A conformational basis for the selective action of ara-adenine

J. theor. Biol. (1977) 67, 499-514

A Conformational Basis for the Selective Action of Ara-Adenine DOUGLAS L. MILES, DANIEL W. MILES, PATRICK REDINGTON AND HENRY EYRING University

Department qf Chemistry, of Utah, Salt Lake City, Utah 84112, U.S.A. (Received 17 December 1976)

The glycosidic “high anti” conformation is postulated to be the conformation required by the enzymes adenosine kinase and inosine phosphorylase. Purine analogs that are stable in this conformation are either effective substrates or inhibitors of these enzymes. Ara-adenine is shown to be highly unstable in the high anti conformation. The inactivity of araadenine as a substrate for both adenosine kinase and inosine phosphorylase is attributed to its inability to assume the high anti conformation specified by these enzymes. That adenosine itself has a local minima in the high anti conformation, as does inosine and guanosine, is required by its ability to inhibit the synthesis of uridylic acid. The minimal cytotoxic properties of ara-adenine is a consequence of its failure, in normal cells, to be converted to the toxic nucleotide form. The ability of ara-adenine to selectively inhibit DNA viruses means that in DNA virus infected cells the conversion of ara-adenine to ara-AMP is facilitated through a mechanism that does not require a substrate high anti conformation. It is apparent that selective antiviral and anticancer nucleoside analogs may be constructed if their conversion to the toxic nucleotide form is prohibited in normal tissues but allowed in cancer cells or virus infected cells. The basis for the selective effects of ara-adenine is that normal cells require a substrate conformation in which ara-adenine is unstable but that certain neoplastic and viral mechanisms for the conversion of ara-adenine to ara-AMP exist which are able to utilize ara-adenine in its stable syn or anti conformations. 1. Introduction

9-/?-D-Arabinofuranosyladenine (ara-A) is an effective antiviral agent and has some potential as an anticancer drug. An increase in activity (Bryson & Connor, 1976; Schwartz, Shipman & Drach, 1976) with decreased selectivity (Schwartz et al., 1976) is realized when the conversion of ara-A to arahypoxanthine (ara-H) by adenosine deaminase is inhibited. Ara-H approximates the antiviral activity of ara-A in the absence of adenosine deaminase 499

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inhibitors. The antiviral and anticancer properties of ara-A depend on its ultimate conversion to ara-ATP (Roy-Burman, 1970). The activation of many cytoxic nucleosides requires the conversion of the nucleoside to the nucleotide (Bennett, Schnebli, Vail, Allan & Montgomery, 1966). This conversion is predominantly associated with the enzymes adenosine kinase and inosine phosphorylase. The minimal toxicity of ara-A coupled with its antiviral and antitumor properties presents a dilemma. This dilemma is that the antiviral effectiveness of ara-A requires conversion of the nucleoside analog to the nucleotide, and the minimal toxicity or selectivity of ara-A requires that this same conversion within normal cells be avoided. The resolution of this dilemma, which must be able to account for the biological effects of ara-A, has a conformational basis. We will show that the high anti glycosidic conformation is required of the substrates adenosine and inosine by adenosine kinase and inosine phosphorylase respectively, and that this conformation is unstable in ara-A. Therefore, in agreement with available experimental results, the failure of arabinosyl analogs to act as substrates for these enzymes accounts for their minimal cytoxic effects to normal tissues and require a unique conversion of ara-A to the nucleotide by DNA virus infected cells or tumor cells that are affected by ara-A. These conversions to the nucleotide, which are unique to the selectively affected diseased cells must be able to form the nucleotide from arabinosyl nucleoside substrates in their stable syn or anti conformations. The selectivity of ara-A and its minimal toxicity are of interest since a primary goal of drug design is the creation of chemotherapeutic agents that will kill virus infections or cancer cells without affecting normal cells. It has been suggested that the low toxicity of ara-A might be due to an inability of normal animal tissues to phosphorylate the drug (Drach, Bus, Schultz & Sandberg, 1974). The suggestion has also been made that ara-A preferentially inhibits a step in nucleotide metabolism catalyzed by a virus specified enzyme (Shipman, Smith, Carlson & Drach, 1976). Ara-A is structurally different from adenosine in only one respect. The 2’-OH group and 2’-hydrogen are interchanged in position. The removal of the 2’-OH group by itself does not lead to antiviral activity since 2’-deoxyadenosine is not known for its anti-DNA virus properties although it is an effective inhibitor of DNA synthesis (Reichard, Canellakis & Canellakis, 1960). The selective antiviral activity of ara-A must reside, then, in the conformational effects of the interchange of the 2’-hydroxy and the 2’hydrogen, and not simply in any effect that deletion of the 2’-OH group may have on enzyme binding. A consideration of normal purine metabolic pathways and of the lack of general toxicity of ara-A requires that ara-A not be directly converted to the

