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An approach to the rational design of new inhibitors for Trypanosoma brucei
Witmans, Cornelis Jacobus
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Witmans, C. J. (1995). An approach to the rational design of new inhibitors for Trypanosoma brucei:
Triosephosphate isomerase Groningen: s.n.
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Section
Introduction
I
8
C
H A P T E R
1
Human African
Trypanosomiasis
INTRODUCTION
A
Introduction
frican trypanosomiasis is endemic in an area, known as the ‘‘tsetse
belt,’’ extending over one third of the African continent and spanning 36 sub-Saharan countries (figure 1.1). The disease may arise, as a
multifocal event throughout equatorial Africa. There are two forms of human African trypanosomiasis or sleeping sickness: gambiense trypanosomiasis, a chronic disease found in Central and West Africa, and
rhodesiense trypanosomiasis, a more acute disease found in East and
southern Africa.1 Fifty million people, living in or around the 200 disease
foci within the tsetse belt, particularly those engaged in hunting, fishing
and farming, are at risk of infection. Only five million people are under
surveillance. The mean prevalence rates are about 1-2%. Currently, about
20,000 new cases are reported each year.1
African trypanosomiasis is caused by subspecies of the genus, t r y p a n o s o m a t i d a e , consisting of uniflagellated parasitic protozoa. T r y p a n o s o m a
b r u c e i b r u c e i was implicated as the cause of ‘‘Nagana’’ (animal trypanosomiasis) in 1894, and trypanosomes were first demonstrated in human ‘‘sleeping sickness’’ in 1902, although the disease was first
described in 1803.1 The subspecies T r y p a n o s o m a b r u c e i r h o d e s i e n s e
causes the acute form of African sleeping sickness in East and southern
Human
to
Human
Cycle
Wild
Animal
Cycle
The Disease and
the Parasite
Figure 1.1
Human African trypanosomiasis is transmitted by tsetse
flies. The darker colored area
is the so-called tsetse belt.
Domestic
Animal
Cycle
Introduction
9
Section I : Introduction
Figure 1.2
Life cycle of the salivarian trypanosome T. brucei.
Vector Control
Figure 1.3
Pyramidal (Lancien-Gouteux)
trap used experimentally by
several rural communities in
Central Africa. The flies enter
the trap, fall into the liquid in
the beaker in the top of the
trap, and drown.
10
Introduction
Africa, whereas the less virulent form of trypanosomiasis in Central and
West Africa, is caused by T r y p a n o s o m a b r u c e i g a m b i e n s e .
The parasites are transmitted to humans by the bite of the tsetse fly or
G l o s s i n a . 1 Part of the life cycle of the parasites takes place in the tsetse
flies. The existence of a developmental cycle for T . b r u c e i in G l o s s i n a
was recognised in 1909.7 In the mammalian bloodstream, the T . b r u c e i
population is pleomorphic, consisting of long, slender, dividing forms, intermediate forms, and short stumpy forms (figure 1.2). The slender forms
rely on aerobic glycolysis, excreting pyruvate as the sole end-product, for
energy generation. Metabolic changes in the stumpy forms probably facilitate their developmental adaptation after ingestion by the fly, whereupon they differentiate and multiply in the tsetse midgut as non-infective
coatless ‘‘procyclic’’ trypanomastigote forms. Procyclic forms migrate to
the salivary glands where, attached to the epithelial cells, they multiply as
a distinct morphological stage, the epimastigote. After cessation of division, the epimastigote starts to express a dense variable surface glycoprotein coat (VSG coat) and this expression leads to detachment and
maturation as non-dividing, infective metacyclic trypanomastigotes.
The VSG coat accounts for about 10 % of the total protein of the bloodstream forms of T . b r u c e i . The variability lies in the fact that the parasite
can shed its coat and build a new one by expressing another surface glycoprotein. The sequential expression of VSGs results in an ever changing
antigenic pattern. Antigenic variation, in the manner and to the extent that
it occurs in the African trypanosomes, is unique to the parasites and appears to be the primary mechanism for evasion of the hosts’ immune responses.7 These conditions arouse deep pessimism about prospects for a
trypanosomiasis vaccine.
