Life Sciences, Vol . 25, pp . 1857-1864 Printed in the U .S .A .
Pergamon Press
THE EFFECTS OF MEFLOQUINE ON ESCHERICHIA COLI Ruth E . Brown, Frank A . Stancato, and Alan D . Wolfe Department of Biological Chemistry, Division of Biochemistry, Walter Reed Army Institute of Research, Washington, D .C . 20012 (Received in final form October 17, 1979) SUMMARY Mefloquine, a quinoline-4-methanol antimalarial drug, also possesses bactericidal activity . Mefloquine causes rapid loss of bacterial viability, cell and spheroplast lysis, cessation of macromolecular synthesis, release of macromolecular constituents, and inhibition of the oxidation of NADH by isolated _E . coli membranes . Results are consistent with the hypothesis that mefloquine is a membrane-active drug . Mefloquine (Figure 1), 1s an antimalarial drug developed under the auspices of the United States Army Medical Research and Development Command to provide effective chemotherapy against the challenge of multiply-drug resistant Plasmodium falciparum (1,2) . Mefloquine suppresses the growth of blood schizonts of _P . falciparum regardless of their sensitivity or resistance to pyrimethamine or chloroquine . The absence of such cross-resistance among these multiply-drug resistant schizonts suggests that the mode of action of mefloquine differs from that of pyrimethamine, which is considered to suppress schizont development through the inhibition of dihydrofolic acid reductase (3), and chloroquine, for which several modes of action have been proposed (4) . Additional reports indicate that mefloquine does not bind to DNA (1,5) . Screening studies in this laboratory, however, have revealed mefloquine to possess bactericidal properties, and the present communication reports the bacteriological portion of an investigation intended to determine the mechanism(s) of action of mefloquine . The results not only indicate that mefloquine affects the function or integrity of _E . coli membranes, but also offers a viable hypothesis for the antimalarial action of mefloquine . MATERIALS AND METHODS Drugs and chemicals were the gifts of the Department of Medicinal Chemistry, WRAIR . Isotopically labelled compounds were purchased from the New England Nuclear Corp ., Boston, Mass . Nitrocellulose filters, type HA, 0 .45 um, were obtained from the Millipore Filter Corp ., Bedford, Mass ., and Instabray from the Yorktown Research Corp ., Hackensack, N .J . Turbidity and absorbance were determined in a Gilford Model 250 spectrophotometer equipped with a Gilford Model 6051 flatbed recorder (Gilford Instrument Labs ., Oberlin, Ohio) . Radioactivity was counted in a Searle Corp . (Des Plaines, Ill .) Mark III liquid scintillation counter . Escherichia coli AT-9, meth - , was grown in the glucose mineral salt medium M-9 (6), supplemented with 20 ug/ml 1-methionine . The effect of drug on cell 0024-3205/79/211857-08$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
185 8
Vol . 25, No . 21, 1979
Effects of Mefloquine on E . CoZi
viability was assayed by colony count on Brain-Heart Infusion Agar incubated at 37 C for 48 hours after serial dilution of culture aliquots and plating . The initial aliquots withdrawn from cultures were diluted one-hundred fold rather than tenfold with isotonic saline to reduce the drug concentration immediately to non-toxic levels . Bacterial uptake and incorporation of isotopically labelled compounds was measured by liquid scintillation counting of cells entrapped on nitrocellulose filters . Culture aliquots of 2 .00 ml were withdrawn for assay, quickly diluted with 2 .00 ml of distilled water or 10% trichloroacetic acid at 4 C, and filtered . Filters were washed with an additional 15 ml of wash solution, and dissolved in Instabray for counting . The concentrations and specific activities of the isotopically labelled ~ ounds were as follows : ( 14 C)-phenylalaiine : 0 .2 uCi /ml, 13 .2 mC /mmol ; co( C)-uracil : 0 .5 uCi /ml, 2 .8 mC i /mmol ; ( H)-deoxyadenosine : 1 .0 uC i /ml, 52 .6 mC i /mmol . Spheroplasts were prepared by the method of Under and Arndt (7), and were suspended in 0 .2 M sucrose containing 0 .5% NaCl and 1 .0 M Tris, pH 9 .0 . The method routinely converted more than 90% of the bacterial population into sphero plasts . Drugs were dissolved in the sucrose-buffer solution, or dimethylsulfoxide . Lysis was determined by recording turbidity at 420 nm . The method of Osborn and Munson (6) was employed to isolate E . coli membranes, while NADH oxidase activity was assayed as described by Osborn et al, (8) .
