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Dec 16, 2015 - Label-Free Bottom-Up Proteomic Workflow for Simultaneously. Assessing the Target Specificity of Covalent Drug Candidates and...
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Label-Free Bottom-Up Proteomic Workflow for Simultaneously Assessing the Target Specificity of Covalent Drug Candidates and Their Off-Target Reactivity to Selected Proteins Yanou Yang, Yue-Zhong Shu,* and W. Griffith Humphreys Bristol-Myers Squibb Research and Development, 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534, United States S Supporting Information *

ABSTRACT: Although designed covalent inhibitors as drug candidates offer several unique advantages over conventional reversible inhibitors, including high potency and the potential for less frequent dosing, there is a general tendency to avoid the covalent mode of action in drug discovery programs due to concerns regarding immune-mediated toxicity that can arise from indiscriminate reactivity with off-target proteins. Therefore, the ability to assess off-target reactivity relative to target specificity is desirable for optimizing covalent drug candidates in the early discovery stage. One concern with current surrogate nucleophile trapping approaches is that they employ a simplistic model nucleophile such as glutathione, which may not reliably reflect the covalent interactions with cellular or extracellular proteins. One way to get a more relevant reactivity assessment is to directly measure the ability of an inhibitor to covalently modify nucelophilic amino acids on biologically relevant proteins, both on- and off-target. In this article, we describe a label-free bottom-up proteomic workflow for simultaneous evaluation of target binding and off-target reactivity of covalent drug candidates to selected proteins at the peptide level. Ibrutinib, a covalent drug targeting the active site of BTK protein, was used as a model compound to demonstrate the feasibility of the workflow. The compound was incubated with a mixture of target protein, Bruton’s tyrosine kinase (BTK), and two abundant proteins in blood, hemoglobin (Hb) and human serum albumin (HSA), and then the ibrutinib modification sites were determined utilizing a bottom-up proteomic approach. A non-BTK specific model compound (1) known to modify cysteine residues was also included. By comparing the extent of off-target modifications to the targeted BTK C481 binding in a wide compound concentration range, we were able to determine the concentration where maximum target binding was achieved with minimal off-target reactivity. The generic label-free bottom-up proteomics workflow described in this article should be useful in the rank order assessment of off-target reactivity vs on-target reactivity of covalent drug candidates in the early drug discovery stage.

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INTRODUCTION Designed covalent inhibitors as drug candidates offer several unique advantages over conventional reversible inhibitors, including high potency resulting from the increased biochemical efficiency through covalent interaction and potential for less frequent dosing thanks to the increased duration of action that outlasts the pharmacokinetics of the drug.1,2 Despite these advantages, there is a general tendency to avoid the covalent mode of action in drug discovery programs due to concerns regarding immune-mediated toxicity caused by the formation of neoantigens. In order to minimize total protein modification, the ability to assess off-target reactivity relative to target specificity is critical for establishing confidence in the safety profile of covalent drug candidates in the early discovery stage.3 To assess off-target binding, a frequently used approach is to measure the reaction rate of a covalent inhibitor with a simple surrogate nucleophile, such as glutathione (GSH) or Nacetylcysteine in buffer.4,5 Since the nucleophilic moiety in a proteinaceous environment can have a significantly different © 2015 American Chemical Society

reactivity profile than when contained in a low molecular weight nucleophile, there have been concerns about the reliability of using thiol trapping agents as a surrogate for offtarget cellular proteins.2 One way to get more relevant offtarget reactivity assessment of a covalent inhibitor is to directly measure its ability to covalently modify nucelophilic amino acids on biologically relevant proteins. Huth et al.6 described an in vitro NMR spectroscopy-based assay (ALARM NMR) to measure the thiol reactivity of discovery compounds using human La antigen protein. This assay has recently been used by Chen et al.7 to assess the off-target reactivity of an irreversible hepatitis C virus (HCV) NS5b polymerase inhibitor. Although a proteinaceous probe was used in this assay, 13C labeled human La antigen protein was required in order to monitor the chemical shift by NMR. The direct analysis of drug modified proteins via global proteomics approaches has proven to be difficult.8 One method Received: November 8, 2015 Published: December 16, 2015 109

