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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 DNPEPcatalyzed 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. Introduction Aminopeptidases are a heterogeneous group of enzymes that catalyze the hydrolysis of N-terminal residues from peptide substrates. Aspartyl aminopeptidase (DNPEP; EC 3.4.11.21) and glutamyl aminopeptidase (ENPEP or aminopeptidase A; EC 3.4.11.7) are the two known acidic residuespecific aminopeptidases present in mammals (Glenner et al., 1962; Wilk et al., 1998; Goto et al., 2006). Mostly found in kidney, lung, and immune cells, ENPEP is a membraneassociated ecto-enzyme belonging to the M1 metallopeptidase family (Wu et al., 1990; Nanus et al., 1993; Goto et al., 2006). ENPEP catalyzes the hydrolysis of angiotensin II (Ang II) to form angiotensin III (Ang III) and is involved in the regulation of systemic blood pressure (Reaux et al., 1999; Mitsui et al., 2003; Wright et al., 2003; Bodineau et al., 2008a) and cancerassociated angiogenesis (Marchio et al., 2004). Whereas the function of ENPEP has been well-studied, the biologic and pathologic roles of DNPEP remain poorly understood. DNPEP belongs to the M18 metallopeptidase family, the members of which are found in all kingdoms of life (Rawlings et al., 2014). The genomes of mammals and most other vertebrate species contain only one M18 metallopeptidaseencoding gene. Sequence identity among mammalian DNPEP orthologs is generally greater than 90%. This strong conservation suggests that DNPEP may play an essential role in cellular metabolism that has remained conserved throughout evolution. DNPEP is a self-compartmentalized, binuclear zinc-containing enzyme that forms a tetrahedron-shaped homododecameric complex (Chaikuad et al., 2012; Chen et al., 2012; Sivaraman et al., 2012). The active site-containing nanocompartment enclosed by the DNPEP tetrahedron is accessible through four ∼20 Å-wide selectivity pores that allow entrance of short peptides. In mammals, DNPEP is expressed in many organ systems with especially high activity in the brain and testis (Wilk et al., 1998). This enzyme is commonly described as cytosolic, although it also exists in a membraneassociated form in some tissues (Cai et al., 2010; Mayas et al., 2012b). A role for DNPEP in regulation of the renin-angiotensin system has been proposed on the basis of its substrate specificity (Wilk et al., 1998; Chen et al., 2012), although its involvement in the renin-angiotensin system has not been examined in vivo. Changes in DNPEP expression and/or activity have been noted in neoplastic disorders such as colon and breast cancers, squamous cell carcinoma, and gliomas (Perez et al., 2009; Martinez-Martos et al., 2011; Mayas et al., 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., This work was supported by Case Western Reserve University School of Medicine; and National Institutes of Health [Grant EY008061 (to K.P.)]. K.P. is the John H. Hord Professor of Pharmacology. Current affiliation: Department of Neurology, College of Medicine, University of Cincinnati, Cincinnati, Ohio. 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-amido4-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 http://molpharm.aspetjournals.org/content/suppl/2014/06/09/mol.114.093070.DC1 Supplemental material to this article can be found at: at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from 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 physiology, 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 aminopeptidase (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 inhibitors of PfM18AAP was previously reported (Pubchem Bioassay: 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 characterization of a set of small-molecule inhibitors of DNPEP. These compounds displayed low micromolar inhibitory activity toward DNPEP-catalyzed hydrolysis of the biologically relevant substrate, Ang II. Some of the compounds were more selective 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. Materials and Methods Chemicals. L-aspartic acid 7-amido-4-methylcoumarin (Asp-AMC), L-glutamic acid 7-amido-4-methylcoumarin (Glu-AMC), andAng II were purchased from Bachem (Torrance, CA). DNPEP Expression and Purification. Bovine DNPEP was expressed in T7 Express BL21Escherichia coli (NewEngland Biolabs, Ipswich, MA) and purified, as previously described (Chen et al., 2012). The concentration and purity of DNPEP were measured by the Bradford 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 synthetic substrate, Asp-AMC, which can be hydrolyzed to produce an AMC fluorophore suitable for the HTS plate reader (Supplemental Fig. 1B). To separate signal from substrate and product, excitation and emission spectra of both Asp-AMC and AMC were measured in a 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 (Supplemental 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 coordinate 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 Cincinnati (UC) compound library by the following: 1) execution of similarity 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 generation and incorporation into the virtual screen, as detailed above. Thirty-three compounds from this screen were experimentally evaluated (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. 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 DNPEPcatalyzed 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 mMAsp-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 FI30min 2FI0min: 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: Activity score5 RFIcp 2RFIenzyme RFIenzyme 2RFIZn 100; where RFIenzyme represents the relative fluorescence intensity of untreated control, RFIcp the relative fluorescence intensity of test 232 Chen et al. at A PE T Jornals on M ay 4, 2017 m oharm .aspeurnals.org D ow nladed from compound-containing sample, and RFIZn the relative fluorescence intensity of the maximum inhibition control. A total of 26,120 compounds from the UC 25,000 Diversity Set in addition to 266 compounds selected from the UC compound collection based on virtual screening and similarity searches was tested at final concentrations of 12.3 mM in the primary HTS. Hits were selected with activity scores #260% (inhibitors), or$130% (activators). These hits were retested in triplicate at final concentrations of 12.3 mM. The potencies of confirmed hits were evaluated in dose-response assays performed in triplicate. Dose-response curves were generated using Genedata Screener Condoseo (Ver. 9.0.0 Standard) Software. EC50 is denoted as 50% effective concentration, that is, 50% inhibitory concentration (IC50) or 50% activation concentration (AC50). Compounds with IC50 or AC50 values #20 mM were selected for further evaluation. The primary, triplicate, and dose-response screens were monitored by two quality control parameters: signal to background (S/B) ratio and the Z9-factor defined, respectively, as: S B ratio5 RFIenzyme RFIZn