Acquired Removability of Aspartic Protease Inhibitors by Direct

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Acquired Removability of Aspartic Protease Inhibitors by Direct Biotinylation Koushi Hidaka, Motoyasu Adachi, and Yuko Tsuda Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00195 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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Acquired Removability of Aspartic Protease Inhibitors by Direct Biotinylation Koushi Hidaka,*a,b Motoyasu Adachi,c and Yuko Tsudaa,b a Faculty

of Pharmaceutical Sciences and b Cooperative Research Center for Life Sciences, Kobe

Gakuin University, Kobe, 650-8586, Japan; c Institute for Quantum Life Science, National Institutes for Quantum and Radiological Science and Technology, Tokai, Ibaraki, 319-1106, Japan KEYWORDS: biotin, cathepsin D, HIV protease, inhibitor, streptavidin

ABSTRACT: Protease inhibitors are used as both research tools and therapeutics. Many of these inhibitors consist of substrate amino acid sequence-derived structure with a transition state mimic to interact with the active site of the protease, suppressing enzymatic activity. However, once they bind, macrodilution or protein denaturation are required to remove them, limiting their usage. In this study, we describe a removable protease inhibitor, which is a directly biotinylated analogue to control the activities of HIV-1 protease and human cathepsin D. In the substrate cleavage assay, we observed that the nanomolar inhibitory activities were lost upon the addition of streptavidin, while the enzymatic activities sufficiently recovered. HIV-1 protease mixed with the removable inhibitor, avoiding autolysis, was still active to be detected by adding streptavidin after one year

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at room temperature. We also observed that the inhibitor was an effective eluent for the simple detection of the activity of proteases purified from human serum and cells. These results demonstrate that direct biotinylation of protease inhibitors could be a novel method for controlling the enzymatic activity from OFF to ON. We proposed the phenomenon that binding equilibrium of inhibitor was shifted from protease to streptavidin with higher affinity, named as “inhibitor stripping action by affinity competition”, or ISAAC. We anticipate that ISAAC could be applicable for preservatives of proteases and activity-based diagnosis of protease related diseases. Furthermore, removable inhibitor to be designed for targeted proteases changing the inhibitor structure may elucidate enzymatic activity in intrinsic form with natural modifications from various biological samples.

INTRODUCTION Protease inhibitors are widely used in research, and many of them are also important as therapeutic agents.1,2 Inhibitors conjugated with fluorescent and reactive groups are called activitybased probes and have been used to visualize protease activity in vivo.3 Peptidomimetic protease inhibitors are designed based on the amino acid sequence of the substrate and incorporate a transition state mimic. Inhibitors have covalent or noncovalent binding interactions with the active site of the protease to suppress enzymatic reaction. Because protease inhibitors exhibit strong binding affinities, from micromolar to picomolar, procedures such macrodilution or protein denaturation are required to remove them, limiting the usage of such inhibitors. Inhibition in one direction arises some risks in the treatment using direct anticoagulants, acquiring reversal antibody agent.4

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The avidin-biotin affinity approach takes advantage of the specific, extremely strong, and noncovalent binding that occurs between these two molecules.5 This affinity was studied for long time to purify biotinylated biomolecules.6 In 1976, the technique was advanced to purify a peptide hormone receptor through immobilization of a biocytin-conjugated adrenocorticotropic hormone peptide to an avidin-conjugated carrier.7 Currently, the use of this affinity method has become more widely available through the development of avidin derivatives, such as streptavidin8 or neutravidin, as well as biotinylation reagents of succinimide esters and maleimides with various spacer structures that are used in the purification, staining, imaging, precipitation of target proteins, and activity-based protein profiling. 9 KNI-10006 (1), shown in Figure 1, is one of a number of peptidomimetic compounds that target HIV-1 protease and malarial plasmepsins. These compounds are based on a design using a phenylnorstatine residue that contains a transition state isostere for the development of anti-HIV and antimalarial agents.10,11 Compound 1 potently inhibits these proteases at sub-nanomolar levels and has moderate affinity to other aspartic proteases, such as pepsin and cathepsin D.12,13 In the initial study, we synthesized affinity-based probes to conjugate biotin using aminocaproyl spacers. Assays using compounds with different spacer lengths revealed that the binding of the target protease in the presence of streptavidin depended strongly on spacer length (Supporting Information, Figure S1). Similar results were reported by other group using PEG or polyproline spacers, leading to the selection of longer ones.14 Therefore, we assumed that although a reduction in enzymatic activity occurs when an inhibitor with a shorter linker is used, such an inhibitor also aids in recovering the protease activity. We then speculated as to what would happen to protease binding if the probe completely lacked a spacer. To answer this question, we synthesized a directly

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biotinylated compound (2) that was shown to be removable to allow for complete control of protease activity.