DOI: 10.1021/acs.chemrestox.5b00460 Chem. Res. Toxicol. 2016, 29, 109−116

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Chemical Research in Toxicology that has been proven successful is through the enrichment of drug labeled proteins using immunoaffinity techniques that recognize the drug.9 The need for antidrug antibody limits this approach to detailed characterization studies. Activity-based protein profiling (ABPP) integrated with click chemistry has emerged as a powerful chemoproteomic platform for evaluating the off-target reactivity of covalent inhibitors in intact cells and animals.10−12 In a recent report,13 Cravatt et al. successfully applied ABPP/click chemistry combined with quantitative MS for global and in-depth analysis of proteins targeted by covalent kinase inhibitors including PF-6274484 and ibrutinib in human cancer cells. This ABPP-SILAC approach can determine what proteins are adducted by covalent inhibitors at the protein level, but it does not provide the evidence on which amino acid residue(s) on the targeted proteins a covalent inhibitor binds, especially when there are more than one possible binding sites on a protein target. Other related approaches have been described that involve a direct conjugation of the reactive drug molecule with a recognition molecule, as exemplified by the work done with amoxicillin− biotin conjugates.14 Although these approaches are very powerful in the global identification of protein targets for covalent inhibitors, the requirement for the synthesis of clickready or biotin-linker probes and questions such as how well the analogues mimic the actual drug behavior may limit their application in off-target activity assessment of covalent drug candidates in the early drug discovery stage. In this article, we describe a label-free bottom-up proteomic workflow for simultaneous evaluation of target binding and offtarget reactivity to selected proteins at the amino acid level. We incubated an unlabeled model covalent drug ibrutinib with a mixture of the target protein, BTK, and two blood-abundant proteins, hemoglobin (Hb) and human serum albumin (HSA), and then directly traced down the modification sites of ibrutinib to the proteins in the mixture utilizing a bottom-up proteomic approach. By comparing the extent of off-target modifications to the targeted C481 binding at various compound concentrations, we were able to determine a concentration window where maximum target binding was achieved with minimal off-target reactivity. The generic label-free bottom-up proteomics workflow described in this article should be useful in the rank order assessment of off-target vs target reactivity of covalent drug candidates in the early drug discovery stage.

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Figure 1. Structures of ibrutinib and 1. solubilized and denatured in 50% TFE containing 5 mM DTT at 50 °C for 60 min as described previously.15 The samples were then alkylated with IAM at a final concentration of 10 mM at room temperature in the dark for 45 min. Samples were diluted 10 times with 50 mM ammonium bicarbonate buffer, followed by digestion. Digestion was carried out using Mass Spec grade Trypsin/Lys-C mix at a 1:20 enzyme-to-protein ratio w/w, at 37 °C for 3 h. Digested samples were stored at −80 °C until LC-MS/MS analysis. Incubation of Ibrutinib or 1 with the Three-Protein Mixture. Ibrutinib (0.01 μM, 0.05 μM, 0.1 μM, 1 μM, 3 μM, 10 μM, and 100 μM) was spiked into the protein mixture with the final protein concentration of 0.5 μM, 2.5 μM, and 2.5 μM for BTK, Hb, and HSA, respectively. For comparison purposes, a known electrophilic compound 1 was also incubated separately at 1 μM, 10 μM, and 100 μM with the three-protein mixture. The incubations were done in triplicate except for the incubation with low ibrutinib concentration (0.01 μM, 0.05 μM, and 0.1 μM), which was done with a single replicate. The total incubation volume was 100 μL. Incubation samples in the absence of compound were included in the experiment as the control. After 60 min of incubation at 37 °C, the incubation samples were precipitated, solubilized, alkylated, and digested by following the same procedures as those described in the Denaturation, Alkylation and Digestion of Protein Mixture section above. UHPLC-HRMS Methods for Peptide Analysis. All digested samples were analyzed using an Accela UHPLC system coupled to a LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Chromatographic separations were performed with an Acquity UPLC BEH 130 C18 1.7 μm (2.1 × 100 mm) column at a flow rate of 0.6 mL/min. The column was eluted with water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B). The UHPLC separation used a linear gradient program of 2% B for 1 min, 2−35% B for 94 min, 35−50% for 15 min, 50−80% for 5 min, and 2% B for 5 min. The column and samples were maintained at temperatures of 55 and 5 °C, respectively. The UHPLC eluent was directly coupled to the LTQ/Orbitrap MS. The LTQ Orbitrap MS was operated in the positive ion electrospray mode using nitrogen as the sheath and auxiliary gases. The heated capillary temperature was set at 330 °C, the capillary voltage was 20 V, the source voltage was 5 kV, and the tube lens voltage was 140 V. The acquisition cycle consisted of a full scan in the orbitrap with a mass range from m/z 250 to 1800 at the resolution of 60,000, followed by 8 data-dependent MS/MS scans acquired in the linear ion trap using collision-induced dissociation (CID). Precursor ions that were detected twice within 20 s were put on a dynamic exclusion list for a period of 45 s. The CID-MS/MS collision energy was set at 35%. The charge state screening was enabled with +1 and unassigned charge states rejected for MS/MS scans. Xcalibur 3.0 was used for both data acquisition and data processing. Extracted ion chromatograms (XICs) were generated with a 10 ppm mass window centered on the exact m/z of peptides of interest. Quan Browser within Xcalibur was used to perform the peptide MS peak integration and peak area reporting. Although a given peptide variation is associated with only one monoisotopic elemental mass, multiple charge states associated with multiple isotopic peaks are often detected. Therefore, the MS peak area of a given peptide is the sum of extracted ion current of all isotopic clusters.

