Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B

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Article

Covalent allosteric inactivation of PTP1B by an inhibitor-electrophile conjugate Puminan Punthasee, Adrian R. Laciak, Andrea Hicks Cummings, Kasi Viswanatharaju Ruddraraju, Sarah M. Lewis, Roman Hillebrand, Harkewal Singh, John J Tanner, and Kent S. Gates Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00151 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 28, 2017

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Biochemistry

Covalent allosteric inactivation of PTP1B by an inhibitorelectrophile conjugate Puminan Punthasee,a Adrian R. Laciak,a Andrea H. Cummings,a Kasi Viswanatharaju Ruddrarajua, Sarah M. Lewis,a Roman Hillebrand,a Harkewal Singh,a John J. Tanner,a,b* and Kent S. Gatesa,b,*

a

University of Missouri

Department of Chemistry 125 Chemistry Building Columbia, MO 65211 b

University of Missouri

Department of Biochemistry 117 Schweitzer Hall Columbia, MO 65211

* To whom correspondence should be addressed: email: [email protected]; phone: (573) 882-6763; FAX: (573) 882-2754

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ACS Paragon Plus Environment

Biochemistry

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ABBREVIATIONS HEPES, 4-(2-hydroxyethyl-1-piperazineethanesulfonic acid; EDTA, ethylenediamine tetraacetic acid; Tris, tris(hydroxymethyl)aminomethane; PTP1B, Protein tyrosine phosphatase 1B; BOP, (benzotriazol-1-yloxy)-tris(dimethylamino)phosphate; Bis-tris, 2[bis-(2-hydroxyethyl)-amino]-2-hydroxymethyl-propane-1,3-diol; (Bis-tris); p-NPP, 4nitrophenyl phosphate disodium salt hexahydrate; DTT, DL-dithiothreitol; EDTA, diethylenediamine

tetraacetic

acid

disodium

monohydrate;

DTPA,

diethylenetriaminepentaacetic acid; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; PEG, polyethylene glycol; PMSF, phenylmethanesulfonyl fluoride; IPTG, isopropylbeta-D-thiogalactopyranoside;

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Biochemistry

ABSTRACT: Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target but it has proven difficult to develop medicinally-useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. The inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of 1.7 ± 0.3 x 102 M-1 min-1. Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B (Hansen S. K.; Cancilla, M. T.; Shiau, T. P.; Kung, J.; Chen, T.; Erlanson, D. A. Biochemistry 2005, 44, 7704-7712). Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus enabling inactivation of the enzyme via selective modification of the allosteric residue C121. Keywords: phosphatase, PTP, affinity labeling, enzyme inactivation, covalent enzyme inactivation

In

recent years, there has been increasing interest in covalent enzyme

inhibitors.1,2 For example, potent and selective enzyme inactivators have been built by

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linking competitive inhibitors to electrophilic functional groups that covalently modify nucleophilic amino acid residues located near the active site.1-7

This “exo-affinity

labeling” strategy, first described many years ago,8-10 has found clinical utility in drugs such as afatinib.1,2 Here we report the synthesis and characterization of an inhibitor-electrophile conjugate designed to serve as an exo-affinity labeling agent against protein tyrosine phosphatase 1B (PTP1B). PTP1B is a validated therapeutic target for the treatment of type 2 diabetes but, to date, extensive efforts have failed to develop medicinally-useful reversible inhibitors.11,12 As a result, it is of interest to explore alternative approaches for the modulation of PTP1B activity.13-18 We employed an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor 2 as a platform for the design of an exo-affinity-labeling agent.11,19 A previous crystallographic analysis of 2a bound to the active site of PTP1B showed that the biaryl moiety protrudes from the active site.19

We anticipated that an electrophilic α-

bromoketone group appended to the biphenyl unit of 2a would be positioned just outside the enzyme active site (Figure 1).20,21 PTP1B contains several amino acids located near the mouth of the active site that have the potential to react with a α-bromoketone group in an exo-affinity labeling scheme, including Asp 48 and Lys 120.20

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Biochemistry

Figure 1. Panel A: Compound 2a bound to the active site of PTP1B. The image was created from pdb 2bgd using Pymol. Panel B: Relationship between the electrophilic α-bromoacetamide group and C121 in the anticipated binding mode of 5a.

We found that the inhibitor-electrophile conjugate 5a (Scheme 1) caused irreversible, time- and concentration-dependent loss of PTP1B activity, consistent with a covalent inactivation mechanism. However, the results of enzyme inactivation kinetics, reversible binding affinity, and mass spectrometric analysis were not consistent with a simple exo-affinity labeling mechanism (Scheme 2, pathway A). Rather, the data pointed to an unusual enzyme inactivation process in which noncovalent binding of the inhibitorelectrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus enabling inactivation of the enzyme via selective modification of an allosteric residue C121 (Scheme 2, pathway B).

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Scheme 1. Synthesis of 5-biphenyl-1,2,5-thiadiazolidin-3-one-1,1-dioxide derivatives. Reagents: i. 4-substituted phenylboronic acid (1.2 equiv), Pd(PPh3)4 (0.05 equiv), 2:1 dimethoxyethane:H2O, 100 ˚C, 30 min (75-80%), ii. TFA, 24 ˚C, 30 min (66-70%), iii. bromoacetyl bromide (5 equiv) in acetone, 24 ˚C, 1 h (for 5a) or acetyl chloride (2 equiv) in acetone, 24 ˚C, 1 h (for 5b).

SH

SH

O Br

121

121

O Br

215

S– NuH

"Ki"

215

S– NuH

O

X

Br

SH 121

215

O

O

S

S– Nu

121

O Br

Pathway A

215

S– NuH

Pathway B

Scheme 2. Possible mechanisms for inactivation of PTP1B by 5a.

