Identification and characterization of novel inhibitors of mammalian aspartyl aminopeptidase

Article abstract of DOI:10.1124/mol.114.093070

Aspartyl aminopeptidase (DNPEP) has been implicated in the control of angiotensin signaling and endosome trafficking, but its precise biologic roles remain incompletely defined. We performed a high-throughput screen of ～25,000 small molecules to identify inhibitors of DNPEP for use as tools to study its biologic functions. Twenty-three confirmed hits inhibited DNPEP-catalyzed hydrolysis of angiotensin II with micromolar potency. A counter screen against glutamyl aminopeptidase (ENPEP), an enzyme with substrate specificity similar to that of DNPEP, identified eight DNPEP-selective inhibitors. Structure-activity relationships and modeling studies revealed structural features common to the identified inhibitors, including a metal-chelating group and a charged or polar moiety that could interact with portions of the enzyme active site. The compounds identified in this study should be valuable tools for elucidating DNPEP physiology. Copyright

Full text of DOI:10.1124/mol.114.093070

Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2014/06/09/mol.114.093070.DC1.html
1521-0111/86/2/231–242$25.00
MOLECULAR PHARMACOLOGY
http://dx.doi.org/10.1124/mol.114.093070
Mol Pharmacol 86:231–242, August 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Identification and Characterization of Novel Inhibitors of
Mammalian Aspartyl Aminopeptidase s
Yuanyuan Chen, Hong Tang, William Seibel, Ruben Papoian,1 Ki Oh, Xiaoyu Li,
Jianye Zhang, Marcin Golczak, Krzysztof Palczewski, and Philip D. Kiser
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio (Y.C., K.O., X.L., J.Z.,
M.G., K.P., P.D.K.); and Drug Discovery Center, College of Medicine, University of Cincinnati, Cincinnati, Ohio (H.T., W.S., R.P.)
Received April 1, 2014; accepted June 9, 2014
ABSTRACT
Aspartyl aminopeptidase (DNPEP) has been implicated in the enzyme with substrate specificity similar to that of DNPEP,
control of angiotensin signaling and endosome trafficking, but identified eight DNPEP-selective inhibitors. Structure-activity
its precise biologic roles remain incompletely defined. We relationships and modeling studies revealed structural features
performed a high-throughput screen of ∼25,000 small mole- common to the identified inhibitors, including a metal-chelating
cules to identify inhibitors of DNPEP for use as tools to study its group and a charged or polar moiety that could interact with
biologic functions. Twenty-three confirmed hits inhibited DNPEP- portions of the enzyme active site. The compounds identified in
catalyzed hydrolysis of angiotensin II with micromolar potency. A this study should be valuable tools for elucidating DNPEP
counter screen against glutamyl aminopeptidase (ENPEP), an physiology.
vertebrate species contain only one M18 metallopeptidase-
Introduction
encoding gene. Sequence identity among mammalian DNPEP
Aminopeptidases are a heterogeneous group of enzymes
orthologs is generally greater than 90%. This strong conser-
that catalyze the hydrolysis of N-terminal residues from
vation suggests that DNPEP may play an essential role in
peptide substrates. Aspartyl aminopeptidase (DNPEP; EC
cellular metabolism that has remained conserved throughout
3.4.11.21) and glutamyl aminopeptidase (ENPEP or amino-
evolution. DNPEP is a self-compartmentalized, binuclear
peptidase A; EC 3.4.11.7) are the two known acidic residue-
zinc-containing enzyme that forms a tetrahedron-shaped
specific aminopeptidases present in mammals (Glenner et al.,
homododecameric complex (Chaikuad et al., 2012; Chen et al.,
1962; Wilk et al., 1998; Goto et al., 2006). Mostly found in
2012; Sivaraman et al., 2012). The active site-containing nano-
kidney, lung, and immune cells, ENPEP is a membrane-
compartment enclosed by the DNPEP tetrahedron is acces-
associated ecto-enzyme belonging to the M1 metallopeptidase
family (Wu et al., 1990; Nanus et al., 1993; Goto et al., 2006).
sible through four ∼20 Å-wide selectivity pores that allow
entrance of short peptides. In mammals, DNPEP is expressed
ENPEP catalyzes the hydrolysis of angiotensin II (Ang II) to
in many organ systems with especially high activity in the
form angiotensin III (Ang III) and is involved in the regulation
brain and testis (Wilk et al., 1998). This enzyme is commonly
of systemic blood pressure (Reaux et al., 1999; Mitsui et al.,
described as cytosolic, although it also exists in a membrane-
2003; Wright et al., 2003; Bodineau et al., 2008a) and cancer-
associated form in some tissues (Cai et al., 2010; Mayas et al.,
associated angiogenesis (Marchio et al., 2004). Whereas the
2012b).
function of ENPEP has been well-studied, the biologic and
A role for DNPEP in regulation of the renin-angiotensin
pathologic roles of DNPEP remain poorly understood.
system has been proposed on the basis of its substrate
DNPEP belongs to the M18 metallopeptidase family, the
specificity (Wilk et al., 1998; Chen et al., 2012), although its
members of which are found in all kingdoms of life (Rawlings
involvement in the renin-angiotensin system has not been
et al., 2014). The genomes of mammals and most other
examined in vivo. Changes in DNPEP expression and/or
activity have been noted in neoplastic disorders such as colon
This work was supported by Case Western Reserve University School of
and breast cancers, squamous cell carcinoma, and gliomas
Medicine; and National Institutes of Health [Grant EY008061 (to K.P.)]. K.P. is
(Perez et al., 2009; Martinez-Martos et al., 2011; Mayas et al.,
the John H. Hord Professor of Pharmacology.
1Current affiliation: Department of Neurology, College of Medicine,
University of Cincinnati, Cincinnati, Ohio.
2012a; Larrinaga et al., 2013). In mice, DNPEP was shown to
be a major target of the chondrocyte-specific microRNA,
Mir140, loss of which led to overexpression of DNPEP and
consequent defects in skeletal development (Nakamura et al.,
dx.doi.org/10.1124/mol.114.093070.
s
This article has supplemental material available at molpharm.aspetjournals.
org.
ABBREVIATIONS: Ang II, angiotensin II; Ang III, angiotensin III; Asp-AMC, L-aspartic acid 7-amido-4-methylcoumarin; Asp-NHOH, aspartic acid
hydroxamate; DMSO, dimethylsulfoxide; DNPEP, aspartyl aminopeptidase; ENPEP, glutamyl aminopeptidase; Glu-AMC, L-glutamic acid 7-amido-
4-methylcoumarin; HTS, high-throughput screen; LC-MS, liquid chromatography-mass spectrometry; MS, mass spectrometry; Pfm18AAP,
Plasmodium falciparum M18 aspartyl aminopeptidase; SAR, structure-activity relationship; S/B, signal to background; UC, University of Cincinnati.
231

232
Chen et al.
Flexstation3 microplate reader (Molecular Devices, Sunnyvale, CA).
