[CANCER RESEARCH 33, 856-858, April 1973] The Molecular Mechanism of Action of Methylthioinosinedicarboxaldehyde on Ribonuclease A as a Model System1

T. C. Spoor, J. L. Hodnett, and A. P. Kimball Department of Biophysical Sciences, University of Houston, Houston, Texas 77004

SUMMARY MATERIALS AND METHODS

The molecular mechanism of action of the antitumor drug, Enzymes. Highly purified bovine pancreatic RNase A methylthioinosine periodate oxidation product, was demon (phosphate free) and Pronase were purchased from Worthing- strated. ton Biochemical Corporation (Freehold, N. J.) and Calbio- This periodate oxidation product (methylthioinosinedicar- chem (Los Angeles, Calif.), respectively. boxaldehyde) inhibited bovine pancreatic ribonuclease A. The MMPR-OP-3SS. Radiopure MMPR-OP-35S was synthesized inhibition was due to Schiffs base formation with e-amino by methods previously reported (10). groups of lysine residues, one of which was in close proximity Enzyme Assay. RNase activity was assayed by the method to the catalytic center of the enzyme. of Kalnitsky et al. (6), with the use of purified yeast RNA (Sigma Chemical Co., St. Louis, Mo.) as substrate. RNase, 7.5 /ug, was preincubated with MMPR-OP or buffer for 15 min at 37°before the addition of 2.5 mg of substrate. Incubations INTRODUCTION were carried out for various times. Absorbance measurements As shown by several studies (2, 5, 7, 11), MMPR-OP2 is a were made on a Beckman Model DB spectrophotometer. potent antitumor and immune suppressive agent. The com MMPR-OP-35S Covalent Binding to RNase A. MMPR-OP- pound produced blocks in the synthesis of DNA and RNA, 3SS, 3.70 Amóles. 5.67 X 10s cpm/jumole, or 14.80 jumóles, and in protein biosynthesis (2, 5, 7, 11). Also, it was used to 5.91 X 10s cpm/Aimole, was added to 0.37 /imole of bovine differentiate glycoprotein biosynthesis into 2 distinct phases pancreatic RNase A, phosphate free, in a final volume of 10 ml (5). Recently, MMPR-OP was utilized as a molecular probe of of distilled water, and the pH was adjusted to 9.0 with 5% the catalytic center of DNA-directed RNA polymerase (10, K2CO3. The reaction mixtures (in duplicate) were incubated 12) and was shown to affinity-label the active center on the ß at 37°for 45 min; the pH was maintained at 9.0 with 5% subunit of the Escherichia coli enzyme (10). The aldehyde K2CO3. After the incubation, 15 mg of NaBH4 were added to groups on the MMPR-OP can (potentially) form reversible each vessel, and the reduction of the Schiffs base was allowed to proceed for 21 hr at 4°.We dialyzed the reaction mixtures Schiffs bases with reactive amino groups of amino acids and proteins. The Schiffs base formed between MMPR-OP and the against running distilled water for 44 hr at 4°to rid the e-amino group of the essential lysine in the catalytic center of solution of unreacted NaBH4 and MMPR-OP-3 5S. The enzyme RNA polymerase was converted to the stable covalent linkage solutions were then freeze dried. The enzymatic activities of by mild reduction with sodium borohydride (10). the RNase:MMPR-OP-3SS derivatives were compared with an In general, MMPR-OP should inhibit any cancer or analogously treated control enzyme. Radioactivity measure "normal" enzyme of nucleic acid metabolism with a lysine in ments were made in a toluene fluor in a Packard Model 3880 or about its catalytic center, limited only by steric unavailabil scintillation spectrometer. Pronase Degradation of RNase:MMPR-OP-35S Derivatives. ity of a lysine to the approach of the reagent. Presumably. MMPR-OP functions as an antitumor agent in vivo in this For a determination of the amino acid(s) of RNase bound by MMPR-OP, 0.5 mg of each derivative was digested for 72 hr manner. This study reports the results of research on bovine with 22.5 jug of Pronase at 37°in 0.02 M phosphate buffer, pH pancreatic RNase A, an enzyme known to have 2 lysines in juxtaposition to the 2 essential histidines in its catalytic center 7.0. The resultant amino acid mixtures were examined by (4). descending paper chromatography in the following 3 solvent systems: l-butanol:acetic acid:water, 4:1:5, (RF lysine = 0.12; RF MMPR-OP = 0.95; RF lysine MMPR-OP = 0.36); 1Supported by Robert A. Welch Grant E-321 and by NIH Grant pyridine:isoamyl alcohol:water:diethylamine, 10:10:7:3 (RF CA-12327. lysine = 0.07; RF MMPR-OP = 0.88; RF lysine - MMPR-OP = 2The abbreviations used are: MMPR-OP, methylthioinosine perio 0.72); and water-saturated phenol (RF lysine = 0.57; RF date oxidation product, methylthioinosinedicarboxaldehyde; RNase, bovine pancreatic RNase A. MMPR-OP = 0.94; RF lysine MMPR-OP = 0.94). The Received November 10, 1972; accepted January 8, 1973. developed chromatograms were cut into serial 1- x 2-cm strips,

856 CANCER RESEARCH VOL. 33

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1973 American Association for Cancer Research. Mechanism of Action ofMMPR-OP and the radioactivity of the individual strips was determined 100 - by liquid scintillation counting in a toluene fluor.