RESULTS Design of a Directly Biotinylated Inhibitor. Prior to synthesizing a biotin-conjugated inhibitor of 1, the position to modify was determined by using X-ray co-crystal structures of HIV-1 protease15, plasmepsin I,16 and histo-aspartic protease HAP.17 These crystallographic structures revealed the binding modes of the 2,6dimethylphenoxy moiety, which was commonly observed on the edges of the protease binding pockets (Figure 1C). A known inhibitor with a 4-amino group at the 2,6-dimethylphenoxy moiety acting as HIV-1 protease inhibitor9 provided an additional reason to modify this position through a peptide bond with biotin. These observations prompted us to synthesize biotinylated 2 as shown in Figure 1A, in which biotin was directly conjugated with the amino group. As expected, the biotin moiety extended from the 4-amino group towards the water exterior and did not interfere with the binding of the inhibitor (Figure 1B and 1C). The head of the biotin moiety appeared to be located close to the side chain of Arg8 residue.

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Figure 1. Directly biotinylated analogues of aspartic protease inhibitors. (A) Chemical structures of compounds 1 to 4. (B) Co-crystal structure of 2 (magenta and green stick) and HIV1 protease (cartoon, PDB 6ixd). (C) Superimposition 2 with 1 (white stick, PDB 3kdb). Biotin moiety (green stick) located just outside of the pocket with the head adjacent to Arg8.

Recovery of Protease Activity. The inhibitory activity of 2 against HIV-1 protease was very strong (Ki value of 0.16 nM) and was equal to that of the parent compound 1.15 Next, we added more than 10 equivalents of streptavidin into the protease/inhibitor mixture. Interestingly, the inhibition observed with 5 nM of compound 2 was almost completely lost, with inhibition reduced to less than 3% and over 97%

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of the enzymatic activity recovered (Figure 2A). In contrast, the activity of HIV-1 protease mixed with 1 was unaltered by the addition of streptavidin. We speculate that this phenomenon can be explained as follows: (i) in the presence of directly biotinylated inhibitor 2, the active site was occluded, eliminating protease activity; and (ii) after an excess amount of streptavidin was added, the molecules of 2 that were dissociated from the protease moved to streptavidin, that is, the equilibrium of the inhibitor-protease complex shifted towards the inhibitor-streptavidin complex, restoring protease activity (Figure 2D). Because the affinity of the biotin analogue for streptavidin (Kd ~10-14) is much higher than that of the inhibitor for the protease (Ki ~10-10), the inhibitor was removed from the protease.

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Figure 2. Removal of biotinylated inhibitors from aspartic proteases. (A) Enzymatic activity of HIV-1 protease (40 ng/mL) with 5 nM compounds 1 and 2 by addition of streptavidin. (B) Human cathepsin D activity with biotinylated inhibitors 2 or 4 (500 nM or 5 nM, respectively) by addition of streptavidin. (C) Repetitive control of HIV-1 protease activity. Substrate cleavage was started by adding the substrate and monitored, followed by addition of 2 (5 nM) at 5min, 10 eq. of streptavidin at 15min, then 2 (50 nM) at 25 min. (D) Proposed mechanism, ISAAC. Binding equilibrium of biotinylated inhibitor was shifted from protease to extremely strong affinity of streptavidin. Error bars represent SEM.

