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Antimicrobial Agents and Chemotherapy, September 2001, p. 2571-2576, Vol. 45, No. 9
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.9.2571-2576.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Virtual Screening of Combinatorial Libraries across
a Gene Family: in Search of Inhibitors of Giardia
lamblia Guanine Phosphoribosyltransferase
Alex M.
Aronov,
Narsimha R.
Munagala,
Irwin D.
Kuntz, and
Ching C.
Wang*
Department of Pharmaceutical Chemistry,
University of California, San Francisco, California 94143-0446
Received 13 February 2001/Returned for modification 2 April
2001/Accepted 18 June 2001
 |
ABSTRACT |
Parasitic protozoa lack the ability to synthesize purine
nucleotides de novo, relying instead on purine salvage enzymes for their survival. Guanine phosphoribosyltransferase (GPRT) from the
protozoan parasite Giardia lamblia is a potential target
for rational antiparasitic drug design, based on the experimental evidence, which indicates the lack of interconversion between adenine
and guanine nucleotide pools. The present study is a continuation of
our efforts to use three-dimensional structures of parasitic phosphoribosyltransferases (PRTs) to design novel antiparasitic agents.
Two micromolar phthalimide-based GPRT inhibitors were identified by
screening the in-house phthalimide library. A combination of
structure-based scaffold selection using virtual library screening across the PRT gene family and solid phase library synthesis led to
identification of smaller (molecular weight, <300) ligands with
moderate to low specificity for GPRT; the best inhibitors, GP3 and GP5,
had Ki values in the 23 to 25 µM range. These results represent significant progress toward the
goal of designing potent inhibitors of purine salvage in
Giardia parasites. As a second step in this process,
altering the phthalimide moiety to optimize interactions in the
guanine-binding pocket of GPRT is expected to lead to compounds with
promising activity against G. lamblia PRT.
| |
INTRODUCTION |
Computer-aided drug design in
combination with combinatorial chemistry approaches, whereby focused or
diverse combinatorial libraries can be designed using computational
methods, is becoming increasingly important in the process of drug
discovery for parasitic targets (7, 11). A number of
groups have reported on the successful design of inhibitors directed
against trypanosomal (2, 4, 15-16), leishmanial
(6), malarial (19), and tritrichomonal (3, 27) targets active in the 10 nM to 50 µM range.
However, with the number of compounds that could be generated by
combinatorial chemistry growing exponentially, it has become apparent
that chemical diversity has surpassed the capacity of high-throughput
screening. In the case of antiparasitics research, which is
concentrated in a limited number of mostly academic labs, the need for
more rapid ligand screening tools has become apparent. Recently, in silico methods for database screening have come to the forefront of
drug discovery (30). By accelerating the screening
process, these methods are able to capitalize on the potential of
virtual combinatorial libraries. While a number of recent reports have focused on structure-based pruning of the virtual combinatorial libraries built around a given preselected scaffold, there has been a
growing trend toward combinatorial scaffold evaluation against a number
of biological targets. Evaluation of binding preferences for
combinatorial libraries across a range of targets could, in principle,
provide information about scaffold generality or selectivity as related
to the target selection (M. L. Lamb, K. W. Burdick, S. Toba,
M. M. Young, A. G. Skillman, X. Zou, J. R. Arnold, and
I. D. Kuntz, unpublished data.).
All protozoan parasites lack the ability to synthesize purine
nucleotides de novo. Instead, they utilize purine salvage pathways to
convert the host organism's purine bases and nucleosides to the
corresponding nucleotides (31). Purine
phosphoribosyltransferases (PRTs) catalyze the
Mg2+-dependent synthesis of purine nucleotides
via reaction of a purine base with
-D-5-phosphoribosyl-1-pyrophosphate (PRPP). Crystal structures of the type I PRTs share a common Rossman's fold and a hood
that is composed primarily of antiparallel 
-sheets positioned around
the enzyme's active site (8, 12, 20-23, 28). Inhibitors of PRTs that are able to block purine salvage in vivo could represent an efficient approach to antiparasite chemotherapy (31,
32).
Giardia lamblia, an anaerobic binucleate flagellated
protozoan that causes intestinal infection, a condition termed
giardiasis in mammals (1), relies primarily on two
independent pathways for its nucleotide synthesis. Adenine PRT and
guanine PRT (GPRT) catalyze the synthesis of AMP and GMP, respectively.
