Myles H. Akabas - US grants

The sense of taste provides animals with a rapid, though limited chemical analysis of a potential food substance. Based on this information the animal decides whether to ingest or expel the substance. In humans, disorders of taste lead to anorexia and subsequent weight loss, which complicates the management of the underlying diseases. The pathophysiology of taste disorders is poorly understood because little is known about the molecular mechanisms involved in the transduction of any taste modality. Our previous studies have demonstrated that bitter taste transduction involves cell surface receptors. Binding of ligand to these receptors stimulates release of calcium from internal stores suggesting the involvement of the inositol triphosphate pathway in the bitter transduction process. The transduction of sweet taste appears to involve a different mechanism. Purification or molecular cloning of the taste receptors would enhance our understanding of the primary events in taste transduction, however, to date none of the taste receptors have been purified or cloned. The long-term goal of this investigation is to elucidate the molecular mechanisms involved in sweet taste transduction. To accomplish this goal, the technique of DNA-mediated gene transfer will be used to establish a mouse cell line expressing the human sweet taste receptor. To assay for expression of the human sweet taste receptor the two sweet proteins, thaumatin and monellin, will be used in an erythrocyte rosetting assay utilizing anti- thaumatin antibodies coupled to the erythrocytes to detect transformed mouse L-cells expressing the human thaumatin-binding sweet taste receptor. Following establishment of the cell line, the gene for the sweet taste receptor will be molecularly cloned from this cell line. The cell line will also facilitate studies of the mechanism of sweet taste transduction to identify second messenger processes involved in the transduction process. The specific goals of this proposal are: 1) To establish a mouse L-cell line expressing the human sweet taste receptor by the technique of DNA- mediated gene transfer; 2) To utilize the cell line to molecularly clone the human sweet taste receptor, and 3) To utilize the cell line to study the mechanism of sweet taste transduction.

DESCRIPTION (provided by applicant): The GABAA receptors are members of the neurotransmitter-gated ion channel gene superfamily that includes nicotinic acetylcholine, serotonin 5-HT3 and glycine receptors. The GABAA receptors mediate fast inhibitory synaptic transmission in the central nervous system. A long term goal of molecular neuroscience has been to understand the structural bases for the functional properties of these receptors and for their modulation by clinically used medicines and by drugs of abuse. Structure-function studies of these receptors took a quantum leap on June 25, 2003 with the publication of a 4Angstrom resolution closed state structure of the homologous 'Torpedo' acetylcholine receptor (AChR) (Miyazawa et al., 2003). At 4A resolution this structure provides a solid foundation for future studies of protein dynamics and agonist-induced conformational changes. At 4Angstrom resolution the peptide backbone path is well defined but the individual amino acid side chain positions are poorly defined. As expected, in the transmembrane (TM) domain each subunit contains four alpha-helical segments (M1, M2, M3, M4) with the M2 segment forming the channel lining and gate. Surprisingly, the helical TM segments extend approximately 10Angstrom above the extracellular membrane surface where they interact with the largely beta-strand, extracellular, agonist-binding domain. A critical interaction between the extracellular and TM domains is via a residue in extracellular Loop 2 and residues in the M2-M3 loop. This proposal will focus on three aspects of GABAA receptor structure: 1) verifying the applicability of the AChR structure to the GABAA receptor, 2) studying the dynamic motion of the channel in the closed state and 3) studying the conformational changes that occur during channel gating from the closed to the open/desensitized states. Aim #1 will test the hypothesis that the AChR structure is a good model for the GABAA receptor by testing predicted proximity relationships between the M2 and M3 and between the M2 and M1 segments within a subunit. Aim #2 will test Unwin and colleagues' gating hypothesis. Aim #3 will probe changes in the M2 segment tertiary and quaternary structure during gating. Aim #4 will probe the tightness of protein packing around the extracellular half of the M2 segment from the 12' to the 27' levels. Aim #5 will probe the mobility and flexibility of the extracellular helical extension of the M2 segment from the 21' to the 27' level. Successful completion of the proposed experiments will either confirm the AChR structure or will provide an experimental basis for refining the structure. In addition, completion of the proposed experiments will provide new insights into the dynamics of the GABAA receptor channel-lining domain in the resting state and as the channel undergoes its agonist-induced conformational changes.

