Biophysical characterization of membrane protein-small molecule interactions

Membrane proteins are account for up to two thirds of known druggable targets. Traditionally, new drugs against this class of proteins have been discovered through HTS. However, not all GPCRs are amenable to traditional screening methods. Recently, fragment-based drug discovery (FBDD) has emerged as a powerful strategy to generate approved drugs against soluble targets. Now, FBDD can be applied to GPCRs with great potential advantages. In recent years, a number of FBDD techniques have been validated for use with GPCRs. The impressive growth in GPCR structure information leads to broad use of structure-based methods for hit discovery and optimization. However, the dynamic nature of GPCRs, and standard issues associated with low level expression and instability during purification, made biophysical and structural characterization of GPCRs particularly difficult. New advances in protein stabilization by using different protein engineering methods and alternative solubilization strategy have shown the potential to facilitate GPCR structural and biophysical studies. The goal of the work described in this thesis was to develop and implement efficient fragment screening methods to discover ligands of GPCRs with novel biological activities, and new advances in receptor production and stabilization to facilitate structural biology of GPCRs in the early stages of drug discovery.UBL - phd migration 201

Exploring Structure-Dynamics-Function Relationship in Proteins, Protein: Ligand and Protein: Protein Systems through Computational Methods

The study focuses on understanding the dynamic nature of interactions between molecules and macromolecules. Molecular modeling and simulation technologies are employed to understand how the chemical constitution of the protein, specific interactions and dynamics of its structure provide the basis of its mechanism of function. The structure-dynamics-function relationship is investigated from quantum to macromolecular-assembly level, with applications in the field of rationale drug discovery and in improving efficiency of renewable sources of energy. Results presented include investigating the role of dynamics in the following: 1) In interactions between molecules: analyzing dynamic nature of a specific non-covalent interaction known as “anion-π [pi]” in RmlC protein. 2) In interactions between molecules and macromolecules: defining the structural basis of testosterone activation of GPRC6A. 3) In disrupting the function using specific substrate interactions: incorporating protein dynamics and flexibility in structure-based drug-discovery approach targeting the prothrombinase coagulation complex. 4) In interactions between macromolecules: elucidating the protein-protein binding and dynamics of electron-transport proteins, Ferrodoxin and Cytochrome c6, with Cyanobacterial Photosystem I

Kvantově chemické pojetí návrhu léčiv

Výpočetní metody jsou nedílnou součástí moderního farmaceutického výzkumu. Počítačový návrh léčiv si klade za cíl snížit čas a náklady spjaté s vývojem léčiva a také detailněji porozumět vazbě inhibitoru k danému biologickému cíli. Kvůli komplikovanosti biologických systémů a potřebě správného popisu nekovalentních interakcí nutných k molekulárnímu rozpoznávání je přesnost běžně používaných molekulově mechanických (MM) metod na hraně spolehlivosti. Na druhou stranu zde vzrůstá tendence používání kvantově mechanických (QM) metod v různých fázích vývoje léčiv díky rostoucím výpočetním možnostem. Tato disertační práce se zabývá aplikací kvantově mechanických metod pro věrný popis mezimolekulových komplexů a jejich interakcí. Tato práce zahrnuje osm původních publikací rozdělených do tří témat a doprovodný text, jenž si klade za cíl zdůraznit některé závěry plynoucí z této práce. V první řadě je vysoce přesnými kvantově mechanickými metodami studována povaha neklasických nekovalentních interakcí, tzv. vazebné interakce pomocí sigma díry. Síla a původ halogenové, chalkogenové a pniktogenové vazby v modelových systémech z rozšířených databází molekul jsou zkoumány přesnou metodou vázaných klastrů (CCSD(T)/CBS) a symetricky adaptovanou poruchovou teorií (SAPT). Druhá část se věnuje třem farmaceuticky...Computational approaches have become an established and valuable component of pharmaceutical research. Computer-aided drug design aims to reduce the time and cost of the drug development and also to bring deeper insight into the inhibitor binding to its target. The complexity of biological systems together with a need of proper description of non-covalent interactions involved in molecular recognition challenges the accuracy of commonly used molecular mechanical methods (MM). There is on the other side a growing interest of utilizing quantum mechanical (QM) methods in several stages of drug design thanks to increased computational resources. This doctoral thesis's topic is the QM-based methodology for the reliable treatement of intermolecular interactions. It consists of eight original publications devided into three topics and an accompanying text that aims to emphasize selected outcomes of the work. Firstly, the nature of nonclassical non-covalent interactions - so called σ-hole bonding - is studied by high-level QM methods. The strength and origin of halogen-, chalcogen- and pnicogen bonded model systems in extended datasets are accurately explored by coupled cluster QM method (CCSD(T)/CBS) and symmetry adapted perturbation theory (SAPT). The second part is devoted to three pharmaceutically...Katedra fyzikální a makromol. chemieDepartment of Physical and Macromolecular ChemistryFaculty of SciencePřírodovědecká fakult