Compound 1 has also been reported to inhibit human cathepsin D.12 Therefore, analogue 2 was tested on cathepsin D to confirm streptavidin stripping. The Ki value of analogue 2 against cathepsin D was determined to be 16 nM. The enzymatic activity of cathepsin D was inhibited by 500 nM of compound 2, 100-fold higher than that to inhibit HIV-1 protease. Cathepsin D activity increased to 82% following the addition of 20 equivalents of streptavidin and was almost completely recovered with the addition of 200 equivalents. Pepstatin A (3) is a standard and highly potent inhibitor of aspartic proteases with low selectivity.18 For comparison, we also synthesized a biotinylated derivative of pepstatin through hydrazide linker. The resulting derivative (4, Figure 1A) inhibited cathepsin D completely, 99% inhibition at 5 nM. Partial recovery of enzymatic activity, up to 28%, was observed following the addition of 20 equivalents of streptavidin. With 200 equivalents of streptavidin, the recovery of enzymatic activity sustained approximately 30%. The repeated inhibition of enzymatic activity is a challenge for truly controlling protease activity. We monitored the substrate cleavage using a portable fluorescence detector, observing that the

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addition of 2 suppressed the protease-mediated substrate cleavage, which was restored by the addition of streptavidin. Subsequently, the further addition of 2, using a 10-fold higher amount, inhibited the protease activity again. The result of repetitive control is one of the unique characteristics of 2 as a removable inhibitor.

Prolonged Stability of Protease Mixed with Removable Inhibitor. One of applications of using a removable inhibitor is for the storage of proteases. HIV-1 protease loses enzymatic activity after 1 week at room temperature due to autolysis, as shown in Figure 3A. In the presence of 2, the restoration of substrate cleavage was confirmed by the addition of streptavidin, even after one year. A protease with five autolysis-inhibiting mutations, WTm5,19 was more stable than one without the mutations. Surprisingly, the rate of substrate cleavage by WTm5 after storage with 2 at room temperature did not change for one year (Figure 3B). Human cathepsin D was unstable and lost enzymatic activity after one day when untreated. This decrease in activity was lessened in the presence of 2, with substrate cleavage detected after three days (Figure 3C). It was difficult to recover cathepsin D activity in the presence of pepstatin derivative 4, with only up to approximately 20% recovered, which was unfavourable to the stability test.

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Figure 3. Prolonged stability of HIV-1 protease and human cathepsin D. (A) Wild-type HIV-1 protease was stored at room temp. for long time with or without 2. Aliquots were sampled, then the reaction was started by adding 10 eq. of streptavidin. (B) Long time storage of wild-type HIV-1 protease (WTm5) containing five mutations, Q7K/L33I/L63I/C67A/C95A, for stability enhancement, with or without 2. (C) Room temp. storage of human cathepsin D with or without 2 or 4. Error bars represent SEM.

Detection of Affinity-Purified HIV-1 Protease Activity. The successful recovery of HIV-1 protease and human cathepsin D activities treated with the removable inhibitor 2 prompted us to use this methodology to detect affinity-bound proteases in more complex mixtures. HIV-1 protease WTm5 was added to human serum and purified using magnetic beads conjugated to 1 through a long spacer (Figure 4A). An increase in fluorescence in a solution eluted using compound 2 was observed following the addition of 10 equivalents of streptavidin, whereas no increase in fluorescence was observed in the absence of streptavidin. The initial velocity varied in a dose-dependent manner (Figure 4B). The activity recovered by ISAAC

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was inhibited by clinical drugs, lopinavir, atazanavir, and duranavir. Affinity binding of cathepsin D from human colon cancer cells, HCT116, was also performed (Figure 4C). A protein was purified from cells after freeze-thaw treatment in the same manner as described above. Substrate cleavage was detected upon the addition of streptavidin that had been suppressed in the presence of 3 (Figure 4D).

Figure 4. Activity detection of affinity-purified protease using ISAAC. (A) Schematic representation of the methodology. Affinity bound protease on magnetic beads was soaked in solution containing 2. Transferred portion of the protease was monitored by

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adding streptavidin and substrate with or without additional inhibitor. (B) Activity of HIV-1 protease purified from human serum. (C) Susceptibility of 5 nM of clinical inhibitors, lopinavir, atazanavir, and darunavir to recovered from 40 μg/mL of HIV-1 protease. (D) Activity of cathepsin D purified from HCT116 cells in the presence of pepstatin A. Error bars represent SEM.

