The World Health Organization estimates that nearly 500,000 people in
sub-Saharan Africa are infected with trypanosomiasis, also known as sleeping
sickness [1]. At present the disease is resurging with prevalence levels
reminiscent of the great epidemic of the 1930s, which killed half a million
people. If untreated African sleeping sickness is always fatal. Available
drug therapy is unsatisfactory because of the need for parenteral administration
,
the emergence of resistant parasites, and severe toxicity - between 5 and
10 % of the patients die as a direct result of melarsoprol administration
[2]. Clearly, better drugs are urgently needed.
Mathematical models show that in the bloodstream form of trypanosomes
glyceraldehyde-3-phosphate exerts considerable control on the glycolytic
flux, and appears therefore to be a good drug design target [4]. The crystal
structures of both the parasite and the human enzyme have been solved [5,6].
While their structures are identical in the active sites, distinct structural
differences occur in and around the binding pocket for the adenosine moiety
of the NAD cofactor. NAD exhibits a weak affinity for both the parasite
and human enzyme, with Km values of 0.45 and 0.04 mM, respectively. Hence,
it is no surprise that adenosine is an even weaker competitive inhibitor,
with a Ki of 50 mM [7]. Despite the widespread prejudice against millimolar
leads we picked adenosine as a starting point for developing selective
inhibitors that would outcompete the cofactor of trypanosomal GAPDH.
Materials and Methods
COMPUTER MODELING
The three-dimensional structures of potential inhibitors were constructed
interactively in the T. brucei GAPDH structure using the molecular
modeling program BIOGRAF [9]. Subsequently, the most promising inhibitors
were docked by Monte Carlo methods with the QXP software [10].
CRYSTALLOGRAPHY
Co-crystals of L. mexicana GAPDH with N6-benzyl-NAD were grown
in sitting drops. Cryo-data to 3.4 A resolution were collected at SSRL
beam line 7-1 and the structure was solved by molecular replacement methods.
Rigid-body refinement of the four enzyme subunits lowered the R-factor
to 30%. Because of the limited resolution, individual atomic positional
refinement was not pursued. Fourfold non-crystallographic symmetry averaging
of only the protein improved the electron density map considerably and
resulted in unambiguous density for the modified cofactor.
ENZYME ASSAYS
GAPDH activity was measured in the direction of NADH formation by monitoring
absorption at 340 nm. The reaction mixture contained 0.8 mM GAPDH and 0.19
mM NAD. All designed compounds were tested against L. mexicana GAPDH.
The best inhibitors were assayed against T. brucei GAPDH.
BIOLOGICAL ASSAYS
Strain 427 T. brucei was obtained from K. Stuart (SBRI, Seattle,
WA) and the bloodstream form of the parasites was cultured in HMI-9 medium
containing 10% fetal calf serum. Murine 3T3 fibroblast cells were grown
in monolayers. The growth of parasite and human cells was determined with
Alamar Blue (TM). Alamar Blue quantitation of T.brucei was verified
to correspond with visual counts determined with a hemacytometer.
Excretion of pyruvate from bloodstream form T. brucei into the
media was determined in cells from a mid-log culture. Pyruvate was quantified
by conversion into lactate with lactate dehydrogenase.
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Results
SELECTIVITY
The NAD adenosine binding environment differs substantially between
T. brucei and human GAPDH. In the parasite enzyme there exists a
hydrophobic cleft adjacent to the ribose O2'. This cleft is absent in the
human enzyme due to a different protein backbone conformation. Hence, this
cleft provides an excellent opportunity to convey selectivity to our designed
inhibitors. Docking studies with various hydrophobic ring systems quickly
showed that only a benzene ring is narrow enough to fit in the cleft. However,
it was not immediately clear how such a ring might be linked to the adenosine
ribose. A link via O2' appeared inappropriate as this hydroxyl is a hydrogen
bond donor to Asp 37. Thus, creating an ether or ester linkage would come
with a high desolvation price. Modeling of various N2' derivatives revealed
that an amide linker would be a proper hydrogen bond donor substitute and
orient the benzene ring into the cleft. Also, it appeared that extra hydrophobic
interactions could be picked up by introducing a methoxy substituent of
the aromatic ring. Figure 1 shows how the designed inhibitor 2'-deoxy-2'-(3-methoxybenzamido)-adenosine
fits beautifully into the selectivity cleft of T. brucei GAPDH.
