Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor-Electrophile Conjugate

Article abstract of DOI:10.1021/acs.biochem.7b00151

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. 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) × 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., and Erlanson, D. A. (2005) Biochemistry 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 allowing inactivation of the enzyme via selective modification of allosteric residue C121.

Full text of DOI:10.1021/acs.biochem.7b00151

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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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Covalent allosteric inactivation of PTP1B by an inhibitor-
electrophile conjugate
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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,*
aUniversity of Missouri
Department of Chemistry
125 Chemistry Building
Columbia, MO 65211
bUniversity of Missouri
Department of Biochemistry
117 Schweitzer Hall
Columbia, MO 65211
* To whom correspondence should be addressed: email: gatesk@missouri.edu; phone:
(573) 882-6763; FAX: (573) 882-2754
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ABBREVIATIONS
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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, 4-
nitrophenyl 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, isopropyl-
beta-D-thiogalactopyranoside;
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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.
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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
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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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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 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 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
S–
S–
"Ki"
215
215
NuH
NuH
O
Br
X
SH
O
121
O
S
S–
215
121
Nu
O
Br
S–
Pathway A
215
NuH
Pathway B
Scheme 2. Possible mechanisms for inactivation of PTP1B by 5a.
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EXPERIMENTAL PROCEDURES
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Materials. Reagents were purchased from the following suppliers: 5-bromo-2-
methyl 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, benzyl mercaptane, alanine,
(benzotriazol-1-yloxy)-tris(dimethylamino)phosphate (BOP), bromoacetyl 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
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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, p-
phenylboronic 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
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and purified as described previously and the concentration of active enzyme in stock
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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 1,1-
dioxide (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
o
100 C 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
1
acetate gave 2a as an off-white solid (780 mg, 79%). H 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); 13C NMR (methanol-d4, 150 MHz) δ 170.1, 157.5, 140.9, 135.6, 130.2, 129.9,
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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.
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Synthesis of 5-(4-methyl-[1,1'-biphenyl]-3-yl)-1,2,5-thiadiazolidin-3-one 1,1-
dioxide (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
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100 C 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
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gave 2b as an off-white solid (73 mg, 62%). H 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,
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2H), 2.50 (s, 3H); C 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 (acetone-
d6, 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.
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Synthesis of 5-(4'-amino-4-methyl-[1,1'-biphenyl]-3-yl)-1,2,5-thiadiazolidin-3-
one 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).
1H 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,
13
2H), 3.35 (s, 2H), 1.89 (s, 3H). C 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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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%
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yield). H 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),
13
4.66 (s, 2H), 4.06 (s, 2H). 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
1
as a brown solid (33 mg, 48% yield). H 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,
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1H), 4.66 (s, 2H), 2.48 (s, 3H), 2.10 (s, 3H). 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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23.9, 17.9; HRMS (ESI-TOF, [M + H]+) m/z calculated for C17H18N3O4S: 360.1018
found 360.1017.
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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.
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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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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:
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(max – min)
y = min +
50 – log[I])(nH)]
1 + 10[(logIC
(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 ion-
exchange 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.
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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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(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.
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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 300SB-
C18, 5 µm particle, 300 Å pore; separation column, 150 mm x 75 µm; 40 mL trap
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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 min-
3% B, 6.1 min-10% B, 27.4 min-70% B, 28.4 min-100% B, 33.4 min-100% B, 37.4 min-
3% 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-LC-
Nanospray MS only spectra acquired over a mass range of 300-3200 Da. Nano-LC-
Nanospray 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.
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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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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.
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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
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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.
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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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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.
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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
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and plunged into liquid nitrogen.
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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.
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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-2-
methylaniline 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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coupled with α-bromoacetyl bromide to generate 5a. The acetyl derivative 5b was
prepared by an analogous reaction with acetyl chloride.
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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
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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 216-
220. 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
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interact with the aromatic and nonpolar residues Tyr46, Val49, Ala217, Ile219, and
Phe182.
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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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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).
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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 pseudo-
first-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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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 (p-
NPP, 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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Table 1. IC50 values for compounds studied in this work. aAssays were conducted with the
substrate p-NPP at its Km value (0.4 mM)19 and Ki values were calculated using the Cheng-
Prusoff equation.39
Reversible binding affinity of 5a, 5b, and 2b for PTP1B. In an exo-affinity-
labeling 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, non-
covalent 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
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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.
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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 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 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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(C32, Table 2). The peptide fragment containing the active site residue C215 was not
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extensively modified by 5a.
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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 p-
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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.
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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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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 exo-
affinity-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 two-
step 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)-1,2,5-
thiadiazolidin-3-one 1,1-dioxide phosphotyrosine isostere in 5a to this site may drive
covalent modification of C121.
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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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