DISCUSSION The biotinylation of ligands is widely used to probe target proteins for bioanalytical applications such as isolation, localization, and diagnostics.6 For this method to succeed, a position for biotin conjugation to the ligand must be determined without sacrificing the binding affinity. We demonstrated that X-ray crystallographic data was helpful in deciding where biotin should be attached to the inhibitor and that direct biotinylation of the inhibitor was succeeded in the generation of compound 2 without losing affinity to the target protease. Protease inhibitors generally bind to the active sites of these enzymes to suppress their activity. If the inhibitor needs to be removed, a denaturant, such as an acidic glycine hydrochloride buffer, must be used to break tertiary structures or the sample must be greatly diluted to decrease the ligand-protein interactions. Therefore, recovering the active form of the protein requires removal of the denaturant and processing to refold and concentrate the protein, which are procedures that can be problematic and prone to failure. In this study, we describe the direct biotinylation of an HIV-1 protease inhibitor that makes possible to control the enzymatic activity from OFF to ON

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through the addition of an excess amount of streptavidin to remove the inhibitor from the protease active site and allow for the protease to cleave its substrate, even in the presence of inhibitor. During the affinity purification of protease, the bound proteins are eluted by denaturant, ligand, and or chemical cleavage of the spacer between the ligand and carrier. The subsequent removal of the denaturant or ligand may require additional tedious procedures. Therefore, such elution steps are not suitable for enzymes to be tested for activity. We demonstrated that the use of ISAAC is applicable to elute the affinity bound protease to detect the enzymatic activity in a small amount of sample with the simple addition of streptavidin and a FRET substrate. The protease activity results obtained using human sera indicates the practicality of detecting proteases in blood samples. The antibody-based detection of proteins related to diseases is a common method for rapid testing kits that target HIV.20,21 Such kits help to control the spread of the infection. In addition to its ease of use, this method allows for HIV antibodies to be detected only two to three months following infection. Nucleic acid amplification testing can be used to detect viral copies in blood plasma only 5 days after infection, but the availability of this high-cost technique is limited.22 Therefore, a novel, rapid, simple, and low-cost detection method is needed. The ISAAC-based methodology presented in this study could be used to measure viral activity based on the detection of viral proteases, and further optimization could reduce the difficulty of diagnosing early-stage HIV infections. This methodology based on ISAAC is simple and is theoretically applicable to most of the reversible inhibitors that target specific proteases. Our results demonstrated the applicability of this method to HIV-1 protease as well as to human cathepsin D. We also presented the ON/OFF/ON/OFF repetitive control using compound 2. The unique characteristics of removable inhibitors could be applied for testing the inhibitory activities of compounds against the recovered activity after affinity purification. The additional inhibition would aid in confirming

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the purified target protease and for testing the susceptibility of clinical drugs before use in treatment.

CONCLUSIONS We developed the directly biotinylated inhibitor bPI-11, another name for 2, to obtain a removable inhibitor. The addition of excess streptavidin masked the nanomolar inhibitory activity of 2, altering the inhibited enzymatic activity. To the best of our knowledge, this is the first report of the repetitive control of protease activity using an inhibitor. The presented simple method can be used to preserve many types of proteases at room temperature for a long time without autolysis. Furthermore, it can be used to investigate the function of natural proteases purified from various biological samples as well as to diagnose protease-related diseases.

EXPERIMENTAL PROCEDURES Protease Inhibition Assay. HIV-1 protease inhibitory activity of the compounds was determined based on the inhibition of a FRET substrate (DABCYL-Ser-Gln-Asn-Tyr-Pro-Ile-ValGln-EDANS, synthesized by solid phase peptide synthesis) cleavage23 using recombinant HIV-1 protease, WTm5 24. In the inhibition assay, 65 μL of 2-(N-morpholino)ethanesulfonic acid (MES)– NaOH buffer (pH 5.5) was mixed with 5 μL of the inhibitor (100 nM) dissolved in DMSO and 20 μL of HIV-1 protease (200 ng/mL) in MES buffer. After incubation for 10 min at 37 °C, the reaction was initiated by addition of 10 μL of the substrate (5 μM) solution. Production of cleaved EDANS substrate was monitored (Ex 355 nm/ Em 500 nm) for 15 min at 37 °C, then, the kinetic slope of was calculated with or without the inhibitor. Average of the percent inhibition was obtained from more than three experiments.

