Difference: VernSchramm (1 vs. 5)
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Public Information on Grants Associated with NYSBC Grant Number: 2R01GM041916-20 Project Title: Transition State Analysis of Enzymatic Reactions PI Information: Name Email Title SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR Abstract: DESCRIPTION (provided by applicant): Transition state (TS) analysis from isotope effects and computational chemistry provides frontier technology for understanding the chemistry of bond change at the instant of enzymatic TS formation. Transition state analogues can be designed from the molecular electrostatic potential surfaces of TSs and have provided unique design parameters for some of the most powerful enzymatic inhibitors known. First and second generation TS analogues for human purine nucleoside phosphorylase (PNP) have advanced from first principles of TS design into human clinical trials for cancer and autoimmune diseases. Third generation PNP inhibitors will be compared to 1st and 2nd generation analogues for binding, structure, thermodynamics and biological lifetimes on PNP in cells. Binding isotope effects are an emerging technology for understanding the geometric and electronic constraints experienced by molecules as they become immobilized at their binding sites on macromolecules, including enzymes and receptors. Binding isotope effects will explore the atomic constraints of substrates and tight- binding TS analogues at the binding sites of human PNP. A surprising diversity of TS structure exists in the same enzyme isolated from different species, establishing the possibility of species-specific TS analogue design. Transition state structures of bacterial 5'- methylthioadenosine nucleosidases (MTANs) will be solved and matched to specific analogues for affinity and structures of reactant and TS-complexes. Biological efficacy of MTAN inhibitors will be analyzed in bacterial quorum sensing pathways. In theory, all enzymatic TSs should be accessible to isotope effect analysis but some provide technical challenges because the chemical step is obscured by non-chemical steps. Human thymidine phosphorylase is a prototype for kinetically difficult TS analyses. TS analysis methods will be established to expose the chemical step by rapid reaction kinetics and altered reaction conditions. Atomic understanding of enzymatic TS chemistry has been developed primarily in enzymes involved in N-ribosyltransferases and deaminases. Expanding the frontier of TS analysis to hydrolysis at carbonyl carbons will be accomplished in the well-known system of HIV-protease and in the important but poorly understood target of human 2'-O-acetyl-ADP-ribosyl esterase. Goals of this research are to push the frontier of enzymatic TS theory to enhance understanding of catalysis and drug design for human targets. PUBLIC HEALTH RELEVANCE: Transition state theory provides an approach to design better drugs for human disease. Expanded methods of drug design will be applied to targets of human disease. Purine nucleoside phosphorylase is a target for leukemia and for autoimmune diseases including psoriasis and tissue transplant rejection; methylthioadenosine phosphorylase is a target for antibiotic-resistant bacteria; thymidine phosphorylase is a target for solid tumors; HIV protease is a target for AIDS infections; and acetyl-ADP-ribosyl hydrolase is a target for diseases of ageing. New methods will be established for the broader application of this theory and the results may lead to new drugs to treat cancer, autoimmunity, bacterial infections and diseases of ageing. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 01-AUG-1989 Project End: 31-JUL-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 1R01CA135405-01A1 Project Title: Transition State Analogues as Modulators of DNA Methylation PI Information: Name Email Title | |||||||||||||||||||||||||||
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| < < | SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR | ||||||||||||||||||||||||||
| > > | SCHRAMM, VERN L. | ||||||||||||||||||||||||||
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| Abstract: DESCRIPTION (provided by applicant): Transition state analogue design is a frontier technology for targeting specific enzymes in human disease. MT-DADMe-Immucillin-A is an orally available transition state analogue inhibitor for human 5'- methylthioadenosine phosphorylase (MTAP). MTAP inhibition slows or prevents the growth of human head and neck, prostate and human lung cancers in mouse xenografts. Normal tissues are not affected and the inhibitor shows no toxicity against normal cells or to mice. The MTAP inhibitor alters metabolites that are expected to change the ability of DNA methyltransferases to methylate DNA. Cancers are commonly caused by mutations that change gene expression patterns and permit the growth and metastases of tumors. Gene expression patterns leading to cancer are governed, in part, by DNA methylation at regions of the genome rich in CpG bases, called CpG islands. The hypothesis for this research is that MTAP inhibitors alter metabolite levels in cancer tissues to inhibit DNA methylation patterns in humans. Loss of methylation for some of the CpG islands near cancer suppression genes is proposed to alter the gene expression patterns of the cancer cells and to slow or prevent cancer cell growth. This hypothesis will be explored in cultured cell lines and mouse xenograft models of the major human malignancies, lung, breast, prostate colon, head and neck and cervical cancers. Results of tumor growth in mouse xenografts will determine if orally available MTAP inhibitors are effective at suppression of the major human cancers and will identify the altered gene expression patterns. The hypothesis also proposes that inhibition of DNA methylation at CpG islands is mediated through DNA methytransferases. Assays of the human methyltransferases in living cultured cancer cells, cell extracts and in purified complexes of human DNA methyltransferases will be coordinated with DNA methylation patterns and gene expression arrays. New MTAP inhibitors will be synthesized to improve efficacy, oral availability and chemical stability. PUBLIC HEALTH RELEVANCE:Human cancers result from loss of control of the DNA regions that act as regulators for cell division. New drug candidates are being developed to restore normal control to these cell regulators. The drugs are then tested to see if they prevent human cancers from growing in cultured human cells and in mice. If successful, these studies could lead to new orally available and non-toxic drugs to treat cancers in humans.
Public Health Relevance: Human cancers result from loss of control of the DNA regions that act as regulators for cell division. New drug candidates are being developed to restore normal control to these cell regulators. The drugs are then tested to see if they prevent human cancers from growing in cultured human cells and in mice. If successful, these studies could lead to new orally available and non-toxic drugs to treat cancers in humans.
Thesaurus Terms:
There are no thesaurus terms on file for this project.
