Molecular and Biochemical Parasitology, 53 (1992) 223-232 © 1992 Elsevier Science Publishers B.V. All tights reserved. / 0166-6851/92/$05.00
223
MOLBIO 01765
The interaction of arsenical drugs with dihydrolipoamide and dihydrolipoamide dehydrogenase from arsenical resistant and sensitive strains of Trypanosoma brucei brucei Alan H. Fairlamb, Keith Smith and Karl J. H u n t e r Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, London, UK (Recieved 14 January 1992; accepted 25 February 1992)
D,L-dihydrolipoamide and D.L-dihydrolipoic acid react to form stable complexes with melarsen oxide with association constants of 5.47 x 10 9 and 4.51 x 10 9 M " J, respectively. These complexes possess 6-membered cyclic dithioarsenite tings which are 10-fold less stable than the 5-membered rings found in the trypanocidal drugs melarsoprol and trimelarsen, but 500fold more stable than the 25-membered macrocyclic ring formed between melarsen oxide and dihydrotrypanothione. L-Lipoic acid concentrations in arsenical sensitive and resistant cloned lines of Trypanosoma brucei brucei have been determined by bioassay using a mutant of Escherichia coli auxotrophic for lipoate. The arsenical resistant strain was found to contain significantly less lipoic acid than the sensitive strain (19.2 + 4.3 and 9.7 + 2.9 pmol (10~ cells)- i, respectively). The activity of the plasma membrane-associated dihydrolipoamide dehydrogenase was found to be slightly, but significantly increased in the arsenical resistant strain (34.7 + 1.4 and 47.8 + 3.7 mU mg- i, respectively). However, the Km for dihydrolipoamide and the inactivation kinetics with melarsen oxide were not significantly different between these strains. Estimates of the ratio of substrate to enzyme are of the order of 12:1 and 6:1 for arsenical sensitive and resistant strains, respectively, suggesting that these components are likely to be intimately associated with each other in the plasma membrane. These findings implicate lipoic acid, but not dihydrolipoamide dehydrogenase, in resistance to arsenical drugs, either through the mechanism of uptake or as the final target of these drugs. Key words: Arsenical drugs; Dihydrolipoamide dehydrogenase; Lipoic acid; Drug resistance; Drug effects; Trypanosoma brucei brucei
Introduction
In 1909 Paul Ehrlich first proposed that trivalent arsenical compounds exert their lethal effects against trypanosomes and spirochaetes by chemical reaction with sulphydryl groups in these pathogenic organisms. However, the first definitive evidence in favour of this theory was obtained in 1923 by Voegtlin and co-workers [1]. Ehrlich's observation that arsenicals had a propensity to combine more avidly with vicinal dithiols rather than simple monothiols ultiCorrespondence address: Alan H.Fairlamb, Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London WCIE 7HT, UK.
mately led to the development of 2,3-dimercaptopropanol (British Anti-Lewisite) as an antidote to the arsenical nerve gases [2]. Subsequently, Friedheim used this compound in the development of melarsoprol (Mel B) as a better tolerated derivative of melarsen oxide [3]. Although the mode of action of the trivalent arsenical drugs such as melarsen oxide and melarsoprol remains to be determined, one primary effect of exposing trypanosomes to these compounds is the formation of a stable adduct (Mel T) [4] between melarsen oxide and the unique intracellular dithiol, dihydrotrypanothione [5]. The other dithiol found in most organisms is dihydrolipoamide, which, along with dihydro-
224
lipoamide dehydrogenase, forms part of the ~oxo-decarboxylase complexes lbr pyruvate and ketoglutarate in mammalian cells. Both of these enzyme complexes are absent from long-slender bloodstream forms of Trypanosoma hrucei [6,7] and therefore dihydrolipoamide dehydrogenase has never been seriously considered as the possible target for arsenical drugs. However, recent reports have shown that these organisms do in fact contain significant amounts of dihydrolipoamide dehydrogenase, which has an unusual localization in the plasma membrane [8]. A similar membrane location has been reported for a second form of dihydrolipoamide dehydrogenase in Escherichia coli [9] where it has been suggested that this enzyme and its substrate may be involved in the transport of maltose into the cell [10]. Given these observations we sought to determine the stability of arsenical drugs with dihydrolipoyl compounds in order to examine the possibility that this metabolite might be involved in arsenical transport and to assess whether arsenicals, free or complexed to lipoamide could inhibit the dihydrolipoamide dehydrogenase activity in arsenical sensitive and resistant trypanosomes.