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Biochemistry

EXPERIMENTAL PROCEDURES Materials. Reagents were purchased from the following suppliers: 5-bromo-2methyl aniline, 5-bromo-2-methoxy aniline, methyl bromoactate, ethyldiisopropylamine, triethylamine, dimethyl formamide, chlorosulfonyl isocyanate, dichloromethane, triethyl amine, trifluoroacetic acid, sodium hydride, 1,2-dimethoxyethane, bis(trisphenyl phosphine)palladium

(II)

dichloride,

thiophenol,

(benzotriazol-1-yloxy)-tris(dimethylamino)phosphate

benzyl (BOP),

mercaptane, bromoacetyl

alanine, bromide,

acetyl chloride, tris(hydroxymethyl)aminomethane (Tris), 2-[bis-(2-hydroxyethyl)amino]-2-hydroxymethyl-propane-1,3-diol (Bis-tris), 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP), DL-dithiothreitol (DTT), diethylenediamine tetraacetic acid disodium

monohydrate

(EDTA·2Na·H2O),

2-[4-(2-hydroxyethyl)piperazin-1-

yl]ethanesulfonic acid (HEPES), diethylenetriaminepentaacetic acid (DTPA), sodium hydroxide, sodium acetate (99+%), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), polyethylene glycol (PEG) 3350, PEG 200, D-(+)-glucose, glycerol, leupeptin trifluoroacetate salt, pepstatin A, and thin layer chromatography plates (TLC) were obtained

from

Sigma-Aldrich

(St.

Louis,

MO),

potassium

carbonate

phenylmethanesulfonyl fluoride (PMSF) 99%, tryptone, yeast extract, LB agar, imidazole, magnesium chloride hexahydrate and sodium chloride were purchased from Fischer Chemical, ampicillin and isopropyl-beta-D-thiogalactopyranoside (IPTG) were purchased from Gold Biotechnology, DNase I was purchased from Roche, pphenylboronic acid and 4-(N-Boc-amino)phenylboronic acid were purchased from Alfa Aesar (Wardhill, MA), Zeba mini centrifugal buffer exchange columns were purchased from Thermoscientific, and Tween® 80 and Triton X-100 were purchased from Pierce

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Biotechnology, and Amicon Ultra centrifugal filter devices were purchased from Millipore. The enzyme consisting of amino acids 1-322 of human PTP1B was expressed and purified as described previously

22

and the concentration of active enzyme in stock

solutions was determined as described by Pregel et. al.23 Compounds 1a and 1b were prepared according to the general synthetic methods described by Black and Kenny for the preparation of 5-(aryl)-1,2,5-thiadiazolidin-3-one 1,1-dioxide derivatives.19,24 Synthesis of 5-(4-methoxy-[1,1'-biphenyl]-3-yl)-1,2,5-thiadiazolidin-3-one 1,1dioxide (2a). A mixture of bromide 1a (1.0 g, 3.11 mmol), K2CO3 (1.3 g, 9.34 mmol) and phenylboronic acid (455 mg, 3.73 mmol) in 15 mL of 1,2-dimethoxyethane and water (2:1 v/v), in a Pyrex tube was degassed by bubbling with nitrogen gas for 5 min at 24 ˚C. To the mixture was added Pd(PPh3)2Cl2 (109 mg, 0.15 mmol), followed by degassing for another 2 min. The reaction tube was sealed and the mixture was heated at 100 oC with stirring for 10 h. The Pd catalyst was removed by filtration, and the filtrate concentrated in vacuo. The resulting basic residue was dissolved in water (15 mL) and washed with ethyl acetate (3 × 20 mL) then acidified to pH 1 with 12 M HCl. The acidified aqueous layer was extracted with ethyl acetate (2 × 25 mL), the combined organic layers dried over anhydrous Na2SO4, filtered, and concentrated by rotary evaporation. Column chromatography on silica gel eluted with 5% acetic acid/ethyl acetate gave 2a as an off-white solid (780 mg, 79%). 1H NMR (methanol-d4, 600 MHz) δ 7.71 (d, J = 2.5 Hz, 1H), 7.66 (dd, J = 8.5, 2.5 Hz, 1H), 7.56-7.58 (m, 2H), 7.42 (t, J = 8.0 Hz, 2H), 7.31 (tt, J = 7.0, 1.5 Hz, 1H), 7.21 (d, J = 8.5 Hz, 1H), 4.54 (s, 2H), 3.93 (s, 3H);

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C NMR (methanol-d4, 150 MHz) δ 170.1, 157.5, 140.9, 135.6, 130.2, 129.9,

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Biochemistry

129.8, 128.3, 127.6, 125.3, 114.3, 56.6, 56.2; HRMS (ESI-TOF, [M + H]+) m/z calculated for C15H15N2O4S: 319.0753, found 319.0762. Synthesis of 5-(4-methyl-[1,1'-biphenyl]-3-yl)-1,2,5-thiadiazolidin-3-one 1,1dioxide (2b). A solution of 1b (120 mg, 0.39 mmol), K2CO3 (216 mg, 1.56 mmol) and phenylboronic acid (72 mg, 0.59 mmol) in 5 mL of 1,2-dimethoxyethane and water (2:1 v/v), in a Pyrex tube was degassed by bubbling with nitrogen gas for 3 minutes at room temperature. To the mixture was added Pd(PPh3)2Cl2 (14 mg, 0.02 mmol), followed by degassing for another 1 min. The tube was sealed and the reaction mixture was heated at 100 oC with stirring for 10 h. The Pd catalyst was removed by filtration and the filtrate concentrated in vacuo. The resulting basic residue was dissolved in water (10 mL), washed with ethyl acetate (3 × 10 mL), and acidified to pH 1 with 12 M HCl. The acidified aqueous solution was extracted with ethyl acetate (2 × 10 mL), the combined organic layers dried over Na2SO4, filtered to remove the drying agent, and concentrated in vacuo. Column chromatography on silica gel eluted with 2% acetic acid/ethyl acetate gave 2b as an off-white solid (73 mg, 62%). 1H NMR (acetone-d6, 500 MHz) δ 7.92 (d, J = 1.5 Hz, 1H), 7.66-7.70 (m, 3H), 7.45-7.48 (m, 3H), 7.37 (t, J = 7.0 Hz, 1H), 4.69 (s, 2H), 2.50 (s, 3H); 13C NMR (acetone-d6, 125 MHz) δ 166.5, 141.1, 140.5, 139.3, 135.2, 132.7, 129.8, 128.6, 128.5, 128.4, 127.6, 57.6, 17.9; HRMS (ESI-TOF, [M + H]+) m/z calculated for C15H15N2O3S: 303.0803, found 303.0796. Synthesis

of

tert-butyl-(3'-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-4'-

methyl-[1,1'-biphenyl]-4-yl)carbamate (3). To a solution of 1b (265 mg, 0.868 mmol) and (4-((tert-butoxycarbonyl)amino)phenyl)boronic acid (247 mg, 1.04 mmol) in 30 mL of 1,2-dimethoxyethane and water (2:1 v/v), in an oven-dried Pyrex tube equipped with a

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stir bar, was added K2CO3 (480 mg, 3.47 mmol). The reaction mixture was degassed by bubbling with nitrogen gas for 5 min before adding Pd(PPh3)2Cl2 (30 mg, 0.04 mmol), followed by degassing for another 1 min. The tube was then sealed and heated at 100 oC with stirring for 16 h.

