Using molecular dynamics for the refinement of atomistic models of GPCRs by homology modeling

Despite GPCRs sharing a common seven helix bundle, analysis of the diverse crystallographic structures available reveal specific features that might be relevant for ligand design. Despite the number of crystallographic structures of GPCRs steadily increasing, there are still challenges that hamper the availability of new structures. In the absence of a crystallographic structure, homology modeling remains one of the important techniques for constructing 3D models of proteins. In the present study we investigated the use of molecular dynamics simulations for the refinement of GPCRs models constructed by homology modeling. Specifically, we investigated the relevance of template selection, ligand inclusion as well as the length of the simulation on the quality of the GPCRs models constructed. For this purpose we chose the crystallographic structure of the rat muscarinic M3 receptor as reference and constructed diverse atomistic models by homology modeling, using different templates. Specifically, templates used in the present work include the human muscarinic M2; the more distant human histamine H1 and the even more distant bovine rhodopsin as shown in the GPCRs phylogenetic tree. We also investigated the use or not of a ligand in the refinement process. Hence, we conducted the refinement process of the M3 model using the M2 muscarinic as template with tiotropium or NMS docked in the orthosteric site and compared with the results obtained with a model refined without any ligand bound.Peer ReviewedPostprint (author's final draft

Computational Algorithms for Predicting Membrane Protein Assembly From Angstrom to Micron Scale

Biological barriers in the human body are one of the most crucial interfaces perfected through evolution for diverse and unique functions. Of the wide range of barriers, the paracellular protein interfaces of epithelial and endothelial cells called tight junctions with high molecular specificities are vital for homeostasis and to maintain proper health. While the breakdown of these barriers is associated with serious pathological consequences, their intact presence also poses a challenge to effective delivery of therapeutic drugs. Complimenting a rigorous combination of in vitro and in vivo approaches to establishing the fundamental biological construct, in addition to elucidating pathological implications and pharmaceutical interests, a systematic in silico approach is undertaken in this work in order to complete the molecular puzzle of the tight junctions. This work presents a bottom-up approach involving a careful consideration of protein interactions with Angstrom-level details integrated systematically, based on the principles of statistical thermodynamics and probabilities and designed using well-structured computational algorithms, up to micron-level molecular architecture of tight junctions, forming a robust prediction with molecular details packed for up to four orders of magnitude in length scale. This work is intended to bridge the gap between the computational nano-scale studies and the experimental micron-scale observations and provide a molecular explanation for cellular behaviors in the maintenance, and the adverse consequences of breakdown of these barriers. Furthermore, a comprehensive understanding of tight junctions shall enable development of safe strategies for enhanced delivery of therapeutics

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

Effect of the cosolutes trehalose and methanol on the equilibrium and phase-transition properties of glycerol-monopalmitate lipid bilayers investigated using molecular dynamics simulations

The influence of the cosolutes trehalose and methanol on the structural, dynamic and thermodynamic properties of a glycerol-1-monopalmitate (GMP) bilayer and on its main transition temperature $$T_m$$ T m is investigated using atomistic molecular dynamics simulations (600 ns) of a GMP bilayer patch (2×8×8 lipids) at different temperatures in the range of 302 to 338 K and considering three different cosolute concentrations. Depending on the environment and temperature, these simulations present no or a single GL $$\rightarrow$$ → LC, LC $$\rightarrow$$ → GL or LC $$\rightarrow$$ → ID transition, where LC, GL and ID are the liquid crystal, gel and interdigitated phases, respectively. The trehalose molecules form a coating layer at the bilayer surface, promote the hydrogen-bonded bridging of the lipid headgroups, preserve the interaction of the headgroups with trapped water and induce a slight lateral expansion of the bilayer in the LC phase, observations that may have implications for the phenomenon of anhydrobiosis. However, this cosolute does not affect $$T_m$$ T m and its dependence on hydration in the concentration range considered. On the other hand, methanol molecules intercalate between the lipid headgroups, promote a lateral expansion of the bilayer in the LC phase and induce a concentration dependent decrease of $$T_m$$ T m , observations that may have implications for the phenomenon of anesthesia. The occurrence of an ID phase in the presence of this cosolute may be viewed as an extreme consequence of lateral expansion. The analysis of the simulations also suggests the existence of two basic conservation principles: (1) the hydrogen-bond saturation principle rests on the observation that for all species present in the different systems, the total numbers of hydrogen-bonds per molecule is essentially constant, the only factor of variability being their distribution among different partners; (2) the densest packing principle rests on the observation that the effective volume per methylene group in the interior of the bilayer is only weakly sensitive to the environment, with values comparable to those for liquid (LC) and solid (ID) alkanes, or intermediate (GL)