Due to a lack of interconversion between the two purine nucleotide
pools, either of the two enzymes could serve as a potential target for giardiasis chemotherapy (33).
G. lamblia GPRT shows little homology with the known
sequences of other purine PRTs (26). It possesses a rather
unique guanine-only specificity, while exhibiting very low activity
with hypoxanthine as a substrate. A recently published high-resolution
X-ray structure of G. lamblia GPRT (23)
demonstrated a number of structural differences between GPRT and other
known PRTs. The purine is stacked between two aromatic residues, Trp180
and Tyr127. While a Trp residue has been also seen at this first
position in Toxoplasma gondii hypoxanthine-guanine-xanthine
PRT (HGXPRT), tyrosine and phenylalanine are present at the
corresponding position in Tritrichomonas foetus HGXPRT and
human hypoxanthine-guanine PRT (HGPRT), respectively. The unusual
substitution is observed at the bottom of the purine binding site, with
Tyr127 taking the place of the typically well-conserved Ile or Leu
residue. Another structural difference can be noted in the position of
the conserved Lys residue, which has been shown to interact with
exocyclic O6 of the purine in all of the known structures of purine
PRTs. Lys152 of GPRT positions its
-NH2 group 6.3 Å away from the O6 of
guanine, in sharp contrast to the typically observed distance of 3 Å,
with two ordered water molecules spanning the distance. Despite the
noted structural differences, the active site preserves the bifocal
character observed in other purine PRTs, formed by the stacking
interaction at the purine binding site and the hydrogen bonding
interactions at the 5'-phosphate binding site of PRTs.
Having previously reported on the successful design of selective
T. foetus HGXPRT inhibitors based on the phthalimide
scaffold (3), we looked into the possibility of designing
G. lamblia-specific GPRT inhibitors. Two bulky
phthalimidocarboxanilides (Fig. 1) were identified as
the most active leads. At this point we decided on the following
two-stage strategy in our quest for GPRT ligands. The first stage would
involve using the virtual and synthesized phthalimide libraries to
focus the search on smaller substituents that would bind to the
5'-phosphate binding site. Following identification of such moieties,
we could then move to design novel scaffolds that would be better
tailored for the GPRT purine binding site than our starting
phthalimides. As part of this project, we will describe the design of
small phthalimide-containing molecules that retain inhibitory activity
against GPRT. Since a decrease in human HGPRT activity has been
demonstrated to be responsible for gouty arthritis and the Lesch-Nyhan
syndrome, the compounds being developed as G. lamblia GPRT
inhibitors should not interfere with mammalian HGPRT activity. Herein
we report on the utility of virtual combinatorial library screening for
the discovery of micromolar inhibitors of G. lamblia GPRT
and on applications of virtual library screening across the
representatives of the target gene family for analyzing species
selectivity in the process of scaffold selection.
| |
MATERIALS AND METHODS |
Materials.
Libraries of phthalimide-containing GPRT
inhibitors were synthesized, purified, and analyzed as reported
previously (3). Guanine and the tetrasodium salt of PRPP
were purchased from Sigma Chemical Co. (St. Louis, Mo.) and are of the
highest purity available.
Enzyme assays.
Isolation (5, 14) and assays
(34) of recombinant G. lamblia GPRT and human
HGPRT from transformed Escherichia coli were performed as
described previously. The substrates were present at the following
concentrations: 20 µM guanine (KM = 16.4 µM) and 1 mM PRPP (KM = 25.6 µM). The
compounds tested were dissolved in dimethyl
sulfoxide-d6, and concentrations were
determined by integration of nuclear magnetic resonance peaks with
methylene chloride as an internal standard. The concentration of
dimethyl sulfoxide in the assays was kept at 10%.
Structure analysis.
The following crystal structures were
used in the virtual library screening comparison: T. foetus
HGXPRT (1hgx) as determined by Somoza et al. (28),
G. lamblia GPRT (1dqn) by Shi et al. (23),
T. gondii HGXPRT (1qk3) by Heroux et al. (13),
Trypanosoma cruzi HGPRT (1tc1) by Focia et al.
(10), E. coli HGPRT (1a95) by Vos et al.