A
part from drug treatment of infected patients the disease is generally
fought via its vector, the tsetse fly. The flies are controlled with
traps and with aerial insecticide spraying. This last measure is quite
costly, of course, and therefore a renewed interest in trapping to control
tsetse fly populations is emerging. In Congo, a conical insecticide-impregnated trap has been developed (figure 1.3). Tsetse flies are attracted
by the shape, color and movements of the trap hanging from a tree. No
tsetse flies were observed over a six month period following its use in
some villages in Couloir and Niari disease foci.
In Ivory Coast, over 15,000 screens impregnated with the insecticide deltamethrin were placed by farmers in strategic sites in an 83 km2 area.
This reduced the tsetse population by 98% within one month at a cost of
US$ 5.50 per hectare.
The possibility to use odours to attract tsetse flies to a trap and thereby increasing its efficiency has been explored in several studies. Octenol, ace-
H u m a n A frican Trypanosom iasis
tone and buffalo urine have been shown to be effective in attracting some
tsetse species. Pyramidal traps (Lancien-Gouteux traps), which attract
tsetse flies with blue-coloured cloth, do not require the use of chemical
attractants or insecticides.6
DRUG TREATMENT
Drug Treatment
T
oday, chemotherapy and chemoprophylaxis of African trypanosomiasis relie essentially on four drugs: p e n t a m id i n e (figure 1.4), s u r a m i n
(figure 1.5), m e l a r s o p r o l (figure 1.6A) and D L - α- d i f l u o r o m e t h y l o r n i t h i n e
(figure 1.7). P e n t a m i d in e is used mainly for prophylaxis. The amidino
groups in the drugs are the reason for their high polarity and hence their
incapability to pass the blood brain barrier. Therefore, it is impossible to
use these drugs against sleeping sickness in a late stage of the disease
when the trypanosomes have entered the brain. S u r a m i n is used for early
disease and m e la r s o p r o l is used for late stage disease when the central
nervous system is infected. D L - α- d i f l u o r o m e t h y l o r n i t h i n e is a new drug
aimed at the inhibition of the polyamine synthesis.
great number of diamidines are now known to be trypanocidal.11
All the diamidines are effective as prophylactic agents, however,
only diminazene (berenil®) is able to effect a cure.
P e n t a m i d i n e (see figure 1.4) was discovered as trypanocidal drug in the
late 1940’s.1 The drug is rapidly absorbed by trypanosomes and at once
exerts a trypanostatic effect on them, both i n v i t r o and i n v iv o . The selective attenuation of the carbohydrate metabolism by inhibiting α-glycerophosphate dehydrogenase, apparently by opposing the coenzymic effect
of spermidine, plays an important role in its mode of action.16 The parasites are finally killed by the host’s defensive forces within some days.
One indirect factor that helps the cure is the following: those parasites
that survive the direct effect of the drug remain infective, but they have a
large number of drug-containing granules. When some parasites die, they
liberate the drug, and also an antigen which causes the host to make ade-
A
Pentamidine
Figure 1.4
Structural formulas of some
diamidines currently under investigation as drug for the treatment of human trypanosomiasis.
D rug Treatm ent
11
Section I : Introduction
quate amounts of antibodies against the remaining trypanosomes. This indirect process is believed to explain the slow onset of the action of the
diamidine drugs, and their prolonged prophylactic effect.15
The diamidines have also severe side effects like hepatoxicity11 and induction of hypotension.12 Vasodilatation mediated by histamine release
seems the most probable cause, since some diamidines, like e.g. p e n t a m id i n e , are known to be potent histamine releasers.12,14 Several other
diamidino compounds have antagonistic activity at α1- and α2-adrenoceptors i n v i t r o . The concentrations at which these compounds antagonise
these receptors is comparable with the concentrations found in plasma of
p e n t a m id i n e in clinical studies. Therefore, blockade of postsynaptic α-adrenoceptors of vascular smooth muscle might be involved in their hypotensive effects.
Apart from severe side effects, resistance to most of these drugs has been
described. Multiple drug resistance of T .v i v a x is prevalent along the coast
in Kenya, and exists in Somalia.13 1,3,5-Triacetylbenzene tris(guanylhydrazone) (figure 1.4) is a new experimental drug based on the structure of
diminazene. Kaminsky and Zweygarth27 studied this drug in a new i n v i t r o assay and found an EC50 value for T .b . b r u c e i between 0.3 to 3.5
µg/ml, whereas diminazene had an EC50 value between 0.046 and 5.263
µg/ml. The variation is caused by different isolates of trypanosomes.