Optimal excitation/emission wavelengths were judged to be 380/460
nm. Dimethylsulfoxide (DMSO), the compound solvent, did not inhibit
DNPEP up to a final concentration of 1% (v/v) (Supplemental Fig. 1D).
Asp-AMC concentrations (50–1000 mM), DNPEP concentrations (0.01–
0.22 mM), and reaction time (0–50 minutes) were individually tested to
optimize the assay while minimizing the HTS costs (Supplemental
Fig. 1, E and F). Day-to-day and plate-to-plate variability of the HTS
assay were within 40%, which is smaller than the threefold difference
suggested by the National Institutes of Health HTS guidelines (http://
www.ncats.nih.gov/research/reengineering/ncgc/assay/criteria/criteria.
html), thus demonstrating the reliability of the HTS assay (Supple-
mental Fig. 1G). The stability of the enzyme under conditions of the
HTS assay was also confirmed (Supplemental Fig. 1H).
Virtual Screen. The DNPEP-aspartic acid hydroxamate coordi-
nate file (PDB accession code 3L6S) was downloaded from the
Research Collaboratory for Structural Bioinformatics Protein Data
Bank (Chaikuad et al., 2012). Docking was performed with the
Schrödinger software suite (Schrödinger Suite 2011: Maestro, version
9.2.109; Schrödinger, New York, NY). Coordinates were prepared for
docking with the Protein Preparation Wizard (Epik version 2.2); the
conformer library was generated using LIGPREP (version 2.4); and
the docking was performed using GLIDE (version 5.7 with SP,
followed by XP Precision). The small database of compounds for the
virtual screen was constructed from the larger University of Cin-
cinnati (UC) compound library by the following: 1) execution of simi-
larity searches using Accelrys’ Pipeline Pilot (Ver 8.0.1.500) on the
aspartic acid hydroxamate ligand from the 3L6S coordinate file; and
2) execution of a range of substructure searches for typical chelating
moieties (e.g., b-hydroxycarbonyls, hydroxamates, catechols, etc.)
and/or aspartyl and glutamyl scaffolds. Because the S1 pocket of 3L6S
was relatively small, compounds with mol. wt. greater than 375 Da
were excluded from the virtual screen. The three-dimensional structures
of this library, including tautomers, alternative protonation states, and
isomers, were generated in Pipeline Pilot prior to conformation gen-
eration and incorporation into the virtual screen, as detailed above.
Thirty-three compounds from this screen were experimentally evalu-
ated (Supplemental Fig. 2), and the most active compounds served as
seed structures for similarity searches in Pipeline Pilot with the top 266
most similar compounds added to the UC Diversity screening set.
2011). A recent suppressor mutant study of a Caenorhabditis
elegans line with endosomal trafficking defects caused by a null
mutation in phosphatidylserine flippase (tat1) revealed that
loss of DNPEP activity corrected the blockade in endosome
cargo sorting and recycling, but not degradation (Li et al.,
2013). These disparate discoveries have not yet allowed a clear,
unified picture of DNPEP physiology to be developed.
Among the various approaches to studying enzyme physi-
ology, manipulation of biologic systems through the use of
selective pharmacological agents allows examination of enzyme
activity loss in an acute setting before the onset of homeostatic
compensation. Additionally, loss of enzyme function can be
readily studied in adult subjects in situations when genetic
ablation of enzyme function is not feasible due to consequent
developmental defects or embryonic lethality. This concern is
pertinent to DNPEP due to its highly conserved nature, broad
expression pattern, and the lethality observed in Plasmodium
falciparum after genetic knockdown of its M18 aspartyl ami-
nopeptidase (PfM18AAP) (Teuscher et al., 2007). Nonselective
metal chelators, reducing agents, and a substrate analog,
aspartic acid hydroxamate (Asp-NHOH) (IC50 5 200 mM),
have been identified as DNPEP inhibitors (Wilk et al., 1998;
Stoermer et al., 2003; Hoffman et al., 2009; Chaikuad et al.,
2012; Chen et al., 2012). However, the lack of selectivity and/
or suboptimal potency of these agents limits their utility for
biologic studies. A high-throughput screen (HTS) for inhib-
itors of PfM18AAP was previously reported (Pubchem Bio-
assay: AID1855) (Schoenen et al., 2010). However, structural
differences in the substrate-binding pockets of mammalian
DNPEP and Pfm18AAP could limit the efficacy of the
identified hit compounds as inhibitors of mammalian DNPEP
(Chen et al., 2012; Sivaraman et al., 2012).
In this study, we report the identification and characteriza-
tion of a set of small-molecule inhibitors of DNPEP. These
compounds displayed low micromolar inhibitory activity to-
ward DNPEP-catalyzed hydrolysis of the biologically relevant
substrate, Ang II. Some of the compounds were more se-
lective for DNPEP than the functionally related enzyme
ENPEP, whereas others were potent inhibitors of both enzymes.
Structure-activity relationship (SAR) analyses and molecular
modeling of the inhibitor-enzyme interactions provided insights
into their mechanism(s) of DNPEP inhibition.
Small-Molecule High-Throughput Screening with
a
Fluorescence-Based Biochemical Assay. Using Asp-AMC as the
substrate, we monitored the effect of test compounds on DNPEP-
catalyzed release of AMC by measuring the fluorescence change over
time. In a 384-well, black-wall, clear-bottom plate, each well was
sequentially loaded with 20 ml 50 mM Tris-HCl (pH 7.5) and 40.65 nl
(12.3 mM final concentration) of test compound in DMSO and 5 ml
50 mg/ml purified DNPEP. After incubation at 37°C for 15 minutes, 25 ml
500 mM Asp-AMC substrate in 50 mM Tris-HCl (pH 7.5) buffer was
added to each well. Fluorescence intensity (Eex/Eem 5 380/460 nm)
was read with the Plate::Vision detector immediately after substrate
was added (FI0 minute) and again after 30 minutes of incubation at 37°C
(FI30 minute). The relative fluorescence intensity (RFI) was defined as
Materials and Methods
Chemicals. L-aspartic acid 7-amido-4-methylcoumarin (Asp-AMC),
L-glutamic acid 7-amido-4-methylcoumarin (Glu-AMC), and Ang II were
purchased from Bachem (Torrance, CA).
DNPEP Expression and Purification. Bovine DNPEP was
expressed in T7 Express BL21 Escherichia coli (New England Biolabs,
Ipswich, MA) and purified, as previously described (Chen et al., 2012).
The concentration and purity of DNPEP were measured by the Brad-
ford assay, Asp-AMC hydrolysis activity assay, Coomassie Brilliant
Blue–stained SDS-PAGE gels, and immunoblotting (Supplemental
Fig. 1, A and C).
HTS Assay Optimization. The HTS assay was modified from a
cuvette-based, fluorometric Asp-p-nitroaniline hydrolysis assay (Chen
et al., 2012). Because the excitation/emission profile of p-nitroaniline
was not compatible with the Plate::Vision detector (Perkin Elmer,
Waltham, MA) used for the HTS, we employed the alternative syn-
thetic substrate, Asp-AMC, which can be hydrolyzed to produce an
AMC fluorophore suitable for the HTS plate reader (Supplemental Fig.
