Ernesto Freire, Professor
Biologyjohns Hopkins University
w400 Wyman Park Building
baltimore, Md 212182680
Grant 3P41RR004328-09S1 from National Center For Research Resources IRG: SSS
Project start date: 1988-08-05
Project end date: 1998-08-04
3P41RR004328-09S1 (1997): $840
Sponsored Links Excellgen http://Excellgen.com
Ernesto Freire, Professor
Johns Hopkins University
w400 Wyman Park Building
baltimore, Md 212182680
Grant 3P41RR004328-05S1 from National Center For Research Resources IRG: SSS
Project start date: 1988-08-05
Project end date: 1993-08-04
3P41RR004328-05S1 (1993): $423
Grants awarded to Ernesto Freire
STRUCTURE-ENERGY CORRELATION IN FOLDING AND RECOGNITION
Ernesto Freire
Johns Hopkins University W400 Wyman Park Building Baltimore, Md 212182680
Grant 5P01GM051362-050004 from National Institute Of General Medical Sciences
Abstract: The realization of structure based molecular design is linked to our ability to accurately estimate or predict protein stabilities or binding affinities from structural considerations. This task essentially reduces to the ability of predicting the Gibbs energy that accounts for the stability of a protein or the binding affinity of a complex from a detailed knowledge of their structure. Recently, it has been realized that the thermodynamic parameters (deltaCp, deltaH, deltaS) that define the Gibbs energy of protein stabilization or binding can be parametrized in structural terms. This empirical approach appears to be promising and to have predictive potential, provided that the structural parametrization is extended and refined. The main goal of this proposal is precisely to extend and refine the existing structural parametrization of the thermodynamic parameters deltaCp, deltaH, and deltaS using systems in which the effects of specific contributions have a higher impact and are therefore easier to identify and candidate. These systems include folding/unfolding of specific mutants of low molecular weight proteins, and the binding of different peptides to specific protein binding sites. The thermodynamic parameters for those events will be measured directly by high sensitivity calorimetric techniques. In our studies, we will consider specific mutants of a 33 amino acid peptide corresponding to the coiled coil (leucine zipper) region of GCN4, and the binding of specific inhibitors to aspartic proteases. The protease systems include endothiapepsin for which the structure of several inhibitor complexes is known at high resolution and the human immunodeficiency virus (HIV) protease for which high resolution structural information is also available. Besides providing information of general significance and applicability, the systems chosen are of intrinsic importance and medical relevance.
Keywords: chemical binding, protein folding, protein structure, thermodynamics, acidity /alkalinity, chemical stability, conformation, dimer, endopeptidase, mutant, peptide, protease inhibitor, structural biology, synthetic peptide, analytical ultracentrifugation, calorimetry, circular dichroism, nuclear magnetic resonance spectroscopy, peptide chemical synthesis
Ernesto Freire, Professor
Johns Hopkins University W400 Wyman Park Building Baltimore, Md 212182680
Grant 5P41RR004328-05 from National Center For Research Resources IRG: SSS
Abstract: The past decade has been marked by spectacular advances in the areas of genetic engineering, computer technology and in the implementation of sophisticated instrumentation for the study of biological macromolecules. These advances have made possible the development of new approaches to the study of the structure- function relationships in macromolecular systems and particularily in complex assemblies of biological macromolecules like protein- protein, protein-membrane, and protein-DNA complexes to name only three examples. Central to the development of a rigorous, quantitative of biological structure and function is a knowledge of the forces and the magnitude of the forces involved in the formation of biological structures as well as the energetics of the molecular interactions associated with biological function. The importance of calorimetry, within this context, is that it is the only technique that allows a direct measurement of the magnitude of the forces and energetics associated with biochemical processes. The Biocalorimetry Center at the Johns Hopkins University, the first of its kind in the country, will have a dual commitment. It will provide a user-oriented facility to aid research scientists in the determination of thermodynamic properties of biological systems using state-of-the-art calorimetric instrumentation (high sensitivity differential scanning calorimetry, isothermal reaction calorimetry and multifrequency calorimetry); and, it will focus in the development of new, supersensitive instruments capable of accurately measuring the energetics of biological processes at the subnanomolar level.
