Metabolism and Mechanism of Action of Formycin B in Leishmania (Purine Metabolism/Formycin A/Allopurinol Riboside) PETRIE RAINEY*T and DANIEL V

Proc. Nati Acad. Sci. USA Vol. 80, pp. 288-292, January 1983 Medical Sciences

Metabolism and mechanism of action of formycin B in Leishmania (purine metabolism/formycin A/allopurinol riboside) PETRIE RAINEY*t AND DANIEL V. SANTI*t§ Departments of Biochemistry and Biophysics and *Pharmaceutical Chemistry and the tDivision of Clinical Pharmacology, Department of Modicine, University of California, San Francisco, California 94143 Communicated by Bruce M. Alberts, September 3, 1982 ABSTRACT Formycin B is a potent inhibitor ofgrowth of the mutates in L. mexicana and L. donovani promastigotes. How- promastigote forms of Leishmania tropical L. mexicana, L bra- ever, adenosine nucleotide counterparts of FoB have not, been ziiensis, andL. donovani The metabolic products formed in these observed in organisms treated with the drug, and it has been organisms are formycin B 5'-monophosphate and formycin A 5'- suggested that the anti-leishmanial effects of FoB may result mono-, di-, and triphosphates, with formycin A 5'-triphosphate from inhibition of adenylosuccinate synthetase by FoB-MP. predominating. In addition, formycin A is extensively incorpo- In this paper we describe experiments that clearly demon- rated into RNA. From the metabolic profile, we conclude that for- strate that FoB is extensively metabolized to corresponding mycin B is first converted to the 5'-monophosphate by the nu- adenosine 5'-nucleotides and subsequently incorporated into cleosidephosphotransferase foundinLeishmania andis subsequently RNA in. L. tropical L. mexicana, and L. donovani. In effect, converted to cytotoxic adenosine nucleotide analogs of formycin nucleoside FoB into 5'- A thatbecome incorporated into RNA. The metabolism and mech- these organisms process the innocuous anism of formycin B appears to be qualitatively similar to that of nucleotides of the highly cytotoxic formycin A (FoA). Further, allopurinol riboside, but quantitative differences and species se- these conversions appear to be more efficient than 'the analo- lectivity suggest that these agents may have a different spectrum gous metabolic processing of allopurinol riboside, and FoB is of as potential anti-leishmanial agents. an effective inhibitor, of some species of Leishmania promasti- activity gotes that are relatively resistant to allopurinol riboside. In recent years, biochemical differences have been uncovered. between Leishmania and their mammalian hosts, that suggest MATERIALS AND METHODS rational approaches towards the design of agents that are se- Materials. FoA, FoB, FoA-MP, and FoA-TPwere purchased lectively toxic towards the parasite. The inosine analog allo- from Calbiochem. [3H]FoA (5 Ci/mmol; 1 Ci = 3.7 X 1010 purinol' riboside serves as a paradigm of one such approach (1) becquerels) was obtained-from Moravek Biochemicals. Bacte- that is relevant to the work described here. As with most inosine rial alkaline phosphatase (39 units/mg) and Crotalus adaman- analogs, allopurinol riboside is not phosphorylated by the ki- teus phosphodiesterase (20 units/mg) were from Worthington; nases normally found in mammalian cells and shows little or no calf intestinal.adenosine deaminase (250 units/mg). and Cro- toxicity; further, because the glycosidic bond is relatively re- talus atrox venom were from Sigma. FoB-MP was prepared in sistant to cleavage the analog is not significantly catabolized. 