Selective Targeting of Extracellular Insulin ... - ACS Publications

Articles pubs.acs.org/acschemicalbiology

Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors Samer O. Abdul-Hay,† Thomas D. Bannister,‡ Hui Wang,‡ Michael D. Cameron,§ Thomas R. Caulfield,† Amandine Masson,† Juliette Bertrand,† Erin A. Howard,† Michael P. McGuire,† Umberto Crisafulli,† Terrone R. Rosenberry,† Caitlyn L. Topper,∥ Caroline R. Thompson,∥ Stephan C. Schürer,§,⊥ Franck Madoux,# Peter Hodder,# and Malcolm A. Leissring*,†,∥ †

Department of Neuroscience, Mayo Clinic Florida, Jacksonville, Florida 32224, United States Department of Chemistry, §Molecular Therapeutics, #Molecular Screening Center, Lead Identification Division, Translational Research Institute, Scripps Research Institute, Jupiter, Florida 33458, United States ∥ Institute for Memory Impairments and Neurological Disorders (UCI MIND), University of California, Irvine, California 92697, United States ⊥ Department of Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine, Miami, Florida 33136, United States ‡

S Supporting Information *

ABSTRACT: Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50app = 63 nM), nontoxic at concentrations up to 100 μM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets.

I

the treatment of diabetes,6 strategies that avoid the inhibition of intracellular pools of IDE will help to minimize undesirable side effects. There is, therefore, a need for experimental probes and pharmacophores that selectively target pools of IDE in different subcellular compartments, particularly the extracellular space. IDE evolved independently from most conventional zincmetalloproteinases7 and consequently possesses a number of distinguishing characteristics. For instance, IDE contains a zincbinding motif (HxxEH) that is inverted with respect to the canonical zinc-metalloproteinase motif (HExxH).8 IDE can also be distinguished pharmacologically from most other zincmetalloproteinases by its sensitivity to thiol-alkylating

nsulin-degrading enzyme (IDE) is an atypical zinc-metallopeptidase that hydrolyzes intermediate-sized peptide substrates in multiple subcellular compartments, including cytosol, mitochondria, and the extracellular space.1 IDE is strongly implicated in the pathogenesis and potential treatment of type 2 diabetes mellitus and Alzheimer’s disease, by virtue of its well-established role in the degradation of two extracellular substrates: insulin and amyloid β-protein (Aβ).2 However, IDE hydrolyzes many intracellular substrates as well, which are implicated in diverse physiological processes.3−5 Currently available genetic and pharmacological tools target all pools of IDE simultaneously; thus, experimental probes that can selectively manipulate distinct pools of IDE will be required to disentangle the distinct roles of intracellular versus extracellular pools of IDE. Moreover, for therapeutic applications, such as inhibiting the breakdown of insulin for © XXXX American Chemical Society

Received: May 6, 2015 Accepted: September 23, 2015

A

DOI: 10.1021/acschembio.5b00334 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

ACS Chemical Biology agents,9−11 such as N-ethylmaleimide (NEM) and iodoacetamide. The most common (nonmitochondrial) isoform of IDE contains 13 cysteine residues,12 and inhibition by thiolmodifying agents such as NEM is known to occur principally via modification of two specific cysteines, C812 and C819, which reside within the C-terminal half of IDE’s unusual bipartite active site.10,11 However, NEM and other thiolalkylating compounds are not suitable for inhibiting IDE in cellbased applications due to broad reactivity with biological nucleophiles, very low IDE potency (with apparent IC50 (IC50app) values > 200 μM), and cellular toxicity at effective concentrations. IDE’s unusual subcellular localization profile is a further distinguishing feature.1 IDE is located principally in the cytosol and also has been described within various intracellular compartments, including endosomes, peroxisomes, and mitochondria.1 On the other hand, IDE is also secreted into the extracellular space via a nonconventional protein export mechanism13 reported to involve exosomes.14 Membraneassociated forms of IDE present on the cell surface also have been described.15 Because these separate pools of IDE degrade different sets of substrates implicated in divergent physiological processes, there is a great need for pharmacological tools that can selectively target intracellular versus extracellular pools of IDE. Herein, we describe the discovery, characterization, and kinetics-based optimization of thiol-targeting IDE inhibitors in the benzoisothiazolone structural class. These compounds exhibit high potency (IC50app values as low as 63 nM), low cellular toxicity, and IDE selectivity, and they act by modifying only the C819 residue of IDE. Notably, by virtue of a unique quasi-irreversible mechanism of inhibition, which is operative only in the oxidizing environment of the extracellular space, these compounds exclusively target extracellular pools of IDE. These novel inhibitors, which are the first drug-like, nonpeptidic IDE inhibitors yet described, represent powerful new tools for dissecting the divergent roles of distinct pools of this biomedically important protease.

Figure 1. Overview of compound screening campaign.

cytotoxicity, and for activity in a cell-based assay utilizing recombinant IDE. A total of 44 hits showed IC50app values < 10 μM (Figure 1), which were subsequently tested using a wellestablished IDE activity assay20 based on a fluorogenic peptide substrate (FRET1) with recombinant wild-type IDE (WT-IDE) or a cysteine-free form of IDE (CF-IDE).11 One family of compounds, the benzoisothiazolones, showed particular promise, with four compounds (compounds 1−4; Table 1) showing encouraging potency in the cell-based IDE assay (IC50app 1−4 μM) and also good potency in our cell-free IDE activity assay (IC50 200−1100 nM). A fifth compound in the class (compound 5) was inactive by hit cutoff criteria (Table 1). Two additional compounds, 6 and 7, were available by purchase, with compound 6 being inactive and compound 7 being active (Table 1). The wide range in potency of this series, apparent in both the cell-based assay (IC50app 1 − >10 μM) and the cell-free assay (IC50app 0.23 − >10 μM) (Table 1), indicated that potency was not solely determined by the presence of the thiol-reactive isothiazolone moiety but was instead dependent on making other productive contacts with IDE. For example, the most potent hit, PubChem CID 2325815 (compound 1 in Table 1), has a morpholine group present at the R1 position. If the benzoisothiazolones were merely acting as nonselective and chemically reactive cysteine traps, without significant cooperative binding to the enzyme, then such electron-donating and bulky ortho substituents would be expected to decrease, rather than increase, the ability of the ligand to interact with IDE. While some electronic effects are apparent (e.g., compare compounds 6 and 7), compound electrophilicity is not the sole factor driving their ability to inhibit IDE.