RESULTS AND DISCUSSION

Inhibition Studies. The results of inhibiting RNase A with various molarities of MMPR-OP are depicted in Chart 1. At 2 min, while the reaction was linear, 10~4, 5X 10~4,and 10~3 M MMPR-OP inhibited RNase 37, 64, and 79%, respectively. The 50% inhibitory molarity of MMPR-OP was about 2 X 1(T4 M. Binding Studies. MMPR-OP-35 S will bind to RNase A, and this binding can be stabilized by mild reduction with NaBH4 (Table 1). When MMPR-OP-35 S was incubated with RNase in a 0 024 10:1 and 40:1 molar ratio, MMPR-OP was found to bind TIME (minutes) covalently in a drug:enzyme ratio of 2 and 8 moles of drug per Chart 2. Enzymatic activities of RNase-MMPR-OP derivatives. Tubes mole of enzyme, respectively. The enzymatic activities of in duplicate (each containing 5.56 X 10 "* ornóleof RNase or RNase these RNase derivatives at molar equivalency (5.56 X 10~4 derivative, 2.5 mg of RNA, and 0.1 M acetate buffer, pH 5.0; 2 ml final jumóle) were compared with RNase carried through the volume) were each incubated at 37°for varying lengths of time, o, binding procedure, but without added drug (Chart 2). There control; + and •¿,respectively,2 and 8 moles MMPR-OP bound per mole enzyme.

400

300

—¿200 o

8 IO

TIME (minutes) 0 10 20 Chart 1. Inhibition of RNase with time by MMPR-OP. Either CENTIMETERS MMPR-OP at various molarities or buffer was preincubated with RNase (7.5 jug) for 15 min at 37°before the addition of 2.5 mg of RNA. Chart 3. Radiochromatographic profile of Pronase digests of RNase derivatives. Aliquots (0.1 ml) of the Pronase digests of RNaserMMPR- Incubations in duplicate were continued for varying lengths of time, o, control; a, A, and +, 1 X 10'\5 X 10'4,and 1 X IO"3 M, MMPR-OP, OP derivatives were chromatographed on Whatman No. 3MM paper and developed in a l-butanol:acetic acidrwater (4:1:5) solvent system. The respectively. developed paper was strip counted, o, RNase:MMPR-OP-3 5S, 8 moles drug per mole enzyme; +, RNase:MMPR-OP-3 5S, 2 moles drug per mole Table 1 enzyme; »,lysine:MMPR-OP-35S, standard. Covalent binding of MMPR-OP to RNase MMPR-OP-35S [3.70 Mmoles (5.67 X 10s cpm/Mtnole) or 14.80 Amóles(5.91 X IO5 cpm/iumole)] was incubated with 0.37 nmole of was a direct correlation between increasing moles of drug RNase at 37°for 45 min in 10 ml of distilled water. The pH was kept bound and increasing inhibition of enzymatic activity. constant at 9.0 with 5% K2CO3. The Schiffs base was reduced by the Interestingly, the RNase:MMPR-OP derivative with 8 moles of addition of 15 mg of NaBH4 in 1 ml of H2O. Reduction was carried out for 21 hr at 4°.Excess MMPR-OP-35S and NaBH4 were eliminated drug bound per mole of enzyme still retained 12% of control by dialysis against running distilled water for 44 hr at 4°. activity. With 2 moles of bound drug, the RNase derivative was inhibited only about 59%. Bovine pancreatic RNase A has 8 Reaction mixture potentially reactive amino groups exposed on its surface, 7 (jumóles)RNase0.37 e-amino groups of lysine residues, and the N-terminal amino Sbound group (1). This correlates well with the RNase:MMPR-OP 5S3.70 (jumóles)0.72 5S:RNase1.95 derivative with 8 moles of drug bound per mole of enzyme. Pronase Degradation of RNase Derivatives. When the ±0.06° ±0.07 radioactive, drug-bound RNase derivatives were degraded with 0.37MMPR-OP-314.80MMPR-OP-353.02 ±0.04MMPR-OP-38.16 ±0.11 Pronase, the only amino acids found bound were lysines a Mean ±S.E. (Chart 3). Chart 3 depicts the Chromatographie profile of the

APRIL 1973 857

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1973 American Association for Cancer Research. T. C. Spoor, J. L. Hodnett, and A. P. K imboli histidine-12 and histidine-119; the 2 amino acids involved as directly in catalysis) (4). In summary, MMPR-OP exerts its inhibitory action on 0.6 RNase by forming a Schiffs base with available reactive amino groups on the surface of the enzyme, one of which lies close to 0.4 n«0.98 the catalytic center.

