Discovery of Potent and Selective Inhibitors of Trypanosoma brucei Ornithine Decarboxylase

Abstract

Human African trypanosomiasis, caused by the eukaryotic parasite Trypanosoma brucei, is a serious health problem in much of central Africa. The only validated molecular target for treatment of human African
trypanosomiasis is ornithine decarboxylase (ODC), which catalyzes the first step in polyamine metabolism. Here, we describe
the use of an enzymatic high throughput screen of 316,114 unique molecules to identify potent and selective inhibitors of
ODC. This screen identified four novel families of ODC inhibitors, including the first inhibitors selective for the parasitic
enzyme. These compounds display unique binding modes, suggesting the presence of allosteric regulatory sites on the enzyme.
Docking of a subset of these inhibitors, coupled with mutagenesis, also supports the existence of these allosteric sites.

Full text

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J Biol Chem. 2010 May 28; 285(22): 16771–16781.
Published online 2010 March 10. doi: 10.1074/jbc.M109.081588
PMCID: PMC2878083
Discovery of Potent and Selective Inhibitors of Trypanosoma brucei Ornithine
Decarboxylase
David C. Smithson, Jeongmi Lee, Anang A. Shelat, Margaret A. Phillips, and R. Kiplin Guy
From the Department of Chemical Biology and Therapeutics , St. Jude Children's Research Hospital, Memphis, Tennessee 38105,
the Graduate Program in Chemistry and Chemical Biology, University of California, San Francisc o, California 94143-2280, and
the Department of Pharmacology, University of Texas Southw estern Medical Center, Dallas, Texas 75390-9041
To whom correspondence should be addressed: 262 Danny Thomas Place, Mail Stop 1000, Memphis, TN 38105-3678., Fax: 901-595-5715;
E-mail: kip.guy@stjude.org.
Perf ormed a portion of this research w hile on appointment as a United States Department of Homeland Security Fellow under the Department
of Homeland Security Scholarship and Fellow ship Program, a program administered by the Oak Ridge Institute for Science and Education for
the Department of Homeland Security through an interagency agreement w ith the United States Department of Energy.
Received November 2, 2009; Revised February 3, 2010
Copyright © 2010 by The American Society f or Biochemistry and Molecular Biology, Inc.
Abstract
Human African trypanosomiasis, caused by the eukaryotic parasite Trypanosoma brucei, is a serious
health problem in much of central Africa. The only validated molecular target for treatment of human
African trypanosomiasis is ornithine decarboxylase (ODC), w hich catalyzes the first step in polyamine
metabolism. Here, we describe the use of an enzymatic high throughput screen of 316,114 unique
molecules to identify potent and selective inhibitors of ODC. This screen identified four novel families of
ODC inhibitors, including the first inhibitors selective for the parasitic enzyme. These compounds
display unique binding modes, suggesting the presence of allosteric regulatory sites on the enzym e.
Docking of a subset of these inhibitors, coupled with mutagenesis, also supports the existence of these
allosteric sites.
Keywords: Cell/Division, Diseases, Enzymes/Decarboxylase, Enzymes/Inhibitors,
Metabolism/Regulation, Organisms/Parasite, Polyamines, Ornithine Decarboxylase
Introduction
Human African trypanosomiasis, caused by the eukaryotic parasite Trypanosoma brucei, is a most
neglected disease in central Africa, with at least 50,000 active cases and 17,000 new cases each year (1).
The disease is fatal if left untreated (2). Current treatments require prolonged drug regimens with drugs
that have unacceptable toxic side effects (3). Additionally, with one exception, current drugs have poorly
understood modes of action (3). The only clinically validated molecular target for treatment of human
African trypanosomiasis is ornithine decarboxylase (ODC), which catalyzes the decarboxylation of
ornithine to produce putrescine, the first step in polyamine metabolism (Fig. 1). The polyamines
putrescine, spermidine, and spermine are known to be necessary for cellular replication (4, 5). Increases
in polyamine concentration have also been linked to carcinogenesis. Therefore, inhibitors of the
polyamine biosynthetic pathway have been extensively investigated as potential chemotherapeutic and
chemopreventative compounds (reviewed in Ref. 6).
In mammalian systems, ODC is tightly controlled via transcriptional, translational, and post-
translational mechanisms (6,–8). It is active as a homodimer and is dependent upon binding to
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pyridoxal 5′-phosphate (PLP), a cofactor shared with many other enzymes (9). The mammalian ODC
protein has one of the shortest known half-lives (∼10–20 min), which is primarily regulated by
antizyme, a polyamine-inducible protein inhibitor that binds to ODC monomers and targets ODC for
degradation (10). The levels of the polyamines are further regulated by inter-conversion of individual
pools and by a highly efficient transport system allowing import and export of polyamines and
intermediates (11). This highly redundant regulation means that mammalian cells are strongly resistant
to changes in polyamine levels. The most widely used inhibitor of mammalian ODC is α-
difluoromethylornithine (DFMO), a highly selective compound that alkylates Cys-360, a catalytic
residue in the ODC active site (12, 13). DFMO is orally available but rapidly cleared (t½ of 1.5 h
(intravenous dosing) to 4 h (oral dosing)) (14). It is relatively nontoxic and can be dosed to extremely
high levels (up to 3.75 g/M) with only minor side effects (15). Despite this, DFMO has largely been
abandoned as a chemotherapeutic due to poor efficacy, w hich has been attributed to the robustness of
the mamm alian polyamine pool. Recently, interest has risen in the use of DFMO as a
chemopreventative in combination with other agents (16,–18).
In T. brucei, polyamine biosynthesis is much simpler (Fig. 1). There are no inter-conversion pathw ays,
and transport of exogenous polyamines plays a lesser role (19). I n addition, T. brucei ODC is much
longer lived than its mammalian counterpart, and levels are not actively regulated (20, 21). The
pathway is regulated in T. brucei apparently via activation of S-adenosylmethionine decarboxylase by
heterodimer formation with a catalytically inactive homolog termed prozyme (22). Prozyme expression
is in turn regulated in response to changes in polyamine levels (23). Depletion of polyamines either by
inhibition of the biosynthetic enzymes or by gene knockdown leads to reduced trypanothione levels and
to cell death (19, 23, 24). DFMO is clinically approved for the treatment of human African
trypanosomiasis, and a new combination with nifurtimox is now the recommended frontline therapy
for late stage Trypanosoma brucei gambiense (25). The mechanism of action of DFMO has been
demonstrated to be the inhibition of polyamine biosynthesis (3). However, the poor pharmacokinetic
behavior of DFMO is a m ajor limiting factor in its use (26). Furthermore, DFMO is only effective
against one of two subspecies of T. brucei causing human disease (27). As the parasite cannot increase
ODC levels in response to polyamine depletion, the development of potent reversible inhibitors may
allow inhibition of ODC to be fruitful in both subspecies (19).
To date, most discovery efforts directed toward ODC have been focused on analogs of ornithine (such as
DFMO), putrescine, or PLP (28). None of these has proved as effective as DFMO for treatment of T.
brucei infections. No large scale efforts to discover novel inhibitors have been reported. The lack of prior
high throughput screening efforts directed at ODC is due in part to the difficulty in assaying its activity.
Classical assay methods utilize capture of CO from radiolabeled ornithine or derivatization of
putrescine followed by high pressure liquid chromatography analysis (29, 30). Neither of these
techniques is tenable for a large scale screening effort.
Therefore, we have optimized an enzyme-linked assay suitable for high throughput screening of ODC.