DESCRIPTION (Taken directly from the application) The long term goal of this research is to elucidate the structural basis of ion conduction, selectivity and gating in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR, the protein which is defective in cystic fibrosis, is a chloride channel whose activity is regulated by phosphorylation and ATP binding. Although the functions of CFTR have been extensively studied, the structure of CFTR, like most other integral membrane proteins, is not well determined. The anion channel is presumably lined, at least in part, by residues from the 12 membrane-spanning segments. The goal of this project is to identify systematically the residue that line the CFTR channel using the scanning-cysteine accessibility method. In this approach reporter cysteines are substituted, one at a time, into putative channel-lining segments. Each cysteine-substitution mutant is expressed in Xenopus oocytes and the water-surface exposure of the cysteine is determined by its ability to react with small, negatively and positively charged, sulfhydryl-specific reagents which are derivatives of methanethiosulfonate. For residues in membrane- spanning segments, we infer that if an engineered cysteine reacts with the reagents then the corresponding wild-type residue is exposed in the channel lumen. By this approach we have already identified 18 channel-lining residues in the M1 and M6 membrane-spanning segments. We will systematically identify the other residues that line the CFTR ion channel, and determine their secondary structure, and the position of the gate, the charge-selectivity filter and the size- selectivity filter. The success of this project will allow us to create a low resolution structural model of the CFTR channel. A model of the channel will provide new insights into the molecular mechanisms underlying ion conduction, selectivity and gating and help to elucidate the mechanism(s) by which disease causing mutations alter CFTR channel function.

DESCRIPTION (provided by applicant): The mission of the Albert Einstein College of Medicine Medical Scientist Training Program (MSTP) is to train physician-scientists who will become future leaders in biomedical and clinical research. It strives to recruit a diverse group of outstanding students and to provide them with rigorous combined medical and research training that prepares them for careers as physician-scientists. Through a flexible and continuously evolving curriculum, the students'are guided with a program that can be tailored to individual needs and interests. The program seeks to provide the trainees with a unique foundation for careers as independent physician- scientists and to facilitate their placement into outstanding postgraduate training programs to enable the next step in their career progression. The training program has 3 phases. In the first 2 years students take an integrated combination of medical, graduate and MSTP-specific courses to provide the didactic foundation for their research and clinical training. They perform research rotations to assist in choosing their thesis research lab. In the program's 2nd phase they perform independent, original research under their mentor's guidance, publish their discoveries and prepare and defend a Ph.D. thesis. In the final phase, they complete their clinical training. The admissions process focuses on academic excellence, prior research experience and enthusiasm for a research career. 124 trainees are in the program, 43% are woman and 15% are members of underrepresented minorities. Since its inception in 1964 as one of the first NIH MD-PhD training programs, 289 trainees have graduated. 224 have completed postgraduate training and published over 10,000 papers, an average of 53 per graduate. 82% have jobs at academic medical centers, research institutes, and NIH or pharmaceutical companies. By various measures the program graduates have achieved outstanding success in their chosen careers and have contributed to the advancement of biomedical research and academic medicine. Based on the quality of our past accomplishments we propose to expand the program, to further integrate graduate and medical training, and increase opportunities for involvement in clinical and translational research in order to prepare a future generation of physician-scientists who will be at the leading edge of biomedical research with the ultimate goal of improving human health and reducing the burden of disease.

DESCRIPTION (provided by the applicant): Malaria is a major public health problem in much of the developing world. Until drug resistance developed, chloroquine was the treatment of choice for Plasmodium falciparum malaria because it was inexpensive, relatively safe and effective. Chloroquine resistance in P. falciparum results from mutations in the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT). PfCRT is an integral membrane protein localized in the parasites' digestive vacuole membrane. Sequence analysis predicts that PfCRT has ten membrane-spanning segments. PfCRT's endogenous function and its molecular interactions with chloroquine are unknown. PfCRT is an ideal anti-malarial drug target because there are no close human PfCRT homologues and because PfCRT knockout produces non-viable parasites implying that PfCRT has an essential role for normal parasite physiology. In order to screen for potential new drugs one needs a functional assay for PfCRT. A major goal of this application is to develop such a functional assay. When expressed heterologously in human embryonic kidney HEK-293 cells we showed that PfCRT is targeted to the lysosomal membrane, the digestive vacuole homologue. This application's goals are divided into two groups. The first group, Aims 1 and 2, will provide a basis for future structure-function studies of PfCRT by determining experimentally the transmembrane topology using epitope tag insertion and selective permeabilization (Aim 1) and by determining whether PfCRT is a dimer in the membrane using FRET (Aim 2). Aims 3 to'5 form the second group of experiments. They are a search for functional assays of PfCRT based on its localization in the lysosomal membrane in our heterologous expression system. These experiments are designed to identify a functional assay for PfCRT or a functional effect of PfCRT expression on lysosomal function. It is the current lack of such an assay and the fact that this is a high risk search that has motivated us to apply for an R21 grant, which is exploratory in nature, rather than an RO1.

