención de la dimerización del receptor de serotonina 5-hidroxitriptamina 1A (5-HT1A) y el receptor galanina 1 (GALR1) que participan en la depresión. Homología de secuencia se utilizó para construir modelos atómicos de estos receptores utilizando las estructuras cristalográficas de los receptores 5-HT 1B y κ de opiáceos, respectivamente. En primer lugar, se identificaron los posibles sitios de unión de zinc para el 5-HT1A usando una sonda molecular. En segundo lugar, los heterodímeros de los dos receptores fueron construidos con diferentes interfaces: TM4 y TM5; TM6 y TM7; TM1 y TM2. El análisis de los 12 sitios de unión de zinc y las interfaces heterodímero sugiere que existe una coincidencia entre los sitios de unión de zinc y las interfaces de heterodimerización que proporcionan una explicación racional para el papel del zinc en los procesos moleculares asociados con la prevención heterodímero.Postprint (published version

Bifunctional ligands of the bradykinin B2 and B1 receptors : an exercise in peptide hormone plasticity

Kinins are the small and fragile hydrophilic peptides related to bradykinin (BK) and derived from circulating kininogens via the action of kallikreins. Kinins bind to the preformed and widely distributed B2 receptor (B2R) and to the inducible B1 receptor (B1R). B2Rs and B1Rs are related G protein coupled receptors that possess natural agonist ligands of nanomolar affinity (BK and Lys BK for B2Rs, Lys-des-Arg9-BK for B1R). Decades of structure-activity exploration have resulted in the production of peptide analogs that are antagonists, one of which is clinically used (the B2R antagonist icatibant), and also non-peptide ligands for both receptor subtypes. The modification of kinin receptor ligands has made them resistant to extracellular or endosomal peptidases and/or produced bifunctional ligands, defined as agonist or antagonist peptide ligands conjugated with a chemical fluorophore (emitting in the whole spectrum, from the infrared to the ultraviolet), a drug-like moiety, an epitope, an isotope chelator/carrier, a cleavable sequence (thus forming a pro-drug) and even a fused protein. Dual molecular targets for specific modified peptides may be a source of side effects or of medically exploitable benefits. Biotechnological protein ligands for either receptor subtype have been produced: they are enhanced green fluorescent protein or the engineered peroxidase APEX2 fused to an agonist kinin sequence at their C-terminal terminus. Antibodies endowed with pharmacological actions (agonist, antagonist) at B2R have been reported, though not monoclonal antibodies. These findings define classes of alternative ligands of the kinin receptor of potential therapeutic and diagnostic value

Mind the Gap - Deciphering GPCR Pharmacology Using 3D Pharmacophores and Artificial Intelligence

G protein-coupled receptors (GPCRs) are amongst the most pharmaceutically relevant and well-studied protein targets, yet unanswered questions in the field leave significant gaps in our understanding of their nuanced structure and function. Three-dimensional pharmacophore models are powerful computational tools in in silico drug discovery, presenting myriad opportunities for the integration of GPCR structural biology and cheminformatics. This review highlights success stories in the application of 3D pharmacophore modeling to de novo drug design, the discovery of biased and allosteric ligands, scaffold hopping, QSAR analysis, hit-to-lead optimization, GPCR de-orphanization, mechanistic understanding of GPCR pharmacology and the elucidation of ligand–receptor interactions. Furthermore, advances in the incorporation of dynamics and machine learning are highlighted. The review will analyze challenges in the field of GPCR drug discovery, detailing how 3D pharmacophore modeling can be used to address them. Finally, we will present opportunities afforded by 3D pharmacophore modeling in the advancement of our understanding and targeting of GPCRs

Formation of Multiple Dimer Interfaces in the Active and Inactive States of a Model G Protein-Coupled Receptor