Keywords: biomedical equipment resource, biophysical chemistry, molecular energy level, nonclinical biomedical equipment, thermodynamics
Project start date: 1988-08-05
Project end date: 1993-08-04
5P41RR004328-05 (1992): $535934
STATISTICAL THERMODYNAMIC STUDIES OF MEMBRANES
Ernesto Freire, Professor
Biologyjohns Hopkins University
w400 Wyman Park Building
baltimore, Md 212182680
Grant 5R01GM037911-09 from National Institute Of General Medical Sciences IRG: PB
Abstract: The major goal of this project is to gain a quantitative understanding of the forces and mechanisms that control the assembly, structural stability and functional properties of membrane proteins. As such, three major aspects of the interactions between membrane proteins and phospholipid bilayer membranes will be studied in detail using an experimental thermodynamic approach 1) The mechanisms and energetics of folding, assembly and stabilization of membrane proteins; 2) The role of the lipid moiety and physicochemical environment on the stability and functional energetics of membrane proteins; and, 3) The energetics of protein insertion into membranes. The research program focuses on two well defined membrane systems a) Cytochrome c oxidase and b) Diphtheria Toxin. Cytochrome c oxidase is a multisubunit enzyme located in the inner mitochondrial membrane. The three largest subunits are synthesized inside the mitochondria and the remaining subunits are imported from the cytoplasm. We are interested in studying the assembly process and structural stability of the enzyme, the role of the phospholipid bilayer matrix in maintaining the structural and functional integrity of the enzyme, the interactions between subunits, the interactions with its substrate cytochrome c, and the interactions with specific lipid components of the inner mitochondrial membrane (e.g. cardiolipin). Diphtheria toxin is composed of two fragments, A (MW=21,000) and B (MW=37,200), joined by a disulfide bridge. The toxin enters the cell by endocytosis, where it is exposed to a pH of approximately 5. The exposure of the toxin to an acidic environment provides the driving force for the membrane insertion and translocation of the toxic A fragment into the cytosol. One of the goals of this project is to understand protein-membrane interactions that result in the penetration of the protein into the bilayer. The goal in this case is to examine the sequence of protein unfolding -->penetration -->translocation -->refolding in vitro and to provide a complete thermodynamic mechanism for this process. This research project utilizes well defined reconstituted systems and a combination of biochemical and biophysical approaches including different types of calorimetry (high sensitivity differential scanning calorimetry, high sensitivity isothermal] titration calorimetry and multifrequency calorimetry), computer assisted quantitative gel electrophoresis, optical spectroscopy, and other spectroscopic techniques
Project start date: 1986-07-01
Project end date: 1996-06-30
5R01GM037911-09 (1994): $189993
5R01GM037911-08 (1993): $182594
5R01GM037911-07 (1992): $176260
TESTICULAR CONTROL OF LH AND FSH SECRETION
Ernesto Freire, Professor
University Of Pittsburgh At Pittsburgh 350 Thackeray Hall Pittsburgh, Pa 15260
Grant 5R01HD016851-03 from National Institute Of Child Health And Human Development IRG: BCE
Abstract: The long term goal of our studies is to elucidate, employing systematic physiologic approaches, the neuroendocrine mechanisms that govern testicular function in the adult rhesus monkey, a representative higher primate. In the present proposal we intend to determine whether the testosterone induced slowing of the frequency of intermittent hypothalamic GnRH secretion is the major mechanism mediating the negative feedback control of gonadotropin secretion by the testis, to determine whether the hypothalamus of the female is endowed with the capacity to respond to the foregoing action of testosterone, to begin to examine the neural mechanisms underlying the testosterone induced retardation of intermittent GnRH secretion, and to evaluate the significance of the episodic nature of testicular testosterone secretion in the negative feedback control of gonadotropin secretion. Remote blood sampling procedures that do not require the use of anesthetics or restraint will be used to collect sequential plasma samples. Hormone concentrations in body fluids will be determined primarily by established radioimmunioassay procedures, and pulsatile patterns of LH, FSH and testosterone secretion will be analyzed by a computer executed algorithm. Hypothalamic lesions and implantations will be conducted using aseptic stereotaxic procedures previously described. These studies will provide important insights into the neuroendocrine control system governing testicular function in higher primates, and may therefore have direct relevance to fertility control and to the treatment of infertility in men.