The Pd catalyst was removed by filtration and the filtrate

concentrated in vacuo to remove 1,2-dimethoxyethane. The solid product obtained in this manner was collected by filtration and washed with ethyl acetate to give 3 as a pale yellow solid (320 mg, 88% yield). 1H NMR (acetone-d6, 300 MHz): δ 7.75 (d, J = 2.0 Hz, 1H,), 7.56 (dt, J = 9.0, 2.0 Hz, 2H), 7.51 (d, J = 9.0 Hz, 2H), 7.48 (dd, J = 9.0, 2.0 Hz, 1H), 7.33 (d, J = 9.0 Hz, 1H), 4.26 (s, 2H), 2.44 (s, 3H), 1.52 (s, 9H). 13C NMR (acetoned6, 75 MHz): δ 176.3, 154.7, 140.6, 140.1, 138.5, 138.3, 135.4, 132.3, 128.0, 127.7, 126.9, 119.7, 80.6, 60.4, 28.7, 18.2; HRMS (ESI-TOF, [M + H]+) m/z calculated for C20H24N3O5S: 418.1437 found 418.1433. Synthesis of 5-(4'-amino-4-methyl-[1,1'-biphenyl]-3-yl)-1,2,5-thiadiazolidin-3one 1,1-dioxide (4). To a flask containing 3 (320 mg, 0.77 mmol) and a stir bar, was added 5 mL of trifluoroacetic acid followed by stirring at room temperature for 20 min. Trifluroacetic acid was evaporated in vacuo, and column chromatography on silica gel eluted with 5% acetic acid/ethyl acetate gave 4 as a pale yellow solid (216 g, 90% yield). 1

H NMR (methanol-d4, 500 MHz): δ 7.61 (d, J = 1.5 Hz, 1H), 7.43 (dd, J = 8.0, 1.5 Hz,

1H), 7.39 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.0 Hz, 1H,), 6.82 (d, J = 8.5 Hz, 2H), 4.27 (s, 2H), 3.35 (s, 2H), 1.89 (s, 3H). 13C NMR (methanol-d4, 125 MHz): 177.1, 147.9, 141.4, 137.7, 137.2, 132.4, 131.4, 128.4, 127.2, 126.9, 117.1, 60.2, 17.9; HRMS (ESI-TOF, [M + H]+) m/z calculated for C15H16N3O3S: 318.0912 found 318.0906.

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Biochemistry

Synthesis of 2-bromo-N-(3'-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-4'methyl-[1,1'-biphenyl]-4-yl)acetamide (5a). To a stirred solution of 7 (220 mg, 0.70 mmol) in acetone was added bromoacetyl bromide (304 µL, 3.48 mmol) dropwise. The resulting mixture was stirred for 30 min at room temperature.

The solvent was

completely removed by rotary evaporation and column chromatography on silica gel eluted with 5% acetic acid-ethyl acetate gave 5a as a pale yellow solid (93 mg, 31% yield). 1H NMR (methanol-d4, 500 MHz) δ 7.89 (d, J = 2.0 Hz, 1H), 7.77 (d, J = 9.0 Hz, 2H), 7.67 (d, J = 9.0 Hz, 2H), 7.65 (dd, J = 8.0, 2.0 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 4.66 (s, 2H), 4.06 (s, 2H).

13

C NMR (methanol-d4, 125 MHz): 175.16, 165.67, 138.97,

137.62, 136.89, 136.00, 131.12, 126.80, 126.57, 125.77, 119.83, 59.00, 16.82; HRMS (ESI-TOF, [M + H]+) m/z calculated for C17H17BrN3O4S: 438.0123 found 438.0118. Synthesis

of

N-(3'-(1,1-dioxido-4-oxo-1,2,5-thiadiazolidin-2-yl)-4'-methyl-

[1,1'-biphenyl]-4-yl)acetamide (5b). To a stirred solution of 4 (60 mg, 0.19 mmol) in dry acetone was added acetyl chloride (30 µL, 0.38 mmol) dropwise. The reaction was stirred at room temperature for 30 min. The solvent was evaporated in vacuo, then water was added and extracted with ethyl acetate (4 × 10 mL). The organic layer was dried over anhydrous Na2SO4, filtered to remove the drying agent, and concentrated in vacuo. Column chromatography on silica gel eluted with 2% acetic acid-ethyl acetate) gave 5b as a brown solid (33 mg, 48% yield). 1H NMR (acetone-d6, 300 MHz): δ 9.26 (s, 1H), 7.89 (d, J = 2.0 Hz, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.66-7.61 (m, 3H), 7.43 (d, J = 8.0 Hz, 1H), 4.66 (s, 2H), 2.48 (s, 3H), 2.10 (s, 3H).

13

C NMR (acetone-d6, 75 MHz): 171.2,

169.1, 141.0, 139.7, 139.2, 136.2, 135.5, 132.8, 128.4, 128.3, 128.1, 121.2, 100.8, 58.0,

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Biochemistry

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23.9, 17.9; HRMS (ESI-TOF, [M + H]+) m/z calculated for C17H18N3O4S: 360.1018 found 360.1017. Inactivation of PTP1B by 5a. Time-dependent enzyme inactivation assays were carried out as described previously.25 Briefly, inactivation assays contained various concentrations of the inactivator along with PTP1B (450 nM) in a buffer composed of Bis-Tris (50 mM) and EDTA (2 mM) containing dithiothreitol (15 µM), Tween 80 (0.05%, v/v), and DMSO (2% v/v) at pH 7.0. The inactivator was introduced into the inactivation mixtures from stock solutions dissolved in DMSO. The mixtures were incubated at 25 °C in a thermostatted hot-block and at selected times aliquots (10 µL) were removed and assayed for remaining PTP1B activity by mixing with a solution (490 µL) containing Bis-tris (50 mM), NaCl (100 mM), DTPA (10 mM), and p-nitrophenyl phosphate (p-NPP, 20 mM) at pH 6.0), followed by incubation at 30 °C for 10 min. The activity assay was quenched by addition of NaOH (500 µL of a 2 M solution in DI water) and the amount of p-nitrophenolate released during 10 min incubation of the activity assay was determined by measurement of the absorbance at 410 nm at room temperature (~24 °C). The inactivation time courses were fit to the equation for a first-order kinetic process to obtain the observed pseudo-first-order inactivation rate constant for each concentration of inactivator. Measurement of IC50 values. Assays for the reversible inhibition of PTP1B (72 nM) contained the test compound (2a, 2b, 5a, or 5b) in Bis-tris (50 mM), NaCl (100 mM), EDTA (2 mM), and p-nitrophenyl phosphate (p-NPP, 0.4 mM), DTT (5 mM), Tween® 80 (0.05% v/v), and DMSO (2% v/v) at pH 7.0. The test compounds were introduced into the solutions as stock solutions dissolved in DMSO. After 20-30 s, the