G protein-coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by seven-transmembrane (7TM) domains connected by intracellular and extracellular loops, an extracellular N-terminus, and an intracellular Cterminus. GPCRs recognize neurotransmitters, sensory molecules and chemotactic agents and are involved in the control of many aspects of metabolism. Since GPCRs play important roles in diverse processes such as pain perception, growth and blood pressure regulation, and viral pathogenesis, GPCRs became important target for therapeutic agents. The tridecapeptide α-factor pheromone (W1H2W3L4Q5L6K7P8G9Q10P11M12Y13) of Saccharomyces cerevisiae and Ste2p, its cognate GPCR, have been used extensively as a model for peptide ligand-GPCR structure and function. The power of yeast genetics has been used to examine the structure and function of Ste2p. Recently, GPCR homodimerization has been demonstrated for many GPCRs, although the role(s) of dimerization in receptor function is disputed. In this dissertation, Ste2p has been used to investigate GPCR dimerization. Part I of this dissertation is an overview of the GPCR structure and its ligandinduced conformational change with specific emphasis on the peptide pheromone α- factor and its receptor Ste2p. Part II of this dissertation is a study originally designed to probe inter-helical interaction between TM1 and TM7 of Ste2p. Site-directed mutagenesis and cysteine cross-linking with targeted residues of Ste2p were carried out. Although the anticipated inter-helical interactions were not identified from this study, the results provided strong evidence for Ste2p dimerization. Part III of this dissertation describes dimer interfaces including TM1 and TM7 of Ste2p. By using the disulfide cross-linking methodology, we studied the participation of specific residues at the intracellular boundary between TM1 and intracellular loop one and the entire TM7 in Ste2p dimerization. The final part of this dissertation contains a study of the participation of the Ste2p N-terminus in homo-dimer formation and the effect of ligand binding on this interaction. This part also includes overall conclusions and suggestions for future experiments that could contribute to an understanding of the dimer interfaces in Ste2p and the role of dimerization in the function of this receptor

Identification of Ligand-Receptor Interactions Between Saccharomyces cerevisiae α-factor Pheromone Receptor (Ste2p) and its Tridecapeptide Ligand

G protein-coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7TM) configuration. These receptors comprise a large and diverse gene family found in fungi, plants, and the animal kingdom. Recent studies with GPCRs have begun to elucidate their importance in many physiological processes, thus various human diseases are associated with GPCR pathology. Although the overall 3D structure of these receptors carry similar features, binding of an extraordinarily diverse array of ligands trigger many different biological pathways. The α-factor receptor (Ste2p) of Saccharomyces cerevisiae belongs to the GPCR family. Upon the α-factor binding to Ste2p, a signal is transduced via an associated guanine-nucleotide binding protein initiating a cascade of events that leads to the mating of haploid yeast cells. As only two GPCRs and two G proteins are encoded in the S. cerevisiae genome, this yeast presents a relatively simple system to study GPCR signal transduction in comparison to mammalian cells that possess hundreds of GPCRs and tens of G proteins. Part I of this dissertation is an overview of GPCRs in general with specific emphasis on the peptide pheromone α-factor and its receptorSte2p. Part II of this dissertation details the design and characterization of a number ofiodinatable α-factor pheromone analogs containing the photo-cross-linkable 4-benzoyl-Lphenylalanine (Bpa) group. One of these analogs [Bpa1, Y3, R7, Nle12, F13] was radioiodinated for detection and used as a probe for cross-linking studies with Ste2p. Chemical (with CNBr & BNPS-skatole) and enzymatic (with Trypsin) cleavage of the receptor/analog complex after the cross-linking was examined to determine the interaction between the α-factor probe and a fragment of the receptor. Data from these digestions indicated that the position one of the α-factor interacts with residues 251 to 294 in the receptor. Similarly Part III of this dissertation describes the design and synthesis of five photoactivatable α-factor analogs that carry Bpa at positions one, three, five, eight, or thirteen. All of these analogs were biotinylated at the ε-amine of the Lys7 for detection and purification purposes. The biological activity (growth arrest assay) and binding affinities of all analogs for Ste2p were determined. Two of the analogs tested, Bpa1 and Bpa5, showed three- to four-fold lower affinity compared to α-factor, whereas Bpa3 and Bpa13 had seven- to twelve-fold lower affinities, respectively. Bpa8 competed poorly with [3H]α-factor for Ste2p. All of the analogs tested had detectable halos in the growth arrest assay indicating that these analogs are α-factor agonists. Cross-linking studies demonstrated that [Bpa1]α-factor, [Bpa3]α-factor, [Bpa5]α-factor and [Bpa13]α-factor were cross-linked to Ste2p; the biotin tag on the pheromone was detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa1, Bpa3, and Bpa13 cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of the pheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5, TM6, and TM7 and portions of EL2 and EL3 close to these TMs. Additionally it was concluded that there is a direct interaction between the position 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. Parts II and III of this dissertation indicate that Bpa-containing α-factor probes are useful in determining contacts between α-factor and Ste2p and initiating mapping of the ligand binding site of the GPCR for its peptide ligand. This dissertation (Part IV) also presents the application of different purification methods and the use of two mass spectrometry instruments for identification of ligandreceptor interactions at the molecular level. Results presented in this part showed that although a single step purification was enough for western blot analyses of the cross-linked receptor fragments, at least a two-step purification and enrichment of the biotinylated peptide fragments were necessary for mass spectrometric studies. MALDITOF experiments showed that the affinity purification of the biotinylated fragments by monomeric avidin beads was successful. Data obtained from CNBr fragments of Bpa1 cross-linked membranes were in agreement with the previous results discussed in Parts II and III of this dissertation suggesting the cross-linking between position one of α-factor and a region of Ste2p covering residues 251 to 294. This part also illustrated that the analyses of the MS/MS data from the cross-linked fragments were more complex than the fragmentation data obtained from biotinylated α-factor; the presence of multiple charge states of fragment ions and unusual fragmentation of branched peptides indicated the necessity of using an instrument with higher resolution. In addition, analyses of the MS/MS data with a customized algorithm would be required to deconvolute the sequence of the cross-linked fragment(s) to identify the cross-linked residue(s) on Ste2p. The final part of this dissertation reviews the overall conclusions and discussion. This part also contains suggestions for future experiments that could help identification of contact points between Ste2p and its peptide ligand α-factor. Additional studies on this GPCR system employing high-resolution mass spectral characterization of fragments should allow identification of residue-to-residue interactions between the analogs used in this study and Ste2p. Such information will aid the mapping of the ligand-binding site of the pheromone receptor and has the potential to provide key insights into peptide ligand mediated activation of GPCRs. This and similar studies may ultimately lead to the discovery of how peptide ligands initiate signal transduction through GPCRs