Keywords: BIOCHEMICAL ENDOCRINOLOGY STUDY SECTION, ENDOCRINOLOGY, HORMONAL REGULATION AND CONTROL (MECHANISMS), PITUITARY-DIENCEPHALON HORMONES, FSH, PITUITARY-DIENCEPHALON HORMONES, LH-ICSH, REPRODUCTIVE SYSTEM MALE, TESTIS, ANDROSTANE SERIES, TESTOSTERONE, NEUROENDOCRINE SYSTEMS, PITUITARY-DIENCEPHALON HORMONES, GONADOTROPINS REGULATING FACTORS, brain metabolism, BRAIN LESIONS SURGICAL, CHEMISTRY, CLINICAL, BLOOD, HISTOLOGY (GENERAL), IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, MAMMALS, PRIMATES, MONKEYS AND APES, OLD WORLD MONKEYS, MACACA SP., M. MULATTA, stereotaxic technique
Project start date: 1982-09-01
Project end date: 1986-07-31
THE ONTOGENY OF GONADOTROPIN SECRETION IN THE MONKEY
Ernesto Freire, Professor
University Of Pittsburgh At Pittsburgh 350 Thackeray Hall Pittsburgh, Pa 15260
Grant 5R01HD013254-06 from National Institute Of Child Health And Human Development IRG: REB
Abstract: The long term goal of our studies is to elucidate the neuroendocrine mechanisms that underlife the ontogeny of gonadotropin secretion in higher primates, including man. In the present proposal we hope to gain an insight into the nature of the extra-gonadal mechanism responsible for the apparent restraint of hypothalamic GnRH secretion during the greater part of prepubertal development and, therefore, for the protracted delay in the onset of puberty in these species. Using the rhesus monkey as an experimental paradigm, we will address the view that the pubertal reactivation of the hypothalamic mechanism responsible for the pulsatile secretion of gonadotropin is triggered by a metabolic or humoral signal. We will determine the role, if any, of neurons sensitive to generalized CNS depression and of opioid neurons in maintaining the prepubertal restraint of gonadotropin secretion. We will complete our studies of the effects of neonatal adrenalectomy on the onteogeny of gonadotropin secretion. In addition, we propose to investigate the role of the fetal testis in determining the early ontogeny of the hypothalamic oscillator that generates the intermittent secretion of GnRH. Blood infusion-withdrawal devices that permit continuous access to the circulation of the rhesus monkey without restraint or anesthesia will be used to conduct servo-controlled cross circulation experiments, or chronically administer drugs and hormones, and to collect sequential blood samples. Routine sera-hematologic techniques will be employed in the cross cirulation experiments to match blood types and to monitor anticoagulation therapy. Hormone concentrations in plasma will be determined by established radioimmunoassay procedures and pulsatile patterns of LH and FSH secretion will be analyzed by computer executed algorithms. The aim of the studies described in the present proposal is to provide the initial insight into the mechanism responsible for the protracted delay in the onset of puberty in man, and thus provide the basis for further and systematic study of this fundamental problem in human development.
Keywords: AGE (ANIMAL), PUBERTY, GROWTH AND DEVELOPMENT, NEUROENDOCRINE SYSTEMS, PITUITARY-DIENCEPHALON HORMONES, LH RELEASING FACTOR, REPRODUCTIVE BIOLOGY STUDY SECTION, ALKALOIDS, MORPHINES, NALOXONE, ANDROSTANE SERIES, TESTOSTERONE, BENZO-ALPHA-PYRONES, WARFARIN, BRAIN, HYPOTHALAMUS, BRAIN, THALAMENCEPHALON, PINEAL BODY, ENDOCRINOLOGY, HORMONAL REGULATION AND CONTROL (MECHANISMS), PITUITARY-ADRENAL AXIS, PITUITARY-DIENCEPHALON HORMONES, FSH, PITUITARY-DIENCEPHALON HORMONES, LH-ICSH, REPRODUCTIVE HORMONES, GONADOTROPINS, REPRODUCTIVE SYSTEM MALE, TESTIS, AGE (ANIMAL), INFANTS NEWBORN, AGE (ANIMAL), MATURE (ADULT), IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, MAMMALS, PRIMATES, MONKEYS AND APES, OLD WORLD MONKEYS, MACACA SP., M. MULATTA, REPRODUCTIVE SYSTEM SURGERY MALE, ORCHIECTOMY, adrenalectomy, computer simulation
Project start date: 1980-04-01
Project end date: 1986-11-30
Sponsored Links Excellgen http://Excellgen.com
CENTER FOR RESEARCH IN REPRODUCTIVE PHYSIOLOGY
Ernesto Freire, Professor
University Of Pittsburgh At Pittsburgh 350 Thackeray Hall Pittsburgh, Pa 15260
Grant 2P30HD008610-12 from National Institute Of Child Health And Human Development IRG: HDPR
Abstract: The broad and long-term objectives of the Center are to elucidate the endocrine and neuroendocrine control systems which govern the reproductive processes in a representative primate, the rhesus monkey. The Center comprises the following project and research resources (cores) The Properties of the Pituitary Glycoprotein Hormones; The Neuroendocrine Control of Reproduction in the Male Rhesus Monkey; Cellular Basis of Control of Gonadotropin Hormone Section by Gonadal Steroids; Control of Folliculogenesis in the Rhesus Monkey; Gonadal Hormone Metabolism in the Rhesus Monkey. The cores are The Primate Research Laboratory (Primate Core), A Steroid Hormone Radioimmunoassay Laboratory, A Polypeptide Hormone Radioimmunoassay Laboratory, and an Administrative Core. The Primate Core houses 280 rhesus monkeys and provides all attendant facilities for their acquisition and care. The Center is also the focus of a major postdoctoral training program in primate reproduction.
