Seleninate in place of phosphate: Irreversible inhibition of protein tyrosine phosphatases

Article abstract of DOI:10.1021/ja804489m

A homotyrosine based seleninic acid irreversibly inhibits protein tyrosine phosphatases by forming a covalent selenosulfide linkage with the active site cysteine sulfhydryl specifically. The details of the event are revealed by model synthetic studies and by kinetic, mass spectrometric, and crystallographic characterization. Copyright

Full text of DOI:10.1021/ja804489m

Published on Web 09/10/2008
Seleninate in Place of Phosphate: Irreversible Inhibition of Protein Tyrosine
Phosphatases
Mohannad Abdo,† Sijiu Liu,‡ Bo Zhou,‡ Chad D. Walls,‡ Li Wu,‡ Spencer Knapp,*,† and
Zhong-Yin Zhang*,‡
Department of Chemistry and Chemical Biology, Rutgers The State UniVersity of New Jersey, Piscataway, New
Jersey 08854, and Department of Biochemistry and Molecular Biology, Indiana UniVersity School of Medicine,
Indianapolis, Indiana 46202
Received June 18, 2008; E-mail: spencer.knapp@rutgers.edu; zyzhang@iupui.edu
Protein tyrosine phosphorylation is a major post-translational
mechanism for cellular signaling in response to growth factors,
hormones, and cytokines. The extent and duration of tyrosine
phosphorylation are tightly regulated by the activities of protein
tyrosine kinases and protein tyrosine phosphatases (PTPs). Similar
to the kinases, the PTPs constitute a large family of enzymes;1
PTP, YopH, resulted in a time- and concentration-dependent loss
of phosphatase activity (Figure 1A). Similar results were obtained
with the mammalian enzymes PTP1B as well as the dual specificity
phosphatases VHR and VHX (Supporting Information and Figure
S1). Inactivation of the PTPs by 3 appeared to be irreversible, as
extensive dialysis and/or buffer exchange of the reaction mixture
failed to restore enzyme activity. Analysis of the pseudo-first-order
rate constant as a function of inhibitor concentration showed that
3-mediated YopH inactivation displayed saturation kinetics (Figure
1B), yielding values for the binding constant KI and the inactivation
malfunction in PTP activity has been associated with human
diseases, including cancer, diabetes and obesity, and autoimmune
disorders. The PTPs share a conserved active site that recognizes
phosphotyrosine (pTyr) and use a common catalytic mechanism
that features a highly nucleophilic Cys residue.2 Among various
promising approaches, the development of small molecule modula-
tors of PTP activity could aid in the study of PTP function in
complex signaling cascades.
Organoselenium compounds3 have been found or induced in
Nature4 and have been studied in the laboratory in all four oxidation
states: selenol, selenenic, seleninic, and selenonic.5 Seleninic acids
(RSeO2H) are unique in that they can be either protonated or
deprotonated6 at physiological pH and can accept Lewis bases at
selenium to form tetracoordinate trigonal bipyramidal adducts
(selenuranes).7 Furthermore, seleninic acids can couple with thiols
over a wide pH range to give a redox product, the mixed
selenosulfide (RSeSR′).8 The anionic seleninate has been advanced
as a bioisostere for carboxylate,9 inasmuch as it possesses a similar
size and charge and can be expected to resist enzymes that process
rate constant ki of 44.5 ( 8.8 µM and 0.243 ( 0.009 min-1
,
respectively. These results suggest that 3 is an active site-directed
affinity agent whose mode of action involves at least two steps:
binding to the PTP active site, followed by covalent modification
of active site residue(s). In further support of this idea, the active
site-directed competitive YopH inhibitors arsenate and p-nitrocat-
echol sulfate were each able to partially protect YopH from
3-mediated inactivation (Figures S2 and S3).
To directly demonstrate that inhibitor 3 inactivates the PTPs by
covalent modification, we analyzed YopH treated with or without
3 using mass spectrometry (Figure S4). The mass of unmodified
YopH PTP domain (residues 163-468) measured by an electrospray
ion trap mass spectrometer was 33 512 Da, which is in close
agreement with the theoretical value (33 513 Da). YopH treated
with 3 showed an altered mass of 33 884 Da. This corresponds to
a mass difference of 372 Da, consistent with the formation of a
mixed selenosulfide covalent adduct between YopH and 3 (the
theoretical mass shift is 371 Da). Although a selenosulfide bond is
normally readily cleaved by DTT in buffer solution,16 the seleno-
sulfide linkage in the modified YopH is comparatively stable,
inasmuch as mM concentrations of DTT failed to reactivate the
inhibitor 3 treated enzyme. Thus, the (PTP)S-SeR′ bond is probably
protected from DTT by being buried in the active site. Finally, 3
failed to modify the catalytically inactive mutant YopH/C403S
(Figure S5), consistent with formation of the covalent adduct
between YopH and 3 at the active site Cys403 explicitly.
-
RCO2-. RSeO2 is also a potential bioisosteric match for the
biologically ubiquitous O-phosphate group (ROPO3-2),10 even
though the latter is doubly charged. We report that, for a PTP
substrate, replacement of the phosphate from tyrosine-OPO3-2 with
-
-CH2SeO2 provides a new inhibitor11 3 that reacts effectively
and irreversibly with the PTPs in a manner consistent with formation
of a covalent selenosulfide adduct.
Seleninic acid 3 was prepared from the homotyrosine derivative12
1 by selenocarboxylate Mitsunobu conversion13 (78% yield) to the
selenoester 2 and then reaction of 2 with DMDO14 in wet acetone
solution at 23 °C; 3 was formed (88%) without any overoxidation
to the selenonate.15 The product was purified by silica chromatog-
raphy and was stable for days stored neat at 23 °C, or in methanol
or DMSO solution. The reaction in wet dichloromethane of 3 (1
equiv) with the sulfhydryl of a model Cys derivative gave the
selenosulfide 4 in 82% yield, thus demonstrating the feasibility of
coupling 3 with the PTP active site Cys residue.
To further define the details of PTP inactivation by 3, we obtained
a 2.3 Å crystal structure of PTP1B ·3 complex, as described in the
Supporting Information and Table S1. The final model for the
PTP1B·3 complex includes PTP1B residues 2-283 and all non-H
atoms in 3. The overall structure of PTP1B ·3 is similar to the
previously determined ligand-free PTP1B structure.17 The major
difference between these two structures is the electron density in
In the PTP catalytic mechanism, the active site Cys sits at the
bottom of the pTyr-binding pocket (i.e., the active site) such that
its Sγ atom is poised 3 Å from the phosphorus atom of the substrate
ready for nucleophilic attack.2 Incubation of 3 with the Yersinia
† Rutgers.
‡ Indiana University.
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2008 American Chemical Society