■

RESULTS AND DISCUSSION To identify novel IDE inhibitors, we conducted a large-scale, cell-based compound screening campaign (Figure 1; PubChem Assay Identifier (AID) 434984) on the NIH Molecular Libraries Small Molecule Repository (MLSMR) library, which, at the time, comprised a collection of ∼325 000 structurally diverse compounds, with most conforming to Lipinski’s rule of five (Ro5).16 The primary screen was conducted in 1536-well format using a well-established, fluorescence polarization-based assay that is a sensitive measure of extracellular IDE activity,17 with nominal compound concentrations of 6 μM. HEK-293 cells, harvested mechanically rather than by trypsinization, were used as an endogenous source of IDE, which is secreted abundantly into the extracellular space.1,18 The performance of the assay in the primary screen was exceptionally good, with Z′ factor values19 routinely exceeding 0.8. Hit cytotoxicity was assessed separately using a luminescence-based HEK cell viability assay. From among the 324 862 compounds tested, 1316 (0.41%) exhibited inhibition exceeding a predetermined cutoff of ∼85% at 6 μM. Available actives were retested in the primary screen to confirm activity and in a number of secondary screens to identify fluorescence artifacts and toxic hits. Thus, 127 hits were triaged in dose−response format for IDE activity in cells, for B


Articles

ACS Chemical Biology Table 1. SAR of Compounds Identified by uHTS and Purchased Variants Thereof

a

NT = not tested.

C812 and C819, are primarily responsible for the thiol sensitivity of the protease10,11 due to their position within the active site (Figure 3A). Consequently, mutant forms of IDE containing only a single cysteine (sC) either at position 812 (sC812) or 819 (sC819) are both completely inhibited by broad-spectrum thiol-alkylating compounds such as NEM11 (Figure 3B). In marked contrast, compound 1 strongly inhibited the single-cysteine mutant sC819, but it had no significant effect on sC812 (Figure 3B). These results support a model wherein IDE inhibition by compound 1 arises not via nonspecific modification of any cysteine but instead via preferential interaction with C819, again suggesting that compound 1 forms productive interactions with the enzyme that augment specific binding proximal to C819. Consistent with this, the dose−response of compound 1 obtained using WT-IDE was statistically indistinguishable from that obtained using sC819 (Supporting Information Figure S2) rather than being shifted to the right, as would be predicted if the compound was reacting nonspecifically with the 13 cysteines within WT-IDE. Preferential interaction with C819 was also predicted by computational docking of compound 1 with the region of IDE containing both C812 and C819 (Supporting Information Figure S3). The potency of irreversible inhibitors is determined by the combined contribution of two distinct kinetic parameters: Ki, the dissociation constant for the reversible interaction of the inhibitor with the enzyme, and kinact, the rate constant for the irreversible reaction, in this case, ring opening and formation of the disulfide bond (Figure 2C). These parameters can be calculated by analyzing the time dependence of enzyme inhibition as a function of inhibitor concentration (see Supporting Information Figure S4). As derived in detail elsewhere,21 kinact represents the theoretical maximum rate constant (i.e., at infinite inhibitor concentration), and Ki represents the concentration of inhibitor at which kobs is 50%

We next sought to elucidate the mechanism of action of compound 1 (CID 2325815) by testing it in a range of assays. To that end, compound 1 was synthesized de novo and purified to >99% purity by final preparative HPLC, to exclude the possible influence of trace impurities, which had given falsepositive results in other compound series. Compound 1 exhibited good potency against WT-IDE (IC50app = 233 nM) while showing no activity against CF-IDE (Figure 2A), indicating that the inhibition depended on the presence of thiols. Consistent with a covalent interaction, inhibition by compound 1 was found to be irreversible in dilution experiments (Figure 2B). We hypothesized that the mechanism of IDE inhibition involves electrophilic attack upon the sulfur atom within the isothiazolone ring by an active-site thiol, resulting in formation of a disulfide bond (Figure 2C). To confirm this, we conducted mass spectrometric analysis of compound 1 after prolonged incubation with a model thiol, Nacetylcysteine (NAC), which yielded results consistent with the proposed mechanism (Figure 2D). Although the mechanism of inhibition involves the irreversible opening of the isothiazolone ring (Figure 2C), the resulting disulfide bond should itself be reversible by reducing agents. Consistent with this prediction, the inhibition of WT-IDE by compound 1 was found to be reversible by treatment with dithiothreitol (DTT) after removal of excess inhibitor by filtration prior to activity testing (Figure 2E). Moreover, as expected for a thiol-reactive compound, no activity was detected in the presence of excess DTT, βmercaptoethanol, or reduced glutathione (Supporting Information Figure S1A). Similarly, even after prolonged incubation of intact cells with compound 1, intracellular pools of IDE were unaffected (Supporting Information Figure S1B), suggesting that the compound is inactivated by the reduced intracellular environment. Among the 13 cysteine residues within the principal (nonmitochondrial) isoform of human IDE,12 two in particular, C