Keywords: ENDOCRINOLOGY, HORMONAL REGULATION AND CONTROL (MECHANISMS), MAMMALS, PRIMATE COLONIES, FACILITIES, RESOURCES, MAMMALS, PRIMATES, MONKEYS AND APES, OLD WORLD MONKEYS, MACACA SP., M. MULATTA, NEUROENDOCRINE SYSTEMS, POPULATION RESEARCH AND TRAINING COMMITTEE, reproduction
Project start date: 1979-09-29
Project end date: 1990-11-30
Ernesto Freire, Professor
Biologyjohns Hopkins University
w400 Wyman Park Building
baltimore, Md 212182680
Grant 5P41RR004328-10 from National Center For Research Resources IRG: SSS
Abstract: The Biocalorimetry Center is the only center of its kind in the United States. It is dedicated to the development of new calorimetric technologies and the application of those technologies in Biomedical Research. It also plays an important role in the training of scientists in the area of calorimetry and in the dissemination of the capabilities of these technologies within the scientific community. Calorimetry is the only technique with the ability to measure the energetics of chemical or biochemical reactions. Since the folding of proteins into their native structures, the degree of advancement of biochemical reactions, the association of biological molecules, the binding of ligands or pathogens to cells or the formation of biological structures in general are dictated by their energetics, calorimetry plays a fundamental role in the development of a complete understanding of these processes and the ability to engineer or modify them. During the last five years, significant advances have been made in these technologies as reflected by the fact that many biological reactions can now be measured using microgram amounts of material. Also, during the last five years, significant advances have been made in the development of new methods of analysis, particularly in the area of Structural Thermodynamics. Continuous developments in this area will bring about the capability to accurately predict the stability of proteins from structural parameters or to predict the binding ability of peptide hormones or other molecules to protein receptors. For the next funding period the development of a new generation of highly sensitive calorimeters is planned. These instruments include a differential scanning calorimeter, multifrequency calorimeter, isothermal titration calorimeter, flow/mix calorimeter and a discrete flow/variable temperature calorimeter. A major effort will be made in the area of Structural Thermodynamics and in the development of a Protein Thermodynamics Database. Also the Biocalorimetry Center is engaged in many collaborative projects with scientists from many laboratories around the country
Keywords: biomedical equipment resource, biomedical resource, calorimetry, nonclinical biomedical equipment, thermodynamics
Project start date: 1988-08-05
Project end date: 1998-08-04
5P41RR004328-10 (1997): $622815
5P41RR004328-07 (1994): $615471
2P41RR004328-06 (1993): $641752
Structure Based Thermodynamic Studies Of HIV-1 Protease
Ernesto Freire, Professor
Biologyjohns Hopkins University
w400 Wyman Park Building
baltimore, Md 21218
Grant 5R01GM057144-11 from National Institute Of General Medical Sciences IRG: ADDT
Abstract: Protease inhibitors are key components in the chemotherapy of HIV/AIDS. Unfortunately, the long term efficacy of protease inhibitors is severely compromised by the appearance of drug-resistant mutations that lower their potency to inadequate levels for effective inhibition and viral suppression. The onset of drug resistance is often accelerated by issues of patient compliance, often aggravated by severe side effects. In addition, the viral subtypes prevalent in Africa, where the vast majority of HIV infections take place, are not the same as the one responsible for the infections in America and Europe. Complicating things even further, a different HIV virus, HIV-2, although less prevalent than HIV-1, is also able to cause AIDS. It is evident, that the development of new protease inhibitors with high potency, effectiveness against different subtypes, low susceptibility to mutations and minimal side effects still remains an urgent goal. The main goal of this project is to develop precise thermodynamic and structural guidelines to develop such inhibitors. The specific goals of this project are - Identification of thermodynamic and structural determinants of extremely high affinity. - Identification of thermodynamic and structural determinants that confer protease inhibitors low susceptibility to mutations and efficacy against different viral subtypes, including HIV-2. - Identification of thermodynamic and structural determinants that lower the affinity of protease inhibitors to unwanted targets and hence improve selectivity and reduce side effects. The goals will be achieved by a combination of experimental thermodynamic measurements (high sensitivity isothermal titration calorimetry and high sensitivity differential scanning calorimetry), structure determination (x-ray crystallography) and structure-based thermodynamic analysis
