Comp. Biochem. Physiol., 1974, VoL 4811, pp. 261 to 273. Pergamon Press. Printed in Great Britain

T H E A C T I O N O F T R Y P A N O C I D A L A R S E N I C A L D R U G S

O N T R Y P A N O S O M A BRUCEI A N D T R Y P A N O S O M . 4 RHODESIENSE

I. W. FLYNN and I. B. R. BOWMAN

Department of Biochemistry, University of Edinburgh Medical School, Teviot Place, Edinburgh, EH8 9AG, U.K.

(Received 13 ffuly 1973)

Abstract--1. Catabolism of glucose by monomorphic Trypanosoma brucei and pleomorphic T. rhodesiense is inhibited by trivalent organic arsenicals. The utilization of tx-oxoglutarate and pyruvate by short stumpy forms of pleomorphic T. rhodesieme is inhibited by similar concentrations of arsenicals.

2. In lysed cell systems, a dual inhibition of pyruvate formation and utiliza- tion is observed. In whole organisms, however, pyruvate oxidase is uninhibited and pyruvate kinase is demonstrated to be a major site of inhibition.

3. The aromatic arsenicals do not inhibit muscle pyruvate kinase, which accounts in part for the specific trypanocidal action of the drugs.

4. Trypanosome pyruvate kinase is inhibited by N-ethyl maleimide and p- chloromercuribenzoate, but not by iodoacetate, iodoacetamide, pentavalent arsenicals or inorganic arsenite. The isofunctional muscle enzyme is equally susceptible to inhibition by p-chloromercuribenzoate.

INTRODUCTION

IT IS NOW evident that the oxidation of keto-acids is the key reaction in arsenite inhibition of respiration, and intensive study by Peters and his group (1946, 1955) has shown that pyruvate oxidase is specifically and markedly inhibited both by inorganic arsenite and by the organic trivalent arsenicals. Despite the early introduction of the organic arsenicals, there is still relatively little information on their metabolic effects compared to those of inorganic arsenite, although the inhibitions by these types of compounds often differ markedly. Although arsenite is trypanocidal, its toxicity precludes its medical use, whereas the trypanocidal organic pentavalent and trivalent arsenicals are used clinically. Aliphatic phenyl- arsenoxides and their reduced counterparts are ineffective against Trypanosoma rhodesiense but have a therapeutic action against T. gambiense infections (Hutchinson & Watson, 1965). However, the latter species develops resistance to tryparsamide (sodium-p-glycinamidophenylarsonate), and this led to the introduction of melaminyl (2,4,6-triamino-S-triazinyl) phenylarsonate (Friedheim, 1940). This compound, known as melarsen, was found to be effective in the treatment of tryparsamide-resistent T. gan~iense (Williamson & Lourie, 1948), to be active as a trypanoeide against T. rhodesiense and to be useful in the treatment of advanced

261

262 I. W. FLYNN AND I. B. R. BOWMAN

human sleeping sickness, where central nervous system involvement requires a drug capable of crossing the "blood-bra in barrier". Despite the continuing use of melarsen as a trypanocide, virtually no studies have been made on the mechanism of action of this drug. In higher organisms, the primary sites of organic arsenical action are still considered to be the ~-ketoacid oxidases, and the trivalent arsenicals are considered as the therapeutically active form. However, this cannot be the site of action in monomorphic bloodstream trypanosomes which lack ~-keto acid oxidase systems (Ryley 1956; Grant & Fukon, 1957). Despite the absence of these enzymes, monomorphic, long, slender trypanosomes are rapidly immobilized by melarsen oxide. In these organisms, then, some other focal point must be the site of action of the organic arsenicals. Chen (1948) and Marshall (1948) produced some evidence that in T. equiperdum and T. evansi, respectively, hexokinase is the most sensitive site in vivo. However, Cantrell (1951, 1953) came to the conclusion that this is not the case. This author found the rates of glucose utilization in control and drug-treated organisms to be identical, although pyruvate production was lower in the presence of mapharsen (m-amino-p-hydroxyphenyl arsenoxide). This work implicates pyruvate kinase as a focal point of arsenical action in T. rhodesiense, both in monomorphic and in pleomorphic strains, the latter having in the short s tumpy form additional sites of action of arsenicals, namely the oxidative decarboxy- lases of pyruvate and ~-oxoglutarate.