90% yield by phosphorylation ofFoB (8) and purified by HPLC Leishmania have a nucleoside phosphotransferase that can cat- on system 1 (see below); the product had a UV! spectrum iden- alyze 5'-phosphorylation of inosine analogs and as a result al- tical to that of FoB and was converted to FoB upon treatment lopurinol riboside is converted to its 5'-monophosphate in-these with alkaline phosphatase or snake venom. HPLC of FoA-TP organisms. Subsequently, allopurinol riboside 5'-monophos- on system 1 showed FoA-DP as an impurity (10%). The FoA- phate is sequentially acted upon by adenylosuccinate synthetase DP was used as a chromatographic marker; it eluted in the re- and lyase (2) to form corresponding adenosine nucleotide ana- gion characteristic of nucleoside diphosphates and was con- logs, which are incorporated into RNA and presumed to be re- verted to FoA upon treatment with alkaline phosphatase or sponsible for the anti-leishmanial activity ofallopurinol riboside snake venom. Chromatographic markers of FoB-DP and FoB- (1). Interestingly, the adenylosuccinate synthetase of Leish- TP were obtained by nitrous acid deamination of FoA-TP (9) as mania may offer a second site ofselectivity, because allopurinol follows: A solution of 0.25 ,umol of FoA-TP in 0.1 ml of 6.5% riboside 5'-monophosphate does not appear to be a substrate (vol/vol) HOAcwas treated with 4mg ofNaNO2 for 1 hr at 750C. for the mammalian enzyme (3, 4). Recently, another inosine HPLC in system 1 gave three UV-absorbing peaks, one each analog, the C-nucleoside formycin B (FoB), has been reported eluting in-regions characteristic ofnucleoside 5'-mono-, di-, and to inhibit the in vitro growth of promastigotes of L. donovani triphosphates and with area ratios of 5:15:80. All compounds and L. mexicana and amastigotes of L. mexicana;. further, pre- had UV spectra identical to the spectrum of FoB, and the com- liminary studies indicate the drug to be therapeutically active pound eluting in the monophosphate region had the same re- in hamsters infected with L. donovani (5). As with allopurinol tention volume as FoB-MP. riboside, FoB has a stable glycosidic bond,. is not phosphoryl- [3H]FoB' was prepared by treating a solution (1.1 ml) con- ated by kinases found in mammalian cells (6), and is remarkably taining 110 nmol of [3H]FoA (0.45 Ci/mmol) and.50 mM nontoxic to animals (7). Although allopurinol- riboside and. FoB KHPO4 (pH 7.4) with 0.12 unit ofadenosine deaminase at 250C are structurally similar, the metabolism and mechanism ofthese for 1 hr. The.product was purified by HPLC on systeml2 which agents in Leishmania have been proposed to differ (5). FoB is gave a single radioactive peak corresponding to authentic FoB; a substrate for the Leishmania nucleoside phosphotransferase in vitro, and formycin B 5'-monophosphate (FoB-MP) accu- Abbreviations: FoB, formycin B; FoB-MP, FoB-DP, and FoB-TP, formycin B 5'-mono-, di-, and triphosphate, respectively; FoA, formycin The publication costs ofthis articlewere defrayed in part by page charge A; FoA-MP, FoA-DP, and FoA-TP, formycin A 5'-mono-, di-, and tri- payment. This article must therefore be hereby marked "advertise- phosphate, respectively. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. § To whom reprint requests should be addressed. 