This assay links the production of CO to the consumption of NADH using phosphoenolpyruvate
carboxylase and malate dehydrogenase (31). The CO produced by ODC is captured as bicarbonate by
the basic reaction buffer and then used by phosphoenolpyruvate carboxylase to carboxylate
phosphoenolpyruvate, generating oxaloacetate. This is then reduced to malate by malate dehydrogenase
in an NADH-dependent fashion allowing the reaction catalyzed by ODC to be monitored by measuring
the decrease in the absorbance of NADH. The optimization of this technique for high throughput
applications will be reported elsewhere. Herein, w e report the use of this method to identify active
compounds from a library of 316,114 unique compounds. This effort led to the discovery of four novel
inhibitory chemotypes possessing previously uncharacterized modes of inhibition. A subset of the
inhibitors appeared to bind to a novel site that was characterized by molecular docking and mutagenesis
techniques.
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Materials
Screening Library
Purification of T. brucei ODC (TbODC) and Human ODC (hODC)
Site-directed Mutagenesis of TbODC
Assay Automation
EXPERIMENTAL PROCEDURES
All chemicals were used as purchased from their vendors. Deionized water was filtered with a
MilliQ Synthesis Ultra-Pure water system (Millipore, Billerica, MA) immediately before use. Infinity
carbon dioxide liquid stable reagent was purchased from Thermo Fisher Scientific (Waltham, MA). L-
Ornithine, PLP, and dithiothreitol (DTT) were purchased from Sigma. DFMO was purchased from
Chem-Impex International (Wood Dale, IL). All plate-based enzymatic assays were performed in 384-
well black-sided, clear-bottomed polystyrene microtiter plates (catalog no. 3702) from Corning Life
Sciences (Acton, MA).
The compound library at St. Jude Children's Research Hospital was assembled from
commercially available collections, including the following: Prestwick Chemical Library (Prestwick
Chemical, Illkirch, France); the LOPAC Collection (Sigma); the Spectrum Collection, NINDS Collection
(National Institutes of Health); Natural Product Collection and Killer Plate Collection (Microsource
Discovery Systems, Gaylordsville, CT); Chemical Diversity (San Diego); ChemBridge (San Diego); Life
Chemicals (Burlington, Ontario, Canada); and Tripos (St. Louis, MO). The library was constructed by
filtering available compounds using a combination of physiochemical metrics chosen to improve
bioavailability and functional group metrics chosen to reduce the likelihood of nonspecific or artifactual
hits (32, 33). The filtered compound list was used to generate maximally diverse clusters by reducing the
compounds to core fragments using the method of Bemis and Murcko (34). The clusters were
prioritized based on the diversity of the existing library. Five to 20 compounds are required per cluster.
Clusters of more than 20 available compounds were preferred, with a maximum of 20 compounds
being purchased from within each cluster.
TbODC and hODC were expressed as N-
terminal His tag fusion proteins in Escherichia coli BL21(DE3) cells as described previously (31).
Protein w as purified by Ni -nitrilotriacetic acid-agarose column followed by Superdex 200 gel
filtration column chromatography. Fractions containing the desired protein were identified by SDS-
PAGE. Those containing ODC were combined and concentrated using an Amicon ultracentrifugal filter
device (10-kDa cutoff, Millipore, UFC901024) to concentrations of ∼40 mg/ml. Y ields of purified
TbODC were generally 7–13 mg/liter of cultured cells. Protein concentration was determined by
Bradford assay. Y ields of purified hODC were ∼2–5 mg/liter of cultured cells.
The S367A and S420A TbODC mutants were produced using the
pODC29 plasmid that encodes the wild-type TbODC with the QuikChange mutagenesis kit
(Stratagene, La Jolla, CA using the following forward primers: 5′-
GTCGTAGG AACTTCTGCCTTTAATGGATTCCAG-3′ and 5′-
CCTTTAATGGATTCCAGGCTCCGACTATTTACTATG-3′ for S396A and S402A, respectively (desired
mutations in boldface type). The TbODC D364E mutant was generated using the standard Kunkel
technique in the Bluescript vector (Stratagene, La Jolla, CA) using the M13 helper phage (Stratagene,
La Jolla, CA) and the Kunkel strain BO265 (35). The primer used for this mutant was 5′-
ATGTGATGGGCTCGAGCAGATAG.
All screening data were generated on a High Resolution Engineering (Woburn, MA)
integrated screening system using Liconic plate incubators (Woburn, MA) and a Stabuli T60 robotic
arm (Stabuli, SC). All automated screening was performed under a nitrogen atmosphere as described
previously (36). Assay solutions were dispensed using Matrix Wellmates (Matrix Technologies, NH)
equipped with 1-μl rated tubes. Plates were centrifuged after all bulk liquid additions using a Vspin plate
centrifuge (Velocity11, Menlo Park, CA). Compound transfers were performed using a 384-well pin tool
equipped with 10-nl slotted hydrophobic surface-coated pins (V&P Scientific, San Diego, CA). This
allowed delivery of ∼25 nl of DMSO stock solution with coefficients of variation of less than 10%. All
absorbance data were measured using an EnVision Multilabel Plate Reader equipped with a 340 nm
narrow bandwidth filter (PerkinElmer Life Sciences, 2100-5740).
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ODC-Phosphoenolpyruvate Carboxylase-Malate Dehydrogenase-linked Assay
Primary Screening Data Analysis and Reaction Rate Calculation
Phosphoenolpyruvate Carboxylase-Malate Dehydrogenase-link ed Assay
This assay was performed under
nitrogen atmosphere as described previously (36). Assay buffer (66 mM Tris, 25 mM NaCl, 8 mM MgSO ,
0.01% Triton X-100, pH 8.05) was prepared daily in water. Plates and compounds were allowed to
equilibrate in the presence of ODC for 20 min before L-Orn was added to start the reaction. Final
primary screening assay conditions were 2.3 mM DTT, 60 μM PLP, 625 μM L-Orn, 150 nM TbODC, 10 μM
test compound, and 60% Infinity CO (v/v) in assay buffer with a final volume of 25 μl. Reaction
progress was monitored by decrease in absorbance at 340 nm using an Envision plate reader
(PerkinElmer Life Sciences) equipped w ith a narrow bandwidth 340 nm filter (PerkinElmer Life
Sciences, 2100-5740).
Compounds for screening were placed in 384-well polypropylene plates (Corning Life Sciences, Acton,
MA) at 10 mM concentrations in DMSO. Sixteen positive controls (DFMO, 1 M in DMSO) and 16
negative controls (DMSO) were placed in a single separate 384-well polypropylene plate and pin-
transferred to test plates after the addition of variable compounds.
Cuvette assays for low throughput re-testing were performed as described above with the following
minor modifications: the final assay volume in cuvettes was 500 μl at 40% Infinity carbon dioxide
liquid stable reagent, 50 μM PLP, 50 μM DTT, 1% DMSO, and varied ornithine concentrations from 10
mM to 100 μM. As with m icroplate assays, assay buffer (66 mM Tris, 25 mM NaCl, 8 mM MgSO , 0.01%
Triton-X, pH 8.05) was prepared fresh daily. Cuvette assays were performed under normal atmosphere
at 37 °C.
Primary screening data analysis was performed
using custom protocols (RISE 3.0) written in Pipeline Pilot (version 7.0, Accelrys) and the R program
(version 2.5.0) (37). Kinetic data from the full 6-min observation (6 points in total) were fit to a linear
model using a robust, iteratively re-w eighted least squares algorithm (“lmrob” function in robustbase R
package, version 0.2-7) that reduces the influence of outliers compared with classical least squares (38).