MATERIALS AND METHODS* Organisms, strain maintenance and parasite preparation

Monomorphic T. brucei, strain TREU 277, and pleomorphic T. rhodesiense, strain EATRO 173, were maintained as in Flynn & Bowman (1973). Removal of blood elements from parasite preparations was carried out by differential centrifugation, sintered glass filtration and erythrocyte precipitation with rabbit anti-rat erythrocyte serum (Flynn & Bowman, 1973). Before use purified trypanosome preparations were routinely checked to ensure motility prior to cell lysis. Spot checks for the absence of LDH activity were carried out to ensure the reliability of the method in removal of blood cells (Dixon, 1966). After purification, cells were washed in glucose-free saline and either used directly or lysed by homogenization at 0°C in 8 vol. of distilled water. This material will be referred to as standard water-lysed material. For investigation of pyruvate kinase, the cell lysate was lyophilized and stored at 4°C.

Measurement of gas exchange Oxygen utilization and carbon dioxide evolution were measured in a conventional

Warburg respirometer (B. Braun & Co., Melsungen) at 37°C with air as the gas phase. Temperature equilibration was carried out for 10 rain prior to the start of the experiments and measurements were terminated by the addition of 0"5 ml 0"33 M-PCA from the side-arm.

* The following abbreviations are used in the text: BSA, bovine plasma albumin; EDTA, ethylenediamine tetracetie acid; PK pyruvate kinase (E.C. 2.7.1.40); LDH, lactate dehydrogenase (E.C. 1.1.1.27); PCA, perchloric acid; TEA, triethanolamine; melamine, 1,3,5-triamino- S-triazine ; melarsen oxide, p-melaminylphenylarsenoxide ; sodium melarsen, disodium p-melaminylphenylarsonate; Mel W, dipotassium p-melaminyl-phenylarseno- dithiosuccinate; PEP, phosphoenolpyruvic acid; L-ec-GP, L-glycerol-3-phosphate.

THE ACTION OF ARSENICAL TRYPANOCIDES 263

Estimation of metabolites Metabolite estimation was carried out essentially as described in Flynn & Bowman

(1973). When incubations were carried out in the presence of potential metabolic inhibitors, coupled enzyme assay systems were standardized in the presence of the same concentrations of inhibitor.

Incubation of cell lysates The standard incubation medium for investigation of lysate metabolism will be referred

to as Minimally Fortified Medium (MFM) and had the following constituents: KCI, 200 #moles; EDTA, 3 #moles; nicotinamide, 25#moles; MgSO~, 20#moles; BSA, 10mg; phosphate buffer, pH 7"4, 67#moles; ADP, 5 #moles; NAD +, 5 #moles; total volume, 3.0 ml.

Pyruvate kinase (PK) The high rate of NADH oxidation in the presence of water-lysed material and the

instability of the enzyme in fresh lysates necessitated the use of lyophilized trypanosomes for assay of PK. Freeze-dried material was resuspended in 1 mM TEA buffer, pH 7"4, homogenized and used within 4 hr of resuspension.

The preferred assay system, dependent upon the oxidation of NADH by LDH in the presence of pyruvate formed by PK, is essentially that of Biicher & Pfleiderer (1962). The standard assay system contained, in a volume of 3"0 ml: TEA buffer, pH 7-4, 100 #moles; MgSO4, 20#moles; KC1, 200#moles; NADH, 0"4#moles; LDH, 5 I.U.; ADP, 1"25 #moles; PEP, 5"0 #moles; enzyme and water to 3-0 ml. The spectrophotometer used was a Unicam model S.P. 800 fitted with a scale-expansion unit and coupled to a slave recorder, Servoscribe model RE211. The cell compartment was maintained at 25 + 0"6°C by means of a circulating water-bath. Initial velocities were estimated by tangential measurement to the recorded curve.