MEFLOOUINE WR 142,490
"NCI
CFa a-(2-Piper idyl)-2,8-bie(trifluoromethyl)-4-quinollnomathonol hydrochloride Figure 1 The structure of mefloquine .
Effects of Mefloquine on E . COZi
Vol . 25, No . 21, 1979
1859
RESULTS Figure 2 shows the influence of graded concentrations of mefloquine upon _E . coli AT-9 growing exponentially in methionine supplemented M-9 medium . A concentration of 2 .8 x 10- M mefloquine had no appreciable effect on growth, while 5 .6 x 10-5 M mefloquine completely inhibited growth, and eventually caused a decrease in turbidity . Additional experiments revealed 50% inhibition to occur at approximately 3 .6 x 10 -5 M mefloquine . Concentrations of drug in excess of 5 .6 x 10-? M caused more rapid and more extensive decreases in turbidity .
1.00 DRUG FREE
Q
0.10
0.010L
30
80
90
120
TIME IN MINUTES Figure 2 The influence of graded concentrations of mefloquine upon the growth and turbidity of subcultures of _E . coli AT-9 . Drug concentrations are listed to the right of the diagram . Figure 3 illustrates the bactericidal property of mefloquine . Upon rapid serial dilution and plating of bacteria subsequent to the addition of the drug to the respective cultures, there occurred an immediate decrease in viability of approximately 34%, with respect to the control culture, at a concentration of 1 x 10 -4 M mefloquine and a decrease in viability in excess of 99% at a concentration of 3 x 10-4+ M drug . Spectrophotometric analysis at 260 nm of exponentially growing cultures
186 0
Effects of Mefloquine on E . COU
Vol . 25, No . 21, 1979
109
W K V É rc W
a y J W U W
10 6
m Q 105
104
Figure 3 The influence of two concentrations of mefloquine upon the viability of E . coli AT-9 . Control :e ; containing drug : . . indicated immediate cessation of nucleic acid synthesis upon addition of bactericidal concentrations of mefloquine . Therefore experiments were undertaken to determine the cause of this inhibition, and the influence of mefloquine upon the uptake of macromolecular precursors . The results of typical experiments are illustrated by Figure 4 . Exponentially growing bacteria were simultaneously exposed to isotopically labelled macromolecular precursors, and a concentration of 1 x 10 -4 M mefloquine, and isotope incorporation measured as a function of time . Uptake and/or incorporation of precursors of protein, DNA, and RN~1 was suppressed within one minute of drug addition . Bacteria prelabelled for 2 / 2 generations and incubated for 4 hours in supplemented M-9 medium containing 10 -4 P1 mefloquine released approximately 50% of their DNA, 40% of their RNA, and 25% of their protein (not shown) . Release of macromolecular constituents may be attributable to either cell lysis, or macromolecular breakdown coupled with cell leakage . The uptake and release of ( 3 H)-2-methylalanine followed a similar pattern : mefloquine immediately suppressed uptake of this amino acid, while preloaded cells rapidly released the compound upon cellular exposure to mefloquine (not shown) . These and other observations suggested that mefloquine caused a rapid destruction or inactivation of a component of the _E . coli cell envelope . This envelope is composed of multilayered inner (cytoplasmic) and outer membranes . The effect of mefloquine upon the cytoplasmic membrane was tested by two methods : 1) spheroplast integrity in the presence of mefloquine, and 2) the influence of mefloquine upon membrane-based oxidation of NADH . Figure 5 illustrates the rapid
Effects of Mefloquine on E . CoZi
Vol . 25, No . 21, 1979
1861
TIME IN MINUTES F igur e 4 The uptake and/or incorporation of constituents of protein, DNA, and RNA by E . coli AT-9 growing exponentially, or exposed to a concentration of 1 x 10 -"4 M mefloquine . Left panel : ( 14 C)-phenylalanine ; center panel : ( 3 H)deoxyadenosine ; right panel : ( 14 C)-uracil . Drug-free :o ; containing drug : . .