Suramin
Figure 1.5
The structural formulas of
trypan red and suramin
12
D rug Treatm ent
P
a u l E h r l i c h (1854-1915) was the first to look for trypanocidal drugs.
In 1904 he cured trypanosome-infected mice with trypan red (figure
1.5), which thereby became the first man-made chemotherapeutic agent.
S u r a m i n (figure 1.5) was discovered in 1920 as drug against trypanosomiasis.15 It is a highly effective analogue of trypan red. The drug was
later used for prophylaxis after it was found that a single dose gave immunity for three months.
The structure of the drug is remarkably specific in that the removal of the
two methyl groups abolishes activity completely. Nevertheless, quite un-
H u m a n A frican Trypanosom iasis
related polyanions, such as dextran sulphate, have also strong trypanocidal activity.
It has long been known that suramin abolishes the infectivity of trypanosomes without affecting their mobility. The electron microscope shows
that the drug strikes hard and specifically at the parasite’s ribosomes,
which lose their polysomal character and form the so-called ‘‘cytoplasmic
granules’’. Fundamentally the attack seems to be on RNA-polymerase;
the nucleus, nucleolus and the kinetoplast remain unaffected and the synthesis of DNA continues.16
S u r a m i n is not suitable for large-scale field use because it has to be administered intravenously and it is sometimes associated with severe side
effects. Like p e n t a m i d i n e it does not pass the blood brain barrier due to
its polar character caused by the sulphonic acid groups that are ionised
under physiological conditions. S u r a m i n is therefore not suitable for
treatment of late stage trypanosomiasis.
T
he first arsenical drug used against human trypanosomiasis was
atoxyl (figure 1.6A). This discovery influenced Ehrlich to commence prolonged experimentation with aromatic arsenicals.15 In 1909 he
discovered that t r i v a l e n t arsenicals were trypanocidal in the test-tube and
Ehrlich suggested that p e n t a v a l e n t arsenicals (like atoxyl) would also be
found active if they were reduced to the t r i v a l e n t drug first. He showed
that p e n t a v a le n t arsenicals were active i n v itro when they were first incubated with reducing tissue. This result explains their activity i n v i v o .
In 1920 a new arsenical drug was discovered: tryparsamide (figure 1.6A),
a pentavalent arsenical drug derived from atoxyl but less toxic. Tryparsamide was effective against the T . b . g a m b i e n s e as it cured the cerebral
form of the disease. Tryparsamide, however, is not effective against T . b .
r h o d e s i e n s e . 16 Many related compounds were subsequently synthesised,
but only a few have found their way into therapeutic use.
Melarsoprol
Figure 1.6
A: The structures of some
arsenical trypanocidal drugs.
B: The structure of the
melarsen oxide-Try(SH)2 derivative (Mel-T).
C: The structure of a melarsoprol analogue currently under
investigation.
D rug Treatm ent
13
Section I : Introduction
A new class of arsenicals, melaminyl-substituted phenylarsonates, of
which melarsen oxide was the first, was introduced by F r i e d h e i m . 17,18
M e l a r s o p r o l (figure 1.6A) was introduced in the 1940s.20 It is used
against the effectors of human sleeping sickness and the animal trypanosomiasis; T . b . b r u c e i, T .b e v a n s i and T . b . e q u i p e r i d u m . M e l a r s o p r o l
is still the drug of choice in late stage trypanosomiasis. It is, however, associated with severe side effects, which are occasionally fatal. M e l a r s o p r o l -resistant trypanosomes have also been reported.
The arsenicals were the first drugs to be recognized as acting through the
formation of covalent bonds. Ehrlich suggested that some arsenicals combine with essential mercapto-groups in the parasite. Arsenicals can bind
to the cysteine thiol groups in proteins. Melarsenoxide was demonstrated
to form a stable adduct with the trypanosomatid metabolite trypanothione; abbreviated as Mel T (figure 1.6B).20 When bloodstream
T .b r u c e i is incubated with either melarsenoxide or m e l a r s o p r o l, Mel T is
the only arsenical derivative detectable in acid soluble extracts of cells.