Chemicals All coenzymes, the potassium salt of PEP and enzymes were obtained from C. P

Boehringer & S6hne, GmbH, Mannheim. Bovine plasma albumin was obtained from Armour Pharmaceuticals Ltd. All other analytical reagents were obtained from BDH Biochemicals Ltd., as were iodoacetate, iodoacetamide and p-chloromercuribenzoate. Sodium melarsen, melarsen oxide and Mel W were gifts from Messrs. May & Baker Ltd., Dagenham, Essex, and p-aminophenylarsenoxide a gift from Parke, Davis Ltd.

RESULTS

T h e inhibitory effects of melarsen oxide and phenylarsenoxide on oxygen utilization by pleomorphic and monomorphic strains are shown in Table 1. In the presence of high concentrations of either drug, the inhibitory effects progressively increased over a 30-min period, possibly due to an action against secondary sites of inhibition which are uninhibited at lower concentrations (I5o values in Table 1). In these cases, the rate of oxygen uptake at the end of the preincubation period is taken as the inhibited rate for calculation of the 150 values. Phenylarsenoxide has an 150 value (concentration for 50 per cent inhibition) in all the systems investigated an order of magnitude lower than the melaminyl derivative. The value in a whole- cell system using glucose is about half that estimated f rom the data of Marshall (1948), but as this author used a whole-blood suspension of 1". evansi and as differ- ences occur even between strains of T. rhodesiense this discrepancy is not surprising.

TA

BLE I

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Str

ain

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O

173,

7

4-8

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sh

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mp

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150

(M)

Ly

sed

cel

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Wh

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Su

bst

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Inh

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rate

P

yru

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Mel

arse

no

xid

e 3

"1+

0'8

×1

0-5

(4)

2"0

+ 0

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-5 (

4) 2

"2 +

0"2

×1

0-5

(5)

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2 +

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1.6

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RE

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ibit

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is g

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+

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dar

d d

evia

tio

n.

THE ACTION OF ARSENICAL TRYPANOCIDES 265

Thus the melaminyl drug and the parent phenylarsenoxide are powerful inhibitors of glucose-supported oxygen uptake and of the oxidative decarboxylations of pyruvate and ~-oxoglutarate. The similarity of the 150 values for the various substrates appeared to indicate a point of action of the arsenicals which was substrate independent. However, the utilization of oxygen with L-~-GP as the substrate is less than 15 per cent inhibited at 2 x 10 -5 M melarsen oxide and 3 x 10 -e M phenylarsenoxide. When the NAD-dependent L-~-GP dehydrogenase was assayed spectrophotometrically again less than 15 per cent inhibition was obtained at the above inhibitor concentrations. Thus in the absence of a cyto- chrome system in these organisms (Flynn, 1971) the two common steps in the pathway of electrons from glucose and the ~-oxo-acids to oxygen, i.e.

NADH+ DHAP+ H + > L-~-GP+ NAD+

and L-~-GP + ~O~ > DHAP + H~O

(Grant & Sargent, 1960), appear insensitive to inhibition by the arsenicals. It has been shown (Flynn, 1971 ; Flynn & Bowman, 1973) that the utilizations of pyruvate and ~-oxoglutarate are confined to single-step oxidative decarboxylations producing acetate and succinate respectively. Thus, as the reoxidation of NADH is arsenical insensitive, the oxidative decarboxylases appear to be target enzymes for these drugs, as in mammalian tissue (Peters et aL, 1946; Peters, 1955). However, the monomorphic strain of T. brucei is equally sensitive to these compounds indicating that the focal point of inhibition in glucose metabolism is not primarily the ~-oxo- acid decarboxylases.

Effects of melarsen oxide on glucose metabolism by cell lysates

At a low concentration of melarsen oxide (1.15 x 10 -5 M) the gas exchange of cell lysates of T. rhodesiense is inhibited while the net glucose utilization is not greatly affected (Table 2). Accompanying this differential effect of the drug is a stimulation of pyruvate formation. The decrease in the utilization of oxygen is the result of the inhibition of two separate processes, the glycolytic breakdown of glucose and the further metabolism of pyruvate. The stimulation of pyruvate formation, despite the slight inhibition of glucose utilization, results from an inhibition of the pyruvate oxidase system. This observation is supported by the inhibition of carbon dioxide formation, which in short stumpy trypanosomes is wholly derived from the decarboxylation of pyruvate (Flynn & Bowman, 1973). However, the net decrease in carbon dioxide production (2.63/~moles) does not equate with the increase in pyruvate formation of only 1.08/,moles. As pyruvate catabolism is confined to a one-step oxidative decarboxylation a second inhibitory point must exist prior to pyruvate in the glycolytic sequence.

