|T H E N I H C A T A L Y S T||J A N U A R Y F E B R U A R Y 2006|
|P E O P L E|
Carole Bewley received her Ph.D. in 1995 in a joint program in chemistry and oceanography from Scripps Institution of Oceanography, University of California, San Diego. She did postdoctoral work in protein NMR in the Laboratory of Chemical Physics, NIDDK, on a Cancer Research Institute fellowship and in 1999 joined the Laboratory of Bioorganic Chemistry, NIDDK, as a tenure-track investigator. She is currently a senior investigator and chief of the Section on Natural Products Chemistry.
My research focuses on three main areas: the discovery and study of biologically active natural products, the design and synthesis of peptide and protein inhibitors of HIV-1 entry, and the discovery and characterization of novel carbohydrate-binding proteins.
Why natural products? Natural products are usually, but not limited to, small organic molecules produced by plants, bacteria, fungi, and lower eukaryotes such as invertebrates, to name a few. Natural products can also include peptides, proteins, and other larger-molecular-weight toxins.
There is abundant evidence that natural products bestow an increased level of fitness on the organism that produce them by providing a means of chemical defense, a primitive equivalent of higher organisms' immune systems.
Having evolved over millions of years to fit into specific receptors and thereby effect biological processes, natural products are endowed with chemical and three-dimensional properties that synthetic molecules may lack.
Natural products therefore represent ideal starting points for identifying inhibitors for, arguably, any biological process.
Thus, in the broadest sense, my laboratory is interested in identifying new natural product structures, especially those found in marine invertebrates and cyanobacteria, that exhibit interesting biological activities; we are also interested in determining the mechanism of action by which they inhibit the system of interest, and, ultimately, in pinpointing the structural basis for their activity.
Two systems that we study intensely and try to inhibit are HIV-1 entry into cells and mycothiol biosynthesis and detoxification in Mycobacterium tuberculosis (MTB), a pathway essential to MTB viability. As chemists, our efforts are divided between discovery-driven and hypothesis-driven research.
We use a variety of techniques to answer questions, including natural products chemistry, synthetic organic chemistry, and NMR spectroscopy for solving chemical and three-dimensional structures and mapping binding sites; biophysical techniques to describe modes and affinities of binding; and, of course, biological assays to provide lead molecules.
Several years ago, we identified a
class of natural products originating from a marine sponge extract in NCI's
Open Repository, that effect mycothiol biosynthesis and detoxification in
MTB. Mycothiol is a small-molecular-weight
thiol unique to actinomycetes that replaces glutathione in this group of bacteria.
These compounds feature an unusual oxygen- and nitrogen-containing spiro-ring system that is key for competitive inhibition of the mycothiol biosynthetic and detoxification enzymes MshB and MCA, respectively.
Using these natural products as structural leads and inspiration, we recently completed the synthesis of a small natural productlike synthetic library whose structures combine important chemical features from the natural products with those important to the substrates. Within these second-generation inhibitors are two compounds that are lethal to MTB at low microgram doses.
In addition to having created a new class of MTB inhibitor, we also have in hand synthetic compounds that can readily be manipulated for further biological studies and can be used as probes for mycothiol metabolism in MTB.
A second example includes our work on novel carbohydrate-binding proteins isolated from marine cyanobacteria, also known as blue-green algae. It is becoming apparent that all cells and most viruses display on their surfaces specific carbohydrate structures or carbohydrate-binding proteins, or both, that are used for attachment, adhesion, and cell-to-cell recognitionespecially noteworthy in the interactions between pathogens and their target cells. Protein-carbohydrate interactions govern or have been implicated in myriad recognition and binding events, such as sperm-egg interactions leading to fertilization, leukocyte homing during the course of inflammation, and trafficking of tumor cells during metastasis.
I became interested in these types of molecules from earlier high-resolution structural and mechanistic studies of cyanovirin-N, a potent HIV-1 fusion-blocking cyanobacterial protein originally discovered by NCI scientists and coincidentally also originating from NCI's Natural Products Repository.
