Metabolism and Mechanism of Action of Formycin B in Leishmania (Purine Metabolism/Formycin A/Allopurinol Riboside) PETRIE RAINEY*T and DANIEL V
Total Page：16
File Type：pdf, Size：1020Kb
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, for- mycin 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.