At higher concentrations of melarsen oxide (2.30-3.45 x 10 -5 M) a marked inhibition of glucose utilization is found. However, the oxygen uptake is signifi- cantly greater than that predicted from the formation of pyruvate and carbon dioxide, indicating an inhibitory point in the glycolytic pathway subsequent to the

266 I . W . FLYNN AND I. B. R. BOWMAN

TABLE 2 - - E F F E C T OF MELARSEN OXIDE ON GLUCOSE METABOLISM BY CELL LYSATES

Concentration Substrate utilized Metabolite produced of melarsen

oxide Glucose Oxygen Pyruvate CO2 ( × 105 M) (#moles) % S* (#moles) % S (#moles) % S (#moles) % s

zero 12"69 100 11"40 100 8"69 100 6"74 1"15 12-14 96 9"56 84 9"77 112 4"11 2-30 8"54 67 7.24 63 8"85 102 2"50 3-45 5"01 39 4-69 41 3-76 43 2"06

100 61 37 31

* % S, Percentage of the uninhibited utilization or production. Standard water-lysed preparations of T. rhodesiense, EATRO 173 (approx.

1"5 mg N), were suspended in M F M with 25/zmoles of glucose and in the presence of varying concentrations of melarsen oxide. Manometric incubations were caried out for 30 min prior to termination by addition of perchloric acid. Other experimental details are as found under Materials and Methods. Pleomorphic composition: 74 per cent short stumpy.

oxidation of glyceraldehyde-3-phosphate, leading to an accumulation of three- carbon acid phosphates. Furthermore, it can be calculated from the data in Table 2 that, in the absence of melarsen oxide, 37 per cent of the glucose used must accumulate as hexose and triose phosphates. With increasing concentrations of melarsen oxide this pool of intermediates decreases, accounting for 27 per cent of the glucose used at 3.45 × 10 -5 M melarsen oxide.

Effect of melarsen oxide on glucose metabolism by whole cells

Table 3 records the effect of varying melarsen oxide concentration on the metabolite balance of pleomorphic T. rhodesiense. When this organism is exposed

TABLE 3 - - E F F E C T OF 1VIELARSEN OXIDE ON GLUCOSE METABOLISM BY WHOLE CELLS

Concentration Substrate utilized Metabolite produced of melarsen

oxide Glucose Oxygen Pyruvate CO ( x 10 e M) (#moles) % S (#moles) % S (#moles) % S (#moles) % s

Zero 8"41 100 8"99 100 10"43 100 3"62 100 0"1 8.11 97 8"57 95 8"82 84 3"54 98 1 6.06 72 6"18 69 6"19 59 2"79 77 3 5.04 60 5"24 58 4"54 43 2"32 64 5 4"54 54 4-90 54 3.27 31 1"91 53

* % S, Percentage of the uninhibited utilization or production. Whole cells of 7". rhodesiense, EATRO 173 (approx. 0'3 mg N), were sus-

pended in saline with 25 #moles of glucose and varying concentrations of melarsen oxide. Manometric incubations were carried out for 30 rain prior to termination with perchloric acid. All other experimental details are as described in Materials and Methods. Pleomorphic composition: 75 per cent short stumpy.

THE ACTION OF ARSENICAL TRYPANOCIDES 267

to the inhibitor, oxygen and glucose utilization and carbon dioxide formation are inhibited in parallel, each of these three parameters having an 150 value of 5-6 x 10 -6 M melarsen oxide. The production of pyruvate is more sensitive to the drug (150 value 1-2 x 10-6 M melarsen oxide) than is its utilization by pyruvate de- earboxylase. Increase in the production of this metabolite, theoretically expected by a specific inhibition of the pyruvate oxidase system, could not be demonstrated in the intact cells at any concentration of melarsen oxide (Fig. 1). This is in contrast

,u mole / 1.2 /u mole glucose

1.0

~8

06

0"~

0.2

A

[Melarsen oxide] (xlO6M|

FIG. 1. Melarsen oxide inhibition of glucose metabolism by whole cells of T. rhodesiense, EATRO 173. Experimental details are as described in Table 3 and under Materials and Methods. Pleomorphic composition: 75-78 per cent short stumpy. Data are expressed as molar ratios with respect to glucose utilization.

