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By N-Methylthiotetrazole-Containing Antibiotics (Hypoprothrombinemia/Vitamin K/Moxalactam/Glutathione/Disulfiram) JAMES J

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By N-Methylthiotetrazole-Containing Antibiotics (Hypoprothrombinemia/Vitamin K/Moxalactam/Glutathione/Disulfiram) JAMES J Proc. Natl. Acad. Sci. USA Vol. 81, pp. 2893-2897, May 1984 Medical Sciences Mechanism of the inhibition of the y-carboxylation of glutamic acid by N-methylthiotetrazole-containing antibiotics (hypoprothrombinemia/vitamin K/moxalactam/glutathione/disulfiram) JAMES J. LIPSKY Departments of Medicine and of Pharmacology and Experimental Therapeutics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Communicated by Victor A. McKusick, January 30, 1984 ABSTRACT Antibiotics that contain a 1-N-methyl-5-thio- A tetrazole (MTT) side group have been associated with hypo- 0 H30C H prothrombinemia. In a detergent-treated rat liver microsomal HO {CH-C-NH * * N-N system, MTT inhibited the carboxylation of the y carbon of COONa -NN CH2-S-C N glutamic acid, a necessary reaction in the synthesis of four of COONa I the clotting factors. In the present work, the inhibition by CH3 MTT was found to be slow in onset, with a lag time of 15 min Moxalactam before significant inhibition occurred. A preincubation of B N-N N-N MTT with the microsomes decreased the lag time and in- 11 11 11 11 N C-S-S-C N creased the extent of inhibition. Glutathione at 1 mM was '% N.11%N' found to markedly decrease the ability of MTT to inhibit this reaction. The disulfide dimer of MTT was a more potent in- CH3 CH3 hibitor of the system than was MTT, with inhibition detected MTT Disulfide Dimer as low as 1 jLM dimer. Disulfiram also inhibited the carboxyl- C ation system. These results indicate that the sulfhydryl group S S 11 11 of MTT is important for the inhibitory effect of MTT and sug- H5C2 C-S-S-C C2H5 gest that a slowly formed metabolite of MTT may be directly responsible for the observed inhibition. The inhibitory mecha- C2H5 C2HN nism of MTT may be analogous to that of disulfiram, which would explain some pharmacologic effects in common with di- Disulfirom sulfiram. In addition, the in vitro observations presented here FIG. 1. (A) The structure of moxalactam. The MTT side group, and a closer examination of the clinical evidence raise the shown in boldface, is attached to the carbon atom at the 3 position of possibility that MTT-containing antibiotic-induced hypopro- the six-membered ring of moxalactam and is found also in cefaman- thrombinemia may not be a vitamin K reversible phenome- dole and cefoperazone. (B) The structure of the disulfide dimer of non. MTT. (C) The structure of disulfiram drawn to emphasize the struc- tural similarity to the MfT disulfide dimer. An increased incidence of hypoprothrombinemia has been associated with the use of several new P-lactam antibiotics, to the 3-position of the six-membered ring of the antibiotic including cefamandole, moxalactam, and cefoperazone (1- (Fig. 1A). The rationale for examining the activity of MTT 3). It was believed that the hypoprothrombinemia was cor- was that it is in a "leaving group" position when the 83-lactam rected by the administration of vitamin K; therefore, two bond of the antibiotic undergoes nucleophilic attack. This mechanisms that relate to vitamin K have been proposed to may occur when the antibiotic interacts with bacterial en- explain the hypoprothrombinemia. One is that these antibi- zymes or nonenzymatically in alkaline solutions (9). Free otics, which are secreted into the bile, kill the intestinal bac- MTT has been found in the plasma of subjects who had been teria that produce vitamin K (4). However, the nutritional given moxalactam (10). We present evidence in this paper role in humans of menaquinones, the bacterial forms of vita- that MTT is a potent inhibitor of the microsomal carboxyl- min K, is uncertain (5), and many antibiotics are secreted ation system and that the mechanism of the inhibition may into the bile but are not associated with an increased inci- be related to the sulfhydryl portion of MTT. dence of hypoprothrombinemia (6). The hypothesis that we and others (6, 7) have proposed is that a common structural METHODS feature of these antibiotics, a 1-N-methyl-5-thiotetrazole Materials. The sodium salt of MTT and its disulfide dimer (MTT) side group, causes a more direct inhibition ofthe vita- were a gift from Hoechst-Roussel Pharmaceuticals (Somer- min K-dependent step in clotting factor synthesis-i.e., the ville, NJ). The pentapeptide L-phenylalanyl-L-leucyl-L-gluta- y-carboxylation of glutamic acid. In support of this hypothe- myl-L-glutamylisoleucine, disulfiram, NADH, dithiothreitol, sis, preliminary findings demonstrated that the MTT side and N-ethylmaleimide were obtained from Sigma, and the group is capable of the in vitro inhibition