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toxic nucleotide by adenosine kinase or by the sequential action of adenosine deaminase, inosine phosphorylase, and hypoxanthine-guanine phosphoribosyltransferase. We have found, contrary to the results of an earlier calculation (Saran, Pullman & Perahia, 1974), that a significant difference between adenosine and ara-A is produced by the interchange of the 2’-hydroxy and the 2’-hydrogen. This difference is that the high anti conformational range (x = 110 to 150 degrees) and part of the syn conformational range (x = 150 to 170 degrees) and not just the conformational range (x = 270 to 360 degrees) is forbidden in the case of ara-A. The high anti conformation is allowed for purines (Thewalt, Bugg & Marsh, 1970; Miles, Inskeep, Townsend & Eyring, 1972) and purine analogs (Singh & Hodgson, 1974; Miles, Miles & Eyring, 1974; Miles, Miles, Redington & Eyring, 1976) some of which are structurally allowed to be effective substrates for purine enzymes such as adenosine kinase (Schnebli, Hill & Bennett, 1967) and inosine phosphorylase (Montgomery, Elliott & Thomas, 1975). The failure of ara-A or analogs of ara-A (Montgomery et al., 1975) to act as substrates for either adenosine kinase or inosine phosphorylase indicates that both of these enzymes require that their substrates, adenosine and inosine respectively, be capable of stability in the high anti conformation. Since normal conversion of ara-A to the toxic nucleotide by purine enzymes is prohibited, the antiviral activity of ara-A requires a viral conversion of ara-A to ara-AMP that does not require a high anti substrate conformation. By considering the purine metabolic pathways, shown in part in Fig. 1, in conjunction with the inherent conformational properties of the substrate analog ara-A, a consistent explanation of the antiviral effects, lack of cytotoxic effects, and absence of general antitumor properties shown by ara-A and ara-H is possible. The substrate glycosidic conformation required by adenosine kinase and inosine phosphorylase is postulated here to be the

Fig. 1. Part of the normal metabolic interconversions that occur with puke and nucleotides.