0, Oxygen utilization; +, pyruvate production, A, COB production.

to the differential effect shown in lysed-cell preparations (Table 2). The lowest concentration of inhibitor which was used (10 -7 M) produced an inhibition of 16 per cent of the formation of pyruvate and had a negligible ( < 5 per cent) inhibitory effect on glucose or oxygen utilization or on CO 2 formation.

In the whole-cell system then, the primary point of inhibition appears to be within the glycolytic sequence. This inhibition cannot lie between glucose-6- phosphate and the triose phosphates, since a decrease in the ratio of oxygen utilized to glucose utilized would be apparent; Fig. 1 shows this ratio to be constant over the entire range of inhibitor concentrations used. It may be shown that the total build-up of glycolytic intermediates in this part of the pathway is 18 + 3 per cent of glucose used, over the whole range of inhibitor concentrations. An accumu- lation of a glycolytic intermediate between 1,3-diphosphoglyceric acid and phospho- enol pyruvate is therefore expected. The constant ratio of COz produced to glucose

268 I. W. FLYNN AND I. B. R. BOWMAN

utilized (Fig. 1) indicates that the pyruvate oxidase system is uninhibited by melarsen oxide in intact cells. The highest concentration of drug used in this series of experiments is 5 x 10 .6 M melarsen oxide; at this drug concentration the utilization of pyruvate by lysed cells (as measured by the uptake of oxygen in the presence of pyruvate as substrate) is 20 per cent sensitive to melarsen oxide. This observation and the observation of Hawking (1938) that arsenicals are concentrated in the trypanosome cell suggest that the lack of inhibition of pyruvate oxidation in the whole cell is due to the inaccessibility of the oxidase system to melarsen oxide.

Closer investigation of metabolite concentrations in the glycolytic pathway between 1,3-diphosphoglyceric acid and pyruvate showed that in the presence of melarsen oxide there was a large increase in PEP (Table 4). Thus, pyruvate kinase appears to be one of the major sites of arsenical inhibition.

TABLE 4---EFFECTS OF MELARSEN OXIDE ON GLUCOSE METABOLISM BY WHOLE CELLS

Standard + Melarsen oxide

Glucose Glucose Inhibition /~moles carbon (%) /~moles carbon (%) (%)

Glucose utilization 8"61 100 5"34 100 38 Oxygen utilization 8'95 -- 5'77 -- 35 Pyruvate production 10"85 63 4'97 47 46 COs production 3"96 8 2"56 8 36 PEP production Approx. 0"35 Approx. 2 1"05 10 3000'o

stimulation

Experimental details as in Table 3, with melarsen oxide at 2 x 10 -6 M. Pleo- morphic composition: 78 per cent short stumpy. Metabolite estimations were carried out as in Materials and Methods. Experimental duration 45 min.

The action of inhibitors on pyruvate kinase

The specific trypanocidal activity of melarsen oxide may depend either on a difference in permeability to the drug between host and parasite cells or on a difference in the target enzyme of the parasite and the isofunctional host enzyme. At least part of the selective action of this drug and its analogues resides in differ- ences between host and parasite pyruvate kinase, as may be seen from Table 5. Melarsen oxide, phenylarsenoxide and p-aminophenylarsenoxide (reduced Atoxyl) are all inhibitory to the trypanosome enzyme but have no effect on rabbit muscle pyruvate kinase. On the other hand, the less specific thiol inhibitor p-chloro- mercuribenzoate is equally effective as an inhibitor of mammalian and trypanosome pyruvate kinase. The inhibitory action of the aromatic arsenicals is confined to the trivalent reduced forms, as sodium melarsen, the pentavalent derivative of melarsen oxide, gives only 17 per cent inhibition at a concentration of ten times the I50 value of the trivalent arsenical. Melamine and p-aminobenzoate are also non-inhibitory

TA

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th

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of

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s g

iven

in

par

enth

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. E

ach

in

div

idu

al

Is0

val

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was

ob

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ed f

rom

no

t le

ss t

han

six

in

hib

ito

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ntr

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ns.