Trypanothione is therefore regarded as a primary target for aromatic
arsenical derivatives in African trypanosomes. Having formed within the
cell, the Mel T complex might act to guide the arsenical drug toward its
ultimate cellular target. Alternatively, the complex might itself be directly
toxic. The latter effect is illustrated by the ability of Mel T to inhibit
Try(S)2 reductase activity.20 A mean intracellular concentration of 17 µM
Mel T could be achieved before cell lysis was observed. This is twice the
K i for Mel T for T . b r u c e i Try(S)2 reductase and this concentration would
have caused considerable inhibition.
Zweygarth and Kaminsky19 reported a new arsenical drug, Mel Cy
(Cymelarsan) (figure 1.6C). Like m e l a r s o p r o l , the drug is in fact a prodrug because the two cysteamine molecules have to be released from Mel
Cy before the compound is active. In chemotherapeutic studies it showed
varying degrees of susceptibility in six different T . b .b r u c e i and T .b . e v a n s i
stocks. The susceptibility varied from 4-27 nM.
DL-α-Difluoromethylornithine
14
D rug Treatm ent
I
n all mammalian cells and in many other eucaryotic cells, decarboxylation of ornithine by ornithine decarboxylase (ODC) is an obligatory
step in the biosynthesis of polyamines such as p u t r e s c i n e , s p e r m id i n e ,
and s p e r m i n e , which are ubiquitous in living cells and are thought to play
important roles in cell division and differentiation.21,22 Hence, inhibition
of this enzyme with specific enzyme-activated irreversible inhibitors such
as D L - α- d i f l u o r o m e t h y l o r n i t h i n e (DFMO) appeared to be an approach to
control abnormalities of these cellular activities (figure 1.7).
The discovery that polyamines in T .b . b r u c e i are also synthesised from
ornithine,23 suggested testing of DFMO against African trypanosomiasis
in rodents infected with T .b . b r u c e i , T . b g a m b i e n s e , T . b . r h o d e s i e n s e and
H u m a n A frican Trypanosom iasis
Figure 1.7
The structural formulas of putrescine, spermine, spermidine.
Putrescine is formed in the
ODC catalysed decarboxylation of ornithine.
DL-α-difluoromethylornithine
(DFMO) is a so-called irreversible mechanism based inhibitor (IMBI). When ODC decarboxylates DFMO, an irreversible inhibitor is formed.
T . b . c o n g o le n s e . Apart from a diminished concentration of polyamines
also inhibition of protein and nucleic acid synthesis and gross morphological changes associated with cytostasis occurred in trypanosomes.24
Trypanosomes are more sensitive to DFMO than mammalian cells.25
They may be more sensitive to the effects of polyamine depletion because they contain the unique glutathione spermidine conjugate dihydrotrypanothione. This is the cofactor of trypanothione reductase, that plays
an essential role in the detoxification of xenobiotics. Since the arsenical
drugs appear to attack this trypanothione metabolism there is a pronounced synergistic effect between Mel B and DFMO.20
It was found that DFMO notably increased host survival and produced
apparent cures. As a result of this, clinical trials were begun in Sudan in
1981 with a dramatically successful outcome. In clinical trials against late
stage T .b . g a m b ie n s e infections in the Ivory Coast, Sudan and Zaire,
T . b .g a m b i e n s e proved to be susceptible to DFMO. In many instances patients had failed one or more courses of melarsoprol before DFMO treatment.26
Bacchi e t a l . 26 tested 16 strains of T . b . r h o d e s i e n s e for susceptibility to
DFMO and seven were partially or totally refractory to this drug. These
results indicate that other novel agents or combinations of agents may be
needed for chemotherapy of the East African disease.
T
he increasing number of pathogenic trypanosomes calls for the development of new drugs. Several biochemical pathways of the parasite’s metabolism are currently under investigation to assess their vulnerability to chemotherapeutic attack. Some lead compounds that have
emerged from these investigations are now in the early stages of development.
C a r n i t h i n e m e t a b o l i s m : carnithine, an essential growth factor derived
from the metabolism of amino acids methionine and lysine, has been
found intracellularly in T . b r u c e i . The compound bromoacetylcarnithine
(BAC) inhibits parasite carnitine metabolism and prolongs the survival of
Other Leads from the
Parasite’s Metabolism
D rug Treatm ent
15
Section I : Introduction
THE ACTION OF SHAM
The aromatic hydroxamic acids, salicylhydroxamic acid and m-chlorobenzhydroxamic acid, are potent
inhibitors of the trypanosomal G3P
oxidase.29 Inhibition, however,
does not harm the bloodstream
form of T.brucei, since the organism has the capability of switching
from an aerobic to an anaerobic
type of glycolysis.