FI30min 2 FI0min:
Each plate included four columns of controls, as follows: 1) untreated
control with maximum enzyme activity, scored as 0%; 2) maximum
inhibition control treated with 0.5 mM ZnCl2, scored as 2100%; 3)
maximum activation control treated with 0.5 mM MnCl2, scored as
1100%; and 4) blank control with no enzyme addition. The relative
activity of each compound was calculated using these controls as
references:
RFIcp 2 RFIenzyme
RFIenzyme 2 RFIZn
Activity score 5
ꢀ 100;
1B). To separate signal from substrate and product, excitation and where RFIenzyme represents the relative fluorescence intensity of
emission spectra of both Asp-AMC and AMC were measured in a untreated control, RFIcp the relative fluorescence intensity of test

Discovery of Small-Molecule DNPEP Inhibitors
233
compound-containing sample, and RFIZn the relative fluorescence described (Goto et al., 2006). The ENPEP sequence used in this work
intensity of the maximum inhibition control. total of 26,120 contained an A473G mutation that resulted in a Q213R amino acid
A
compounds from the UC 25,000 Diversity Set in addition to 266
compounds selected from the UC compound collection based on virtual
substitution. The A473G change in ENPEP is a naturally occurring
polymorphism found in ∼6% of the human population. The Q213R
screening and similarity searches was tested at final concentrations of amino acid substitution was previously shown to have no effect on
12.3 mM in the primary HTS. Hits were selected with activity scores
ENPEP catalytic properties or expression (Tonna et al., 2008). One liter
#260% (inhibitors), or $130% (activators). These hits were retested in of Sf9 cells at a density of 1–2 ꢀ 106 cells/ml was infected with 20–30 ml
triplicate at final concentrations of 12.3 mM. The potencies of confirmed P3 baculovirus and then cultured at 28°C with 120 rpm shaking
hits were evaluated in dose-response assays performed in triplicate. for 48–72 hours. The culture was harvested, and cells and debris
Dose-response curves were generated using Genedata Screener Con- were sedimented by centrifugation. ENPEP was purified from the
doseo (Ver. 9.0.0 Standard) Software. EC50 is denoted as 50% effective
supernatant by ammonium sulfate fractionation and Ni-affinity and gel
concentration, that is, 50% inhibitory concentration (IC50) or 50% filtration chromatography. The resulting preparation was .95% pure
activation concentration (AC50). Compounds with IC50 or AC50 values as judged by Coomassie-stained SDS-PAGE gels. About 2 mg purified
#20 mM were selected for further evaluation. The primary, triplicate, ENPEP was obtained from a 1 L culture.
and dose-response screens were monitored by two quality control
parameters: signal to background (S/B) ratio and the Z9-factor defined,
respectively, as:
ENPEP enzymatic activity was assayed using the fluorogenic
substrate Glu-AMC (Bachem) in the presence of 1 mM CaCl2, as
previously described (Goto et al., 2006). The excitation and emission
wavelengths were 380 nm and 460 nm, respectively. The Km value for
the preparation was ∼0.4 mM consistent with previous reports (Goto
et al., 2006). kcat was ∼57 seconds21, which is somewhat higher than
that previously described, possibly due to differences in the protein
purification methods employed.
ꢀ
RFIenzyme
RFIZn
S
Bꢀ ratio 5
and
The counter screen of DNPEP inhibitors against ENPEP was
conducted in an assay solution consisting of 10 ng/ml purified ENPEP,
1 mM CaCl2, 0.36 mM Glu-AMC, and 10 mM test or control compound
(final concentrations) in a total volume of 50 or 100 ml. ZnCl2, a strong
inhibitor of ENPEP activity, was used as a positive control, whereas
a DMSO control was used to measure baseline ENPEP activity. The
enzyme was preincubated with the test compound for 10 minutes at
37°C prior to initiation of the reaction by addition of substrate. The
change in fluorescence was measured every 30 seconds for 5 minutes.
The absolute quantity of AMC product generated was determined
from AMC standards measured alongside the experimental samples.
The degree of inhibition is presented as the difference in activity
between the experimental and DMSO control samples. Inhibition by
10 mM ZnCl2, which was essentially complete, was set to 2100%
activity. The DMSO control was scored as 0% activity S/B ratio and
Z9-factor were used to evaluate the assay quality.
SDenzyme 1 SDZn
Meanenzyme 2 MeanZn
Z9 5 1 2 3 ꢀ
ꢀ ðZhanget al:; 1999Þ;
where S.D. and Mean are the standard deviations and means of the
RFI readings.
Orthogonal Assay for Hit Confirmation. To differentiate true
hits from false positives, we tested the active compounds from the
HTS in a mass spectrometry (MS)-based Ang II hydrolysis assay.
Each well in a 384-well plate was loaded with 10 ml diluted compound
in 50 mM Tris-HCl (pH 7.5) (to achieve a final compound con-
centration of 10 mM) and 10 ml 10 mg/ml purified DNPEP. Mix-
tures were incubated for 15 minutes at 37°C, and then the reaction
was initiated by addition of 20 ml 0.5 mM Ang II in 50 mM Tris-HCl
(pH 7.5). After 30 minutes of incubation at 37°C, the reaction was
terminated by addition of 40 ml 40% acetic acid. Mixtures in each
well were transferred to sample tubes and subjected to liquid
chromatography-mass spectrometry (LC-MS) for Ang II and Ang III
quantification, as described previously (Chen et al., 2012). An 1100
series Agilent (Santa Clara, CA) high-performance liquid chromatog-
raphy instrument equipped with an XBridge BEH300 C4 column
(Waters, Milford, MA) was used to fractionate samples, and eluates
were injected into a LXQ linear ion trap mass spectrometer (Thermo
Scientific, Waltham, MA) at a flow rate of 0.2 mL/min. Ang II and Ang
III eluted at about the same time from the C4 column in a 50%
acetonitrile and 50% H2O mobile phase. Ang III concentration was
quantified by measuring the chromatogram peak areas corresponding to
the 11 and 12 ions of Ang II (AreaAng II) and Ang III (AreaAng III):
MS Analysis of Compound Mass. Selected compounds were
obtained as powders from the UC compound library and dissolved in
water at concentrations of 1 mM for MS analysis. Solutions were directly
injected into a LXQ linear ion trap mass spectrometer (Thermo
Scientific) using an ESI source operating in positive ion mode.
Results
Overview. The discovery of DNPEP modulators began
with a fluorescence-based HTS of ∼25,000 chemically diverse
compounds in addition to 266 compounds selected based on
virtual screening. Hits from the primary screen were then
tested by an orthogonal, MS-based assay to quantify their
inhibition toward DNPEP-catalyzed Ang II hydrolysis. Con-
firmed hits were then tested for their selectivity in a counter
screen against ENPEP, which has substrate specificity
similar to that of DNPEP and is a therapeutic target for
hypertension and angiogenesis. SAR and molecular modeling
studies were performed on the hits to understand their
essential chemical features and probable mode of binding to
DNPEP (Fig. 1A).
