The Inhibition of Enzymes by Drugs and Pesticides Thomas H. Cmmartie Stauffer Chemical Company. Richmond, CA 94804
Many medicinal drugs and many pesticides have biologically important functions as a direct result of their effect on enzymes. Enzymes are large proteins (polymers of a-amino acids) which enable specific chemical reactions to occur a t reasonable rates a t moderate temperatures and a t near neutral pH (1, 2). The reactions catalyzed by enzymes are responsible for such important biochemical processes as the light-driven reductionof Con to organic sugars, the utilization of sugars in cellular energy production, and the synthesis of hiopolymers such as proteins, polysaccharides, and polynucleotides. Consequently, the growth and replication of every living organism depends on the proper and coordinated functioning of a large number of enzymes. Some very simple biochemical entities such as viruses can exist for long periods in an inactive state without using enzymes. However, viruses must invade a living host cell and take over its metabolism (control its enzymes) to carry out the reactions necessary to produce more virus particles. Considering the crucial role played by enzymes in almost all biochemical processes, i t should not be surprisina that compcunds which alter the catalytic activity of ~nzsm&can have prufound effects on cells and then on whole nraanisms. Molecules like these can either increase the rate (acsvate) or decrease the rate (inhibit or inactivate) a t which enzymes function. In fact, such compounds produced naturally within cells represent one important way in which enzymes are regulated. The activity of enzymes must be carefully controlled so that several enzymes can work together in sequence to convert an initial substance through several chemical reactions to a final product with the greatest efficiency. In additiun, enzymes at thr beginning of such pathways are often inhibited by the final pruduct of the pathway so that this first enzyme (and theretore the whole pathway) van be turned off when enough of the tinal has heen produced. Clearly then, compounds which affect the activity of enzymes have the potential to cause dramatic alterntiuns in cellular metabolic activity, and these changes may he either hmeficial or harmful to the organism. I11 addltion r o compounds produced within an organism, enzymes can he affrctrd hs some comoounds auulird .. irom an external source if such compoundsbenetrate the organism and enter the cells. I t makes no difference whether these compounds are natural products or are produced synthetically-inhibition of a particular enzyme will result in the same changes in metabolism and wiil have the same final effects on the organism. When the organism is man and the objective of the application is relief from disease, the compounds are called drugs. When the organism is other than man and the objective is the control of a pest, the compounds are called pesticides. In biochemical terms, drugs and pesticides are vew similar, as are the processes hv which such compounds are identified and studied. ~ l t h o u ~not h all drugs or pesticides function by changing the catalytic activity of enzymes, enough do work in this way to provide examples of a variety of kinds of enzyme inhibition. Since enzyme inhibitors and enzyme activators cause a change in the rate of an enzyme-catalyzed reaction, kinetic studies are the most important experiments used to study these compounds. The topic of enzyme kinetics is not particularly difficult (3), hut it quickly becomes complex when an inhibitor and several reactants must be considered. In this
paper enzyme kinetics will not he discussed, but it is important to remember that the conclusions drawn about how inhibitors interact with enzymes depend primarily on careful kinetic studies. Enzyme catalysis alwavs occurs at an actiue site, a small region or pocket on the surface of the enzyme which has evolved to have the proper three-dimensional arrangement of the protein to hold the suhstrares in thr uptimum configuration fur reaction and to provide any necessary catalyticgroups (such as acids or bases) precisel; where they can do the most good. The decrease in rate caused by an enzyme inhibitor occurs as a consequence of the binding of the inhibitor to give an enzyme-inhibitor complex which has less catalytic activity than the free enzyme. Most enzyme inhibitors bind a t the active site and prevent the substrates from hindina and undereoina reaction. However, some inhihitors bindat sites remove; from the active site and cause inhibition indirectly by chanaina the shape of the active site or the positionof-impor= catalytic groups. There are a variety of experimental tests to distinguish active-site directed inhibitors from others. Enzyme inhibitors fall usually into one of two classes, reversible or irreversible, according to whether the inhibitor can dissociate from the enzyme-inhibitor complex to regenerate the free enzyme. Reversible inhibitors interact with their target rnzymes by the same noncovalent binding forces which hold nwst substrates to enzymes during catitlysis (21. The inhibitiun results immediatelv un mixine the inhibitor ~ ~ ~ with the enzyme, and the inhibition can c e reversed by reducing the concentration of the inhibitor. This can be done by simpie dilution or by separating the small inhibitor from the very~. large enzyme by filtration throueh size selective membranes or columns. ~
