(Received for publication, June 7, 1995)
From the
The compound 4-(fluoromethyl)phenyl phosphate (FMPP), recently
shown to be a mechanism-based inhibitor of prostatic acid phosphatase
(Myers, J. K., and Widlanski, T. S.(1993) Science 262,
1451-1453), was examined for its effect on calcineurin. This
compound inhibits calcineurin in a time-dependent, first order manner.
Inactivation with [H]FMPP led to a specific
labeling of the catalytic subunit with a stoichiometry of 0.75 mol of
label/mol of protein. A related substrate, 4-methylphenyl phosphate, is
able to protect calcineurin from FMPP-mediated inhibition. Scavenging
nucleophiles, such as cysteine, do not affect the rate of inhibition
when included in the reaction. In addition, extensive dialysis
indicates that inhibition is essentially irreversible. These results
demonstrate that FMPP inactivates calcineurin in a mechanism-based
fashion by forming a covalent adduct with calcineurin A, the catalytic
subunit.
Calcineurin, also known as protein phosphatase 2B, is a
Ca- and calmodulin-dependent protein phosphatase
consisting of a 59-kDa catalytic subunit (calcineurin A) and a 19-kDa
Ca
binding subunit (calcineurin B). The A subunit
shows extensive homology with the family of serine/threonine protein
phosphatases that also includes protein phosphatases 1 and 2A (1) , whereas calcineurin B is a member of the family of
Ca
-binding proteins that includes calmodulin,
troponin C, and parvalbumin and is presumed to have a regulatory
function.
By use of the immunosuppressant drugs cyclosporin A and FK506, calcineurin has recently been identified as having a role in the T-cell receptor signal transduction pathway. These drugs bind to distinct intracellular receptors (2, 3, 4) and form a complex that binds to and inhibits the phosphatase activity of calcineurin(5, 6, 7) . Inhibition of calcineurin in T-lymphocytes prevents the formation of an active transcription factor necessary for the production of the cytokine interleukin-2(8, 9) . The discovery of calcineurin inhibition by cyclosporin A-cyclophilin and FK506-FKBP has heightened interest in the enzyme, with many labs focusing research on the details of drug-mediated inhibition and aspects related to the enzymatic mechanism. Currently nothing is known about the active site environment.
Recent studies (10, 11) ()have described a pair of
structurally similar inhibitors of protein-tyrosine phosphatases that
appear to act as mechanism-based inhibitors. These novel phosphatase
inhibitors, 4-(fluoromethyl)phenyl phosphate (FMPP) (
)and
4-(difluoromethyl)phenyl phosphate (DFPP), are substrates that undergo
transformation after enzyme-catalyzed hydrolysis to yield a reactive
intermediate, presumably a quinone methide, that inactivates the enzyme
by forming a covalent bond to an active site residue. Because p-nitrophenyl phosphate and tyrosine phosphate are substrates
for calcineurin, the fluoromethylphenyl phosphates, as analogues, might
be hydrolyzed by it as well.
In this study we present data that identify FMPP as a mechanism-based inhibitor of calcineurin. The inhibitor produced a time-dependent, first order loss of calcineurin activity that was saturable, with stoichiometric and specific labeling of calcineurin A, the catalytic subunit. Addition of an alternate substrate protected the enzyme from inactivation. Exogeneous nucleophiles had no effect on inactivation, indicating that the inactivation event is inaccessible to solvent nucleophiles and was therefore occurring at the active site. In addition, FMPP-treated enzyme remained completely inactive for a period of several days, demonstrating that the inhibition is essentially irreversible. This report represents the first demonstration of a mechanism-based inhibitor for calcineurin.
where is the enzyme activity at time t,
is the enzyme activity at time zero, k
is the rate constant for inactivation, K
is the Michaelis constant for the inhibitor, and
[I] is the inhibitor concentration. Plots of ln
/
versus time at different
concentrations of inhibitor should yield a series of first order rate
constants (k
) for inactivation according to :
A double-reciprocal plot () will give a nonzero
intercept of -1/k, if the effect is
saturable in [I], and a slope of
-K
/k
:
Figure 1:
Double-reciprocal plot of FMPP
inhibition of calcineurin. Values of k were
determined from the slope of plots of ln (
/
) versus time. The data are the means ± standard error of
three separate experiments.
Figure 2: Substrate protection of calcineurin from FMPP-mediated inactivation. Assays were done as described under ``Experimental Procedures.'' A, effect of FMPP on calcineurin activity in the presence and absence of PMPP. Squares, 40 mM FMPP; filled circles, 57 mM FMPP; open circles, 40 mM FMPP + 100 mM PMPP; triangles, 57 mM FMPP + 57 mM PMPP. B, log plot showing concentration dependence of PMPP protection from FMPP-mediated inactivation. All assays were done in the presence of 40 mM FMPP. Open circles, 0 mM PMPP; open squares, 1 mM PMPP; triangles, 5 mM PMPP; filled squares, 10 mM PMPP; filled circles, 50 mM PMPP.