Institution: YESHIVA UNIVERSITY
500 W 185TH ST
NEW YORK, NY 10033
Fiscal Year: 2008
Department: BIOCHEMISTRY
Project Start: 11-SEP-2008
Project End: 31-JUL-2013
ICD: NATIONAL CANCER INSTITUTE
IRG: DMP
Grant Number: 5R01CA072444-12 Project Title: Ricin-Mechanism, Transition State and Inhibitor Design PI Information: Name Email Title SCHRAMM, VERN L. | |||||||||||||||||||||||||||
| Deleted: | |||||||||||||||||||||||||||
| < < | SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR | ||||||||||||||||||||||||||
| Abstract: DESCRIPTION (provided by applicant): Ricin immunoconjugates are in clinical trials as anticancer agents. However their use leads to vascular leak syndrome, an unacceptable side effect. It is proposed to avoid this side effect by the design and production of powerful inhibitors that bind tightly to unwanted ricin, inactive it and prevent it from damaging normal tissue. The transition state structure of ricin A-chain is known and is being used to design powerful transition state analogue inhibitors. Mimics of the transition state will be chemically synthesized and made into stem-loop RNA or RNA/DNA hybrids containing mimics of the ricin A- chain transition state. In addition to the transition state structure, inhibitor design will be guided by x-ray crystallography and NMR. Covalently closed circular RNA, DNA and RNA/DNA hybrids containing chemically stable elements of transition state features are being developed. A second goal of this research is to develop new methods to detect ricin catalytic activity. These detection methods are important for measuring the amount of ricin-linked immunochemotherapy agents in the blood of patients undergoing ricin therapy. It is the catalytic activity of ricin that is toxic to humans. Methods that detect ricin catalytic activity would also be useful to detect ricin in case it is used as a bioterrorism agent. The broader significance of this work is to provide new catalytic insights into enzymes that process RNA. Methods developed to solve the transition state structure of ricin will be used to investigate the transition state structures of tRNA deaminase (TadA?) and mismatched double-stranded RNA adenylate deaminase (ADAR-1) and to design transition state analogues for these enzymes. In summary, the results of these studies are proposed to provide; 1) sensitive methods for the detection of ricin activity, 2) powerful transition state analogue inhibitors of ricin with long biological lifetimes, 3) transition state structures and powerful transition state analogue inhibitors for TadA? and ADAR-1. Crystallization and high-resolution NMR studies of inhibitors with the target proteins has potential for additional mechanistic insights into this developing area of nucleic acid enzymology. Ricin is a powerful toxin obtained from castor beans. It is being used in clinical trials for cancer therapy. When it becomes too active, it begins to damage normal tissue as well as the cancer. This research proposes a way to make an antidote to reduce damage to normal tissue. Ricin is also a bioterrorism threat. A similar antidote would be useful to prevent damage to populations exposed to ricin during a terrorism episode.
Public Health Relevance:
This Public Health Relevance is not available.
Thesaurus Terms:
There are no thesaurus terms on file for this project.
Institution: YESHIVA UNIVERSITY
500 W 185TH ST
NEW YORK, NY 10033
Fiscal Year: 2008
Department: BIOCHEMISTRY
Project Start: 15-SEP-1997
Project End: 31-JUL-2012
ICD: NATIONAL CANCER INSTITUTE
IRG: SBCA
Grant Number: 2R01AI049512-06A1 Project Title: Purine Pathways and Inhibitor Design in Plasmodium PI Information: Name Email Title | |||||||||||||||||||||||||||
| Changed: | |||||||||||||||||||||||||||
| < < | SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR | ||||||||||||||||||||||||||
| > > | SCHRAMM, VERN L. | ||||||||||||||||||||||||||
| Deleted: | |||||||||||||||||||||||||||
| < < | d134 1 | ||||||||||||||||||||||||||
| Abstract: DESCRIPTION (provided by applicant): Plasmodium falciparum is the leading cause of death from malaria, taking the lives of over a million children and causing clinical illness in 300 to 500 million people each year. The parasite has acquired resistance against most antimalarials and new drugs are required. P. falciparum is a purine auxotroph, requiring purine salvage from human erythrocytes for survival. Using the frontier technology of transition state analysis, the transition state structures of P. falciparum purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) have been solved and used to design transition state analogue inhibitors to match a their transition states. These inhibitors block their respective pathways and kill parasites cultured in human erythrocytes, but do not cure infections of Plasmodium yoelii in mice, an animal model of the disease. Metabolite labeling patterns and mouse studies have established that new pathways remain to be discovered and targeted. Inhibitor design against two critical targets will be assisted by solving the transition state structures of P. falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), the most critical step in purine synthesis. In the essential pathway of malarial de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) is the essential first step to form all pyrimidine nucleotides. These transition state structures will be solved by frontier methods coupling kinetic isotope effects and quantum chemistry. A new generation of inhibitors will be patterned on these transition states and tested against parasites cultured in human erythrocytes and in the mouse model of P. yoelii infection. Purine salvage and synthetic pathways in parasites and their interruption with inhibitors will be investigated with purine precursors with specific radioisotope labels. The ultrasensitive method of accelerator mass spectrometry (AMS) will be used to follow normal pathways of purine salvage without perturbing normal pools in cultured cells and in mouse infections. The AMS approach has revealed uncharacterized pathways of purine salvage and these will be defined in metabolic, enzymatic and inhibitor approaches. Antimalarials that block purine salvage or pyrimidine synthesis may be useful therapeutics as single agents or in combination with agents targeted against other pathways. Simultaneous blocking of two targets decreases the ability of mutational escape by the parasite. PUBLIC HEALTH RELEVANCE Malaria is an infectious disease cause by parasites spread by mosquitoes in tropical regions of the developing world. Approximately one million children die each year from the disease and current drugs are losing their efficiency because of acquired antibiotic resistance by the parasites. This research proposes new ways to treat malaria by discovered new ways to kill the parasites without harming the human host and by exploring new drugs.