photometrically by titration with 5.5'dithiobis(2-nitrobenzoic acid) in 1.0 M Tris buffer, pH 8.0, using an extinction coefficient of 14.14 mM i, cm -I at 412 nm [13]. pAminophenyldichioroarsine (APDCA) was synthesised by a standard procedure [14]. The arsenical compounds melarsen oxide (Mel O) and melarsoprol (Arsobal, Mel B), were kind gifts from Specia, Rhone-Poulenc.
Determination of association constants ./br dihydrolipoic acid and dihydrolipoamide with melarsen oxide. Varying amounts ofdihydrolipoic acid or dihydrolipoamide were mixed with either melarsen oxide or melarsoprol (Mel B) (10 mM stock solutions in N.N'-dimethylformamide) to give a final concentration of 1 mM arsenical (20% (v/v) aqueous propylene glycol was used as diluent to maintain solubility of all the components in the mixture). After incubation at room temperature for 15 min, samples were separated by HPLC on a Beckman ODS 5p ion-pair column (4.6 mm x 25 cm) essentially as described previously only using solvent C (50% (v/v) npropanol in 0.25% (Li +) camphor sulphonate, pH 2.64) in place of the previously described solvent B [4]. Melaminophenylarsenicals and their adducts were detected by their absorbance at 280 nm.
Materials and Methods
Organisms. Bloodstream Trypanosoma brucei ($427.118) was maintained in rats and isolated and purified by standard procedures [11]. A cloned arsenical resistant line designated $427 cRU15 derived from this clone (see accompanying paper) was similarly maintained. Both clones were stored at - 7 0 ° C as stabilates in 10% (v/v) glycerol. Reagents. D,L-Dihydrolipoic acid and D,Llipoamide were purchased from Sigma Chemic a i C o . D,L-dihydrolipoamide was prepared by reduction of D,L-lipoamide with sodium borohydride [12] and after recrystallization, stored at -20:~C. Immediately before use, concentrations of dithiols were determined spectro-
Lipoic acid content. Total lipoic acid was determined after acid hydrolysis by bio-assay with an E. coli strain auxotrophic for lipoate [15]. Briefly, trypanosome extracts were prepared by suspending 3 × 10s cells in 0.3 ml of 6 N HCI containing 3 mg bovine serum albumin. Before heating, the solution was deoxygenated by passing a stream of nitrogen over the surface of the liquid for 20 min. Samples were hydrolysed for 2 h at 120°C, taken to dryness under a stream of nitrogen, redissolved in 200 mM (K ~) phosphate buffer, pH 7.0 and filtered to remove suspended solids. Samples were then assayed as in [15] with hydrolysed bovine serum albumin as blank and lipoic acid hydrolysed in bovine serum albumin as standard.
225
Enzyme and protein assays. Trypanosome extracts were prepared by uitrasonication in 50 mM (K ~) phosphate buffer, 2 mM EDTA, pH 7.0, followed by brief centrifugation (10000 rev./min, 3 min) to remove unbroken cells. Dihydrolipoamide dehydrogenase activity was determined spectrophotometrically at 25°C in 50 mM ( K * ) phosphate buffer/2 mM EDTA, pH 7.0 containing 1 mM N A D and 0.4 mM D,L-dihydrolipoamide by monitoring the increase in absorbance at 340 nm [16]. For the inactivation studies with arsenicals, trypanosome extracts were incubated in assay buffer at 4°C with 200 /~M melarsen oxide or paminophenyldichloroarsine in the presence and absence of N A D H . Aliquots were removed at intervals and residual enzyme activity determined by 50-fold dilution into the above assay mixture. Protein was determined using Coomassie blue reagent (Bio-Rad) [:7].