(29), and human HGPRT (1hmp) by Eads et al.
(8). Positions of the purine/pseudopurine ligands
(e.g., guanine, immucilin G phosphate, formycin B, etc.) were used to
align the structures. The Sybyl (version 6.5; Tripos Associates, St.
Louis, Mo.) software package was used for display and analysis of the
structures and to compute all atom Amber charges for the proteins.
Virtual library construction.
UC_Select (25) in
combination with the Daylight version of the Available Chemicals
Directory was used to identify original reagent sets, as well as for
the elimination of reagents that had unattractive chemical or
pharmaceutical properties. Similarity and superstructure searches of
the Available Chemicals Directory were performed with Daylight's
Merlin system (version 4.61; Daylight Chemical Information Systems,
Inc., Santa Fe, N.M.), using a Tanimoto similarity metric and
Daylight's hashed connectivity fingerprints. Sybyl's CONCORD module
was used to build the virtual reagent library. The virtual library was
prepared within Sybyl using in-house SPL scripts, and
Gasteiger-Marsili charges were computed.
Docking.
DOCK4.01 (9) was used to screen the
virtual phthalimide libraries against the target PRTs. In every case,
the previously described (3) three-step docking procedure
was used involving (i) library preorientation, followed by (ii) rigid
docking and (iii) flexible scoring. Spheres were chosen by
superimposing phthalimide on the purine or pseudopurine atom positions
derived from the respective crystal structures as reported previously
(3). The docking parameters were held constant for runs
across the PRT gene family and will be available upon request.
| |
RESULTS AND DISCUSSION |
Choice of scaffold and chemistry.
Having previously reported
on the successful design of selective submicromolar T. foetus HGXPRT inhibitors based on the phthalimide scaffold, we
looked into the possibility of designing inhibitors that would target a
related enzyme, G. lamblia GPRT. Due to the specificity
difference between the T. foetus and G. lamblia
PRTs, phthalimide would not be generally expected to serve as an
efficient scaffold in the context of the GPRT binding site. The imide
portion of the molecule mimics the
C6-N1-C2
of xanthine, one of the natural substrates of HGXPRT. It could be
expected to form unfavorable interactions with the backbone carbonyls
of Asp181 and Asp187, which directly interact with the exocyclic
N2 of guanine. However, due to the general
similarity in the shape of the active sites between the T. foetus and G. lamblia PRTs, we decided that screening of our in-house phthalimidocarboxanilide database for inhibitory activity against GPRT could potentially result in identification of a
starting point for our lead discovery efforts. This approach resulted
in identification of two active molecules (Fig. 1). Both phthalimide
derivatives contained bulky fused ring systems that most likely derived
much of their binding energy from burying the large hydrophobic
substituents. We set out to design smaller, less lipophilic molecules
that would retain their activity against GPRT.
Virtual library construction and evaluation.
As the first
step, UC_Select (25) was used to perform initial database
mining. Lipinski "druglikeness" criteria (17) served as a filter, followed by visual inspection and removal of unreactive molecules, to produce three separate reagent sets: 599 anilines, 460 primary amines, and 298 secondary amines (for a more detailed description of the procedure, see reference 3). Virtual
phthalimide libraries were prepared as described in Materials and
Methods. The anilide, primary amide, and secondary amide libraries were treated independently.
Six publicly accessible crystal structures of purine PRTs were used to
put together a target library. All of the structures
chosen have the
trademark PRT flexible loop in the "open" conformation
or
disordered (in the case of
T. foetus HGXPRT). This loop
has
been shown to act as a flap that covers the catalytic pocket to
shield the oxocarbonium transition state from nucleophilic attack
by
bulk solvent (
21-22). As a control, two purine PRT
structures
with the loop in the "closed" conformation were also
chosen for
docking: human HGPRT (1bzy) (
24) and
Plasmodium falciparum HGXPRT
(1cjb) (
22). In
the case of the control structures we observed
that >98% of the
library members received a positive score, i.e.,
were classified as
nonbinders (data not shown). However, that
would be inconsistent with
our previously published results (
3)
showing that human
HGPRT was inhibited by most phthalimides in
the 25 to 250 µM range.
This result suggests that the inhibitors
bind to the open or partially
open form of the
enzyme.