Exploiting Photoisomerization: Spectroscopy on a Carotenoid Sensor and Retinal Proteins

Light-based methodologies enjoy popularity due to their non-invasive nature. In particular in the field of optogenetics, where genetic targeting of neurons permits not only simultaneous imaging of a large number of cells but also optical control of neuronal activity. For this, ion channels or pumps are inserted into the membrane which are activated by light. A deep biophysical understanding of the optogenetic systems is key for their successful application. In this thesis, I present a new member in the family of organic voltage sensors. I demonstrate that in a single lipid bilayer environment, the carotenoid Zeaxanthin has a linear and reversible spectral Raman response to an electric field applied across the membrane. The underlying mechanism is an increased photoisomerization rate resulting in a higher 13-cis population which is detected via a characteristic vibrational band at 1130 cm−1. Channelrhodopsin-2 (ChR2) is a frequently used protein in optogenetics to silence neuronal activity. By variation of amino acid side chains, we found experimental evidence for ground-state heterogeneity in the hydrogen bond interactions of the retinal protonated Schiff base (PSB). We have identified with Raman spectroscopy two spectral components of the C=N–H mode of the PSB at 1661 and 1665 cm−1, representing hydrogen bonds to different amino acid side chains. These two interactions of the PSB could be essential for a voltage-sensing mechanism in ChR2. In a pioneering approach we combined time-resolved absorption spectroscopy with serial femtosecond X-ray crystallography to scrutinize mechanistic details of sodium pumping in Krokinobacter eikastus rhodopsin 2 (KR2). Using an infrared-emitting quantum cascade laser (QCL), we verified that crystalline KR2 exhibits reaction kinetics similar to those observed in its detergent solubilized form. Hereupon, we have identified a previously proposed transient sodium binding site during the O intermediate where the sodium is coordinated by the amino acid side chains of N112 and D251. The findings regarding the ion transport mechanism in KR2 will facilitate the design of protein variants for an optogenetic application. Bistable G-protein coupled receptors (GPCRs) have two thermally stable conformations and are a promising class of rhodopsins which have the potential to serve as an optogenetic switch. We were able to conduct a first biophysical characterization of the invertebrate jumping spider rhodopsin-1 (JSR1). We propose a model of the two-photon reaction based on spectroscopic results. During these reactions, the Schiff base stays protonated implying that a deprotonation is not a prerequisite for the function of bistable GPCRs. A proposed mediating water molecule as part of the counterion complex in the inactive conformation is identified by Raman spectroscopy and later confirmed by an X-ray crystallographic structure. In conclusion, this thesis provides insights into the mechanistic details of established and upcoming optogenetic tools. These results will help to adapt their biophysical properties better suiting the needs of application.Lichtbasierende Methoden erfreuen sich aufgrund ihrer nicht-invasiven Eigenschaft großer Beliebtheit. Im Besonderen in der Optogenetik, wo Neuronen genetisch modifiziert werden um nicht nur die simultane Beobachtung einer großen Anzahl von Neuronen, sondern auch optische Kontrolle von neuronaler Aktivität zu ermöglichen. Hierzu werden Ionenkanäle oder -pumpen in die Membran gebracht, die durch Licht aktiviert werden können. Ein tiefes Verständnis von optogenetischen Systemen ist eine Schlüsselvoraussetzung für eine erfolgreiche Anwendung. In dieser Arbeit präsentiere ich einen Neuzugang in die Familie der organischen Spannungssensoren. Ich demonstriere, dass das Karotenoid Zeaxanthin, eingebracht in eine einzelne Lipiddoppelschicht, eine lineare und reversible Reaktion zeigt, wenn ein elektrisches Feld über die Membran angelegt wird. Der zugrunde liegende Mechanismus ist eine größere Population an 13-cis Isomeren, hervorgerufen durch eine erhöhte Photoisomerationsrate. Dies führt zu einem Anwachsen einer charakteristischen Vibrationsbande bei 1130 cm−1. Kanalrhodopsin-2 (ChR2) wird regelmäßig in der Optogentik genutzt um neuronale Aktivität zu verhindern. Durch Variation von Aminosäurenseitenketten liefern wir Beweise für eine Heterogenität in der Wasserstoffbrückeninteraktion der protonierten Schiffschen Base (PSB) im Grundzustand. Wir konnten mit Raman Spektroskopie zwei spektrale Komponenten in der PSB Vibrationsmode (C=N–H) bei 1661 und 1665 cm−1 identifizieren, die jeweils eine Wasserstoffbrücke zu einer anderen Aminosäurenseitenkette darstellen. Diese zwei Interaktionen könnten von Bedeutung für einen Spannungsmessungsmechanismus in ChR2 sein. Um den Natriumpumpmechanismus von Krokinobacter eikastus rhodopsin 2 (KR2) zu untersuchen, haben wir in einer Pionierarbeit zeitaufgelöste Absorbtionspektroskopie mit Röntgenkristallographie kombiniert. Die Benutzung eines Quantumkaskadenlasers (QCL) ermöglichte es uns sicher zu stellen, dass kristallines KR2 vergleichbare Reaktionskinetiken aufweist als in Detergens gelost. Wir konnten daraufhin eine im Vorfeld postulierte vorübergehende Natriumbindungsstelle während des O Intermediats zwischen den Seitenketten von N112 und D251 identifizieren. Die Ergebnisse über den Ionentransportmechanismus werden die Konzipierung von Proteinvarianten für eine optogenetische Anwendung erleichtern. Bistabile G-Protein-gekoppelte Rezeptoren (GPCRs) haben zwei thermisch stabile Konformationen und sind eine vielversprechende Klasse von Rhodopsinen für einen optogentischen Schalter. Wir konnten eine erste biophysikalische Charakterisierung von jumping spider rhodopsin-1 (JSR1) durchführen. Wir schlagen, basierend auf spektroskopischen Ergebnissen, ein Model einer zwei-Photonen Reaktion vor. Während dieser Reaktionen bleibt die SB protoniert. Dies impliziert, dass eine Deprotonierung keine Voraussetzung für die Funktion von bistabilen GPCRs ist. Ein angenommenes Wassermolekül als Teil des Konterionennetzwerks konnte mit Raman Spektroskopie detektiert werden, was später durch eine Röntgenstruktur bestätigt wurde. Zusammenfassend bietet diese Arbeit Einblicke in die Mechanismen von etablierten sowie neuen optogenetischen Werkzeugen. Die Resultate werden dazu beitragen, ihre biophysikalischen Eigenschaften an die Erfordernisse der Anwendung anzupassen