288 Downloaded by guest on September 25, 2021 Medical Sciences: Rainey and Sand Proc. NatL Acad. Sci. USA 80 (1983) 289 rechromatography of the purified material demonstrated that at 1,000 x g) and resuspended at a density of 2-5 X 107 cells less than 0.01% of[3H]FoA could have been present in the prep- per ml in fresh medium containing 3.0 ,M [3H]FoB (0.45 Ci/ aration. mmol). At specified times cells were harvested by centrifuga- L. mexicana (Tres Bracos, strain 9) and L. tropica (Iran, strain tion and washed with 1 ml of ice-cold phosphate-buffered sa- 252) promastigotes were obtained from S. Meshnik (Rockefeller line. The pellet was suspended in 100 jul ofcold 0.6 M trichloro- Univ.); L. donovani (Khartoum; WR 378) and L. braziliensis acetic acid containing [14C]alanine (120,000 dpm/ml) as a (Panama; WR 477) promastigotes were obtained from L. Hen- marker for dilution. After 10 min at 0-40C, the suspension was dricks (Walter Reed Army Institute of Research). centrifuged to give the acid-soluble and acid-insoluble fractions. HPLC Analyses. HPLC analyses were performed on a Hew- The acid-soluble fraction was carefully aspirated and neu- lett-Packard 1084 instrument equipped with a detector mea- tralized by extraction with 0.13 ml of 0.5 M tri(n-octyl)amine suring A2u and a peak integrator. Peaks were identified and in Freon-113 (11) for 30 sec with intermittent Vortex mixing. quantitated by comparison to authentic standards. The systems Aftercentrifugation, the lower (organic) phase was aspirated and employed were as follows: System 1 used a Partisil Sax column discarded. The radioactivity in a portion of the neutralized (4.6 x 250 mm; Altex); the flow rate was 3 ml/min; isocratic phase was measured and the [14C]alanine present was used to elution with 18 ml of 7 mM NH4PO4 (pH 3.85) was followed correct for dilution during manipulations (10); the remainder by a 90-ml linear gradient to 250 mM NH4PO4/5W0 mM KCl of the neutralized acid-soluble extract was analyzed within 1 hr (pH 4.5) and then a 30-ml isocratic elution with the latter buffer. by HPLC on system 1 or kept at -80'C until analysis. Fractions As described (10), nucleotides elute in regions characteristic of containing radioactivity were adjusted to pH 7.4 with 1 M Tris the number ofphosphate groups they possess, and this system and treated with C. atrox venom (1 mg/ml) at 370C for 18 hr. was used for initial separation and tentative identification of Solutions were deproteinized with 0.1 vol of6 M trichloroacetic metabolites of FoB as well as nucleotide pool analysis. A com- acid and neutralized by extraction with 1.2 vol of 0.5 M tri(n- plete description of the retention behavior of naturally occur- octyl)amine/Freon. The resultant radioactive nucleosides were ring nucleotides on this system has been given (10). The reten- identified as either FoA or FoB by HPLC on system 2. tion behavior offormycin derivatives on system 1 are as follows For analysis of incorporation of formycin into nucleic acids, (retention volumes in parentheses): FoA (3 ml), FoB (4 ml), the acid-insoluble pellet was sequentially washed with 1 ml of FoA-MP (12 ml), FoB-MP (37 ml), FoB-DP (63 ml), FoA-DP 0.1 M trichloroacetic acid (00C) and four 0. 1-ml portions of70% (65 ml), FoA-TP (101 ml), FoB-TP (105 ml). System 2 used Lich- (vol/vol) ethanol (-200C), collecting the pellet by centrifuga- rosorb RP-18 (4.6 x 250 mm; Altex) and a 100-ml linear gradient tion (15,000 X g, 30 sec, 4°C) after each wash. The pellet was of 0-50% aqueous methanol as eluant; flow rate was 2 ml/min. dissolved in 200 ,ul of 0.3 M KOH and kept at 250C for 18 hr This system was used for separation and identification of FoB to hydrolyze RNA and, after chilling in an ice bath, 30 ,ul ofcold (RV, 21 ml) and FoA (RV, 46 ml) subsequent to the digestion 6 M trichloroacetic acid was added to precipitate DNA. After ofnucleotide metabolites isolated on system 1. The RV of FoA centrifugation (15,000 X g, 30 sec, 4°C), the supernatant was was somewhat variable, depending on the salt concentration, aspirated and the pellet was washed with three 100-,l portions and all separations of[3H]FoA and [3H]FoB were performed in ofice-cold 0.1 M