The three virtual libraries were successively docked to all of the
target structures. The results for 100 top-scoring
compounds
from each run are shown in Fig.
2. We have previously
speculated
(
3) that the phthalimide scaffold could
potentially have built-in
selectivity for
T. foetus HGXPRT
over human HGPRT, since the human
enzyme does not act on xanthine.
Indeed, based on our virtual
screening data, the tritrichomonal enzyme
has the highest level
of susceptibility to inhibition by phthalimides,
with
T. gondii HGXPRT as the second highest on our PRT list.
Of the three HGPRTs
on the list, the human enzyme appears to be the
most susceptible,
while the trypanosomal isozyme would be predicted to
bind poorly
to the compounds in the three libraries. Based on the
virtual
performance of the phthalimides against the two HGXPRT enzymes
in the panel, one would also anticipate a similar showing against
the
malarial enzyme. Given the kinetic parameters for
P. falciparum HGXPRT (29 µM for xanthine) (37),
phthalimides represent a good
potential scaffold for designing
inhibitors of the malarial enzyme.
In conclusion, the variation in the
library performance across
the targets could be deconvoluted into (i)
scaffold preference
based on substrate specificity for the purine
binding site and
(ii) structural differences within the 5'-phosphate
binding pocket.
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FIG. 2.
Comparative performance of phthalimide-based virtual
libraries across the PRT gene family. 1, T. foetus
HGXPRT; 2, G. lamblia GPRT; 3, T. gondii
HGXPRT; 4, T. cruzi HGPRT; 5, E. coli
HGPRT; and 6, human HGPRT. The following letters refer to libraries: a,
phthalimidocarboxanilides; b, primary phthalimidocarboxamides; c,
secondary phthalimidocarboxamides. The y axis represents
absolute DOCK score values for the top 100 scoring members of
individual libraries. Actual scores were negative in all cases.
|
|
Evaluation of consensus substituents.
For G. lamblia GPRT, the primary amide library would be predicted to be
the most active of the three virtual libraries tested. In all three
cases, based on the results of virtual library screening, building
selectivity into the system would be expected to be one of the major
challenges. As the first step in our drug design effort against
G. lamblia GPRT (see the introduction), the top 10% of the
scorers in both the anilide and primary amide libraries were visually
evaluated. A number of consensus substitutions shared by most of the
top scorers were identified, and the best ligands were chosen for
synthesis. In the case of the anilide library, a small substituent at
the meta position in the aromatic ring was predicted to be
beneficial; most top scorers had a methyl or a chloro substituent
placed at that position. A p-fluoro group was also found to
be prevalent among the top scoring anilides. A number of different
substituents were placed at the ortho position, occupying a
region proximal to the flexible loop. The o-chloro group was chosen as representative of that class of compounds. In the primary amide library, a consensus substituent was a
para-substituted phenethyl group, with small substituents
predicted to be best at this position.
A straightforward chemical scheme implemented to prepare
the consensus substituted phthalimides (Fig.
3) has been
described
previously. Briefly, we used trimellitic anhydride as a
starting
material, which upon conversion to 4-carboxyphthalimide was
attached
to the trityl chloride-functionalized resin. Target compounds
were produced on solid support and were cleaved from the resin
in the
final step. Six compounds bearing consensus substitutions
were
prepared, and the results are shown in Table
1. Four of
six compounds prepared showed GPRT inhibitory activity comparable
to
that of the lead compounds 1 and 2. GP5 was one of the most
active and
also the smallest anilide prepared. According to our
model, it forms a
hydrogen bond with Asp125 of GPRT. As predicted,
a small hydrophobic
substituent at the
para position in the phenethyl
group was
well tolerated in the primary amide series, with GP3
being fivefold
more active than the unsubstituted
N-phenethyl
amide
(A. M. Aronov and C. C. Wang, unpublished data). Both GP3
and
GP5 were shown to act as competitive inhibitors of GPRT, with
Ki values of 23 and 25 µM, respectively
(data not shown).
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FIG. 3.
Chemical scheme for preparation of the consensus
substituted phthalimides. a,
(NH4)2CO3, melt; b, trityl chloride
resin, DIPEA, CH3CN; c, amine library, PyBrOP, DIPEA,
DMA, 5 days; d, TFA, 3 h. The sphere represents resin support.
|
|
The model for the binding of GP3 and GP5 within the active site of
G. lamblia GPRT is shown in Fig.
4. We
decided to prepare
a number of analogues to test this binding
hypothesis (Table
2).