nucleosides

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high anti conformation. Ara-A’s failure to act as a substrate for normal adenosine kinase (Drach et al., 1974; Schnebli et al., 1967) and inosine phosphorylase (Montgomery et al., 1975; LePage & Junga, 1965) accounts for its minimal cytotoxic (Bennett, Shannon, Allan 8 Arnett, 1975; Allan & Bennett, 1973; Schabel, 1968; Wilkerson, Finley, Finley & Ch’ien, 1973) and antitumor (Brink & LePage, 1964a,b; Bennett et af., 1975) effects, which are dependent on these normal pathways leading to the nucleotide. The effectiveness of ara-A requires conversion of the nucleoside analog to the nucleotide (Brink & LePage, 1964a,b; Bennett et al., 1975) and its degree of selectivity requires that this conversion occur within diseased cells affected by the drug without substantial egress of the nucleotide from the cell. The selective action of ara-A against DNA viruses (S~pman et al,, 1976; Shannon, Westbrook & Schable, 1974) clearly indicates that a viral or viral influen~d conversion of at-a-A to ara-AMP occurs and that normal adenosine kinase is not capable of making this conversion. The limited anticancer effectiveness of ara-A is prima facie evidence that the types of cancer as well as the DNA viruses affected by ara-A are capable of a unique conversion of ara-A to ara-AMP. The toxicity of the 3’,5’-cyclic nucleotide of ara-A (Sidwell et al., 1973) is a further indication of a unique phosphoryIation of ara-A. The slow dephosphorylation of ara-AMP (LePage, Lin, Orth & Gottlieb, 1972) and its increased toxicity associated with its slow ~netration of cell membranes (Plunkett & Cohen, 1975; Cohen, 1975; Plunkett, Papi, Ortiz & Cohen, 1974) imply that the use of ara-nucleotides rather than aranucleosides will result in less favorable therapeutic ratios, unless significant conversion of the nucleotide to the nucleoside occurs before the ara-nucleotide is either utilized or converted to a toxic form. The equivalent therapeutic indices found for ara-HMP and ara-A (Allen et al., 1975) may be accounted for by such a conversion. The minimal cytotoxic and antitumor effects associated with the use of ara-A are reversed if ara-A is used with an adenosine deaminase inhibitor (Cass & Au-Yeung, 1976; Plunkett et al., 1975). This may be caused by an increased conversion of ara-A to the nucleotide within cancer cells unable to utilize ara-H (Brink & LePage, 1964a,b) followed by in~ltration of ara-nucleotides from the cancer cell into the host tissues (Ho, 1976). 2. Calculations The iterative extended Hiickel theory (IEHT) method (Rein, Clarke & Harris, 1970) was used to calculate the preferred glycosidic conformation of ara-A. We have previously applied this method to calculations on nucleosides (Miles et al., 1974; Miles et al., 1976). The structure and net atomic

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charges calculated for ara-A are shown in Fig. 2. Utilizing co-ordinates determined by X-ray crystallography (Bunick & Voet, 1974), we rotated the base moiety in 20 degree increments about the Cl’-N9 glycosidic bond and calculated the total energy of the molecule in each conformation. A plot of the variation in total energy of the molecule with rotation about the glycosidic bond is shown in Fig. 3. For comparison we used a Lennard-Jones 6-12 type potential function including an electrostatic term, to calculate the glycosidic conformational energy preferences for ara-A, 2’-deoxyadenosine and 2-C-methyladenosine. The coordinates used for 2’-deoxyadenosine and 2-C-methyladenosine are those for ara-A except that the 2’-OH group has

Fig. 2. Structure and net atomic charges calculated for ara-A. The inactive isomer is shown.

been replaced by a hydrogen or a methyl group. The net atomic charges used for the electrostatic interaction terms for these molecules were those determined by an IEHT calculation except that charges were assigned to the 2’-methyl group. These charges were 0.10 for the carbon and O-05 for each hydrogen. The Lennard-Jones results for 2-C-methyIadenosine are plotted in Fig. 3 as is an IEHT calculation on 2’-deoxyadenosine. In Fig. 4 is plotted the Lennard-Jones calculations on both ara-A and 2’-deoxyadenosine. The van der Waals’ radii (R) and energies for interacting pairs at I = R were taken from the literature (Lakshminarayan & Sasisekharan, 1969. A dielectric constant of 1-O was used.

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Fig. 3. Variation in energy of ara-A ( -) of 2-C-Methyl-2’ deoxyadenosine (- . - . -)

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calculated using the IEHT method and using the Lenhard-Jones method.

)O

Fig. 4. Variation in energy of am-A the Lennard-Jones method.

( -)

and 2’-deoxyadenosine

(-.