Designing Peptidomimetics

The concept of a peptidomimetic was coined about forty years ago. Since then, an enormous effort and interest has been devoted to mimic the properties of peptides with small molecules or pseudopeptides. The present report aims to review different approaches described in the past to succeed in this goal. Basically, there are two different approaches to design peptidomimetics: a medicinal chemistry approach, where parts of the peptide are successively replaced by non-peptide moieties until getting a non-peptide molecule and a biophysical approach, where a hypothesis of the bioactive form of the peptide is sketched and peptidomimetics are designed based on hanging the appropriate chemical moieties on diverse scaffolds. Although both approaches have been used in the past, the former has been more widely used to design peptidomimetics of secretory peptides, whereas the latter is nowadays getting momentum with the recent interest in designing protein-protein interaction inhibitors. The present report summarizes the relevance of the information gathered from structure-activity studies, together with a short review on the strategies used to design new peptide analogs and surrogates. In a following section there is a short discussion on the characterization of the bioactive conformation of a peptide, to continue describing the process of designing conformationally constrained analogs producing first and second generation peptidomimetics. Finally, there is a section devoted to review the use of organic scaffolds to design peptidomimetics based on the information available on the bioactive conformation of the peptide.Postprint (author's final draft

The effects of opioids on the peripheral terminals of rat and guinea pig sensory neurons