trichloroacetic acid. The supernatant and first the presence ofunlabeled markers. System 3 used a Lichrosorb washing were combined and extracted with 1.2 vol of 0.5 M RP-18 column (4.6 x 250 mm) eluted with 50 ml of a linear tri(n-octyl)amine/Freon as described above to give the RNA gradient of 0-25% methanol containing 5 mM sodium hexane- hydrolysate, and the radioactivity in a portion was measured to sulfonate/5 mM acetic acid, pH 3.6; flow rate was 2 ml/min. quantitate incorporation offormycin into RNA. The pellet was This system was used to separate and quantitate nucleoside redissolved in 300 ,ul ofcold 0.3 M KOH, precipitated with 30 components of RNA and to confirm the identity of FoA and Al of ice-cold 6 M trichloroacetic acid, and washed with three FoB. Pertinent compounds had the following retention behav- 100-,l portions ofice-cold 0.1 M trichloroacetic acid to ensure ior on system 3 (retention volumes in parentheses): uridine (12 complete removal of acid-soluble material. The residue was ml), guanosine (21 ml), cytidine (28 ml), adenosine (32 ml), FoB dissolved in 100 ,u of 0.3 M KOH and neutralized with acetic (20 ml), FoA (40 ml). acid, and radioactivity in DNA was measured. The RNA hy- Cell Culture. L. tropica, L. mexicana, L. braziliensis, and drolysate was adjusted to pH 8.5 by the addition of1 M Tris and L. donovani promastigotes were grown at 26°C in medium 199 incubated with alkaline phosphatase (0.15 unit/ml), C. ada- (GIBCO) supplemented with 20% fetal calf serum, 25 mM manteus phosphodiesterase (0.15 unit/ml), and MgCl2 (10 mM) Hepes (pH 7.4), and gentamycin at 0.1 ug/ml. Doubling times for 24 hr at 250C. The resultant nucleosides were analyzed by were 9-12 hr and maximal cell density was reached at ca. 2 X HPLC on system 3. 107 cells per ml for L. tropica and L. braziliensis, 7 X 107cells per ml for L. donovani, and 108 cells per ml for L. mexicana. RESULTS Cell cultures were maintained by thrice weekly passage into Effect of FoB on the Growth ofLeishmania Promastigotes. fresh medium and were counted by using a Coulter Counter The growth inhibitory effects ofFoB did not vary greatly among ZBI. Cells were routinely examined microscopically for their the species ofLeishmania. Concentrations of FoB that resulted well being (motility and morphology), and all cultures used in in a 50% reduction in the growth rates of L. tropica, L. mexi- experiments were verified to be free of contaminating organ- cana, L. braziliensis, and L. donovani were 0.14, 0.18, 0.30, isms by Gram stain and culture on blood agar at 37°C for 48 hr. and 0.20 ,uM, respectively. When L. tropica promastigotes For growth inhibition studies, cells were seeded at ca. 0.5 to were exposed to 1 ,M FoB, no increase in cell number was 1 X 106 cells per ml and treated with various concentrations of observed after as long as 3 weeks. When observed under the the drug. Cell number was determined at least once per day light microscope over this period, cells initially elongated and over a period of 6-7 days. EC50values refer to the concentration then became spherical and immobile, but with distinctly rec- of drug that inhibited the growth rate ofcells by 50%. The val- ognizable flagella; ultimately, most cells appeared as spherical, ues reported are the average of three to five separate deter- amorphous objects presumed to be dead. However, even after minations in which the SEMs were less than 20% ofthe mean. 3 weeks in 1 ,uM FoB, a few cells remained motile and transfer Metabolism of FoB. Leishmania promastigotes in midloga- to drug-free medium resulted in restoration of growth. When rithmic phase growth were harvested by centrifugation (4 min cells