of the y-carboxyl- NaH14CO2 was from New England Nuclear. Vitamin K1 ation of glutamic acid in a detergent-treated rat liver micro- was obtained as Aquamephyton (phylloquinone) from Merck somal system (8). This system contains enzymes capable of Sharp & Dohme. Male Sprague-Dawley rats, 250-300 g, the reduction of vitamin K and the carboxylation of glutamic were obtained from Harland Sprague-Dawley (Walkersville, acid. The MTT side group is attached by a methylene linkage MD). Microsome Preparation. Rat liver microsomes were pre- The publication costs of this article were defrayed in part by page charge pared and the carboxylation ofglutamic acid was determined payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: MTT, 1-N-methyl-5-thiotetrazole. 2893 Downloaded by guest on September 26, 2021 2894 Medical Sciences: Lipsky Proc. NatL Acad. Sci. USA 81 (1984) by a modification of the method of Houser et al. (11). Rats Table 1. Effect on inhibition of an additional complement of were fasted for 72 hr prior to decapitation and removal ofthe reaction components and glutathione liver. Livers were homogenized, 33% (wt/vol), in 250 mM sucrose/0.08 M KCI/25 mM imidazole, pH 7.4, at 20C. The Amount added, % inhibition, homogenate was centrifuged at 10,000 x g for 10 min, and Component ,Umol mean ± SEM the resulting supernatant was centrifuged at 100,000 x g for Control 61 ± 3 60 min. The pellet from this centrifugation was resuspended NADH 1.16 64 ± 2 in the imidazole buffer to which had been added 1.5% (vol/ Pentapeptide 0.33 71 ± 3 vol) Triton X-100. The final volume of this solution was NaHCO3 0.23 76 ± 5 equal to the volume of the supernatant of the initial centrifu- Vitamin K 0.03 66 ± 7 gation. This suspension was centrifuged at 100,000 x g for 60 Glutathione 0.58 67 ± 4 min, and the supernatant was taken as the microsomal prep- aration. tivity was slow in onset, but significant inhibition began Carboxylation Assay. The carboxylation assay mixture, about 15 min after the start of the reaction. When MTT was unless otherwise indicated, contained: the substrates, L- preincubated with the microsomal system 15 min prior to the phenylalanyl-L-leucyl-L-glutamyl-L-glutamylisoleucine at initiation of the reaction with vitamin K, the onset of the 1.45 mM and 10 pCi (1 Ci = 37 GBq) of NaH14CO2 (43 inhibition was more rapid and the degree of inhibition was mCi/mmol; final concentration, 0.4 mM); 500 1.l of micro- greater. somal preparation (average = 3.5 mg of protein); and 2 mM To determine if this inhibition was due to depletion of one NADH. Ten microliters of inhibitor was added to give a final or more of the reaction components, additional reaction volume of 580 ,ul. The pentapeptide, NADH, and inhibitor components were added individually to the microsomal sys- were added as solutions in imidazole buffer. After a 5-min tem containing 1 mM MTT 15 min after beginning the reac- preincubation at 22°C, reactions were initiated by the addi- tion. None of the reaction components examined reversed tion of 25 ,ug of vitamin K1 unless otherwise indicated in the the inhibition (Table 1). text. The reaction mixture was incubated for 30 min at 22°C, Since greater inhibitory activity was observed with a 15- the reaction was stopped by the addition of 0.5 ml of 10% min preincubation of MTT with the microsomal system (Fig. trichloroacetic acid, and the mixture was centrifuged. The 2), the effect of the duration of the preincubation on inhibi- supernatant containing the radioactive peptide was removed tion was examined. The inhibitory activity of 0.3 mM MTT and flushed with CO2 gas for 15 min. An 0.8-ml aliquot was increased in a linear manner with increasing time of prein- subjected to liquid scintillation counting to determine 14CO2 cubation (Fig. 3). incorporation into the peptide. Protein determinations were These results suggested that an interaction between MTT performed by the method of Lowry et al. (12). and the microsomes plays some part in the ultimate inhibi- tion of the y-carboxylation. The sulfhydryl group present on RESULTS MTT might play a role in such an interaction. Therefore, the The rate of y-carboxylation by the microsomal system is effect of glutathione on the ability of MTT to inhibit the v- shown in Fig. 2. After an initial lag period of about 2 min, carboxylation system was examined. Glutathione was added this uninhibited rate was fairly linear over the 30-min incuba- to the reaction mixtures at the time of addition of MTT. In tion time. In the presence of 0.7 mM MTT, inhibition of ac- the presence of 1 mM glutathione, there was a considerable decrease in the ability of MTT to inhibit the reaction (Fig. 4). 100 _ With glutathione present at 1 mM, it required 40 mM MTT to achieve 30% inhibition of the carboxylation system, whereas 90 only 0.3 mM MTT was required in the absence of glutathi- one.
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