During aerobic glycolysis every triosephosphate is converted to pyruvate and one molecule of NAD+ is
reduced to NADH. NADH is reoxidised to NAD+ by glycerol-3-phosphate dehydrogenase through the
conversion of DHAP to G3P.
Due to the inhibition of G3P oxidase, the G3P concentration rises in
the glycosome because the glyco-
Intermezzo I
somal membrane constitutes a true
barrier for the phosphorylated intermediates of the glycolytic pathway.
The reversed reaction of glycerol
kinase becomes important now.
This causes the formation of one
molecule of ATP from G3P and
ADP.
In case that excess of glycerol is
present, glycerol kinase is inhibited
by its own substrate and the formation of ATP ceases. Hence anaerobic-like glycolysis is no longer
possible and the parasite dies because its energy supply is blocked.
trypanosome infected mice.1 BACs mode of action is still not understood
and further studies will be conducted on this compound.
M i c r o t u b u l e s : Compounds that disrupt the tubulin structures making up
the parasite’s chemical cytoskeleton are under study. A number of phenothiazines are active i n v i t r o but have not shown efficacy i n v i v o .
G l y c o l y t i c e n z y m e s : Trypanosomes are extremely vulnerable to inhibition
of their glycolytic metabolism. This was first observed by Clarkson and
Brohn29 when they treated rats that were infected with trypanosomes, simultaneously with salicylhydroxamic acid (SHAM) and glycerol. They
observed that the trypanosomes disappeared within 5 minutes from the
bloodstream of the rats. However, they reappeared after several days because all the SHAM and glycerol was removed from the host’s bloodstream before the parasites, hidden in the tissues and organs, were
reached (see Intermezzo I: The action of SHAM).
Hence the glycolytic metabolism seems to be a useful target metabolism
for future trypanocidal drugs. All glycolytic enzymes have been isolated
and are being assessed for their potential as target enzyme. One of these
enzymes, i . e . Triosephosphate Isomerase (abbreviated as TIM), is the target enzyme for the study presented in this thesis.
16
D rug Treatm ent
H u m a n A frican Trypanosom iasis
TRYPANOSOMAL GLYCOLYSIS
T
Trypanosomal Glycolysis
he African trypanosomes belonging to the b r u c e i subgroup are characterised by an unusual flexibility of their single large mitochondrion. In the insect stage, the mitochondrion is fully developed and respiratory chain phosphorylation provides the main source of energy. In the
bloodstream and tissue fluids of the vertebrate host, however, mitochondrial biogenesis can be completely repressed. The mitochondrion regresses into a p r o -mitochondrion that lacks a functional Kreb’s cycle and
the mitochondrial oxidative phosphorylation. The parasite depends entirely on glycolysis for energy production.28 The dependence on an inefficient energy provision is compensated by an extreme high glucose consumption.30 In trypanosomes a unique form of compartmentalisation is
found: most of the enzymes of the glycolytic pathway are contained
within an organelle, the glycosome. These organelles in T . b r u c e i bloodstream trypanomastigotes are extremely homogeneous in size (0.3 µm)
and they represent 4% of the total cell volume and 8% of the total protein
content. It has been estimated that each trypanosome contains between
200 and 300 glycosomes.