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) using

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The anti conformational range lies between x = - 90 to +90 degrees and the syn conformational range lies between x = + 90 to +270 degrees. For convenience we have altered this convention (Sundaralingham, 1975) so that values of x between +70 and + 150 degrees are identified as the high anti range. A cis planar arrangement of 01 ‘-Cl’-N9-C8 corresponds to a dihedral angle of zero degrees. When looking along the glycosidic (Cl’ - N9) bond, a positive rotation corresponds to a clockwise rotation of the far group. 3. Discussion of Results The IEHT calculated global minimum corresponds to the conformation found in the solid state (Bunick & Voet, 1974), which is the anti conformation. In addition to the global minimum found in the anti range, a minima was found in the syn conformational range close in energy to that at the anti conformation. The Lennard-Jones calculations show that the syn conformation is slightly more stable than the anti conformation. Energy differences of such small magnitudes are meaningless since molecules undergo internal adjustments of geometry not accounted for in calculations on rigid molecules. By both the IEHT and Lennard-Jones methods large barriers are found in the high anti conformation and in the syn conformation. These large barriers are due to steric interactions involving the base moiety and the 2’-hydroxy group. Removal of the 2’-hydroxy group and replacing it with a hydrogen, retaining the same geometry elsewhere in the molecule results in a substantial reduction of the high anti and syn barriers. Adenosine is able to exist in the high anti conformation as does both inosine and guanosine (Thewalt et al., 1970; Miles et al., 1972). The existence of sizeable barriers to rotation were not demonstrated in the high anti conformation but only in the syn conformation for ara-A calculations done earlier (Saran et al., 1974). The earlier calculation utilized coordinates for the base moiety from one source which were attached to coordinates for the ribose moiety from another source. The orientation that must be chosen for the Cl’-N9 bond direction must be regarded as an independent variable capable of seriously affecting the rotational profile obtained by any calculational procedure. 4. Conformation

and Purine Interconversions

In Fig. 1 is a portion of the normal metabolic pathways for purine interconversions with each enzymic conversion labeled as to the substrate conformation required for the reaction. The glycosidic conformations required

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by AMP kinase, AMP aminohydrolase, adenosine nucleotidase, and adenylosuccinate lyase are indicated in the figure. They were determined in various systems (Hampton & Sasaki, 1973; Hampton, Harper & Sasaki, 1972u,b) that may correspond to the substrate conformations specified by similar enzymes in humans. However, important differences exist in the metabolism of ara-AMP by man and by mice (LePage et al., 1972) which suggests some caution in applying information obtained from other enzyme sources to the same enzymes in man. The required glycosidic conformation for adenosine deaminase is the anti conformation (Hampton et al., 1972a,b; Ikehara & Fukui, 1974). We propose that the enzymes adenosine kinase and inosine phosphorylase require a high anti conformation so that the conversion of ara-A to the nucleotide by normal metabolic pathways is precluded because of the instability of ara-A in this conformation. Adenosine interferes with pyrimidine synthesis by inhibiting the enzyme OMP pyrophosphorylase (Ishii & Green, 1973) and the enzyme OMP decarboxylase upon conversion to AMP (Beardmore & Kelley, 1974). Purines, purine analogs and pyrimidine analogs that are stable in the high anti conformation are effective inhibitors of these enzymes (Beardmore et al., 1974; Saenger & Suck, 1973; Miles, et al., unpublished results). The high anti energy minima found for inosine, guanosine and the analog ribavirin (Thewalt et al., 1970; Miles et al., 1972; Miles et al., 1976) is also available to adenosine since the dominant base-ribose interactions that determine the preferred glycosidic orientation are similar for adenosine, inosine, guanosine and ribavirin throughout the anti and high anti conformational ranges. The conformational adjustments that result in solid state high anti conformations in inosine and guanosine (Thewalt et al., 1970) have only recently been reported for adenosine (Neidle, Kuhlbrandt & Achari, 1976). The ability of adenosine to behave like high anti analogs in the inhibition of pyrimidine biosynthesis indicates that adenosine has an energetic minima in the high anti conformational range. The conformational energy preferences calculated for nucleosides are dependent on the crystal structure chosen. Alternative crystal structures will result in different rotational profiles. Ribavirin in one crystal form has energy minima in the high anti and high syn conformations and in another crystal form it is calculated to be stable in the normal anti and syn conformations (Miles et al., 1976). Ara-A and ara-H are inactive as substrates for adenosine kinase and inosine phosphorylase respectively (Bennett et al., 1975; Schnebli et al., 1967; Montgomery et al., 1975; Lindberg, Klenow & Hansen, 1967; LePage, Khaliq & Gottlieb, 1973). The role of enzyme-substrate conformative response is important since there is some difference between these two

