Chapter 1
Biosynthesis and Utilization of Isoprenoids Overview of Research Directions
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James M . Trzaskos Du Pont Merck Pharmaceutical Company, Experimental Station, Wilmington, DE 19880-0400
Mevalonic acid biosynthesis and its subsequent metabolism into a plethora of isoprenoid compounds has reemerged as a prominent field of study in chemical and biochemical science. The conversion of mevalonic acid into key intermediates of cellular metabolism, as structural components of cellular membranes, as natural products of significant commercial u t i l i t y , and as natural protectants against infection and disease highlights the significance of t h i s biochemical pathway. Additionally, the isoprenoid pathway has served as the target for important therapeutics and agrochemicals used to combat such diverse diseases such as fungal infections and infestations, hypercholesterolemia, and atherosclerosis. The contents of this volume will expand on these topics with details on the chemical, biochemical, and molecular biological progress made in this field presented at two recent ACS symposia. I have been given the assignment to provide an overview of the s c i e n t i f i c d i r e c t i o n s i n the isoprenoid f i e l d . For t h i s purpose, I have r e f l e c t e d upon recent symposia proceedings and have written my thoughts i n a manner which I believe demonstrate the breadth of science involved i n furthering our understanding and s p e c i f i c knowledge of the isoprenoid pathway. Contributing authors to t h i s volume were s o l i c i t e d from speakers at the ACS National Meeting held A p r i l 14-19, 1991 and the ACS/FASEB Symposium on the Biosynthesis and U t i l i z a t i o n of Isoprenoids (Reductase V) held A p r i l 18-20, 1991 i n A t l a n t a , GA. These two symposia attracted over 400 p a r t i c i p a n t s to a combined t o t a l of 15 s c i e n t i f i c sessions held over the course of one week. Reflected i n t h i s volume are many diverse aspects of isoprenoid
0097-6156/92/0497-0002$06.00/0 © 1992 American Chemical Society
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
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metabolism which have at t h e i r root a common biosynthetic pathway. The l a s t compilation of such a wide range of topics associated with isoprenoids appeared i n 1981 i n a two volume set e n t i t l e d "Biosynthesis of Isoprenoid Compounds," edited by John W. Porter and Sandra L. Spurgeon. That work d e t a i l e d the major metabolic processes associated with isoprenoid biosynthesis which should serve the reader as a foundation and reference f o r entry into t h i s field.
The Biochemical Pathway t o Polyisoprenoids Enzymology of the Pathway. Formation of Mevalonic Acid. Our seeming ease of acceptance that mevalonic acid serves as the immediate precursor to the diverse s t r u c t u r a l class we c a l l isoprenoids overshadows the extensive e f f o r t and work which lead to t h i s remarkable discovery ( i ) . Indeed emphasis i n recent years has focused on the d e t a i l e d enzymology of mevalonic acid formation from 3/7-hydroxy3-methyglutaryl coenzyme A (HMG-CoA) catalyzed by the enzyme HMGCoA reductase (J2) . In f a c t , the basis f o r past symposia i n t h i s s e r i e s (5), and the reason f o r the Reductase V s u b t i t l e i n the t i t l e f o r the ACS/FASEB symposium, has been to review progress made i n understanding the reductase biochemically and to exploit these findings through chemical applications. I t i s i r o n i c , therefore, that only seven presentations at these symposia involved discussions of reductase s p e c i f i c a l l y . These include elucidation of mevalonic acid formation v i a two related HMG-CoA reductase isozymes i n plants. The r e l a t i o n s h i p of these two enzymes to themselves and the o v e r a l l isoprenoid pathway should be forthcoming with cloning of the two proteins and continued work on t h e i r biochemistry. In mammalian systems, the importance of HMG-CoA reductase as a therapeutic target continues to d e l i v e r increasingly more potent i n h i b i t o r s . Alternative approaches to blocking mevalonate production v i a regulation of reductase gene expression with oxygenated s t e r o l s also remains an active research area. We should expect to see new developments i n t h i s f i e l d f o r years to come. Isomerase, Polyprenyl Sythetase, and Prenyl Transferases. The molecular mechanisms underlying formation and u t i l i z a t i o n of the isoprenoid unit i s b e n e f i t t i n g from molecular b i o l o g i c a l approaches. Cloning of the enzymes with subsequent s i t e directed mutagenesis i s leading to a greater understanding of the reaction mechanisms f o r the isomerase and prenyl transferase enzymes. Advances i n our understanding of gene regulation are also being made i n mammalian systems through investigation of the prenyl transferase enzymes. Important new discoveries can be anticipated as the sequence homology as well as d i v e r s i t y of polyprenyl synthetases from d i f f e r e n t species become evident. The functional s i g n i f i c a n c e of various protein domains w i l l be unraveled i n the coming years. Applications of t h i s information i n the design and synthesis of new chemical e n t i t i e s with u t i l i t y i n the agrochemical and pharmaceutical industries should be anticipated and w i l l c e r t a i n l y advance t h i s f i e l d .