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The active HIV-1 protease concentration was titrated using atazanavir, determined as 1.0 nM. The velocities under several substrate concentrations were analyzed and fitted to the MichaelisMenten equation to obtain Km value as 9.6 μM. Ki value was determined by testing under six inhibitor concentrations fitting the curve to Morrison’s equation of tight binding inhibitor25. Inhibition of recombinant human cathepsin D (R&D Systems, Inc., Minneapolis, USA) was analyzed based on cleavage of a substrate (MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-LeuLys(Dnp)-D-Arg-NH2, Peptide Institute, Inc., Osaka, Japan). The reaction mixture consisting 65 μL of sodium acetate buffer (pH 3.5), 5 μL of inhibitor in DMSO, and 20 μL of cathepsin D (400 ng/mL) in sodium acetate buffer was pre-incubated for 10 min at 37 °C. The enzyme reaction was initiated by addition of 10 μL of the substrate (100 μM) solution. Production of cleaved MOCAc fragment was monitored (Ex 320 nm/ Em 405 nm) for 15 min at 37 °C, then, the kinetic slope of was calculated with or without the inhibitor. Average of the percent inhibition was obtained from more than three experiments. The active cathepsin D concentration was titrated using pepstain A, determined as 46.1 pM. The velocities under several substrate concentrations were analyzed and fitted to the Michaelis-Menten equation to obtain Km value as 25.7 μM. Ki value was determined by testing under six inhibitor concentrations fitting the curve to Morrison’s equation of tight binding inhibitor. Protease Activity Assay with ISAAC. A mixture consisting 55-65 μL of MES buffer (pH 5.5), 5 μL of inhibitor (100 nM) in DMSO, and 20 μL of HIV-1 protease (200 ng/mL) in MES buffer was added to 0-10 μL of streptavidin (13.7 μg/mL, FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan) in MES buffer. After incubation for 10 min at 37 °C, the reaction was initiated by addition of 10 μL of the FRET substrate (5 μM) solution. Production of the cleaved EDANS

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substrate was monitored as described above. Average of the percent inhibition was obtained from more than three experiments. Inhibition of human cathepsin D was analyzed based on cleavage of a substrate for cathepsin D as mentioned above. The reaction mixture consisting 55-65 μL of sodium acetate buffer (pH 3.5), 5 μL of inhibitor (100 nM compound 2 or 20 nM compound 4) in DMSO, and 20 μL of cathepsin D (400 ng/mL) in sodium acetate buffer was added to 0-10 μL of streptavidin (137 μg/ mL). After incubation for 10 min at 37 °C, the reaction was initiated by addition of 10 μL of the FRET substrate (100 μM) solution. Production of cleaved MOCAc fragment was monitored for 15 min at 37 °C as mentioned above. The kinetic slope of was calculated with or without the inhibitor. Average of the percent inhibition was obtained from more than three experiments. Repetitive Control of HIV-1 Protease Activity. 20 μL of HIV-1 protease WTm5 (200 ng/mL) in MES buffer (pH 5.5) was added to 79 μL of MES buffer and incubated for 10 min at 37 °C. The reaction was initiated by adding 1 μL of the FRET substrate (500 μM) in DMSO solution and monitored the cleaved EDANS fragment using a handheld fluorometer (Ex 365 nm/ Em 440-470 nm) for 35 min. 1 μL of compound 2 (500 nM) in DMSO, streptavidin (13.7 μg/mL) in MES buffer, and 2 (5 μM) in DMSO were added in sequence at 5 min, 15min, and 25 min, respectively. For comparison, HIV-1 protease activity was also measured without addition of inhibitor and streptavidin. Average of the percent inhibition was obtained from more than three experiments. Affinity Purification of HIV-1 Protease Using Streptavidin Agarose Column. A mixture of 649 μL of MES (pH 5.5) buffer, 11.6 μL of recombinant HIV-1 protease (4.3 mg/mL) pre-mixed with 120 μL of sodium acetate buffer (pH5.0) and with 50 μL of biotinylated analogues (6-9, 1 mM) DMSO solution, 12.5 μL of porcine pepsin (4.0 mg/mL) premixed with 50 μL of pepstatin A (1 mM) DMSO solution, 6.5 μL of human albumin (7.7 mg/mL) was loaded to HiTrapTM