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enzymes. Adenosine kinase is unable to act upon either 2’-deoxyadenosine or ara-A while inosine phosphorylase can operate on 2’-deoxyinosine at a reduced rate but is still unable to operate on ara-H (LaPage & Junga, 1965; Bennett, Allan, Carpenter & Hill, 1976). The stabilization of the C2’ endo conformation by the removal of the Z-OH group (Sundaralingam, 1975) causes interactions that destabilize the high anti conformational range. This effect can be strengthened or reduced by other structural modifications (Montgomery et a/., 1975). (A)

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KINASE

Ara-A is known to be ineffective as a substrate for adenosine kinase as is 2’-deoxyadenosine. The I& for both of these molecules is about 1000 times that of adenosine (Lindberg et al., 1967). The very low activity attributed to adenosine kinase for ara-A may be due to the use of impure enzyme preparations that contain 2’-deoxyadenosine kinase azaadenosine. The inactivity of B-arabinofuranosyl-8-azaadenosine (/I-ara-&azaA) as a substrate for adenosine kinase (Montgomery et al., 1975) is due to the prohibition of the required high anti conformation to this analog. The activity, although reduced, of 8-aza+2’-deoxyadenosine (Montgomery et a/., 1975) is evidence that the 2’-OH group is not required for binding to adenosine kinase. This analog is inactive, however, with inosine phosphorylase and adenosine kinase from Ehrlich cells (Frederiksen, 1964). The 2-aza analog of 2’-deoxyadenosine may be a substrate for adenosine kinase while a similar analog of ara-A is not (Montgomery et al., 1975). Some structural (Bennett & Hill, 1975) and electronic (Kaneti, 1973) requirements that must be met by potential adenosine kinase substrates have been proposed. Bennett & Hill (1975) proposed that a potential substrate should have a 2’-hydroxy group trans to the purine moiety and that a considerable degree of freedom of rotation about the Cl’-N9 bond should exist. Kaneti (1973) found that there was a correlation between electronic properties of the nitrogen at the 3 position of the purine base and the rates of phosphorylation found for many analogs of adenosine. However, no correlation was found between substrate binding to the enzyme and the electronic properties of the base moiety. Adenosine analogs such as formycin and 8-azaadenosine, which are stable in the high anti conformation, are phosphorylated at a faster rate than predicted by the correlation between electronic properties and phosphorylation rate found for other adenosine analogs. Structural modifications of the purine moiety that decrease the net atomic charge at N3 should increase the activity of those analogs as substrates for adenosine kinase according to Kaneti (1973). The activity of 2-azapurines and of 2-fluoro adenosine as substrates for adenosine kinase support this