Articles

ACS Chemical Biology

Figure 2. Characterization of the mechanism of action of compound 1. (A) Inhibition of IDE by compound 1 is cysteine-dependent. (B) Compound 1 acts irreversibly. One nanomolar IDE is effectively inhibited by high concentrations of reversible inhibitor Ii123 (30 nM), compound 1 (3 μM), or NEM (2 mM) but not by “low” (100-fold lower) concentrations. However, upon incubation of 100 nM IDE with “high” inhibitor concentrations, followed by 100-fold dilution of the IDE/inhibitor mixture, IDE is inhibited by irreversible compounds (compound 1 and NEM) but not by reversible inhibitor Ii1. (C) Proposed reaction mechanism. Note that reaction with thiols results in breakage of the isothiazolone ring (irreversible) and the formation of a disulfide bond (reversible). (D) Confirmation of proposed reaction mechanism by mass-spectrometry. Compound 1 was reacted for 3 days with equimolar N-acetylcysteine (NAC) and then analyzed by ESI-MS, yielding a reactant and product of the expected masses. (E) Following treatment of IDE with compound 1 (5 μM) and removal of excess inhibitor by filtration, inhibition is reversible by treatment with DTT (0.5 mM), consistent with the proposed reaction mechanism depicted in (C).

Figure 3. Compound 1 interacts selectively with C819. (A) Outer surface (left) and internal chamber (right) of IDE highlighting the relative position of C812 and C819 to the active-site Zn atom. (B) Mutants of IDE containing only a single cysteine at position 812 (sC819) or 819 (sC819) are both effectively inhibited by NEM, but only sC819 is effectively inhibited by compound 1. Note that both compounds inhibit wild-type (WTIDE) but not cysteine-free IDE (CF-IDE). *p < 0.05.

of kinact. For compound 1, Ki and kinact were determined to be 3.52 μM and 2.05 min−1, respectively (Table 2). By comparison of the assay values for compounds 6 and 7 (Table 1), we hypothesized that the potency of compound 1 could be improved by introducing an electron-withdrawing

group to the phenyl ring of the benzoisothiazolone moiety. A fluorine group would be predicted to promote thiol reactivity, accelerating the irreversible step and therefore increasing kinact. To test this empirically, we synthesized derivatives of compound 1 with an F or CF3 group present (compounds 8 D


Articles

ACS Chemical Biology Table 2. SAR of Derivatives of Compound 1 and Kinetic Properties Thereof

Cmpd

R1

Ki (μM)

1 8 9

H F CF3

3.52 3.62 3.73

kinact (min−1)

IC50app (μM) cellfree

IC50app (μM) cell-based

Ki (μM) CF-IDE

2.05 13.4 15.3

0.21 0.063 0.071

1.4 0.11 0.12

5.00 5.16 5.32

Table 3. Biochemical, Chemical, and Pharmacological Properties of Compound 8

CID molecular formula IC50app in vitro, FRET1 (nM) IC50app in vitro, insulin (nM) IC50app in cells, FAβB (nM) Ki (μM) kinact (min−1) MW cLogP cLogD7.4 H donors H acceptors tPSA (Å2) HAa count LEb BEIc SEId Ro5 compliant? chemical stability in PBS solubility in assay buffer (μM) Liver Microsome Stability human/rat/mouse (min) HEK toxicity EC50

and 9, respectively) (Table 2 and Scheme 1). As hypothesized, the added groups gave significant (3- to 12-fold) improvements in potency relative to compound 1 (Table 2). Notably, these increases in potency were entirely attributable to increases in kinact (Table 2). To establish this point by an alternative method, the Ki values for compounds 1, 8, and 9 were determined using CF-IDE (where only the reversible interaction would be operative due to the lack of cysteines), which were not found to differ significantly from one another (Table 2). Although they were not statistically different from each another, the latter values were all nominally higher than the Ki values obtained for WT-IDE, presumably due to the substitution of serine at position 819 in CF-IDE, which is larger and less hydrophobic than the native cysteine residue. Compound 8, which exhibits lower IC50app, MW, and LogP values than those of compound 9, was further profiled, with the properties presented in Table 3. In particular, compound 8 inhibited the degradation of insulin with a potency (IC50app = 82 ± 20.2 nM) similar to that obtained with FRET1 and Aβ, showed no toxicity in cells up to 100 μM, and showed minimal reactivity with cysteines in a proteome-wide, activity-based protein-profiling assay22 (Supporting Information Figure S5). To highlight a key functional property of compound 8, namely, its ability to selectively inhibit extracellular pools of IDE, the compound was compared to two other IDE inhibitors, Ii123 and 6bK,6 in terms of their ability to inhibit extracellular and intracellular pools of IDE in BV-2 cells, a microglial cell line that expresses abundant IDE24 (see Methods). While extracellular IDE was inhibited strongly by all compounds

57390068 C21H22FN3O5S2 63 ± 8.1 (n = 5) 82 ± 20.2 (n = 4) 110 ± 12 (n = 4) 3.62 ± 0.86 (n = 5) 13.4 ± 2.7 (n = 5) 479.5 2.4 2.0 0 9 113 32 0.243 15 4.91 yes ≫48 h 5.1 >120/50/24 >100 μM

a HA = heavy atoms (non-hydrogen atoms). bLE = ligand efficiency (ΔG/HA), calculated using Ki value (units of kcal mol−1 HA−1). cBEI = binding efficiency index (pIC50app/MW, kDa). dSEI = surface efficiency index (pKi/tPSA/100 Å2).

tested (Figure 4A), intracellular pools of IDE were found to be inhibited by Ii1 and 6bK but not by compound 8 (Figure 4B). Additionally, chemo-informatic analyses establish that compound 8 is fully compliant with multiple criteria for druglikeness, including Lipinski’s Ro516 and Jorgensen’s rule of 3 (Ro3)25 as well as the Ghose26 and Veber27 filters, criteria that are not satisfied by previously described IDE inhibitors (Supporting Information Table S1). Compound 8, also