Drug Addition
A420
.7
0-----
.6
0---- .
Polymywin
Mafloquine
.4
0
0 .5
1 .0 1 .5 Time in Minutes Figure 5
20
2 .5
The influence and specificity of mefloquine on spheroplasts of E . coli AT-9 . The turbidity of a 1 .8 ml aliquot of spheroplasts wits determined and recorded, 0 .2 ml of drug or drug solvent added, and the mixture was stirred manually for 10 seconds . Turbidity was recorded at 420 nm as a time function .
186 2
Effects of Mefloquine on E . Coli
Vol . 25, No . 21, 1979
lytic effect of mefloquine upon spheroplasts of E . coli , and for comparison, also presents the influence of polymyxin B, a membrane-active drug (9,10), previously shown to lyse spheroplasts (11) . Both drugs caused a decrease in turbidity indicative of spheroplast lysis, although the kinetics of turbidity reduction varied between mefloquine and polymyxin B, suggestive of different mechanisms of action . In contrast, pyrimethamine failed to lyse spheroplasts . Although not depicted, quinine, quinacrine, and chloroquine failed to lyse spheroplasts . Twenty minute incubation of spheroplasts with concentrations of 1 x 10 -4 M mefloquine or polymyxin B resulted in 39% and 41% lysis, respectively . The cytoplasmic membrane of E . coli is presently thought to contain more than eight enzymes, among them NADH oxidase (6) . After isolation of the total _E . coli membrane fraction (6,8), the influence of mefloquine on the oxidation of NADH was determined . Figure 6 depicts the influence of graded concentrations of mefloquine on the oxidation of NADH (8) . Increasing drug concentrations progressively reduced the rate of NADH oxidation, and the highest drug concentration employed, 1 .5 x 10-4 M, decreased the oxidation rate by 93% . It is noteworthy that enzyme inhibition by mefloquine occurred in a concentration range identical to its antibacterial effects .
INFLUENCE OF MEFLOQUINE ON MEMBRANE BOUND NADH OXIDASE 100
CONTROL .5X10-5M 2
80
NADH OXIDIZED
5X10- 5M
60 40
IXIO' 4 M
20 1 .5X10' 4 M 50
100 150 200 TIME IN SECONDS
250
Figure 6 The oxidation of NADH by E . coli membrane fractions in the absence of mefloquine, and in the presence of the designated concentrations of drug . Reactions were assayed spectrophotometrically at 340 nm as a function of time . The volume of the reaction mixture was 2 .0 ml, and the mixture contained 0 .05 M Tris-HC1, pH 7 .5, membrane fraction (protein content 66 ug/ml) (12), and 1 x 10 -4 M NADH . The order of addition was : buffer, membrane fraction, drug, and NADH . Permutation of the order of addition did not change the extent of inhibition . Total disappearance of the NADH absorbance at 340 nm connoted 100% oxidation .