Figure
1: Binding mode model of 2'-deoxy-2'-(3-methoxybenzamido)adenosine
to T. brucei GAPDH, with the solvent accessible surface shown as
dots. The 3-methoxybenzamido moiety fits in the selectivity cleft formed
by Met 38 and Val 205*.
Enzyme inhibition studies showed that 2'-deoxy-2'-(3-methoxybenzamido)adenosi
ne
inhibits T. brucei GAPDH 45 times better than adenosine. More importantly
,
no evidence of human GAPDH blocking could be detected. Thus, we succeeded
in obtaining a selective inhibitor [7].
AFFINITY BY SCREENING
Having solved the selectivity problem, we then focused on improving
the affinity of our lead [11]. Modeling showed that the introduction of
a 2-thienyl at position 8 of the purine ring would bury a major part of
the Leu 112 side chain (Figure 1). After synthesis we discovered that a
180 fold gain in affinity with respect to adenosine was obtained this way.
Unfortunately, 8-adenine substituents appeared in computro to be sterically
incompatible with 2'-(3-methoxybenzamido) because they clash with the amido
oxygen. Experimental evidence for this conclusion came after we subsequently
synthesized 2'-deoxy-2'-(3-methoxybenzamido)-8-(2-thienyl)adenosine. This
compound was only a millimolar inhibitor, worse than its mono-substituted
parent compounds.
Subsequently, we shifted our attention to the N6 position of the purine.
This atom is adjacent to two hydrophobic areas on the protein surface,
one formed by the side chains of Leu 112, Phe 113 and Arg 91, another one
by Met 38 and Arg 91 (Figure 1). Therefore, we purchased five N6-adenosine
derivatives with hydrophobic substituents for screening, and synthesized
nine others. Incorporating N6 into an amide function appeared to be detrimental,
but several amines worked fine. Especially N6-benzyl-adenosine looked promising
with a 10-fold affinity gain over adenosine.
Figure
2: Modeled binding mode of N6-benzyl-adenosine to T. brucei
GAPDH, with the solvent accessible surface shown as dots. The N6-benzyl
group fits in a hydrophobic environment. Note that an alternative binding
model is possible in which the benzyl fits in between the Met 38 and Arg
91 side chains.
The benzyl substituent of N6-benzyl-adenosine can be modeled into the
binding site in two different orientations (Figure 2). We resolved this
dilemma by a crystal structure determination. Because we co-crystals of
parasite GAPDH with N6-benzyl-adenosine failed to grow, we synthesized
the N6-benzyl-NAD analogue and obtained crystals. In the experimental
structure with L. mexicana GAPDH the N6-benzyl is sandwiched between
the side chains of Met 39 and Arg 92, corresponding to Met 38 and Arg 91
in T.brucei (Figure 3).
Figure
3: Experimental binding mode of N6-benzyl-NAD to L.mexicana
GAPDH.
AFFINITY BY DESIGN
N6-Benzyl-adenosine formed the basis for further affinity improvement
by design. A search in the Available Chemicals Database 95.2 revealed the
commercial availability of 1,124 benzylamines of which 88 appeared suitable
for reaction with 6-chloropurine riboside. All 88 were modeled as N6-adenosine
derivatives in the T. brucei binding site. Poorly fitting molecules
were rejected. After similarity clustering we decided on the synthesis
of six of them. All six proved to be more potent than N6-benzyl-adenosine
(Table 1). The best compound was N6-(1-naphtalenemethyl)adenosine, with
a 333-fold gain in affinity over adenosine.