Studies in vivo and in vitro suggest that opioids can modulate nociceptive signals by interacting with receptors on peripheral neurons. We investigated the peripheral actions of mu (μ), delta (δ), and kappa (κ) opioid agonists using an electrophysiological model of inflammatory-type nociception. Dorsal horn convergent neurons were recorded extracellularly in the halothane anesthetized intact adult rat. Subcutaneous injection of formalin into the hindpaw receptive field of these neurons results in two distinct phases of cell firing. Neither morphine, exogenous ligand for the μ receptor, nor the δ agonist Tyr-D-Ser-(tbu)-Gly-Phe-Leu-Thr (DSTBULET) influenced the formalin response when administered peripherally into the paw. The κ-selective ligand U50488H produced a dose-dependent, naloxone-reversible inhibition of both phases of formalin-induced activity which does not result from leakage of the drug into the systemic circulation. Intrathecal administration of μ and δ, but not κ, opioids has previously been shown to inhibit the biphasic formalin response in the adult rat. Our data suggest that different types of opioid receptors may be important in the periphery and spinal cord. There is some indication that opioid receptor populations are different in adult and neonatal rat spinal cord. For example there are functional μ and κ, but not δ, opioid receptors in an in vitro model of nociceptive activity in the neonatal rat spinal cord. We looked at these apparent developmental differences in binding assays in which opioid receptors in the two tissues were characterized by measurements of ligand binding to crude membrane fractions. Results from binding studies agreed well with fictional studies, in that δ opioid binding sites were not detected on neonatal rat spinal cord membranes. Levels of κ binding were higher in the neonate than in the adult. Novel continuous clonal cell lines with some characteristics of nociceptive dorsal root ganglion (DRG) neurons were tested as a potential model system for the action of opioids on primary afferent nerve fibers. Two of the cell lines expressed δ, but not μ or κ, opioid binding sites. We could not detect effects of δ opioids on potassium currents (as measured by 86Rubidium efflux) or on the release of substance P-like immunoreactivity (SP-LI). We concluded that these cell lines were not good models for studying opioid action on sensory neurons. Measurement of SP-LI release from guinea pig cardiac right ventricular slices did provide a useful model to study peripheral actions of opioids. Formalin (0.2%), capsaicin (100 nM-3μM), and a depolarizing concentration of potassium (100 mM K+) increased the outflow of SP-LI from heart slices. Agonists at μ, δ, and κ opioid receptors inhibited K+-stimulated release and these effects were reversed by naloxone to differing degrees. High concentrations of μ and κ ligands, in their own right, increased the outflow of SP-LI, and these results are compared to previous reports of opioid excitation. Formalin-evoked SP-LI release from heart slices was subject to modulation by opioids. These results agreed well with in vivo results, in that SP-LI release evoked by formalin was not inhibited by μ or δ opioid agonists, but was sensitive to blockade by the κ ligand U50488H. We have demonstrated effects of opioids on the peripheral terminals of sensory neurons in two different models. Peripheral κ, but not μ or δ, receptors were important in modulating formalin-induced effects both in vivo and in vitro. Central κ receptors, at least in adult rats, have been shown not to influence the formalin response in vivo to the same extent as μ and δ receptors. In vitro μ, δ, and κ opioids modulated responses to K+ depolarization of sensory neurons at the peripheral terminals. These results provide strong evidence that peripheral opioid receptors can modulate nociceptive signals

A Review of the Updated Pharmacophore for the Alpha 5 GABA(A) Benzodiazepine Receptor Model

An updated model of the GABA(A) benzodiazepine receptor pharmacophore of the alpha 5-BzR/GABA(A) subtype has been constructed prompted by the synthesis of subtype selective ligands in light of the recent developments in both ligand synthesis, behavioral studies, and molecular modeling studies of the binding site itself. A number of BzR/GABA(A) alpha 5 subtype selective compounds were synthesized, notably alpha 5-subtype selective inverse agonist PWZ-029 (1) which is active in enhancing cognition in both rodents and primates. In addition, a chiral positive allosteric modulator (PAM), SH-053-2'F-R-CH3 (2), has been shown to reverse the deleterious effects in the MAM-model of schizophrenia as well as alleviate constriction in airway smooth muscle. Presented here is an updated model of the pharmacophore for alpha 5 beta 2 gamma 2 Bz/GABA(A) receptors, including a rendering of PWZ-029 docked within the alpha 5-binding pocket showing specific interactions of the molecule with the receptor. Differences in the included volume as compared to alpha 1 beta 2 gamma 2, alpha 2 beta 2 gamma 2, and alpha 3 beta 2 gamma 2 will be illustrated for clarity. These new models enhance the ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists at the Bz BS of GABA(A) receptors

Treatment with opioid antagonists and mTOR inhibitors

Embodiments of the invention provide methods of treating a disorder or disease characterized by cellular proliferation and migration by co-administering a synergistically effective amount of an mTOR inhibitor and a μ-opioid receptor antagonist