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Biochemistry

amount of PTP1B activity in the assays was assessed by measuring the increase in absorbance at 410 nm as a function of time resulting from the enzyme-mediated conversion of p-NPP to p-nitrophenolate. The fraction of remaining PTP1B activity relative to a control assay containing no test compound, and only the DMSO vehicle, was plotted versus the log of compound concentration. The data was fitted to the equation for dose-response using GraphPad Prism:

y = min +

(max – min) 1 + 10

[(logIC50 – log[I])(nH)]

(Eqn 1)

Where max is the maximum activity value, min is the minimum activity value and nH is the Hill coefficient. Mass Spectrometric Analysis of PTP1B inactivated by 5a. Thiol-free PTP1B (230 µL of a 7 mg/mL solution) in 5 mL of buffer (50 mM Bis-Tris, 2 mM EDTA, 15µM DTT, 0.01% (v/v) Tween® 80, 5% (v/v) DMSO, pH 7) containing compound 5a (100 µM) was incubated at 25°C until only ~20% of the enzyme activity remained. A control sample without 5a was prepared and followed simultaneously. Following inactivation of PTP1B, the samples were concentrated using Amicon Ultra-4 centrifugal filters (10K, Millipore) for 30 min at 4800 rcf and 11°C. Ion Exchange Buffer A (4 mL, 0.03 M NaCl, 100 mM HEPES, 1 mM EDTA, 0.1% β-ME, 10% glycerol, pH 7.2) was added to Amicon centrifugal filters and the samples were concentrated again at 4800 rcf and 11 °C. Meanwhile, mini anion exchange columns spin columns (Pierce, catalog #90010) were prepared with ion-exchange buffer A according to the manufacturer’s protocol. The concentrated protein was then loaded onto the mini anion exchange column and centrifuged at 2000 x g for 5 min. The sample was washed with an additional 100 µL of

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buffer A and centrifuged again. The detergent-free protein was then eluted with ionexchange buffer B (100 µL, 0.3 M NaCl, 100 mM HEPES, 1 mM EDTA, 0.1% β-ME, 10% glycerol, at pH 7.2). The sample was then buffer-exchanged using mini-centrifugal columns (Pierce) with 100 µL of a buffer composed of 50 mM Bis-Tris, 2 mM EDTA, and 600 µM DTT pH 7. The samples were stored at –20 ˚C prior to mass spectrometric analysis. For LC-Nanospray QTOF MS-MS/MS analysis of intact PTP1B modified by 5a, protein samples were centrifuged at 20000 x g rcf at room temperature. An aliquot of each

sample

was

diluted

1/25

with

sample

diluent

(30/970/1,

V/V/V,

acetonitrile/water/99% formic acid), briefly centrifuged, and transferred to an autosampler vial. Fractionations of the sample were carried out on Agilent G4240-63001 SPQ105 Protein Chip II (Zorbax 300 SB-C8, 5 µm particle, 300 Å pore; separation column, 43 mm x 75 µm; 40 nL trap column). Nano-LC-Nanospray MS performed on Agilent 6520A QTOF mass spectrometer with a Chip Cube source (G4240A). A 0.1 µL portion of each sample was injected into carrier solvent (30/970/1, V/V/V, acetonitrile/water/99% formic acid) flowing at 3 µL/min for transfer and enrichment on the trap column in the Chip. At 8 min post-injection, the flow from trap column was directed to the analytical column and sample components were separated at 0.3 µL/min flow rate using a linear gradient of increase % solvent B (900/100/1, V/V/V acetonitrile/water/99% formic acid) for the following steps: 8 min-3% B, 10 min-35% B, 37.5 min-70% B, 40 min-90% B, 40 min-90% B, 47 min-3% B, 48 min-3% B (enrichment), 50 min-3% B (enrichment), 60 min-3% B (enrichment). Positive ion electrospray spectra were acquired at capillary potential of 2325 V. Heated nitrogen gas

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Biochemistry

(365 °C), introduced at a flow rate of 4 L/min, assisted desolvation of sample ions. The Fragmentor, Skimmer, and Octapole1 RF Vpp potentials were 175 V, 65 V, and 750 V, respectively. Data analysis was performed with Agilent Mass Hunter software. For digested protein samples, each protein sample (20 µL) was denatured in 2 N urea (10 µL, 8 N) and 1 N ammonium bicarbonate buffer (10 µL, pH 8). Iodoacetamide (IAA) (1 µL, 62.5 mM) was added to alkylate free cysteine residues. After alkylation, excess IAA was neutralized with 20 mM DTT (in 0.25 N ammonium bicarbonate buffer, pH 8) and the samples were allowed to sit at room temperature for at least 5 min. Meanwhile, trypsin (Promega V511A modified TPCK-treated porcine trypsin) was freshly prepared (according to V511 prep) at a concentration of 1 µg/µL in 50 mM acetic acid. To each sample of PTP1B (40 µg) was added wt/wt 1/25 trypsin/protein, 1.6 µL of Trypsin stock solution (1.6 µg). The samples were allowed to digest for 15 h in an incubator at 37°C. Following digestion, the samples were acidified by addition of formic acid (1 µL, 88% formic acid) to inhibit any remaining tryptic activity. 10 µL aliquots were taken from each digested sample and frozen prior to LC-MS analysis. For nano-LC-nanospray QTOF MS-MS/MS analysis of trypsin digested protein samples, each trypsin digested sample, a 10 µL aliquot of frozen digest was thawed on ice

before

analysis

and

mixed

with

sample

diluent

(30/970/1,

V/V/V,

acetonitrile/water/99% formic acid (5a treated: 121 µL sample diluent; control: 110 µL sample diluent) to give 1.9 pmol/peptide/µL. Nano-LC-Nanospray MS was performed on an Agilent 6520A with a Chip Cube source (G4240A). Fractionations of the samples were carried out on an Agilent G4240-62006 peptide separations chip (Zorbax 300SBC18, 5 µm particle, 300 Å pore; separation column, 150 mm x 75 µm; 40 mL trap