DESCRIPTION (provided by applicant): Each year millions of people undergo general anesthesia so that surgeons can perform life-saving operations. For most patients general anesthesia is safe and effective, however, side effects, such as nausea and vomiting, and complications, such as cardiovascular instability or cognitive dysfunction, increase anesthesia-associated morbidity. Understanding the molecular basis of general anesthetic action may lead to the identification of newer and safer general anesthetics. The long term goal of this project is to elucidate the molecular bases of general anesthesia. Propofol, a widely used intravenous general anesthetic, is the major focus of this project. At concentrations used clinically, propofol interacts with many proteins, however, a major target for its anesthetic effects is the GABA-A receptors. This project will focus on propofol's interactions with GABA-A receptors. In order to understand the molecular basis of propofol's action we must identify the amino acids that form its binding sites, elucidate the conformational changes that it induces, and determine how these conformational changes modulate receptor function. Our ability to accomplish these goals took a quantum leap forward with the recent publication of the homologous acetylcholine binding protein (AChBP) and nicotinic acetylcholine (ACh) receptor structures. These structures provide a solid foundation for structure-based studies of anesthetic interactions with GABA-A receptors. This structural information will guide our experimental design and the interpretation of our results. We propose the following Specific Aims: 1) To identify the residues that lie in close proximity to the propofol binding site near the beta subunit Met286 position and to determine the orientation of propofol in the binding site using sulfhydryl- reactive propofol derivatives. 2) To probe the extent of conformational changes induced by potentiating concentrations of propofol in the M2 segment that lines the ion channel and forms the channel gate. These experiments will serve the dual purposes of verifying the applicability of the ACh-derived structural information to GABA-A receptors and placing structure-function studies of anesthetic interactions with ion channels on a sound structural basis. The successful completion of this project will provide dynamic information on propofol's interactions with GABA-A receptors and new insights into the molecular basis of general anesthetic action. They may provide a rational basis for design of new, safer general anesthetics.

DESCRIPTION (provided by applicant): The racial and ethnic diversity of the American population is not reflected in the biomedical research workforce or in the faculty at research universities and medical schools. Lack of intensive research experiences during their undergraduate studies leaves many underrepresented minority students unprepared to apply to highly competitive Ph.D. and MD-Ph.D. programs. The goal of this Post-Baccalaureate Research Education Program (PREP) is to provide underrepresented minority students who want to pursue a career in biomedical research with the intensive research experience and academic enrichment to make them competitive applicants to rigorous Ph.D. and MD-Ph.D. programs. Einstein has a long history of successfully mentoring underrepresented minority students through both its Ph.D. and MD-Ph.D. programs. We will use a layered mentoring system to support the PREP trainees and integrate them into the Einstein community. Each trainee will have a URM Ph.D. and MD-Ph.D. peer mentor, a faculty Program Advisor and a research mentor. The program will begin with a two week Orientation and Boot Camp period. Trainees will work with their Program Advisor and research mentor to formulate an Individual Development Plan (IDP). The mentored research project will emphasize the process of hypothesis-based research, the excitement and joy of scientific discovery, and the persistence and creativity needed to achieve success. The academic component of the program will include: 1) workshops on study skills/time management and library/bibliographic search techniques, 2) a graduate course in Biomedical Research Methods and Analysis, 3) a month long Medical Correlation and 4) a weekly PREP journal club. The course will provide a conceptual understanding of the major experimental approaches used in modern biomedical research. The PREP Journal Club will teach critical reading of the literature. Throughout the program special emphasis will be placed on development of oral, written and poster communication skills through lectures, workshops and individual practice. Trainees will perfect their professional skill through Career & Professional Development workshops and perform videotaped mock interviews to improve their ability to communicate their strengths in the interview process for Ph.D. or MD-Ph.D. programs. In collaboration with the Hispanic Center of Excellence, trainees will be involved in community outreach through visits to local Bronx high schools with a high percentage of URM students. This combination of didactic and experiential learning will prepare the applicants to apply successfully to highly competitive Ph.D. and MD-Ph.D. programs. We expect that more than 75% of the trainees will matriculate and successfully complete such programs. This will expand the diversity of our own graduate programs and ultimately the faculty at Einstein and beyond.