Scheme 1. Synthetic Route Used for the Generation of Compounds 1, 8, and 9a

a

Reagents and conditions: (a) (1) Et3N, morpholine; 91%; (2) H2, Pd/C; 85%. (b) (1) NaOH, PhCH2Br; 92% for compound 1, 88% for compound 8, 94% for compound 9; (2) (COCl)2, CH2Cl2. (c) (1) EtNiPr2, CH2Cl2; (2) PhI(OCOCF3)2, TFA, CH2Cl2, preparative HPLC purification; 51% for compound 1, 40% for compound 8, 43% for compound 9. E


Articles


have developed a potent and mechanistically distinctive IDE inhibitor that selectively targets extracellular pools of IDE, compound 8. This compound is the first small-molecule, nonpeptidic, drug-like inhibitor of IDE, is the first nontoxic, irreversible IDE inhibitor yet described, and shows selectivity not just for IDE but also for a specific cysteine residue within the protease’s active site (C819). Importantly, by utilizing a quasi-irreversible mode of inhibition, compound 8 exclusively inhibits the extracellular pool of IDE. Compound 8, therefore, will be highly useful for future studies investigating the relative contribution of intracellular versus extracellular pools of IDE to different physiological processes and pathological conditions.

■

METHODS

Compound Screening. Detailed descriptions of the overall screening campaign (AID 434984), including the primary cell-based activity screen (AID 434962), confirmatory activity screens (AIDs 435028, 463220, 588712), cytotoxicity assays (AIDs 449730, 463221, 588709), cell-free activity assays (AIDs 588711 and 624067), counterscreens for fluorescence artifacts (AIDs 588718 and 624353), and confirmatory activity assays with a fluorogenic peptide substrate using WT-IDE (AIDs 624066 and 624340) or CF-IDE (AID 624338) are available from PubChem via the Internet at http://pubchem.ncbi. nlm.nih.gov. Insulin degradation was quantified using a homogeneous time-resolved fluorescence-based assay (CisBio Bioassays) as described.23 DTT-free solutions were used for all assays. Tests for Irreversible Inhibition. Activity assays were conducted using the FRET1 substrate essentially as described20 under three different conditions: (1) using 1 nM WT-IDE in the presence of “high” inhibitor concentrations (30 nM Ii1; 3 μM compound 1, 2 mM NEM); (2) using 1 nM WT-IDE in the presence of “low” inhibitor concentrations (100-fold lower, or 0.3 nM Ii1, 30 nM compound 1, 20 μM NEM); and using 100 nM WT-IDE first incubated with “high” inhibitor concentrations for 30 min and then diluted 100-fold (to 1 nM WT-IDE and “low” inhibitor concentrations) prior to execution of the activity assay. Irreversible inhibitors in the latter condition retain their inhibitory potential despite dilution to the low concentration. Tests for Reversible Inhibition. Quantification of reversible inhibition to CF-IDE was performed using the FRET1 substrate (5 μM) as described.20 Briefly, dose−response curves were conducted to obtain IC50app values for each compound, which were converted to Ki values using the Cheng−Prussoff equation incorporating the experimentally determined KM value for FRET1 hydrolyzed by CFIDE (16.6 ± 1.1 μM). Confirmation of Reaction Mechanism by Mass-Spectrometry. Compound 1 and NAC were combined in PBS at equimolar concentrations (50 mM) and incubated at 22 °C for 3 days. The masses of the constituents were subsequently analyzed by electrospray ionization mass-spectrometry as described.29 Tests for Efficacy in the Presence of Reducing Agents and on Intracellular Pools of IDE. IDE activity in the presence of DTT, βME, and GSH was assayed as described,20 using the FRET1 substrate. Intracellular and extracellular IDE activities were quantified in HEK and BV-2 cell lines. For experiments with HEK cells, confluent cell monolayers were incubated for 36 h in the presence of compound 1 (10 μM) or vehicle (DMSO). The conditioned medium was collected for quantification of extracellular IDE activity. After washing the cells three times in PBS, intracellular pools of IDE were recovered by incubating intact cells for 30 min in a hypotonic solution (50 mM Tris-HCl) at 4 °C and collecting them by scraping, followed by mechanical disruption by extrusion three times through a 30G hypodermic needle and centrifugation at 10 000g for 10 min at 4 °C. For experiments with HEK cells, IDE activity in the latter supernatant and in the conditioned medium was quantified in the absence or presence of DTT (1 mM) as described.17 Experiments with BV-2 cells were conducted essentially as above, except that cells were treated for 16 h with inhibitor concentrations of 30 μM. Cell extracts were diluted only modestly (1:200 v/v) so that even reversible inhibitors, if cell-

Figure 4. Compound 8 selectively targets extracellular IDE. Effects of compound 8 versus Ii1 and 6bK on extracellular (A) and intracellular (B) pools of IDE evaluated in BV-2 cells (see Methods). IDE activity was detected using a fluoresceinated and biotinylated Aβ peptide (FAβB) as described.17 Note that, in contrast to Ii1 and 6bK, compound 8 does not affect intracellular pools of IDE. Data are mean ± SEM for three replicates. *p < 0.05.

designated ML345, has been accepted as a molecular probe by the NIH Molecular Probe Center Network.28 The benzoisothazalone-based IDE inhibitors described in this study, in addition to being the first small-molecule, nonpeptidic, truly drug-like inhibitors of IDE yet developed, are of special interest because of their unique, quasi-irreversible mechanism of action, which confers a number of unusual characteristics. While the benzoisothiazolones are cysteinemodifying agents, they differ markedly from NEM or other conventional thiol-alkylating agents in being significantly more potent (e.g., >1000-fold more potent than NEM) and nontoxic in long-term cell culture (e.g., CC50 > 100 μM in HEK-293 cells treated for 48 h). These two features, in turn, appear to be attributable to a unique combination of properties. First, compound 8 exhibits strong affinity not just for IDE as a whole but more specifically for a particular cysteine within the active site of IDE (C819). Because the selectivity of these compounds depends on the particular ensemble of substituents present, these groups may act both by facilitating binding to the region surrounding C819 in IDE and possibly also by sterically blocking interactions with off-target thiols. Second, the rate of the irreversible step in the reaction (kinact) is relatively slow (∼2 to 13 min−1). This property should serve to minimize the adventitious interaction with off-target thiols, limiting productive interactions to those involving a considerable residence time. Third, in contrast to the essentially irreversible S−C bond formed by thiol-alkylating agents, the S−S bond made by the compounds in this series is reversible. This feature likely also contributes to the lack of toxicity, by making any off-target interactions that do occur relatively transient. Notably, because these compounds are inactivated under the reducing conditions present in the cytosol, their activity is limited to the extracellular space, thus greatly reducing the number of potentially toxic off-target interactions. The latter property also distinguishes compound 8 from previously described inhibitors of IDE by enabling it to selectively target extracellular pools of IDE. In conclusion, through a high-throughput compound screening campaign, medicinal chemistry optimization, and coordinated biochemical mechanistic- and kinetics-based studies, we F


