actions of furine analogs: enzyme specificity studies as a ... · enzyme specificity and to an...

(CANCER RESEARCH 29, 2448-2453, December 1969]

Actions of Furine Analogs: Enzyme Specificity Studiesas a Basis for Interpretation and Design

Gertrude B. Elion

Wellcome Research Laboratories, Burroughs Wellcome & Co., Inc., Tuckahoe, New York 10707

Summary

The importance of quantitative studies of the specificities ofpurine-metabolizing enzymes are discussed. These data notonly enable one to make predictions concerning the metabolicdisposition and effectiveness of a drug in vivo, but they alsohelp to identify its loci of action. They may also serve as abasis for understanding the selective toxicity of a compoundand aid in the design of new antimetabolites.

Introduction

The goal of the molecular chemotherapist is to understandnot only where an agent acts, but how it exerts an effect thatis more damaging to one type of cell than to another. Much isknown about the biosynthesis of nucleic acids, the regulatorymechanisms which influence it, and some of the loci at whichpurine antagonists act. However, the reasons for selectivetoxicity of these antimetabolites to tumor cells remainobscure. A knowledge of the enzyme levels and pool sizes ofvarious metabolites in tumors and normal tissues may help toanswer this question. The utilization of such data will depend,however, on quantitative information about the specificstructural requirements for substrates and inhibitors of theseenzymes. It is the purpose of this paper to analyze some of thedata on the specificities of purine-anabolizing enzymes and toillustrate how this information can be used to design newcompounds and to interpret the activities of known antimetabolites.

Phosphoribosyltransferases

It is now generally recognized that, in order to interfere withnucleic acid synthesis, a purine analog must, in most cases,first be anabolized to the nucleotide level (24). For purinebases, conversion to the ribonucleotide is accomplished by thephosphoribosyltransferases. At least two phosphoribo-syltransferases, adenine and hypoxanthine-guanine, occurwidely in mammals. Systematic studies on the specificities ofboth of these enzymes have recently been carried out in ourlaboratories. Most of the information to date is concernedwith binding to the enzyme (KÂ¡)although in some cases thevelocity of conversion to nucleotide has also been measured.Both types of information are important in designing futureanalogs, since it is necessary to know whether the inability of acompound to serve as a substrate for these enzymes is due to

poor binding or to some structural feature which prevents thecatalytic reaction.

Studies with the hypoxanthine-guanine phosphoribosyl-transferase from human red cells (37) have shown that the bestbinding (KÂ¡= 2.4 X 1CT6Mis obtained with purines which

have a double-bonded oxygen or sulfur in the 6-position and ahydrogen or amino group in position-2. Methylation of thesecompounds at N-l reduces binding only 10-fold, whereasalkylation on any other ring nitrogen reduces it at least1000-fold. Purines with single-bonded substituents atposition-6, e.g., methoxyl, methylthio, chloro, hydrogen, andamino, are very poorly bound. The 2-amino group enhancesbinding somewhat, but a 2-hydroxyl group decreases itmarkedly, e.g., xanthine has a KÂ¡= 2.5 X lo"4 M. The effects

of changes in the imidazole ring (Table 1) indicate that thepresence of a nitrogen at position-7 of the purine ring isimportant for binding to this enzyme, whereas the 9-nitrogenis less so. [The poor binding of the 7-hydroxytriazolo(4,5-d)pyrimidine at pH 7.7 may be the result of its high acidity.Unlike the other analogs it would be essentially completelyionized at pH 7.7.' ]

Table 1

HN

KÂ¡X IO3 M (pH 7.7, 38Â°C)

NNNNCCCNNCNCNCNSNN0.00240.091.00.046*1.01.3Â°

Binding constants of hypoxanthine analogs to hypoxanthine-guaninephosphoribosyltransferase.

"T. A. Krenitsky, S. N. Neil.G. B. Elion,and G. H.Hitchings. Adenine

Phosphoribosyltransferase from Monkey Liver. Specificity and Properties. 244: 4774-4784, 1969.

8-Azaguanine (5-amino-7-hydroxytriazolo(4,5-d)pyrimidine) wasfound to be a substrate for the hypoxanthine phosphoribosyltransferasefrom Brewer's yeast at pH 6.0 but not at pH 7.4 (46); for the hog liver

enzyme, the pH optimum was reported to be 7.0 (57).