were treated with 10 tLM or greater FoB, the aberrant Downloaded by guest on September 25, 2021 290 Medical Sciences: Rainey and Santi Proc. Nad Acad. Sci. USA 80 (1983) Table 1. Nucleotide pools and FoB metabolites in Leishmania promastigotes after treatment with 0.3 pM FoB for 8 hr Concentration, pmol/106 cells 0.10 L. tropica L. mexicana L. donovani Metabo- With With With lite Control FoB Control FoB Control FoB II NAD 33 34 24 14 10 8 AMP 7.4 10 8.6 8.7 13 10 as 0.05 x UDP-Hex 41 63 32 38 33 36 E ADP 13 11 30 20 15 12 -0 GDP 10 6.7 13 7.5 6.9 8.9 UTP 35 40 37 57 27 23 CTP 13 16 12 13 12 10 ATP 180 180 180 170 140 100 0 GTP 37 44 35 33 40 34 FoB-MP 1.99 0.41 1.73 Retention volume, ml FoA-MP 0.22 0.41 0.29 FoA-DP 0.68 1.41 2.34 FIG. 1. HPLC of the acid-soluble extract of L. tropica promasti- FoA-TP 9.39 9.77 17.1 gotes treated with 3.0 1tM [3H]FoB for 8 hr. The extract from 1 x 107 cells was separated on system 1. UDP-Hex, UDP-hexose. In RNA FoA 1.36 2.05 2.65 morphological changes occurred more rapidly; by 7 days, only Adenosine/ immobile, spherical particles were observed and growth could FoA 110 90 85 not be re-initiated by passage into drug-free medium. We in- terpret these results to indicate that low concentrations of FoB Datawere obtained from corresponding experiments depicted in Fig. are 2. Data obtained by HPLC/UV are presented to two significant fig- leishmanistatic towards promastigotes, whereas higher con- ures. FoB metabolites, which were quantitated by liquid scintillation centrations are leishmanicidal. A similar observation has been counting, are presented to three significant figures. UDP-Hex corre- made with Leishmania amastigotes (5). sponds to UDP-glucose, UDP-galactose, or both as determined by RV. Metabolism of FoB in Leishmania When promastigotes of L. tropica, L. mexicana, and L. donovani were incubated with 3.0 ,uM [3H]FoB, HPLC of the acid-soluble extracts gave five in Leishmania promastigotes treated with 3.0 ,tM [3H]FoB over radioactive peaks. Fifg. 1 shows the HPLC profile ofL. tropica an 8-hr period, and Table 1 provides the amounts accumulated that was exposed to [ H]FoB for 8 hr; HPLC profiles ofL. nwx- after 8 hr. Total metabolites initially accumulate at approxi- icana and L. donovani treated under similar conditions were mately the same rate in all species. By 2 hr, near maximal ac- qualitatively similar. The peak eluting earliest (RV, 4 ml) was cumulation is achieved in L. mexicana and L. tropica, whereas identified as unmodified FoB by chromatography with an au- metabolites continue to accumulate in L. donovani for up to 8 thentic sample on HPLC systems 1 and 2. The other radioactive hr. Throughout the period ofexposure, FoA-TP is the predom- peaks were identified, in order of their elution, as FoA-MP, inant metabolite and, by 4 hr, accounts for 65-70% of the in- FoB-MP, FoA-DP, and FoA-TP by the following criteria: First, tracellular metabolites. By 8 hr, the FoA-TP is present at levels the radioactive metabolites were shown to chromatograph with equivalent to 5-18% ofthe intracellular ATP (Table 1). FoA-MP authentic standards on system 1. Second, each radioactive peak and FoA-DP constitute 8-15% of the total metabolites and, in was collected and treated with C. atrox venom to convert it to general, parallel the accumulation of FoA-TP. Accumulation of the corresponding nucleoside, which was then identified as FoB-MP reaches maximal concentrations within 1-2 hr and either FoA or FoB on HPLC system 2. then gradually declines as other metabolites accumulate. The Fig. 2 shows the kinetics ofaccumulation ofFoB metabolites intracellular concentrations of FoB-MP that accumulate in L.