Results
Interaction of lipoyl derivatives with arsenical drugs. The reduced forms of lipoic acid and lipoamide react to form stable complexes that can be readily separated by reversed phase HPLC (Fig. 1). When an equimolar amount of D,L-dihydrolipoamide is mixed with melarsen oxide, all of the arsenical (Fig. I A) is converted into 2 new derivatives with retention times of 27.5 and 35.7 min (Fig. IC). A similar result is obtained on mixing equimolar D,L-dihydrolipoic acid and melarsen oxide, forming derivatives with retention times of 32.5 and 38.9 min (not shown). The ratio of the areas of the 2 new peaks is similar for both lipoyl derivatives (4.00 + 0.37 (N = 6) and 3.96 + 0.46 (N--6) for dihydrolipoamide and dihydrolipoic acid, respectively) and presumably represent the Dand L-diastereoisomers of the lipoyi-arsenical complexes. To test whether the stability constants for these adducts were similar to that of melarso-
T
0"1 AU
0'1 AU
1
1 J
A
J
B
E _
L
A
r
i
0
25
50
0
TIME, rain
. J f
j
25
TIME,
50 min
Fig. 1. Interactionof melarsenoxideand melarsoprolwith dihydrolipoamide.Details of the HPLC techniquesare describedin the methods. Trace A: melarsen oxide (10 nmol); trace B: dihydrolipoamide(10 nmol); trace C: melarsen oxide plus dihydrolipoamide (10 nmol of each); trace D: melarsoprol (10 nmol); trace E: dihydrolipoamide (10 nmol); trace F: melarsoprol plus dihydrolipoamide(10 nmol of each).
226
prol, equimolar mixtures of melarsoprol (Fig. I D) and dihydrolipoamide (Fig. I E) were incubated for 15 rain before analysis by HPLC. The mixture (Fig. IF) shows a decrease in the peak area for melarsoprol (the overlapping peaks of the diastereoisomers elute at 21.2 and 22.2 min) and is associated with the formation of the lipoamide-arsenical adducts identified in Fig. IC. Similar results were obtained with dihydrolipoic acid (not shown) indicating that the stability constants for the lipoyl-derivatives with melarsen oxide must be of the same order of magnitude as melarsoprol. In contrast, addition of equimofar dihydrotrypanothione to melarsoprol resuits in no adduct formation, consistent with the > 1000-fold difference in stability complexes previously reported [4]. In the case of the monothiols, glutathione and cysteine, no new adducts could be separated by HPLC even when added in 20-fold excess (not shown), consistent with previous findings [4]. In order to determine the stability complexes of the lipoyl adducts, melarsoprol was mixed with varying amounts of dihydrolipoamide or dihydrolipoic acid and the mixture allowed to come to a new equilibrium:
[MEL LI KLIP --
[MEL O][LIP] [MEL B] a n d KBAI. =
[MEL O][BAL] Therefore, eliminating [MEL O]: gl.IP
[MEL L][BAL]
KBAL
[MEL B][LIP]
Since the amount of MEL L complex formed results in an equivalent amount of BAL release, then [MEL L] = [BAL]. Substituting for [BAL] and rearranging: KI.IP
[MEL L] 2 -
[MEL B][LIP] KBAI.