2448 CANCER RESEARCH VOL. 29

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Enzyme Specificities

Known substrates for hypoxanthine-guanine phosphoribo-syltransferase are: hypoxanthine, guanine, xanthine, 6-mer-captopurine, thioguanine, 8-azaguanine, and 4-hydroxy-pyrazolo(3,4-d)pyrimidine (allopurinol) (37, 40, 46, 57). Noribonucleotide formation has been detected using this enzymewith adenine (37, 46), purine (46), 6-methylpurine (46), uricacid (37), azathioprine (37), or 4,6-dihydroxypyrazolo(3,4-d)

pyrimidine (37).Studies on the adenine phosphoribosyltransferase from

monkey liver2 have pointed up some interesting differences

between it and the hypoxanthine enzyme. The naturalsubstrate, adenine, is bound very tightly (Km = 6.9 X IO"7 M),

but every other- compound examined has a KÂ¡at least 40 timesgreater. Substitution of one of the hydrogens of the 6-aminogroup by another amino group (i.e., hydrazinÃ¶) or a hydroxyl(i.e., hydroxylamino) results in a 100-fold decrease in binding.If a small straight-chain alkyl group (e.g., methyl, ethyl, orn-propyl) replaces one of the hydrogens, the Kf is raised to10~4 M; higher alkyl groups, branched-chain alkyl, benzyl, andphenyl groups raise the KÂ¡to IO"3 M or higher. Among the

compounds which do not have a nitrogen attached to the6-position, purine 6-aldoxime is bound best (KÂ¡= 5 X lo"5 M),

and purine and 6-methylpurine are bound better (KÂ¡= 5 Xlo"4 M) than compounds with a chloro, cyano, hydroxyl,mercapto, carboxy, or carboxamido group (KÂ¡^>lo"3 M).

The requirement for a hydrogen at position-2 of the adeninemolecule is fairly specific although the 2-fluoro compound hasa moderately low A/ (2.8 X IO"5 M). The poor binding of

adenine derivatives with a chloro, methyl, amino, hydroxyl, ormercapto group at position-2 suggests that there is a spatiallimitation at this position. However, the better binding of2-methylthioadenine (KÂ¡ = 2.6 X lo"4 M) than of2-methyladenine (KÂ¡= 8.7 X IO"4 M) indicates that size is not

the only factor. A bulky group at position-8 does not interferemarkedly with binding. Both the 8-methyl and8-m-nitrophenyl derivatives of adenine have KÂ¡values around 6X 1CTSM.

Variations in the 5-membered ring of the purine (Table 2)produce somewhat different specificities for the adeninephosphoribosyltransferase than for the hypoxanthine enzyme.Here, binding with the pyrazolo(3,4-d)pyrimidine (where Yand Z are N) is better than for any of the other variants tested,whereas the thiazolopyrimidine binds very poorly.

Structural requirements for binding and for the catalyticreaction may be quite different. Thus, 2,6-diaminopurinebinds poorly (KÂ¡> IO"3 M), but the Vm for its transformation

to nucleotide is approximately 60% that of the Vm foradenine2 (12). The binding of 4-aminopyrazolo(3,4-d) pyrimidine is better (KÂ¡= 2.7 X lo"5 M) than that of 2,6-diamino

purine, but its Vm is only 2.3% of the Vm for adenine.However, at a substrate concentration of 2.5 X IO"5 M (a

concentration which might realistically be attained in vivo),2,6-diaminopurine and 4-aminopyrazolo (3,4-d) pyrimidine areanabolized at 8 and 12% respectively of the rate for adenine.

Table 2

NH

Kj X IO3 M (pH 7.7, 38Â°C)

NNNCCCNCNCNNSNN0.00069>110.0270.49

2T. A. Krenitsky, S. N. Neu, G. B. Elion, and G. H. Hitchings.

Adenine Phosphoribosyltransferase from Monkey Liver. Specificity andProperties. J. Biol. Chem., 244: 4779-4784, 1969.

Binding constants of adenine analogs to adeninephosphoribosyltransferase.

Thus, despite its relatively poor binding to the enzyme,2,6-diaminopurine can be converted to its nucleotide at a ratesimilar to that of 4-aminopyrazolo(3,4-d) pyrimidine.

Other Anabolic Enzymes

Quantitative data on other anabolic enzymes are stillsomewhat fragmentary. A few analog nucleotides have beenstudied with inosinate dehydrogenase (2, 29, 30, 51),adenylosuccinate synthetase (3), guanylate kinase (43-45),phosphoribosylpyrophosphate amidotransferase (42), andribonucleotide reducÃase(18, 56), but no systematic structure-activity relationships have been established. With adenosinekinase there has been considerable work with cells in tissueculture and with cell-free extracts (8) but very littlequantitative determination of binding constants. Substratespecificities with the isolated, partially purified enzyme (34,53) have yielded valuable information. This enzyme is ofparticular interest and importance because, in general, itssubstrates are nucleosides that are not substrates for nucleo-side phosphorylase and are, consequently, not formed fromnor degraded to free purines. Adenosine kinase may, therefore,provide a route to nucleotides that is not otherwise availablefor some compounds. The phosphorylation of 9-0-D-arabino-syladenine to the triphosphate level in vivo (15), the conversion of the 2'-deoxyriboside and 3'-deoxyriboside of