25 L. tropics

20-

UII 15

ELlo 0

Time, hr FIG. 2. Accumulation of formycin metabolites inLeishmania promastigotes treated with 3.0 gM [3H]FoB. m, FoB-MP; A, FoA-MP; v, FoA-DP; o, FoATP; e, FoA incorporated into RNA; *, total metabolites. Downloaded by guest on September 25, 2021 Medical Sciences: Rainey and Santi Proc. Natd Acad. Sci. USA 80 (1983) 291 mexicana are somewhat lower than in L. tropica and L. dono- nucleotide of FoA. No other metabolites were observed, nor vani. When exposure to FoB is prolonged to 24 hr, all metabolite was incorporation into DNA detectable. From this, we conclude levels decline, as do the FoA-TP/FoA-DP and ATP/ADP ra- that the metabolism of FoB in Leishmania occurs as shown in tios; we presume these changes mark the onset of cellular de- Fig. 3. The initial formation of FoB-MP is presumed to be cat- generation as observed morphologically. alyzed by the Leishmania nucleoside phosphotransferase which When Leishmania spp. were treated with 3.0 /.M [3H]FoB has been shown to catalyze the in vitro phosphorylation of FoB there was a progressive increase in the incorporation ofradio- (5) as well as the related pyrazolopyrimidine nucleoside allo- activity into RNA (Fig. 2) which, by 8 hr, reached levels of 1.4- purinol riboside (1). The subsequent conversion of FoB-MP to 2.7pmol/106 cells (Table 1). Degradation ofthe RNA to its com- FoA-TP follows the established pathway for conversion of IMP ponent nucleosides and HPLC analysis revealed that the ra- to ATP. Because FoB is claimed not to be phosphorylated in dioactivity was exclusively associated with FoA. Further, the mammalian cells (6), formation ofFoB-MP in Leishmania is the molar ratio ofFoA to adenosine in RNA was quite high at 1:110 first step in the metabolism of FoB that contributes to its se- in L. tropica, 1:90 in L. mexicana, and 1:85 in L. donovani. The lectivity. The Leishmania adenylosuccinate synthetase also higher incorporation of FoA into L. donovani is in accord with shows a broader specificity for IMP analogs than does the cor- the lower levels of ATP pools found in this species after treat- responding mammalian enzyme (2-4) and may serve as an ad- ment with FoB (see below). Incorporation ofradioactivity into ditional site for selectivity. DNA was below the limits of our detection (<0.01 pmol/106 The EC50 values we have determined for growth inhibition cells). offour species of Leishmania promastigotes by FoB are in the Effect ofFoB on Nucleotide Pool Sizes in Leishmania. A typ- submicromolar range. When Leishmania spp. are exposed to ical HPLC/UV profile of the nucleotide pools in 7 X 106 L. 3.0 ,uM FoB, the aforementioned metabolites accumulate rap- tropica cells is shown in Fig. 1. Although the pool sizes are idly. FoA-TP accounts for 65-70% ofthe total metabolites and small, a number of nucleotides can be identified and quanti- by 8 hr reaches levels equivalent to 5-17% of the intracellular tated. Table 1 shows the effect on intracellular nucleotides after ATP concentration. The fact that FoA metabolites greatly ex- treatment of Leishmania promastigotes with 3.0 AuM FoB for ceed the intracellular concentration of FoB-MP suggests that 8 hr. With L. tropica, the major effects of FoB on the nucleotide the latter is a quite good substrate for the adenylosuccinate syn- pool sizes are the increase in UDP-hexose, which expands by thetase and lyase of Leishmania. With the data now available, some 50%, and the 33% reduction in the GDP pool. In L. mex- the previously reported inhibition ofadenylosuccinate synthe- icana there are significant reductions in the pool sizes ofNAD, tase by FoB-MP (5) is best explained by its participation as an ADP, and GDP and an increase in the UTP pool. The major alternative substrate, followed by rapid conversion to FoA-MP effect in L. donovani is the ca. 30% reduction in the ATP pool, by adenylosuccinate lyase present in the crude extract used. which occurs within 2 hr and remains throughout the 8-hr ex- FoA is also extensively incorporated into RNA to give, after 8 posure. It