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Glutathione (L-g-glutamyl-L-cysteinylglycine, GSH), the main non-protein
thiol in cells, plays a key role in defense against free radicals, peroxides,
and electrophiles. We have made potent and specific inhibitors of GSH
synthesis (e.g. buthionine sulfoximine, BSO) that cause GSH depletion
in vivo.
Studies with BSO and other inhibitors have defined the
inter- and intra-organ pathways of GSH turnover and have elucidated the role of GSH in cell survival. BSO-mediated GSH depletion
sensitizes tumor cells to alkylating agents, redox cycling drugs and
radiation therapy. We have recently cloned and expressed human g-glutamylcysteine synthetase
(g-GCS), the first and rate-limiting
enzyme of GSH biosynthesis, and are exploring the chemical synthesis and
mechanism of action of several novel inhibitors. We are also investigating the synthesis and use of novel g-GCS inhibitors directed at
pathologic microbes (e.g., Salmonella and Trypanosomes). Inhibitors of the mechanistically related enzyme, glutamine synthetase,
are of interest as possible anti-tuberculosis drugs.
In separate studies we are examining the enzymology, physiology and
pharmacology of nitric oxide (NO), a free radical formed by five
election oxidation of a guanidinium nitrogen of L-arginine.
Cytokine-induced macrophages form large amounts of NO as part of their
cytotoxic armament whereas endothelial cells form NO more slowly as a
vascular smooth muscle relaxant and inhibitor of platelet aggregation
and adhesion. We have prepared a series of N-substituted arginine analogs
as inhibitors of NO synthesis and have begun to "map" the active sites
of macrophage and endothelial cell NO synthase in order to design
cell-type specific inhibitors. We have also developed novel non-amino acid inhibitors of the enzyme. In collaboration with other
investigators, we have shown that (i) basal NO synthesis contributes
to normal blood pressure homeostatasis, (ii) NO synthesis can be
limited by arginine availability, (iii) overproduction of NO accounts
for the hypotension seen in septic (endotoxic) shock or seen following
administration of tumor necrosis factor, interleukin-1a , or interleukin-2. The potentially lethal
hypotension caused by septic shock or cytokine injection was reversed
by giving NO synthesis inhibitors. Most recently, we have established
that NO and its products have a direct inhibitory effects on GSH
biosynthesis and have developed several isoform selective, inreversible NO synthase inhibitors.
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Selected Publications
"Nitric Oxide Synthases: Properties and Catalytic Mechanism", O.W. Griffith
and D.J. Stuehr, Annual Review of Physiology, 57, 707-736
(1994).
"S-Alkyl-L-Thiocitrullines:
Potent Stereoselective Inhibitors of Nitric Oxide
Synthase with Strong Pressor Activity in Vivo", K. Narayanan,
L. Spack, K. McMillan, R.G. Kilbourn, M.A. Hayward, B.S. Masters, and
O.W. Griffith, Journal of Biological Chemistry, 270, 11103-11110
(1995).
"L-Thiocitrulline: A Stereospecific, Heme-Binding Inhibitor of Nitric
Oxide Synthases", C. Frey, K. Narayanan, K. McMillan, L. Spack,
S.S. Gross, B.S. Masters, and O.W. Griffith, Journal of Biological
Chemistry, 269, 26083-26091 (1995).
"Nitric oxide synthase: Characterization by electron paramagnetic
resonance of the interactions of L-arginine and L-thiocitrulline with
the heme cofactor region of nitric oxide synthase", J.C. Salerno,
C. Frey, K. McMillian, R. Williams, B.S.S. Masters, and
O.W. Griffith. Journal of Biological Chemistry, 270, 27423-27428
(1995).
"NO in septic shock: good, bad or ugly?
Learning from iNOS knockouts", S.S. Gross, R.G. Kilbourn, and O.W.
Griffith, Trends in Microbiology, 4, 47-49 (1996).
"Inhibition of endothelial cell amino acid
transport System y+ by arginine analogs that inhibit nitric oxide synthase", K.K. McDonald, R. Rouhani, M.E. Handlogten, E.R. Block,
O.W. Griffith, R.D. Allison, and M.S. Kilberg, Biochimica et Biophysica
Acta, 1324, 133-141 (1997).
"Design of nitric oxide synthase inhibitors
and their use to reverse hypotension associated with cancer immunotherapy", O.W. Griffith and R.G. Kilbourn. Advances in Enzyme
Regulation, 37, 171-194 (1997).