The slope values from kinetic data fits w ere taken as end points that w ere then used for the calculations
of all other plate statistics. Only plates passing the minimum Z-prime and Z-factor thresholds of 0.4 and
0.4, respectively, were accepted. Initial screening hits were determined on a plate-by-plate basis by
identifying compounds with activities that were simultaneously outliers from the negative control and
variable compound populations. The outlier cutoffs were calculated as the upper fourth plus 1.5 times
the fourth spread (the upper fourth and fourth spread are similar to the third quartile and interquartile
range, respectively), which corresponds to a p value ∼0.005 for normal distributions. However, such
cutoff criteria are more robust to population deviations from normality (39). For calculations of Z-
scores, 16 positive and 16 negative controls were used unless otherwise stated. Z-scores were calculated
as described previously (40). Reaction rate was changed from absorbance units/min to millimolar
NADH/min using an extinction coefficient of 6.349 absorbance units mM cm for NADH and an
approximate path length of 0.4 cm for assays performed at 25 μl of final volume in a 384-well plate.
Dose-response data were fit using a nonlinear regression to a four-parameter sigmoidal curve model,
resulting in fit values for maximum and minimum responses, Hill slope, and EC .
For assay of the linking enzymes, assay
buffer (66 mM Tris, 25 mM NaCl, 8 mM MgSO , 0.01% Triton X-100, pH 8.05) was prepared daily using
water. Compounds were allowed to equilibrate in the presence of enzymes for 20 min before substrate
was added.
Final assay concentrations were 2.3 mM DTT, 60 μM PLP, 0.75 mM sodium bicarbonate, 60% Infinity
carbon dioxide liquid stable reagent, and 0.01% Triton X-100. Reaction progress was monitored by
decrease in absorbance at 340 nm using an Envision plate reader (PerkinElmer Life Sciences) equipped
with a narrow bandwidth 340 nm filter (PerkinElmer Life Sciences, 2100-5740) for 10 min with time
points taken every minute. Data from m inutes 2–7 were fit to a linear model using statistical methods
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Radiolabeled Ornithine ODC As say
Reductive Assay
Reversibility Assay
Kinetic Analysis and Dose-response Analysis
Computational Chemistry and Molecular Modeling
as described below.
CO released from L-[1- C]ornithine by the decarboxylation activity
of TbODC was directly measured as described previously (41, 42) at pH 7.5 at 37 °C in the absence or
presence of inhibitors. I C values were determined in an 8-point dose-response curve performed in
singlicate. Data were fit to a simple IC model as described previously (43).
Reductive activity of all hits was determined using a high throughput assay based on the
detection of molecules capable of reducing resazurin (catalog no. R7017, Sigma) to resorufin as
described previously (44). Briefly, 25 μl of assay solution (5 μM resazurin, 50 m M HEPES, 50 mM NaCl,
pH 7.5, 50 μM DTT, prepared immediately prior to usage) was added to a black 384-well polystyrene
plate (catalog no. 3573, Corning, Lowell, MA) using a Matrix WellMate (Thermo Fisher, Hudson, NH).
Test compounds were added by pin transfer using 10-nl hydrophobic coated pins (FP1CS10H, V&P
Scientific, Inc., San Diego) to a final concentration of 10 μM. Test plates were incubated in the dark at
room temperature for 60 min, and fluorescence intensity was read on an Envision (PerkinElmer Life
Sciences) with excitation = 560 nm and emission = 590 nm. Each compound was read in
quadruplicate, and the signals were averaged to generate relative activity levels. Compounds that
differed significantly from DMSO levels as determined by use of in-house statistical software (RI SE 3.0)
were scored as being potentially redox-active.
Reversibility assays w ere performed by dialysis. ODC (1500 nM) was incubated with
inhibitors at 10× IC concentrations for 40 min in assay buffer. Samples were then dialyzed in assay
buffer overnight using M 3000 cutoff Slide-A-Lyzer Mini dialysis units (Pierce) and assayed at 600 μM
Orn, 60 μM PLP, and 150 nM ODC. Percent activity recovered was defined as the difference between the
ratio of the dialyzed rate to the uninhibited rate and the ratio of the inhibited (10× IC ) rate to the
uninhibited rate. Compounds were labeled reversible if >90% activity was recovered.
Mode of inhibition for ornithine was determined by monitoring
the reaction rate in the presence of increasing substrate concentration (0–10 mM Orn in the presence of
60 μM PLP) with varying concentrations of inhibitors (0, IC , 3× IC , and 4× I C ). For PLP mode
of inhibition studies, ornithine concentration was held constant at 600 μM, whereas PLP was varied
from 0 to 600 μM. All kinetic data were gathered from experiments performed in 384-well format, as
described above. The data were used in Lineweaver-Burk analysis followed by fitting the data to the
appropriate Michaelis-Menten inhibition equation (competitive, mixed competition with varied α
values, noncompetitive, or uncompetitive) for determination of K values (45). I n specific cases reported
in this study, the mode of inhibition and the K values were confirmed by analysis using the cuvette-
based assay. K value for ornithine was calculated by the fitting data to the Michaelis-Menten equation
with variable K and V values. Data were fit using GraphPad Prism 4.03 (GraphPad Software, La
Jolla, CA).
Reaction rates for dose-response data were calculated as described below. Rates were then normalized
to DMSO and DFMO (1 mM) controls, and sigmoidal curves with variable slopes (four-parameter fit)
were fit using G raphPad Prism 4.03 or custom protocols in Pipeline Pilot (version 7.0, Accelrys).
Structural data used in modeling experiments were taken
from the Protein Data bank (1QU4, apo-TbODC; 1D7K, apo-hODC) (13, 46). All molecular modeling
was performed using molecular operating environment (version 2007.09, Chemical Computing G roup,
Inc.). Site Finder, a geometric method for finding potential binding pockets similar to LigSite (47), was
used to identify potential binding sites. The analysis was run with 1.4 and 1.8 Å probe radii for
hydrophobic and hydrophilic probes, respectively. A minimum site size of 25 α-spheres was used, and a
minimum bounding sphere radius of 2.5 Å w as used to eliminate smaller pockets unlikely to bind with
drug-like molecules. Dummy atoms were placed at the center of the α-spheres generated by Site Finder
and used as superposition targets for docking calculations.
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Primary Screen Results
Secondary Testing
Docking studies were also performed using molecular operating environment (version 2007.09).
Docking was performed using rigid ligand and receptors, using the Alpha Triangle placement method
with 80,000 maximum generations for each ligand conformation. Scoring was done using the London
dG scoring function. This is a five-parameter function taking into account rotational and translational
entropy, ligand flexibility, hydrogen bonding, metal ligations, and desolvation energies. The predefined
ligand conformer library w as generated using the systematic conformational search function in
molecular operating environment w ith a 10 kcal/mol cutoff. Results were visualized in PyMOL (version
0.99, Delano Scientific LLC), and receiver-operator curve scores were generated in Pipeline Pilot
(version 7.0, Accelrys).
RESULTS
For the purposes of identifying novel inhibitors of TbODC, a collection of 316,114
unique molecules was tested at St. Jude Children's Research Hospital. The compounds were screened at
10 μM against the TbODC-phosphoenolpyruvate carboxylase-malate dehydrogenase-linked enzyme
system. Plates with Z′ values of less than 0.4 were rejected for the purposes of identifying hits. This
somewhat liberal data quality cutoff was chosen to maximize the number of hits chosen for further
examination, because preliminary examination of the data showed that the hit rate was quite low. After
filtering for poor Z′, 625 plates, with an average Z′ of 0.52 and an average Z-factor of 0.47, containing a
total of 240,000 unique compounds remained. These plates were used for selecting hits. Hits were
picked using robust methods corresponding roughly to using a p value cutoff of 0.005, resulting in 883
primary hits (0.3% hit rate) (39). No minimum activity was required for characterization as a “hit.”