Vol . 25, No . 21, 1979
Effects of Mefloquine on E. Coli
1863
DISCUSSION Mefloquine possesses bactericidal and lytic properties which have been primarily related to an interaction with a bacterial membrane or membranes . The drug, as a function of its concentration, causes an immediate loss of bacterial viability, rapidly suppresses uptake of macromolecular precursors, inhibits nuleic acid and protein synthesis, lyres spheroplasts, and inhibits an enzyme located in the cytoplasmic membrane . Inhibition of macromolecular synthesis, conversely, appears to be a secondary effect of the drug, since mefloquine does not bind to DNA (1,5), and only marginally suppressed synthesis of polyphenylalanine when tested in an amino acid polymerization system (unpublished results) . The influence of mefloquine superficially resembled that of polymyxin B, a membrane poison which also has been reported to inhibit bacterial respiratory processes .and components (13) . These effects are considered to be a consequence of drug-membrane interaction, but it should be noted that antimycin A, an inhibitor of the membrane bound b group of cytochromes (14), also lyres spheroplasts and inhibits respiration supported membrane transport of 2-methylalanine (15) . The molecular binding sites of these drugs have not been precisely defined, but the extent to which they influence biological processes and structures may be in part a consequence of their specific membrane binding loci . The relevance of the antibacterial effects of mefloquine to its antimelarial action is pertinent . Morphological studies (2) have indicated that mefloquine damages permeability barriers or controls in plasmodia . 'Cytoplasmic' re gions of trophozoites of _P . falciparum, and, in addition, of merozoites of _P . vivax , swelled upon administration of mefloquine to infected monkeys (2) . Ultimately all internal structure disappeared, a result perhaps analogous to lysis of bacteria and spheroplasts by mefloquine . Both antimycin A and mefloquine cause the disappearance, in P . ber hei, of the energy dependent aggregation of pigment by chloroquine (1,16,17,18 . It is of interest that the enzyme NADH oxidase has been found (19) on the outer membrane of _P . lophurae . However, the significance of mefloquine inhibition of NADH oxidase must await not only knowledge of the physiological role of this enzyme, but also an analysis of the influence of mefloquine on additional membrane-bound enzymes . Partition studies have shown uninfected erythrocytes to concentrate mefloquine from plasma by a factor of 1 .7 (20), while preliminary studies in this laboratory suggest parasitized erythrocytes to additionally concentrate meflo quine by a factor in excess of 1 .5 . The mefloquine concentration in the plasma of a human volunteer was observed (21) to spike at 0 .85 ug/ml for a single 500 mg dose, and therefore the concentration of mefloquine in parasitized erythrocytes may well be in excess of 5 .7 x 10-6 M, one sixth the concentration required to achieve 50% inhibition of bacterial growth . It should be noted that mefloquine doses up to 1 .5 gms (22) have been employed with human volunteers . In addition, within the infected erythrocyte, 49% of the mefloquine was found to be membrane associated (20) . Thus pharmacological studies tend to support the hypothesis suggested by the present results . ACKNOWLEDGMENTS The authors wish to thank Drs . Brian Hansen and B .P . Doctor for their interest and advice, Dr . Melvin Heiffer and his colleagues of the Department of Medicinal Chemistry, the Walter Reed Army Institute of Research, for their interest and generosity, and Miss Janet Davies and Mr . Clarence Emery for their expert technical assistance .