"Measurements of total plasma nitrite and nitrate in pediatric
patients with the systemic inflammatory response syndrome",
L. Spack, P.L. Havens, and O.W. Griffith, Critical Care Medicine,
25, 1071-1078 (1997).
"L-amino acid oxidase (LOX) modulation of melphalan activity against
intracranial glioma", K. Moynihan, G.B. Elion, C. Pegram, C.J. Reist,
D. Wellner , D.D. Bigner, O.W. Griffith, and H.S. Friedman, Cancer
Chemotherapy & Pharmacology, 39, 179-186 (1997).
"Characterization of
5-oxo-L-prolinase in normal and tumor tissues of humans and rats: a potential new target for biochemical modulation
of glutathione", X. Chen, R.L. Schecter, O.W. Griffith, M.A. Hayward, L.C. Alpert, and G. Batist.
Clinical Cancer Research, 4, 131-138 (1998).
"N5-(1-Imino-3-butenyl)-L-ornithine. A
neuronal isoform selective mechanism-based inactivator of nitric oxide
synthase", B.R. Babu and O.W. Griffith, Journal of Biological Chemistry,
273, 8882-8889 (1998).
"Expression
purification of human gamma-glutamylcysteine synthetase", I.
Misra, O. W. Griffith, Protein Expression and Purification, 13,
268-276 (1998)
"Design of isoform-selective
inhibitors of nitric oxidase synthase", B.R. Babu and O.W. Griffith,
Current Opinion in Chemical Biology, 2, 491-500 (1998).
"The Enzymes of Glutathione Synthesis: g-Glutamylcysteine Synthetase",
O.W. Griffith and R.T. Mulcahy, Advances in Enzymology and Related Areas of Molecular
Biology, 73, 209-267 (1999).
"L-Arginine Binding to Nitric Oxide Synthase: The Role of H-bonds to the
Non-reactive Guanidinium Nitrogens", B.R. Babu, C. Frey and O.W. Griffith,
Journal of Biological Chemistry, 274, 25218-25226 (1999).
"Adaptation to Chronic Hypoxia Confers Tolerance to Subsequent Myocardial
Ischemia by Increasing Nitric Oxide Production",
J.E. Baker, P. Holman, B. Kalyanaraman, O.W. Griffith and K.A. Pritchard, Jr.,
Annals of the New York Academy of Sciences, 874, 236-253, (1999).
"Chronic myocardial hypoxia increases nitric oxide synthase and decreases caveolin-3.",
Y. Shi, K.A. Pritchard, Jr, P. Holman, P. Rafiee, O.W. Griffith, B. Kalyanaraman, J.E. Baker.
Free Radic Biol Med, 29 (8), 695-703 (2000)
"Implications for isoform-selective inhibitor design derived from the binding mode of bulky isothioureas to the heme domain of endothelial nitric-oxide synthase.",
C.S. Raman, H. Li, P. Martasek, B.R. Babu, O.W. Griffith, B. S. Masters, T.L. Poulos.
J Biol Chem, 276 (28), 26486-91 (2001)
"In vivo proton (H1) magnetic resonance spectroscopy for cervical carcinoma.",
J.R. Allen, R.W. Prost, O.W. Griffith, S. J. Erickson, B. A. Erickson.
Am J Clin Oncol, 24 (5) 522-529 (2001)
"Escherichia coli gamma-glutamylcysteine synthetase. Two active site metal ions affect substrate and inhibitor binding.",
Kelly BS, Antholine WE, Griffith OW.
J Biol Chem, 277 (1), 50-58 (2002)
"Glutaminase and the control of airway pH: yet another problem for the asthmatic lung?",
O.W. Griffith
Am J Respir Crit Care Med, 165(1), 1-2 (2002)
"Non-heme iron protein: a potential target of nitric oxide in acute cardiac allograft rejection.",
G.M. Pieper, N.L. Halligan, G. Hilton, E.A. Konorev, C.C. Felix, A.M. Roza, M.B. Adams, O.W. Griffith
Proc Natl Acad Sci U S A, 100, 3125-3130 (2003)
"Structural Characterization and Kinetics of Nitric-oxide Synthase Inhibition by Novel N5-(Iminoalkyl)- and N5-(Iminoalkenyl)-ornithines.",
L.E. Bretscher, H. Li, T.L. Poulos, O.W. Griffith
J Biol Chem, 278, 46789-46797 (2003)
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