I
I
I
12 --
I O ~
M E L O + LIP + B A L ~ - - - M E L L + M E L B
8 where MEL O = melarsen oxide; LIP = dihydrolipoate or dihydrolipoamide; BAL = 2,3-dimercaptopropanol and MEL L and MEL B represent the adducts with lipoate or lipoamide and dimercaptopropanol, respectively. Samples were analyzed by HPLC and the concentration of each arsenical component in the mixture determined from the response factors obtained from standards. The equilibrium concentration of dihydrolipoate or dihydrolipoamide, [LIP], can be calculated by subtracting the amount of lipoyl-arsenical adduct, [MEL L], formed from the initial amount of dihydrolipoate added. The relative affinity constant for dihydrolipoamide or dihydrolipoate (KLxp/KBAL)can now be calculated from the following equations:
4
0
l 0
i 8O
I
I 160
[MEL e] [uP] Fig. 2. Determination of association constants for dihydrolipoamide and dihydrolipoic acid adducts with melarsen oxide (KLw) relative to melarsoprol (KaAL)- The derivation of the equations and the method used are described in the text. The concentrations of reactants and products are given as nmol applied to the HPLC system. Open circles: dihydrolipoamide; closed circles: dihydrolipoic acid. The slopes of each line are the ratios of association constants, KL,,/KBAL (0.068 and 0.057 for dihydrolipoamide and dihydrolipoic acid, respectively).
227 TABLE I Association constants of melaminophenylarsenical derivatives Dithiol
Adduct
Atoms in ring
Ka ( x 107M- i)
Dimercaptopropanol a Dimercaptosuccinate a Dihydrolipoamide h Dihydrolipoic acid b Dihydrotrypanothione a Glutathione c Cysteine ¢
MEL B (melarsoprol) MEL W (trimelarsen) MEL L,CONH, MEL LcooH MELT
5 5 6 6 25 -
7930 4500 547 451 1.21 unstable unstable
aDetermined by titration of free thiol in equilibrium mixtures, data from ref. 4. bDetermined by exchange between test thiol and melarsoprol by HPLC. CAdduct formation can be demonstrated by UV spectrophometry, but the products are not stable enough for separation by HPLC.
Thus, plotting [MEL L]2 against [MEL B][LIP] should yield a straight line with a slope equal to KLIp/KBA L. This relationship was found to hold for both dihydrolipoamide and dihydrolipoate (Fig. 2) giving slopes of 0.068 and 0.057, respectively. Since the association constant for melarsoprol has previously been determined to be 7.93 x 101° M - l [4], the absolute stability constants can be calculated. Table I lists the mean stability complexes for the D,L-lipoyl arsenicals which are both about 15-fold less stable than melarsoprol, but about 500-fold more stable than that of melarsen oxide with dihydrotrypanothione. Lipoic acid and dihydrolipoamide dehydrogenase content of T. brucei. Total lipoic acid content was measured in acid hydrolysates of arsenical sensitive and resistant strains of T. brucei by bioassay using an E. coli strain auxotrophic for lipoic acid. In three separate trypanosome preparations, the arsenical sensi-
tive cells contained significantly more lipoic acid than the resistant cell line (Table II). Although the mean L-lipoate concentration showed some variation between experiments (up to 2-fold), within experiments there was a consistent and highly significant 50% reduction in lipoate concentration in resistant cells. The difference in overall mean concentrations of L-lipoate in three separate experiments was also highly significant (19.22 versus 9.71 pmol (10 s cells) -I for sensitive and resistant cells, respectively). These results indicate that lipoic acid represents a minor fraction of the total intracellular low molecular weight thiols since the total glutathione (i.e., free and conjugated to spermidine) is at least 100-fold greater (2.04 nmol (10 s cells)-1) [20]. Paradoxically, as reported in the accompanying paper [20] the dihydrolipoamide dehydrogenase content was found to be significantly increased in the arsenical resistant strain (34.7 versus 47.8 mU mg -I for
TABLE II Lipoic acid content of arsenical sensitive and resistant T. brucei Sensitive
Resistant
Ratio (R/S)
pb
(pmol (lO s cells)- i)~ Expt. 1 Expt. 2 Expt. 3
14.85 ± 0.10 c 23.59 + 0.78 19.13 ± 2.72
6.50 ± 1.75 12.43 + 0.68 10.00 + 0.29
0.44 0.53 0.52
223
MOLBIO 01765
The interaction of arsenical drugs with dihydrolipoamide and dihydrolipoamide dehydrogenase from arsenical resistant and sensitive strains of Trypanosoma brucei brucei Alan H. Fairlamb, Keith Smith and Karl J. H u n t e r Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, London, UK (Recieved 14 January 1992; accepted 25 February 1992)