6-methylaminopurine to their respective monophosphates byextracts from Ehrlich ascites cells (54), and the ability of thecarbocyclic analog of adenosine to act as a substrate foradenosine kinase (6) indicate that changes in the sugar arepermissible for substrates of this enzyme. Adenosine kinasealso phosphorylates the ribonucleosides of the adenine analogsof the variety of condensed pyrimidine systems, e.g., pyrazolo-(3,4-d)pyrimidine (53), pyrazolo(4,3-d)pyrimidine (FormycinA) (32), pyrrolo(2,3-d)pyrimidine (Tubercidin) (13, 53), andtriazolo(4,5-d)pyrimidine (53). Further quantitative information on the specificity of adenosine kinase would be extremelyuseful in the design of new adenosine analogs.

The presence of a nucleoside kinase activity different fromadenosine kinase has been reported in a thioguanine-resistant

DECEMBER 1969 2449



Gertrude B. Elion

line of Ehrlich ascites carcinoma (47), but it is not certainwhether this represents several different enzymes or adenosinekinase of altered specificity.

Catabolic Enzymes

For the catabolic enzymes, quantitative data have beenaccumulating for a number of years, and the tempo of thiswork has been increasing, e.g., xanthine oxidase (5, 10, 11,22,27, 39), nucleoside phosphorylase (35, 36, 38), adenosinedeaminase (14, 17, 19, 28, 38, 48), guanase (4, 33, 41, 49),and adenase (31). These studies have added greatly to ourknowledge concerning the effects of structural changes onenzyme specificity and to an understanding of why potentiallyactive compounds often fail to be successful drugs.

Analogs of Hypoxanthine

Some examples may serve to illustrate how a knowledge ofenzyme specificities may be applied. Two analogs of hypoxan-thine with widely different properties are 6-mercaptopurineand allopurinol. Both, like hypoxanthine, are substrates forhypoxanthine phosphoribosyltransferase (37), purine nucleoside phosphorylase (35), and xanthine oxidase (22, 26, 55).The binding constants for each are shown in Table 3.6-Mercaptopurine mimics hypoxanthine closely for all threeenzymes. It can compete successfully with hypoxanthine forthe phosphoribosyltransferase, and it binds better to thisanabolic enzyme than to the two catabolic enzymes. Moreover, the binding constants are in the range of concentrationswhich may be achieved in vivo with a dose of about 2 mg/kg,the dose which is used clinically. Allopurinol, on the otherhand, binds 5000 times as well to xanthine oxidase (22) asto either the phosphoribosyltransferase (37) or the nucleo

side phosphorylase (36). Although the Vm for the transformation of allopurinol to its ribonucleotide can be calculatedto be 24% of guanine (37), the binding to the phosphoribosyltransferase is very poor (Km = 1 X lo"3 M). Since blood

levels of allopurinol after therapeutic doses are seldom ashigh as 5 X IGT5 M (25),allopurinol cannot displace hypo

xanthine from the phosphoribosyltransferase. Consequently,little or no allopurinol ribonucleotide is formed under invivo conditions. On the other hand, allopurinol is anexcellent substrate for and inhibitor of xanthine oxidase invivo as well as in vitro (22). Since it is bound 100 times aswell as 6-mercaptopurine is to xanthine oxidase, it canprevent the oxidation of 6-mercaptopurine to thiouric acidand thereby increase the amount of 6-mercaptopurineavailable for conversion to thioinosinic acid. This results ina potentiation of the antitumor activity of 6-mercaptopurine

by allopurinol (23).

Adenine-metabolizing Enzymes

To examine which groups may substitute for the 6-aminogroup in binding to adenine-metabolizing enzymes, 6-methyl-thiopurine, 6-chloropurine, and their ribonucleosides havebeen compared with adenine and adenosine (Table 4). Forthree enzymes, nucleoside phosphorylase, xanthine oxidase,and adenosine kinase, the 6-methylthio group behaves like the6-amino group. On the other hand, adenine phosphoribosyltransferase and adenosine deaminase can readily differentiatebetween these two groups. Consequently, 6-methylthiopurine

R. Pomales and G. B. Elion, unpublished observations.

Table3EnzymeHypoxanthine

phosphoribosyltransferaseNucleoside phosphorylaseXanthine oxidaseHypoxanthineK/XM2.4

X IO"61 X 10s3 X 10~66-MercaptopurineKj-XM3.9

X Id"67.3 X 10~s1.8 X Id"5AllopurinolXV

XMin-3

in'3 .1.9X107

Binding constants of hypoxanthine anologs.