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prediction. The biological activity of 2-aza adenosine (Montgomery et al., 1975) indicates that it is a substrate for both adenosine kinase and inosine phosphorylase. Replacing the -CH group at the 2 position of the base by a nitrogen reduces the net negative charge at the N3 position from -0,170 to -0.134 electrons according to calculations of net atomic charges in adenine and 2-azaadenine made using the IEHT method. The net atomic charge at N3 in 2-fluoroadenine is calculated to be -0.143. We suggest that a reduction in negative charge at N3 diminishes the destabilizing interactions between the nitrogen at the 3 position of the base and the ribose when the substrate is in the conformational range specified by adenosine kinase, which is the high anti conformation. Thus we see that 2-aza purine nucleosides will stabilize the high anti conformation required by adenosine kinase and should result in increased phosphorylation of 2-aza purine nucleoside analogs. Purine nucleoside analogs that are inherently more stable in the high anti conformation as a result of the replacement of the -CH group at the 8 position of the base by a nitrogen are readily phosphorylated. 8-Azaadenosine and formycin are effective substrates for adenosine kinase because the structural modification at the 8 position (replacement of -CH with a nitrogen) stabilizes the high anti conformation required by adenosine kinase. The stabilization of the high anti conformation realized with these analogs is greater than the destabilization of the high anti conformation attributable to the increased NZribose interactions. This accounts for the failure of analogs modified in the five-membered ring portion of the purine base to obey the correlations found by Kaneti (1973) for all other adenosine analogs that he investigated. That the enzyme adenosine kinase requires a high anti glycosidic conformation of potential substrates accounts for the increase in phosphorylation rates associated with decreased electronic interactions involving the nitrogen at the 3 position. Other structural modifications which stabilize the required high anti conformation can account for the exceptions to the correlation of N3 electronic properties and experimental phosphorylation rates. The effect of N3-ribose interactions on adenosine kinase activity is expressed through the effect these interactions have on conformation. The tram rule of Bennett & Hill (1975) is not able to account for the inactivity of the a-arabinofuranoside of 2-azaadenine (ar-ara-2-azaA). We suggest that the activity of u-ara-8-azaA as a substrate for adenosine kinase depends on its preference for a high anti conformation. Both S-azaadenosine and a-ara-8-azaA are substrates for adenosine kinase (Schnebli, et al., 1967; Bennett, Allan, Hill, Thomas & Carpenter, 1975). %Azaadenosine is phosphorylated more rapidly than adenosine (Schnebli et al., 1967) and cc-ara-8-azaA is phosphorylated more rapidly than c+araA (Bennett et al., 1976). This is in agreement with the greater stability of 8-aza purine

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analogs in the high anti conformation compared with adenosine and a-araA. The inactivity of cx-ara-Zazaadenine can be explained as follows. This analog has the lone electron pair at the N2 position oriented like the oxygen at the 6 position in @nosine which is not a substrate for adenosine kinase. An amino group is not required at the 6 position of the substrate for adenosine kinase activity since replacement of the amino group by a hydrogen results in increased activity (Schnebli et al., 1967). The inactivity of a-ara-2-azaadenine may be attributed to destabilizing or inactivating interactions between the adenosine kinase enzyme and the N2 lone electron pair. These interactions should be similar to those that occur between the enzyme and the oxygen at the 6 position of inosine and which prevent the enzymic reaction. The N2 lone electron pair of a-ara-2-azaadenine and the oxygen at the 6 position of inosine are able to assume similar spatial relationships with the 05’ and 03’ oxygens of their respective arabinose and ribose moieties. The apparent conversion of 2-fluoro-2’-deoxyadenosine to the nucleotide by adenosine kinase (Parks & Brown, 1973) is further evidence that the 2’-OH group is not structurally required for activity, but exerts a conformational effect to decrease activity. Other structural changes such as replacement of the CH group at the 2 position of the base by a nitrogen, are able to counter the conformational effect of removal of the 2’-OH group and restore activity. This is not the case with ara-purine nucleoside analogs, which remain inactive with respect to adenosine kinase or inosine phosphorylase when other structural modifications are made. The activity of 2’-C-methyladenosine as an effective antivaccina agent in mice (Walton, Jenkins, Nutt, Holly & Nemes, 1969) is of interest. From Fig. 3 we see that ara-A and 2’-C-methyladenosine are conformationally similar. The activity of 2’-C-methyladenosine against other DNA viruses has not been investigated to our knowledge. Such activity, similar to that of ara-A may be expected not only against the vaccina virus but against other DNA viruses as well. Both ara-A and 2’-C-methyladenosine have been reported to be toxic to KB cells (Schwartz et al., 1976; Walton, Jenkins, Nutt, Zimmerman & Holly, 1966; Goldin, Wood & Engle, 1968). Ara-A is three to six times more toxic (Schwartz et al., 1976) to DNA synthesis in viralinfected KB cells than in uninfected KB cells. The degree of selectivity of 2’-C-methyladenosine has not been investigated to our knowledge. The higher toxicity of ara-A and ara-H in KB cell systems (Schwartz et al., 1976) than in other cell systems (Bennett et al., 1975; Drach et al., 1974) indicates that a mechanism for phosphorylation other than an adenosine kinase which requires a substrate high anti conformation is operating in KB cells. The metabolism of ara-A to phosphates of adenosine as well as phosphates of ara-A in KB cells indicates that ara-A is cleaved to give adenine in these