This technique is more completely described under “Experimental Procedures.”
To further characterize the hits, samples of each unique compound were cherry-picked and subjected to
full dose-response studies using a dilution series of 10 points in a 1:3 dilution steps (top = 100 μM). Of
the initial 883 compounds picked, 189 displayed a saturating dose response in the ODC-
phosphoenolpyruvate carboxylase-malate dehydrogenase system, whereas 310 displayed partial dose
responses. Upon retesting, 384 of the initially identified hits were inactive, giving a validated hit rate of
43.4%. None of the compounds detectably affected the phosphoenolpyruvate carboxylase-malate
dehydrogenase system at concentrations up to 100 μM concentrations. Comparison of inhibited reaction
rates at both 150 and 300 nM ODC showed that all compounds were acting via inhibition of O DC and
that ODC inhibition was the rate-limiting step in the coupled assay system. The remaining validated hits
were filtered to remove those containing potentially undesirable chemical moieties, such as highly
reactive groups or metal-containing compounds, and checked for commercial availability, leaving 179
commercially available validated hits. These validated hits were re-ordered from their suppliers. At the
same time, expanded sets were designed, centered on promising compound series, and sourced from the
same vendors. This produced a total of 260 compounds that were subjected to the secondary assay
panel.
The identity and purity of all 260 re-ordered compounds were validated using ultra
high pressure liquid chromatography-mass spectrometry (48). The average purity was 79%, and any
compound less than 90% pure was rejected from further analysis. Seventy five of the compounds failed
purity or identity checks. Stock solutions were prepared at a putative concentration of 10 mM in DMSO,
and the concentrations were confirmed using chemiluminescent nitrogen detection (49). The remaining
compounds were re-tested in the primary assay to validate their activities. Of the 185 pure compounds
tested, 76 showed activity in the primary assay. At this point, the compounds were also screened versus
hODC and at high (300 nM) concentrations of TbODC to determine selectivity and verify that O DC
inhibition was the rate-limiting step in the system. Inhibition of TbODC was determined to be rate-
limiting in all cases. The compounds were also screened for redox activity to eliminate nonspecific
radical based inhibitors (44). A representative compound from each scaffold series was also tested using
the radiolabeled ornithine assay to confirm inhibition of TbODC in an orthogonal assay system. The

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Novel Inhibitors of ODC
Identification of Potential Inhibitor Binding Sites on TbODC
representative compound was also tested for reversibility by dialysis. Of the 76 compounds active in the
primary assay, only 7 were acceptably active in all assays. The results of these are summarized in
Table 1. The active compounds represented four scaffold classes. Following confirmation of activity in
this fashion, the compounds were screened at varying L-Orn concentrations (0.31, 1.3, 2.5, 5.0, and 10
mM) as well as varying PLP concentrations (0.5, 1.5, 1, 3, 5, and 10 μM) to determine the mode of
inhibition and K values. These compounds, spanning four scaffold series, along with small structure-
activity groups are discussed below.
The first well behaved inhibitor discovered during the course of this effort
(compound 1, Table 1, and Fig. 2) was a bisbiguanide compound. This compound was quite potent, with
a K value of 2.7 μM, and displayed competitive inhibition with respect to ornithine but noncompetitive
inhibition with respect to PLP. However, despite its resemblance to the dithioamidines, it was not a
selective inhibitor of TbODC, inhibiting hODC with a similar potency. This, as well as the difference in
the mode of inhibition with respect to PLP, suggests that it has a different binding mode than the
dithioamidines. This compound was reversible and inactive in the redox assay. No close analogs w ere
available for structure-activity relationship studies.
The second and third chemotypes were the benzthiazoles (compounds 2 to 5, Table 1, and Fig. 3) and
the indoles (compounds 5 and 6, Table 1, and Fig. 4) that were moderately potent, with K values of
14.0 and 27.1 μM, respectively. Both chemotypes were nonselective inhibitors, inhibiting both hODC and
TbODC. Both chemotypes displayed uncompetitive inhibition with respect to PLP. The benzthiazoles
were uncompetitive with respect to ornithine, whereas the indoles were noncompetitive. Both
chemotypes were also reversible inhibitors of TbODC. Although there was only a single commercially
available active member in each of these re-ordered series, the preliminary structure-activity
relationships from the screening collection suggest a specific binding interaction is responsible for the
inhibition. This is particularly true for the benzthiazoles, which were heavily represented in the
compound library. After compound 2 was verified as a promising compound, all compounds containing
the benzthiazole core were cherry-picked from our screening collection and subjected to full dose-
response analysis. Although the small amounts of available compound prevented determination of K
values for these compounds, the IC values at 625 μM Orn, 60 μM PLP, and 150 nM TbODC suggest
that very little modification to the scaffold is acceptable. The structures and I C values for these
compounds can be found in the supplemental material. To maintain activity, the 4-position ethoxy
group must be present. Moving this group to the 6-position on the benzthiazole ring or substitution with
a methoxy group eliminates activity. Furthermore, the p-propoxy group on the phenyl amide moiety
must also be preserved. This group can be substituted with a p-isopropoxyl moiety with a similar
potency being maintained, but all other substitutions on this ring were inactive. In the case of the indole
compounds, only one close analog could be obtained for testing. This compound, SJ000360927, showed
that the nitrile group was necessary for activity.
The final novel chemotype discovered was the dithioamidines (compounds 8 to 15, Table 1, and Fig. 5).
These compounds were both selective and potent inhibitors of TbODC, with the most potent showing a
K of 3.6 μM. Importantly, this compound has no detectable effect on hO DC up to 100 μM. The
compounds exhibit competitive inhibition w ith respect to ornithine and uncompetitive inhibition with
respect to PLP. They also displayed reversible inhibition. A reasonable preliminary structure-activity
relationship exists in the series. To be active, these compounds must contain two thioamidine moieties
connected by a flexible linker of at least four atoms. Longer linkers were tolerated, with no upper limit
on linker length being detected within the test set. These compounds were not active in the redox assay
and are among the most potent reversible inhibitors of TbODC known. I nterestingly, the anti-
trypanosomal drug pentamidine (11) was also part of this inhibitor group, but it does not appear to act
by this mechanism in w hole cells.
Because the active sites of TbODC and hODC are
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highly homologous, it seemed unlikely that the TbODC selective dithioamidines were binding at the
active site, despite the fact that they w ere competitive with ornithine (21, 50). Therefore, an effort was
made to identify other potential binding sites on the surface of the TbODC homodimer. Site Finder was
used to identify three major possible binding sites. The first of these (site 1) is a pocket just below the
active site bounded by Arg-337, His-333, Gly-201, and Pro-245. The second (site 2) is a groove at the
dimer interface just above the active site bounded by Lys-173, Val-168, Pro-297, and Phe-179. The final
site (site 3) is a small, relatively deep pocket above and behind the active site bound by Asp-364, Ser-
396, and Ser-402, with a possible entrance to the protein hydrophobic core defined by Asn-92, Asp-38,
Gln-401, and Glu-36. After computational identification, each site was utilized for DOCKing using a
pre-generated conformer library containing all active dithioamidines as well as inactive analogs. Sites 1
and 2 were both predicted to be poor binding sites in comparison with the active site for all active
compounds in the test set. However, site 3 was predicted to be a better binding site for the
dithioamidines than the active site. Furthermore, site 3 yielded better discrimination between active and
inactive dithioamidines than did any other site, with a receiver-operator characteristic area under curve
score of 0.99 versus scores of ∼0.91 at the three other sites. The receiver-operator characteristic area
under curve score is the probability that the assay will rank a randomly chosen true positive ahead of a
randomly chosen true negative. A score of >0.5 in this metric represents discriminatory ability. Binding
at site 3 would also explain the selectivity seen with this scaffold series. Residues at both possible
entrances to the site are unconserved between TbODC and hODC. The apo structure of this site and the
top docked pose of the dithioamidine 9 at this site are pictured in Fig. 6, a and b, respectively.