EXPERIMENTAL SECTION

Materials. BTK protein was obtained from Life Technologies (Grand Island, NY). Human hemoglobin, human serum albumin, trifluoroethanol (TFE), ammonium bicarbonate, dithiothreitol (DTT), and iodoacetamide (IAM) were purchased from SigmaAldrich (St. Louis, MO, USA). Ibrutinib and 2-(methylsulfonyl)benzo[d]thiazole (1) (Figure 1) are available from Ryan Scientific, Inc. (Mt. Pleasant, SC), trypsin/lysC was purchased from Promega (Madison, WI), and formic acid, HPLC grade water, and acetonitrile were purchased from EMD chemicals (Gibbstown, NJ, USA). Preparation of the Three-Protein Mixture. BTK protein in storage buffer was dialyzed for 3 h against a 100 mM phosphate buffer (pH 7.4) using Slide-A-Lyzer MINI dialysis devices (Pierce Biotechnology) equipped with a membrane with a molecular weight cutoff of 10,000. The three-protein mixture was prepared by mixing postdialysis BTK protein with human serum albumin and human hemoglobin in PBS buffer at a molar ratio of 1:5:5. Denaturation, Alkylation, and Digestion of Protein Mixture. Proteins in the mixture were precipitated out in triplicate using ProteoExtract Protein Precipitation Kit (EMD Millipore, Billerica, MA) by following the vendor’s instructions. The protein pellets were 110

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Chemical Research in Toxicology Table 1. Free Cysteine Containing Peptides from BTK, Hb, and HSA Detected after Trypsin/Lys-C Digestion protein BTK

Hb

HSA

Cys position C63 C145, C154, C155 C165 C337 C464 C481 C502 C506 C527 C633 alpha C104 beta C93 beta C112 C34

monoisotopic elemental massa (charge)b

peptides ITCVETVVPEK ITCVETVVPEKNPPPER YHPCFWIDGQYLCCSQTAK NAMGCQILENR HYVVCSTPQSQYYLAEK LVQLYGVCTK QRPIFIITEYMANGCLLNYLR FQTQQLLEMCK DVCEAMEYLESK NCLVNDQGVVK VYTIMYSCWHEK LLSHCLLVTLAAHLPAEFTPAVHASLDK GTFATLSELHCDK GTFATLSELHCDKLHVDPENFR LLGNVLVCVLAHHFGK ALVLIAFAQYLQQCPFEDHVK

1273.6588 1964.0037 2433.0340 1304.5965 2071.9673 1179.6322 2584.3294 1424.6792 1472.6163 1244.6183 1615.7163 3023.6267 1477.6871 2585.2333 1775.9869 2489.2777

(2+) (2+, 3+, 4+) (2+, 3+) (2+, 3+) (2+, 3+) (2+) (3+) (2+, 3+) (2+, 3+) (2+, 3+) (2+, 3+) (3+, 4+, 5+) (2+, 3+) (3+, 4+) (2+, 3+, 4+) (2+, 3+, 4+)

