|
|
New Faculty Members
Paul Berti
Enzyme Mechanisms & Inhibition
Paul Berti obtained his Ph.D. at McGill University, and
comes to us following a postdoctoral fellowship in the Department of Biochemistry at the Albert Einstein College of Medicine in New York City.
His research seeks to understand how enzymes work. Enzymes catalyze reactions by binding to the substrate and stabilizing the transition state of the reaction - the highest energy point on the reaction pathway. Using kinetic isotope effects (KIEs), it is possible to determine the structure of the transition state in exquisite detail - showing precisely what the enzyme does in order to cause catalysis. Such an understanding of medically and commercially important enzymes can be used to design inhibitors, or to catalyze novel reactions.
By stabilizing the transition state, enzymes reduce the activation energy of a reaction, increasing its rate. It is deduced that the short-lived substrate transition state binds to the enzyme more strongly than reactant or products. A stable compound which mimics the transition state can therefore provide a potent inhibitor. However, the transition state is difficult to characterize because of its short lifetime - less than 1013 s. Standard structural techniques cannot be used. Instead, one must use some kinetic method, such as KIEs. KIEs report on the change in an atom's environment between the reactant and the TS. KIEs from substrates labeled at many positions give a highly detailed picture of the TS of an enzyme catalyzed reaction. The TS structure for ricin-catalyzed hydrolysis of an adenylate residue in RNA is shown below. Knowing the TS structure of a reaction is the first step in designing TS mimics as inhibitors.
All organisms possess elaborate DNA repair machinery to maintain the integrity of the genome. MutY is an enzyme which catalyzes an important base excision repair (BER) reaction, necessary for repairing the (8-oxo-G):A base pair mismatch. 8oxoG is one of the major products of oxidative DNA damage, e.g. from ionizing radiation. A is often misincorporated at the site of the oxidatively damaged G residue. MutY hydrolyses the adenine base from A in the first step of a multi-step repair process that replaces A with C. Dr. Berti wants to understand how MutY catalyzes this hydrolysis reaction.
Change in positive charge (grey) in ricin-catalyzed hydrolysis of adenylate in RNA. The reactant (left) is protonated and stabilized by the enzyme to form the transition state (right).There are only two enzymes known that catalyze a carboxyvinyl transfer reaction, MurA and AroA. Both occur in bacteria and both are antibiotic targets. AroA is part of the biosynthetic pathway for aromatic amino acids. It is also present in the malaria parasite, and in plants, where it is the target of the herbicide glyphosate (the active ingredient in Roundup™). MurA catalyzes the first committed step in peptidoglycan (part of the bacterial cell wall) biosynthesis and is the target of the antibiotic fosfomycin. Although inhibitors exist for both enzymes, there is a pressing need for better ones. Fosfomycin is often ineffective because of antibiotic-induced resistance, and does not work at all on MurA in such important organisms as M. tuberculosis. Glyphosate is a good herbicide, but is not an antibiotic. The goal is to elucidate the catalytic mechanisms of MurA and AroA, then exploit that knowledge to design inhibitors that will be effective as antibiotics.
John Valliant
Synthetic, Medicinal & Radio-pharmaceutical Chemistry
John Valliant obtained his Ph.D. at McMaster University under the supervision of Colin Lock
and Russell Bell. He returns to us following a postdoctoral fellowship at the Harvard School of Medicine.
Dr. Valliant's research is concerned with the development of new inorganic pharmaceuticals including novel radioimaging and radiotherapeutic agents. Modern drug discovery techniques provide a rational, systematic approach to these efforts - in contrast to the serendipitous means with which traditional inorganic drugs were discovered. Both solution and solid phase synthetic methods are employed.
Inorganic pharmaceuticals currently being developed include new gold compounds for use in the treatment of rheumatoid arthritis. The biological activities of these new complexes are evaluated through interaction with a network of collaborators. Technetium (Tc) chemistry is another area of active interest. 99mTc is a short half-life metastable state of the 99Tc nucleus. It is the most widely used diagnostic agent in nuclear medicine. Its chemistry is conveniently studied using 99Tc which, unlike 99mTc, can be handled safely in mg quantities. Technetium has a diverse chemistry with known compounds having oxidation states from -I to +VII and coordination numbers up to nine. New radioimaging agents will result from a better understanding of the chemistry of technetium. New complexes of technetium are consequently under investigation. Dr. Valliant has recently developed a novel synthetic method to prepare NO containing precursors of both 99Tc and 99mTc. This research involves active collaboration with industry. For example, Tc/Re RP128 was originally developed by Resolution Pharmaceuticals to image sites of infectious and non-infectious inflammation.