Virtual Screen, Compound Selection, and Primary
HTS. To identify pharmacological regulators of DNPEP by
HTS, a biochemical HTS assay was developed using fluores-
cence emission as a readout of the enzyme activity. Due to the
preference of DNPEP for N-Asp–containing peptides, we
employed L-aspartic acid 7-amido-4-methylcoumarin (Asp-
½AngIIIꢁ
½AngIIIꢁ
AreaAngIII
5
5
:
½AngIIꢁinitial ½AngII 1 AngIIIꢁ AreaAngIII 1 AreaAngII
Reactions were optimized to ensure Ang II hydrolysis was linear over
time, so that the endpoint product/initial substrate ratio represented
the initial reaction velocity of the enzyme. As in the fluorogenic HTS
assay, the DMSO control was scored 0%, the 0.5 mM ZnCl2-treated
control was scored as 2100%, and the 0.5 mM MnCl2-treated control
was scored as 1100%. Confirmed hits were identified as those with
activity scores #250% in triplicate readings and IC50 # 20 mM in
dose-response assays. Quality of the orthogonal assays was monitored
by the S/B ratio, Z9-factor, and
SDhit
Meanhit
%CV 5
ꢀ 100%:
ENPEP Expression, Purification, and Activity Assay. Human
ENPEP was expressed in Sf9 insect cells essentially as previously AMC) as the substrate for the HTS (Fig. 1B). DNPEP-catalyzed

234
Chen et al.
Fig. 1. Overview of DNPEP inhibitor discovery. (A) Flowchart of DNPEP inhibitor discovery and characterization. (B) The peptidomimetic, fluorogenic
substrate Asp-AMC was used for the HTS assay. Hydrolysis of this compound by DNPEP yields a fluorescent product, AMC, which can be readily
quantified in high-throughput format. (C) The orthogonal assay used MS to quantify enzymatic production of Ang III from Ang II.
hydrolysis of this compound generates the fluorescent compound activity scores less than 260% and more than 130% were
AMC, which can be distinguished from intact Asp-AMC by its identified as DNPEP inhibitors and activators, respectively
red-shifted excitation/emission profile. The known DNPEP in- (Supplemental Table 1). The hit rates for inhibitors and
hibitor and activator, ZnCl2 (0.5 mM) and MnCl2 (0.5 mM), were activators were 1.09% and 0.84%. Quality control param-
used as the 2100% and 1100% controls, respectively, and the eters for the primary HTS are shown in Supplemental Table 2.
enzyme-only condition was set as the 0% change in activity Comparison of the distributions of compound activities from
control. The final assay quality was characterized by a Z9-factor of unbiased screening using the 25,000 compound Diversity Set
0.87, a signal-to-background ratio (S/B) of 247, and coefficient of and the pool from virtual screen revealed a 7.6-fold enriched
variation of 3.9%.
hit rate of 8.3% for the latter group of compounds (Fig. 2B).
To enrich the HTS library with potential DNPEP inhibitors, There were 367 of 397 DNPEP inhibitors and 219 of 222
a virtual screen was undertaken based on known DNPEP DNPEP activators available in the UC compound collection
crystal structures. By comparing the structures of ligand-free for triplicate and dose-response confirmation tests. Hits were
DNPEP and DNPEP in complex with the weak inhibitor/ first re-screened in triplicate at 12.3 mM with independent
substrate analog Asp-NHOH (Chaikuad et al., 2012; Chen aliquots of the test compounds, which confirmed 282 DNPEP
et al., 2012), we found that the substrate-binding pocket was inhibitors and 3 activators (Fig. 2C; Supplemental Tables 3
larger than Asp-NHOH and could potentially accommodate and 4). The compound activity distribution of the triplicate
bulkier groups. The active site also contained two positively screen showed a dramatic enrichment of hits (76.8% and 1.4%
charged regions, the metal center where hydrolysis is catalyzed for inhibitors and activators), demonstrating the high quality
and Lys370, which is located in the S1 pocket of DNPEP and of the primary HTS screen. Confirmed hits were next an-
conserved in all acidic residue-specific M18 aminopeptidases. A alyzed by dose-response assays performed in triplicate with
search of the entire .350,000 UC compound library revealed 10 inhibitor concentrations obtained by two-fold serial di-
33 inhibitor candidates. Some of these molecules contained lutions. From this screen, 281 inhibitors and 2 activators of
hydroxamic, sulfonic/phosphonic, or aspartic/glutamic acid DNPEP were confirmed with EC50 values #20 mM, repre-
groups as potential metal-chelating moieties. Nine of the 33 senting 99.6% and 66.7% retention rates, respectively (Sup-
compounds were hits from the Pfm18AAP inhibitor screen plemental Tables 5 and 6). The EC50 distribution of these hits
(Schoenen et al., 2010) (Pubchem Bioassay: AID1855). These is shown in Fig. 2D. In total, 36 inhibitors were identified that
33 compounds then were tested in the proposed HTS assay displayed submicromolar potency. Z9-factors for the primary,
system to assess its reliability as well as the quality of the triplicate, and dose-response screens using the Asp-AMC hy-
virtual screening procedure (Supplemental Fig. 2). The small- drolysis assay were 0.74 6 0.03, 0.79 6 0.05, and 0.78 6 0.03,
scale screen of these 33 compounds at 10 mM identified three which demonstrated the reliability and quality of these screens
hits with greater than 50% inhibition toward DNPEP activity (Supplemental Tables 2, 4, and 6).
(Fig. 2A; Supplemental Fig. 2). These three hits together with
MS-Based Orthogonal Screen. The primary fluorescence-
Asp-NHOH (Fig. 2A) were then used as seeds for a similarity based assay allowed simple and robust HTS of the large
and three-dimensional structure-based search of the UC com- chemical library selected for this study. However, com-
pound library, which yielded 266 compounds with high docking pounds that are strongly fluorescent, colored, poorly soluble,
scores to the DNPEP active site.
or redox-active could give false-positive readings due to their
The primary HTS was performed using the UC 25,000 ability to interfere with the fluorescence emission readout of
compound Diversity Set (26120 compounds actually tested) the assay. These compounds needed to be distinguished from
and 266 compounds selected from the virtual screen. Compounds true hits before further investigation (Simeonov et al., 2008;
were tested at a final concentration of 12.3 mM. Compounds with Baell and Holloway, 2010; Soares et al., 2010). To identify true

Discovery of Small-Molecule DNPEP Inhibitors
235
Fig. 2. The primary HTS for DNPEP
inhibitors. (A) DNPEP inhibitors identi-
fied in a small-scale Asp-AMC hydrolysis
screen with predicted high affinity for the
DNPEP active site. These compounds were
used as search seeds to identify related
compounds in the 350,000 UC chemical
library with potentially greater efficacy and
potency. This search yielded 266 com-
pounds that were added to the 25,000 UC
Diversity Set for the HTS. (B) Sorted
activity plot of the 26,368 compounds from
the 25,000 UC Diversity Set (black dots)
and virtual screen (red dots) in the Asp-
AMC hydrolysis assay. Each compound
was tested at 12.3 mM. The inset table
summarizes the primary HTS results. (C)
Sorted activity plot of 586 hits, including
367 DNPEP inhibitors and 219 activators
identified in the primary HTS, tested in
triplicate at 12.3 mM in the Asp-AMC
hydrolysis assay. Mean activity score (Mean)
and S.D. (residual) are shown as black and
red dots, respectively. Results are summa-
rized in the inset table. (D) A sorted EC50
(i.e., IC50 and AC50 for inhibitors and
activators) plot of 285 hits, including 282
DNPEP inhibitors and 3 activators, con-
firmed in the dose-response Asp-AMC
hydrolysis screen. Summarized results
are presented in the inset table.