Molecular dynamics study of the allosteric control mechanisms of the glycolytic pathway

There is a growing body of interest to understand the regulation of allosteric proteins. Allostery is a phenomenon of protein regulation whereby binding of an effector molecule at a remote site affects binding and activity at the protein‟s active site. Over the years, these sites have become popular drug targets as they provide advantages in terms of selectivity and saturability. Both experimental and computational methods are being used to study and identify allosteric sites. Although experimental methods provide us with detailed structures and have been relatively successful in identifying these sites, they are subject to time and cost limitations. In the present dissertation, Molecular Dynamics Simulations (MDS) and Principal Component Analysis (PCA) have been employed to enhance our understanding ofallostery and protein dynamics. MD simulations generated trajectories which were then qualitatively assessed using PCA. Both of these techniques were applied to two important trypanosomatid drug targets and controlling enzymes of the glycolytic pathway - pyruvate kinase (PYK) and phosphofructokinase (PFK). Molecular Dynamics simulations were first carried out on both the effector bound and unbound forms of the proteins. This provided a framework for direct comparison and inspection of the conformational changes at the atomic level. Following MD simulations, PCA was run to further analyse the motions. The principal components thus captured are in quantitative agreement with the previously published experimental data which increased our confidence in the reliability of our simulations. Also, the binding of FBP affects the allosteric mechanism of PYK in a very interesting way. The inspection of the vibrational modes reveals interesting patterns in the movement of the subunits which differ from the conventional symmetrical pattern. Also, lowering of B-factors on effector binding provides evidence that the effector is not only locking the R-state but is also acting as a general heat-sink to cool down the whole tetramer. This observation suggests that protein rigidity and intrinsic heat capacity are important factors in stabilizing allosteric proteins. Thus, this work also provides new and promising insights into the classical Monod-Wyman-Changeux model of allostery

Chemistry & Chemical Biology 2013 APR Self-Study & Documents

UNM Chemistry & Chemical Biology APR self-study report, review team report, response to review report, and initial action plan for Spring 2013, fulfilling requirements of the Higher Learning Commission

Desarrollo In silico de sensores fluorescentes para disecar vías de señalización celular