Using a combination of multidimensional heteronuclear NMR techniques, isothermal titration calorimetry, and an HIV-1 fusion assay, we showed that cyanovirin-N contains two novel carbohydrate-binding motifs encoded into a single polypeptide chain that can bind with nanomolar affinities a small disaccharide ligand identical to the terminal arms of branched N-linked oligomannose structures.
This result was unheard of at the time: Carbohydrate-binding proteins almost universally bind their saccharide ligands with very weak affinities (high micromolar to millimolar) and typically oligomerize to augment avidity and selectivity. Not only was the demonstrated specificity and affinity unprecedented, the studies also demonstrated that cyanovirin-N exerts its potent antiviral activity through high-affinity interactions with high-mannose structures that are abundant on the HIV surface envelope glycoprotein gp120. Furthermore, cyanovirin-N exhibited a novel three-dimensional fold that to date cannot be placed into other known protein families.
A logical extension of these findings has led to a second large component of our research that is devoted to the discovery of other novel carbohydrate-binding proteins. These molecules are fascinating because they greatly expand our knowledge and understanding of protein-carbohydrate recognition and the structural and dynamic features that are necessary for high-affinity carbohydrate recognition. They also provide potentially valuable reagents for inhibiting or probing virus-cell or pathogen-cell interactions.
We have recently published structural and biological studies on MVL, another cyanobacterial protein that potently blocks HIV-1 entry, albeit through carbohydrate-mediated interactions that are entirely distinct from those of cyanovirin-N.
Identification of a second protein with novel, high-affinity carbohydrate-binding properties and antiviral activity ensures that these organisms and the natural products they produce will continue to hold our interest.
Druey received his M.D. degree from Rush Medical College
in Chicago in 1987. After completing a residency in internal medicine at The
New York Hospital/Weill Cornell Medical Center in New York in 1990, he joined
NIH in 1991 as a clinical associate in the Allergy
and Immunology Training Program in NIAID and went on to complete his postdoctoral
training in the B-cell
Molecular Immunology Section of the Laboratory
of Immunoregulation. In 1997, he became acting head of the Molecular
Signal Transduction Section of the Laboratory
of Allergic Diseases (LAD), NIAID.
As a kid with asthma, I wanted to understand how ordinary things that other people seemed to have no problem withmy dog, the horses I rode, the dusty barn, lawn mowing made it so difficult for me to breathe. Initially, I pursued a career pathmedicineto help others with this disease. But the more I counseled and treated patients with asthma, the more I felt compelled to understand what caused it on a molecular level.
Asthma is a collection of symptoms including wheezing and shortness of breath. And although there are characteristic lung abnormalities such as hypercontractility of bronchial smooth muscle and extensive lung inflammation induced by allergen exposure, there is no single known etiology of asthma.
After my clinical training in allergy and immunology at the NIH Clinical Center and during postdoctoral training with John Kehrl in the Laboratory of Immunoregulation, NIAID, I investigated signal transduction pathways in the immune system.
In particular, I became interested in G proteincoupled receptors (GPCRs), which are by far the most common cell-surface receptors in the mammalian genome. These receptors rely on a molecular switchthe heterotrimeric G protein, which cycles between GDP- and GTP-bound formsto transmit their signals. In asthma, GPCRs control not only the contractility of bronchial smooth muscle but also entry of inflammatory cells into the lung.
During the course of this postdoctoral work, I was instrumental in the discovery of a new family of regulators of G proteinmediated signal transduction. These regulators of G protein signaling, or RGS proteins, help determine the amplitude and timing of GPCR signaling in response to extracellular ligands.They bind to the a subunit of the G protein and accelerate its rate of GTP hydrolysis.
This large family of proteins (more than 25 members in mammalian cells) exhibits some promiscuity. That is, most RGS proteins bind similar G protein substrates although several RGS proteins are expressed in the same cell. Therefore, we asked how individual RGS proteins regulate specific GPCR pathways and what functions these proteins might have in the pathogenesis of asthma.
During my time as a tenure-track investigator in the LAD, I tried to address some of these issues, starting with straightforward biochemical questions. For example, how is the activity of certain RGS proteins regulated? We found key residues shared by many of these proteins that were sites of phosphorylation or palmitoylation.