D,L-dihydrolipoamide and D.L-dihydrolipoic acid react to form stable complexes with melarsen oxide with association constants of 5.47 x 10 9 and 4.51 x 10 9 M " J, respectively. These complexes possess 6-membered cyclic dithioarsenite tings which are 10-fold less stable than the 5-membered rings found in the trypanocidal drugs melarsoprol and trimelarsen, but 500fold more stable than the 25-membered macrocyclic ring formed between melarsen oxide and dihydrotrypanothione. L-Lipoic acid concentrations in arsenical sensitive and resistant cloned lines of Trypanosoma brucei brucei have been determined by bioassay using a mutant of Escherichia coli auxotrophic for lipoate. The arsenical resistant strain was found to contain significantly less lipoic acid than the sensitive strain (19.2 + 4.3 and 9.7 + 2.9 pmol (10~ cells)- i, respectively). The activity of the plasma membrane-associated dihydrolipoamide dehydrogenase was found to be slightly, but significantly increased in the arsenical resistant strain (34.7 + 1.4 and 47.8 + 3.7 mU mg- i, respectively). However, the Km for dihydrolipoamide and the inactivation kinetics with melarsen oxide were not significantly different between these strains. Estimates of the ratio of substrate to enzyme are of the order of 12:1 and 6:1 for arsenical sensitive and resistant strains, respectively, suggesting that these components are likely to be intimately associated with each other in the plasma membrane. These findings implicate lipoic acid, but not dihydrolipoamide dehydrogenase, in resistance to arsenical drugs, either through the mechanism of uptake or as the final target of these drugs. Key words: Arsenical drugs; Dihydrolipoamide dehydrogenase; Lipoic acid; Drug resistance; Drug effects; Trypanosoma brucei brucei
Introduction
In 1909 Paul Ehrlich first proposed that trivalent arsenical compounds exert their lethal effects against trypanosomes and spirochaetes by chemical reaction with sulphydryl groups in these pathogenic organisms. However, the first definitive evidence in favour of this theory was obtained in 1923 by Voegtlin and co-workers [1]. Ehrlich's observation that arsenicals had a propensity to combine more avidly with vicinal dithiols rather than simple monothiols ultiCorrespondence address: Alan H.Fairlamb, Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London WCIE 7HT, UK.
mately led to the development of 2,3-dimercaptopropanol (British Anti-Lewisite) as an antidote to the arsenical nerve gases [2]. Subsequently, Friedheim used this compound in the development of melarsoprol (Mel B) as a better tolerated derivative of melarsen oxide [3]. Although the mode of action of the trivalent arsenical drugs such as melarsen oxide and melarsoprol remains to be determined, one primary effect of exposing trypanosomes to these compounds is the formation of a stable adduct (Mel T) [4] between melarsen oxide and the unique intracellular dithiol, dihydrotrypanothione [5]. The other dithiol found in most organisms is dihydrolipoamide, which, along with dihydro-
224
lipoamide dehydrogenase, forms part of the ~oxo-decarboxylase complexes lbr pyruvate and ketoglutarate in mammalian cells. Both of these enzyme complexes are absent from long-slender bloodstream forms of Trypanosoma hrucei [6,7] and therefore dihydrolipoamide dehydrogenase has never been seriously considered as the possible target for arsenical drugs. However, recent reports have shown that these organisms do in fact contain significant amounts of dihydrolipoamide dehydrogenase, which has an unusual localization in the plasma membrane [8]. A similar membrane location has been reported for a second form of dihydrolipoamide dehydrogenase in Escherichia coli [9] where it has been suggested that this enzyme and its substrate may be involved in the transport of maltose into the cell [10]. Given these observations we sought to determine the stability of arsenical drugs with dihydrolipoyl compounds in order to examine the possibility that this metabolite might be involved in arsenical transport and to assess whether arsenicals, free or complexed to lipoamide could inhibit the dihydrolipoamide dehydrogenase activity in arsenical sensitive and resistant trypanosomes.