According to the model, placing an
o-amino substituent instead
of the
m-hydroxyl is
not expected to significantly alter activity,
while small hydrophobic
substitutions at the
ortho position were
disallowed due to
unfavorable interactions with Asp125 and the
backbone carbonyl of
Tyr127. Indeed, the amino-substituted analog
GP11 was only moderately
less active than GP5, while addition
of an
o-methyl group in
GP13 had a deleterious effect on activity.
In the primary amide analog
series, the model predicted that a
larger hydrophobic substituent was
somewhat disfavored at the
para position, while a group
containing two or more heavy atoms
could not be accommodated within the
5'-phosphate binding pocket
of GPRT, clashing with C
of Asp125 and
C
of Ile134. A bromine
introduced at the
para position in
GP9 faired worse than the chloro
substitution in GP3, while GP10 did
not possess any appreciable
GPRT inhibitory activity. Placing small
hydrophobic substituents
at the
meta position did not yield
any improved binders. The most
active compounds from both series showed
little preference for
G. lamblia GPRT when tested for
inhibition of the human HGPRT
(Table
3), with measured
selectivity ranging from 1.6-fold to
>7-fold.
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FIG. 4.
Modeled binding mode of GP3 (red) and GP5 (blue) within
the active site of G. lamblia GPRT (23).
Electrostatic potential surface shown for GPRT (negative potential in
red, positive in blue) was generated using MOLCAD (Sybyl, version 6.5;
Tripos Associates).
|
|
Overall, the experimental findings agree with the proposed model. The
most apparent feature of the predicted binding modes
for the two
inhibitor series is that in both cases the aromatic
ring positions
itself in the 5'-phosphate binding site, proximal
to the conserved
Ser130 and Thr133, while the phthalimide does
not appear to have a
single binding mode. The phthalimide scaffold
is not optimal for the
purine binding site, with the best compounds
reported herein being
approximately 1- to 2-orders-of-magnitude-weaker
binders than the best
rationally designed phthalimide-based
T. foetus HGXPRT
inhibitors described previously. These compounds
show only low to
moderate specificity for
G. lamblia GPRT. However,
this
scaffold can be used in the initial stage of the design of
GPRT
inhibitors, serving as a vehicle to rapidly optimize the
N-amide
portion of the inhibitors. The second part of our GPRT
inhibitor design
strategy is going to involve using the optimized
N-amide moieties of
GP3 and GP5 to go back and design a more selective
scaffold that would
occupy the purine binding portion of the active
site. For example, a
library of synthetically accessible carboxylic
acids coupled to the
m-aminophenol or
p-chlorophenethylamine could
be
screened in silico to produce better alternatives to phthalimide
in the
context of the guanine-binding cleft of GPRT. The results
of this
investigation will be reported in due
course.
In conclusion, we have used virtual combinatorial library screening
across the representatives of the PRT gene family to make
in silico
predictions of combinatorial library performance against
a panel of
related enzymes. This approach has been applied to
the discovery of
micromolar inhibitors of
G. lamblia GPRT. Phthalimide-based
GPRT inhibitors displayed low to moderate selectivity for the
parasite
enzyme. Extension of the virtual screening matrix may
prove useful in
the process of scaffold selection for the purine
binding
site.
| |
ACKNOWLEDGMENTS |
We thank Paul Ortiz de Montellano for his support and insights in
the course of this project. We are also grateful to Kenneth Foreman for
helpful discussions.
This work was supported by the National Institutes of Health (grant
AI-19319 to C.C.W. and grant GM31497 to I.D.K.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pharmaceutical Chemistry, 513 Parnassus Ave., Box 0446, University of California, San Francisco, CA 94143-0446. Phone: (415) 476-1321. Fax:
(415) 476-3382. E-mail: ccwang{at}cgl.ucsf.edu.
Present address: Vertex Pharmaceuticals Inc., Cambridge, MA 02139.
| |
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Antimicrobial Agents and Chemotherapy, September 2001, p. 2571-2576, Vol. 45, No. 9
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.9.2571-2576.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