Mechanistic insights and in silico studies on selected G protein-coupled receptors implicated in HIV and neurological disorders.

Doctoral Degree. University of KwaZulu-Natal, Durban.G protein-coupled receptors (GPCRs) are the largest membrane protein receptor superfamily involved in a wide range of physiological processes. GPCRs form the major class of drug targets for a diverse array of pathophysiological conditions. Consequently, GPCRs are recognised as drug targets for the treatment of various diseases, including neurological disorders, cardiovascular conditions, oncology, diabetes, and HIV. The recent advancement in GPCR structure resolutions has provided novel avenues to understand their molecular basis of signal transduction, ligand recognition and ligand-receptor interactions. These advances provide a framework for the structure-based discovery of new drugs in targeting GPCRs implicated in the pathogenesis of various human diseases. In this thesis, the interactions of inhibitors at two dopamine receptor subtypes and C-C chemokine receptor 5 (CCR5) of the Class A GPCR family were investigated. Dopamine receptors and CCR5 are validated GPCR targets implicated in neurological disorders and HIV disease, respectively. The lack of structural information on these receptors limited our comprehension of their antagonists’ structural dynamics and binding mechanisms. The recently solved crystal structures for these receptors have necessitated further investigations in their ligand-receptor interactions to obtain novel insights that may assist drug discovery towards these receptors. This thesis comprehensively investigated the binding profiles of atypical antipsychotics (class I and class II) at the first crystal structure of the D2 dopamine receptor (D2DR). The class I antipsychotics exhibited binding poses and dynamics different from the class II antipsychotics with disparate interaction mechanistic at D2DR active site. The class II antipsychotics were remarkably observed to establish a recurrent and vital interaction with Asp114 via strong hydrogen bond interactions. Furthermore, compared to class I antipsychotics, the class II antipsychotics were found to engage favourably with the deep hydrophobic pocket of D2DR. In addition, the structural basis and atomistic binding mechanistic of the preferential selective inhibition at D3DR over D2DR were explored. This study investigated two small molecules (R-VK4-40 and Y-QA31) with substantial selectivity (> 180-fold) for D3DR over D2DR. The selective antagonists adopted shallow binding modes at D3DR while demonstrating a deep hydrophobic pocket binding at D2DR. Also, the vital roles and contribution of critical residues to the selective binding of R-VK4-40 and Y-QA31were identified in D3DR. Structural and binding free energy analyses further discovered distinct stabilising effects of the selective antagonists on the secondary architecture and binding profiles of D3DR relative to D2DR. Furthermore, the atomistic molecular interaction mechanism of how slight structural modification between novel derivatives of 1-heteroaryl-1,3-propanediamine (Compd-21 and - 34) and Maraviroc significantly affects their binding profiles toward CCR5 were elucidated. This study utilised explicit lipid bilayer molecular dynamics (MD) simulations and advanced analyses to explore these inhibitory disparities. The thiophene moiety substitution common to Compd-21 and -34 was found to enhance their CCR5-inhibitory activities due to complementary high-affinity interactions with residues critical for the gp120 V3 loop binding. The study further highlights the structural modifications that may improve inhibitor competitiveness with the gp120 V3 loop. Finally, structure-based virtual screening of antiviral chemical database was performed to identify potential compounds as HIV-1 entry inhibitors targeting CCR5. The identified compounds made pertinent interactions with CCR5 residues critical for the HIV-1 gp120-V3 loop binding. Their predicted in silico physicochemical and pharmacokinetic descriptors were within the acceptable range for drug-likeness. Further structural optimisations and biochemical testing of the proposed compounds may assist in the discovery of novel HIV-1 therapy. The studies presented in this thesis provide novel mechanistic and in silico perspective on the ligand-receptor interactions of GPCRs. The findings highlighted in this thesis may assist in further research towards the identification of novel drug molecules towards CCR5 and D2-like dopamine receptor subtypes.List of thesis publications on page vi-vii. Research Output on page viii-ix