Table 1: Inhibition of L.mexicana GAPDH by N6-adenosine derivatives
N6-substituent IC50(µM)
benzyl 4,2002-methylbenzyl 700
3-methylbenzyl 750
1,2,3,4-tetraH-1-naphtyl 360
1-naphtalenemethyl 150
2-[2-(hydroxymethyl)phenylthio]-benzyl 340
diphenylmethyl 240
COMBINING SELECTIVITY WITH AFFINITY
Several of the N6-substituents were combined with the 2'-(3-methoxybenzamido)
substituent, leading to potent and selective inhibition (Table 2).
Table 2: Selective parasite GAPDH inhibition by N6-substituted
2'-deoxy-2'-(3-methoxybenzamido)adenosine derivatives (IC50 in µM)
N6-substituent L. mexicana T. brucei humanbenzyl 16 159 >530a2-methylbenzyl 4 40 >270a
1-naphtalenemethyl 0.2 2 >200a
a insoluble above and non-inhibitory at stated concentration
In particular the 1-naphtalenemethyl derivative affords submicromolar
inhibition. Its modeled binding mode is shown in Figure 4. None of the
designed compounds inhibited the human GAPDH to any degree at submillimolar
concentrations, their upper solubility limit.
Figure
4: Modeled binding mode of 2'-deoxy-2'-(3-methoxybenzamido)-N6- (1-naphtalen
e-methyl)adenosine
to L.mexicana GAPDH.
BIOLOGICAL ACTIVITY
Several of our designed compounds were tested for their potency in inhibiting
parasite growth. Encouragingly, the ranking of the ED50 values thus obtained
and the IC50 values in the enzyme inhibition assay was identical. The most
potent GAPDH inhibitor, produced 50% growth inhibition of bloodstream
form T. brucei at a concentration of 30 µM. Simultaneously,
pyruvate excretion by the parasite was shut down in a dose-dependent manner.
Importantly, none of our designed GAPDH inhibitors was toxic for the mammalian
cells we assayed at concentrations up to 0.05 mM.
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Discussion and Conclusion
The present study underlines the value of a structure-based approach
to dramatically improve the affinity of a 50 millimolar lead. Such leads
are generally ignored by the pharmaceutical industry, which insists on
micromolar leads. The fact that we obtained five orders of magnitude affinity
gain in one round of drug design shows that more optimism may be warranted.
Also, the current results demonstrate that selectivity can be built into
inhibitors.
During the course of this project many collegues have expressed concerns
of using adenosine as a scaffold because of nature's ubiquitous use of
this moiety in cofactors and co-substrates, such as NAD, FAD, ATP, etc.
However, our 200 nM inhibitor's shape is a very significant departure from
that of unmodified adenosine. In fact, it may be considered structurally
as different from adenosine as an HIV-protease inhibitor from poly-alanine.
Furthermore, we have tested our best inhibitor against several ATP- and
NAD-dependent enzymes, such as phosphoglycerate kinase, lactate dehydrogenase
and glycerol-3-phosphate dehydrogenase, and found no inhibition.
Our optimism is further corroborated by the first in vitro test results.
The most potent GAPDH inhibitors were most toxic for the parasites and
blocked pyruvate production. None of these effects were seen in the parallel
studies with mammalian cells. While the current results are highly suggestive
we cannot rule out the possibility that our inhibitors kill the parasites
by other mechanisms. Studies to resolve this issue are underway.
ACKNOWLEDGEMENTS
Thanks go to the following collaborators at the University of Washington:
Alex Aronov and Dr. Michael Gelb for synthesis of the inhibitors and enzymatic
assays, Dr. Stephen Suresh for the crystal structure determination of the
complex between N6-benzyl-NAD and L. mexicana GAPDH, Dr. Frederick
Buckner for assessing the potency of the inhibitors in vitro, and Dr. Wim
Hol for leading the project. This project has also benefited in earlier
stages from contributions by Dr. Paul Michels, Dr. Veronique Hannaert and
Dr. Fred Opperdoes (Christian de Duve Institute of Cellular Pathology,
Brussels, Belgium), Dr. Serge Van Calenbergh, Dr. Arthur Van Aerschot and
Dr. Piet Herdewijn (REGA Institute, Leuven, Belgium).
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