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den

ote

s n

ot

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ibit

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s n

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t 5

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.

tO

O~

270 I. W. FLYNN AND I. B. R. BOWMAN

(Table 5) indicating that the functional group in the enzymic inhibition is the arsenoxide residue. Inorganic arsenite and the alkylating reagents iodoacetate and iodoacetamide are non-inhibitory to the trypanosome PK. Hence the trypanocidal effect of these inhibitors (Ryley, 1956; Williamson, 1959) must be due to inhibition at a different site, perhaps glyceraldehyde-3-phosphate dehydrogenase.

DISCUSSION From the known nature of the metabolism of the monomorphic trypanosomes,

it is unexpected that the organic arsenicals exert any profound trypanocidal effect. The bloodstream form of T. brucei, strain TREU 277, and the long slender form of T. rhodesiense, strain EATRO 173, do not oxidase pyruvate or ~-oxoglutarate and cell lysates of these organisms contain no demonstrable pyruvate oxidase activity (Flynn & Bowman, 1973). In the absence of a tricarboxylic acid cycle and cyto- chrome system in these organisms, inhibition of other enzymes known to be inhibited by phenylarsenoxide and its derivatives such as cytochrome oxidase (Kreke et al., 1950) malate dehydrogenase and succinoxidase (Barron & Singer, 1945) cannot be responsible for the trypanocidal activity of arsenicals. The rapidity of the trypanocidal effect indicates a site of action in the energy-trapping systems of the organism, thus eliminating other known susceptible enzymes such as fl- amylase (Ghosh, 1958) and fl-hydroxybutyrate dehydrogenase (Singer & Barron, 1945) as the focal point of arsenical inhibition in monomorphic trypanosomes.

In lysed-cell preparations of pleomorphic strains, the further metabolism of pyruvate is more susceptible to inhibition at a low concentration of melarsen oxide (1.15 × 10 -5 M) than is the production of pyruvate (Table 2). At higher concentra- tions of the trypanocide (3.45 × 10 -5 M) pyruvate production and formation are both inhibited, indicating the presence of multiple inhibitory sites. The utilization of both pyruvate and a-oxoglutarate are one-step reactions (Flynn & Bowman, 1973) and are both directly susceptible to inhibition by the aromatic arsenicals (Table 1) in parallel with the isofunctional enzymes of mammalian systems (Peters et al., 1946). The inhibition of glucose utilization may be due to a secondary inhibition by the drug of hexokinase or due to a decrease in the net availability of ATP as a result of pyruvate kinase inhibition. The dual inhibition of pyruvate formation and utilization observed in the lysed-cell system could not be demon- strated in whole-cell suspensions (Table 3). Both ~-oxo-acid oxidases are known to be active in vivo: the pleomorphic types which contain these enzymes can survive utilizing c~-oxoglutarate as the sole extracellular energy source (Vickerman, 1965; Flynn & Bowman, 1973). Thus the failure to demonstrate the primary inhibition of pyruvate oxidation in vivo is presumably due to the relative inaccessibility of the mitochondrial a-oxo-acid oxidases compared to the cytoplasmic pyruvate kinase.

It therefore appears that the site of action of the arsenical trypanocides is constant in monomorphic and pleomorphic strains of Trypanosoma, and relatively independent in vivo of the c~-oxo-acid oxidases in short stumpy forms of the natural infections. One site of action has been narrowed down to pyruvate kinase, as a result of the demonstration of PEP accumulation under inhibition and confirmed by

THE ACTION OF ARSF~qICAL TRYPANOCIDEfl 271

direct assay of pyruvate kinase in the presence of melarsen oxide and related inhibitors (Tables 4 and 5). These conclusions are consistent with those of Cantrell (1951, 1953), who also demonstrated an inhibitory point in the glycolytic chain between glucose-6-phosphate and pyruvate, showing a decrease in pyruvate formation without inhibition of glucose utilization in whole cells of T. equiperdum. The differences between the conclusions of Cantrell and those of Chen (1948), who, using lysed preparations of T. equiperdum, demonstrated the sensitivity of hexokinase to the arsenicals, are also borne out by the present work.