15


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column) and the sample analyzed using instrument software version B.04.00. Samples were injected (0.1 µL for 5a treated; 0.2 µL for control) on LC-MS into carrier solvent (30/870/1, V/V/V, acetonitrile/water/99% formic acid) flowing at 4 µL/min for transfer and enrichment on the trap column of the Chip. At 5.9 min post-injection, the flow from trap column was directed to the analytical column and sample components were separated at 0.4 µL/min flow rate using a linear gradient of increase % solvent B (900/100/1, V/V/V/ acetonitrile/water/99% formic acid) for the following steps: 5.9 min3% B, 6.1 min-10% B, 27.4 min-70% B, 28.4 min-100% B, 33.4 min-100% B, 37.4 min3% B (enrichment), 52.4 min-3% B (enrichment). Positive ion electrospray spectra were acquired at capillary potential of 2100 V. Heated nitrogen gas (365 °C), introduced at a flow rate of 4 L/min, assisted desolvation of sample ions. The Fragmentor, Skimmer, and Octapole1 RF Vpp potentials were 175 V, 65 V, and 750 V, respectively. Nano-LCNanospray MS only spectra acquired over a mass range of 300-3200 Da. Nano-LCNanospray MS plus MS/MS: MS spectra were acquired over a mass range of 300-3000 Da at a rate of 0.5 seconds per spectrum and MS/MS spectra acquired over the mass range of 60-3000 Da at the same rate. MS and MS/MS data processed with the “Find by Molecular Feature” program in Agilent Mass Hunter Qualitative Analysis Suite. MS/MS data was exported and database searched on the Mascot search engine (Matrix Science) using compound lists. Expression and Purification of PTP1B (1-321 domain). The enzyme was prepared by a modification of a protocol described previously.22,26 The PTP1B (1-321) plasmid was transformed into E. coli BL21(DE3) cells and plated on LB agar containing ampicillin (50 µg/mL). The plate was incubated at 37 ºC overnight and a single colony of

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Biochemistry

the transformant was picked to inoculate 10 mL starter culture made of 1% tryptone, 0.5% yeast extract, and 2% glucose. This was incubated overnight at 37 ºC with constant shaking at 250 rpm. The starter culture was used to inoculate 1 L of media, separated into two 2L flasks containing 500 mL of LB with 50 ug/mL of ampicillin and 0.2% glucose. The cells were allowed to shake constantly for at 37 ºC and 250 rpm. After two hours of cell growth (OD at 600 nm of ~ 0.4), the cells were induced with 0.4 mM IPTG. Cells were harvested after an additional 5 hours (OD ~ 1) by centrifugation at 4 ºC and 3500 rpm and resuspended in Buffer A (20 mM Tris, 150 mM NaCl, 10% glycerol pH 7.5) with DNase. The cell pellet was quick-frozen into liquid nitrogen for later use. Frozen cells were thawed at 4 ºC in the presence of the following protease inhibitors: 10 µM leupeptin, 1 µM pepstatin A, and 1 mM PMSF. Cells were stirred for 15- 20 minutes at 4 ºC followed by disruption using French press. Unbroken cells and debris were removed by centrifugation using an SS34 rotor for 60 min at 17,000 rpm. The supernatant was collected and subjected to a second centrifugation step for 30 min at 17,000 rpm using an SS34 rotor. The resulting supernatant was used for further purification by immobilized metal-ion affinity chromatography (Ni2+-charged HiTRAP; GE Healthcare). After loading the protein, the resin was washed with 4 column volumes of Buffer A supplemented with 30 mM imidazole. The fractions were eluted by a linear gradient of 30-1000 mM imidazole. Fractions containing PTP1B were pooled and mixed with Tobacco Etch Virus protease (1 mg of protease per 40 mg of PTP1B) and 1 mM tris(2-carboxyethyl)phosphine (TCEP). The sample was incubated for 8 h at 20 ºC and then dialyzed against buffer A. The dialyzed protein was again loaded onto the Ni2+ charged column using buffer A. Tag-free PTP1B was collected in both the flow-through

17


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and by elution at 30 mM imidazole. The purified protein was dialyzed into 10 mM Tris, 25 mM NaCl, 1 mM EDTA, 1 mM TCEP pH 7.5. Finally, the protein was concentrated and distributed into thin-walled PCR tubes, quick-frozen in liquid nitrogen, and stored at –80 ºC. Preparation of the C32S mutant of PTP1B. The mutant C32S PTP1B was created by inserting codons for either serine into PTP1B 1-298 plasmid stored in DH5α using a QuickChange Kit (Stratagene). The clone for the mutant enzyme was confirmed by DNA sequencing. Expression and purification of the mutant enzyme was carried out with some slight modifications by following the protocol for expression and purification of PTP1B.26 The PTP1B mutant plasmids were transformed in E. coli BL21AI cells and plated on LB Agar containing ampicillin (50 µg/mL). The plates were incubated at 37 °C overnight and one colony was picked and placed in a 10 mL starter culture made of 1% tryptone, 0.5% yeast extract, and 2% glucose. The tube was incubated at 37 °C with constant shaking at 250 rpm overnight. The starter culture was separated into 4 flasks containing 500 mL of LB with ampicillin (50 µg/mL) and glucose (0.2%). The flasks were incubated at 37°C and shaken constantly at 250 rpm until the OD reached ~0.4. Once the OD reached 0.4, IPTG (0.4 mM) was added to start expression of the mutant PTP1Bs. The media was placed back in the shaker for another 3-4 h at which point the OD had reached ~1. The cells were then harvested centrifugation at 4°C and 1000 rcf before resuspending them in Buffer A (50 mM HEPES, 500 mM NaCl, 0.5 mM DTT, pH 7.5). The cells were cracked by use of a French press and unbroken cells were removed by centrifugation for 60 min at 5000 rcf. The supernatant was collected and centrifuged again for 30 min at 5000 rcf. The supernatant was then purified by immobilized metal-ion