Computational modeling of cationic lipid bilayers in saline solutions

Based on computer simulations performed at single-molecule resolution, the effects of monovalent NaCl salt on cationic DMTAP/DMPC (dimyristoyltrimethylammoniumpropane/dimyristoylphosphatidylcholine) lipid bilayer systems are discussed. The monograph reviews, revises and expands the previously published work on how NaCl affects the structural and electrostatic [1] and the dynamic [2] properties of these systems. The effects of NaCl depended qualitatively on the cationic DMTAP lipid fraction. When the fraction was low, NaCl had a notable effect of the structural properties of the bilayer, decreasing the area per lipid, increasing the tail order, reorienting the DMPC head groups, and increasing the average electrostatic potential difference over the head group region. At high DMTAP fraction there was scarcely an effect when NaCl was added. The reason for this dichotomy was the ability of the Na+ ions to bind with the DMPC lipid carbonyl oxygens at low DMTAP fraction and to tie 2 to 4 DMPCs into a dynamic complex. At high DMTAP fraction the binding of Na+ was prevented by the high positive surface charge of the bilayer. The lateral diffusion of Na+ ions within the carbonyl region had two qualitatively different modes. Na+ ions bound to a DMPC diffused very slowly, whereas the free Na+ ions traveled rapidly within the carbonyl region. The combined effect of the two motions appeared as Na+ ions hopping from one DMPC carbonyl oxygen to the next

Application of fragment-based drug discovery to membrane proteins

Membrane proteins are an interesting class due to the variety of cellular functions and their importance as pharmaceutical targets, but they pose significant challenges for fragment-based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. Using the recently developed Target Immobilized NMR Screening (TINS) approach, we screened 1,200 fragments for binding to the enzyme Disulphide bond forming protein B. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 uM, which could be categorized as cofactor binding inhibitors or mixed model inhibitors of both cofactor and substrate protein interaction. Our results establish the utility of fragment-based methods in the development of inhibitors of membrane proteins, making a wide variety3of important membrane bound pharmaceutical targets amenable to such an approach. We first tested the immobilization procedure on G protein coupled receptors and ion channels. Furthermore, we used Nanodiscs, an alternative solubilization strategy, to solubilize teh protein without detergents. This resulted in less broad NMR signals, less non-specific binding issues, and identification of the binders from the original screen, proving that the nanodisc solubilization technique is compatible with TINS.Medicinal Chemistr

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

Biophysical Insights into Peptide and Alcohol Perturbations on Biomimetic Membranes

Biological membranes exist in every domain of life. Life exists due to the presence of these special structures for which we take for granted. They are composed of fatty lipids and workhorse proteins and act as the premier interface of biological processes. Due to the sheer quantity and complexity within their thin boundary, studying their actions and properties pose challenges to researchers. As a result, simplified biomembrane mimics are employed regularly. We will use several types of biomembrane mimics to understand fundamental properties of membranes. In the present thesis, we also attempt to move beyond the canonical structure-based theories upon which a majority of biophysical studies are predicated upon. This has been the case as structural quantities still greatly inform on the conditions of the bilayer system, yet the exact lipid distribution and movement are less studied. We will focus upon the movement and organization of phospholipids using a bounty of biophysical techniques, such as small angle neutron scattering, molecular dynamics, and more. The results will be interpreted to show how phospholipid mobility fits into the greater membrane framework