El campo de fuerza para simulaciones de grano grueso y multiescala denominado SIRAH viene siendo desarrollado en la última década por el grupo de Simulaciones Biomoleculares del Institut Pasteur de Montevideo. Posee parámetros para proteínas, ácidos nucleicos, lípidos, glicanos y han incorporado estudios de modificaciones post-traduccionales. Debido a que los campos de fuerza necesitan estar en continua actualización para mantenerse vigentes e incorporar parámetros de nuevas especies moleculares, durante esta tesis se colaboró en la actualización del campo de fuerzas SIRAH2.0 y se desarrollaron parámetros de iones divalentes como zinc, magnesio y se extendió la aplicabilidad de los iones calcio que ya se encontraban parametrizado. A su vez, era de suma importancia verificar la capacidad de SIRAH para predecir proteínas intrínsecamente desestructuradas (IDPs), extendiendo la aplicabilidad del campo de fuerzas sin necesidad de introducir cambios relevantes en la parametrización. A su vez, abordé el estudio y desarrollo de sensores genéticamente codificados para nucleótidos cíclicos como 3 ́5 ́-adenosín/guanosín monofosfato cíclico (AMPc/GMPc respectivamente). La búsqueda de sensores genéticamente codificados ha permitido el estudio de manera no invasiva y en tiempo real de la compartimentalización de señales subcelulares. Para ello, nuestro grupo ya había desarrollado un biosensor primero en su clase, en la que un módulo fluorescente es insertado dentro del dominio de enlace a AMPc de la subunidad regulatoria de la proteína PKA (Proteína Quinasa dependiente de AMPc) mientras el segundo se encuentra en su extremo C-terminal. De esta manera, el extremo N-terminal de la cadena polipeptídica puede ser fusionado a cualquier proteína de interés. Esta arquitectura novedosa mantiene la funcionalidad de dicha proteína y garantiza la localización del sensor en el punto de interés sin deber confiar en la colocalización. Teniendo este precedente, en la presente tesis se desarrolla la construcción de un sensor para GMPc, cuyo dominio de unión a nucleótido cíclico es PKG (Proteína Quinasa dependiente de GMPc). Por otro lado, gracias al primer sensor de AMPc, se pudo establecer que la compartimentalización mínima en el sarcómero puede ser establecida en una escala de nanómetros. Por lo que, en este proyecto de investigación de doctorado, se busca construir la unidad estructural mínima de un signalosoma de la cadena beta adrenérgica en el sarcómero del músculo cardíaco para entender la disposición de las diferentes proteínas, el volumen que ocupan y la capacidad de moléculas como el AMPc para llegar a sus proteínas blanco. Gracias a esto y estudios del Dr. Baillie del Grupo de Molecular Pharmacology en Glasgow University, pudimos evidenciar los sitios de contacto entre proteínas como las troponinas, PKAs y fosfodiesterasas. Estos resultados llevan al primer ejemplo de un modelo 3D del denominado “islas de AMPc”, vagamente propuesto hace tres décadas aproximadamente, pero, hasta el momento, no bien definido en términos estructurales. Estos resultados llevan a una predicción de tamaños relativos y concentraciones que muestran al músculo cardíaco como una pieza de relojería donde todo debe trabajar de manera unísona y precisa. Finalmente, la experiencia obtenida durante mis estudios doctorales nos llevó a proponer un procedimiento simplificado pero general para generar sensores fluorescentes para nucleótidos cíclicos con una afinidad arbitraria por su ligando. Vale la pena destacar, que el procedimiento de diseño puede basarse en conceptos simples, sin la necesidad de ser un experto computacional, utilizando servidores disponibles en línea

APS Science 2007.

This report provides research highlights from the Advanced Photon Source (APS). Although these highlights represent less than 10% of the published work from the APS in 2007, they give a flavor of the diversity and impact of user research at the facility. In the strategic planning the aim is to foster the growth of existing user communities and foresee new areas of research. This coming year finds the APS engaged in putting together, along with the users, a blueprint for the next five years, and making the case for a set of prioritized investments in beamlines, the accelerator, and infrastructure, each of which will be transformational in terms of scientific impact. As this is written plans are being formulated for an important user workshop on October 20-21, 2008, to prioritize strategic plans. The fruit from past investments can be seen in this report. Examples include the creation of a dedicated beamline for x-ray photon correlation spectroscopy at Sector 8, the evolution of dedicated high-energy x-ray scattering beamlines at sectors 1 and 11, a dedicated imaging beamline at Sector 32, and new beamlines for inelastic scattering and powder diffraction. A single-pulse facility has been built in collaboration with Sector 14 (BioCARS) and Phil Anfinrud at the National Institutes of Health, which will offer exceptionally high flux for single-pulse diffraction. The nanoprobe at Sector 26, built and operated jointly by the Argonne Center for Nanoscale Materials and the X-ray Operations and Research (XOR) section of the APS X-ray Science Division, has come on line to define the state of the art in nanoscience

Microgravity science & applications. Program tasks and bibliography for FY 1995

This annual report includes research projects funded by the Office of Life and Microgravity Sciences and Applications, Microgravity Science and Applications Division, during FY 1994. It is a compilation of program tasks (objective, description, significance, progress, students funded under research, and bibliographic citations) for flight research and ground based research in five major scientific disciplines: benchmark science, biotechnology, combustion science, fluid physics, and materials science. Advanced technology development (ATD) program task descriptions are also included. The bibliography cites the related principle investigator (PI) publications and presentations for these program tasks in FY 1994. Three appendices include a Table of Acronyms, a Guest Investigator index and a Principle Investigator index