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cells (Schwartz et al., 1976; Bennett et al., 1975). This is in contrast to the general inactivity of adenosine with purine nucleoside phosphorylases (Parks & Agarwal, 1972) and of ara-A with adenosine phosphorylase (Bennett, Vail, Chumley & Montgomery, 1966). The possibility that Formycin A can inhibit adenosine phosphorylase just as Formycin B inhibits inosine phosphorylase (Crabtree & Senft, 1974) means that the simultaneous use of Formycin A and ara-A in KB cells may result in improved selectivity for ara-A and that ara-A analogs modified so that there is a hydrogen at the 7 position of the base would not be substrates for adenosine phosphorylase in KB cells. Arabinosyl-6-mercaptopurine (ara-MP), which is an analog of inosine, is not converted to the toxic nucleotide by KB cells (Loo, Lu dz Gottlieb, 1973). The non-toxicity of ara-MP to both KB cells and to mice (Goldin et al., 1968; Loo et al., 1973; Kimball, LePage 8z Bowman, 1964) contrasts with the toxicity to KB cells of 2’-deoxy-6 mercaptopurine ribonucloside and 6-MP (Goldin et al., 1968). The arabinosyl derivative of 6-methyl mercaptopurine (ara-MeMP) is also non-toxic to KB cells (Goldin et al., 1968) but its anticancer properties have not been investigated to our knowledge. The lack of toxicity of these analogs indicate, that like ara-A, they are not substrates for adenosine kinase or inosine phosphorylase because the substrate high anti conformation required is not possible. (B)

SUBSTRATE

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PHOSPHORYLASE

Purine nucleoside phosphorylases are able to cleave the glycosidic bonds of inosine, guanosine, xanthosine and their deoxyribose derivatives (Parks & Agarwal, 1972). The 2’-OH group is not required for activity @Page & Junga, 1965). The 2’-deoxy nucleosides are less active as substrates (Bennett et al., 1976). Ara-analogs are not substrates for inosine phosphorylase (Brink & LePage, 1964a,b; Montgomery et al., 1975; LePage & Junga, 1965). The excretion in the urine of ara-nucleosides (LaPage et al., 1973; Loo et al., 1973) requires that the conversion of these analogs to the free bases by inosine phosphorylase does not occur (Brink & LePage, 1964@). Ara-A and ara-H, over a period of time are also metabolized to ara-Xanthine (ara-X) which is also excreted in the urine as the nucleoside (Borondy, Mourer, Drach, Chang & Glazko, 1973). The required conformation for inosine phosphorylase is the high anti conformation denied to ara-analogs. The ability of Formycin B to act as a strong competitive inhibitor of erythrocyte purine nucleoside phosphorylase (Sheen, Kim & Parks, 1968) is further evidence that the high anti conformation is required by inosine phosphorylase. Both Formycin and Oxoformycin B are stable in the high anti conformation (Miles et al., 1974; D. L. Miles et al., unpublished results) and Formycin B is expected to be conformationally similar and prefer a