is ofinterest to note that, compared to other species, hr, a molar ratio ofadenosine to FoA of85-110, depending on more FoA-TP accumulates in L. donovani (equivalent to 18% the species. of the ATP present); FoA is also more efficiently incorporated Knowing the metabolism ofFoB in Leishmania, we may now into RNA in this species. The intracellular concentrations of consider the likely mechanisms for its cytotoxic effects. As pre- NAD, AMP, ADP, and GTP are also reduced in L. donovani viously suggested (5), competition between FoB-MP and IMP (>15%), but the significance of these changes is questionable for adenylosuccinate synthetase could result in purine starva- due to the small amounts being measured. tion. This may account for the 30% decrease in ATP pools and 15% decrease in GTP pools in L. donovani treated with FoB. DISCUSSION However, purine nucleotide depletion in L. nmxicana appears too low to account for cytotoxicity, and purine starvation is not The results described here clearly demonstrate that the metab- observed in L. tropica treated with FoB. In fact, there are no olites of FoB in L. tropica, L. mexicana, and L. donovani pro- common changes in nucleotide pool sizes ofLeishmania treated mastigotes are FoB-MP, FoA-MP, FoA-DP, and FoA-TP. Fur- with FoB that would point to a central mechanism ofthis drug ther, the drug is incorporated into RNA exclusively as the that involves perturbation of nucleotide metabolism. Most 0 0 H H NH21 H HN N H ~rN\ N N N~ QNN N 2

HOH2C o H203POH2C 0 H203POH2C

HO OH HO OH HO OH

FoB FoB-MP FoA-MP

3 w FoA- DP 4 - FoA-TP 5 - FoA-RNA

FIG. 3. Proposed metabolism of FoB in Leishmania. The enzymes assumed to be involved are 1, nucleoside phosphotransferase; 2, adenylosuccinate synthetase/lyase; 3, AMP kinase; 4, nucleoside diphosphokinase; 5, RNA polymerase. Downloaded by guest on September 25, 2021 292 Medical Sciences: Rainey and Santi Proc. Natt Acad. Sci. USA 80 (1983) likely, the cytotoxic effects of FoB result from the metabolites than that of IMP (2). Thus, the differences in the metabolites of FoA that accumulate in Leishmania. FoA itself is cytotoxic ofallopurinol riboside and FoB that accumulate in Leishmania to a variety of cells, and the intracellular metabolites are es- can be explained by a more facile conversion of FoB-MP to FoA- sentially identical to those we observe in Leishmania treated MP by the adenylosuccinate synthetase/lyase of these organ- with FoB (6, 12, 13). The diversity of the biochemical effects isms. The preceding comparison of the metabolic products of of FoA metabolites suggests multiple sites of action, primarily FoB and allopurinol riboside suggests that the differing anti- involving FoA-TP and RNA containing FoA. FoA-TP substi- leishmanial properties of these drugs, as well as the species tutes for ATP in a wide variety of important enzymic reactions variation in the effects of allopurinol riboside, are directly re- (14) and inhibits the initiation of DNA transciption (15). Incor- lated to the intracellular concentration of the corresponding poration of FoA into RNA inhibits the release ofnewly synthe- ATP analogs or the extent of their incorporation into RNA. If sized RNA from ternary transcription complexes (15) and in- this is correct, it should be possible to design related analogs terferes with RNA processing (16). Thus, although the exact thatwillbe more efficiently processed through this pathway and mechanism remains unresolved, it is most logical to conclude thus provide even more effective anti-leishmanial agents. that anti-leishmanial activity of FoB resides in its ability to serve as a precursor to nucleotides of FoA and subsequent incorpo- Note Added in Proof. Studies on the metabolism ofFoB in L. donovani ration into RNA. promastigotes have recently been reported (17) and are in accord with In many respects, the anti-leishmanial effects of FoB are the results described here. to analogous those reported for allopurinol riboside. However, This investigation received financial support from the UNDP/ there are notable