RTc (min) 23.5 22.6 43.2 21.7 25.5 27 73.7 33.7 38.6 19.6 30.0 63.7 25.5 41.7 57.1 70.0

a

Calculated monoisotopic elemental mass. The observed monoisotopic elemental mass is within 10 ppm of calculated values. bObserved charge state. cAverage retention time if multiple spectra were detected for the same peptide. Database Searching. The acquired MS/MS spectra were searched against the refseq human database using SORCERERSEQUEST software (Sage-N Research Inc. Milpitas, CA). The adduction of ibrutinib and 1 and S-carbamidomethylation of cysteine (+57.02146 Da), deamination of asparagine and glutamine (+0.9840), and oxidation of methionine and histidine (+15.99492 Da) were specified as variable modifications. The mass shifts with ibrutinib and 1 modifications were calculated as 440.1961 and 132.9986, respectively, which is based on the mass shift of GSH adducts formed in human liver microsomal incubation as reported previously.16 Less than two missed cleavages by trypsin were also considered in the search. Peptide mass tolerance was set at ±10 ppm. Fragment mass tolerance was set at ±1.0 Da. The peptide probability requirement was to be equal to or above the value which gives 1% FDR in peptide identification.

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only interested in free cysteine containing peptides. BTK has a total of 12 free cysteine residues including targeted active site cysteine C481. Human HSA has only one free cysteine (C34) residue available for adduction, while all other cysteine residues are joined together by disulfide bonds.21 In human hemoglobin, the α-subunit contains one free cysteine residue (α-C104), and the β-subunit contains two free cysteine residues (β-C93 and βC112).22 In order to detect the peptides containing all these free cysteine residues, we optimized our workflow using a control three-protein mixture with no inhibitor present. Our initial attempt with trypsin digestion resulted in a very low signal for the C481-containing peptide (data not shown). Among the different digestion enzymes tested, trypsin/Lys-C digestion gave the most intense signal for the C481 containing peptide QRPIFIITEYMANGCLLNYLR and led to the successful detection of peptides covering all the free cysteine residues from all three proteins, as shown in Table 1. On the basis of all the peptides detected for the three proteins (list of peptides detected for BTK, Hb and HSA), the sequence coverage is 74.8%, 76.5%, and 85.8% for BTK, HSA, and Hb, respectively. Identification of Adducted Peptides. After establishing a workflow for identifying all the peptides of interests from the three proteins in the mixture as described above, we then appled this workflow to detect/identify ibrutinib adduction sites on these three proteins. We incubated the three protein mixture at fixed concentrations (BTK/Hb/HSA = 0.5:2.5:2.5 μM) with ibrutinib or 1 at 100 μM concentration. The proteins in the incubation were then prepared by the same procedure as established with the control three-protein mixture. The tryptic peptide mix of these incubation samples were analyzed by LCMS/MS and followed by targeted database searching to specifically look for peptides with the addition of C 25 H 24 N 6 O 2 (+440.1961) for ibrutinib and C 7 H 3 NS (132.9986) for 1 to cysteine residues. The peptide probability requirement was set to be equal to or above the value which gives 1% FDR in peptide identification. On the basis of these criteria, a total of 14 modified peptides corresponding to 12 cysteine residues from BTK and Hb were identified in the incubation with 100 μM ibrutinib. The sequences and monoisotopic elemental masses of these peptides are shown

RESULTS AND DISCUSSION

Detection of Peptides of Interest. In addition to target protein BTK, we included two high abundance blood proteins, hemoglobin (Hb) and human serum albumin (HSA), in the protein mixture to assess the off-target reactivity of ibrutinib. As a matter of fact, Hb and HSA have been used as biomarkers of exposure to exogenous and endogenous electrophiles owing to their inherent advantages of high abundance in blood, lack of repair, and relatively long residence times.17−19 Of the many nucleophilic amino acid residues available on Hb and HSA for substitution reactions with electrophiles, free thiols in cysteine residues are attractive candidates because of their strong nucleophilicity. C34 in HSA provides the largest fraction of free thiol in blood serum (80%) and accounts for the majority of the antioxidant activity of albumin, being the preferential plasma scavenger of reactive species such as hydrogen peroxide, superoxide anion, hydroxyl radical, peroxynitrite and phospholipid hydroperoxides.20 In order to detect peptides adducted by ibrutinib, we sought to develop a workflow allowing us to detect all the peptides of interest from the three proteins (BTK, Hb and HSA) in a mixture where a ratio of 1:5:5 for BTK/Hb/HSA was used. This arbitrary ratio of target to off-target proteins was set to reflect some degree of in vivo abundance but not compromise the sensitivity of analytical methodology. Since ibrutinib can only modify free cysteine residues, but not cysteine residues occupied by the disulfide bond or already adducted, we were 111