Articles

ACS Chemical Biology penetrant, would be sufficiently concentrated to be active in the final assay. IDE activity in conditioned medium and cell extracts was quantified using the avidin-agarose precipitation version of a wellestablished Aβ degradation assay described previously.17 Determination of Kinetics of Irreversible Inhibition. Activity assays were conducted using recombinant WT-IDE (2 nM) and FRET1 substrate (5 μM) in the presence of different concentrations of inhibitors. Reactions were monitored continuously at 3 s intervals immediately after addition of inhibitor as described.20 Observed rate constants (kobs) were obtained by fitting curves to the resulting data, which were subsequently plotted as a function of inhibitor concentration. Hyperbolic curves were fitted to the latter data to obtain kinact and Ki as described,30 according to the following formula

kobs =

Shimadzu preparative HPLC instrument was used for final compound purification. Analytical HPLC data used an Agilent Eclipse XDB-C18 column, 4.6 × 150 mm. The HPLC solvents used were acetonitrile and water with 0.1% formic acid added to each mobile phase as the pH modifier. HRMS data was collected on samples of previously undescribed compounds 8 and 9 at the University of Illinois, using TOF and ESI mass-spectrometry. 5-Fluoro-2-(2-morpholin-4-yl-5-morpholin-4-ylsulfonylphenyl)-1,2-benzothiazol-3-one (8). This compound was synthesized in a convergent manner, in six steps overall, with the longest linear sequence being four steps, as summarized in Scheme 1. The overall yield of the process (after preparative HLPC purification of the final product) is 26%. The 400 MHz 1H NMR spectrum is depicted in Supporting Information Figure S6, and the analytical HPLC, in Figure S7. Step 1. A mixture of sulfonyl chloride A (2.36 g, 10 mmol) and triethylamine (5.06 g, 50.0 mmol) in CH2Cl2 (50 mL) was treated with dropwise with morpholine (2.61g, 30 mmol) at RT. After addition was complete, the reaction was stirred at RT for 14 h, quenched with saturated NH4Cl, and extracted with ethyl acetate. The combined organic extracts were washed with brine and dried over Na2SO4. The solvent was removed, and the residue was purified by flash column chromatography (hexanes/ethyl acetate = 1:1, Rf = 0.15) to afford 3.261 g (91%) of compound B as a yellow solid. HRMS [M + H]+: calcd for C14H19N3O6S, 357.1; found, 358.0; 1H NMR (400 MHz, CDCl3) δ (ppm) 3.03−3.06 (m, 4H), 3.21−3.23 (m, 4H), 3.76−3.79 (m, 4H), 3.87−3.89 (m, 4H), 7.18 (d, J = 8.8 Hz, 1H), 7.79 (dd, J = 2.0, 8.8 Hz, 1H), 8.17 (d, J = 2.0 Hz, 1H). Step 2. To a solution of compound B (488 mg, 1.37 mmol) in THF (10 mL) and MeOH (10 mL) was added Pd/C (10%, 50 mg). The reaction mixture was then stirred under atmosphere of H2 for 5 h, filtered, and concentrated to afford 378 mg (85%) of compound C as white solid. HRMS [M + H]+: calcd for C14H21N3O4S, 327.1; found, 328.1. Step 3. A solution of acid D (344 mg, 2.0 mmol) in MeOH (12 mL) was treated with NaOH (160 mg, 4.0 mmol). The reaction mixture was stirred at RT for 10 min. The solvent was removed to afford a solid, which was dissolved in acetone (18 mL). Benzyl bromide (376 mg, 2.2 mmol) was added. The reaction was sonicated for 5 min and then stirred at 0 °C for 1 h. The precipitate was collected by vacuum filtration. The solid was dissolved in H2O and then treated with 1 N HCl. The precipitate was collected by vacuum filtration and dried in air, affording 462 mg (88%) of compound E as a white solid. 1H NMR (400 MHz, acetone-d6) δ (ppm) 4.25 (s, 2H), 7.27−7.36 (m, 4H), 7.45−7.47 (m, 2H), 7.54−7.57 (m, 1H), 7.70− 7.73 (m, 1H). Steps 4 and 5. A suspension of acid E (570 mg, 2.17 mmol) in CH2Cl2 (24 mL) was treated with (COCl)2 (441 mg, 3.48 mmol) and a drop of DMF at 0 °C under an atmosphere of N2. The reaction was stirred at 0 °C for 30 min and then at RT for 3 h. The solvent was removed. The residue was dissolved in CH2Cl2 (24 mL). Compound C (781 mg, 2.39 mmol) was added and cooled to 0 °C. Diisopropylethylamine (841 mg, 6.51 mmol) was added dropwise. The reaction mixture was stirred at RT overnight, quenched with H2O, and extracted with CH2Cl2. The combined organic extracts were washed with brine and dried over Na2SO4. The solvent was removed, and the residue was purified by flash column (hexanes/ethyl acetate = 1:1, Rf = 0.20) to afford 1.04 g (84%) of compound F as a yellow solid. HRMS [M + H]+: calcd for C28H30FN3O5S2, 571.1; found, 572.0; 1H NMR (400 MHz, CDCl3) δ (ppm) 2.93−2.95 (m, 4H), 3.13−3.15 (m, 4H), 3.78−3.83 (m, 8H), 4.05 (s, 2H), 7.07−7.09 (m, 3H), 7.19− 7.20 (m, 3H), 7.30−7.33 (m, 2H), 7.42−7.45 (m, 1H), 7.58−7.60 (m, 2H), 8.92 (br s, 1H); 19F NMR (400 MHz, CDCl3) δ (ppm) −111.6. Step 6. A mixture of [bis(trifluoroacetoxy)iodo]benzene (PIFA, 1.02 g, 2.36 mmol) and TFA (0.415 g, 3.64 mmol) in CH2Cl2 (18 mL) was cooled to 0 °C under an atmosphere of N2. A solution of compound 6 (1.04 g, 1.82 mmol) in CH2Cl2 (35 mL) was added dropwise. The reaction mixture was stirred at 0 °C for 30 min and RT 30 min and then refluxed for 40 h. The crude product was concentrated, and the residue was purified by flash column