photometrically by titration with 5.5'dithiobis(2-nitrobenzoic acid) in 1.0 M Tris buffer, pH 8.0, using an extinction coefficient of 14.14 mM i, cm -I at 412 nm [13]. pAminophenyldichioroarsine (APDCA) was synthesised by a standard procedure [14]. The arsenical compounds melarsen oxide (Mel O) and melarsoprol (Arsobal, Mel B), were kind gifts from Specia, Rhone-Poulenc.
Determination of association constants ./br dihydrolipoic acid and dihydrolipoamide with melarsen oxide. Varying amounts ofdihydrolipoic acid or dihydrolipoamide were mixed with either melarsen oxide or melarsoprol (Mel B) (10 mM stock solutions in N.N'-dimethylformamide) to give a final concentration of 1 mM arsenical (20% (v/v) aqueous propylene glycol was used as diluent to maintain solubility of all the components in the mixture). After incubation at room temperature for 15 min, samples were separated by HPLC on a Beckman ODS 5p ion-pair column (4.6 mm x 25 cm) essentially as described previously only using solvent C (50% (v/v) npropanol in 0.25% (Li +) camphor sulphonate, pH 2.64) in place of the previously described solvent B [4]. Melaminophenylarsenicals and their adducts were detected by their absorbance at 280 nm.
Materials and Methods
Organisms. Bloodstream Trypanosoma brucei ($427.118) was maintained in rats and isolated and purified by standard procedures [11]. A cloned arsenical resistant line designated $427 cRU15 derived from this clone (see accompanying paper) was similarly maintained. Both clones were stored at - 7 0 ° C as stabilates in 10% (v/v) glycerol. Reagents. D,L-Dihydrolipoic acid and D,Llipoamide were purchased from Sigma Chemic a i C o . D,L-dihydrolipoamide was prepared by reduction of D,L-lipoamide with sodium borohydride [12] and after recrystallization, stored at -20:~C. Immediately before use, concentrations of dithiols were determined spectro-
Lipoic acid content. Total lipoic acid was determined after acid hydrolysis by bio-assay with an E. coli strain auxotrophic for lipoate [15]. Briefly, trypanosome extracts were prepared by suspending 3 × 10s cells in 0.3 ml of 6 N HCI containing 3 mg bovine serum albumin. Before heating, the solution was deoxygenated by passing a stream of nitrogen over the surface of the liquid for 20 min. Samples were hydrolysed for 2 h at 120°C, taken to dryness under a stream of nitrogen, redissolved in 200 mM (K ~) phosphate buffer, pH 7.0 and filtered to remove suspended solids. Samples were then assayed as in [15] with hydrolysed bovine serum albumin as blank and lipoic acid hydrolysed in bovine serum albumin as standard.
225
Enzyme and protein assays. Trypanosome extracts were prepared by uitrasonication in 50 mM (K ~) phosphate buffer, 2 mM EDTA, pH 7.0, followed by brief centrifugation (10000 rev./min, 3 min) to remove unbroken cells. Dihydrolipoamide dehydrogenase activity was determined spectrophotometrically at 25°C in 50 mM ( K * ) phosphate buffer/2 mM EDTA, pH 7.0 containing 1 mM N A D and 0.4 mM D,L-dihydrolipoamide by monitoring the increase in absorbance at 340 nm [16]. For the inactivation studies with arsenicals, trypanosome extracts were incubated in assay buffer at 4°C with 200 /~M melarsen oxide or paminophenyldichloroarsine in the presence and absence of N A D H . Aliquots were removed at intervals and residual enzyme activity determined by 50-fold dilution into the above assay mixture. Protein was determined using Coomassie blue reagent (Bio-Rad) [:7].