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Table 2. Summary of Covalently Modified Peptides Detected in a Three-Protein Mixture Incubated with 100 μM Ibrutinib protein BTK

Hb

HSA

Cys position C63 C145 C154 C155 C165 C337 C464 C481 C502 C506 C527 C633 Alpha C104 Beta C93 Beta C112 C34

peptidesa

monoisotopic elemental massb (charge)c

RTd(min)

mass accuracye (ppm)

ITCVETVVPEK ITCVETVVPEKNPPPER YHPCFWIDGQYLCCSQTAK YHPCFWIDGQYLCCSQTAK YHPCFWIDGQYLCCSQTAK NAMGCQILENR HYVVCSTPQSQYYLAEK LVQLYGVCTK QRPIFIITEYMANGCLLNYLR FQTQQLLEMCK DVCEAMEYLESK NCLVNDQGVVK VYTIMYSCWHEK LLSHCLLVTLAAHLPAEFTPAVHASLDK GTFATLSELHCDK GTFATLSELHCDKLHVDPENFR LLGNVLVCVLAHHFGK ALVLIAFAQYLQQCPFEDHVK

1656.8371 (3+) 2347.1882 (4+) no adduction detected 2816.2149 (4+)

64.2 58.8

2.1370 4.0900

70.9 71.8 66.2 58.8 67.9 92.0

2.1780 2.0915 2.8360 3.5283 7.0200 3.6580

76.3 62.2

2.6415 2.8348

84.8 64.5 67 80.3

6.7000 2.7315 2.6829 1.5420

1687.7761 (2+, 3+) 2455.1506 (3+, 4+) 1562.8180 (3+) 2967.5151 (3+, 4+) no adduction detected 1855.7960 (3+) 1627.7977 (2+, 3+) no adduction detected 3406.8244 (4+) 1860.8671 (2+, 3+, 4+) 2968.4161 (3+, 4+) 2159.165 (3+, 4+) no adduction detected

a Modified cysteines are underlined. bCalculated monoisotopic elemental mass. The observed monoisotopic elemental mass is within 10 ppm of calculated values. cObserved charge state. dAverage retention time if multiple spectra were detected for the same modified peptide. eAverage mass accuracy if multiple spectra were detected for the same modified peptide.

Table 3. Summary of Covalently Modified Peptides Detected in a Three-Protein Mixture Incubated with 1 protein BTK

Hb

HSA

Cys position C63 C145 C154 C155 C165 C337 C464 C481 C502 C506 C527 C633 alpha C104 beta C93 beta C112 C34

peptidesa

monoisotopic elemental massb (charge)c 1349.6393 (2+) 2039.9873 (3+) 2509.0197 (3+)

ITCVETVVPEK ITCVETVVPEKNPPPER YHPCFWIDGQYLCCSQTAK YHPCFWIDGQYLCCSQTAK YHPCFWIDGQYLCCSQTAK NAMGCQILENR HYVVCSTPQSQYYLAEK LVQLYGVCTK QRPIFIITEYMANGCLLNYLR FQTQQLLEMCK DVCEAMEYLESK NCLVNDQGVVK VYTIMYSCWHEK LLSHCLLVTLAAHLPAEFTPAVHASLDK GTFATLSELHCDK GTFATLSELHCDKLHVDPENFR LLGNVLVCVLAHHFGK ALVLIAFAQYLQQCPFEDHVK

1380.5775 2147.9513 1255.6131 2660.3167 1500.6615 1548.6010 1320.5986 1691.6984 3099.6148 1553.6693 2661.2177 1851.9704 2565.2641