These modifications directly affected the activity of a prototypical RGS protein, RGS16, by altering its subcellular localization or stability. Using genetic screening and assessment of various signaling pathways, we identified new binding partners for this and other RGS proteins that may also regulate RGS activity or implicate them in unique functions outside of the G protein realm.
The basic structure-function studies have set the foundation for us now to ask how certain RGS proteins control GPCR activity in individual cell types from normal and asthmatic lung. For example, mast cells are crucial initiators of the allergic process in the lung. Mast cells bind allergens, which then cross-link membrane-bound IgE antibody and cause the mast cells to degranulate and release inflammatory mediators. These compounds set off an allergic cascade culminating in bronchial hyperreactivity.
Surprisingly, we have found that mouse mast cells deficient in RGS13 exhibit markedly enhanced degranulation to IgE-allergen stimulation in vitro and dramatically increased anaphylaxis responses in vivo. Thus, RGS13 may normally suppress IgE-mediated allergic reactions, which are not known to be G-protein dependent. These results suggest that RGS proteins may control multiple intracellular signaling networks.
I believe these research efforts address critical problems in understanding allergic diseases generally and asthma specifically. In the near future, we plan to focus on understanding how RGS proteins control contractility of bronchial smooth muscle and the activation and migration of immune cellsboth integral to the development of pathological abnormalities found in asthma.
Alexander Pletnev earned his Ph.D. in chemistry in 1983 from the Institute of Molecular Biology and Genetics, USSR Academy of Sciences in Novosibirsk. In 1990, he received his Doctorate of Sciences Degree in biochemistry and molecular biology from the Institute of Molecular Biology, USSR Academy of Sciences. He joined NIAID in 1991 as a visiting scientist and initiated a successful research program to develop vaccines against diseases caused by flaviviruses. In 1997, he became a tenure-track investigator in the Laboratory of Infectious Diseases, NIAID, and is currently a senior investigator.
My long-term interest in tickborne encephalitis stemmed from the high prevalence of this disease in Europe and Asia due to the highly neurovirulent tick-borne encephalitis viruses (TBEV). This illness is rare in North America. TBEV is transmitted to various mammal species and causes human disease of varying severity, with up to 30 percent mortality. Most of these viruses are "select agents" (assigned to biosafety level 3 or 4), based on their high lethality and their potential for human infection by the oral or aerosol route.
Currently, a vaccine produced by formalin inactivation of TBEV is available in Europe, but multiple inoculations are needed for effective immunity, and the breadth of its protective effect has been questioned. The goal of my research program is to develop a safe live attenuated virus vaccine that provides durable immunity after a single inoculation against the most neurovirulent members of the TBEV complex.
We developed a novel approachchimerizationfor the construction of live attenuated flavivirus vaccines. This pioneering strategy was based on conservation among flaviviruses of genome organization, number of viral proteins, replicative strategy, gene expression, virion structure, and morphogenesis. Specifically, this strategy involves the construction of a viable antigenic chimera from two heterologous flavivirusesthe structural protein genes of a full-length cDNA clone of a non-neuroinvasive partner (mosquito-borne dengue type 4 virus; DEN4) are replaced by the corresponding structural protein genes of another flavivirus that is neuroinvasive and against which protective immunity is sought. Because the structural proteins induced neutralizing protective antibodies, chimeric virus can be used as vaccine against the donor of the structural proteins.
I found that chimerization of TBEV or Langat virus (LGT; a member of the TBEV complex) with DEN4 completely ablated detectable neuroinvasiveness (the ability of virus to spread from peripheral tissues to the central nervous system, where it produces fatal encephalitis). Chimeras were immunogenic in mice and able to induce resistance against challenge with TBEV or LGT. Chimeric viruses were also attenuated, immunogenic, and efficacious in rhesus monkeys.
To determine the safety, infectivity, and immunogenicity of the LGT/DEN4 vaccine, a Phase I clinical trial in healthy adults was initiated at the Johns Hopkins School of Public Health Center for Immunization Research in Baltimore.