? DESCRIPTION (provided by applicant): Infection with unicellular eukaryotic Plasmodium species parasites causes malaria. P. falciparum causes the most virulent form of malaria. Currently, artemisinin combination therapy (ACT) is the treatment of choice for infected individuals. The rise of artemisinin resistant P. falciparum in Southeast Asia makes it imperative to develop new antimalarial drugs. Malaria parasites are purine auxotrophs. They transport purine precursors from the host erythrocyte into the parasite via the P. falciparum Equilibrative Nucleoside Transporter 1 (PfENT1). In the parasite, purine salvage pathway enzymes modify the purine precursors to form the nucleotides needed for RNA and DNA synthesis and other cellular processes. At purine concentrations found in human blood (<10 µM), PfENT1 knockout parasites are not viable in culture. Thus, PfENT1 inhibitors may function as potent antimalarial drugs. The goal of this project is to explore the therapeutic hypothesis that inhibition of PfENT1 will kill malaria parasites and provide a novel target for antimalarial drug development. We have developed a simple, robust yeast cell growth assay and used it in a high throughput screen (HTS) to identify PfENT1 inhibitors. 5-fluorouridine (5-FUrd) kills wild type Saccharomyces cerevisiae. Mutant fui1? yeast that lack the endogenous plasma membrane purine/uridine nucleoside transporter are 100 times more resistant to 5-FUrd. PfENT1 transports 5-FUrd. In the presence of 125 µM 5-FUrd, PfENT1-expressing fui1? yeast will only grow if a PfENT1 inhibitor is present to prevent 5-FUrd uptake. In 384 well plates, the Coefficient of Variation was <6.2%, Signal Window > 12, and the Z' score > 0.80, indicating a highly robust assay. We screened a 64,500 compound library and identified 171 hits. We tested nine of the top hits in a series of secondary assays. All nine inhibited [3H]adenosine uptake into both PfENT1-expressing yeast and into erythrocyte-free trophozoite stage P. falciparum with IC50 values in the 2 - 40 nM range. The nine compounds, five distinct chemical scaffolds, do not kill yeast but do kill P. falciparum parasites in culture with IC50 values in the 5 - 55 µM range. The goals of this application are 1) to improve the potency and selectivity of the PfENT1 inhibitors through medicinal chemistry; 2) to define the mechanism of action of the inhibitors and their impact on parasite biology and growth at various life cycle stages; 3) to test the efficacy of the inhibitorsin a mouse malaria model; and 4) to identify the inhibitor binding site and the conformation of PfENT1 to which the inhibitors bind. Successful completion of this project will determine the utility of targeting PfENT1 for antimalarial drug development and may identify compounds suitable for further development.

Project Summary/Abstract Neither the biomedical research workforce nor the faculty at research universities and medical schools reflect the racial and ethnic diversity of the American population. In part, this is due to the limited number of underrepresented minority (URM) students who enter PhD and MD-PhD programs. Lack of substantive research experiences during undergraduate studies leaves many URM students unprepared to apply to competitive graduate programs. The goal of this Post-Baccalaureate Research Education Program (PREP) is to provide URM and disabled students, who want to pursue a career in biomedical research, with the intensive research experience and academic enrichment necessary to make them competitive applicants to rigorous PhD and MD-PhD programs. Einstein has a long history of successfully mentoring URM students through both its PhD and MD-PhD programs. The Einstein PREP has four major components designed to prepare trainees to apply successfully to PhD or MD-PhD programs: 1) an intensive, mentored laboratory research experience, 2) a didactic component to increase trainees' scientific knowledge and prepare them for interdisciplinary research, 3) an oral and written communications skills enhancement program, and 4) a layered mentoring system to provide the trainees with the support network they need to flourish in the program and beyond. These four components are interwoven throughout the year long program. The program begins with a two week Orientation during which trainees select their research mentor. The mentored research project emphasizes the process of hypothesis-based research, the excitement and joy of scientific discovery, and the persistence and creativity needed to achieve success. Trainees work with the Program Directors and research mentor to formulate an Individual Development Plan. Each trainee is assigned either a URM PhD or MD-PhD student peer mentor. The Einstein Minority Scientist Association will run a monthly PREP Journal Club and other social and scientific activities. The Program Directors will meet with the trainees on a weekly basis for informal and didactic sessions. This combination of didactic and experiential learning will prepare the applicants to apply successfully to highly competitive PhD and MD-PhD programs. We expect that more than 75% of our trainees will matriculate and successfully complete such programs. To date, 19 trainees have entered the program, 12 have completed and all are enrolled in PhD or MD-PhD programs, i.e., 100% success. Of the 7 trainees currently in the program, one applying to MD-PhD programs already has an acceptance; five applying to PhD programs already have multiple interview invitations. We expect that all will successfully matriculate into graduate programs. The seventh trainee will spend a second year in the program while she applies to MD-PhD programs. Our PREP alumni will ultimately expand the US biomedical workforce diversity.