(2+, 3+) (3+) (2+, 3+) (3+, 4+) (2+, 3+) (2+, 3+) (2+) (2+, 3+) (3+, 4+, 5+) (2+, 3+) (3+, 4+) (2+, 3+, 4+) (2+, 3+, 4+)

RTd(min)

mass accuracye (ppm)

45.6 40.2 58.6 59.6 60.0 48.4 43.4 51.4 83 62.2 62.4 38.5 53.5 75.2 47.4 57.2 66.7 86.6

2.4121 3.0674 3.3334 3.5757 2.8268 2.7285 3.1587 2.9311 3.7233 3.2615 4.8031 2.312 2.9198 3.4481 3.1407 2.6442 3.3807 3.5012

a

Modified cysteines are underlined. bCalculated monoisotopic elemental mass. The observed monoisotopic elemental mass is within 10 ppm of the calculated values. cObserved charge state. dAverage retention time if multiple spectra were detected for the same modified peptide. eAverage mass accuracy if multiple spectra were detected for the same modified peptide.

The data from peptides C481 are shown as an example (QRPIFIITEYMANGCLLNYLR) (Figure 2). In order to ascertain whether the adducted peptides were covalent inhibitor related, we checked the presence of these peptides in the control incubations. None of these peptides were detected in the control incubation samples. Instead, the IAM adducted variation of these peptides were all detected in control incubation samples. Furthermore, since adducting with ibrutinib or 1 increases the hydrophobicity of the peptides, the adducted peptide variations should elute later than the corresponding IAM modified variations in UPLC. As expected, modified variations were found to always elute later than the corresponding IAM modified variations (Table 1−3). Also since ibrutinib is more hydrophobic than 1, peptides modified

in Table 2. In comparison, all 16 free cysteine residues from the three proteins in the incubation mixture were adducted by 1 at 100 μM compound concentration (Table 3), indicating that ibrutinib is somewhat more specific than 1. This is not surprising since the initial inherent reversible binding affinity of ibrutinib with BTK leads to the placement of the electrophile at or near the nucleophile at the active site of the target protein, which in theory should enable less chemically reactive warheads to be utilized in the covalent inhibitor design.2 The mass accuracy tolerance of measured vs calculated m/z was set as less than 10 ppm for the assignment of modified peptides, and most of them were well below 5 ppm. Each modified peptide identified by the database search is supported by analysis of the CID fragmentation pattern (Figure S2−S33). 112

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Figure 2. MS data from C481 containing peptide QRPIFIITEYMANGCLLNYLR modified by ibrutinib. (A) Full scan accurate mass acquired in the orbitrap for one of its charge states; (B) CID fragment ion spectra acquired on m/z = 742.8856 in the ion trap; (C) proposed main CID fragmentation route.

Table 4. Modification Percentage at Different Compound Concentrationsa,b ibrutinib (μM) protein BTK

Hb

HSA

Cys position C63 C145 C154 C155 C165 C337 C464 C481 C502 C506 C527 C633 alpha C104 beta C93 beta C112 C34

peptides ITCVETVVPEK YHPCFWIDGQYLCCSQTAK YHPCFWIDGQYLCCSQTAK YHPCFWIDGQYLCCSQTAK NAMGCQILENR HYVVCSTPQSQYYLAEK LVQLYGVCTK QRPIFIITEYMANGCLLNYLR FQTQQLLEMCK DVCEAMEYLESK NCLVNDQGVVK VYTIMYSCWHEK LLSHCLLVTLAAHLPAEFTPAVHASLDK