o 0.2 ACKNOWLEDGMENTS o _) 0.0 We wish to thank Mr. P. R. Loy of the Department of Biophysical Sciences for supplies of the MMPR-OP and the lysine:MMPR-OP-35S -0.2 standard. 4.0 3.5 30 -LOGen REFERENCES Chart 4. Hill plot of RNase inhibition by MMPR-OP. MMPR-OP 1. Barnard, E. A. Ribonucleases. Ann. Rev. Biochem., 38: 677-732, (various molarities) or 0.1 M acetate buffer, pH 5.0, was preincubated with RNase (7.5 Mg)for 15 min at 37°before the addition of 2.5 mg of 1969. RNA in a final volume of 2.0 ml. The tubes in duplicate were then 2. Bell, J. P., Faures, M. L., LePage, G. A., and Kimball, A. P. incubated for an additional 2 min at 37°.Logv/(V - v) is the log of the Immunosuppressive and Antitumor Activity of the Periodate Oxidation Product of ß-D-Ribosyl-o-methylthiopurine. Cancer rate of the inhibited reaction divided by the rate of the uninhibited reaction minus the rate of the inhibited reaction. -Log [/] is the Res., 28: 782-787, 1968. negative log of the inhibitor concentration. 3. Bell, J. P., and Gisler, R. H. Distribution and Metabolism of the Periodate Oxidation Product of 9-(3-D-Ribosyl-6-methylthiopurine. Biochem. Pharmacol., 18: 2103-2114, 1969. Pronase digests placed at the origin of Whatman No. 3MM 4. Blow, D. M., and Steitz, T. A. X-Ray Diffraction Studies of paper and developed for 40 cm in l-butanol:acetic acid:water Enzymes. Ann. Rev. Biochem., 39: 63-100, 1970. (4:1:5) along with the standard lysine:MMPR-OP derivative. 5. Cronenberger, J. H., and Kimball, A. P. Studies on a Model The lysine:MMPR-OP-35S of the standard and the Pronase Immunological System. Cancer Res., 31: 1257-1260, 1971. digests was at 14 to 15 cm (Rf = 0.36) in this solvent system. 6. Kalnitsky, G., Hummel, J. P., and Dierks, C. Some Factors Which The Pronase digests contained no unreacted MMPR-OP-3 5S, Affect the Enzymatic Digestion of Ribonucleic Acid. J. Biol. since this had been dialyzed away from the RNase derivatives. Chem., 234: 1512-1516, 1959. Counts at the origin represented undegraded enzyme and 7. Kimball, A. P., Wilson, M. J., Bell, J. P., and LePage, G. A. oligopeptides. The above result was also obtained in 2 other Inhibition of Thymidylate Kinase and DNA Polymerase by the solvent systems (see "Materials and Methods"). Loy (9) had Oxidation Product of ß-D-Ribosyl-6-methylthiopurine. Cancer Res., 28: 661-665, 1968. shown earlier that MMPR-OP appeared to have specificity for 8. Loftfield, R. B., and Eigner, E. A. Molecular Order of Participation lysine and a lysine homolog, ornithine. Also, Bell and Gisler of Inhibitors (or Activators) in Biological Systems. Science, 164: (3) showed that the compound would bind to glycine. These 3 305-308, 1969. amino acids are related, in that no méthylènegroups lie 9. Loy, P. R. Studies on the Periodate Oxidation Product of beyond their reactive amino groups. However, ornithine is not iì-D-Ribosyl-6-methylthiopurine, M.S. Thesis, University of Hous a protein amino acid, and the amino group of glycine in a ton, Houston, 1970. protein is tied up in the peptide bond except when it is 10. Nixon, J., Spoor, T., Evans, J., and Kimball, A. Affinity Labeled E. TV-terminal. The TV-terminal group of RNase is lysine (1,4). coli DNA Directed RNA Polymerase. Biochemistry, 11: HUI Plot. Although MMPR-OP binds to RNase to inhibit the 4570-4573, 1972. 11. Spoor, T., and Kimball, A. P. Mammalian DNA-Dependent RNA enzyme, there still remained the possibility that a lysine(s) was Polymerase: Inhibition of a Mammalian Enzyme by Methylthioino- bound that was not in the proximity of the active center. For sine Dialdehyde. Proc. Soc. Exptl. Biol. Med., 136: 605-607, this reason. Hill Plots were done so that we might determine 1971. whether a critical lysine(s) was involved (8). The results of 1 12. Spoor, T., Persico, F., Evans, J., and Kimball, A. P. Recognition such study is shown in Chart 4. The Hill plot has a slope of 1, Sites on or About the Catalytic Center of DNA-Dependent RNA which shows that 1 lysine is bound in the vicinity of Polymerase. Nature, 227: 57-59, 1970.

858 CANCER RESEARCH VOL. 33

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1973 American Association for Cancer Research. The Molecular Mechanism of Action of Methylthioinosinedicarboxaldehyde on Ribonuclease A as a Model System

T. C. Spoor, J. L. Hodnett and A. P. Kimball

Cancer Res 1973;33:856-858.

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