As far as is known, the only pathway available for production of metabolic energy in 2". brucei and T. rhodesiense is glycolytic degradation of glucose. Thus the enzyme responsible for a net production of ATP in these organisms is pyruvate kinase. This enzyme would therefore make an ideal target for chemotherapeutic attack, as an inhibition at this site reduces the net energy gain to zero. Evidence has been presented in the present work to show that this is in fact the primary site of action of melarsen oxide. Furthermore, the inability of the aromatic arsenicals to inhibit pyruvate kinase from mammalian muscle (Table 5) may be held in support of the specific trypanocidal action of these drugs. However, the muscle enzyme has neither homotropic nor heterotropic allosteric properties whereas the parasite enzyme is aUosterically activated by both PEP and fructose- 1,6-diphosphate and thus the parasite kinase is much more closely related to the allosteric isoenzyme from host liver which is inhibited by the arsenical drugs (Flynn, 1971). Also the lack of effect of melamine on trypanosome pyruvate kinase and the comparable activities of melarsen oxide, phenylarsenoxide and p- amino-phenylarenoxide indicate that the development of the melaminyl series of arsenicals has not markedly affected the toxicity at the site of action, but has possibly served to increase the differential uptake of the drugs by parasite and host cells. In the absence of differential permeability, little therapeutic benefit would be obtainable from this series of compounds, despite their inactivity against the mammalian muscle enzyme.

The inability of arsenite to inhibit trypansome PK indicates the requirement for an organic residue to stabilize the drug-enzyme complex. Both host muscle and parasite enzymes are thiol dependent as evidenced by the potent action of p- chloromercuribenzoate (Table 5), thus bearing out the original postulate of Voegtlin et al. (1923) that arsenical trypanocidal action results from reaction with intra- cellular thiol groups. Pentavalent arsenicals have no aiSnity for thiols. Hence the slight sensitivity of trypanosome PK to the pentavalent analogue of melarsen oxide, sodium melarsen, may be due to a low concentration of the reduced trivalent form present as an impurity or to reduction under the experimental conditions. The in vivo trypanocidal effects of the pentavalent arsenicals therefore lie in their activation by reduction, as suggested by Crawford (1947), despite some evidence that this expected reduction does not occur to any marked extent (Crawford & Levvy, 1947; Frost, 1967). When melarsen oxide is administered as the dimercapto- succinate derivative (Mel W) the trypanocidal activity is apparent by what is presumably a direct thiol exchange with the PK as Mel W is directly inhibitory

272 I. W. FLYNN AND I. B. R. BOWMAN

(Table 5). The P K of T. brucei has been purified and extensively investigated. Its kinetic properties and interaction with the arsenicals will be published elsewhere.

T h e position of monomorphic strains as experimental analogues of natural p leomorphic infections is also clarified by this work. Most screening tests are carried out on old laboratory monomorphic strains which have acquired certain stable characteristics. T h e increased virulence found in these strains, associated with either art increased or decreased sensitivity to drugs, may be thought to give a misleading impression of therapeutic value. However, the mode of action of at least the aromatic arsenicals appears to be independent of the morphological state. Also the response of the P K of monomorph ic and pleomorphic strains is identical with respect to their inhibition by thiol reagents including the arsenicals. T h e convenience of using such adapted strains therefore appears justifiable in terms of the interpretat ion and investigation of arsenical action.

Acknowledgements--This work was supported by grants from the Trypanosomiasis Panel, Overseas Development Administration, Foreign & Commonwealth Office. We thank Miss Marion Thomson for technical assistance.

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Key Word Index'---Trypanosoma rhodesiense ; Trypanosoma brucei ; carbohydrate metabol- ism; arsenicals; melarsen oxide; pyruvate kinase; ~-oxoglutarate oxidase; pyruvate oxidase.