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Streptavidin HP (GE Healthcare, Germany) equilibrated with MES buffer. After incubation for 5 min at room temperature, the column was washed with 5 mL of MES buffer containing 10% DMSO. The bound proteins were eluted with 2 mL of MES buffer containing 10% DMSO with compound 1 (0.2 mM) and 6 mL of glycine-HCl buffer (0.1 M, pH 2.2). First 1.5 mL of the washing solution and the whole elute were condensed using centrifugal filter device (Amicon® Ultra), diluted with 500 μL of water twice, measured up to 50 μL. The concentrated solution was added to sample buffer solution with reducing agent (Cat. No. 09499-14, Nacalai, Kyoto, Japan), heated and analyzed by SDS-PAGE using tricine buffer and the gels were stained by Coomassie Brilliant Blue. The protein content of the concentrated eluted solution was estimated using NanoOrange® quantification reagent. Affinity Purification Using Magnetic Beads and ISAAC. Slurry of magnetic beads with Nhydroxysuccinimide ester (25 μL NHS Mag SepharoseTM, GE Healthcare, Germany) was coupled with intermediate 5, washed, and equilibrated with 500 μL of MES buffer. A mixture of 1 to 10 μL of recombinant HIV-1 protease (0.1 mg/mL) and 100 μL of human serum was diluted with 190 to 199 μL of MES buffer was incubated with the beads at room temperature. After 30 min, the liquid was removed, and the beads were washed 500 μL of MES buffer three times. Then, 60 μL of MES buffer containing 2.5% DMSO with compound 2 (25 nM) was added and incubated for 5 min, the liquid was collected. A mixture consisting 55 μL of MES buffer, 5 μL of DMSO, and 20 μL of the eluate was added to 10 μL of streptavidin (137 μg/ mL). After incubation for 10 min at 37 °C, the reaction was initiated by addition of 10 μL of the above-mentioned FRET substrate (5 μM) solution. Production of the cleaved EDANS substrate was monitored (Ex 355 nm/ Em 500 nm) for 15 min at 37 °C, then, the kinetic slope of was monitored.

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HCT116 cells (ECACC 91001005) were cultured in McCoy’s 5A medium containing 10% fatal bovine serum at 37°C, then, dissociated by TrypLETM Express, collected and washed by D-PBS(-). The pellet (27.8  103 cells) were frozen and thawed, mixed with 300 μL of D-PBS(-). The suspending solution was diluted with 300 μL of sodium acetate buffer (pH5.0). The NHS magnetic beads (25 μL) were coupled with 5, washed, and equilibrated with 300 μL of sodium acetate buffer. The cellular suspension was incubated with the beads at room temperature for 30 min, the liquid was removed, and the beads were washed 300 μL of sodium acetate buffer three times. Then, 60 μL of sodium acetate buffer containing 2.5% DMSO with compound 2 (2.5 μM) was added and incubated for 5 min, the liquid was collected. A mixture consisting 60 μL of sodium acetate buffer (pH 3.5), 5 μL of DMSO with or without pepstatin (100 nM) and 20 μL of the eluate was added to 5 μL of streptavidin (137 μg/ mL). After incubation for 10 min at 37 °C, the reaction was initiated by addition of 10 μL of the abovementioned cathepsin D substrate solution (100 μM). Production of cleaved MOCAc fragment was monitored for 15 min at 37 °C as mentioned above. X-Ray Crystallographic Analysis of WTm5 Complexed with 2. Preparation of WTm5 for crystallization was performed as reported.19 WTm5 bPI-11 complex was prepared with a 2-fold molar excess of ligand to protease at 2.0 mg/mL protein concentrations. The complex was crystallized using hanging drop vapor diffusion method by mixing in equal volumes (2 μl each) of protease solution and crystallization buffer containing 50 mM sodium acetate buffer at pH 5.0 and 20% saturated ammonium sulfate. Glycerol was used for cryo protectant at final concentration of 30% (w/v). The obtained crystals were flash-frozen under a nitrogen gas cryo-stream (95 K), and subjected to X-ray at BL5A beamline in the High Energy Accelerator Research Organization KEK Photon Factory (Tsukuba, Japan). Data for the complex crystal were processed to the resolution of

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1.00 Å resolution by using HKL2000.26 The structure of the complex was refined to a crystallographic R-factor of 13.2% (free R-factor = 15.0%) using program PHENIX 1.9.27

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI: 10.1021/acs.bioconj-chem.XXXXXXX. X-Ray diffraction data collection and refinement statistics, affinity purification of HIV-1 protease using probes, synthetic scheme and details of compounds, including Table S1, Figure S1, Scheme S1 (PDF)

AUTHOR INFORMATION Corresponding author *[email protected]

ORCID Koushi Hidaka: 0000-0002-8956-6996

Notes

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Bioconjugate Chemistry

The authors declare no competing financial interests.