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Vol . 25, No . 21, 1979
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 .
W . PETERS, R.E . HOWELLS, J. PORTUS, B .L . ROBINSON, S . THOMAS, AND D .C . WARHURST, Ann . Trop . Med . Parasit . 71, 407-418 (1977) . L .H . SCHMIDT, R. CROSBY, J. RASCO, AND D . VAUGHN, Antimicro. Ag . and Chem . 13, 1011-1030 (1978) . R . FERONE, J. BURCHALL, and G. HITCHINGS, J . Mol . Pharm . _5, 49-59 (1969) . F .E . 11A11N, Antibiotics _III, 58-78, Springer-Verlag, Berlin, Heidelberg, New York (1975) . M .W . DAVIDSON, B .G . GRIGGS, JR ., D .W . BOYKIN, AND W.D . WILSON, Nature 254, 632-634 (1975) . M .J . OSBORN AND R . MUNSON, Methods in Enzymology XXXI , Part A, 642-653 Acad . Press, New York, San Fransisco, London, (1974) . N.D . ZINDER AND W .F . ARNDT, Proc . Nat . Acad . Sci . U.S .A . _42, 586-590 (1956) . M .J . OSBORN, J.E . GANDER, E . PARISI, AND J . CARSON, J. Biol . Chem . 247, 3962-3972 (1972) . B .A . NEWTON, J. Gen . Microbiol . 9, 54-64 (1953) . B .A . NEWTON, J. Gen . Microbiol . _10, 491-499 (1954) . 11 . TEUBER and J . BADER, FENS Letters _1, 75-78 (1977) . O .H . LOWRY, N.J . ROSEBROUGH, A.L . FARR, and R.J . RANDALL, J. Biol . Chem . 193, 265-1,75 (1951) . P1 . TEUBER, Arch . Microbioi. 100, 131-144 (1974) . R.E . BASFORD, H .D . TISDALE, J. L . GLENN, and D .E . GREEN, Biochim. Biophys . Acta 24, 107-115 (1957) . R. MARQUIS, J. Bacteriol . 89, 1453-1459 (1965) . D .C . WARHURST, B .L . ROBINSON, R .E . HOWELLS, and W . PETERS, Life Sciences _10, 761-771 (1971) . C .A . H(?1EWOOD, D.C . WARHURST, W . PETERS, AND V .C . BAGGALEY, Proc . Helm . Soc . Wash ., 382-386 (1972) . A. EINHEBER, D.M . PALMER, AND M . AIKAWA, Exp. Parasit . _40, 52-61 (1976) . S.G . LANGRETH, Bull . WHO 55, 171-178 (1977) . J .Y . MU, Z .H . ISRAILI, ANDP.G . DAYTON, Drug Met . and Disp . _3, 198-210 (1975) . J.M . GRINDEL, P .F . TILTON, AND R.D . SHAFFER, J. Pharm. Sci . _66, 834-837 (1977) . C .M . TRENHOLME, R.L . WILLIAMS, R .E . DESJARDINS, H. FRISCHER, P.E . CARSON, K.H . RIECKMANN, C .G . CANFIELD, Science 190, 792-794 (1975) .
Pergamon Press
THE EFFECTS OF MEFLOQUINE ON ESCHERICHIA COLI Ruth E . Brown, Frank A . Stancato, and Alan D . Wolfe Department of Biological Chemistry, Division of Biochemistry, Walter Reed Army Institute of Research, Washington, D .C . 20012 (Received in final form October 17, 1979) SUMMARY Mefloquine, a quinoline-4-methanol antimalarial drug, also possesses bactericidal activity . Mefloquine causes rapid loss of bacterial viability, cell and spheroplast lysis, cessation of macromolecular synthesis, release of macromolecular constituents, and inhibition of the oxidation of NADH by isolated _E . coli membranes . Results are consistent with the hypothesis that mefloquine is a membrane-active drug . Mefloquine (Figure 1), 1s an antimalarial drug developed under the auspices of the United States Army Medical Research and Development Command to provide effective chemotherapy against the challenge of multiply-drug resistant Plasmodium falciparum (1,2) . Mefloquine suppresses the growth of blood schizonts of _P . falciparum regardless of their sensitivity or resistance to pyrimethamine or chloroquine . The absence of such cross-resistance among these multiply-drug resistant schizonts suggests that the mode of action of mefloquine differs from that of pyrimethamine, which is considered to suppress schizont development through the inhibition of dihydrofolic acid reductase (3), and chloroquine, for which several modes of action have been proposed (4) . Additional reports indicate that mefloquine does not bind to DNA (1,5) . Screening studies