Public Health Relevance:
This Public Health Relevance is not available.
Thesaurus Terms:
There are no thesaurus terms on file for this project.
Institution: YESHIVA UNIVERSITY
500 W 185TH ST
NEW YORK, NY 10033
Fiscal Year: 2008
Department: BIOCHEMISTRY
Project Start: 01-APR-2001
Project End: 30-APR-2013
ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
IRG: DDR
Grant Number: 5P01GM068036-059001 Project Title: Chemistry Core PI Information: Name Email Title SCHRAMM, VERN L. | |||||||||||||||||||||||||||
| Deleted: | |||||||||||||||||||||||||||
| < < | SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR | ||||||||||||||||||||||||||
| Abstract: Core A - 'The Chemistry Core'. The Chemistry Core' supports the production of specialized samples that are needed by the various experimental Projects of the Program Project. There are three components to this: (1). The overexpression and purification of proteins and mutants as required. This work will be carried out at the protein production facility at the AECOM that has been developed for the New York Consortium Structural Genomics Project, funded by NIGMS. (2) The synthesis of protein samples containing site-specific incorporated stable isotopes (13C, 15N, 2H). This will be carried out at Rockefeller University under the direction of Tom Muir. His lab has been a pioneer of the protein semisynthesis technique, Expressed Protein Ligation, which allows any target protein to be assembled in vitro from two or more segments, which can be recombinant or synthetic in origin. (3) The synthesis of small molecules as required that incorporate stable isotopes (13C, 15N, 2H) site specifically. Small molecule synthesis will be performed at Carbohydrate Chemistry, Natural Products Processing Group, Industrial Research Limited, Lower Hutt, New Zealand. Dr. Richard Furneanx, director of the Carbohydrate Chemistry Group, will oversee this effort.
Public Health Relevance:
This Public Health Relevance is not available.
Thesaurus Terms:
biomedical facility, peptide chemical synthesis, protein engineering
small molecule
biotechnology, protein purification, radiotracer
Institution: YESHIVA UNIVERSITY
500 W 185TH ST
NEW YORK, NY 10033
Fiscal Year: 2008
Department:
Project Start:
Project End:
ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
IRG: ZRG1
Grant Number: 5P01GM068036-050002 Project Title: Coordination of Protein Dynamics and Chemistry in PNP PI Information: Name Email Title | |||||||||||||||||||||||||||
| Changed: | |||||||||||||||||||||||||||
| < < | SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR | ||||||||||||||||||||||||||
| > > | SCHRAMM, VERN L. | ||||||||||||||||||||||||||
Abstract: Purine nucleoside phosphorylase (PNP) catalyzes phosphorolysis of 6-oxypurine nucleosides and deoxynucleosides. The transition state structure is oxacarbenium-like from kinetic isotope effects and transition state analogues (Immucillins) designed from this structure bind with pM affinity. Crystal structures have been solved with substrate, product and transition state analogues. The hypothesis emerging for catalysis is formation of an oxacarbenium ion transition state by neighboring group interactions from the 5'-hydroxyl of the ribosyl group and the enzyme-bound phosphate nucleophile. The catalytic site places neighbor oxygens the ribosyl 04', assisting electron contribution from the ribosyl group to the leaving group. This geometry supports an 'electronic promoting vibration' where protein groups fluctuate to bring oxygens closer, promoting electron expulsion. Computational chemistry dynamics (Schwartz, Project 4) will identify groups associated with this dynamic. Catalytic site mutations predicted to disrupt the promoting vibration will be made and tested. Isotope-edited infrared spectroscopy (Callender, Project 1) has established strong spectral bands associated with the phosphate nueleophile and the leaving group interactions. We propose time-resolved spectral analysis to correlate changes in protein dynamics, catalytic site chemistry, pH, leaving group and nucleophile interactions. T-jumps of dynamic equilibrium mixtures PNP with substrates and products will be induced by laser on a fast time scale followed by time-resolved monitoring of each parameter. Caged H+ will be used to initiate pH jumps to examine chemical and structural perturbations through proton donor/acceptor sites. Caged phosphate will be used to convert PNP.Immucillin to PNP.Immucillin.PO4, followed by isotope-edited following of the structural changes associated with slow-onset tight binding to resemble a transition state complex (Dyer, Project 3). Time-resolved spectra will be examined from psec to min time scales to follow local and global dynamics. Preliminary results establish rich IR spectral signatures for PO4 and leaving group interactions. These results will provide novel insights for the relationship between protein dynamics, ligand interactions and dynamics in catalysis.
Public Health Relevance:
This Public Health Relevance is not available.
Thesaurus Terms:
active site, catalyst, chemical reaction, electronic spectra, enzyme activity, intermolecular interaction, protein transport, purine nucleoside phosphorylase
conformation, enzyme structure, protein binding, protein structure function
animal tissue, combinatorial chemistry, high performance liquid chromatography, human tissue, infrared spectrometry, laser, nuclear magnetic resonance spectroscopy, protein purification, radiotracer, relaxation spectrometry
Institution: YESHIVA UNIVERSITY
500 W 185TH ST
NEW YORK, NY 10033
Fiscal Year: 2008
Department:
Project Start:
Project End:
ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
IRG: ZRG1
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Revision 414 Jul 2009 - Main.DavidCowburn
My Links
Personal PreferencesUncomment preferences variables to activate them (remove the #-sign). Help and details on preferences variables are available in TWikiPreferences.