Keywords: antiAIDS agent, drug design /synthesis /production, drug screening /evaluation, endopeptidase, human immunodeficiency virus 1, protease inhibitor, protein structure, thermodynamics, virus protein chemical binding, drug resistance, enzyme activity, gene mutation, genetic strain, human immunodeficiency virus 2 X ray crystallography, calorimetry, spectrometry
Project start date: 1998-03-01
Project end date: 2010-02-28
5R01GM057144-11 (2008): $494296
5R01GM057144-10 (2007): $493906
5R01GM057144-09 (2006): $519019
2R01GM057144-08A1 (2005): $531310
3R01GM057144-08A1S1 (2005): $27093
Sponsored Links Excellgen http://Excellgen.com
STRUCTURE BASED THEMODYNAMIC STUDIES OF HIV-1 PROTEASE
Ernesto Freire
Johns Hopkins University, W400 Wyman Park Building, Baltimore, Md 21218
Grant 5R01GM057144-13 from National Institute Of General Medical Sciences
Abstract: Protease inhibitors are essential components in the chemotherapy of HIV-1 infection. Despite their success, the long-term efficacy of antiretroviral therapies is continuously hindered by the emergence of viral strains that exhibit resistance to protease inhibitors. The onset of drug resistance is often accelerated by therapy lapses associated with the occurrence of severe side effects in patients undergoing highly active anti-retroviral therapy (HAART). In addition, the viral subtypes prevalent in Africa, where the vast majority of HIV infections take place, are not the same as the one responsible for the infections in America and Europe. Complicating things even further, a different HIV virus, HIV-2, although less prevalent than HIV-1, is also able to cause AIDS. It is clear that successful protease inhibitors would have to maintain appropriate potency against a wide range of target variability. As of today, the FDA has approved nine protease inhibitors for clinical use. While all of them target the active site pocket of the same enzyme, they do so with different potency, different resistance profiles and different selectivity towards unwanted targets. An ideal inhibitor should have extremely high potency against the wild type protease, exhibit low susceptibility to protease mutations associated with drug resistance and not interfere with human targets, thus minimizing side effects. The main goal of this project is to develop precise thermodynamic and structural guidelines to develop such inhibitors. The specific goals of this project are - Development of thermodynamic and structural rules aimed at achieving extremely high affinity. - Identification of thermodynamic and structural determinants that confer protease inhibitors low susceptibility to mutations and efficacy against different viral subtypes, including HIV-2. - Development of thermodynamic and structural rules aimed at limiting the affinity of protease inhibitors to unwanted targets and hence improving selectivity. The goals will be achieved by a combination of experimental thermodynamic measurements (high sensitivity isothermal titration calorimetry and high sensitivity differential scanning calorimetry), structure determination (x-ray crystallography) and structure-based thermodynamic analysis. More than 30 million people in the world are infected with HIV/AIDS and more than 2 million die each year. Despite their initial success, antiretroviral therapies are hindered by the emergence of drug resistant viral strains and by the occurrence of severe side effects. The main goal of this project is to develop precise guidelines for the development of antiretrovirals, especially protease inhibitors, characterized by extremely high potency, low susceptibility to drug resistance and minimal side effects
Keywords: AIDS; AIDS Virus; AIDS/HIV; AIDS/HIV problem; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immune Deficiency Syndrome Virus; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; Acquired Immunodeficiency Syndrome Virus; Active Sites; Adverse effects; Affinity; Africa; Americas; Antiproteases; Antiretroviral Therapy, Highly Active; Calorimetry; Calorimetry, Differential Scanning; Clinical; Compliance behavior; Crystallographies; Crystallography; Development; Differential Scanning Calorimetry; Differential Thermal Analysis, Calorimetric; Drug resistance; Drug resistant viral; Effectiveness; Endopeptidase Inhibitors; Enzymes; Esteroproteases; Europe; Exhibits; Genetic Alteration; Genetic Change; Genetic defect; Goals; Guidelines; HAART; HIV; HIV Infections; HIV-1; HIV-1 Protease; HIV-2; HIV-I; HIV-II; HIV/AIDS; HIV/AIDS problem; HIV1; HIV1 protease; HIV2; HTLV-III; HTLV-III Infections; HTLV-III-LAV Infections; HTLV-IV; Highly Active Antiretroviral Therapy; Human; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type