Enrichment plots (ROC plots) of the docking results for the three alternate sites, as well as the active
site, can be found in supplemental material.
As the DOCKing studies suggested that site 3 might represent the bone fide site for the dithioamidenes,
this site was examined in greater detail. The majority of the high scoring dock poses involved favorable
interactions of one thioamidine moiety with Asp-364, a negatively charged residue at the bottom
binding site. The second thioamidine moiety assumed placements allowing a wide variety of interactions
with charged or polar groups in and around the binding pocket. Previous studies have shown that Asp-
364 is critical to enzyme function and that substitution with alanine renders the enzyme inactive (51).
To test our binding hypothesis, a more conservative mutant (D364E) was used to evaluate the role of
the residue in the binding of the dithioamidine compounds. Although this mutant did have dramatically
increased K (60 versus 0.37 mM) and decreased k values (0.02 versus 7 s ), some activity was still
measurable. This mutant was then used to test compounds 1 and 8, two competitive inhibitors with
similar K values emerging from our studies that possess theoretically different binding modes. The non-
TbODC selective inhibitor 1 was able to inhibit 20 μM D364E TbODC with a K value of 10.5 ± 0.7 μM,
whereas compound 8 had no detectable effect up to 100 μM, indicating that Asp-364 does play a role in
the inhibitory activity of the dithioamidine compounds. The results of these assays, along with steady
state kinetic data for all enzymes used in the comparison, are included in Table 2.
Two other mutants, S402A and S396A, at one possible entrance to the binding site were also tested. The
S402A mutant expressed as an insoluble aggregate, and S396A had no detectable effect on binding of
compound 1 or 8. However, in light of the fact that many energetically close docked poses of compound
8 placed the second thioamidine moiety at the entrance defined by Asp-38, this is not unexpected.
To further evaluate the effect of the D364E mutation on the geometry of site 3, the residue was virtually
modified using the apo structure 1QU4 as a template. After performing a rotomer search and
minimizing all residues within 8 Å of the mutation using the AMBER99 force field, no large scale
structural changes were seen. However, Glu-364 is able to move closer to the backbone amine of Thr-
359 and assume a more optimal hydrogen bonding position (2.10 versus 2.44 Å). Furthermore, Glu-
364 is able to move w ithin hydrogen bonding distance of Lys-169, a residue located in a loop whose
flexibility is thought to be important for proper functioning of the catalytic cycle (52). This movement
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also orients the carboxylate of the residue aw ay from the thioamidine groups of the inhibitors, making
the binding interaction less favorable.
DISCUSSION
This study was the first large scale screening effort to discover new chemotypes for ODC inhibition.
Screening a large chemical library has led to the discovery of several potent and selective inhibitors,
including the first known inhibitors that are selective for TbODC over the highly homologous hODC.
We also report the first nonsubstrate, nonproduct-based inhibitors of ODC. The identification of
pentamidine, a known weakly binding inhibitor of TbODC (K > 30 μM), as a hit compound in the
primary screen shows that our assay was sensitive (53). This suggests that most reversible ODC
inhibitors present in our screening library with K values below 30 μM are likely to have been identified.
The four classes of inhibitors identified in this screening effort represent novel chemotypes for O DC
inhibition. They also possess novel modes of inhibition. The benzthiazoles, typified by compound 2, are
the most potent ornithine uncompetitive inhibitors known for ODC and are also uncompetitive with
respect to PLP. Although the exact binding modes of these compounds are unknown, it is unlikely to be
at the active site, because addition of substrate increases the potency of the inhibitor. I t is known that
binding of ornithine stabilizes the dimerization of ODC (54). This suggests that the benzthiazoles bind to
the ODC homodimer. Although other uncompetitive inhibitors of ODC have been characterized
previously, their K values have been in the millimolar range (55), significantly weaker than compound
2, which possesses a K of 12.6 to 15.4 μM. Identification of the binding site for this inhibitor would allow
further optimization with the potential for an improvement in binding affinity. Because the compound
is nonselective for TbODC over hODC, it is also likely that the binding site is conserved between the
human and trypanosomal enzym es.
The indole compound 6 is noncompetitive with respect to ornithine and uncompetitive with respect to
PLP, suggesting a binding mode that differs from the benzthiazoles and is not at the active site. Because
this compound is nonselective for TbODC versus hODC, the binding site must be conserved between the
two enzymes. Although the presence of an electrophilic nitrile that is required for activity suggests the
possibility of a covalent mechanism of action, the fact that inhibition is completely reversible indicates
any modification is reversible. As with compound 2, the identification of this binding site would prove
useful in the further development of novel ODC inhibitors for both TbODC and hODC.
Two classes of potent competitive inhibitors were also discovered during the screen. The first of these,
exemplified by compound 1 (alexidine), is bisbiguanides. Reports of the antimicrobial activity of
alexidine date back to the 1950s (56), and it has antifungal activity and has been assayed as a potential
chemotherapeutic compound (57, 58). Alexidine represents an interesting molecule for use in
characterizing ODC more fully. Although it is a competitive inhibitor, alexidine is too large to fit
completely within the active site, suggesting that the binding mode is more than a simple interaction
with a single active site. Prior studies have suggested that ODC is susceptible to allosteric modulation by
Gly-418, another large, basic antimicrobial compound (59). However, Gly-418 binds very weakly (K of
3–8 mM), severely hampering mechanistic studies. Alexidine, with the much lower K of 3.8 μM,
represents a much better opportunity to investigate the modulation of ODC through allosteric binding
sites.
The final class of inhibitors discovered in this screening effort is the most interesting. The dithioamidine
series and compound 8 in particular represent the first selective inhibitors for TbODC. Compound 8 is a
known inhibitor of nitric-oxide synthase and is one of the most potent reversible inhibitors of O DC
known, with a K of 3.6 μM. This compound is structurally significantly different from the simple
putrescine and ornithine analogs previously reported as reversible inhibitors of ODC (28, 60), suggesting
that either the active site of O DC tolerates larger compounds or that these compounds are not binding
at the active site. Because compound 8 is not highly functionalized, it may be possible for medicinal
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chemistry efforts to improve its potency. This proposition is supported by the work of Tidwell and co-
workers (61, 62), who have previously reported similar diamidine scaffolds as anti-trypanosomal
compounds with unknown mechanism and have shown clear structure-activity relationships within
these compound series. In addition, other diamidine compounds such as berenil have been shown to be
effective inhibitors of S-adenosylmethionine decarboxylase, another enzyme in the polyamine
biosynthetic pathway (53). This raises the possibility that diamidine or dithioamidine compounds could
be developed that are able to inhibit both TbODC and S-adenosylmethionine decarboxylase, effectively
shutting off both rate-limiting steps in the polyamine biosynthetic pathway.