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
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REGULATION OF ISOPENTENOID METABOLISM Points of Divergence Along the Pathway. Formation of polyisoprenoids provides the basis f o r the d i v e r s i t y associated with the isoprenoid pathway. H i s t o r i c a l l y , Ruzicka masterfully solved the s t r u c t u r a l d i v e r s i t y of many of the c y c l i c mono ( C ) -, sesqui ( C ) - , and d i ( C ) terpenoids through the application of Wallach's "isoprenoid r u l e " ( i ) . Today t h i s work continues as questions of d i v e r s i t y at the enzymatic l e v e l are beginning to be answered. In the area of terpenoid biosynthesis, key questions remain as to how the seemingly related cyclase enzymes impart unique c y c l i z a t i o n processes to a common a c y c l i c substrate? How do the cyclases impose conformational r e s t r a i n t s ? How are reaction intermediates s t a b i l i z e d ? What prevents destruction of the enzyme through autoalkylation reactions? Answers to these questions are being pursued and w i l l be forthcoming with p u r i f i c a t i o n , cloning and mutagenesis studies of the enzymes involved. Progress toward our understanding of diverse reaction mechanisms i s also being made i n the s t e r o l branch of the pathway. Our understanding of squalene formation and the importance of squalene synthetase as a regulatory enzyme i n s t e r o l biosynthesis should advance more quickly with the molecular cloning of the yeast enzyme. S i m i l a r l y , advances on squalene c y c l i z a t i o n mechanisms using p u r i f i e d preparations w i l l aid i n i n h i b i t o r design. This i s an extremely competitive area of research because of the importance which the synthetase plays i n cholesterol biosynthesis. I n h i b i t o r s of t h i s enzyme should serve as useful therapeutics to control elevated serum cholesterol l e v e l s (see below). Further along the pathway, i t has been shown that novel c a t a l y t i c processes are associated with s t e r o l formation. The 14aformyloxy-lanost-8-en-3/7-ol intermediate i n lanosterol demethylation has been i s o l a t e d and defines a new reaction mechanism catalyzed by a cytochrome P-450 species. Application of mechanistic data of t h i s sort has lead to the design of extremely strong enzyme i n h i b i t o r s directed at the demethylase and other s t e r o l transforming enzymes more d i s t a l i n the pathway. This i s p a r t i c u l a r l y true i n the plant and fungal areas where i n h i b i t o r y molecules can be expected to f i n d application i n various a g r i c u l t u r a l settings f o r either fungal or weed control. 1 Q
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Isoprenoid Natural Products Rubber, Terpenoids, and Carotinoids. The importance of the isoprenoid pathway i s not only evident through advances i n our understanding of basic science and enzymology, but also i n the key metabolic role and commercial potential of the end-products of the divergent pathways. Rubber i s perhaps the most obvious example of a commercial application of end products of polyisoprene biosynthesis. Optimization of rubber production through genetic engineering and biotechnology w i l l c e r t a i n l y lead to better sources and expanded growth opportunities f o r t h i s material. S i m i l a r l y , application of biotechnology to the growth and production of high carotenoid-containing algae i s leading to natural sources f o r these materials which h i s t o r i c a l l y have been produced through organic synthesis. A greater demand f o r carotenoids i n the pharmaceutical industry and as food colorants are the major factors behind these new process developments. An expanded use of terpenoids can also be invisioned with novel application i n disease control.