modulators of DNPEP activity, we employed a LC-MS–based were retested in a single experiment shown in Fig. 3C that
method that directly measures products of DNPEP-catalyzed yielded 26 compounds with repeatable activity scores #250%,
hydrolysis of the physiologic peptide substrate, Ang II representing an 81.3% confirmation rate. The average S.D. for
(Chen et al., 2012) (Figs. 1C; 3A). As shown in Fig. 3B, 281 this screen was 5.9 6 4.6%, and the Z9-factor was 0.63, both of
inhibitors and 2 activators of DNPEP were tested in triplicate which confirmed the assay reliability (Supplemental Ta-
at 10 mM by the Ang II hydrolysis assay. Due to the large ble 9). Potencies of these confirmed 26 inhibitory compounds
quantity of hits to be tested and the limitation of LC- were then tested using a 10-concentration dose-response assay
MS capacity, the test was separated into nine individual performed in triplicate. Twenty-three compounds showed IC50
experiments with eight repeats of each control in each ex- values #20 mM (Fig. 3D; Table 1), thus confirming their
periment. A total of 35 DNPEP inhibitors was confirmed by activities as true DNPEP inhibitors. Quality control parameter
the first orthogonal screen, whereas no activators were active values for the dose-response assays are shown in Supple-
in this assay, which is not surprising given the inherent mental Table 10.
general difficulty in finding compounds that stimulate en-
Counter Screen against ENPEP. ENPEP exhibits sim-
zyme activity (Bishop and Chen, 2009; Mast et al., 2014) ilar substrate preferences to those of DNPEP. However, there
(Supplemental Tables 7 and 8). The low enrichment of hits are fundamental differences in the active site structures of these
confirmed from the orthogonal screen can be attributed to two enzymes that could make selective chemical inhibition of
two main factors. First, there are false positives arising from one enzyme over the other achievable (Chen et al., 2012; Yang
various assay interference effects, as described above. Sec- et al., 2013). Selective inhibitors of DNPEP will be useful probes
ond, the specificity constant for Ang II hydrolysis by DNPEP to study the biology of this enzyme. Additionally, dual inhibitors
(kcat/Km 5 76.3 ꢀ 103 M21ꢀS21) is much higher than that of of both DNPEP and ENPEP could also be useful for further
Asp-AMC (kcat/Km 5 1.3 ꢀ 103
M
21ꢀS21) so that either development potentially as therapeutic agents for treatment of
a higher inhibitor concentration or higher DNPEP-binding hypertension ( Bodineau et al., 2008b; Marc et al., 2012;
affinity could be required for the inhibitors to exert detect- Balavoine et al., 2014). Therefore, we performed a counter
able effects. Indeed, the activity distribution chart revealed screen to test the confirmed 23 DNPEP inhibitors for
that 98 of the 248 inactive compounds retained inhibitory their activities toward ENPEP. Human ENPEP containing
activities in the range of 250% to 220%, which is significantly a C-terminal 6-His tag was expressed in Sf9 insect cells as a
above the level of error in the assay, consistent with the latter secreted protein (Goto et al., 2006). The purified protein ex-
explanation (Fig. 3B). To eliminate systematic errors, the DNPEP hibited catalytic parameters similar to those described pre-
inhibitors identified in the nine Ang II hydrolysis experiments viously (Goto et al., 2006) (Fig. 4, A and B). Activities of the 23

236
Chen et al.
Fig. 3. Quantification of product of Ang II hydrolysis by LC-MS–based orthogonal assay. (A) Mass spectra of peptides present in the reaction mixtures
after the 30-minute incubation. Two sets of +1, +2, and +3 ions present in the top panel correspond to Ang II (substrate, labeled black) and Ang III
(cleavage product, marked in red). Production of Ang III was completely abolished in a sample treated with 0.5 mM ZnCl2 (bottom spectrum). (B) The
sorted activity plot of 284 hits (282 inhibitors and 2 activators) identified in the primary HTS and retested by the Ang II hydrolysis assay at 10 mM in
triplicate. Results are summarized in the inset table. (C) The repeat of Ang II hydrolysis assay to confirmed hits (32 available of 35 total) selected from
triplicate screen shown in (B) by one-plate experiment, at 10 mM in triplicate. Results are summarized in the inset table. (D) The sorted IC50 plot for 23 of
the most potent inhibitors. Each compound was tested at 10 different doses. Curve fitting was performed using the Hill equation.
DNPEP inhibitors toward ENPEP were plotted in comparison 406365. There were no major additional peaks in their spectra,
with their activities toward DNPEP in Fig. 4C. The S/B ratio indicating their purity. The mass spectrum of compound
and Z9-factor for this assay were 313 and 0.56, respectively. 406365 showed an ion at the expected mass (m/z 5 435) as
This counter screen revealed a number of compounds that well as additional peaks at m/z values of 432 and 437, in-
displayed strong inhibition toward both enzymes, whereas dicating that the sample was probably not pure. Freshly made
others were quite selective for DNPEP. To semiquantitatively aqueous stocks of compounds 93293, 98897, 897927, and
describe their specificity, the 23 compounds were grouped as 406365 all inhibited DNPEP in a dose-dependent manner
DNPEP-selective ActivityDNPEP 2 ActivityENPEP (# 250%, 8 with IC50 values very similar to those shown in Fig. 5 and
compounds), partially DNPEP-selective (250% , ActivityDNPEP 2 Table 1, thus confirming their activity toward DNPEP. Com-
ActivityENPEP , 220%, 6 compounds), and dual inhibitors pounds 241320, 705521, and 374081 also were active, but only
(ActivityDNPEP 2 ActivityENPEP $ 220%, 9 compounds), ac- when dissolved in alkaline buffers possibly due to poor aqueous
cording to the difference in activity scores between the two solubility at neutral pH.
enzymes (Table 1; Fig. 5).