k inact[I]

(

[I] + K i 1 +

[S] KM

)

where [I] is the inhibitor concentration, [S] is the substrate concentration (5 μM in kinetic experiments), and KM is the Michaelis constant for the substrate (21.32 μM) as determined previously.31 Curve fits and quantitative analyses were conducted using Prism 5.0 (GraphPad Software, Corp.). Computational Docking. Docking was performed using Glide (v. 5.6) within the Schrödinger software suite (Schrödinger, LLC).32 The starting conformation of ligands was obtained by the method of PolakRibière conjugate gradient (PRCG) energy minimization with the Optimized Potentials for Liquid Simulations (OPLS) 2005 force field33 for 5000 steps or until the energy difference between subsequent structures was less than 0.001 kJ mol−1 Å−1.32 Our docking methodology has been described previously,34−36 and the scoring function utilized as well as the rationale for its selection is described elsewhere.37 Briefly, in order to generate the grids for docking, the ions, molecular refracting molecules, and 1,4-diethylene dioxide were removed from the IDE crystal structure (PDB code 3E4A).23 Schrödinger’s SiteFinder module was used to determine grid placement for the region of C812 and C819. The binding site was generated via multiple overlapping grids with a default rectangular box. Then, a larger composite grid was generated such that both C812 and C819 were contained within the grid. Using this grid, compound 1 was docked using the Glide algorithm within the Schrödinger suite as a virtual screening workflow (VSW). The docking proceeded from lower precision through SP docking and Glide extra precision (XP) (Glide, v. 5.6, Schrödinger, LLC).38,39 The top 10 000 poses were ranked for best scoring pose, and unfavorable scoring poses were discarded. Each conformer was allowed to have multiple orientations in the site. Site hydroxyls, such as in serines and threonines, were allowed to move with rotational freedom. The induced-fit docking method was utilized within Schrödinger suite to allow larger side-chain reorientation, as needed. Hydrophobic patches were utilized within the VSW as an enhancement. Top favorable scores from dockings of compound 1 yielded 10 poses. XP descriptors were used to obtain atomic energy terms like hydrogen-bond interaction, electrostatic interaction, hydrophobic enclosure, and pi−pi stacking interaction that result during the docking run.38,39 Figures were generated using Maestro, the built-in graphical user interface of the Schrödinger software suite (v. 5.6) (Schrödinger, LLC). Chemoinformatics Analyses. To evaluate the drug-likeness of compounds, physicochemical and other drug-related properties, including compliance with Lipinski’s Ro5 and Jorgensen’s Rule of 3 (Ro3), were calculated using QikProp25 (v. 4.0) within the Schrödinger software suite (Schrödinger, LLC). Adherence to the Ghose and Veber filters was calculated as described.26,27 Synthesis of Compounds. All chemical reagents and solvents were acquired from commercial vendors. Reactions were monitored by LC-MS (Thermo/Finnegan LCQ Duo ion trap system with MS/MS capability). An Agilent 1200 analytical HPLC was used for quantitative purity assessment. Teledyne-Isco “combiflash” automated silica gel MPLC instruments were used for chromatographic purifications. A Brüker 400-MHz NMR instrument was used for NMR analysis. A G



■

chromatography (hexanes/ethyl acetate = a gradient of 1:1 to 1:4) and then by preparative HPLC to afford 343 mg (40%) of compound 8 (ML345) as a white solid. HRMS [M + H]+: calcd for M + H = C21H23FN3O5S2, 480.1; found, 479.9; HRMS [M + H]+: calcd for M + H = C21H23FN3O5S2, 480.1063; found, 480.1060; 1H NMR (400 MHz, CDCl3) δ (ppm) 2.98−3.01 (m, 8H), 3.67−3.71 (m, 8H), 7.11 (d, J = 8.8 Hz, 1H), 7.36−7.41 (m, 1H), 7.48−7.52 (m, 1H), 7.63 (dd, J = 2.4, 8.4 Hz, 1H), 7.70 (dd, J = 2.4, 8.0 Hz, 1H), 7.84 (d, J = 2.0 Hz, 1H); 19F NMR (400 MHz, CDCl3) δ (ppm) −115.4. Purity was measured to be >98% (LC-MS analysis, confirmed by analytical HLPC analysis; HPLC purity data is shown in Figure S7). 2-(2-Morpholin-4-yl-5-morpholin-4-ylsulfonylphenyl)-5-(trifluoromethyl)-1,2-benzothiazol-3-one (9). Synthesis was carried out in the same manner as reported for compound 8 (Scheme 1). HRMS [M]+: calcd for M = C22H22F3N3O5S2, 529.0953; found, 529.0952. Purity was measured to be >98% (LC-MS analysis, confirmed by analytical HLPC analysis).