1

3

10

compd 1 (μM) 100

1

10

100

2.6 ± 0.9

96.5 ± 1.6

1.4 ± 0.9 0.4 ± 0.2 6.0 ± 3.9 2.2 ± 0.5 0.3 ± 0.1 97.4 ± 0.6

1.0 ± 0.3

1.0 ± 0.7 5.0 ± 3.0

1.0 ± 0.5 0.4 ± 0.1 84.2 ± 16.4

GTFATLSELHCDK

98.1 ± 0.8

4.2 1.3 3.1 6.2 1.7 1.9 5.0 0.8

± ± ± ± ± ± ± ±

3.0 0.8 2.1 1.4 1.0 1.4 1.5 0.6

0.2 ± 0.3 3.5 + 2.4

9.7 ± 5.3

34.1 ± 4.7

0.8 ± 0.7

9.5 ± 6.3

4.8 ± 6.7

LLGNVLVCVLAHHFGK

41.6 ± 12.4 29.0 ± 18.2

0.1 ± 0.1

ALVLIAFAQYLQQCPFEDHVK

4.0 ± 3.5 5.7 ± 3.4 4.1 ± 2.4 8.4 ± 3.5 23.5 ± 14.4 12.1 ± 5.9 23.2 ± 7.4 76.5 ± 10.5 13.7 ± 6.0 12.8 ± 8.6 35.9 ± 8.3 7.8 ± 4.8 2.2 ± 3.2

2.4 ± 3.3

a

The percentage was calculated by dividing the MS peak area of the ibrutinib-modified peptide by the combined MS peak area from the same peptide with both ibrutinib and IAM modifications. bn = 3.

demonstrates the importance of binding affinity and access to the protein nucleophile in determining the covalent binding characteristics of molecules with inherently reactive moieties. In addition to free cysteine, other amino acid residues in HSA, including lysine, arginine, and histidine, have been reported to be modified by nucleophiles.9,24 Although we included these amino acid residues in our database search as targeted modification sites, no adduction on these amino acid residues were identified. This can be explained by the higher reactivity of cysteine thiols toward ibrutinib and 1 and the

by ibrutinib eluted later than the corresponding peptides modified by 1. C34 of HSA was reported to be a strong nucleophilic center, which is thought to play a role in the antioxidant activity of albumin. However, C34 of HSA was not modified by ibrutinib even at a compound concentration of 100 μM, although it was modified by 1 at the same concentration. According to X-ray crystal structural characterization of HSA, C34 is located at the surface of the HSA molecule with its Sγ atom oriented toward the interior and surrounded by side chains of Pro35, His39, Val77, and Tyr84.21,23 The differential binding to this residue 113

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Figure 3. Comparison of extracted ion chromatograms of C481 containing peptide QRPIFIITEYMANGCLLNYLR modified by ibrutinib (m/z = 743.1340, 4 charges, RT = 92.0 min) or IAM (m/z = 862.7847, 3 charges, RT = 73.7 min) after incubating with ibrutinib at 50 nM (A), 100 nM (B), and 1 μM (C).

was very specific. Even at 1 μM ibrutinib concentration (2:1 ratio of drug:BTK), the majority of C481 was adducted (Table 4), while adduction to other cysteine residues by ibrutinib was not detected at this concentration. At 100 μM compound concentration, the specificity of the compound had decreased; however, the extent of modification to most off-target cysteine residues by ibrutinib is well below 10%. This concentration dependent specificity is consistent with a previous report.13 In comparison, 1 did not show specificity to any cysteine residues, including the target C481 peptide. This is not surprising since 1 was not designed as a covalent inhibitor to target BTK protein. For all of the cysteine residues except the target cysteine C481, the extent of modification by 1 was always higher than that by ibrutinib, further confirming that ibrutinib is more specific than 1. How the concentration dependent specificity of ibrutinib translates to a more complete in vitro system or an in vivo system is difficult to predict, and the clinical plasma Cmax of ibrutinib is approximately 3 μM. Among all the off-target cysteine residues present in the protein mixture, beta C93 of Hb seems to be the most reactive one. It is the only cysteine residue adducted by 1 at 1 μM compound concentration. Furthermore, it is the only off-target cysteine residue adducted by ibrutinib at 3 μM compound concentration. This is consistent with previous report where only beta C93 was found to be modified when the ratio of electrophile to Hb protein was low enough.26 For the ibrutinib concentration range of 1−100 μM, we did not observe significant differentiation in terms of the extent of modification for target cysteine where the majority of C481 was modified even at the lowest tested concentration of 1 μM. In order to explore the target saturation vs drug concentration relationship further, lower ibrutinib concentrations of 10−100 nM were explored and the modification percentage determined as described above. The estimated modification percentages were 2.6%, 18.9%, and 34.5% for 10 nM, 50 nM, and 100 nM, respectively. The extracted ion chromatograms of IAM modified and ibrutinib modified C481 containing peptide