Chemical Properties of Identified Hits. According to
Confirmation of Chemical Composition and Activity. their chemical similarities, the 23 confirmed DNPEP inhibitors
To confirm the identity of the most selective and potent hits were clustered into 9 groups, named A through I (Fig. 5;
from each promising chemical class shown in Fig. 5, we ob- Table 1). Notably, many of these compounds contain two neg-
tained compounds 93293, 98897, 241320, 705521, 897927, atively charged or polar groups separated by four to six covalent
374081, and 406365 as powders from the UC chemical library bonds. Catechol/Benzenetriol, aminothiazole, amide, carboxyl-
and analyzed them by MS. The principle observed ion exactly ate, furan, rhodanine, polycyclic (hydro)quinones, and sulfonic
matched the expected mass for all of these compounds except acid groups were especially prevalent among the identified

Discovery of Small-Molecule DNPEP Inhibitors
237
TABLE 1
Summary of hit activities from the primary high-throughput screen, orthogonal, and counter screen assays
DNPEP
ENPEP
Glu-AMC
Hydrolysis
Chemical
Groups
Asp-AMC Hydrolysis
Ang II Hydrolysis
Number
Compound Identification
D
Activity Score
(10 mM)
Activity
a
Activity Score (12.3 mM)
IC50 (mM)
Activity Score (10 mM)
IC50 (mM)
Score
A
1
2
93293b
392425b
897833c
98897b
295.0 6 1.4
287 6 1.4
1.4
5.0
293.9 6 0.6
289.6 6 0.3
255.6 6 11.3
294.3 6 0.9
292.7 6 2.4
293.3 6 0.6
291.8 6 5.2
278.9 6 4.5
293.0 6 1.3
292.2 6 2.3
291.0 6 2.4
250.1 6 6.5
259.3 6 8.9
291.7 6 3.2
280.5 6 4.6
290.7 6 3.6
256.0 6 9.7
294.0 6 1.1
293.0 6 2.4
288.9 6 2.5
275.3 6 4.0
290.7 6 4.3
287.2 6 8.7
0.1
0.81
12
277.8 6 9.7
287.4 6 2.5
6.5 6 11.1
271.9 6 2.2
284.4 6 1.1
245 6 7.2
228.8 6 8.7
4.5 6 3.9
289 6 0.8
5.5 6 3.4
245.8 6 4.9
222.4 6 3.1
243.2 6 2.9
217.3 6 2.5
55.5 6 9.6
272.8 6 8.9
20.2 6 7.5
283.9 6 2.0
244.8 6 5.3
261 6 4.2
20.8 6 6.6
264.9 6 3.8
292.7 6 1.0
216.1
22.2
262.1
222.4
28.3
248.3
263.0
283.4
24.0
297.7
245.2
227.7
216.1
274.4
2136.0
217.9
255.8
210.1
248.2
227.9
274.5
225.8
5.5
B
C
3
283 6 0.2
1.7
4
295.7 6 0.4
263.8 6 2.4
284.4 6 1.0
274.5 6 5.2
282.5 6 0.9
283 6 0.7
287.4 6 1.2
273.6 6 1.4
281.8 6 0.2
268.1 6 2.5
274.4 6 3.1
274.7 6 0.7
272.3 6 0.8
281.7 6 0.7
293.4 6 0.2
274.6 6 1.2
262.8 6 2.3
278.8 6 0.8
277.4 6 1.5
277.8 6 1.0
0.54
5.8
2.4
4.4
0.16
0.83
2.7
2.3
3.1
1.3
0.40
2.0
0.26
5.0
1.7
4.7
3.6
8.3
18
5.7
3.6
1.4
17
2.1
4.2
6.2
3.0
6.0
2.8
5
769078b
702610
241320c
705521c
936427b
374081c
405279
914971
920521b
897927c
251431c
347749b
252185c
505901b
923254
773587
406365c
899189
312792b
6
D
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
10.2
5.7
E
F
G
1.6
2.0
3.2
0.84
7.7
8.0
H
I
5.7
3.2
3.2
Ang II, angiotensin II; Asp-AMC, L-aspartic acid 7-amido-4-methylcoumarin; DNPEP, aspartyl aminopeptidase; ENPEP, glutamyl aminopeptidase; Glu-AMC, L-glutamic
acid 7-amido-4-methylcoumarin.
aD Activity score = activity scoreDNPEP 2 activity scoreENPEP, where activity scoreDNPEP is for Ang II hydrolysis.
bDual inhibitors of DNPEP and ENPEP.
cDNPEP-selective inhibitors.
inhibitors (Fig. 5). Catechol and rhodanine are known metal groups A, C, and D displayed submicromolar potencies and
chelators; sulfonic acid or related phosphonic acid–containing com- maximum inhibitory effects greater than 89% (Fig. 5).
pounds have previously been identified as metallo-aminopeptidase
As shown in Fig. 5, DNPEP-selective inhibitors marked
inhibitors (Mucha et al., 2010); and carboxylate and amide with an asterisk and dual inhibitors marked with a double
groups resemble features of DNPEP substrates. Compounds dagger each shared broadly similar chemical characteristics,
899189 and 312792 (Fig. 5, group I) possess reactive, electro- with some exceptions. Amide or amide-like hydrozones (897833,
philic benzoquinone groups that most likely make them non- 705521, and 406365), amino carboxylic acids (241320, 374081,
specific enzyme inhibitors. Four compounds with identification and 406365), sulfonic acids (897927 and 251431), and com-
numbers 93293, 392425, 769078, and 241320 belonging to pound 252185 are DNPEP-selective inhibitors that are not
Fig. 4. Counter screen of hits toward ENPEP. (A) Coomassie-Blue–stained SDS-PAGE gel and immunoblot of purified human ENPEP. (B) Michaelis
Menten kinetics of Glu-AMC hydrolysis by ENPEP. The inset shows the calculated Km and kcat values. (C) Comparison of hits toward DNPEP in Ang II
hydrolysis over ENPEP in Glu-AMC hydrolysis in activity score at 10 mM (sorted by activity scores toward ENPEP). Differences in activity score toward
DNPEP over ENPEP (Dactivity score) are shown in Table 1. Hits with Dactivity scores #250%, between 250% and 220%, or $220% were defined as
DNPEP-selective, DNPEP-preferential, and dual inhibitors, respectively.

238
Chen et al.
Fig. 5. Layout of confirmed DNPEP inhibitors clustered in groups A–I. On top are the functional groups found in the identified DNPEP inhibitors. Each
compound is displayed by its chemical structure, the compound identification, and its activities toward DNPEP (Ang II hydrolysis assay) and ENPEP
(Glu-AMC hydrolysis assay) at 10 mM. Concentrations are IC50 values obtained from dose-response assays. Activity scores #250% are marked in red.