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
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S t e r o l s . Although I have already mentioned that s t e r o l transforming enzymes serve as targets f o r i n h i b i t o r design, I think i t i s equally important to point out the u t i l i t y of s t e r o l s as commercial products. Through a combination of c l a s s i c a l fermentation technology coupled with molecular biology, genetics and bioengineering, i t i s now possible to accumulate v i r t u a l l y any isoprenoid generated along the pathway. As an example, yeast have been selected which over-produce s t e r o l s to l e v e l s up to 20% of the dry c e l l weight without s i g n i f i c a n t growth impairment or harm to the organism. Thus, the use of yeast as a feedstock f o r isoprenoid precusors used i n organic synthesis of vitamins, s t e r o i d hormones, or novel a p p l i c a t i o n can be anticipated i n the years ahead. Extension of t h i s technology, which r e s u l t s i n accumulation of isoprenoid products, to other branches of the pathway could lead to organisms with altered isoprenoid end-product p r o f i l e s with multiple a p p l i c a t i o n . For instance, i t may be possible to design insect r e s i s t a n t plants through changes i n the balance of endproducts derived from the isoprenoid pathway. I t can be invisioned that t o x i c i t y may be imparted to a parasite without a l t e r a t i o n i n host productivity by the appropriate s e l e c t i o n of a host endproduct isoprenoid p r o f i l e , a s t e r o i d perhaps obtained through bioengineered metabolic blockade. Thus, a p o t e n t i a l l y environmentally safe method f o r insect control may be f e a s i b l e through knowledge and manipulation of the isoprenoid pathway. Advances of t h i s type w i l l c e r t a i n l y be made within the next few years. Commercial Applications Agrochemicals. Fungal Growth Control. H i s t o r i c a l l y the s t e r o l branch of the isoprenoid pathway has served as a valuable target f o r fungicide development. Enzyme i n h i b i t o r s directed at squalene epoxidation, s t e r o l 14a-demethylation, and s t e r o l nuclear bond rearrangements ( s t e r o l Δ +Δ -isomerase) have i d e n t i f i e d the allylamines, the azoles, and the morpholines as useful s t r u c t u r a l classes f o r c o n t r o l l i n g pathogenic fungi (4) . These bountiful and successful discoveries have created an intense search for new and novel i n h i b i t o r s of the s t e r o l biosynthetic pathway. Application of highly s p e c i f i c , directed research at new enzyme targets such as 2,3-oxidosqualene c y c l i z a s e , s t e r o l 4-demethylase, and the s t e r o l C-24 a l k y l a t i o n sequence i s ongoing along with searches for better i n h i b i t o r s of " c l a s s i c a l " enzyme targets. Application of highly sophisticated i n h i b i t o r design strategies using knowledge of enzyme reaction mechanisms i s leading to extremely potent and p o t e n t i a l l y useful agents. These high energy intermediates (HEI) block c a t a l y s i s i n a mechanism s p e c i f i c manner and act as enzyme traps. One can expect t h i s work to lead to highly useful agents f o r the agrochemical sector i n keeping with the past t r a d i t i o n of s t e r o l biosynthesis i n h i b i t o r as fungicides. 8
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Herbicides. Our understanding of the enzymology of s t e r o l biosynthesis i s becoming increasing more r e f i n e d . We are beginning to see that the subtle differences between species may allow f o r
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
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designing unique chemical i n h i b i t o r s with exacting s p e c i f i c i t y . However, the commonalty of the biochemical process, and the necessity of isoprenoid products to support metabolic functions, i s defining a new use for s t e r o l biosynthesis i n h i b i t o r s as herbicides and plant growth regulators. Again, the o l d and seemingly well characterized fungicide i n h i b i t o r s are leading the way. We are f i n d i n g that these agents are losing t h e i r defined s p e c i f i c i t y with age. The apparent unwanted s i d e - e f f e c t of a c t i v i t y against host pathways i s actually defining targets f o r future herbicide development. Indeed, unique agents can be envisioned which maintain the desired potency toward the isoprenoid biosynthetic pathway, but which undergo novel transformations p r i o r to i n h i b i t i o n , or display novel uptake properties which can e s t a b l i s h s p e c i f i c i t y and plant s e l e c t i v i t y while maintaining the proven mechanism of action. Agents which target the isoprenoid pathway i n t h i s manner can be expected to be developed as herbicides i n the future. Pharmaceuticals. Antimycotics. As i n the previous section on agrochemical fungicide development, the enzymes involved i n ergosterol biosynthesis have served as targets for pharmaceutical antimycotic development. In t h i s application, the s t e r o l 14a-demethylase has