The remarkable selectivity for TbODC versus hODC displayed by the dithioamidines is likely due to the
binding of these compounds to a secondary site, located behind the active site and bounded by TbAsp-
364, TbSer-396, and TbSer-402, with a second possible entrance defined by TbAsn-92, TbAsp-38,
TbGln-401, and TbGlu-36. Binding at this site may explain selectivity, because the residues at the
entrance to the site are not conserved between the trypanosomal and the human enzymes. In hODC,
TbSer-402 is replaced by hArg-402, and TbAsn-92 is substituted by hLys-92. Examination of the crystal
structure of the human enzyme (1D7K) shows these changes place greater positive charge density at the
binding site entrances and, in the case of the TbSer-402 entrance, completely occlude access to the
binding pocket. Both molecular modeling experiments and mutagenesis data support the idea that
TbAsp-364, a residue known to be important for ODC catalytic activity, is important for the inhibitory
properties of the dithioamidines. However, crystallographic data will be necessary to confirm this
hypothesis and establish the exact binding modes of the inhibitors.
Asp-364 is conserved across a wide range of eukaryotic ODC enzymes, indicating an important role for
maintaining enzymatic functionality (51). The residue is uniquely positioned at the center of interaction
between two important loops. The first loop, in which Asp-364 resides, contains both Asp-361 and Cys-
360. Asp-361 is an active site residue involved in the stabilization of the terminal amine of ornithine
during substrate binding. The precise positioning of the substrate by Asp-361, along w ith Asp-332 and
the backbone carbonyl of Tyr-331, has been hypothesized to be necessary for the substrate binding.
Cysteine 360, the residue thought to be responsible for protonating the anion generated by
decarboxylation of ornithine, would also likely be highly sensitive to small movements (63). The second
loop, which interacts with Asp-364, contains Lys-169 (directly hydrogen bonded to Asp-364) and Leu-
166, a residue that has been predicted to interact with the carboxylate of L-ornithine (52). This loop is
known to be flexible and that flexibility is thought to be important in the ODC catalytic cycle.
Perturbations in the positioning of Asp-364 would affect the populations of conformers available to this
loop. In short, Asp-364 is at the center of what is likely to be a highly dynamic set of hydrogen bond
interactions involving many residues known to be vital to enzymatic function. The importance of its
positioning is supported by mutagenic data for this residue, where substitution with an alanine renders
the enzym e inactive, and even the relatively conservative substitution with glutamic acid increases K
by more than 175-fold and decreases k by 280-fold.
The relatively small number of inhibitors discovered in this screen and the similarity of these inhibitors
to polyamines underscore the difficulty of developing inhibitors for this enzyme. The bulk of available
evidence, including the results of this screen, suggests that the small and charged active site of ODC is
unable to bind conventional drug-like molecules. However, this study has shown that TbODC is
susceptible to inhibition by compounds likely to be acting at allosteric sites. Furthermore, allosteric
inhibition of this enzyme may allow it to escape the stabilization typically seen in the presence of other
reversible inhibitors in mammalian systems.
In conclusion, we report the use of a high throughput assay to screen over 300,000 molecules and
identify potent and selective inhibitors of TbODC. These inhibitors are likely to bind at novel nonactive
site locations on the enzyme and represent valuable tools for further development of more drug-like
inhibitors of this clinically relevant drug target.
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Supplementary Material
Supplemental Data:
This work was supported, in whole or in part, by National Institutes of Health Grant R01 AI34432 (to M. A. P.). This
work was also supported by the American Lebanese Syrian Associated Charities, St. Jude Children's Research
Hospital, and Welch Foundation Grant I-1257 (to M. A. P.).
The on-line version of this article (available at http://www.jbc.org) contains supplemental material.
The abbreviations used are:
ODC ornithine decarboxylase
PLP pyridoxal 5′-phosphate
DFMO α-difluoromethylornithine
DTT dithiothreitol
Orn ornithine
TbODC T. b rucei ODC
hODC human ODC.
REFERENCES
1. World Health Organization (2006) African Trypanosomiasis (Sleeping Sickness), Fact Sheet, 259 Ed.,
World Health Organization Media Centre, Geneva.
2. Pays E., Nolan D. P. (1998) Mol. Biochem. Parasitol. 91, 3–36. [PubMed: 9574923]
3. Barrett M. P., Boykin D. W., Brun R., Tidwell R. R. (2007) Br. J. Pharmacol. 152, 1155–1171.
[PMCI D: PMC2441931] [PubMed: 17618313]
4. Russell D., Snyder S. H. (1968) Proc. Natl. Acad. Sci. U.S.A. 60, 1420–1427. [PMCI D: PMC224936]
[PubMed: 4299947]
5. Abdel-Monem M. M., Newton N. E., Weeks C. E. (1974) J. Med. Chem. 17, 447–451.
[PubMed: 4830542]
6. Casero R. A., Jr., Marton L. J. (2007) Nat. Rev. Drug Discov. 6, 373–390. [PubMed: 17464296]
7. I wami K., Wang J. Y ., Jain R., McCormack S., Johnson L. R. (1990) Am. J. Physiol. 258, G308–
G315. [PubMed: 2106270]
8. Childs A. C., Mehta D. J., Gerner E. W. (2003) Cell. Mol. Life Sci. 60, 1394–1406.
[PubMed: 12943227]
9. Amadasi A., Bertoldi M., Contestabile R., Bettati S., Cellini B., di Salvo M. L., Borri-Voltattorni C.,
Bossa F., Mozzarelli A. (2007) Curr. Med. Chem. 14, 1291–1324. [PubMed: 17504214]
10. Coffino P. (2001) Nat. Rev. Mol. Cell Biol. 2, 188–194. [PubMed: 11265248]
11. Wallace H. M., Fraser A. V., Hughes A. (2003) Biochem. J. 376, 1–14. [PMCID: PMC1223767]
[PubMed: 13678416]
12. Metcalf B. W., Bey P., Danzin C., Jung M. J., Casara P., Vevert J. P. (1978) J. Am. Chem. Soc. 100,
2551–2553.
13. Grishin N. V., Osterman A. L., Brooks H. B., Phillips M. A., Goldsmith E. J. (1999) Biochemistry 38,
15174–15184. [PubMed: 10563800]
14. Carbone P. P., Douglas J. A., Thomas J., Tutsch K., Pomplun M., Hamielec M., Pauk D. (2000) Clin.
Cancer Res. 6, 3850–3854. [PubMed: 11051229]
*
3

5/31/13
Discovery of Potent and Selective Inhibitors of Trypanosoma brucei Ornithine Decarboxylase
www.ncbi.nlm.nih.gov/pmc/articles/PMC2878083/?report=printable
12/20
15. G riffin C. A., Slavik M., Chien S. C., Hermann J., Thompson G., Blanc O., Luk G. D., Baylin S. B.,
Abeloff M. D. (1987) Invest. New Drugs 5, 177–186. [PubMed: 3115911]
16. Meyskens F. L., Jr., McLaren C. E., Pelot D., Fujikawa-Brooks S., Carpenter P. M., Hawk E., Kelloff
G., Lawson M. J., Kidao J., McCracken J., Albers C. G., Ahnen D. J., Turgeon D. K., G oldschmid S.,
Lance P., Hagedorn C. H., Gillen D. L., Gerner E. W. (2008) Cancer Prev. Res. 1, 32–38.
[PMCI D: PMC2562024]
17. Simoneau A. R., G erner E. W., Nagle R., Ziogas A., Fujikawa-Brooks S., Yerushalmi H., Ahlering T.
E., Lieberman R., McLaren C. E., Anton-Culver H., Meyskens F. L., Jr. (2008) Cancer Epidem.
Biomark. Prev. 17, 292–299.