Table 4

Adenine 6-Methylthiopurine 6-ChloropurineEnzymeEnzyme

Adenine phosphoribosyltransferase

Nucleoside phosphorylase

Xanthine oxidaseKt

X MSubstrate6.9

X IO"7 +

6.6 XId"3ix id"5 +A,X

M>1

X Id"3

1.1 X Id"4Substrate

KÂ¡XM>1

XlÃ²"3+

7.3 X Id"6Substrate9(-)?+

RibonucleosidesAdenosine kinase 1.8 X Id"6

Adenosine deaminase 5 X Id

5X 10sÂ»Id"4 7.1 X IO""4

Binding constants and substrate specificities for enzymes metabolizing adenine analogs.





is not converted to the ribonucleotide (7), whereas6-methylthiopurine ribonucleoside is (7, 16, 53). A somewhatdifferent situation occurs in the 6-chloro compounds.Chloropurine ribonucleoside is a substrate for adenosinedeaminase (17, 20) as well as for adenosine kinase (8, 53).Since the available data suggest that this ribonucleoside is notcleaved by purine nucleoside phosphorylase, the extent of itsconversion of 6-chloropurine ribonucleotide in vivo woulddepend on its relative binding and rate of reaction with thekinase and the deaminase. The poor binding of 6-chloropurineto adertine phosphoribosyltransferase2 and its tight binding to

xanthine oxidase (21) suggest that this purine would not beconverted to a ribonucleotide very efficiently. However, whenxanthine oxidase is inhibited, e.g., by allopurinol, theantitumor effect of 6-chloropurine is potentiated (23),implying that 6-chloropurine is, indeed, a substrate for adeninephosphoribosyltransferase.

Thioinosinic Acid

The inhibitory effects of thioinosinic acid on a number ofthe enzymes of purine anabolism have been investigated (2, 3,30, 42, 51). The data (Table 5) suggest that the mostvulnerable enzymes are inosinate dehydrogenase and phospho-ribosyl-pyrophosphate amidotransferase, for which the KÂ¡values for thioinosinic acid are in the concentration rangeachieved in vivo. If the pool sizes of inosinate in a cell werevery low, however, the inhibition of adenylosuccinate synthe-tase might also play an important role in the inhibitoryactivity. Recent investigations of acid-soluble pool sizes ofvtumors in our laboratory suggest that some 6-mercaptopurine-sensitive tumors do, indeed, have lower inosinate pools than6-mercaptopurine-resistant tumors.4 Differences in the inosin

ate polls of tumor and normal tissue may provide a partialexplanation for the selective antitumor effects of thioinosinicacid5. The mechanism of action of 6-mercaptopurine, how

ever, cannot be explained entirely on the basis of theinhibitory effects of thioinosinic acid. There is evidence thatthioinosinic acid is converted to both 6-methylthiopurineribonucleotide (1) and thioguanylic acid (52). Each of thesenucleotides inhibits phosphoribosyl-pyrophosphate amidotransferase (42); thioguanylic acid also inhibits inosinatedehydrogenase (29,44) and guanylate kinase (43â€”45).

Table 5

Inosinic acid ThioinosinicacidEnzymeKm X M KÂ¡XMInosinate

dehydrogenase 1.4 X 10Phosphoribosyl-pyrophosphate

amidotransferaseAdenylosuccinate synthetase 3 X 103.6

XIO"64.4

X 10"*3 X10"*Binding

constants of inosinic and thioinosinic acids.

L. Carrington and G. B. Elion, unpublished observations.5The data of Salser and Balis (50) and of Bennett (9) suggest that

such differences do exist between the inosinate pools of Sarcoma 180and liver or intestine.

Conclusion

The aims of systematic investigations of the specificities ofthe purine-metabolizing enzymes are several. With respect to

any individual compound, one would like to be able to predictwith reasonable accuracy the overall fate of the compound invivo, as well as the concentrations required to exert a desiredeffect. One would also hope that knowledge of the effects ofvarious ring structures and substituent groups on enzymebinding would lead to the more rational synthesis of newinhibitors. Finally, it might be possible to evaluate the relativeimportance of the various loci of action of a cytotoxiccompound and to utilize this biochemical knowledge todetermine which are the critical reactions in the survival ofvarious cell types. When this knowledge is integrated with thedata on enzyme levels and pool sizes, it may be possible todescribe in molecular terms the reasons for the selective effectsof 6-mercaptopurine and other purine analogs.

Acknowledgments

I am indebted to Dr. G. H. Hitchings for his helpful advice andstimulating discussion.

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1969;29:2448-2453. Cancer Res Gertrude B. Elion Basis for Interpretation and DesignActions of Purine Analogs: Enzyme Specificity Studies as a

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