gained prominence as a key enzyme f o r c o n t r o l l i n g c l i n i c a l l y important pathogenic fungi. Enhanced o r a l b i o a v a i l a b i l i t y , improved pharmacokinetics and greater safety are permitting the broad application of new agents to an increasing patient population. In comparison to older, f i r s t and second generation agents, these new compounds appear to be more s e l e c t i v e and s p e c i f i c f o r the fungal s t e r o l 14a-demethylase than f o r the mammalian counterpart which also shows s e n s i t i v i t y to azole i n h i b i t i o n . A d d i t i o n a l l y , the new azole antimycotics seem less promiscuous toward other s t e r o i d metabolism cytochrome P-450 enzymes which enhances t h e i r s i d e - e f f e c t p r o f i l e enormously. These advances serve as a testimonial to the type of progress one can anticipate for other therapeutic agents which target the isoprenoid pathway, and which are currently under development. Hypocholesterolemic Agents. Elevated serum cholesterol has been shown to be a prominent factor i n the etiology of coronary heart disease and atherosclerosis. Thus, the search f o r safe and e f f e c t i v e agents to lower blood cholesterol has dominated pharmaceutical research i n recent years. The cholesterol biosynthetic pathway has served as the major focus of t h i s research for obvious reasons. I n h i b i t i o n of the rate l i m i t i n g enzyme i n the pathway, HMG-CoA reductase, with the s t a t i n class of therapeutics has proven to be an extremely e f f e c t i v e way to block cholesterol production with ensuing serum cholesterol reduction. Cholesterol biosynthesis blockade at t h i s early step i n isoprenoid production, however, also leads to depletion of other key isoprenoids derived from mevalonic acid. Although no obvious, l i f e - t h r e a t e n i n g t o x i c i t i e s have surfaced with these therapeutics to date, the p o t e n t i a l remains. A d d i t i o n a l l y , despite the high e f f i c a c y of the s t a t i n s , i t i s necessary to use them i n combination with other
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
1. TRZASKOS
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agents to obtain maximum serum cholesterol lowering i n c e r t a i n patients with extreme hypercholesterolemia. Thus, the hunt continues for more and better agents to block cholesterol formation. The t a c t i c s involve directed synthesis toward squalene synthetase i n h i b i t o r s which would target the cholesterol branch of the isoprenoid pathway s p e c i f i c a l l y . A l s o , mimicry of n a t u r a l l y occurring oxysterol regulators of HMG-CoA reductase, i s proving to be affective i n producing novel agents to f i g h t elevated cholesterol l e v e l s . One can expect to see pharmaceutical drug discovery providing us with numerous agents to control and manipulate cholesterol production. Many of these agents w i l l f i n d u t i l i t y i n the c l i n i c a l s e t t i n g , while others w i l l be valuable t o o l s for the basic s c i e n t i s t to use i n d i s s e c t i n g the isoprenoid pathway i n greater d e t a i l . NEW HORIZONS Extension of Pathway D i v e r s i t y . Prenylated P r o t e i n s . An emerging area of intense biochemical and chemical research was born with the r e a l i z a t i o n that proteins can be modified p o s t - t r a n s l a t i o n a l l y by polyisoprenoid attachment. Protein prenylation serves as an i n i t i a l step i n a processing sequence which includes carboxy terminal p r o t e o l y s i s , prenylcysteine transmethylation, and membrane association. Unique protein prenylation sequences have been defined such as the Caax sequence found i n p 2 1 along with others found i n a number of small molecule weight G-proteins and nuclear lamanins. Incorporation of farnesyl or geranylgeranyl groups v i a highly s p e c i f i c protein prenyl transferase enzymes which recognize the protein prenylation motifs are s t a r t i n g to be described. The p o t e n t i a l for therapeutic and agrochemical development which targets these enzymes and processes i s sure to be r e a l i z e d i n the coming years. I t can also be anticipated that new findings w i l l emerge from these studies which w i l l contribute to advances i n the areas of natural products, enzymology, biochemistry, and chemistry of the isoprenoid pathway. Thus, the r i c h t r a d i t i o n of the isoprenoid pathway can be expected to continue with novel advances contributing to our general s c i e n t i f i c knowledge for years to come. r a e
LITERATURE CITED 1. 2. 3. 4.
Nes, W.R. and McKean, M.L. Biochemistry of Steroids and Other Isopenteroids. University Park Press, Baltimore, MD 1977. 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase; Sabine, J. R., Ed.; CRC Press, Boca Ratan, FL 1983. Geelan, M.J.H., Gibson, D.M., and Rodwell, V.W. FEBS Lett. 1986, 201, 183-186. Recent Trends in the Discovery, Development and Evaluation of Antifungal Agents, Fromtling, R. Α . , E d . ; J.R. Prous Science Publishers, Barcelona, Spain 1987.
RECEIVED December 26, 1991
Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