ACKNOWLEDGEMENTS We are sincerely grateful to Dr. Y. Kiso for his generous encourage on biotinylation of peptides. We thank Dr. M. Kameoka for guiding the use of magnetic beads, Mrs. S. Tokai and Mr. T. Hamada for helping the initial study on the affinity probes with aminocaproyl spacers. Protease inhibitors, atazanavir, darunavir, and lopinavir were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH. This research was supported by grants from JSPS KAKENHI (25460163 and 18K05344) and from Kobe Gakuin University Research Grant C (to K.H.). The synchrotron radiation experiments were performed at the BL-5A of KEK-PF under the approval of the Photon Factory Program Advisory Committee (Proposal No. 2016G516).

ABBREVIATIONS DABCYL, 4-((4-dimethylamino)phenylazo)benzoic acid; EDANS, 5-((2aminoethyl)amino)naphthalenesulfonic acid; DMSO, dimethyl sulfoxide; HAP, histo-aspartic protease; HIV, human immunodeficiency virus; ISAAC, inhibitor stripping action by affinity competition; MES, 2-(N-morpholino)ethanesulfonic acid

REFERENCES

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(1) Leung, D., Abbenante G., and Fairlie G. P. (2000) Protease inhibitors: current status and future prospects. J. Med. Chem. 43, 305–341. (2) De Clercq, E. (2014) Current race in the development of DAAs (direct-acting antivirals) against HCV. Biochem. Pharmacol. 89, 441–452. (3) Berger, A. B., Vitrono, P. M., and Bogyo, M. (2004) Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery. Am. J. Pharmacogenomics 4, 371–381. (4) Pollack, C. V., Reilly, P. A., Ryn, J., Eikelboom, J. W., Glund, S., Bernstein, R. A., Dubiel, R., Huisman, M. V., Hylek, E. M., Kam, C.-W., et al. (2017) Idarucizumab for dabigatran reversal Full cohort analysis. N. Eng. J. Med. 377, 431–441. (5) Green, N. M. (1975) Avidin. Adv. Protein Chem. 29, 85–133. (6) Wilchek, M. and Bayer, E. (1988) The avidin-biotin complex in bioanalytical applications. Anal. Biochem. 171, 1–32. (7) Hofmann, K. and Kiso, Y. (1976) An approach to the targeted attachment of peptides and proteins to solid supports. Proc. Natl. Acad. Sci. 73, 3516–3518. (8) Weber, P. C., Wendoloski, J. J., Pantoliano, M. W., and Salemme, F. R. (1992) Crystallographic and thermodynamic comparison of natural and synthetic ligands bound to streptavidin. J. Am. Chem. Soc. 114, 3197–3200.