The glycolytic enzymes involved in the conversion of glucose and glycerol into phosphoglycerate constitute more than 90% of the organelle’s
protein content. The sequestering of the glycolytic enzymes within an organelle may contribute to the high glycolytic flux by locally increasing
the substrate concentrations. Indeed, tunnelling of glycolytic metabolites
through the glycosomes has been demonstrated in intact T . b r u c e i cells. It
has been shown that under conditions of low ionic strength, after disrupting the glycosomal membrane, all glycolytic enzymes, except PGI, stick
together and behave as members of a large multienzyme complex.31
According to Opperdoes32 the glycosomal membrane constitutes a barrier
to most of the glycolytic intermediates and cofactors. In T . b r u c e i the intracellular concentration of the glycolytic intermediates is not dissimilar
to that in cells from other eukaryotes. Since the glycosomes represent
only 4% of the cellular volume and contain 20-30% of the glycolytic intermediates, the concentration of the glycolytic intermediates must be at
least five times higher than in the cytosol. The concentration of both the
active sites and glycolytic metabolites have been estimated to be in the
millimolar range in the glycosome. Since each of the enzymes has a Km
for its respective substrate of the same order of magnitude, a high portion
of the glycolytic metabolites must be in the protein-bound form. This is a
further advantage of compartmentation of the glycolysis.32 It allows the
trypanosome to maintain an extremely high glycolytic flux with a relatively small amount of protein. The glycolytic flux through the T .b r u c e i
cell is 0.08 µmol glucose per minute per milligram protein, and for each
The Glycosome
T rypanosom a l G lycolysis
17
Section I : Introduction
Figure 1.8
G-6-P: glucose-6-phosphate;
FDP: fructose-1,6-diphosphate;
DHAP: dihydroxyaceton phosphate; DGAP: D-glyceraldehyde-3-phosphate; G3P: glycerol-3- phosphate; 1,3-DPGA:
1,3-diphosphoglyceric acid;3PGA: 3-phosphoglycerate; Pi:
inorganic phosphate; GAPDH:
glyceraldeyde-3-phosphate dehydrogenase; TIM: triosephosphate isomerase.
molecule of pyruvate produced one molecule of ATP is synthesized (figure 1.8).32
The Glucose
Metabolism
18
Trypanosomal Glycolysis
T
he glucose metabolism in the bloodstream-form trypanosome differs
from glycolysis in eukaryotes in a number of respects: (a ) Trypanosomes lack a lactate dehydrogenase and therefore the NADH generated in
the glycolysis is reoxidized by molecular oxygen via a dihydroxyacetone
phosphate (DHAP): glycerol-3-phosphate (G3P) shuttle combination
with a terminal G3P oxidase in the mitochondrion. This oxidase reacts
with oxygen without the intervention of pyridine nucleotide coenzymes
or cytochromes. Its high activity and specificity for G3P is sufficient to
account for the high rate of respiration of bloodstream trypanosomes; (b )
pyruvate rather than lactate is the end-product of glycolysis. It is excreted
into the host’s bloodstream; (c ) under anaerobic conditions, glucose is
converted quantitatively into equimolar amounts of pyruvate and glycerol.28,32
Since the production and consumption of ATP and NAD are balanced in
the glycosome, this organelle is not involved in the net ATP synthesis.
This occurs in the cytosol, where 3-phosphoglyceric acid (3PGA) is converted into pyruvate. Similarly, no net change in NAD takes place in the
glycosome during aerobic glycolysis because the NADH produced in the
glycosome is reoxidised by glycerol-3-phosphate dehydrogenase which
reduces DHAP to G3P which, in its turn, is reoxidised to DHAP in the
mitochondrion by the terminal G3P oxidase (figure 1.8).32
Under anaerobic conditions, glucose is metabolised at the same rate as
under aerobic conditions, with the formation of equimolar amounts of
pyruvate and glycerol. Because of the absence of oxygen, the terminal
G3P oxidase in the mitochondrion cannot convert G3P to DHAP. This re-
H u m a n A frican Trypanosom iasis
sults in a drastic increase of the G3P concentration in the glycosome
which causes a reversal of the glycerol-kinase reaction. A G3P:ADP transphosphorylase activity of glycerol kinase has indeed been found in T .
b r u c e i and the relatively high specific activity of glycerol kinase, provides for the synthesis of glycerol and ATP from G3P and ADP. Moreover, glycerol kinase functions indeed under true equilibrium conditions
in absence of oxygen, rather than catalyzing the phosphorylation of glycerol to G3P.33 This is facilitated by the ability of the ATP/ADP ratio inside the glycosome to change independently from that in the cytosol.
Therefore, under anaerobic conditions as well, no net change in ATP and
NAD occurs in the glycosome during glucose metabolism. No ATP is
consumed and the dismutation of glucose continues to produce 3-phosphoglycerate (3PGA) which is excreted in the cytosol to be converted to
pyruvate with the concomittant production of ATP.
T
wo phases can be recognised in the glycolytic pathway: an ATP consuming phase and an ATP producing phase. In the ATP consuming
phase two molecules of ATP are required for the production of glucose-6phosphate and of fructose-1,6-bisphosphate.39,40
In figure 1.8 triosephosphate isomerase (TIM) is located roughly in the
middle of the glycolytic cascade, at the end of the ATP consuming phase.