■

REFERENCES

(1) Leal, M. C., and Morelli, L. (2013) Insulysin, in Handbook of Proteolytic Enzymes (Rawlings, N. D., and Salvesen, G., Eds.) 3rd ed., pp 1415−1420, Academic Press, Boston, MA. (2) Hersh, L. B. (2006) The insulysin (insulin degrading enzyme) enigma. Cell. Mol. Life Sci. 63, 2432−2434. (3) Shii, K., and Roth, R. A. (1986) Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease. Proc. Natl. Acad. Sci. U. S. A. 83, 4147− 4151. (4) Edbauer, D., Willem, M., Lammich, S., Steiner, H., and Haass, C. (2002) Insulin-degrading enzyme rapidly removes the beta-amyloid precursor protein intracellular domain (AICD). J. Biol. Chem. 277, 13389−13393. (5) van Endert, P. (2011) Post-proteasomal and proteasomeindependent generation of MHC class I ligands. Cell. Mol. Life Sci. 68, 1553−1567. (6) Maianti, J. P., McFedries, A., Foda, Z. H., Kleiner, R. E., Du, X. Q., Leissring, M. A., Tang, W. J., Charron, M. J., Seeliger, M. A., Saghatelian, A., and Liu, D. R. (2014) Anti-diabetic activity of insulindegrading enzyme inhibitors mediated by multiple hormones. Nature 511, 94−98. (7) Makarova, K. S., and Grishin, N. V. (1999) The Zn-peptidase superfamily: functional convergence after evolutionary divergence. J. Mol. Biol. 292, 11−17. (8) Becker, A. B., and Roth, R. A. (1992) An unusual active site identified in a family of zinc metalloendopeptidases. Proc. Natl. Acad. Sci. U. S. A. 89, 3835−3839. (9) Williams, F. G., Johnson, D. E., and Bauer, G. E. (1990) [125I]insulin metabolism by the rat liver in vivo: evidence that a neutral thiol-protease mediates rapid intracellular insulin degradation. Metab., Clin. Exp. 39, 231−241. (10) Malito, E., Ralat, L. A., Manolopoulou, M., Tsay, J. L., Wadlington, N. L., and Tang, W. J. (2008) Molecular bases for the recognition of short peptide substrates and cysteine-directed modifications of human insulin-degrading enzyme. Biochemistry 47, 12822−12834. (11) Neant-Fery, M., Garcia-Ordonez, R. D., Logan, T. P., Selkoe, D. J., Li, L., Reinstatler, L., and Leissring, M. A. (2008) Molecular basis for the thiol sensitivity of insulin-degrading enzyme. Proc. Natl. Acad. Sci. U. S. A. 105, 9582−9587. (12) Leissring, M. A., Farris, W., Wu, X., Christodoulou, D. C., Haigis, M. C., Guarente, L., and Selkoe, D. J. (2004) Alternative translation initiation generates a novel isoform of insulin-degrading enzyme targeted to mitochondria. Biochem. J. 383, 439−446. (13) Zhao, J., Li, L., and Leissring, M. A. (2009) Insulin-degrading enzyme is exported via an unconventional protein secretion pathway. Mol. Neurodegener. 4, 4. (14) Bulloj, A., Leal, M. C., Xu, H., Castano, E. M., and Morelli, L. (2010) Insulin-degrading enzyme sorting in exosomes: a secretory pathway for a key brain amyloid-beta degrading protease. J. Alzheimers Dis. 19, 79−95. (15) Goldfine, I. D., Williams, J. A., Bailey, A. C., Wong, K. Y., Iwamoto, Y., Yokono, K., Baba, S., and Roth, R. A. (1984) Degradation of insulin by isolated mouse pancreatic acini. Evidence for cell surface protease activity. Diabetes 33, 64−72. (16) Lipinski, C. A., Lombardo, F., Dominy, B. W., and Feeney, P. J. (2001) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Delivery Rev. 46, 3−26. (17) Leissring, M. A., Lu, A., Condron, M. M., Teplow, D. B., Stein, R. L., Farris, W., and Selkoe, D. J. (2003) Kinetics of amyloid betaprotein degradation determined by novel fluorescence- and fluorescence polarization-based assays. J. Biol. Chem. 278, 37314− 37320. (18) Shen, Y., Joachimiak, A., Rosner, M. R., and Tang, W. J. (2006) Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism. Nature 443, 870−874.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00334. Drug-like properties of compound 8 vis-à-vis other IDE inhibitors; efficacy of compound 1 in the presence of reducing agents and on intracellular IDE activity; doseresponse curves for compound 1 using WT-IDE and sC819; computational docking of compound 1 with the active site of human IDE confirms preferential interaction with C819; examples of kinetic data used to determine Ki and kinact; proteome-wide activity-based profiling of compound 8 for cysteine reactivity; 400-Hz 1 H NMR spectrum for compound 8; analytical HPLC of compound 8. (PDF).

■

Articles

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

This work was supported by grant DA024888 from the National Institutes of Health and grant 7-11-CD-06 from the American Diabetes Association to M.A.L. and by grant U54 MH084512 from the National Institutes of Health to T.D.B., H.W., M.D.C., S.C.S., F.M., and P.H. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We gratefully acknowledge the assistance of B. Mercer, K. Emery, and J. Ferguson for assistance with the management of data and submission to PubChem, H. Rosen and W. Roush for program support, S. Newlove for purification of recombinant IDE, and J.P. Maianti, A. Saghatelian, and D. Liu for providing 6bK.