in this laboratory, however, have revealed mefloquine to possess bactericidal properties, and the present communication reports the bacteriological portion of an investigation intended to determine the mechanism(s) of action of mefloquine . The results not only indicate that mefloquine affects the function or integrity of _E . coli membranes, but also offers a viable hypothesis for the antimalarial action of mefloquine . MATERIALS AND METHODS Drugs and chemicals were the gifts of the Department of Medicinal Chemistry, WRAIR . Isotopically labelled compounds were purchased from the New England Nuclear Corp ., Boston, Mass . Nitrocellulose filters, type HA, 0 .45 um, were obtained from the Millipore Filter Corp ., Bedford, Mass ., and Instabray from the Yorktown Research Corp ., Hackensack, N .J . Turbidity and absorbance were determined in a Gilford Model 250 spectrophotometer equipped with a Gilford Model 6051 flatbed recorder (Gilford Instrument Labs ., Oberlin, Ohio) . Radioactivity was counted in a Searle Corp . (Des Plaines, Ill .) Mark III liquid scintillation counter . Escherichia coli AT-9, meth - , was grown in the glucose mineral salt medium M-9 (6), supplemented with 20 ug/ml 1-methionine . The effect of drug on cell 0024-3205/79/211857-08$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
185 8
Vol . 25, No . 21, 1979
Effects of Mefloquine on E . CoZi
viability was assayed by colony count on Brain-Heart Infusion Agar incubated at 37 C for 48 hours after serial dilution of culture aliquots and plating . The initial aliquots withdrawn from cultures were diluted one-hundred fold rather than tenfold with isotonic saline to reduce the drug concentration immediately to non-toxic levels . Bacterial uptake and incorporation of isotopically labelled compounds was measured by liquid scintillation counting of cells entrapped on nitrocellulose filters . Culture aliquots of 2 .00 ml were withdrawn for assay, quickly diluted with 2 .00 ml of distilled water or 10% trichloroacetic acid at 4 C, and filtered . Filters were washed with an additional 15 ml of wash solution, and dissolved in Instabray for counting . The concentrations and specific activities of the isotopically labelled ~ ounds were as follows : ( 14 C)-phenylalaiine : 0 .2 uCi /ml, 13 .2 mC /mmol ; co( C)-uracil : 0 .5 uCi /ml, 2 .8 mC i /mmol ; ( H)-deoxyadenosine : 1 .0 uC i /ml, 52 .6 mC i /mmol . Spheroplasts were prepared by the method of Under and Arndt (7), and were suspended in 0 .2 M sucrose containing 0 .5% NaCl and 1 .0 M Tris, pH 9 .0 . The method routinely converted more than 90% of the bacterial population into sphero plasts . Drugs were dissolved in the sucrose-buffer solution, or dimethylsulfoxide . Lysis was determined by recording turbidity at 420 nm . The method of Osborn and Munson (6) was employed to isolate E . coli membranes, while NADH oxidase activity was assayed as described by Osborn et al, (8) .
MEFLOOUINE WR 142,490
"NCI
CFa a-(2-Piper idyl)-2,8-bie(trifluoromethyl)-4-quinollnomathonol hydrochloride Figure 1 The structure of mefloquine .
Effects of Mefloquine on E . COZi
Vol . 25, No . 21, 1979
1859
RESULTS Figure 2 shows the influence of graded concentrations of mefloquine upon _E . coli AT-9 growing exponentially in methionine supplemented M-9 medium . A concentration of 2 .8 x 10- M mefloquine had no appreciable effect on growth, while 5 .6 x 10-5 M mefloquine completely inhibited growth, and eventually caused a decrease in turbidity . Additional experiments revealed 50% inhibition to occur at approximately 3 .6 x 10 -5 M mefloquine . Concentrations of drug in excess of 5 .6 x 10-? M caused more rapid and more extensive decreases in turbidity .
