(Received for publication, August 24, 1995; and in revised form, November 28, 1995)
From the
We present here for the first time a method for determining the rate constants associated with slow binding inhibition of prostaglandin H synthase (PGHS). The rate constants were determined by a method using initial steady-state conditions, which minimize the impact of catalytic autoinactivation of the enzyme. The currently available methods for determining the kinetic constants associated with slow binding enzyme inhibition do not distinguish between rate decreases due to enzyme inhibition or due to autoinactivation of the enzyme. A mathematical model was derived assuming a rapid reversible formation of an initial enzyme-inhibitor complex (EI) followed by a slow reversible formation of a second enzyme-inhibitor complex (EI*). The two enzyme inhibitor complexes are assumed to be in slow equilibrium. This method was used to evaluate the kinetic parameters associated with the binding and selectivity of the nonsteroidal antinflammatory drugs (NSAIDs), flurbiprofen and indomethacin.
The K values associated with the formation of the first reversible
complex (EI) for flurbiprofen with PGHS1 and PGHS2 were 0.53
± 0.06 and 0.61 ± 0.08 µM, respectively; the
rate constants for the forward isomerization, k
,
into the second reversible complex (EI*) were 0.97 ±
0.99 and 0.11 ± 0.01 s
, respectively, and
rates of the reverse isomerization from EI*, k
, were 0.031 ± 0.004 and 0.0082
± 0.0008 s
, respectively. Indomethacin was
estimated to form the EI complex with the same affinity for
both PGHS1 and PGHS2, 10.0 ± 2.8 µM and 11.2
± 2.0 µM, respectively, and dissociate from EI* at approximately the same rate 0.0011 ± 0.0002
s
and 0.0031 ± 0.0003 s
,
respectively. However, the rate of isomerization into EI* from EI was much greater for PGHS1 than PGHS2, 0.33 ± 0.08
s
as compared with 0.034 ± 0.004
s
. These results show that the overall affinity for
the inhibition of PGHS1 versus PGHS2 was 30-fold greater for
indomethacin (K
* = 0.032 ±
0.005 and 1.02 ± 0.08 µM, respectively) and 3-fold
greater for flurbiprofen (K
* =
0.017 ± 0.002 and 0.045 ± 0.004 µM,
respectively). The results also show that for both PGHS1 and PGHS2,
flurbiprofen was bound tighter to the initial EI complex than
indomethacin; however, the rate of dissociation from EI* was
slower for indomethacin than flurbiprofen. The rate of the forward
isomerization to EI* is primarily responsible for the
selectivity of both NSAIDs for PGHS1. This analysis shows the
quantitative importance of the different kinetic parameters upon the
overall binding affinity of these NSAIDs and should greatly assist in
our understanding of the structural interactions that promote
enzyme-inhibitor binding.
The treatment of pain and inflammation by the inhibition of
prostaglandin formation has been successfully accomplished with many
currently marketed NSAIDs ()including indomethacin,
flurbiprofen, ibuprofen, and aspirin. These therapeutic agents are
thought to elicit their action by the inhibition of prostaglandin H
synthase. However, severe gastrointestinal irritation is also observed
with the administration of these compounds, limiting their usage. This
irritation has been associated with the inhibition of prostaglandin
formation in the gastrointestinal tract(1) . The recent
discovery of an inducible form of prostaglandin H synthase (PGHS2) has
renewed interest in discovering new NSAIDs that will be better
tolerated(2) . This isoform is induced at the sites of
inflammation (3) . The working hypothesis is that selective
inhibition of the inducible enzyme will block the inflammation and the
pain associated with the inflammation without serious gastrointestinal
side effects(4, 5) . To this end, a great deal of
effort is being spent to discover PGHS2 selective compounds.
