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Turning back the hands of time: is aging a disease? Progress is being made in the search for anti-aging compounds.

According to the standard drug design paradigm, a drug therapy is targeted for a particular disease according to well-established procedures, such as to:

* Obtain an X-ray structure of the relevant protein and ligand (small-molecule) complex that activates the disease symptoms;

* Develop small-molecule inhibitors through high-throughput screening using combinatorial libraries or virtual screening programs or both;

* Optimize the "lead molecules" by chemical modification to improve their biological activity.

But how does one target the "disease" called aging? (Most people who would argue that aging is not a disease are under 50!) The problem is that aging actually is a spectrum of degenerative changes and is by its nature multifactorial. According to the Free Radical Theory of Aging (FRTA), the superoxide free radical generated as a one percent by-product of normal metabolism is converted through acid-base and redox chemistry into the more active oxidizing agent hydrogen peroxide. [H.sub.2][O.sub.2], which can be deactivated by the enzymes catalase and/ or glutathione peroxidase, may however go on to form, usually through interaction with transition metals, the highly oxidizing hydroxyl radical (HO *). HO * can then attack any biological substrate, including lipid, protein, carbohydrate, or DNA, leading to undesirable end products such as lipid peroxides, protein carbonyls, or hydroxylated DNA adducts, with a resulting loss of biological function. These compounds and others represent biomarkers for the situation generically termed "oxidative stress." The FRTA basically says that the cumulative effects and progressive damage caused by oxidative stress correlate with biological aging. So it makes sense to intervene chemically to try to reduce free-radical-induced damage.

There are two obvious pathways for intervention:

* Try to reduce the rate of initiation of superoxide free radicals;

* Try to interrupt chain reactions propagated by free radicals already present.

Since the metabolic errors are proportional to the rate of metabolism, slowing the metabolic rate should produce proportionally fewer free radicals. Simplifying metabolism to glucose + [O.sub.2] + ADP [right arrow] C[O.sub.2] + [H.sub.2]O + ATP, reducing caloric intake (less glucose), or reducing oxygen consumption (living at high altitude) should produce fewer free radicals in the initiation step. Indeed, caloric restriction (CR) has been shown to increase maximum lifespan in rodents and other animals by 25 percent or more as well as reduce tumour incidence in the older animals. CR may also work for humans, but it takes a long time to do the experiment! Nevertheless, the CR society believes that there is no reason why caloric restriction should not apply to humans, and an increasing number of people are using themselves as subjects for the experiment (see The persistence of this atypical behaviour remains to be seen, but we are all aware of the reverse problems associated with the increase in obesity.

Assuming that radicals have already been generated, the next approach is to try antioxidant therapy to reduce the damage the radicals may cause. Four years ago, with the help of an NSERC Strategic Project Grant, we began a series of experiments aimed at retarding the effects of aging by the systematic design, synthesis, and testing of novel antioxidants. Because of the lack of an obvious genetic target, we began by examining effective naturally occurring chemical antioxidants such as vitamins C and E. We refined existing theoretical methods so we could accurately predict the chemical behaviour of antioxidants, based on the bond dissociation enthalpy (BDE) of the weakest, and hence most reactive, bond in each molecule (1). We determined that an optimum "design window" for the weak bond in a lipopbilic antioxidant would be between 68-77 kcal [mol.sup.-1]--weaker than the bond in [alpha]-tocopherol (the most active antioxidant component of vitamin E) but stronger than the OH bond in ascorbate anion. The reason for this choice lies in the biochemistry of an antioxidant. After it becomes oxidized to form a radical, it must be reduced to regenerate anew the antioxidant. Vitamin C provides the reducing power.

Within this design window we saw many synthetic possibilities. Others were contributed by our colleagues Tony Durst, FCIC (University of Ottawa), and Ross Barclay, FCIC (Mount Allison University). Early on in this work, we settled on catechols (or ortho-hydroxyphenols) as the most promising target for the synthetic work. The reason for this is simple--catechol has a BDE which lies a full 9 kcal [mol.sup.-1] below phenol (phenol has a BDE of 87 kcal [mol.sup.-1]) and thus, with no other functional groups, catechol itself is almost in the design window. Adding other electron donors such as methyl to the benzene ring results in a variety of molecules within the window. Some of these were synthesized by 2001 and they behaved exactly as predicted: lower BDE, faster reaction rate with free radicals (2, 3).