Figure 3: Effect of exogeneous nucleophiles on FMPP inhibition of calcineurin. Assays were done as described under ``Experimental Procedures.'' Open circles, no added nucleophiles; filled circles, 62.5 mM cysteine; triangles, 50 mM FMPP; squares, 62.5 mM cysteine + 50 mM FMPP.
Figure 4: Irreversible inactivation of calcineurin by FMPP. Assays were done as described under ``Experimental Procedures,'' and enzyme activity was determined at the indicated times. Circles, enzyme incubated with 75 mM PMPP; squares, enzyme incubated with 60 mM FMPP; triangles, enzyme incubated with both 75 mM PMPP and 60 mM FMPP. The experiments with FMPP and FMPP plus PMPP were done in duplicate.
Mechanism-based inhibitors of protein phosphatases are
relatively new compounds. Two examples of these that show promise for
unraveling aspects regarding the catalytic mechanisms of protein
phosphatases are FMPP(10) and DFPP (11) (Fig. 5, 1 and 2, respectively). FMPP has been
shown to be a mechanism-based inhibitor of human prostatic acid
phosphatase, a broad specificity phosphatase(10) . DFPP, a
similar compound, has been shown to inactivate both prostatic acid
phosphatase and the protein-tyrosine phosphatase SHP(11) .
Experiments in our lab with a related compound, 4-trifluoromethylphenyl
phosphate (Fig. 5, 3), indicated that it was an inhibitor of
calcineurin, although the kinetics of inactivation were more complex
than anticipated. (
)We therefore decided to test FMPP as a
mechanism-based inhibitor of calcineurin.
Figure 5: Structures of phenyl phosphate derivatives: FMPP (1), DFPP (2), 4-(trifluoromethyl)phenyl phosphate (TFPP, 3), and AMPP (4).
In order to show that a compound inhibits via mechanism-based inhibition, a number of criteria need to be satisfied. These include a time-dependent, first order loss of activity; the observance of saturable inhibition kinetics; substrate protection; the lack of effect of solvent nucleophiles on the rate of inactivation; the covalent and stoichiometric attachment of the inhibitor to the enzyme; and the irreversiblity of inactivation(18) . Inhibition of calcineurin by FMPP meets the above criteria, indicating that inactivation is occurring at the active site via enzyme-mediated generation of a reactive species.
Inactivation of calcineurin by FMPP is rapid, with greater than 70%
inactivation occurring within the first 30 s of incubation with FMPP at
concentrations of 20 mM or higher. In all cases the
inactivation was first order with a rate of inactivation proportional
to the amount of inhibitor. A nonzero intercept on the ordinate of a
double-reciprocal plot indicated saturable inhibition occurring at a
rate of 8.82 min. This rate constant is likely to be
comprised of several rate constants, including those representing
hydrolysis of the phosphate ester as well as any other chemical or
enzymatic steps leading to a covalent enzyme adduct. This value is
comparable with k
values of 7-35
min
for aryl phosphate esters substituted at the para position (Table 1) and indicates that the
rate-limiting step for inactivation of calcineurin by FMPP is likely to
be hydrolysis of the phosphate ester.
The stoichiometry of
inactivation was determined using [H]FMPP. In
these experiments, inactivation with [
H]FMPP led
to near stoichiometric labeling of calcineurin, with the majority of
the label incorporated into the catalytic subunit. Some label was also
found associated with calcineurin B and calmodulin. Although this could
represent nonspecific labeling of these proteins, it could also
indicate that they are close enough to the active site to allow
labeling by inhibitor diffusing out of the active site. Preliminary
mass spectrometry experiments have indicated that FMPP-inactivated
calcineurin A has a mass approximately 115 daltons larger than
untreated enzyme (expected mass change is 106 daltons), whereas the
mass of calcineurin B, as well as that of calmodulin, is unchanged
(data not shown).
If FMPP inactivates in a mechanism-based fashion, an alternate substrate should compete with the inhibitor for access to the active site and protect calcineurin from inactivation, with the rate of inactivation inversely proportional to the substrate concentration. PMPP is identical in structure to FMPP, with the exception of an isosteric substitution of a hydrogen atom by fluorine. When FMPP was incubated with calcineurin at varying concentrations of PMPP, the enzyme was protected from inactivation, and this protection was dependent on the concentration of PMPP (Fig. 2B). A concentration of 1 mM PMPP provided little protection against 40 mM FMPP, 10 mM PMPP protected up to 60% of the activity over a period of 20 min, and 50 mM PMPP provided almost complete protection during the time course of the assay.