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Biochemistry

affinity chromatography (Ni2+-charged HiTRAP; GE Healthcare). The fractions were eluted using Buffer B at 3% (Buffer A with 1 M imidazole). The fractions were pooled and dialyzed into Buffer Q (50 mM Tris, 25 mM NaCl, and 2 mM DTT at pH 8.5). The protein was then purified again on a Q-sepharose column with a NaCl gradient (0-1 M) in Buffer Q and the fractions were collected, and pooled. The proteins was concentrated using Amicon centrifugal filters (30K, Millipore) at 5000 rcf at 4°C for 20 min. The protein was diluted with Buffer Q to the desired concentration and then 100 µL samples were aliquoted into 500 µL eppendorf tubes, quick-frozen in liquid nitrogen, and stored at –80 °C. Crystal structure determination. Crystallization trials were performed at 4º C using sitting drop vapor diffusion and a 10 mg/mL stock solution of PTP1B (1-321). A reservoir condition, similar to some previously published,27,28 was found to grow PTP1B crystals (0.1 M MgCl2, 0.1 M Bis-Tris pH 6.0, and 27% (w/v) PEG 3350). Rod-shaped crystals appeared, and were used as a micro-seed stock for further optimization trials. Diffraction quality crystals were grown using 0.1 M MgCl2, 0.1 M Bis-Tris pH 6.3-6.5, and 23-27% (w/v) PEG 3350, with micro-seeding by streaking the drop with horse hair. Hexagonal rod crystals were grown at 4º C for a week, and then moved to 20º C to allow the crystals to grow bigger. Crystals of PTP1B complexed with 5b were prepared by soaking PTP1B crystals at room temperature. The crystals were first cryoprotected using 25% (w/v) PEG 3350, 0.1 M Bis-Tris pH 6.4, 0.1 M MgCl2, and 15% (w/v) PEG 200. Then the crystals were soaked with a solution containing the cryobuffer supplemented with 2 mM of 5b. The soak time was varied from 30 to 45 min. Crystals were picked up with Hampton loops

19


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and plunged into liquid nitrogen. A 2.1 Å resolution X-ray diffraction data set was collected at ALS beamline 4.2.2 using a Taurus-1 CMOS detector in shutterless mode. The data set consisted of 900 images covering a rotation range of 180° with an exposure time of 360 seconds. The detector distance was 300 mm.

The data sets were processed with XDS29 and

AIMLESS30 via CCP4i.31 The crystal has space group P3121 with unit cell dimensions of a = b = 88.6 Å and c = 103.8 Å; there is 1 molecule in the asymmetric unit. Refinement was initiated using a model of PTP1B structure (PDB code 2CM8) with Cys215 modified to an alanine. Molecular replacement phasing as implemented in CCP4i MolRep32 was used. PHENIX33 was used for refinement and COOT34 was used for model building. The structures were validated using the Protein Data Bank (PDB) validation server and MolProbity.35 The coordinates and structure factor amplitudes have been deposited in the PDB under accession code 5T19. Data collection and refinement statistics are listed in Table S1.

RESULTS AND DISCUSSION Synthesis of the inhibitor-electrophile conjugate 5a and control inhibitor 5b. Preparation of the desired inhibitor-electrophile conjugate 5a started with 5-bromo-2methylaniline and followed the general route described by Black et al. for the preparation of 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide derivatives (Scheme 1).19,24

Suzuki-

Miyaura reactions on the bromoaryl compounds 1 were used to forge the biaryl linkages in 2 and 3. Treatment of 3 with trifluoroacetic acid gave the arylamine 4 that was

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Biochemistry

coupled with α-bromoacetyl bromide to generate 5a. The acetyl derivative 5b was prepared by an analogous reaction with acetyl chloride. Crystallographic analysis of 5b bound to the active site of PTP1B. Previous studies elucidated the structure of the complex between 2a and PTP1B19, but the analogous structural data for the methyl analog 2b was not available. Because our work employed 2b as a platform for the design of an exo-affinity labeling agent, we felt it was important to determine whether this analog bound to PTP1B in a manner similar to 2a. Thus, we sought a crystal structure of PTP1B complexed with 5b Electron density representing 5b was evident in the active site near Cys215 (Figure 2A). There is no significant difference between the binding modes of 5b and 2a19, as seen in the superposition of the two structures (Figure 2B). The substitution of a methyl group at the ortho-position of the first phenyl group still gave the desired orthogonal orientation between the heterocycle and phenyl group.19,36 The addition of the acetamide group to the para-position of the biphenyl did not alter the position of the inhibitor in the active site. Electron density for the carbonyl oxygen and the methyl group in the acetyl group was weak, indicative of rotation disorder about the C–N bond. With a closed conformation of WPD loop, 5b enjoys many of the same interactions with the enzyme as 2a19,36. The sulfone oxygens hydrogen bond to the guanidinium side chain of Arg221 and the backbone nitrogens of P-loop residues 216220. The carbonyl oxygen of the 1,2,5-thiadiazolidin-3-one 1,1-dioxide ring hydrogen bonds to the backbone nitrogen of Phe182 and the side chain nitrogen of Gln266. The methylene residue of the heterocycle and the aromatic rings of the biphenyl group

21


Biochemistry

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interact with the aromatic and nonpolar residues Tyr46, Val49, Ala217, Ile219, and Phe182.

Figure 2. Structure of PTP1B complexed with 5b. (A) Electron density and environment of 5b. The mesh represents a simulated annealing Fo-Fc omit map (3σ). The inset in the upper left shows a different orientation of the ligand (3σ). Due to lack of electron density, the acetyl group is not included in the coordinates deposited in the PDB. It is included here for completeness. (B) Superposition of PTP1B complexed with 5b (gray) and 2a (cyan, PDB code 2BGD). Covalent inactivation of PTP1B by 5a. Our experiments employed the catalytic domain of recombinant human PTP1B (residues 1-322). We found that incubation of 5a with PTP1B (450 nM) in a buffer composed of Bis-Tris (50 mM) and EDTA (2 mM) containing dithiothreitol (15 µM) and Tween 80 (0.05%, v/v) at pH 7.0 and 25 °C caused time- and concentration-dependent inactivation of the enzyme (Figure 3A). The plot of the observed pseudo-first-order rate constants (kobs) versus the concentration of 5a