In the original NSERC proposal, we intended to study the antioxidant properties in micelles, as a simulated cell model, and then to test the promising candidates in animals. Other colleagues including David Miller (Carleton University) pointed out the fallacy of this approach. Prior to any consideration of using animals, you first need to study toxicity of your compounds in cell culture and demonstrate efficacy. With assistance from Miller and Bill Willmore (Carleton University) we set up a cell culture facility at Carleton to test the new antioxidants.

Originally, we used human leukemia cultures (HL-60 strain) and later PC12 (adrenal) cell cultures, which had been selected to produce all adherent cell type. We quickly learned the many practicalities involved in the choice of cell type (somewhat arcane, but cells that adhere to a plate have advantages). We had to deal with exasperating problems of optimal growth and reproducibility including unexpected problems such as genetic drift. After a difficult start-up period, cell growth became satisfactory. We then had to pose the following questions:

* What is the toxicity of our antioxidants to the cells, and how do we measure toxicity?

* What is a suitable oxidative stressor to choose in our cell line?

* How strong are the protective effects of the antioxidant, i.e. the resistance to oxidative stress?

We assumed that according to the FRTA, if we could substantially reduce the amount of oxidative stress present, then the cells (organism) would possibly live longer. Certainly they could be expected to have a more healthy old age.

By 2002, cell testing was in full swing. We looked at about eight synthetic antioxidants, as well as some well-known reference antioxidants such as resveratrol (in wine), epigallocatechin gallate (in tea), and solubilized forms of vitamin E. At this stage, we rediscovered some well-known problems with catechols. Through the loss of the two exchangeable H-atoms in the OH groups, they become quinones, via formation of the semiquinone. Symbolizing the catechol as Q[H.sub.2], semiquinone as QH* and quinone as Q, the antioxidant behaviour comes from the reaction Q[H.sub.2] + LOO* [right arrow] QH* + LOOH, which breaks the chain reaction of lipid peroxidation (here LOO* could he a lipid peroxyl radical, which is a chain carrier, and LOOH is the lipid hydroperoxide product).

However, catechols also have a "dark side." They can act as pro-oxidants. First, the semiquinone QH* has a low pK and will ionize at biological pH into its anion [Q.sup.*-]. Then the electron transfer reaction [Q.sup.8-] + 02 [right arrow] Q + 02 converts molecular oxygen into superoxide anion. The quinone is then reduced by the one-electron reducing enzyme cytochrome P450 reductase and a redox cycle begins. This is a chain reaction using molecular oxygen as the reagent and the quinone as the catalyst. The result is ultimately the production of [H.sub.2.0.sub.2] (via disproportionation from superoxide, or from the enzyme superoxide dismutase that converts superoxide into hydrogen peroxide plus oxygen), which in high enough concentrations is toxic to cells.

By 2003, we saw that our growing array of synthetic catechols were proving to be strong hydrogen peroxide producers and were much too toxic to consider as potential anti-aging drugs. Our chemical antioxidants had fallen victim to biological enzymes that turned them into powerful pro-oxidants! We had to change direction, to look for catechols that had a low BDE for loss of the first (antioxidant) H-atom, but a high BDE for loss of the second (redoxcycle generating) H-atom. A possible solution was provided by Barclay and Durst via the family of naphthalenediols. 1,2- and 1,4-naphthalenediol are very active pro-oxidants, however 1,8- and 2,3-naphthaleuediol are not; the latter two do not lose the second H-atom to form quinones at all. The reason for this difference has to do with loss of aromaticity in the benzene ring adjacent to the dicarbonyl groups. When aromaticity is lost, as in the 2,3-naphthoquinone, a heavy energy penalty is paid and the result is stabilization of the intermediate semiquinone. Durst prepared the substituted compound 1,4-dipropyl-2,3-naphthalenediol (termed DPND), which is now looking promising in our battery of tests on cells. First, unlike the single-ring catechols that autoxidize in water and are strong peroxide generators in cells, DPND does not and is not. Unlike the catechols or 1,4-naphthalenediol, DPND is relatively non-toxic to cells. Finally, it has shown significant resistance to oxidative stress, increasing the viability of cells subject to such stress.