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Substrate Analog InhlbRors Structural analogs of the reactants or products of an enzyme-catalyzed reaction make up one large and very important class of reversible inhibitors. By definition, reactants and products (which are reactants for the reverse reaction) of an enzyme-catalyzed reaction must be able to bind to the active site of the enzyme. Small changes in structure often result in compounds which are still able to hind to the active site hut are not capable of undereoine reaction. i.e. enzvme inhibitors. One o f t h e best known examples of a reactant analog withutility as a drug is provided by the sulfonamides. the sulfa drugs which revoiutibnized thetreatment of man; bacterial infections in the late 1930's ( 4 ) . Mammals obtain the essential B-vitamin folic acid from the diet and have an enerw-dependent system for absorbing this charged compound into cells. Many bacteria and some malaria parasites, however, cannot absorb this material and must make it de novo. One step in the biosvnthesis of folic acid reauires D aminobenzoic'acid as oneieactant (Fig. l). ~ulfanilamihe inhibits this reaction bv bindine to the enzvme in nlace of D aminobenzoic acid. he bacterFa cannot make sufficient {alic acid for normal arowth and reoroduction. and anv infection is therefore contained. In some cases, inhibitors which are analogs of reactants can he very potent, as illustrated by the anticancer drug methotrexate (Fig. 2). All cells require that folic acid, however obtained, be reduced to tetrahydrofolic acid. This form of the compound is required as a cofactor by a number of Volume 63
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Transltlon State Analog lnhlbltors Despite a few examples of reactant analogs like methotrexate which are very potent, reactants and products of enzyme-catalyzed reactions do not generally make good models for inhibitors. Enzymes bave not evolved to bind tightly to reactants and products, otherwise these products would never come off the enzyme. The structure that an enzyme does usually hind tightly is the transition state for the reaction (6). The function of an enzyme is to provide a lower energy barrier between the reactants and the products. If an enzyme binds to the transition state much better than to the reactants or products, the difference in binding energy can be applied to lowering this energy barrier and accelerating the reaction. The transition state of a reaction is inherently unstablr and has only a fleeting existence, hut a stable mimicuf the transirion stateshould hind eiiicientls to the enzyme and he a potent inhibitor. Examoles of transition state analogs that function as an enzyme inhibitors are the glyphosateItype (N-(phosphonomethy1)glycines) herbicides and plant-growth regulators that must be applied to plant foliage but are effective against almost all plants. These compounds bave been reported to block the biosynthesis of the ring system of aromatic amino acids, a process which occurs in plants but not in animals. The snecific tareet enzvme is enol~vruvvl -~ ., , shikimate-3-ohosphatesynthasc, which catalyres theadditionof phosphoenol ~ P F PtIo n cvclic nln)hd {Fir. 3 1 .The mechanism wruvate . may invol've de;elopkent of a ~ a r t i apositive l charge on Cp of P E P followed bv attack of the alcohol on C2 (7). When protonated on the nitrogen, glyphosate is a stable analog of the positively charged transition state and blocks reaction of P E P with the alcohol. This prevents biosynthesis of aromatic amino acids, and this inhibition may he responsible for the death of the treated plants. There are many other enzymes which use P E P as a reactant which are not inhibited by glyphosate, presumably because they do not generate the same positively charged transition state. A major drawback to the use of reversible inhibitors of enzymes as drugs or pesticides is the waning of inhibition as the concentration of inhibitor falls due to excretion or me-
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Figure 1. Mode of action of the suifonamidedrugs.
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DHFR = Dihydrofolme Reductose
Figure 2. inhibition of dihydrofoiatereductase by methotrsxate important enzymes. Some of these enzymes incorporate 1carbon units into purines and thymine, nucleic acids which are required in large quantity by rapidly growing cancerous cells. The conversion of folic acid to tetrahydrofolic acid is catalyzed by folate reductase, and this enzyme binds methotrexate about 50,000 times more tightly than it does the normal suhstrate folic acid (5)? Since there is little folic acid in cells to start, methotrexate can completely prevent the reduction to tetrahvdrofolic acid and thereby kills the cells. Unfortunately, methotrexate is ultimately toxic to all cells and cannot be used in prolonged therapy.
Glyphasate
EPSPS
i
Enoylpyruvyl~hikimote-3-phorphote
Tronrifion
Stote?
rynthoae
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Methotrexate dissociates from the reductase very slowly and so has been referred to as a "pseudoirreversible" inhibitor by some researchers. 766
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Figure 3. inhibition of ammatic amino acid biosynthesis by
glyphmate.
ODC
Ornithmnc decorboxrlors
Figure 4. Made of action of penicillins.