Results
Interaction of lipoyl derivatives with arsenical drugs. The reduced forms of lipoic acid and lipoamide react to form stable complexes that can be readily separated by reversed phase HPLC (Fig. 1). When an equimolar amount of D,L-dihydrolipoamide is mixed with melarsen oxide, all of the arsenical (Fig. I A) is converted into 2 new derivatives with retention times of 27.5 and 35.7 min (Fig. IC). A similar result is obtained on mixing equimolar D,L-dihydrolipoic acid and melarsen oxide, forming derivatives with retention times of 32.5 and 38.9 min (not shown). The ratio of the areas of the 2 new peaks is similar for both lipoyl derivatives (4.00 + 0.37 (N = 6) and 3.96 + 0.46 (N--6) for dihydrolipoamide and dihydrolipoic acid, respectively) and presumably represent the Dand L-diastereoisomers of the lipoyi-arsenical complexes. To test whether the stability constants for these adducts were similar to that of melarso-
T
0"1 AU
0'1 AU
1
1 J
A
J
B
E _
L
A
r
i
0
25
50
0
TIME, rain
. J f
j
25
TIME,
50 min
Fig. 1. Interactionof melarsenoxideand melarsoprolwith dihydrolipoamide.Details of the HPLC techniquesare describedin the methods. Trace A: melarsen oxide (10 nmol); trace B: dihydrolipoamide(10 nmol); trace C: melarsen oxide plus dihydrolipoamide (10 nmol of each); trace D: melarsoprol (10 nmol); trace E: dihydrolipoamide (10 nmol); trace F: melarsoprol plus dihydrolipoamide(10 nmol of each).
226
prol, equimolar mixtures of melarsoprol (Fig. I D) and dihydrolipoamide (Fig. I E) were incubated for 15 rain before analysis by HPLC. The mixture (Fig. IF) shows a decrease in the peak area for melarsoprol (the overlapping peaks of the diastereoisomers elute at 21.2 and 22.2 min) and is associated with the formation of the lipoamide-arsenical adducts identified in Fig. IC. Similar results were obtained with dihydrolipoic acid (not shown) indicating that the stability constants for the lipoyl-derivatives with melarsen oxide must be of the same order of magnitude as melarsoprol. In contrast, addition of equimofar dihydrotrypanothione to melarsoprol resuits in no adduct formation, consistent with the > 1000-fold difference in stability complexes previously reported [4]. In the case of the monothiols, glutathione and cysteine, no new adducts could be separated by HPLC even when added in 20-fold excess (not shown), consistent with previous findings [4]. In order to determine the stability complexes of the lipoyl adducts, melarsoprol was mixed with varying amounts of dihydrolipoamide or dihydrolipoic acid and the mixture allowed to come to a new equilibrium:
[MEL LI KLIP --
[MEL O][LIP] [MEL B] a n d KBAI. =
[MEL O][BAL] Therefore, eliminating [MEL O]: gl.IP
[MEL L][BAL]
KBAL
[MEL B][LIP]
Since the amount of MEL L complex formed results in an equivalent amount of BAL release, then [MEL L] = [BAL]. Substituting for [BAL] and rearranging: KI.IP
[MEL L] 2 -
[MEL B][LIP] KBAI.