C O M M U N I C A T I O N S
the carbonyl oxygen in the methyl ester and the side chain of
Gln262. The phenyl ring of 3 is engaged in extensive hydrophobic
interactions with the residues lining the active site cavity, including
Tyr46, Ser216, Ala217, and Arg221 (Figures 2B and S6). Hydro-
phobic interactions are also observed between the benzylic meth-
ylene next to the selenium and the side chain of Arg221, while the
carbonyl carbon and the methyl group in the methyl ester interacts
with the side chains of Tyr46 and Val49, respectively. Finally, the
tert-butyl group in 3 also makes contacts with Tyr46 (Figures 2B
and S6). These noncovalent interactions likely contribute to the
efficient PTP inactivation by 3.
The kinetic, mass spectrometry, and X-ray crystallographic data
together demonstrate that 3 is an effective pTyr surrogate that can
specifically modify the PTP active site Cys residue by forming a stable
selenosulfide linkage. It is worthwhile to note that the kinetic
parameters KI and ki for inhibitor 3-mediated YopH inactivation (44.5
µM and 0.243 min-1) compare very favorably to those determined
for the previously described R-bromobenzyl phosphonate based probe
(4.1 mM and 0.11 min-1).19 These observations highlight the potential
for developing seleninate based small molecule probes to modulate
PTP activity in signaling and in diseases.
Figure 1. Kinetic analysis of YopH inactivation by 3 at 25 °C and pH 6.
(A) Time and concentration dependence of inhibitor 3-mediated YopH
inactivation. (B) Concentration dependence of the pseudo-first-order rate
constants kobs for 3-mediated YopH inactivation.
Acknowledgment. Dedicated to Prof. J. Meinwald on the
occasion of his 80th birthday. We are thankful to the Prusoff
Foundation for partial financial support at Rutgers and to Rohm
and Haas Co. for a graduate assistantship to M.A. The work at
Indiana University was supported by NIH CA126937.
Supporting Information Available: Experimental details for 2-4,
and description of PTP inhibition assays, mass spectrometry, and
crystallographic characterization of PTP1B·3. This material is available
free of charge via the Internet at http://pubs.acs.org.
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