■

ABBREVIATIONS CF-IDE, cysteine-free IDE; IDE, insulin-degrading enzyme; MLSMR, Molecular Libraries Small Molecule Repository; NAC, N-acetylcysteine; NEM, N-ethylmaleimide; Ro5, Lipinski’s rule of five; WT-IDE, wild-type IDE H


Articles

ACS Chemical Biology (19) Zhang, J. H., Chung, T. D., and Oldenburg, K. R. (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J. Biomol. Screening 4, 67−73. (20) Cabrol, C., Huzarska, M. A., Dinolfo, C., Rodriguez, M. C., Reinstatler, L., Ni, J., Yeh, L.-A., Cuny, G. D., Stein, R. L., Selkoe, D. J., and Leissring, M. A. (2009) Small-molecule activators of insulindegrading enzyme discovered through high-throughput compound screening. PLoS One 4, e5274. (21) Mallender, W. D., Szegletes, T., and Rosenberry, T. L. (1999) Organophosphorylation of acetylcholinesterase in the presence of peripheral site ligands. Distinct effects of propidium and fasciculin. J. Biol. Chem. 274, 8491−8499. (22) Bachovchin, D. A., Zuhl, A. M., Speers, A. E., Wolfe, M. R., Weerapana, E., Brown, S. J., Rosen, H., and Cravatt, B. F. (2011) Discovery and optimization of sulfonyl acrylonitriles as selective, covalent inhibitors of protein phosphatase methylesterase-1. J. Med. Chem. 54, 5229−5236. (23) Leissring, M. A., Malito, E., Hedouin, S., Reinstatler, L., Sahara, T., Abdul-Hay, S. O., Choudhry, S., Maharvi, G. M., Fauq, A. H., Huzarska, M., May, P. S., Choi, S., Logan, T. P., Turk, B. E., Cantley, L. C., Manolopoulou, M., Tang, W. J., Stein, R. L., Cuny, G. D., and Selkoe, D. J. (2010) Designed inhibitors of insulin-degrading enzyme regulate the catabolism and activity of insulin. PLoS One 5, e10504. (24) Qiu, W. Q., Walsh, D. M., Ye, Z., Vekrellis, K., Zhang, J., Podlisny, M. B., Rosner, M. R., Safavi, A., Hersh, L. B., and Selkoe, D. J. (1998) Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation. J. Biol. Chem. 273, 32730−32738. (25) Jorgensen, W. L., and Duffy, E. M. (2002) Prediction of drug solubility from structure. Adv. Drug Delivery Rev. 54, 355−366. (26) Ghose, A. K., Viswanadhan, V. N., and Wendoloski, J. J. (1999) A knowledge-based approach in designing combinatorial or medicinal chemistry libraries for drug discovery. 1. A qualitative and quantitative characterization of known drug databases. J. Comb. Chem. 1, 55−68. (27) Veber, D. F., Johnson, S. R., Cheng, H. Y., Smith, B. R., Ward, K. W., and Kopple, K. D. (2002) Molecular properties that influence the oral bioavailability of drug candidates. J. Med. Chem. 45, 2615−2623. (28) Bannister, T. D., Wang, H., Abdul-Hay, S. O., Masson, A., Madoux, F., Ferguson, J., Mercer, B. A., Schurer, S., Zuhl, A., Cravatt, B. F., Leissring, M. A., Hodder, P. (2010) ML345, A Small-Molecule Inhibitor of the Insulin-Degrading Enzyme (IDE), in Probe Reports from the NIH Molecular Libraries Program, NIH, Bethesda, MD. (29) Abdul-Hay, S. O., Sahara, T., McBride, M., Kang, D., and Leissring, M. A. (2012) Identification of BACE2 as an avid β-amyloiddegrading protease. Mol. Neurodegener. 7, 46. (30) Rosenberry, T. L., Sonoda, L. K., Dekat, S. E., Cusack, B., and Johnson, J. L. (2008) Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter. Biochemistry 47, 13056−13063. (31) Song, E. S., Mukherjee, A., Juliano, M. A., Pyrek, J. S., Goodman, J. P., Jr., Juliano, L., and Hersh, L. B. (2001) Analysis of the subsite specificity of rat insulysin using fluorogenic peptide substrates. J. Biol. Chem. 276, 1152−1155. (32) Mohamadi, F., Richards, N. G. J., Guida, W. C., Liskamp, R., Lipton, M., Caufield, C., Chang, G., Hendrickson, T., and Still, W. C. (1990) Macromodelan integrated software system for modeling organic and bioorganic molecules using molecular mechanics. J. Comput. Chem. 11, 440−467. (33) Jorgensen, W. L., and Tiradorives, J. (1988) The OPLS Potential Functions for Proteins - Energy Minimizations for Crystals of Cyclic-Peptides and Crambin. J. Am. Chem. Soc. 110, 1657−1666. (34) Caulfield, T. R., and Devkota, B. (2012) Motion of transfer RNA from the A/T state into the A-site using docking and simulations. Proteins: Struct., Funct., Genet. 80, 2489−2500. (35) Loving, K., Salam, N. K., and Sherman, W. (2009) Energetic analysis of fragment docking and application to structure-based pharmacophore hypothesis generation. J. Comput.-Aided Mol. Des. 23, 541−554. (36) Vivoli, M., Caulfield, T. R., Martinez-Mayorga, K., Johnson, A. T., Jiao, G. S., and Lindberg, I. (2012) Inhibition of prohormone

convertases PC1/3 and PC2 by 2,5-dideoxystreptamine derivatives. Mol. Pharmacol. 81, 440−454. (37) Friesner, R. A., Murphy, R. B., Repasky, M. P., Frye, L. L., Greenwood, J. R., Halgren, T. A., Sanschagrin, P. C., and Mainz, D. T. (2006) Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J. Med. Chem. 49, 6177−6196. (38) Salam, N. K., Nuti, R., and Sherman, W. (2009) Novel method for generating structure-based pharmacophores using energetic analysis. J. Chem. Inf. Model. 49, 2356−2368. (39) Caulfield, T., and Medina-Franco, J. L. (2011) Molecular dynamics simulations of human DNA methyltransferase 3B with selective inhibitor nanaomycin A. J. Struct. Biol. 176, 185−191.

I


Selective Targeting of Extracellular Insulin ... - ACS Publications

Recommend Documents