differences between these drugs that are wor- World Bank/WHO Special Programme for Research and Training in thyofconsideration. First, the effects ofallopurinol riboside and Tropical Diseases and the Smith, Kline and Beckman Corporation. P.R. FoB on the growth of Leishmania promastigotes differ signifi- received partial support from U.S. Public Health Service Training cantly. It has been reported that the inhibitory effect of allo- Grant GM 07546. purinol riboside varies greatly among species of Leishmania promastigotes (1): L. braziliensis was the most sensitive, L. don- 1. Nelson, D. J., Lafon, S. W., Tuttle, J. V., Miller, W. H., Miller, ovani was intermediate, and L. mexicana was relatively resis- R. L., Krenitsky, T. A., Elion, G. B., Berens, R. L. & Marr, J. J. (1979)J. Biol Chem. 254, 11544-11549. tant. Furthermore, reduction of growth rate by allopurinol ri- 2. Spector, T., Jones, T. E. & Elion, G. B. (1979) J. Biol. Chem. boside requires 18-21 hr to become fully established and the 254, 8422-8426. drug is reportedly leishmanistatic. In contrast, there is little 3. Spector, T. & Miller, R. L. (1976) Biochim. Biophys. Acta 445, variation in the susceptibility of Leishmania species towards 509-517. FoB, which shows EC50 values of0.1-0.3 uM in L. tropica, L. 4. Elion, G. B. (1978) in Handbook ofExperimental Pharmacology, mexicana, L. donovani, and L. braziliensis. We have not ob- eds. Kelley, W. N. & Wiener, I. M. (Springer, New York), Vol. 51, pp. 485-514. served lag periods before the onset of inhibition of growth 5. Carson, D. A. & Chang, K.-P. (1981) Biochem. Biophys. Res. caused by FoB and, although some organisms survived pro- Commun. 100, 1377-1383. longed exposure to low concentrations ofthe drug, higher con- 6. Umezawa, H., Sawa, T., Fukagawa, Y., Hommo, I., Ishizuka, centrations were clearly leishmanicidal. Second, although the M. & Takeuchi, T. (1967) J. Antibiotics (Tokyo) 20, 308-316. metabolism of allopurinol riboside and FoB are qualitatively 7. Ishizuka, M., Sawa, T., Hori, S., Takazama, H., Takeuchi, T. similar, they differ significantly in quantitative aspects. As & Umezawa, H. (1968)J. Antibiotics (Tokyo) 21, 5-12. 8. Yoshikawa, M., Kato, T. & Takenishi, T. (1969) Bull Chem. Soc. shown here, when Leishmania promastigotes are treated with Jpn. 42, 3503-3508. 3 ,uM FoB, the major metabolite is FoA-TP, which reaches lev- 9. Suzuki, S. & Marumo, S. (1960)J. Antibiotics (Tokyo) 14, 34-38. els of 10-20 pmol/106 cells, and FoA replaces about 1% ofthe 10. Pogolotti, A. L., Jr., & Santi, D. V. (1982) Anal Biochem. 126, adenosine residues in RNA. At 6-fold higher concentrations of 335-345. allopurinol riboside, the major metabolite in Leishmania is al- 11. Khym, J. Y. (1975) Clin. Chem. 21, 1245-1252. lopurinol riboside 5'-monophosphate, which accumulates to 12. Suhadolnik, R. J. (1979) Nucleosides as Biological Probes (Wiley, New York), pp. 169-183. give intracellular concentrations in the millimolar range. The 13. Caldwell, I. C., Henderson, J. F. & Paterson, A. R. P. (1969) corresponding ATP analog, 4-aminopyrazolo(3,4-d)pyrimidine Can. J. Biochem. 47, 901-908. ribonucleoside 5'-triphosphate, is a minor metabolite that ac- 14. Ward, D. C., Cerami, A., Reich, E., Acs, G. & Altwerger, L. cumulates to the extent of0.2 and 3 pmol/106 cells in L. mex- (1969)J. Biol Chem. 244, 3243-3250. icana and L. donovani, respectively. Further, 4-aminopyra- 15. Darlix, J. L., Fromageot, P. & Reich, E. (1971) Biochemistry 10, zolo(3,4-d)ribonucleoside replaced only 1 in 1,700 adenosine 1525-1531. 16. Majima, R., Tsutsumi, K., Suda, H. & Shimura, K. (1977) J. residues of RNA after 24-hr incubation. As previously noted, Biochem. (Tokyo) 82, 1161-1166. FoB-MP binds to Leishmania adenylosuccinate synthetase 17. Nelson, D. J., Lafon, S. W., Jones, T. E., Spector, T., Berens, some 10-fold more tightly then does IMP (5), whereas the K, R. L. & Marr, J. J. (1982) Biochem. Biophys. Res. Commun. 108, ofallopurinol riboside 5'-monophosphate is some 25-fold higher 349-354. Downloaded by guest on September 25, 2021