IV; Human immunodeficiency virus 1; Human immunodeficiency virus 2; Human, General; Immunodeficiency Virus Type 1, Human; Immunodeficiency Virus Type 2, Human; Immunologic Deficiency Syndrome, Acquired; Infection; LAV-2; LAV-HTLV-III; Lymphadenopathy-Associated Virus; Man (Taxonomy); Man, Modern; Measurement; Mutation; Patient Compliance; Patient Cooperation; Patients; Peptidase Inhibitors; Peptidases; Peptide Hydrolase Inhibitors; Peptide Hydrolases; Peptide Peptidohydrolase Inhibitors; Predisposition; Protease Antagonists; Protease Inhibitor; Protease, HIV-1; Proteases; Proteinase Inhibitors; Proteinases; Proteolytic Enzymes; Resistance; Resistance profile; Resistant profile; Side; Structure; Susceptibility; T-Lymphotropic Virus Type III Infections, Human; Thermodynamic; Thermodynamics; Titrations; Treatment Compliance; Treatment Side Effects; Viral; Virus-HIV; anti-retroviral therapy, highly active; antiretroviral therapy; base; chemotherapy; compliance cooperation; drug resistant; drug resistant virus; genome mutation; human T cell leukemia virus III; human T lymphotropic virus III; improved; inhibitor; inhibitor/antagonist; patient adherence; public health relevance; resistance to Drug; resistant; resistant to Drug; side effect; success; therapy adverse effect; therapy compliance; therapy cooperation; treatment adverse effect
Relevance: More than 30 million people in the world are infected with HIV/AIDS and more than 2 million die each year. Despite their initial success, antiretroviral therapies are hindered by the emergence of drug resistant viral strains and by the occurrence of severe side effects.The main goal of this project is to develop precise guidelines for the development of antiretrovirals, especially protease inhibitors, characterized by extremely high potency, low susceptibility to drug resistance and minimal side effects
Project start date: 1998-03-01
Project end date: 2013-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PA-07-070
5R01GM057144-13 (2010): $485668
2R01GM057144-12 (2009): $478079
ASSEMBLY AND INHIBITION THERMODYNAMICS
Ernesto Freire, Dr.
Drexel University, Office Of Research, Philadelphia, Pa 19104
Abstract: Principal Investigator/Program Director (Last, First, Middle) Chaiken, Irwin M / Freire, Ernesto The viral envelope glycoprotein gp120 is responsible for the initial events in HIV-1 infection. The binding of gp120 to the cell surface receptor CD4 induces a series of allosteric events that culminate with the fusion of the viral and cell membranes. The entire series of events defines different intervention points at which the process can be interrupted, therefore providing various opportunities for inhibitor development. These intervention points define two classes of inhibitors compounds that competitively inhibit the binding of gp120 to its cellular partners and compounds that block allosteric signaling and downstream activation. The rational design and optimization of.inhibitors directed at any of these intervention points requires a precise knowledge of the structural energetics and conformational stability of gp120, its binding interactions, allosteric pathways and potential sites for inhibitor targeting. In addition, since the vast majority of HIV-1 infection occurs in Sub-Saharan Africa, where the main viral subtypes are C and A rather than the B subtype responsible for the infection in the United States and Western Europe, it is important that inhibitors are effective against those subtypes since they differ by as much as 30% in their genomes, including gp120. these are the main issues addressed in this project and can be summarized in the following specific aims 1. Thermodynamic characterization of the structural stability and cooperative linkage between binding sites in gp120 by utilizing a combination of microcalorimetric and structure-based thermodynamic studies. 2. Development of thermodynamic guidelines for competitive inhibitors. How do we inhibit gp120 binding without triggering the allosteric activation cascade? 3. Development of thermodynamic guidelines for allosteric inhibitors. Identification of potential binding sites that can be targeted for blocking gp120 allosteric pathways. 4. Characterization of the allosteric inhibitor BMS-806 and analogs, the only gp120 inhibitor in clinical studies. Identification of the BMS-806 binding site. Identification of the critical functionalities in BMS- 806. Development of BMS-806-like inhibitors based upon different chemical scaffolds. 5. Development of thermodynamic guidelines for gp120 inhibitors that are effective against different HIV-1 subtypes and exhibit low susceptibility to potential drug resistant mutants. The studies involve a combination of experimental thermodynamic measurements (isothermal titration calorimetry and differential scanning calorimetry) and structure-based thermodynamic analysis