DNPEP-selective inhibitors and dual inhibitors are marked with asterisks and double daggers, respectively.
rigidly planar. Conversely, most dual inhibitors were planar a known pan assay-interference moiety (Mendgen et al., 2012)
molecules residing in chemical groups A, C, G, and I that that has been reported active in a significant number of HTS
featured catechol-aminothiazole, rhodanine-enylfuran, fluo- assays, causing concerns about the specificity of compounds
rescein, and quinone derivatives. Exceptions to this general containing this chemical group (Carter et al., 2001; Voss et al.,
trend included compounds 347749 (group F) and 936427 2003; Carlson et al., 2006; Powers et al., 2006; Baell and
(group D) that are nonplanar. Interestingly, compound 251431 Holloway, 2010). However, these compounds share the common
acted as a stimulator of ENPEP boosting its activity 1.5-fold features seen in other DNPEP inhibitors with two functional
(Fig. 5E).
groups bridged by four to six covalent bonds, suggesting that
Among the submicromolar potent inhibitors, only compound they could exert a DNPEP-selective effect with further modi-
241320 (group D) was DNPEP-selective, whereas 93293, fications. The fluorescein derivatives in group G are fluorescent
392425 (group A), and 769078 (group C) inhibited both DNPEP compounds that are commonly used as dyes. Despite concerns
and ENPEP. Compounds 93293 and 392425 (group A inhib- about these compounds interfering with the primary screening
itors) contain metal-chelating catechol and thiazole groups also assay, their inhibitory effect toward DNPEP was confirmed in the
found in E. coli methionine aminopeptidase (MetAP) inhibitors orthogonal, MS-based assay, thus assuring that they are genuine
(Wang et al., 2008; Chai and Ye, 2010). Compound 769078 inhibitors. However, their bulky, planar structures and ortho-
represents group C dual inhibitors containing a rhodanine semiquinone moieties suggest that these compounds are non-
moiety linked to furan ring by an alkene spacer. Rhodanine is specific inhibitors and thus will not undergo further development.

Discovery of Small-Molecule DNPEP Inhibitors
239
SAR Analysis of DNPEP Inhibitors. We used five suggesting the sulfur moiety is the immediate metal-chelating
strong DNPEP inhibitors (241320, 347749, 98897, 769078, atom of the rhodanine-containing group. The furan ring could
and 702610) as seeds to search for related compounds in the not be replaced with thiophene or pyridine (e.g., compounds
UC compound library. Similar compounds identified by molec- 980083, 496193, 240994, and 240991). A variety of groups,
ular fingerprint comparisons were manually inspected to ensure including methyls (e.g., compounds 770885 and 911299),
all selected compounds retained the putative metal-chelating halogens (e.g., compounds 98875, 769080, and 1001360),
functional groups expected to interact with the DNPEP metal carboxylate (compound 98871), nitrate (compound 769641),
center (92 compounds; Supplemental Figs. 3 and 4). The methoxyl (compound 98897), and even bulky groups such as
inhibitory activity of these compounds toward DNPEP was chlorophenyl (compound 397172) or morpholine (compound
then tested in triplicate at a final concentration of 10 mM by the 954647) could be linked at position 2 of the furan ring while
Asp-AMC hydrolysis assay. A total of 25 of 92 tested compounds maintaining reasonable inhibitory activity toward DNPEP.
was identified as DNPEP inhibitors with activity scores #260 This indicates that the vinylfuran group may be bound in the
(27.2% hit rate) (Supplemental Figs. 3 and 4). The Z9-factor from larger S19-S29 pocket of the enzyme, which can accommodate
the similar compound test was 0.96, demonstrating the re- bulkier groups compared with the S1 pocket. In agreement
liability of this assay (Supplemental Table 11).
with this hypothesis, compound 897927 (Fig. 5E) contains
Compound 241320 was the most potent DNPEP-specific a group C compound scaffold (typical of dual inhibitors) linked
inhibitor (IC50 5 0.26 mM), and an activity test of its neighboring to a bulky benzylsulfonic acid moiety that confers DNPEP
compounds (Supplemental Fig. 3) revealed that DNPEP in- selectivity. Therefore, the 2-furan extension may be consid-
hibition by this scaffold could tolerate minor modifications on ered as a modification site for further development of group C
the serine group, including deletion of the 3-hydroxyl group, compounds to increase their DNPEP selectivity.
addition of a methyl group, deletion of the carboxylate group, or
replacement of the carboxylate with a hydroxyamide moiety
(compounds 503659, 247266, 247275, and 93984). However,
Discussion
halogenations of the catechol ring abolished DNPEP inhibi-
In this study, we identified 23 DNPEP inhibitors with IC50
tory activity (compounds 520266 and 130024), suggesting values ,20 mM, among which 8 exhibited selectivity for
that the inhibitory effect of the catechol is due to its DNPEP over ENPEP. An additional 25 DNPEP inhibitors
interaction with the DNPEP metal center. These related were identified from a chemical similarity search based on
active compounds retain the most essential features of 5 selected inhibitors. Together, these compounds displayed
compound 241320 and are likely to be DNPEP-selective.
significant chemical diversity and could be clustered into
Compound 347749, the only nonplanar dual inhibitor, is 9 families based on their chemical structures. Most of the in-
comprised of threonine coupled to a rhodanine ring. Activities hibitors have in common a putative metal coordinating moiety
of its neighboring molecules (Supplemental Fig. 3) showed linked by four to six covalent bonds to a negatively charged or
that compound 347749 is relatively intolerant of chemical polar group. The most common metal-coordinating groups
changes for maintenance of its inhibitory activity. Modifica- found were catechol and rhodanine rings. SAR analysis of all
tions of the threonine group mostly ablated DNPEP inhibitory DNPEP inhibitors from the initial and hit enrichment screens
activity of the molecule. However, compound 279700 with showed that DNPEP inhibitors could occupy at least two
threonine replaced with 4-bromoaniline retained weak DNPEP distinct positions in the active site of the enzyme in addition to
inhibitory activity. Despite concerns about nonspecific effects of coordinating the metal center. Small negatively charged
rhodanine compounds, the SAR results indicate that compound groups could occupy the S1 pocket of the active site consisting
347749 could be selective for DNPEP and ENPEP.
of a small, positively charged pocket, whereas more bulky
Compounds 769078 and 98897 are strong dual inhibitors groups could interact with the putative S19-S29 sites of the
containing rhodanine rings with rigid hydrophobic vinyl furan peptide-binding groove (Fig. 6A). The group A and C dual
extensions (Fig. 5). Compound 702610 is a DNPEP preferred inhibitors are rigid and could bind to the zinc centers of both
inhibitor, grouped in the same chemical cluster with 769078 DNPEP and ENPEP with similar affinity. These DNPEP and
and 98897, but containing a 4-amino-thioxo-1,2,4-triazinone ENPEP dual inhibitors could be useful as pan-blockers of
ring in place of rhodanine. The exocyclic NH2 group and ad- aspartyl aminopeptidase activity, but their lack of selectivity
jacent carbonyl and thiocarbonyl moieties most likely form a would be a concern in cell-based or in vivo experiments.
strong metal-chelating functionality. The neighboring search Conversely, the group D and E DNPEP-selective inhibitors in
and activity test of group C compounds (Supplemental Fig. 4) addition to possessing strong chelator functionalities have
showed they tolerate geometrical isomerizations and even extensions with rotatable bonds that could conform to the
alkene linker extension or truncation while still retaining DNPEP active site and cause selective binding.