taholism. Additional doses of the compound are then required to maintain the desiredlevel of inhibition. This problem can be circumvented hv the use of irreversible inhihitors, compounds which permanently inactivate their target enzymes. Irreversible inhibition often requires some time after mixing to develop fully because i t usually results from the formation of a stable covalent bond between the inhihitor and the enzyme. The inhibitor cannot dissociate from the enzyme regardless of the concentration of free inhibitor present, and only the biosynthesis of new enzyme can restore the catalysis. Affinny Labels A very important subgroup of irreversible enzyme inhibitors are known as affinity labels. These are molecules which are structurallv similar to a reactant of the tareet enzvme " hut also contain a chcmicallv rearrive functional grnup. The ul'finits lnbel arts initially hke a huhstrate analuc and binds specif~callyto the activ; site of the enzyme, b;t then the reactive moietv makes a stable covalent bond with an amino acid group in or near the active site. An example of such an inactivator is provided by the p-lactam antibiotics exemplified by penicillins (Fig. 4). Bacteria which are susceptible to penicillins have a complex, rigid cell wall which is built up from layers of various polymeric materials which are crosslinked by short chains of amino acids. The final step in the ~roductionof these essential crosslinks is the formation of a peptide bond between a free amino group a t the terminus of a De~tidefraement of one laver and the carboxvl rroun between two Ganines which end a peptide fragment on an adiacent laver. This reaction is catalvzed bv a transacvlase (so called because i t exchanges one acyl group for anocher). Penicillin resembles the terminal alanine-alanine group and binds to the transacylase. The 4-membered p-lactam ring is easily opened because of steric strain and is attacked bv an amino group in the active site of the enzyme which is essential for normal catalysis. The resulting adduct is stable so that the ring-opened penicillin stays on the enzyme and prevents formation of the crosslinks necessary to complete the cell wall. The bacterium cannot then withstand the high osmotic pressure within the cell and bursts (8).
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Sulclde Inactlvators Although affinity labels can he highly specific for their target enzymes, they have the problem that their reactive functionality may he attacked and destroyed before the compound can reach its site of action. Any such reaction would lower the amount of material available to inactivate the desired target and might also have undesirable side effects. I t would certainly he more efficient to have enzyme
Figure 5. Suicide Inactivation of wnimine decarboxylase.
inactivators which are chemically unreactive until after they have hound to the desired enzyme. This trick can be accomplished if the inactivator is designed so that the reactive functionality is uncovered only after the inhibitor is bound and the enzvme heains to react with it. Such comnounds haw been called mechanism-based inactivators, k,.,'inhihtors, or suicide substrates. Theie inhihirors reauire not onlv that they bind to the active site of the enzymeabut also that the enzyme be ahle to start a normal catalytic sequence which results in the formation of areactive structure already bound to the enzyme. If this intermediate is sufficiently reactive so that almost all of it covalently hinds to the enzyme and little is released into solution, the mechanismbased inhibitor can be verv selective for its tareet enzvme. a-I)itluoromethylornithine(Fig. 5 ) has been iuund recentI s to he useful in the treatment of African slee~ine . sickness. adisease caused by a trypanosome parasite carried by tsetsd flies. The parasites Droduce ~utrescine(which is necessarv h r the bi;synthesis.ot' pn&i and nucleic acids) only b?. the decarboxvlstion oi oraithine. This conversion is caralyzed by ornithine decarhoxylase by amechanism which uses the coenzyme pyridoxal phosphate to assist in the release of . . CO. from the suhsrrate ( 9 ) . ct-Uitl~lorunieth~.lornithine is chemically stahle in solution hut i i a potent suiride inactivator oi rhii enzyme becnuse it can generare a highly reactive, conjugated intermediate hy elimination of F- during catalysis. This intermediate ran be attacked bv an as \.et unidrntified group on the enzyme to give a stable, covalent adduct which blocks the active site of the enzyme. Without a constant source of putrescine the parasite fails to grow and multi~lv,and the disease is rapidlv relieved. .Mth&h there are some other ways in which enzymes can be inhihired, the ti~reguin: examples illustrate the major caregories of enLyme inhibitors. Many uther compounds u s d in medicinal and agricultural chemistry also work by changing the nrrivity of enqmes, and there is consid~rable current effort t,, diicwer enzyme inhihitors which are more efficient, more selective, and more useful in the control of disease and of pests. For these efforts to succeed, there must be continued oroeress in understandine how enzvmes function and howenz&es catalyzing the same reaction in different organisms can he inhibited selectively.
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Literature Clted (11 Splittgerher. A. C. J. Cham Edur. 1984 20.30. (2) H ~G G ' ~' E ~ Catsly& ~E ~ ~ ~and Re&atitinana', ~ . Aesdddic NNNY Y Y ~ 19,82. (3) Cornelius, R.D. J. Chem.Educ. 1986.20,30. (I) Shepherd. R. G."Medicinal Chemistry". 3rd 4. Bergcr. ; A,, Ed., Wiley-Interscience: New Y a k , 1970; Pt 1. (6) Bak8r.B. R."MedieinalChemis~y",3rd ed.: Bcrger,A.. Ed., Wiley-lntcraeience: New
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Y0.k. 1970, Pf 1. (61 Wolfendcn,R.Aeent.Chefl.Re~. 1972,5.10. (71 Anton.D. L.:Hedstrom, L.;Fish,S.M.:Ahele,R.H.Bioehemistry 1983,22,6903. (8) Hwuer, J. R. E.;Stedmsn, R. J . "Medicinal Chemiatr)", 3rd rdi Dnssr, A,, PA; Wiley-lnlerseienee: New York. 1970. Pt 1. (91 ~ ~ ~ ~ h i , ~ . ~ . ; ~ ~ t h ~ ~ , ~ . ~ . ; ~ u t n e r , S . H . ; ~ c ~ ~ ~ ~ , P . P . ; S j a e r d s m a , ~ . S c i a n 210,332.