1.00 DRUG FREE
Q
0.10
0.010L
30
80
90
120
TIME IN MINUTES Figure 2 The influence of graded concentrations of mefloquine upon the growth and turbidity of subcultures of _E . coli AT-9 . Drug concentrations are listed to the right of the diagram . Figure 3 illustrates the bactericidal property of mefloquine . Upon rapid serial dilution and plating of bacteria subsequent to the addition of the drug to the respective cultures, there occurred an immediate decrease in viability of approximately 34%, with respect to the control culture, at a concentration of 1 x 10 -4 M mefloquine and a decrease in viability in excess of 99% at a concentration of 3 x 10-4+ M drug . Spectrophotometric analysis at 260 nm of exponentially growing cultures
186 0
Effects of Mefloquine on E . COU
Vol . 25, No . 21, 1979
109
W K V É rc W
a y J W U W
10 6
m Q 105
104
Figure 3 The influence of two concentrations of mefloquine upon the viability of E . coli AT-9 . Control :e ; containing drug : . . indicated immediate cessation of nucleic acid synthesis upon addition of bactericidal concentrations of mefloquine . Therefore experiments were undertaken to determine the cause of this inhibition, and the influence of mefloquine upon the uptake of macromolecular precursors . The results of typical experiments are illustrated by Figure 4 . Exponentially growing bacteria were simultaneously exposed to isotopically labelled macromolecular precursors, and a concentration of 1 x 10 -4 M mefloquine, and isotope incorporation measured as a function of time . Uptake and/or incorporation of precursors of protein, DNA, and RN~1 was suppressed within one minute of drug addition . Bacteria prelabelled for 2 / 2 generations and incubated for 4 hours in supplemented M-9 medium containing 10 -4 P1 mefloquine released approximately 50% of their DNA, 40% of their RNA, and 25% of their protein (not shown) . Release of macromolecular constituents may be attributable to either cell lysis, or macromolecular breakdown coupled with cell leakage . The uptake and release of ( 3 H)-2-methylalanine followed a similar pattern : mefloquine immediately suppressed uptake of this amino acid, while preloaded cells rapidly released the compound upon cellular exposure to mefloquine (not shown) . These and other observations suggested that mefloquine caused a rapid destruction or inactivation of a component of the _E . coli cell envelope . This envelope is composed of multilayered inner (cytoplasmic) and outer membranes . The effect of mefloquine upon the cytoplasmic membrane was tested by two methods : 1) spheroplast integrity in the presence of mefloquine, and 2) the influence of mefloquine upon membrane-based oxidation of NADH . Figure 5 illustrates the rapid
Effects of Mefloquine on E . CoZi
Vol . 25, No . 21, 1979
1861
TIME IN MINUTES F igur e 4 The uptake and/or incorporation of constituents of protein, DNA, and RNA by E . coli AT-9 growing exponentially, or exposed to a concentration of 1 x 10 -"4 M mefloquine . Left panel : ( 14 C)-phenylalanine ; center panel : ( 3 H)deoxyadenosine ; right panel : ( 14 C)-uracil . Drug-free :o ; containing drug : . .
Drug Addition
A420
.7
0-----
.6
0---- .
Polymywin
Mafloquine
.4
0
0 .5
1 .0 1 .5 Time in Minutes Figure 5
20
2 .5
The influence and specificity of mefloquine on spheroplasts of E . coli AT-9 . The turbidity of a 1 .8 ml aliquot of spheroplasts wits determined and recorded, 0 .2 ml of drug or drug solvent added, and the mixture was stirred manually for 10 seconds . Turbidity was recorded at 420 nm as a time function .
186 2
Effects of Mefloquine on E . Coli
Vol . 25, No . 21, 1979
lytic effect of mefloquine upon spheroplasts of E . coli , and for comparison, also presents the influence of polymyxin B, a membrane-active drug (9,10), previously shown to lyse spheroplasts (11) . Both drugs caused a decrease in turbidity indicative of spheroplast lysis, although the kinetics of turbidity reduction varied between mefloquine and polymyxin B, suggestive of different mechanisms of action . In contrast, pyrimethamine failed to lyse spheroplasts . Although not depicted, quinine, quinacrine, and chloroquine failed to lyse spheroplasts . Twenty minute incubation of spheroplasts with concentrations of 1 x 10 -4 M mefloquine or polymyxin B resulted in 39% and 41% lysis, respectively . The cytoplasmic membrane of E . coli is presently thought to contain more than eight enzymes, among them NADH oxidase (6) . After isolation of the total _E . coli membrane fraction (6,8), the influence of mefloquine on the oxidation of NADH was determined . Figure 6 depicts the influence of graded concentrations of mefloquine on the oxidation of NADH (8) . Increasing drug concentrations progressively reduced the rate of NADH oxidation, and the highest drug concentration employed, 1 .5 x 10-4 M, decreased the oxidation rate by 93% . It is noteworthy that enzyme inhibition by mefloquine occurred in a concentration range identical to its antibacterial effects .