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(9) (a) Speers, A. E. and Cravatt, B. F. (2004) Chemical Strategies for Activity‐Based Proteomics. ChemBioChem 5, 41–47. (b) Willems, L. I., Overkleeft, H. S., and van Kasteren, S. I. (2014) Current Developments in Activity-Based Protein Profiling. Bioconjugate Chem. 25, 1181–1191. (10) Hidaka, K., Kimura, T., Abdel-Rahman, H. M., Nguyen, J.-T., McDaniel, K. F., Kohlbrenner, W. E., Molla, A., Adachi, M., Tamada, T., Kuroki, R., et al. (2009) Small-sized human immunodeficiency virus type-1 protease inhibitors containing allophenylnorstatine to explore the S2' pocket. J. Med. Chem. 52, 7604–7617. (11) Hidaka, K., Kimura, T., Ruben, A. J., Uemura, T., Kamiya, M., Kiso, A., Okamoto, T., Tsuchiya, Y., Hayashi, Y., Freire, E., et al. (2008) Antimalarial activity enhancement in hydroxymethylcarbonyl (HMC) isostere-based dipeptidomimetics targeting malarial aspartic protease plasmepsin. Bioorg. Med. Chem. 52, 7604–7617. (12) Nezami, A., Kimura, T., Hidaka, K., Kiso, A., Liu, J., Kiso, Y., Goldberg, D. E., and Freire, E. (2003) High-affinity inhibition of a family of Plasmodium falciparum proteases by a designed adaptive inhibitor. Biochemistry 42, 8459–8464. (13) Kiso, A., Hidaka, K., Kimura, T., Hayashi, Y., Nezami, A., Freire, E., and Kiso Y. (2004) Search for substrate-based inhibitors fitting the S2′ space of malarial aspartic protease plasmepsin II. J. Peptide Sci. 10, 641–647. (14) Sato, S., Kwon, Y., Kamisuki, S., Srivastava, N., Mao, Q., Kawazoe, T., and Uesugi M. (2007) Polyproline-rod approach to isolating protein targets of bioactive small molecules: Isolation of a new target of indomethacin. J. Am. Chem. Soc. 129, 873–880.

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(15) Kawasaki, Y., Chufan, E. E., Lafont, V., Hidaka, K., Kiso, Y., Amzel, L. M., and Freire, E. (2010) How much binding affinity can be gained by filling a cavity? Chem. Biol. Drug Des. 75, 143–151. (16) Bhaumik, P., Horimoto, Y., Xiao, H., Miura, T., Hidaka, K., Kiso, Y., Wlodawer, A., Yada, R. Y., and Gustchina, A. (2011) Crystal structures of the free and inhibited forms of plasmepsin I (PMI) from Plasmodium falciparum. J. Struct. Biol. 175, 73–84. (17) Bhaumik, P., Xiao, H., Parr, C. L., Kiso, Y., Gustchina, A., Yada, R. Y., and Wlodawer A. (2009) Crystal structures of the histo-aspartic protease (HAP) from Plasmodium falciparum. J. Mol. Biol. 388, 520–540. (18) Umezawa, H., Aoyagi, T., Morishima, H., Matsuzaki, M., and Hamada, M. (1970) Pepstatin, a new pepsin inhibitor produced by Actinomycetes. J. Antibiot. 23, 259–262. (19) Adachi, M., Ohhara, T., Kurihara, K., Tamada, T., Honjo, E., Okazaki, N., Arai, S., Shoyama, Y., Kimura, K., Matsumura, H., et al. (2009) Structure of HIV-1 protease in complex with potent inhibitor KNI-272 determined by high-resolution X-ray and neutron crystallography. Proc. Natl. Acad. Sci. 106, 4641–4646. (20) Brauer, M., De Villiers, J. C., and Mayaphi, S. H. (2013) Evaluation of the Determine™ fourth generation HIV rapid assay. J. Virol. Methods 189, 180–183. (21) Kawahata, T., Nagashima, M., Sadamasu, K., Kojima, Y., and Mori, H. (2013) Evaluation of an immunochromatographic fourth generation test for the rapid diagnosis of acute HIV infection. Kansenshogaku Zasshi 87, 431–434.

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(22) Schreiber, G. B., Busch, M. P., Kleinman, S. H., and Korelitz, J. J. (1996) The risk of transfusion-transmitted viral infections. N. Engl. J. Med. 334, 1685–1690. (23) Matayoshi, E. D., Wang, G. T., Krafft, G. A., and Erickson, (1990) J. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science 247, 954–958. (24) Matsumura, H., Adachi, M., Sugiyama, S., Okada, S., Yamakami, M., Tamada, T., Hidaka, K., Hayashi, Y., Kimura, T., Kiso, Y., et al. (2008) Crystallization and preliminary neutron diffraction studies of HIV-1 protease cocrystallized with inhibitor KNI-272. Acta Cryst. F64, 1003–1006. (25) Morrison, J.F. (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochim. Biophys. Acta 185, 269–286. (26) Otwinowski, Z. and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode, in Methods in Enzymol. Volume 276 (Carter, C. W. Jr, Sweet, R. M., Eds.) pp 307–326, Academic Press: New York. (27) Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev, A., Zwart, P. H., and Adams, P. D. (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr., 68, 352–367.

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