Wing Ming Keung

Tel: 432 4001

Fax: 566 3137

Research Overview

Alcohol Abuse/Alcoholism

Investigators in alcohol research have long sought a pharmaceutical agent that would selectively suppress craving for alcohol. However, the lack of knowledge on the biochemical basis for the urge to drink alcohol and a truly predictive animal model for human alcohol abuse/dependence have made this an elusive goal. We have chosen a relatively unconventional approach to the problem by taking advantage of the vast clinical experience of earlier generations in China, where for millennia folk medicines have been used apparently safely and effectively for the treatment of "alcohol addiction."

This approach has led to the discovery of daidzin, the active principle of an ancient Chinese herbal treatment (Radix puerariae, RP) for "alcohol addiction," which selectively suppresses alcohol intake by alcohol preferring Syrian golden hamsters. Since then, the antidipsotropic activity of crude RP and purified or synthetic daidzin have been confirmed independently by us and others in Wistar rats, Fawn Hooded rats, and the genetically bred alcohol preferring P rats under various experimental conditions including two-lever choice, two-bottle free-choice, limited access, and alcohol-deprived paradigms.

Unlike other known antidipsotropic agents, daidzin was discovered based solely on empirical clinical information rather than any preconceived theories or hypotheses derived from animal data. This unique property of daidzin not only has further strengthened our belief that it and/or one or more of its congeners could be developed into safe and efficacious agents for the treatment of alcohol abuse/dependence in humans, but also provides a powerful tool to identify and characterize the elusive biochemical pathway(s) that regulate alcohol consumption.

We have now identified the mitochondrial monoamine oxidase (MAO) - aldehyde dehydrogenase (ALDH-2) pathway as a potential site of action for daidzin and shown that it inhibits the second step of this pathway. Using a series of structural analogs of daidzin, we have revealed a positive correlation between the alcohol intake suppressive activities of these analogs and their abilities to increase the MAO/ALDH-2 activity ratio in isolated hamster liver mitochondria. On the basis of this finding, we have (i) proposed that a biogenic aldehyde derived from the action of MAO may play an important role in mediating the antidipsotropic action of daidzin, (ii) developed a simple and rapid in vitro assay as a primary screening assay for potential drug candidates, and (iii) opened the door for detail investigations of the mechanistic basis underlying the use and abuse of alcohol.

Synthesis of better and more efficacious analogs of daidzin is another objective of daidzin research. Using a semi-rational approach based on SAR established in earlier studies, we have designed and synthesized large number of analogs that are more potent and/or with better pharmacokinetic properties than daidzin. The pharmacological profiles of these new lead compounds are now being investigated. To design and synthesize even more potent antidipsotropic agents based on the proposed site of action of daidzin mentioned above, we have begun to explore new approaches based on computer modeling using "molecular docking" programs.

Metallothionein-III and Alzheimer's Disease

Metallothioneins (MTs) are a family of cysteine rich metal binding proteins discovered here at harvard Medical School by Dr. Bert L. Vallee more than 40 years ago. The chemical and physical properties of MT have been thoroughly investigated. However, its precise biological function(s) remains a subject of intensive investigation.

Metallothionein-III (MT-III) is a brain-specific isoform of MT. Like other members of the MT family, it contains 20 cysteine residues in conserved positions and binds zinc and copper. While astrocytes are the major cell type in which this protein was detected, its presence in neuronal cells has also been demonstrated. Unlike other MTs, MT-III is not induced by metals, glucocorticoids, or interferon. Further, MT-III is functionally unique - it inhibits the growth promoting effect of Alzheimer's disease (AD) brain extracts on primary cultures of rat embryonic cortical neurons. In fact, MT-III was originally isolated and identified as a Growth Inhibitory Factor (GIF) from normal brains. MT-III levels decrease in astrocytes surrounding damaged areas of the brain of neurodegenerative diseases such as AD, Parkinsonism, amyotrophic lateral sclerosis (ALS), and Down's syndrome (DS) but increase in reactive astrocytes in the vicinity of an old cerebral infarct, stab wounds, and NMDA-induced excitotoxic brain injury. These findings suggest an important role for MT-III in neuronal regeneration and degeneration. To date, the molecular nature of the growth promoting factor(s) in and the molecular mechanism by which MT-III suppresses the growth promoting effect of AD brain extracts are still unknown.

Formation of fibrillar deposits of amyloid peptides (consisting mainly of A_1-42 and A_1-40) in the brain is one of the defining pathological features of AD and many believe that abnormal A accumulation and/or deposition play an important role in the pathogenesis of AD. The direct involvement of A and/or aggregates of A in neurodegeneration is suggested by the observation that fibrillar A is neurotoxic, inducing neuronal cell death in culture and neuronal loss in vivo. In primary cultures of hippocampal neurons, two seemingly opposing effects of A have been observed - it is neurotrophic for differentiating neuronal precursor cells but is neurotoxic for mature neurons. The growth inhibitory activity of MT-III may stem from its ability to antagonize the neurotrophic effect of A which is apparently rich in AD brains.

To identify and characterize the physiological function(s) of MT-III in general and its potential role in the pathogenesis of AD and other neurodegenerative diseases in particular, we have launched a new program to investigate the potential function(s) of MT-III in neuro-degeneration and/or regeneration. Preliminary results indicate that MT-III attenuates both the trophic and toxic effects of A on neuronal cells in culture and modifies both quantitatively and qualitatively A aggregation in vitro. Better understanding of the molecular mechanism underlying MT-III's action on the nerve cells and A will shed light on the biological function of MT-III and provide valuable information for the identification of new agents useful for prevention and/or treatment of AD.

Selected Publications

Keung WM, Vallee BL. Daidzin and daidzein suppress free-choice ethanol intake by Syrian golden hamsters. Proc. Natl. Acad. Sci. U.S.A. 1993; 90:10008-10012.

Keung WM, Lazo O, Kunze L, Vallee BL. Daidzin suppresses ethanol consumption by Syrian golden hamsters without blocking acetaldehyde metabolism. Proc. Natl. Acad. Sci. USA. 1995; 92:8890-8993.

Keung WM, Lazo O, Kunze L, Vallee BL. Potentiation of the bioavailability of daidzin by an extract of Radix puerariae. Proc. Natl. Acad. Sci. USA. 1996; 93:4284-4288.

Keung WM, Klyosov AA, Vallee BL. Daidzin inhibits mitochondrial aldehyde dehydrogenase and suppresses ethanol intake of Syrian golden hamsters. Proc. Natl. Acad. Sci. USA. 1997; 94:1675-1679.

Keung WM, Vallee BL. Daidzin and its antidipsotropic analogs inhibit serotonin and dopamine metabolism in isolated mitochondria. Proc. Natl. Acad. Sci. USA. 1998; 95:2198-2203.

Rooke N, Li DJ, Li J, Keung WM. The Mitochondrial Monoamine Oxidase - Aldehyde Dehydrogenase Pathway - A Potential Site of Action of Daidzin. J. Med. Chem. 2000; 43(20):4169-4179.

Keung WM. Biogenic aldehyde(s) derived from the action of monoamine oxidase may mediate the antidipsotropic effect of daidzin. Chemico Biological Interactions 2001; 130(132):921-932.

Irie Y, Keung WM. Metallothionein-III Antagonizes the Neurotoxic and Neurotrophic Effects of Amyloid Peptides. Biochem. Biophys. Res. Commun. 2001; 282:416-420.

Related Patents

US patents 5,204,369; 5,624,910; 5,886,028; 6,121,010.