It lies between the reaction steps catalysed by aldolase and by glyceraldehydephosphate dehydrogenase. Its role in glycolysis is the conversion of
dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde-3-phosphate
(DGAP).
Triosephosphate
Isomerase
The conversion of DGAP to pyruvate yields two ATP molecules. Since
the production of fructose-1,6-bisphosphate consumes two ATP molecules and because only DGAP is utilised in the glycolytic pathway there
would be no net ATP synthesis if not all of the triose units were converted to pyruvate, with concomitant production of ATP. TIM ensures
that the glycolysis is an efficient energy producing pathway; it provides
for the complete catabolism of the whole hexose unit rather than just half
of it.39 Hence, the conversion of DHAP to DGAP by TIM is a key step in
glycolysis. TIM seems therefore a suitable target enzyme for future trypanocidal drugs.
T rypanosom a l G lycolysis
19
Section I : Introduction
An Approach to the rational Design of New Inhibitors for T. Brucei TIM
AN APPROACH TO THE RATIONAL DESIGN OF NEW
INHIBITORS FOR T. B RUCEI TIM
T
Introduction
he study presented in this thesis is part of a larger multidisciplinary
project with the common objective of rational development of new
drugs against African trypanosomiasis. The glycolytic pathway was chosen as target system.
The strategy was that of a so-called rational drug design cycle (figure 1.9)
as outlined by Hol in 1986.38 After the isolation of the glycolytic enzymes, attempts were made to crystallize them and subsequently the Xray structures of the enzymes were determined. These three-dimensional
structures were the input in the rational drug design cycle. The structures
were studied and potential ligands were designed.
Following the design, some potential inhibitors were synthesised and
tested for inhibitory activity. If active, the inhibitors were diffused into
the crystals of the glycolytic enzymes for which they were designed. In
this way a crystal of the complex of the inhibitor with the target enzyme
was obtained. Subsequently, the X-ray structure of this complex was
solved. The data obtained were evaluated and used in the design of new
inhibitors aiming at better inhibitory activity.
Scope of this thesis
he scope of this thesis is the investigation of a rational approach to
the design of new inhibitors of TIM. This enzyme had been isolated
together with the other glycolytic enzymes present in the glycosome and
it was the first enzyme that gave suitable crystals for an X-ray analy-
T
Figure 1.9
A"Rational Drug Design Cycle"
employing three-dimensional
structures of target proteins
and their complexes with putative ligands designed rationally
with the aim of becoming a
drug lead.
20
An Approach to the rational D esign of New Inhibitors for T. Brucei TIM
H u m a n A frican Trypanosom iasis
sis.36,37 An X-ray structure of high resolution has been obtained and this
structure was studied with computer modelling techniques. The aim of
these studies was the rational design of new inhibitors for this enzyme.
A serious disadvantage of TIM as target enzyme is the fact that there is a
strong homology in terms of primary and tertiary structure of the active
site of TIMs of various species (see chapter 2). This means that an inhibitor designed to bind solely in the active site, is an inhibitor with a comparable affinity for TIMs of all species.
In order to make the inhibitor specific for the trypanosomal enzyme, additional affinity contributions need to be looked for outside the active site
in areas specific for the enzyme of this species. The specific amino acid
sequence nearest to the active site, the so-called selectivity area, is still
15Å away from the center of the active site. To span this distance is not a
trivial task. The objective of this thesis is to design inhibitors that have an
anchor in the active site and an extension outside the active site, in order
to reach the selectivity area. The general requirements for these new selective inhibitors are being outlined in the next chapter (page 37). The investigation started with a known inhibitor to which some modifications
were made. These new, potential inhibitors were synthesized and tested
for inhibitory activity. This investigation is described in Chapter 3. In
Chapter 4 ensuing studies involving further modifications in order to extend the inhibitors away from the active site of the enzyme are described.
The design and synthesis of an entirely new series of potential inhibitors
designed d e n o v o are described in Chapter 5.
Finally, some variations were made on irreversibly binding inhibitors described in the literature. These inhibitors were subsequently combined
with one of the inhibitors described in Chapter 5. This study is described
in Chapter 6. In Chapter 7 the conclusions of this study are reported.
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