18. Shantz L. M., Levin V. A. (2007) Amino Acids 33, 213–223. [PubMed: 17443268]
19. Xiao Y., McCloskey D. E., Phillips M. A. (2009) Eukaryot. Cell 8, 747–755. [PMCI D: PMC2681613]
[PubMed: 19304951]
20. Ghoda L., Phillips M. A., Bass K. E., Wang C. C., Coffino P. (1990) J. Biol. Chem. 265, 11823–11826.
[PubMed: 2365702]
21. Phillips M. A., Coffino P., Wang C. C. (1987) J. Biol. Chem. 262, 8721–8727. [PubMed: 3036823]
22. Willert E. K., Fitzpatrick R., Phillips M. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 8275–8280.
[PMCI D: PMC1895940] [PubMed: 17485680]
23. Willert E. K., Phillips M. A. (2008) PLoS Pathog. 4, e1000183. [PMCID: PMC2562514]
[PubMed: 18949025]
24. Fairlamb A. H., Henderson G . B., Bacchi C. J., Cerami A. (1987) Mol. Biochem. Parasitol. 24, 185–
191. [PubMed: 3114634]
25. Priotto G ., Kasparian S., Mutombo W., Ngouama D., Ghorashian S., Arnold U., Ghabri S., Baudin
E., Buard V., Kazadi-Kyanza S., Ilunga M., Mutangala W., Pohlig G., Schmid C., Karunakara U.,
Torreele E., Kande V. (2009) Lancet 374, 56–64. [PubMed: 19559476]
26. Milord F., Pépin J., Loko L., Ethier L., Mpia B. (1992) Lancet 340, 652–655. [PubMed: 1355219]
27. Iten M., Matovu E., Brun R., Kaminsky R. (1995) Trop. Med. Parasitol. 46, 190–194.
[PubMed: 8533023]
28. Bey P., Danzin C., Jung M. (1987) in Inhibition of Polyamine Metabolism, Biological Significance
and Basis for New Therapies (McCann P. P., Pegg A. E., Sjoerdsma A., editors. eds) p. 32, Academic
Press, San Diego.
29. Bitonti A. J., McCann P. P., Sjoerdsma A. (1982) Biochem. J. 208, 435–441. [PMCID: PMC1153981]
[PubMed: 6818954]
30. Thyssen S. M., Libertun C. (2002) Neurosci. Lett. 323, 65–69. [PubMed: 11911991]
31. Osterman A., Grishin N. V., Kinch L. N., Phillips M. A. (1994) Biochemistry 33, 13662–13667.
[PubMed: 7947774]
32. Shelat A. A., Guy R. K. (2007) Curr. Opin. Chem. Biol. 11, 244–251. [PubMed: 17524728]
33. Hann M., Hudson B., Lewell X., Lifely R., Miller L., Ramsden N. (1999) J. Chem. I nf. Comp. Sci. 39,
897–902.
34. Bemis G. W., Murcko M. A. (1996) J. Med. Chem. 39, 2887–2893. [PubMed: 8709122]
35. Kunkel T. A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 488–492. [PMCI D: PMC397064]

5/31/13
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www.ncbi.nlm.nih.gov/pmc/articles/PMC2878083/?report=printable
13/20
[PubMed: 3881765]
36. Smithson D. C., Shelat A., Baldwin J., Phillips M. A., G uy K. R. (2010) Assay Drug Dev. Technol., in
press. [PMCI D: PMC3098607]
37. Team R. D. (2007) R: A Language and Environment for Statistical Computing, R Foundation for
Statistical Computing, Vienna.
38. Rousseeuw P., Croux C., Todorov V., Ruckstuhl A., Salibian-Barrera M., Maechler M. (2006)
robustbase: Basic Robust Statistics, R package 0.2–7 Ed.
39. Hoaglin D. C., Mosteller F., Tukey J. W., editors. (eds) (1983) Understanding Robust and
Exploratory Data Analysis, pp. 57–64, John Wiley & Sons, Inc., New Y ork.
40. Zhang J. H., Chung T. D., Oldenburg K. R. (1999) J. Biomol. Screen. 4, 67–73.
[PubMed: 10838414]
41. Pegg A. E., McG ill S. (1979) Biochim. Biophys. Acta 568, 416–427. [PubMed: 486492]
42. Lee J., Michael A. J., Martynowski D., Goldsmith E. J., Phillips M. A. (2007) J. Biol. Chem. 282,
27115–27125. [PubMed: 17626020]
43. Baldwin J., Michnoff C. H., Malmquist N. A., White J., Roth M. G., Rathod P. K., Phillips M. A.
(2005) J. Biol. Chem. 280, 21847–21853. [PubMed: 15795226]
44. Lor L. A., Schneck J., McNulty D. E., Diaz E., Brandt M., Thrall S. H., Schwartz B. (2007) J.
Biomol. Screen. 12, 881–890. [PubMed: 17579124]
45. Segel I. H. (1975) Enzyme Kinetics, Behavior and Analysis of Rapid Equilibrium and Steady-state
Enzyme Systems, pp. 249–250, John Wiley & Sons, Inc., New Y ork.
46. Almrud J. J., Oliveira M. A., Kern A. D., G rishin N. V., Phillips M. A., Hackert M. L. (2000) J. Mol.
Biol. 295, 7–16. [PubMed: 10623504]
47. Hendlich M., Rippman F., Barnickel G. (1997) J. Mol. Graphics 15, 359–363.
48. Lemoff A., Yan B. (2008) J. Comb. Chem. 10, 746–751. [PubMed: 18698828]
49. Y an B., Zhao J., Leopold K., Zhang B., Jiang G. (2007) Anal. Chem. 79, 718–726.
[PubMed: 17222042]
50. Steglich C., Schaeffer S. W. (2006) Infect. Genet. Evol. 6, 205–219. [PubMed: 16344004]
51. Osterman A. L., Kinch L. N., G rishin N. V., Phillips M. A. (1995) J. Biol. Chem. 270, 11797–11802.
[PubMed: 7744828]
52. Jackson L. K., Baldwin J., Akella R., Goldsmith E. J., Phillips M. A. (2004) Biochemistry 43, 12990–
12999. [PubMed: 15476392]
53. Bitonti A. J., Dumont J. A., McCann P. P. (1986) Biochem. J. 237, 685–689.
[PMCI D: PMC1147045] [PubMed: 3800910]
54. Solano F., Peñafiel R., Solano M. E., Lozano J. A. (1985) FEBS Lett. 190, 324–328.
[PubMed: 3930296]
55. Henley C. M., 3rd, Mahran L. G., Schacht J. (1988) Biochem. Pharmacol. 37, 1679–1682.
[PubMed: 3377830]
56. Rose F. L., Swain G. (1956) J. Chem. Soc. 4422–4425.
57. G anendren R., Widmer F., Singhal V., Wilson C., Sorrell T., Wright L. (2004) Antimicrob. Agents

5/31/13
Discovery of Potent and Selective Inhibitors of Trypanosoma brucei Ornithine Decarboxylase
www.ncbi.nlm.nih.gov/pmc/articles/PMC2878083/?report=printable
14/20
Chemother. 48, 1561–1569. [PMCID: PMC400561] [PubMed: 15105106]
58. Yip K. W., Ito E., Mao X., Au P. Y ., Hedley D. W., Mocanu J. D., Bastianutto C., Schimmer A., Liu
F. F. (2006) Mol. Cancer Ther. 5, 2234–2240. [PubMed: 16985057]
59. Jackson L. K., G oldsmith E. J., Phillips M. A. (2003) J. Biol. Chem. 278, 22037–22043.
[PubMed: 12672797]
60. Garvey E. P., Oplinger J. A., Tanoury G . J., Sherman P. A., Fowler M., Marshall S., Harmon M. F.,
Paith J. E., Furfine E. S. (1994) J. Biol. Chem. 269, 26669–26676. [PubMed: 7523409]
61. Athri P., Wenzler T., Ruiz P., Brun R., Boykin D. W., Tidwell R., Wilson W. D. (2006) Bioorg. Med.
Chem. 14, 3144–3152. [PubMed: 16442293]
62. Mathis A. M., Holman J. L., Sturk L. M., Ismail M. A., Boykin D. W., Tidwell R. R., Hall J. E.
(2006) Antimicrob. Agents Chemother. 50, 2185–2191. [PMCID: PMC1479144] [PubMed: 16723581]
63. Jackson L. K., Brooks H. B., Osterman A. L., Goldsmith E. J., Phillips M. A. (2000) Biochemistry 39,
11247–11257. [PubMed: 10985770]
Figures and Tables
FIGURE 1.