Even though inactivation of calcineurin occurs in a time-dependent fashion and substrate protects against inactivation, it does not prove that FMPP is a mechanism-based inhibitor. One other possibility is that the inhibitor forms a reactive species in solution, independent of catalysis, that is preferentially directed toward the active site. Another possibility is that enzyme activity is required to form a product that upon release into solution is able to form a reactive species that indiscriminantly inactivates the enzyme. One way to distinguish between these two cases and one in which an electrophilic species is formed and reacts at the active site is to include an excess of a scavenger nucleophile in the reaction buffer. Any reactive species either formed in solution or released from the enzyme will react with this nucleophile and be quenched before it can reassociate with the enzyme in a nonspecific manner. When either cysteine, KCN, or NaSCN were present in the assay, calcineurin was still inhibited by FMPP, indicating that the reactive species was sequestered from solvent, presumably at the active site. Additional evidence for sequestering of the reactive species at the active site is provided by experiments with AMPP (Fig. 5, 4). AMPP can be hydrolyzed by calcineurin, but because acetate is a poor leaving group, elimination of acetate is slower than that of fluoride. The initial hydrolysis product can then be released into solution before a reactive quinone methide is formed. AMPP had little inhibitory effect on calcineurin, indicating that if a reactive species forms from this substrate, it quickly becomes deactivated by solvent.
If FMPP is inactivating calcineurin by forming a chemically stable adduct with an active site residue, the inactivation should be irreversible. Extensive dialysis (119 h) with several changes of buffer did not reverse inactivation of calcineurin by FMPP. To show that prolonged dialysis was not incompatible with recovery of activity, a sample of calcineurin dialyzed in parallel, but in the presence of PMPP instead of FMPP, remained active. This sample showed a gradual loss of activity with time, with greater than 70% activity remaining after 119 h. A second control sample, with both PMPP and FMPP present, showed an initial drop in activity, as expected due to the presence of FMPP, but after 17 h of dialysis its activity paralled that for the uninactivated enzyme (Fig. 4).
In the case of inactivation by both FMPP and DFPP, it is believed that after the enzyme catalyzes phosphate ester hydrolysis, elimination of fluoride occurs, generating a quinone methide(10) . This reactive electrophile can then be attacked by a nearby nucleophile, resulting in a covalent modification (Fig. 6). If fluoride elimination is rapid it may occur before release of product from the active site, resulting in covalent modification of an active site residue. The data presented indicate that FMPP is indeed a mechanism-based inhibitor of calcineurin.
Figure 6:
Scheme showing the proposed mechanism of
inhibition of calcineurin by FMPP. The mechanism involves hydrolysis of
the phosphate ester with subsequent formation of the quinone methide at
the active site and attack by an active site nucleophile. k represents the rate constant for
hydrolysis of the phosphate ester, k
represents the rate constant for elimination of flouride ion
and the generation of the reactive quinone methide intermediate, k
is the rate constant for the release
of the phenol, and k
is the rate
constant for release of the quinone
methide.
As a reagent that inactivates by reaction with active site residues, FMPP will be useful for identifying calcineurin residues that participate in substrate binding and/or catalysis; experiments with this aim in mind are currently in progress. At present, there is nothing known about the active site of calcineurin or other enzymes in the serine/threonine protein phosphatase family such as protein phosphatases 1 and 2A. Given the fact that these members share extensive homology, it is likely that they catalyze phosphate ester hydrolysis in a mechanistically similar fashion. A comparison of the primary sequences of calcineurin with protein phosphatases 1 and 2A indicates an active site domain of >200 residues and, within this domain, six regions of very high conservation consisting of approximately 60 amino acids that probably represent either active site residues or structurally important regions that have been conserved during evolution(19, 20, 21) .
Since the
discovery of its involvement in T-cell activation, calcineurin has
become the focus of a number of studies aimed at determining its
structure and function. Certainly one of the aims of these studies is
to design novel calcineurin inhibitors that retain immunosuppressive
activity but lack the toxic side effects noted for these powerful
transplantation drugs. With a K for inactivation
in the millimolar range, FMPP is not an ideal inhibitor for
calcineurin. However, the K
of 44.4 mM does compare with K
values for substrates
with analogous structure (Table 1). The fact that certain
phosphopeptide and phosphoprotein substrates of calcineurin have K
values in the micromolar
range(22, 23, 24) indicates that additional
structural elements could be utilized and, using FMPP as a model,
incorporated to provide increased binding affinity for novel, potent,
and specific calcineurin inhibitors.