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Biochemistry

revealed a linear relationship consistent with a second-order inactivation process. The slope of the line provided a rate constant for the inactivation of PTP1B by 5a of 1.7 ± 0.3 x 102 M-1 min-1 (Figure 3B). Filtration of the inactivated enzyme through Sephadex G-50 to remove the excess inactivator did not result in the return of enzyme activity, again consistent with covalent modification of the enzyme (Figure S1). We also carried out experiments providing evidence that the loss of enzyme activity was not a result of promiscuous aggregate-based inhibition (Figure S2).37 For example, the rate at which 5a inactivated PTP1B was not significantly altered when carried out in the presence of a different detergent. The observed pseudo-first-order rates of inactivation by 5a (100 µM) were 2.8 ± 0.3 x 10-2 min-1 under our standard conditions and 2.4 x 10-2 min-1 when 0.001% (v/v) Triton X100 was substituted for Tween-80 (0.05% v/v) (Figure S2). Similarly, 10-fold higher concentrations of the enzyme did not significantly alter the rate at which 5a inactivated the enzyme. The observed pseudofirst-order rate of inactivation by 5a (100 µM) was 2.8 ± 0.3 x 10-2 min-1 under our standard inactivation conditions (450 nM PTP1B) and 2.6 ± 0.7 x 10-2 min-1 at an enzyme concentration of 4.5 µM (Figure S2). Together, the results provided evidence that 5a inactivated PTP1B via covalent modification of the enzyme.38 Neither the acetyl control compound 5b (Figure 3A), nor the parent inhibitor 2a caused time-dependent loss of enzyme activity. This was consistent with a mechanism involving alkylation of the enzyme by the bromoacetyl unit in 5a.

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Biochemistry

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Figure 3. Inactivation of PTP1B by 5a. Panel A: Time courses for the inactivation of PTP1B by various concentrations of 5a. Inactivation assays were carried out as described previously.25 Inactivation time courses were measured for 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 µM concentrations of 5a. For clarity, only time courses for 50, 100, 200, 300, 500 µM concentrations of 5a are shown (from top to bottom). The upper data set is the time course for incubation of the enzyme with the control compound 5b and is similar to control assays containing enzyme alone. Pseudo-first-order rate constants (kobs) were obtained from the time courses by non-linear curve fitting. Panel B: A plot of kobs versus concentration of 5a. The slope of the resulting line corresponds a second-order inactivation rate constant (kinact) of 1.7 ± 0.3 x 102 M-1 min-1 (R2 = 0.98).

Figure 4. IC50 values for the reversible inhibition of PTP1B by compounds used in this study. IC50 values: 2a, 4.8 ± 0.2 µM, Hill coefficient = 0.99 (r2 = 0.9999); 5a, 54 ± 1 µM, Hill coefficient = 1.02, (r2 = 0.9999); 2b, 76.7 ± 9.3 µM, Hill coefficient = 1.02 (r2 = 0.9999). PTP1B was added to assay solutions containing various concentrations of test compounds, p-nitrophenyl phosphate (pNPP, 0.4 mM), DMSO (4%, v/v), Bis–Tris (50 mM), EDTA (2 mM), DTT (5 mM), Tween 80 (0.05%). After 2 min, the enzyme activity was monitored by measuring increase in absorbance at 410 nm as a function of time resulting from the conversion of p-NPP to p-nitrophenol.

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Biochemistry

a

Table 1. IC50 values for compounds studied in this work. Assays were conducted with the 19 substrate p-NPP at its Km value (0.4 mM) and Ki values were calculated using the Cheng39 Prusoff equation.

Reversible binding affinity of 5a, 5b, and 2b for PTP1B. In an exo-affinitylabeling process, it is anticipated that a plot of the observed rate of enzyme inactivation versus concentration will display saturation kinetics resulting from equilibrium association of the agent with the enzyme active site prior to covalent reaction (Scheme 2, Pathway A).38 In principle, the half-maximal rate of enzyme inactivation should mirror the Ki of the inhibitor core that is used (assuming that the rate of covalent enzyme modification is slow relative to kon and koff for noncovalent association).38 The Ki for the parent inhibitor core (2b) used in our design was expected to be in the lower micromolar range.19 Importantly, we saw no evidence for saturation kinetics in the inactivation of PTP1B by 5a (Figure 3). This made it important to characterize the reversible, noncovalent binding affinity of 5a for PTP1B. We first characterized the parent inhibitor core 2b, finding an IC50 of 77 ± 9 µM against PTP1B.

The inhibitor-electrophile

conjugate 5a showed a similar IC50 value of 54 ± 1 µM (Figure 4). In the case of 5a, enzyme activity was measured immediately after mixing the compounds with PTP1B to minimize time-dependent, covalent inactivation of the enzyme during the experiment (Figure 4). The acetyl derivative 5b showed an IC50 value of 64 ± 7, similar to 2b and 5a (Table 1, Figure S3). For comparison, we measured the IC50 of 2a and obtained a value

25


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of 4.8 ± 0.2 µM, closely matching that reported by Black et al. for this compound.19 Clearly, the α-bromoacetamide-containing inhibitor-electrophile conjugate 5a retained noncovalent binding affinity for PTP1B, in the low micromolar range. Yet, curiously, enzyme inactivation by this compound did not display saturation kinetics across this concentration range. Mass spectrometric analysis of PTP1B inactivated by 5a. The absence of saturation kinetics (Figure 3) suggested that 5a did not inactivate PTP1B via the anticipated exo-affinity labeling mechanism (Scheme 2, Pathway A). Fortunately, mass spectrometric analysis of the inactivated protein provided insight regarding the mechanism by which 5a inactivated PTP1B.

For mass spectrometric analysis, the

enzyme was inactivated by 5a (10 equiv, 1 h) in our standard buffer composed of BisTris (50 mM) and EDTA (2 mM) containing dithiothreitol (15 µM) and Tween 80 (0.05%, v/v) at pH 7.0 and 25 °C and the modified enzyme purified by passage through ion exchange resin.

Nanospray QTOF-mass spectrometric analysis of unmodified

PTP1B gave the expected mass of 34997.9 Da (Figure 5A), while the protein treated with 5a revealed a major new signal at 35355.3 Da corresponding to modification with 1 equiv of 5a (Figure 5B).

A second, less intense, signal was observed corresponding to

modification of the enzyme with two equivalents of 5a (Figure 5B). LC-ESI-MS was used to analyze the peptide fragments generated by tryptic digestion of native PTP1B and PTP1B treated with 5a. The results provided evidence that 5a modified the fragment composed of amino acids 121-131 (Table 2). Further MS/MS analysis indicated that 5a was attached to cysteine 121 within this fragment (C121, Figure 6). A weaker signal was observed corresponding to modification of the tryptic fragment containing cysteine 32

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Biochemistry

(C32, Table 2). The peptide fragment containing the active site residue C215 was not extensively modified by 5a.