Keywords: AIDS Virus; Address; Africa South of the Sahara; Binding; Binding (Molecular Function); Binding Sites; C-C CKR-5; C-C Chemokine Receptor Type 5; CC Chemokine Receptor 5; CC-CKR-5; CC-CKR5; CCCKR5; CCR-5; CCR5 Protein; CD195 Antigen; CHEMR13; CKR-5; CKR5; CMKBR5; CXC-R4; CXCR-4; CXCR4; CXCR4 gene; Calorimetry; Calorimetry, Differential Scanning; Cell Communication and Signaling; Cell Signaling; Cell Surface Receptors; Cell membrane; Chemicals; Chemokine (C-C Motif) Receptor 5; Chemokine (C-C) Receptor 5; Clinical Research; Clinical Study; Combining Site; Cytokines, Chemotactic; Cytoplasmic Membrane; D2S201E; Development; Differential Scanning Calorimetry; Differential Thermal Analysis, Calorimetric; Drug resistance; Envelope Glycoprotein gp120, HIV; Event; Exhibits; FB22; Genome; Glycoproteins; Guidelines; HIV Envelope Protein gp120; HIV-1; HIV-1 Fusion Co-Receptor; HIV-I; HIV1; HM89; HSY3RR; HTLV-III gp120; Homologous Chemotactic Cytokines; Human immunodeficiency virus 1; Immunodeficiency Virus Type 1, Human; Infection; Intercrines; Intervention; Intervention Strategies; Intracellular Communication and Signaling; Knowledge; LAP3; LCR1; LESTR; Measurement; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; NPY3R; NPYR; NPYRL; NPYY3R; Pathway interactions; Plasma Membrane; Predisposition; Principal Investigator; Process; Programs (PT); Programs [Publication Type]; Reactive Site; Receptors, CCR5; Receptors, CKR5; SIS cytokines; Series; Signal Transduction; Signal Transduction Systems; Signaling; Site; Structure; Sub-Saharan Africa; Subsaharan Africa; Susceptibility; Thermodynamic; Thermodynamics; Titrations; United States; Viral; Western Europe; analog; base; biological signal transduction; chemoattractant cytokine; chemokine; conformation; conformational state; design; designing; drug resistant; env Protein gp120, HIV; gp120; gp120 ENV Glycoprotein; gp120(HIV); human T cell leukemia virus III; human T lymphotropic virus III; inhibitor; inhibitor/antagonist; interventional strategy; mutant; pathway; plasmalemma; programs; resistance to Drug; resistant to Drug; scaffold; scaffolding
Budget start date: 1-AUG-2010
Budget end date: 31-JUL-2011
5P01GM056550-15_0010 (2010): $380048
3P01GM056550-14S2_0010 (2009): $49200
STRUCTURE BASED THERMODYNAMIC STUDIES OF HIV-1 PROTEASE
Ernesto Freire, Professor
Biologyjohns Hopkins University
w400 Wyman Park Building
baltimore, Md 212182680
Grant 2R01GM057144-04 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: One of the most serious side effects associated with the therapy of HIV-1 infection is the appearance of viral strains that exhibit resistance to protease inhibitors. The main goal of this project is to understand the molecular mechanisms by which specific mutations in the HIV- 1 protease elicit inhibitor resistance and to incorporate that knowledge in the development of new strategies for drug design. A related goal is to understand the effectiveness of inhibitors against protease molecules from different HIV- 1 subtypes. These proteases contain amino acid variations at several locations, including some that have been associated with drug resistance. The main questions that this project will answer are 1) How do specific mutations in the protease molecule preferentially lower the binding affinity of inhibitors relative to the substrate? What are the thermodynamic and structural basis of resistance? 2) How effective are current inhibitors against proteases from different HIV- 1 subtypes? While subtype B is the predominant in the United States and has been the target for drug design, other subtypes are prevalent in Africa, Asia and some European countries, and account for most of the HIV-l infections worldwide. 3) Is the thermodynamic origin of the forces that define the binding affinity of inhibitors related to their susceptibility to resistance-causing mutations? Is it possible to identify protease inhibitors with drastically different enthalpy/entropy balances but similar affinities? Do they respond to resistance-causing mutations in a different way? 4) Is it possible to develop molecular design strategies that explicitly consider the susceptibility to resistance causing mutations? These questions will be answered by a combination of experimental thermodynamic measurements (high sensitivity isothermal titration calorimetry and high sensitivity differential scanning calorimetry), structure determination (x-ray crystallography) and structure-based thermodynamic calculations
Keywords: antiAIDS agent, aspartic endopeptidase, drug design /synthesis /production, drug resistance, drug screening /evaluation, human immunodeficiency virus 1, protease inhibitor, protein structure, thermodynamics chemical binding, enzyme activity, gene mutation, genetic strain X ray crystallography, calorimetry, spectrometry