DNPEP inhibitory activity; however, addition of alkylation of
Catechols are known to coordinate divalent metal ions by
the alkene linkage of furan to rhodanine abolished activity virtue of their acidic ortho-hydroxyl groups. A recent HTS
(compounds 921127, 926399, and 921314). Introduction of screening campaign to develop inhibitors against Pfm18AAP,
polar groups on the N3 atom of the rhodanine ring, for exam- a potential drug target for the treatment of malaria, identified
ple amines or carboxylic acids, was tolerated (compounds a number of catechol-containing inhibitory molecules (Schoenen
910179, 966958, 521685, 943988, and 994188). Conversely, et al., 2010). In fact, the best-in-class probe identified in
methyl, ethyl, or isobutyl alkylation generally abolished in- that screen, ML369 (Supplemental Fig. 5A), features a cate-
hibitory activity (e.g., compounds 938731, 920776, 917820, chol moiety linked to a piperidine-tetrahydroquinoline ring
374601, and 771512). Selective modifications to and even system. Catechols have also been identified as inhibitors of
opening of the rhodanine ring with deletion of the carbonyl methionine aminopeptidase, which, like DNPEP, also contains
group (compound 96270) did not ablate the inhibitory activity, a binuclear zinc center. A structure of E. coli methionine

240
Chen et al.
Fig. 6. Modeling of selected inhibitors in the DNPEP active site. (A) Structure of the DNPEP active site (PDB accession code 3VAT) showing the
binuclear zinc center (gray spheres) in relation to the S1 site and the peptide-binding groove of the enzyme, which is partially formed by additional
subunits of the DNPEP complex (shown in surface representation). Lys370, present in the S1 site of the enzyme, confers selectivity by interacting with the
negatively charged N-terminal Asp or Glu residue of DNPEP peptide substrates. Models of (B) compound 241320 and (C) compound 347749 binding to
the DNPEP active site. The model in (B) was docked based on the structure of methionine aminopeptidase in complex with a catechol-containing
inhibitor (Wang et al., 2008), whereas the model in (C) was placed assuming that the exocyclic double-bonded sulfur atom is the relevant metal chelator
in this compound. Both models were energy minimized using the Crystallography and NMR System program (Brunger, 2007). Distances are given in
angstroms.
aminopeptidase in complex with a catechol-containing com- ring for maintenance of inhibitory activity toward DNPEP.
pound revealed the mode of interaction of the catechol ring with An increase or decrease in the linker by one carbon atom
the binuclear zinc center (Wang et al., 2008). In this structure, substantially reduced or abolished activity against both
one of the hydroxyl groups of the catechol replaced water as DNPEP and Pfm18AAP. Interestingly, modifications to the
a bridging ligand, whereas the other interacted with one of the heteroaromatic ring with or without changes in the linker
zinc ions enforced by the active site shape. A second mode of portion of the molecule were observed to substantially alter
binding whereby both hydroxyl groups serve as metal bridging the IC50 values of these compounds toward DNPEP without
ligands with the plane of the catechol group perpendicular changing their activity toward Pfm18AAP. These data support
to the inter-metal axis might also be possible depending upon the proposition that differences in residues lining the peptide-
the active site structure. Modeling of the catechol-containing binding groove between these two enzymes where the bulky
compound 241320 into the active site structure of mammalian heterocyclic rings most likely bind can be exploited to achieve
DNPEP suggests that it could adopt a mode of binding similar selectivity.
to the methionine aminopeptidase-catechol complex (Fig. 6B).
The metal-binding capacity of rhodanine is well-known
The alternative binding mode with perpendicular catechol from its use as a copper-detecting agent in histologic studies
and intermetal planes may not be feasible due to steric (Lindquist, 1969). Direct binding of rhodanine to the zinc
clashes with surrounding active site residues. Compound center of anthrax lethal factor, a metallopeptidase, was
241320 also features a serinyl side chain that appears to be previously reported (Forino et al., 2005), and ligand-docking
sufficiently small in size to accommodate the S1 pocket of studies suggest that it can also bind the zinc center of matrix
DNPEP, with the carboxylate and hydroxyl groups interact- metalloproteases (Hu et al., 2008). In these studies, the
ing with the surrounding Lys370 side chain thus mimicking rhodanine was observed or predicted to coordinate zinc
the Asp side chain of peptide substrates (Fig. 6, A and B). through its endocyclic sulfur atom. Based on steric consid-
Several compounds from the current HTS as well as the erations, we hypothesize that rhodanine is more likely to
Pfm18AAP HTS are catechols linked to bulky groups that interact with the binuclear center of DNPEP via its exocyclic
would be unable to fit in the S1 pocket. Instead, these groups rather than endocyclic sulfur atom. Density function theory
most likely bind in the putative peptide-binding groove of the calculations predict that the highest occupied molecular
enzyme or one of the other small pockets located in close orbital and negative electrostatic potential of the rhodanine
proximity to the metal center. Notably, residues within 8 Å of ring localize predominantly to the exocyclic sulfur atom
the DNPEP and Pfm18AAP metal centers, including those (Mendgen et al., 2012), which supports the proposed role of
that make up the S1 pocket, are absolutely conserved, this site in metal coordination. Compound 347749 can be
whereas differences can be found in the peptide-binding modeled into the DNPEP active site in a sterically acceptable
grooves of these two enzymes. The previously reported counter fashion with the exocyclic sulfur binding in the bridging
screen of Pfm18AAP inhibitors against human DNPEP ligand position between the zinc ions and the negatively
(hM18AAP, AID588696) identified 25 compounds active charged theoninyl side chain occupying the S1 pocket via an
against human DNPEP as defined by IC50 values ,100 mM. ionic interaction with Lys370 and hydrogen-bonding interac-
Data from this screen provide additional SAR information tions with surrounding protein groups. In this orientation, the
for M18 aminopeptidase inhibitors. The compound used as endocyclic sulfur atom of the rhodanine ring can form
a basis for this counter screen SAR analysis (CID 23724194) a favorable interaction with the Met435 side chain sulfur
consisted of a catechol ring linked by an aminoethyl chain atom (Fig. 6C). Rhodanine has a notorious reputation in the
to an acridine heteroaromatic ring (Supplemental Fig. 5B). field of HTS drug discovery due to its tendency to display
This compound was intolerant to changes in the catechol activity in a diverse range of HTS assays (Baell and Holloway,

Discovery of Small-Molecule DNPEP Inhibitors
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DNPEP over ENPEP. Additionally, a screen of 16 compounds
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Acknowledgments
The authors thank Dr. Leslie T. Webster, Jr., for critically
reviewing the manuscript.
Authorship Contributions
Participated in research design: Chen, Tang, Seibel, Papoian,
Palczewski, Kiser.
Conducted experiments: Chen, Tang, Seibel, Oh, Li, Golczak, Kiser.
Performed data analysis: Chen, Tang, Seibel, Zhang, Golczak,
Palczewski, Kiser.
Mucha A, Drag M, Dalton JP, and Kafarski
P (2010) Metallo-aminopeptidase
Wrote or contributed to the writing of the manuscript: Chen, Kiser.
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Address correspondence to: Dr. Krzysztof Palczewski, Department of
Pharmacology, School of Medicine, Case Western Reserve University, 10900
Euclid Avenue, Cleveland, Ohio 44106-4965. E-mail: kxp65@case.edu or
Dr. Philip D. Kiser, Department of Pharmacology, School of Medicine, Case
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J Biol Chem 288:25638–