Related Topics
Public Information on Grants Associated with NYSBC Grant Number: 2R01GM041916-20 Project Title: Transition State Analysis of Enzymatic Reactions PI Information: Name Email Title SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR Abstract: DESCRIPTION (provided by applicant): Transition state (TS) analysis from isotope effects and computational chemistry provides frontier technology for understanding the chemistry of bond change at the instant of enzymatic TS formation. Transition state analogues can be designed from the molecular electrostatic potential surfaces of TSs and have provided unique design parameters for some of the most powerful enzymatic inhibitors known. First and second generation TS analogues for human purine nucleoside phosphorylase (PNP) have advanced from first principles of TS design into human clinical trials for cancer and autoimmune diseases. Third generation PNP inhibitors will be compared to 1st and 2nd generation analogues for binding, structure, thermodynamics and biological lifetimes on PNP in cells. Binding isotope effects are an emerging technology for understanding the geometric and electronic constraints experienced by molecules as they become immobilized at their binding sites on macromolecules, including enzymes and receptors. Binding isotope effects will explore the atomic constraints of substrates and tight- binding TS analogues at the binding sites of human PNP. A surprising diversity of TS structure exists in the same enzyme isolated from different species, establishing the possibility of species-specific TS analogue design. Transition state structures of bacterial 5'- methylthioadenosine nucleosidases (MTANs) will be solved and matched to specific analogues for affinity and structures of reactant and TS-complexes. Biological efficacy of MTAN inhibitors will be analyzed in bacterial quorum sensing pathways. In theory, all enzymatic TSs should be accessible to isotope effect analysis but some provide technical challenges because the chemical step is obscured by non-chemical steps. Human thymidine phosphorylase is a prototype for kinetically difficult TS analyses. TS analysis methods will be established to expose the chemical step by rapid reaction kinetics and altered reaction conditions. Atomic understanding of enzymatic TS chemistry has been developed primarily in enzymes involved in N-ribosyltransferases and deaminases. Expanding the frontier of TS analysis to hydrolysis at carbonyl carbons will be accomplished in the well-known system of HIV-protease and in the important but poorly understood target of human 2'-O-acetyl-ADP-ribosyl esterase. Goals of this research are to push the frontier of enzymatic TS theory to enhance understanding of catalysis and drug design for human targets. PUBLIC HEALTH RELEVANCE: Transition state theory provides an approach to design better drugs for human disease. Expanded methods of drug design will be applied to targets of human disease. Purine nucleoside phosphorylase is a target for leukemia and for autoimmune diseases including psoriasis and tissue transplant rejection; methylthioadenosine phosphorylase is a target for antibiotic-resistant bacteria; thymidine phosphorylase is a target for solid tumors; HIV protease is a target for AIDS infections; and acetyl-ADP-ribosyl hydrolase is a target for diseases of ageing. New methods will be established for the broader application of this theory and the results may lead to new drugs to treat cancer, autoimmunity, bacterial infections and diseases of ageing. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 01-AUG-1989 Project End: 31-JUL-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 1R01CA135405-01A1 Project Title: Transition State Analogues as Modulators of DNA Methylation PI Information: Name Email Title SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR d85 1 Abstract: DESCRIPTION (provided by applicant): Transition state analogue design is a frontier technology for targeting specific enzymes in human disease. MT-DADMe-Immucillin-A is an orally available transition state analogue inhibitor for human 5'- methylthioadenosine phosphorylase (MTAP). MTAP inhibition slows or prevents the growth of human head and neck, prostate and human lung cancers in mouse xenografts. Normal tissues are not affected and the inhibitor shows no toxicity against normal cells or to mice. The MTAP inhibitor alters metabolites that are expected to change the ability of DNA methyltransferases to methylate DNA. Cancers are commonly caused by mutations that change gene expression patterns and permit the growth and metastases of tumors. Gene expression patterns leading to cancer are governed, in part, by DNA methylation at regions of the genome rich in CpG bases, called CpG islands. The hypothesis for this research is that MTAP inhibitors alter metabolite levels in cancer tissues to inhibit DNA methylation patterns in humans. Loss of methylation for some of the CpG islands near cancer suppression genes is proposed to alter the gene expression patterns of the cancer cells and to slow or prevent cancer cell growth. This hypothesis will be explored in cultured cell lines and mouse xenograft models of the major human malignancies, lung, breast, prostate colon, head and neck and cervical cancers. Results of tumor growth in mouse xenografts will determine if orally available MTAP inhibitors are effective at suppression of the major human cancers and will identify the altered gene expression patterns. The hypothesis also proposes that inhibition of DNA methylation at CpG islands is mediated through DNA methytransferases. Assays of the human methyltransferases in living cultured cancer cells, cell extracts and in purified complexes of human DNA methyltransferases will be coordinated with DNA methylation patterns and gene expression arrays. New MTAP inhibitors will be synthesized to improve efficacy, oral availability and chemical stability. PUBLIC HEALTH RELEVANCE:Human cancers result from loss of control of the DNA regions that act as regulators for cell division. New drug candidates are being developed to restore normal control to these cell regulators. The drugs are then tested to see if they prevent human cancers from growing in cultured human cells and in mice. If successful, these studies could lead to new orally available and non-toxic drugs to treat cancers in humans. Public Health Relevance: Human cancers result from loss of control of the DNA regions that act as regulators for cell division. New drug candidates are being developed to restore normal control to these cell regulators. The drugs are then tested to see if they prevent human cancers from growing in cultured human cells and in mice. If successful, these studies could lead to new orally available and non-toxic drugs to treat cancers in humans. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 11-SEP-2008 Project End: 31-JUL-2013 ICD: NATIONAL CANCER INSTITUTE IRG: DMP Grant Number: 5R01CA072444-12 Project Title: Ricin-Mechanism, Transition State and Inhibitor Design PI Information: Name Email Title SCHRAMM, VERN L. SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR Abstract: DESCRIPTION (provided by applicant): Ricin immunoconjugates are in clinical trials as anticancer agents. However their use leads to vascular leak syndrome, an unacceptable side effect. It is proposed to avoid this side effect by the design and production of powerful inhibitors that bind tightly to unwanted ricin, inactive it and prevent it from damaging normal tissue. The transition state structure of ricin A-chain is known and is being used to design powerful transition state analogue inhibitors. Mimics of the transition state will be chemically synthesized and made into stem-loop RNA or RNA/DNA hybrids containing mimics of the ricin A- chain transition state. In addition to the transition state structure, inhibitor design will be guided by x-ray crystallography and NMR. Covalently closed circular RNA, DNA and RNA/DNA hybrids containing chemically stable elements of transition state features are being developed. A second goal of this research is to develop new methods to detect ricin catalytic activity. These detection methods are important for measuring the amount of ricin-linked immunochemotherapy agents in the blood of patients undergoing ricin therapy. It is the catalytic activity of ricin that is toxic to humans. Methods that detect ricin catalytic activity would also be useful to detect ricin in case it is used as a bioterrorism agent. The broader significance of this work is to provide new catalytic insights into enzymes that process RNA. Methods developed to solve the transition state structure of ricin will be used to investigate the transition state structures of tRNA deaminase (TadA?) and mismatched double-stranded RNA adenylate deaminase (ADAR-1) and to design transition state analogues for these enzymes. In summary, the results of these studies are proposed to provide; 1) sensitive methods for the detection of ricin activity, 2) powerful transition state analogue inhibitors of ricin with long biological lifetimes, 3) transition state structures and powerful transition state analogue inhibitors for TadA? and ADAR-1. Crystallization and high-resolution NMR studies of inhibitors with the target proteins has potential for additional mechanistic insights into this developing area of nucleic acid enzymology. Ricin is a powerful toxin obtained from castor beans. It is being used in clinical trials for cancer therapy. When it becomes too active, it begins to damage normal tissue as well as the cancer. This research proposes a way to make an antidote to reduce damage to normal tissue. Ricin is also a bioterrorism threat. A similar antidote would be useful to prevent damage to populations exposed to ricin during a terrorism episode. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 15-SEP-1997 Project End: 31-JUL-2012 ICD: NATIONAL CANCER INSTITUTE IRG: SBCA Grant Number: 2R01AI049512-06A1 Project Title: Purine Pathways and Inhibitor Design in Plasmodium PI Information: Name Email Title SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR d134 1 Abstract: DESCRIPTION (provided by applicant): Plasmodium falciparum is the leading cause of death from malaria, taking the lives of over a million children and causing clinical illness in 300 to 500 million people each year. The parasite has acquired resistance against most antimalarials and new drugs are required. P. falciparum is a purine auxotroph, requiring purine salvage from human erythrocytes for survival. Using the frontier technology of transition state analysis, the transition state structures of P. falciparum purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) have been solved and used to design transition state analogue inhibitors to match a their transition states. These inhibitors block their respective pathways and kill parasites cultured in human erythrocytes, but do not cure infections of Plasmodium yoelii in mice, an animal model of the disease. Metabolite labeling patterns and mouse studies have established that new pathways remain to be discovered and targeted. Inhibitor design against two critical targets will be assisted by solving the transition state structures of P. falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), the most critical step in purine synthesis. In the essential pathway of malarial de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) is the essential first step to form all pyrimidine nucleotides. These transition state structures will be solved by frontier methods coupling kinetic isotope effects and quantum chemistry. A new generation of inhibitors will be patterned on these transition states and tested against parasites cultured in human erythrocytes and in the mouse model of P. yoelii infection. Purine salvage and synthetic pathways in parasites and their interruption with inhibitors will be investigated with purine precursors with specific radioisotope labels. The ultrasensitive method of accelerator mass spectrometry (AMS) will be used to follow normal pathways of purine salvage without perturbing normal pools in cultured cells and in mouse infections. The AMS approach has revealed uncharacterized pathways of purine salvage and these will be defined in metabolic, enzymatic and inhibitor approaches. Antimalarials that block purine salvage or pyrimidine synthesis may be useful therapeutics as single agents or in combination with agents targeted against other pathways. Simultaneous blocking of two targets decreases the ability of mutational escape by the parasite. PUBLIC HEALTH RELEVANCE Malaria is an infectious disease cause by parasites spread by mosquitoes in tropical regions of the developing world. Approximately one million children die each year from the disease and current drugs are losing their efficiency because of acquired antibiotic resistance by the parasites. This research proposes new ways to treat malaria by discovered new ways to kill the parasites without harming the human host and by exploring new drugs. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 01-APR-2001 Project End: 30-APR-2013 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: DDR Grant Number: 5P01GM068036-059001 Project Title: Chemistry Core PI Information: Name Email Title SCHRAMM, VERN L. SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR Abstract: Core A - 'The Chemistry Core'. The Chemistry Core' supports the production of specialized samples that are needed by the various experimental Projects of the Program Project. There are three components to this: (1). The overexpression and purification of proteins and mutants as required. This work will be carried out at the protein production facility at the AECOM that has been developed for the New York Consortium Structural Genomics Project, funded by NIGMS. (2) The synthesis of protein samples containing site-specific incorporated stable isotopes (13C, 15N, 2H). This will be carried out at Rockefeller University under the direction of Tom Muir. His lab has been a pioneer of the protein semisynthesis technique, Expressed Protein Ligation, which allows any target protein to be assembled in vitro from two or more segments, which can be recombinant or synthetic in origin. (3) The synthesis of small molecules as required that incorporate stable isotopes (13C, 15N, 2H) site specifically. Small molecule synthesis will be performed at Carbohydrate Chemistry, Natural Products Processing Group, Industrial Research Limited, Lower Hutt, New Zealand. Dr. Richard Furneanx, director of the Carbohydrate Chemistry Group, will oversee this effort. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: biomedical facility, peptide chemical synthesis, protein engineering small molecule biotechnology, protein purification, radiotracer Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2008 Department: Project Start: Project End: ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 5P01GM068036-050002 Project Title: Coordination of Protein Dynamics and Chemistry in PNP PI Information: Name Email Title SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR Abstract: Purine nucleoside phosphorylase (PNP) catalyzes phosphorolysis of 6-oxypurine nucleosides and deoxynucleosides. The transition state structure is oxacarbenium-like from kinetic isotope effects and transition state analogues (Immucillins) designed from this structure bind with pM affinity. Crystal structures have been solved with substrate, product and transition state analogues. The hypothesis emerging for catalysis is formation of an oxacarbenium ion transition state by neighboring group interactions from the 5'-hydroxyl of the ribosyl group and the enzyme-bound phosphate nucleophile. The catalytic site places neighbor oxygens the ribosyl 04', assisting electron contribution from the ribosyl group to the leaving group. This geometry supports an 'electronic promoting vibration' where protein groups fluctuate to bring oxygens closer, promoting electron expulsion. Computational chemistry dynamics (Schwartz, Project 4) will identify groups associated with this dynamic. Catalytic site mutations predicted to disrupt the promoting vibration will be made and tested. Isotope-edited infrared spectroscopy (Callender, Project 1) has established strong spectral bands associated with the phosphate nueleophile and the leaving group interactions. We propose time-resolved spectral analysis to correlate changes in protein dynamics, catalytic site chemistry, pH, leaving group and nucleophile interactions. T-jumps of dynamic equilibrium mixtures PNP with substrates and products will be induced by laser on a fast time scale followed by time-resolved monitoring of each parameter. Caged H+ will be used to initiate pH jumps to examine chemical and structural perturbations through proton donor/acceptor sites. Caged phosphate will be used to convert PNP.Immucillin to PNP.Immucillin.PO4, followed by isotope-edited following of the structural changes associated with slow-onset tight binding to resemble a transition state complex (Dyer, Project 3). Time-resolved spectra will be examined from psec to min time scales to follow local and global dynamics. Preliminary results establish rich IR spectral signatures for PO4 and leaving group interactions. These results will provide novel insights for the relationship between protein dynamics, ligand interactions and dynamics in catalysis. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: active site, catalyst, chemical reaction, electronic spectra, enzyme activity, intermolecular interaction, protein transport, purine nucleoside phosphorylase conformation, enzyme structure, protein binding, protein structure function animal tissue, combinatorial chemistry, high performance liquid chromatography, human tissue, infrared spectrometry, laser, nuclear magnetic resonance spectroscopy, protein purification, radiotracer, relaxation spectrometry Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2008 Department: Project Start: Project End: ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1
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| > > | Public Information on Grants Associated with NYSBC Grant Number: 2R01GM041916-20 Project Title: Transition State Analysis of Enzymatic Reactions PI Information: Name Email Title SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR Abstract: DESCRIPTION (provided by applicant): Transition state (TS) analysis from isotope effects and computational chemistry provides frontier technology for understanding the chemistry of bond change at the instant of enzymatic TS formation. Transition state analogues can be designed from the molecular electrostatic potential surfaces of TSs and have provided unique design parameters for some of the most powerful enzymatic inhibitors known. First and second generation TS analogues for human purine nucleoside phosphorylase (PNP) have advanced from first principles of TS design into human clinical trials for cancer and autoimmune diseases. Third generation PNP inhibitors will be compared to 1st and 2nd generation analogues for binding, structure, thermodynamics and biological lifetimes on PNP in cells. Binding isotope effects are an emerging technology for understanding the geometric and electronic constraints experienced by molecules as they become immobilized at their binding sites on macromolecules, including enzymes and receptors. Binding isotope effects will explore the atomic constraints of substrates and tight- binding TS analogues at the binding sites of human PNP. A surprising diversity of TS structure exists in the same enzyme isolated from different species, establishing the possibility of species-specific TS analogue design. Transition state structures of bacterial 5'- methylthioadenosine nucleosidases (MTANs) will be solved and matched to specific analogues for affinity and structures of reactant and TS-complexes. Biological efficacy of MTAN inhibitors will be analyzed in bacterial quorum sensing pathways. In theory, all enzymatic TSs should be accessible to isotope effect analysis but some provide technical challenges because the chemical step is obscured by non-chemical steps. Human thymidine phosphorylase is a prototype for kinetically difficult TS analyses. TS analysis methods will be established to expose the chemical step by rapid reaction kinetics and altered reaction conditions. Atomic understanding of enzymatic TS chemistry has been developed primarily in enzymes involved in N-ribosyltransferases and deaminases. Expanding the frontier of TS analysis to hydrolysis at carbonyl carbons will be accomplished in the well-known system of HIV-protease and in the important but poorly understood target of human 2'-O-acetyl-ADP-ribosyl esterase. Goals of this research are to push the frontier of enzymatic TS theory to enhance understanding of catalysis and drug design for human targets. PUBLIC HEALTH RELEVANCE: Transition state theory provides an approach to design better drugs for human disease. Expanded methods of drug design will be applied to targets of human disease. Purine nucleoside phosphorylase is a target for leukemia and for autoimmune diseases including psoriasis and tissue transplant rejection; methylthioadenosine phosphorylase is a target for antibiotic-resistant bacteria; thymidine phosphorylase is a target for solid tumors; HIV protease is a target for AIDS infections; and acetyl-ADP-ribosyl hydrolase is a target for diseases of ageing. New methods will be established for the broader application of this theory and the results may lead to new drugs to treat cancer, autoimmunity, bacterial infections and diseases of ageing. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 01-AUG-1989 Project End: 31-JUL-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 Grant Number: 1R01CA135405-01A1 Project Title: Transition State Analogues as Modulators of DNA Methylation PI Information: Name Email Title SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR | |||||||||||||||||||||||||||||||||||||||||