I
I
I
12 --
I O ~
M E L O + LIP + B A L ~ - - - M E L L + M E L B
8 where MEL O = melarsen oxide; LIP = dihydrolipoate or dihydrolipoamide; BAL = 2,3-dimercaptopropanol and MEL L and MEL B represent the adducts with lipoate or lipoamide and dimercaptopropanol, respectively. Samples were analyzed by HPLC and the concentration of each arsenical component in the mixture determined from the response factors obtained from standards. The equilibrium concentration of dihydrolipoate or dihydrolipoamide, [LIP], can be calculated by subtracting the amount of lipoyl-arsenical adduct, [MEL L], formed from the initial amount of dihydrolipoate added. The relative affinity constant for dihydrolipoamide or dihydrolipoate (KLxp/KBAL)can now be calculated from the following equations:
4
0
l 0
i 8O
I
I 160
[MEL e] [uP] Fig. 2. Determination of association constants for dihydrolipoamide and dihydrolipoic acid adducts with melarsen oxide (KLw) relative to melarsoprol (KaAL)- The derivation of the equations and the method used are described in the text. The concentrations of reactants and products are given as nmol applied to the HPLC system. Open circles: dihydrolipoamide; closed circles: dihydrolipoic acid. The slopes of each line are the ratios of association constants, KL,,/KBAL (0.068 and 0.057 for dihydrolipoamide and dihydrolipoic acid, respectively).
227 TABLE I Association constants of melaminophenylarsenical derivatives Dithiol
Adduct
Atoms in ring
Ka ( x 107M- i)
Dimercaptopropanol a Dimercaptosuccinate a Dihydrolipoamide h Dihydrolipoic acid b Dihydrotrypanothione a Glutathione c Cysteine ¢
MEL B (melarsoprol) MEL W (trimelarsen) MEL L,CONH, MEL LcooH MELT
5 5 6 6 25 -
7930 4500 547 451 1.21 unstable unstable
aDetermined by titration of free thiol in equilibrium mixtures, data from ref. 4. bDetermined by exchange between test thiol and melarsoprol by HPLC. CAdduct formation can be demonstrated by UV spectrophometry, but the products are not stable enough for separation by HPLC.
Thus, plotting [MEL L]2 against [MEL B][LIP] should yield a straight line with a slope equal to KLIp/KBA L. This relationship was found to hold for both dihydrolipoamide and dihydrolipoate (Fig. 2) giving slopes of 0.068 and 0.057, respectively. Since the association constant for melarsoprol has previously been determined to be 7.93 x 101° M - l [4], the absolute stability constants can be calculated. Table I lists the mean stability complexes for the D,L-lipoyl arsenicals which are both about 15-fold less stable than melarsoprol, but about 500-fold more stable than that of melarsen oxide with dihydrotrypanothione. Lipoic acid and dihydrolipoamide dehydrogenase content of T. brucei. Total lipoic acid content was measured in acid hydrolysates of arsenical sensitive and resistant strains of T. brucei by bioassay using an E. coli strain auxotrophic for lipoic acid. In three separate trypanosome preparations, the arsenical sensi-
tive cells contained significantly more lipoic acid than the resistant cell line (Table II). Although the mean L-lipoate concentration showed some variation between experiments (up to 2-fold), within experiments there was a consistent and highly significant 50% reduction in lipoate concentration in resistant cells. The difference in overall mean concentrations of L-lipoate in three separate experiments was also highly significant (19.22 versus 9.71 pmol (10 s cells) -I for sensitive and resistant cells, respectively). These results indicate that lipoic acid represents a minor fraction of the total intracellular low molecular weight thiols since the total glutathione (i.e., free and conjugated to spermidine) is at least 100-fold greater (2.04 nmol (10 s cells)-1) [20]. Paradoxically, as reported in the accompanying paper [20] the dihydrolipoamide dehydrogenase content was found to be significantly increased in the arsenical resistant strain (34.7 versus 47.8 mU mg -I for
TABLE II Lipoic acid content of arsenical sensitive and resistant T. brucei Sensitive
Resistant
Ratio (R/S)
pb
(pmol (lO s cells)- i)~ Expt. 1 Expt. 2 Expt. 3
14.85 ± 0.10 c 23.59 + 0.78 19.13 ± 2.72
6.50 ± 1.75 12.43 + 0.68 10.00 + 0.29
0.44 0.53 0.52