much shorter incubation time and lower compound concentration used in the experiments. Concentration Dependent Specificity. In order to determine the extent of modification at different compound concentrations, we incubated the protein mixture at fixed concentrations (BTK/Hb/HSA = 0.5:2.5:2.5 μM) with ibrutinib or 1 at a concentration range of 1 μM−100 μM. The estimated adduction percentage was then determined based on the MS peak areas of modified and unmodified variations in the extracted ion chromatograms for a given free cysteine containing peptide in the same incubation sample. The unmodified portion of a given peptide is actually adducted with alkylating reagent IAM since any cysteine residues, which were not adducted with ibrutinib or 1 during the incubation, would have been adducted with IAM during the alkylation step. The combination of ibrutinib or 1 adducted and the IAM adducted variation for the same peptide reflects the total peptide. Assuming the MS responses from different peptide variations are the same, the MS peak areas in the extracted ion chromatograms of a given peptide can be used to estimate the adduction percentage, as shown in Table 4. Although this MS-based approach has been used previously,15,25 one concern with calculating the percentage of modification using MS peak area was that the MS ionization efficiency for drug bound vs nondrug bound peptides may be different and/or the ionization for different variations of the same peptide might be different. Therefore, the modification percentage shown in Table 4 can only be used to roughly rank order the extent of modification by different compounds. As shown in Table 4, the extent of modification by both ibrutinib and 1 is clearly dependent on compound concentration. For all of the modified cysteine residues, the higher the compound concentration was, the higher was the extent of modification. Among the 12 cysteine residues adducted by ibrutinib at 100 μM compound concentration, only five were detected at 10 μM. Also, some of the peptides were detected as adducted by 1 only at the highest concentration of 100 μM. As expected, the modification of C481 of BTK protein by ibrutinib 114

DOI: 10.1021/acs.chemrestox.5b00460 Chem. Res. Toxicol. 2016, 29, 109−116

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QRPIFIITEYMANGCLLNYLR at different ibrutinib concentrations are shown in Figure 3. The relationship between incubation time and extent of modification for different cysteine residues was not specifically investigated in this article. However, based on our results, it is clear that the incubation time of 1 h used in our experiments allowed differentiation in the extent of modification between different cysteine residues for the range of compound concentrations tested.

ABBREVIATIONS GSH, glutathione; BTK, Bruton’s tyrosine kinase; Hb, hemoglobin; HSA, human serum albumin; TFE, trifluoroethanol; IAM, iodoacetamide; DTT, dithiothreitol; UHPLC/ HRMS, ultrahigh pressure liquid chromatography/high resolution accurate mass spectrometry; FDR, false discovery rate; MS, mass spectrometry

CONCLUSIONS We have developed a label-free bottom-up proteomic workflow for simultaneous evaluation of target binding and off-target reactivity of covalent drug candidates. After incubating the model compound ibrutinib with BTK and two high abundance blood proteins, Hb and HSA, we were able to directly trace the modification sites from the three proteins at the peptide level. When the compound concentration was ≤1 μM, only the target cysteine C481 was adducted by ibrutinib, demonstrating the specificity of ibrutinib binding. Modification to C481 was detected even at 10 nM ibrutinib concentration. In comparison, the strong electrophile 1, which was not designed to target BTK, did not show specificity toward any BTK cysteine residues, confirming that the specificity of ibrutinib toward C481 is highly dependent on the high affinity for the active site. As expected, the specificity was concentration dependent: offtarget cysteine residues were also modified when ibrutinib concentration was increased. For example, at 100 μM ibrutinib concentration, a total of 13 modified peptides from 11 off-target cysteine residues were identified. Furthermore, the extent of modification for a given cysteine also increased as the compound concentration increased. This label-free bottom-up proteomics workflow clearly shows promise as a potential tool to access and rank order on- and off-target reactivity for covalent drug discovery.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00460. MS/MS spectra and fragmentation table for the adducted peptides (PDF) List of peptides detected for BTK, Hb, and HSA (XLSX)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 609-818-5652 E-mail: [email protected]. Funding

This study was fully financed by Bristol-Myers Squibb Company. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Qing Xiao and Ashok Dongre for providing access to the SORCERER-SEQUEST data base and their technical help in database searching. The authors also acknowledge Qian Ruan, Joseph Tino, Aberra Fura, and James Burke for helpful discussions. 115

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