Figure 5. Deconvoluted nanospray QTOF-MS analysis of PTP1B and PTP1B modified with 5a. Panel A: native PTP1B with a mass of 34997.92 Da for the intact protein and two PTP1B fragments corresponding to residues 1-184 and 185-298 at 22307.90 Da and 12690.96 Da. Panel B: PTP1B treated with 5a displays prominent signals with mass increases of 357.4 amu and 714.8 amu, consistent with single and double modification by 5a.

Compound 5a inactivates C32S mutant PTP1B with a rate constant similar to the native enzyme.

The covalent modification of C121 revealed by the mass

spectrometric analysis can explain the inactivation of PTP1B by 5a. Indeed, Hansen et al.,

showed

that

modification

of

C121

by

4-(aminosulfonyl)-7-fluoro-2,1,3-

benzoxadiazole 6 caused an approximately 85% decrease in enzyme activity.40 However, our mass spectrometric data revealed C32 as a secondary site modified by 5a. This posed the question of whether modification of C32 by 5a might contribute to the inactivation of PTP1B. To consider this issue, we generated the C32S mutant of PTP1B. First, we found that the mutant enzyme retained catalytic activity toward the substrate p27


Biochemistry

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nitrophenyl phosphate that is comparable to that of the native enzyme. Second, we found that inactivation of the C32S mutant by 5a proceeded at a rate very similar to inactivation of the native enzyme by this compound.

The observed pseudo-first-order rate of

inactivation of the wild-type enzyme by 5a (200 µM) was 5.0 ± 0.7 x 10-2 min-1, while that for inactivation of the C32S mutant under these conditions was 4.8 ± 1.0 x 10-2 min-1. This provided evidence that modification of C32 was not necessary for the inactivation of native PTP1B by 5a.

Figure 6. LC-MS/MS analysis of the 5a-modified tryptic fragment 121CAQYWPQKEEK131. The generated b- and y-type fragment ions confirmed the sequence of the peptide and the location of 5a on Cys121. Fragments modified by 5a are denoted by an asterisk.

Table 2. Modification of various cysteine-containing tryptic fragments in PTP1B by 5a.

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Biochemistry

CONCLUSIONS We find that the inhibitor-electrophile conjugate 5a causes covalent inactivation of PTP1B; however, the evidence suggests that this does not occur via a simple exoaffinity-labeling mechanism.

Instead, the α-bromoacetamide unit of 5a covalently

modifies Cys121 of the enzyme, a residue that is not in close proximity to the active site. In fact, crystallographic analyses show that Cys121 lies approximately 20 Å away from the active site – a distance that cannot be spanned by 5a when bound at the active site of the enzyme (Figures 1 and 2). Importantly, there is no indication that enzyme dynamics can bring Cys121 near the enzyme active site41,42 The results can be explained by a twostep mechanism (Scheme 2, pathway B) in which 5a first associates non-covalently with the active site of PTP1B. Rather than leading to covalent inactivation of the enzyme, reversible binding of 5a to the active site protects the catalytic residue Cys215 from covalent modification that might typically be induced by electrophilic agents such as the α-bromoketone group.21,22,43,44 With the active site nucleophile C215 protected from alkylation, C121 evidently is the next most reactive site for covalent modification by the α-bromoacetamide unit of 5a. A secondary phosphotyrosine binding site near C121 has been described previously;36 and weak noncovalent binding of the 5-(aryl)-1,2,5thiadiazolidin-3-one 1,1-dioxide phosphotyrosine isostere in 5a to this site may drive covalent modification of C121. Previous work provides evidence that covalent modification of the “allosteric” C121 residue can inactivate PTP1B.40 The term “allosteric residue” has been used in this context to describe an amino acid distant from the active site, whose covalent modification results in loss of protein function.40 C121 is far removed from the active

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site of PTP1B, yet Hansen et al. showed that modification of this residue by 4(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole

6

caused

an

approximately

85%

inactivation of the enzyme.40 These authors suggested that covalent modification of C121 disrupts the normal network of interactions between C121, Y124, and the critical H214 residue adjacent to the active site nucleophile C215.40 It remains unclear why, in the work of Hansen et al, the catalytic residue C215 was apparently not modified by 6. Perhaps the stereoelectronic requirements for an SNAr reaction between C215 and 6 cannot be accommodated within the deep and narrow active site of PTP1B. Recent work from Bishop’s group described a similar allosteric inactivation of PTP-SHP2 that involves modification of the residue homologous to C121 in PTP1B.45 It is interesting to note that, in crystal structures of both PTP1B and SHP2, these allosteric cysteine thiol groups appear to be buried just below the surface of the protein. Clearly, normal solution dynamics provide access to conformations in which these cysteine thiol groups are accessible to electrophilic reagents. Enzyme inhibition resulting from covalent modification of cysteine residues distant from the active site, in some cases, can provide an interesting alternative to traditional competitive inhibitors.13,45-54 Our results provide an unusual example in which noncovalent binding of an inhibitor-electrophile conjugate to the active site of PTP1B protects the highly reactive catalytic residue C215 from covalent modification, thus enabling inactivation of the enzyme via alkylation of the distal allosteric residue C121 (Scheme 2, pathway B).

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Biochemistry

NH2 O S O

O

N O

Br

N F

6

7

ASSOCIATED CONTENT Supporting Information Crystallographic data collection and refinement statistics, evidence that inactivation by 5a is irreversible, evidence that inactivation by 5a is not aggregate-based, and reversible inhibition of PTP1B by 5b. AUTHOR INFORMATION Corresponding Authors *K.S.G.: email, [email protected]; phone, (573) 882-6763; FAX, (573) 882-2754 *J.J.T.: email, [email protected]; phone, (573) 884-1280; FAX, (573) 882-2754 Funding We are grateful to the Diabetes Action Research and Education Foundation (Grant number 276) for partial financial support of this work. MALDI mass spectrometry was conducted at the Charles W. Gehrke Proteomics Center at the University of Missouri and we thank Beverly DaGue for assistance with these experiments. We thank Dr. Jay Nix for help with data collection and processing at the Advanced Light Source beamline 4.2.2. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DEAC02-05CH11231. Notes

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The authors declare no competing financial interest. We thank Dr. Derek Seiner for identifying the crystallization conditions used in some experiments.

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For table of contents use only O

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PTP1B

Covalent allosteric inactivation of PTP1B by an inhibitor-electrophile conjugate Puminan Punthasee, Adrian R. Laciak, Andrea H. Cummings, Kasi Viswanatharaju Ruddraraju, Sarah M. Lewis, Roman Hillebrand, Harkewal Singh, John J. Tanner* and Kent S. Gates*

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Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B

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