As a logical extension of our strategy for the development flavivirus vaccines, I have added West Nile virus (WN), a mosquito-borne flavivirus, to my research agenda. WN was chosen for study because this agent recently entered the United States for the first time and spread rapidly throughout North America, where it has produced severe neurological disease in humans, domestic animals, and birds. A high degree of attenuation for mice, geese, horses, and monkeys was achieved by chimerization of WN with DEN4. Despite the high level of attenuation in mice and monkeys, both the WN/DEN4 chimera and its deletion mutant WN/DEN4D30 induced a high titer of neutralizing antibodies and provided complete protection of animals against lethal WN challenge.
Currently, the WN/DEN4D30 vaccine is under evaluation for safety and immunogenicity in a clinical trial in healthy volunteers. In order to prevent sporadic or epidemic encephalitis caused by the other neurotropic flaviviruses such as St. Louis encephalitis, Powassan, or Japanese encephalitis virus, I plan to develop vaccines using the chimerization approach that has been successful for WN and TBEV viruses.
Jun "Jim" Zhang
Jun "Jim" Zhang obtained his medical degree from the Shanghai Medical University, China, in 1988, followed by an internship at the International Peace Maternity and Child Hospital in Shanghai. He was later certified by the U.S. Educational Commission for Foreign Medical Graduates (ECFMG). He received his Ph.D. degree in epidemiology from the University of North Carolina at Chapel Hill in 1994. He conducted research with both Family Health International in North Carolina and the World Health Organization in Geneva, Switzerland. He was an assistant professor at the Mount Sinai School of Medicine in New York before joining the Epidemiology Branch at NICHD in 1997 as a tenure-track investigator. He is currently a senior investigator.
My research focus has been on obstetric and perinatal issues that affect a large number of pregnant women and address the safe and efficacious clinical management of women in labor. Highlights of my research include the following studies:
Empirical evaluation of the effect of epidural analgesia for labor pain on labor progress and the need for operative interventions such as Caesarean or forceps delivery. We found that epidural analgesia use does not result in an increased risk of prolonged labor, Caesarean delivery, or other unfavorable events during labor and delivery compared with intravenous analgesia.
Empirical evaluation of the existing labor curve currently used by clinicians with regard to labor progression or failure and the need for clinical intervention. Our study showed that the current diagnostic criteria of labor protraction and arrest were too stringent for contemporary obstetric populations, leading to excessive use of Caesarean delivery.
Empirical evaluation of labor progression and risk of Cesarean delivery in electively induced labor. We found that nulliparous women with an unfavorable cervix whose labor was induced had a high rate of labor arrest and a threefold increased risk of Caesarean delivery compared with nulliparous women with spontaneous onset of labor. Our study called for judicious use of labor induction in women delivering their first babies.
Randomized clinical trial on medical management with misoprostol for early pregnancy failure (or miscarriage). Our trial demonstrated that misoprostol is a safe, effective, well-tolerated, and inexpensive alternative to surgical management for early pregnancy failure.
Today, more than one in four pregnant women in the United States deliver by Caesarean section, and the rate continues to rise. I am launching a large observational study to describe labor patterns in contemporary obstetric U.S. populations, with the aim of combating this rising Caesarean rate and identifying appropriate times to perform Caesarean delivery warranted by labor arrest. Findings from this study are anticipated to have a direct impact on obstetric practice.
In addition, identifying and diagnosing fetal growth restriction has been a long-standing challenge in modern obstetric and perinatal research.
Pivotal to understanding the dynamics of human fetal growth and defining normal and abnormal fetal growth is the development of standards for fetal anthropometric parameters measured longitudinally throughout gestation. These parameters can be used to develop interval velocity curves and can be customized for physiological and genetic factors.
I am developing a prenatal ultrasound study to establish a U.S. national standard for normal fetal size and growth velocity at various gestational ages; the study will also provide a basis for an individualized standard for optimal fetal growth to improve the precision with which fetal growth restriction or excessive growth is diagnosed. As part of the study, I will also develop a new formula to improve fetal weight estimation by ultrasound and collect biological samples to study the causes of idiopathic fetal growth restriction.
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