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| > > | Abstract: DESCRIPTION (provided by applicant): Transition state analogue design is a frontier technology for targeting specific enzymes in human disease. MT-DADMe-Immucillin-A is an orally available transition state analogue inhibitor for human 5'- methylthioadenosine phosphorylase (MTAP). MTAP inhibition slows or prevents the growth of human head and neck, prostate and human lung cancers in mouse xenografts. Normal tissues are not affected and the inhibitor shows no toxicity against normal cells or to mice. The MTAP inhibitor alters metabolites that are expected to change the ability of DNA methyltransferases to methylate DNA. Cancers are commonly caused by mutations that change gene expression patterns and permit the growth and metastases of tumors. Gene expression patterns leading to cancer are governed, in part, by DNA methylation at regions of the genome rich in CpG bases, called CpG islands. The hypothesis for this research is that MTAP inhibitors alter metabolite levels in cancer tissues to inhibit DNA methylation patterns in humans. Loss of methylation for some of the CpG islands near cancer suppression genes is proposed to alter the gene expression patterns of the cancer cells and to slow or prevent cancer cell growth. This hypothesis will be explored in cultured cell lines and mouse xenograft models of the major human malignancies, lung, breast, prostate colon, head and neck and cervical cancers. Results of tumor growth in mouse xenografts will determine if orally available MTAP inhibitors are effective at suppression of the major human cancers and will identify the altered gene expression patterns. The hypothesis also proposes that inhibition of DNA methylation at CpG islands is mediated through DNA methytransferases. Assays of the human methyltransferases in living cultured cancer cells, cell extracts and in purified complexes of human DNA methyltransferases will be coordinated with DNA methylation patterns and gene expression arrays. New MTAP inhibitors will be synthesized to improve efficacy, oral availability and chemical stability. PUBLIC HEALTH RELEVANCE:Human cancers result from loss of control of the DNA regions that act as regulators for cell division. New drug candidates are being developed to restore normal control to these cell regulators. The drugs are then tested to see if they prevent human cancers from growing in cultured human cells and in mice. If successful, these studies could lead to new orally available and non-toxic drugs to treat cancers in humans.
Public Health Relevance: Human cancers result from loss of control of the DNA regions that act as regulators for cell division. New drug candidates are being developed to restore normal control to these cell regulators. The drugs are then tested to see if they prevent human cancers from growing in cultured human cells and in mice. If successful, these studies could lead to new orally available and non-toxic drugs to treat cancers in humans.
Thesaurus Terms:
There are no thesaurus terms on file for this project.
Institution: YESHIVA UNIVERSITY
500 W 185TH ST
NEW YORK, NY 10033
Fiscal Year: 2008
Department: BIOCHEMISTRY
Project Start: 11-SEP-2008
Project End: 31-JUL-2013
ICD: NATIONAL CANCER INSTITUTE
IRG: DMP
Grant Number: 5R01CA072444-12 Project Title: Ricin-Mechanism, Transition State and Inhibitor Design PI Information: Name Email Title SCHRAMM, VERN L. SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR Abstract: DESCRIPTION (provided by applicant): Ricin immunoconjugates are in clinical trials as anticancer agents. However their use leads to vascular leak syndrome, an unacceptable side effect. It is proposed to avoid this side effect by the design and production of powerful inhibitors that bind tightly to unwanted ricin, inactive it and prevent it from damaging normal tissue. The transition state structure of ricin A-chain is known and is being used to design powerful transition state analogue inhibitors. Mimics of the transition state will be chemically synthesized and made into stem-loop RNA or RNA/DNA hybrids containing mimics of the ricin A- chain transition state. In addition to the transition state structure, inhibitor design will be guided by x-ray crystallography and NMR. Covalently closed circular RNA, DNA and RNA/DNA hybrids containing chemically stable elements of transition state features are being developed. A second goal of this research is to develop new methods to detect ricin catalytic activity. These detection methods are important for measuring the amount of ricin-linked immunochemotherapy agents in the blood of patients undergoing ricin therapy. It is the catalytic activity of ricin that is toxic to humans. Methods that detect ricin catalytic activity would also be useful to detect ricin in case it is used as a bioterrorism agent. The broader significance of this work is to provide new catalytic insights into enzymes that process RNA. Methods developed to solve the transition state structure of ricin will be used to investigate the transition state structures of tRNA deaminase (TadA?) and mismatched double-stranded RNA adenylate deaminase (ADAR-1) and to design transition state analogues for these enzymes. In summary, the results of these studies are proposed to provide; 1) sensitive methods for the detection of ricin activity, 2) powerful transition state analogue inhibitors of ricin with long biological lifetimes, 3) transition state structures and powerful transition state analogue inhibitors for TadA? and ADAR-1. Crystallization and high-resolution NMR studies of inhibitors with the target proteins has potential for additional mechanistic insights into this developing area of nucleic acid enzymology. Ricin is a powerful toxin obtained from castor beans. It is being used in clinical trials for cancer therapy. When it becomes too active, it begins to damage normal tissue as well as the cancer. This research proposes a way to make an antidote to reduce damage to normal tissue. Ricin is also a bioterrorism threat. A similar antidote would be useful to prevent damage to populations exposed to ricin during a terrorism episode. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 15-SEP-1997 Project End: 31-JUL-2012 ICD: NATIONAL CANCER INSTITUTE IRG: SBCA Grant Number: 2R01AI049512-06A1 Project Title: Purine Pathways and Inhibitor Design in Plasmodium PI Information: Name Email Title SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR d134 1 Abstract: DESCRIPTION (provided by applicant): Plasmodium falciparum is the leading cause of death from malaria, taking the lives of over a million children and causing clinical illness in 300 to 500 million people each year. The parasite has acquired resistance against most antimalarials and new drugs are required. P. falciparum is a purine auxotroph, requiring purine salvage from human erythrocytes for survival. Using the frontier technology of transition state analysis, the transition state structures of P. falciparum purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) have been solved and used to design transition state analogue inhibitors to match a their transition states. These inhibitors block their respective pathways and kill parasites cultured in human erythrocytes, but do not cure infections of Plasmodium yoelii