Sch em at ic of th e poly am in e bio sy nthetic pat h way in T. b ru cei. The follo wing ab brev iatio ns are used :
S-adeno sy lmethio nine dec arbo x ylase (SAMDC), S-adenosy lmethio nine (S-A doMet), 5 ′-methy lthioadeno sine
(MTA), de c ar box ylate d S-adenosy lmethio nine (DC-S-AdoMet), putre sc ine (Put), spe rmidine (Spd), spermidine
sy nthase (SpdS), DFMO, glutathiony l spermidine sy nthase (GSS), try pano thio ne sy nthase (TryS), try panothione
reductase (Try R), and reactiv e ox ygen species (ROS).
TABLE 1

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Inhibitors of TbODC
All values are in micromolar unless otherwise noted.
* K values were determined by global fitting of raw rate data to appropriate Michaelis-Menten
equations and are expressed as fit value followed by 95% confidence limits. K values were determined
using data from three separate triplicate experiments.
** IC values were determined at 37 °C at 400 μM L-Orn as described under “Experimental
Procedures.” Data were fit to simple IC models.
*** Primary HTS hit compound is shown. All other compounds were part of reordered analog series.
**** αK value from uncompetitive inhibition model is shown.
‡ Single point is active at 100 μM. All IC values were determined at isokinetic conditions (1.5× K L-
Orn, 60 μM PLP) as described under “Experimental Procedures.” IC values are expressed as the mean
of three measurements taken in triplicate ± S.D. ODC concentrations were 150 nM for all experiments.
High and low ODC rates were determined at 150 and 300 nM ODC, respectively, in the presence of 625
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μM L-Orn. NT indicates not tested; NA indicates not active.
FIGURE 2.
Bisbiguanide in h ibitor data. a and b, Line we av er-Burk plots for o rnithine versus co mpo und 1 and PLP
ve rsus 1. ♦ = 10 μM, ▾ = 6 μM, ▴ = 4 μM, and ● = uninhibit ed. c, spe c ies se lectiv ity analy sis for com po und 1. Data
for TbODC (▾) and hODC (■) enzy me-linked assay s were c o llec ted at 22 °C in 3 84-well plates. Data were fitte d to a
four-parameter sigmo idal dose respo nse for determination of IC values. A ll data were c o llec ted under
isokine tic co nditions at 1 .5 × K for o rnithine. d, rev ersibility of co mpo und 1. TbODC was incub ated with
inhibitor for 1 h follo wed by ov ernight dialy sis into assay buffer. Data were collected as described unde r
“Ex pe rimental Procedures.”
FIGURE 3.
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Ben zt h iazole in h ibit or dat a. Sele c tiv ity curv es and rev ersibility data fo r these com po unds can be fo und in
the supplemental material. Lineweav e r-Burk plot for o rnithine versus co mpo und 2 and PLP v ersus 2. ♦ = 28 μM,
▾ = 9.5 μM, ▴ = 3.2 μM, and ● = uninhibite d.
FIGURE 4.

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Indole in h ibito r dat a. Selec tiv ity c ur v e s and rev ersibility data for the se compounds can b e found in the
supplemental mater ial. Lineweav er-Burk plot for ornithine ve rsus c o mpound 6 and PLP versus 6. ♦ = 40 μM, ▾ =
13 μM, ▴ = 4.4 μM, and ● = uninhibited.
FIGURE 5.
Dithioam idine in h ib it or data. a and b, Linewe av er-Bur k plots for o rnithine versus compo und 8 and PLP
ve rsus 8. ♦ = 40 μM, ▾ = 13 μM, ▴ = 4.4 μM, and ● = uninhibited. c, spe c ies se lectiv ity analy sis for compo und 8.

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Data for TbODC (▾) and hODC (■) enzy me-linked assay s wer e co llec ted at 22 °C in 384-well plates. Data were
fitte d to a fou r-param eter sigmo idal dose response for determinatio n o f IC v alue s. All data were collected
under iso kinetic conditions at 1 .5× K for o rnithine. d, rev ersibility of co mpo und 8. TbODC was inc ubated with
inhibitor for 1 h follo wed by ov ernight dialy sis into assay buffer. Data were collected as described unde r
“Ex pe rimental Procedures.”
FIGURE 6.
Proposed dithioam idin e-bindin g site. a, TbODC bound to DFMO. DFMO is in purple; PLP is salmo n; activ e
site residues are w hite, and residues that form the pr opo sed binding site are indicate d as follo ws: Asp-364 is
color ed yellow and for ms the botto m of the binding site, and mag enta re sidue s form the two po ssible entrances
to the pro po sed dithioamidine -binding site. b, co mpo und 9 do c ked into the pro posed binding site o n Tb ODC
and super impo se d with the apo human ODC struc ture. TbODC residues are orange, and hODC residues are ligh t
blue. A sp -364 is yellow , and activ e site re sidue s are white, DFMO is purple, and PLP is salm on. Note that Ser-
40 2 from TbODC is no t c o nserv ed in hODC, and that the opening to the pro posed site is completely occluded in
the human enzy me. This c o uld help ac c ount for the remar kab le selectiv ity seen with the dithioamidine series o f
inhibitors.
TABLE 2
Steady state kinetic analysis of TbODC and hODC enzymes with inhibition data
All data were collected at 37 °C using the linked enzyme assay as described under “Experimental
Procedures.” All IC values were determined at isokinetic conditions (400 μM L-Orn for WT-TbODC
and S396A-TbODC, 60 mM L-Orn for D364E TbODC, 150 μM L-Orn for WT-hODC, and 60 μM PLP was
used for all experiments). IC values were determined with the cuvette-based enzyme-linked assay
using the same protocol as for the CO assay. ODC concentrations were 150 nM for all experiments
except for D364E, in which 20 μM ODC was used to compensate for the low k of the mutant. Values
in parentheses are K values in micromolars. K values were obtained as described under “Experimental
Procedures.” A value of ≫100 indicates no detectable effect at the maximum concentration of 100 μM.
ODC protein K (L-Orn ) k
k/K (L-
Orn ) Com pou n d 1 IC Com pou n d 8 IC
mMsμMμM
Wild ty pe
TbODC
0.37 ± 0.03 7 .0 ± 0.2 18.9 5.1 ± 0.6 (2 .7 ± 1 .2) 6.2 ± 0.8 (3 .6 ± 1 .5)
D364E TbODC 55.2 ± 4.6 0.01 5 ±
0.001
0.00027 22.6 ± 2.6 (1 0.5 ±
0. 7 )
≫100 (≫100)
S396A TbODC 0.27 ± 0.0 3 10.0 ± 0 .4 37 .0 6.9 ± 1.1 12.9 ± 0.5
Wild ty pe hODC 0.1 7 0 ±
0.01 5
5.3 ± 0.2 31 .2 15.9 ± 3.8 ≫100
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