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high anti conformation. The minimal toxicity of ara-A and ara-H (Bennett et al., 1975) requires that enzymic reactions leading to toxic nucleotides in normal cells be inoperative. The inability of inosine phosphorylase to utilize arabinosyl analogs is caused by the instability of these analogs in the required high anti conformation. 5. Application to the Design of Selective Antiviral and Anticancer Drugs The prohibition of the high anti conformation in ara-A, demonstrated by our conformational energy calculations, supports the proposal that ara-A is not converted by normal cells to the nucleotide (Drach et al., 1974), and that adenosine is converted to the nucleotide when in the high anti conformation denied to ara-A. The antiviral effectiveness of ara-A is dependent on its conversion to the nucleotide. Therefore a viral mechanism, not requiring substrates in the high anti conformation and distinguishable therefore from normal adenosine kinase, is able to convert ara-A to ara-AMP. The minimal toxicity associated with ara-A is attributable to the failure of normal adenosine kinase or inosine phosphorylase to operate on ara-analogs that are not stable in the high anticonformations required for enzyme activity. Anticancer effects that are realized with ara-A are attributable to a conversion to the nucleotide occurring within the cancer cell itself. For those types of cancer against which ara-A is selectively effective, alternate or less specific routes for conversion of ara-A to ara-AMP must exist. Cancer types that rely to a significant extent on salvage mechanisms and the utilization of preformed purines are most likely to respond to treatment with ara-A. The conformational properties of ara-A and in particular the prohibition of the high anti conformation, provide estimates for not only its metabolic fate, but may serve to indicate key metabolic differences between normal, viral and cancer cell metabolism that depend on substrate conformation. We have recently determined that the high anti conformation or a syn conformation close to that around x = 150 degrees is required for the conversion of IMP to XMP (Miles et al., 1976). Thus the conversion of ara-HMP to araXMP is prohibited. The substrate conformation required by adenylosuccinate synthetase has not been determined, but this enzyme from rabbit muscle is able to operate on ara-HMP (Spector & Miller, 1976) which indicates that the enzyme requires that the substrate be in the conventional anti or syn conformations, that are energetically allowed to ara-HMP. The inactivity of adenylosuccinate synthetase with XMP and 8-aza-IMP (Spector & Miller, 1976), which are stable in the syn and high anti conformations respectively,

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indicate that the anti conformation may be required by the enzyme. The predominant conversion of Gaza-inosine which prefers the high anti conformation, to polynucleotides of guanosine rather than of adenosine in various cells (Bennett 8zAllan, 1976) supports the concept that the conversion of IMP to XMP requires a high a&i conformation (Miles et al., 1976) and that the conversion of IMP to adenylosuccinate requires another conformation, probably the anti conformation. The conversion of ara-A or ara-H to the nucleotide by viral or neoplastic mechanisms is required if selective biological effects are to be realized. The establishment that metabolic conversions unique to, or more efficient in diseased cells exist, suggests that antiviral and anticancer drugs be designed so that they are converted to the toxic nucleotide form primarily by viral or neoplastic mechanisms. Such chemical agents will have a high degree of selectivity. The distribution of an antiviral or anticancer molecule between normal and abnormal metabolic mechanisms for phosphorylation or conversion to the nucleotide, will determine its degree of selectivity. A favorable distribution, as occurs with ara-A, is attributable to the effect of conformation on these competing mechanisms. 6. Summary The arguments in favor of a conformational basis for the antiviral potency and minimal cytotoxic effects of ara-A have been put forth. The conformational effect of removal of the 2’-OH group of adenosine and of the aramodification are similar in that both structural changes destabilize the high anti glycosidic conformation. Other structural or enzymic mechanisms can modify the conformational effect of a 2’-OH group deletion but the strong steric effect and destabilizing interactions of the ara-modification on glycosidic conformation is not reversible. Of critical importance is the concept that the expression of biochemical effects by a nucleoside analog depends on the manner in which it is converted to the nucleotide. The conformational preclusion of the high anti conformation in ara-A, which is required by both adenosine kinase and inosine phosphorylase is a prima facie explanation of ara-A’s lack of cytotoxic and antitumor effects and requires an alternate mechanism for its conversion to the nucleotide in order to explain the antiviral effects expressed by ara-ATP. That diverse and desirable biological effects may be achieved with nucleoside analogs designed so as to direct their conversion to toxic nucleotides has not been generally recognized. The expression of biological effects by nucleoside analogs, whether antiviral or anticancer, and the degree of non-toxicity to normal cells is capable

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SELECTIVITY

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