Project start date: 1998-03-01
Project end date: 2005-02-28
2R01GM057144-04 (2001): $328744
3R01GM057144-04S1 (2001): $42919
5R01GM057144-07 (2004): $428423
3R01GM057144-06S1 (2003): $75851
5R01GM057144-06 (2003): $329787
STRUCTURE AND FUNCTION OF GLYCOLIPID RECEPTORS
Ernesto Freire, Professor
Biologyjohns Hopkins University
w400 Wyman Park Building
baltimore, Md 212182680
Grant 5R01NS024520-09 from National Institute Of Neurological Disorders And Stroke IRG: BBCB
Abstract: Gangliosides are important components of the cell plasma membrane, especially in nervous tissue where they constitute about 10% of the total lipid. It has been found that gangliosides are involved in the recognition machinery of the cell, cell adhesion, cell-cell interactions and that they serve as receptors for glycopeptide hormone, neuropeptides, bacterial toxins and viruses. This research project is directed to study at the molecular level a major aspect of ganglioside function, namely, the role of gangliosides as cell surface receptors using well defined experimental systems. The molecular details and interactions of cholera toxin (CT) and the heat-labile enterotoxin of E. coli with their receptor ganglioside G(m1) will be studied using an experimental thermodynamic approach. The structural stability of these two proteins and their inter- and intra-subunit interactions will be studied in detail. Particularly, functionally relevant molecular interactions will be identified and their energetics measured using state-of-the-art ultra high sensitivity calorimetric techniques. The modulation of cooperative interactions within the toxin molecule by ganglioside G(M1) lead to 1) the attachment of the toxin binding subunits to the cell surface; 2) the release of the toxic subunits from the binding subunits of the toxin molecules; and, 3) the subsequent penetration of the toxic subunits into the interior of the target membrane. These studies will provide a detailed quantitative of the functional energetics of a specific ganglioside receptor
Keywords: chemical structure function, cholera toxin, enterotoxin, ganglioside, membrane activity, receptor, thermodynamics acidity /alkalinity, chemical stability, lipid bilayer membrane, membrane structure, molecular site, protein structure, protonation, receptor binding, temperature calorimetry, circular dichroism, computer simulation
Project start date: 1986-07-01
Project end date: 1996-02-29
5R01NS024520-09 (1994): $199648
Sponsored Links Excellgen http://Excellgen.com
5R01NS024520-08 (1993): $194401
2R01NS024520-07 (1992): $183493
STRUCTURE BASED THERMODYNAMIC STUDIES OF HIV1 PROTEASE
Ernesto Freire, Professor
Biologyjohns Hopkins University
w400 Wyman Park Building
baltimore, Md 212182680
Grant 5R01GM057144-03 from National Institute Of General Medical Sciences IRG: ARRD
Abstract: Adapted from applicant´s ) This grant application is focused on elucidating the molecular basis of clinical resistance to HIV-1 PR inhibitors. Resistant mutants of HIV-1 PR show lower affinity for inhibitors, yet are still able to process viral polyprotein substrates in the course of viral replication. The amino acid sequences of resistant mutants are known from clinical studies. In order to elucidate the molecular basis of resistance to HIV-1 protease inhibitors, the complete binding energetics of substrate and inhibitors to the HIV-1 PR (wt; resistance mutants) will be measured by high sensitivity reaction calorimetric techniques and standard spectroscopic assays. Structure-based thermodynamic analysis will be performed using a novel approach developed in ´s laboratory. This information will be used to develop a structural map of the binding energetics of substrates and inhibitors. The structural map will identify the energetic contributions from each group in the protease, substrate or inhibitor molecules to the overall binding affinity. This map will also identify enthalpic and entropic contributions to the binding energetics, and dissect the entropy contributions into conformation and solvent-related components. Differences in the type and strength of interactions will be evaluated and used to identify why some specific mutations affect the binding affinity of the substrate and inhibitor in different ways
Keywords: antiAIDS agent, aspartic endopeptidase, drug resistance, human immunodeficiency virus 1, protease inhibitor, protein structure, thermodynamics gene mutation calorimetry, spectrometry
Project start date: 1998-03-01
Project end date: 2001-02-28
5R01GM057144-03 (2000): $267655
5R01GM057144-02 (1999): $259952
1R01GM057144-01 (1998): $271566
Ernesto Freire, Professor
Johns Hopkins University W400 Wyman Park Building Baltimore, Md 212182680
Grant 5P41RR004328-09 from National Center For Research Resources IRG: SSS
Project start date: 1988-08-05
Project end date: 1998-08-04
5P41RR004328-09 (1996): $599246