Thus, in 2004, after much testing, we believe that we have a new family of lead compounds with good biological activity. They are currently being tested:

* For toxicity in hepatic cells (Peter O'Brien, University of Toronto);

* In an "animal-on-a-chip" model (Michael Shuler, Cornell University);

* In cortical neurons (Michael Poulter and Bruce Pappas, Carleton University);

* For longevity and oxidative stress experiments on nematodes (Chandra Srinivasan, California State University, Fullerton).

Our second lead compound is the 1,8-naphthalenediol provided by Barclay, along with derivatives of this structure, and so far this family of compounds also has desirable chemical and biological properties.

But even if lead compounds are effective at reducing oxidative stress, can chemical antioxidants increase longevity? Recent experiments by C. K. Lee and co-workers (4) compared the effects of the antioxidants [alpha]-lipoic acid and coenzyme [Q.sub.10] to caloric restriction on the longevity of mice. They monitored the expression of 9,977 genes via DNA microarrays in these experiments, and showed that although all three approaches showed reduced markers of oxidative stress, only CR increased maximum life span (by 13 percent). Further, they showed that only CR prevented the all-important age-related changes in mitochondrial energy metabolism.

From these experiments on gene expression, we now have an additional consideration in the study of aging. We can think about a new target for drug design--how can we maintain energy metabolism at youthful levels? We have come full circle--we can now return to the paradigm of structure-based drug design with new molecular targets. Thus by combining the development of molecules to reduce oxidative stress, along with identification of critical energy-related proteins, we have the beginnings of a rational approach to slow the aging process.


I would like to thank the many colleagues involved in this project, and particularly the students Mihaela Flueraru, MCIC, and Alex Chichirau, MCIC, who developed the cell culture facility, and Leo Chepelev who did many of the theoretical calculations.


(1.) J. S. Wright, E. R. Johnson and G.A. DiLabio, "Predicting the Activity of Phenolic Antioxidants: Theoretical Method, Analysis of Substitutent Effects and Application to Major Families of antioxidants," J. Amer. Chem. Soc. 123, (2001), pp. 1173-1183.

(2.) M. C. Foti, E. R. Johnson, M. R. Vinqvist, J. S. Wright, L. R. C. Barclay and K. U. Ingold, "Naphthalene Diols: A New Class of Antioxidants. Intramolecular Hydrogen Bonding in Catechols, Naphthalene Diols and their Aryloxyl Radicals," J. Org. Chem. 67, (2002), pp.5190-5196.

(3.) H. H. Hussain, G. Babic, T. Durst, J. S. Wright, M. Flueraru, A. Chichirau and L. L. Chepelev, "Development of Novel Antioxidants: Design, Synthesis and Reactivity," J. Org. Chem. (2003), pp.68, 7023-7032.

(4.) C. K. Lee, T. D. Pugh, R. G. Klopp, J. Edwards, D. B. Allison, R. Weindruch and T. A. Prolla, "The Impact of [alpha]-Lipoic Acid, Coenzyme [Q.sub.10], and Caloric Restriction on Life Span and Gene Expression Patterns in Mice," Free Rad. Biol. Med. (2004), 36, pp. 1043-1057.

James S. Wright, MCIC, is Chancellor's Professor of Chemistry at Carleton University's Ottawa-Carleton Chemistry Institute.
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Title Annotation:research for new anti oxidents
Comment:Turning back the hands of time: is aging a disease?
Author:Wright, James S.
Publication:Canadian Chemical News
Geographic Code:1USA
Date:Jul 1, 2004
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