INFLUENCE OF MEFLOQUINE ON MEMBRANE BOUND NADH OXIDASE 100
CONTROL .5X10-5M 2
80
NADH OXIDIZED
5X10- 5M
60 40
IXIO' 4 M
20 1 .5X10' 4 M 50
100 150 200 TIME IN SECONDS
250
Figure 6 The oxidation of NADH by E . coli membrane fractions in the absence of mefloquine, and in the presence of the designated concentrations of drug . Reactions were assayed spectrophotometrically at 340 nm as a function of time . The volume of the reaction mixture was 2 .0 ml, and the mixture contained 0 .05 M Tris-HC1, pH 7 .5, membrane fraction (protein content 66 ug/ml) (12), and 1 x 10 -4 M NADH . The order of addition was : buffer, membrane fraction, drug, and NADH . Permutation of the order of addition did not change the extent of inhibition . Total disappearance of the NADH absorbance at 340 nm connoted 100% oxidation .
Vol . 25, No . 21, 1979
Effects of Mefloquine on E. Coli
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DISCUSSION Mefloquine possesses bactericidal and lytic properties which have been primarily related to an interaction with a bacterial membrane or membranes . The drug, as a function of its concentration, causes an immediate loss of bacterial viability, rapidly suppresses uptake of macromolecular precursors, inhibits nuleic acid and protein synthesis, lyres spheroplasts, and inhibits an enzyme located in the cytoplasmic membrane . Inhibition of macromolecular synthesis, conversely, appears to be a secondary effect of the drug, since mefloquine does not bind to DNA (1,5), and only marginally suppressed synthesis of polyphenylalanine when tested in an amino acid polymerization system (unpublished results) . The influence of mefloquine superficially resembled that of polymyxin B, a membrane poison which also has been reported to inhibit bacterial respiratory processes .and components (13) . These effects are considered to be a consequence of drug-membrane interaction, but it should be noted that antimycin A, an inhibitor of the membrane bound b group of cytochromes (14), also lyres spheroplasts and inhibits respiration supported membrane transport of 2-methylalanine (15) . The molecular binding sites of these drugs have not been precisely defined, but the extent to which they influence biological processes and structures may be in part a consequence of their specific membrane binding loci . The relevance of the antibacterial effects of mefloquine to its antimelarial action is pertinent . Morphological studies (2) have indicated that mefloquine damages permeability barriers or controls in plasmodia . 'Cytoplasmic' re gions of trophozoites of _P . falciparum, and, in addition, of merozoites of _P . vivax , swelled upon administration of mefloquine to infected monkeys (2) . Ultimately all internal structure disappeared, a result perhaps analogous to lysis of bacteria and spheroplasts by mefloquine . Both antimycin A and mefloquine cause the disappearance, in P . ber hei, of the energy dependent aggregation of pigment by chloroquine (1,16,17,18 . It is of interest that the enzyme NADH oxidase has been found (19) on the outer membrane of _P . lophurae . However, the significance of mefloquine inhibition of NADH oxidase must await not only knowledge of the physiological role of this enzyme, but also an analysis of the influence of mefloquine on additional membrane-bound enzymes . Partition studies have shown uninfected erythrocytes to concentrate mefloquine from plasma by a factor of 1 .7 (20), while preliminary studies in this laboratory suggest parasitized erythrocytes to additionally concentrate meflo quine by a factor in excess of 1 .5 . The mefloquine concentration in the plasma of a human volunteer was observed (21) to spike at 0 .85 ug/ml for a single 500 mg dose, and therefore the concentration of mefloquine in parasitized erythrocytes may well be in excess of 5 .7 x 10-6 M, one sixth the concentration required to achieve 50% inhibition of bacterial growth . It should be noted that mefloquine doses up to 1 .5 gms (22) have been employed with human volunteers . In addition, within the infected erythrocyte, 49% of the mefloquine was found to be membrane associated (20) . Thus pharmacological studies tend to support the hypothesis suggested by the present results . ACKNOWLEDGMENTS The authors wish to thank Drs . Brian Hansen and B .P . Doctor for their interest and advice, Dr . Melvin Heiffer and his colleagues of the Department of Medicinal Chemistry, the Walter Reed Army Institute of Research, for their interest and generosity, and Miss Janet Davies and Mr . Clarence Emery for their expert technical assistance .
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