When exposed to thermal neutrons, 10B decomposes into a high energy alpha particle and lithium ion which cause irreparable tissue damage in a 10 micrometer range. Boron Neutron Capture Therapy (BNCT) is based on this reaction together with selective binding of certain boron compounds in diseased tissues. Carboranes (C2B10H12) are clusters of boron and carbon atoms which can be derivatized with a biomolecule to concentrate 10B within cancer (or arthritis) cells. These clusters are chemically stable and show resistance to degradation in vivo. Dr. Valliant's research involves synthesizing carborane complexes of transition metals which mimic biologically active compounds. These new coordination complexes are used for simultaneous imaging and therapeutic destruction of cancer tumors and arthritic tissue.
Solid phase synthesis and combinatorial chemistry have revolutionized the drug discovery process. Dr. Valliant has adapted these techniques, originally developed to investigate organic drugs, to the development of novel, targeted radiopharmaceuticals and inorganic pharmaceuticals. The research involves developing new solid-phase reactions which can subsequently be used to generate libraries of inorganic and radiopharmaceutical compounds. Interesting candidate compounds which are identified by testing in vitro are then synthesized on a larger scale and tested in vivo.
McMaster has world-class facilities for developing the chemistry of radiopharmaceuticals including a 5 MW nuclear reactor, a cyclotron and labs designed to handle most types of radionuclides.
Yingfu Li
In Vitro Evolution of Catalytic DNA and Other Functional Nucleic Acids
Yingfu Li obtained his Ph.D. at Simon Fraser University, and comes to us following a
postdoctoral fellowship in the Department of Biology at Yale University.
Dr. Li's research is concerned with the creation and understanding of DNA molecules with catalytic properties.
The repetitive and extraordinarily stable polynucleotide chains of DNA serve as an ideal storage system for genetic information. Best known for its helical structure and its relatively inert character, DNA also can be compelled through "in vitro selection" to catalyze a surprising variety of chemical reactions including porphyrin metallation, DNA phosphorylation, DNA capping and DNA ligation. These artificial DNA enzymes generate large chemical rate enhancements and demonstrate precise substrate recognition much like their counterparts made of protein or RNA. Dr. Li's studies of prototypic DNA enzymes indicate that DNA has substantial untapped potential for intricate structure formation which could be exploited for novel chemical and biological catalysis.
In vitro selection methodology, the principal tool of Dr. Li's laboratory, allows directed isolation and evolution of deoxyribozymes (also known as DNA enzymes or DNAzymes) from random-sequence DNA libraries containing up to 1016 distinct molecules. The approach uses directed test-tube selection which mimics Darwinian evolution. New DNA sequences with specific catalytic properties are thereby uncovered. Combinatorial, computational and structural analysis provide means of engineering these deoxyribozymes with precise folding characteristics, substrate specificities, and thermal stabilities. Characteristics are tailored for specific chemical, biological, or medicinal applications.
Rational enzyme design demands a detailed understanding of enzyme catalysis - enzyme structures, catalytic mechanisms and principles of enzyme-substrate recognition. Upon isolation of catalytic DNA molecules, Dr. Li's laboratory is concerned with detailed structural and mechanistic investigations of molecular recognition of substrates by deoxyribozymes.
Also of interest is the study of DNA aptamers. These single-stranded DNA molecules have specific tight binding to intended ligands (small molecules or proteins, etc.). They provide the basis for new diagnostic and biosensing tools. For example, DNA aptamers can be designed with unique spectroscopic properties to facilitate their use as biosensors.
Another direction of Dr. Li's research is the development of DNA-binding drugs through in vitro selection and combinatorial screening of random-sequence DNA libraries, and libraries of small organic and inorganic compounds. The goal is to design DNA-binding molecules with inhibitory effects on DNA replication and translation and consequent anti-cancer properties. The development of new anti-tumor agents is clearly very much of interest.
Next article ...
Previous article ...
|