in mice, an animal model of the disease. Metabolite labeling patterns and mouse studies have established that new pathways remain to be discovered and targeted. Inhibitor design against two critical targets will be assisted by solving the transition state structures of P. falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), the most critical step in purine synthesis. In the essential pathway of malarial de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) is the essential first step to form all pyrimidine nucleotides. These transition state structures will be solved by frontier methods coupling kinetic isotope effects and quantum chemistry. A new generation of inhibitors will be patterned on these transition states and tested against parasites cultured in human erythrocytes and in the mouse model of P. yoelii infection. Purine salvage and synthetic pathways in parasites and their interruption with inhibitors will be investigated with purine precursors with specific radioisotope labels. The ultrasensitive method of accelerator mass spectrometry (AMS) will be used to follow normal pathways of purine salvage without perturbing normal pools in cultured cells and in mouse infections. The AMS approach has revealed uncharacterized pathways of purine salvage and these will be defined in metabolic, enzymatic and inhibitor approaches. Antimalarials that block purine salvage or pyrimidine synthesis may be useful therapeutics as single agents or in combination with agents targeted against other pathways. Simultaneous blocking of two targets decreases the ability of mutational escape by the parasite. PUBLIC HEALTH RELEVANCE Malaria is an infectious disease cause by parasites spread by mosquitoes in tropical regions of the developing world. Approximately one million children die each year from the disease and current drugs are losing their efficiency because of acquired antibiotic resistance by the parasites. This research proposes new ways to treat malaria by discovered new ways to kill the parasites without harming the human host and by exploring new drugs. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: There are no thesaurus terms on file for this project. Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 01-APR-2001 Project End: 30-APR-2013 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: DDR Grant Number: 5P01GM068036-059001 Project Title: Chemistry Core PI Information: Name Email Title SCHRAMM, VERN L. SCHRAMM, VERN L. vern@aecom.yu.edu PROFESSOR Abstract: Core A - 'The Chemistry Core'. The Chemistry Core' supports the production of specialized samples that are needed by the various experimental Projects of the Program Project. There are three components to this: (1). The overexpression and purification of proteins and mutants as required. This work will be carried out at the protein production facility at the AECOM that has been developed for the New York Consortium Structural Genomics Project, funded by NIGMS. (2) The synthesis of protein samples containing site-specific incorporated stable isotopes (13C, 15N, 2H). This will be carried out at Rockefeller University under the direction of Tom Muir. His lab has been a pioneer of the protein semisynthesis technique, Expressed Protein Ligation, which allows any target protein to be assembled in vitro from two or more segments, which can be recombinant or synthetic in origin. (3) The synthesis of small molecules as required that incorporate stable isotopes (13C, 15N, 2H) site specifically. Small molecule synthesis will be performed at Carbohydrate Chemistry, Natural Products Processing Group, Industrial Research Limited, Lower Hutt, New Zealand. Dr. Richard Furneanx, director of the Carbohydrate Chemistry Group, will oversee this effort. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: biomedical facility, peptide chemical synthesis, protein engineering small molecule biotechnology, protein purification, radiotracer Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2008 Department: Project Start: Project End: | |||||||||||||||||||||||||||||||||||||||||
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| > > | Abstract: Purine nucleoside phosphorylase (PNP) catalyzes phosphorolysis of 6-oxypurine nucleosides and deoxynucleosides. The transition state structure is oxacarbenium-like from kinetic isotope effects and transition state analogues (Immucillins) designed from this structure bind with pM affinity. Crystal structures have been solved with substrate, product and transition state analogues. The hypothesis emerging for catalysis is formation of an oxacarbenium ion transition state by neighboring group interactions from the 5'-hydroxyl of the ribosyl group and the enzyme-bound phosphate nucleophile. The catalytic site places neighbor oxygens the ribosyl 04', assisting electron contribution from the ribosyl group to the leaving group. This geometry supports an 'electronic promoting vibration' where protein groups fluctuate to bring oxygens closer, promoting electron expulsion. Computational chemistry dynamics (Schwartz, Project 4) will identify groups associated with this dynamic. Catalytic site mutations predicted to disrupt the promoting vibration will be made and tested. Isotope-edited infrared spectroscopy (Callender, Project 1) has established strong spectral bands associated with the phosphate nueleophile and the leaving group interactions. We propose time-resolved spectral analysis to correlate changes in protein dynamics, catalytic site chemistry, pH, leaving group and nucleophile interactions. T-jumps of dynamic equilibrium mixtures PNP with substrates and products will be induced by laser on a fast time scale followed by time-resolved monitoring of each parameter. Caged H+ will be used to initiate pH jumps to examine chemical and structural perturbations through proton donor/acceptor sites. Caged phosphate will be used to convert PNP.Immucillin to PNP.Immucillin.PO4, followed by isotope-edited following of the structural changes associated with slow-onset tight binding to resemble a transition state complex (Dyer, Project 3). Time-resolved spectra will be examined from psec to min time scales to follow local and global dynamics. Preliminary results establish rich IR spectral signatures for PO4 and leaving group interactions. These results will provide novel insights for the relationship between protein dynamics, ligand interactions and dynamics in catalysis. Public Health Relevance: This Public Health Relevance is not available. Thesaurus Terms: active site, catalyst, chemical reaction, electronic spectra, enzyme activity, intermolecular interaction, protein transport, purine nucleoside phosphorylase conformation, enzyme structure, protein binding, protein structure function animal tissue, combinatorial chemistry, high performance liquid chromatography, human tissue, infrared spectrometry, laser, nuclear magnetic resonance spectroscopy, protein purification, radiotracer, relaxation spectrometry Institution: YESHIVA UNIVERSITY 500 W 185TH ST NEW YORK, NY 10033 Fiscal Year: 2008 Department: Project Start: Project End: ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1 | |||||||||||||||||||||||||||||||||||||||||
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Revision 116 Jul 2008 - Main.TWikiRegistrationAgent
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