Previous work by Rome and Lands (6) with ram PGHS1 revealed
that many PGHS inhibitors, including indomethacin, inhibited the enzyme
in a time-dependent manner. The mechanism of the time-dependent
inhibition was subsequently postulated to be associated with a slow
binding mechanism involving a slow reversible isomerization of the
initial enzyme-inhibitor complex (EI) to a second
enzyme-inhibitor complex (EI*) (see ) (7, 8) . Although the inhibition was reported to be
reversible, the rate of the reverse isomerization from EI*
(dissociation rate) was not addressed in those reports. The only
reported attempt to determine the reverse isomerization rates was by
Walenga and co-workers (9) , who observed slow reversible
inhibition of human platelet cyclooxygenase by indomethacin and
determined the t for the recovery of enzymatic
activity to be between 100 and 200 min(9) . This was
considerably shorter than the t
of 4-5
days for human platelet. In this manuscript we present a method to
determine the rate constants associated with slow binding inhibition of
PGHS1 and PGHS2, which minimizes the impact of the substrate-dependent
autoinactivation upon the slow binding inhibition kinetics. This method
allows for the first time the determination of the intrinsic
equilibrium affinity of slow binding inhibitors for PGHSs, as defined
by K
*, and the impact of the individual
components of the reaction dynamics upon the overall inhibition.
Figure 1: Reaction profile showing the consumption of oxygen with time after the addition of 200 µM arachidonic acid with PGHS2. Curve 1, control; curve 2, preincubation with 25 µM indomethacin for 20 s; curve 3, preincubation with 25 µM indomethacin for 2 min. Inset, the first derivative spectra of the first minute of the oxygen consumption curves. The average of the 5 minimum points was used to determine the maximum initial velocity.
In order to determine the rate constants associated with
inhibition of PGHS by NSAIDs, an approach was needed to minimize the
impact of the substrate catalyzed rate of enzyme autoinactivation. The
oxidation of arachidonic acid to prostaglandin H by PGHS is
associated with the consumption of 2 mol of oxygen consumed for every
mol of product formed. The continuous progress of the reaction can be
measured by monitoring the consumption of oxygen. The oxygen
consumption profile (Fig. 1) is characterized by a rapid,
protein-dependent decrease in oxygen concentration after addition of
the substrate, arachidonic acid. The rate of decrease in oxygen
concentration decreases until all the enzyme is inactivated. The
maximum velocity, v
, (
)obtained in
each reaction was used for the kinetic evaluations in order to minimize the effect of enzyme inactivation. We assume that at
the maximum velocity no enzyme has been inactivated. The maximum
velocity was determined from the first derivative of the oxygen
consumption versus time profile (Fig. 1, inset).
The next issue in developing a method to estimate
the rate constants was to determine the appropriate kinetic model of
inhibition. The v associated with oxygen
consumption was observed to decrease in a time-dependent manner
following preincubation of indomethacin and flurbiprofen with both
human PGHS enzymes, as reported previously(7) . The reactions
were initiated with 200 µM arachidonic acid, a
concentration greater than 40-fold excess of the apparent K
. Under these conditions, all enzyme-inhibitor
complexes that are in rapid equilibrium with free enzyme should rapidly
bind the excess substrate and catalyze product formation. The
inhibition will be the result of enzyme-inhibitor complex that is not
in rapid equilibrium with free enzyme. Plots of the percent activity
remaining versus preincubation time show a sharp initial
decrease in activity, which eventually reaches a plateau (Fig. 2). This characteristic was also reported for the ram
enzyme with indomethacin and flurbiprofen, where 4 and 6% residual
activity, respectively, was observed to always remain(7) . More
recently Quellet and Percival (11) reported 0.6 and 13%
residual activity remaining following inhibition of human PGHS2 by
indomethacin and flurbiprofen, respectively. The residual activity
(plateau activity) appears to saturate as inhibitor concentrations are
increased. This observation is consistent with a mechanism where even
at saturation a certain percentage of the inhibitor bound enzyme is in
rapid equilibrium with substrate. The enzyme-inhibitor complex in rapid
equilibrium with substrate (termed EI) must also be in slow
equilibrium with another enzyme-inhibitor complex (termed EI*)
in order to observe reversible time-dependent inhibition. These
observations are consistent with the proposed mechanism of slow binding
inhibition, first termed by Morrison(12) , where the inhibitor
initially binds to the enzyme to form an equilibrium complex (EI), which in turn slowly isomerizes to another reversible
enzyme-inhibitor complex (EI*) (). The amount of
residual activity will then depend on the equilibrium between EI and EI*.
Figure 2: Time-dependent inhibition of human PGHS1 and PGHS2 by indomethacin and flurbiprofen. Individual data points were determined from the first derivative of the oxygen consumption assay after preincubation of enzyme with inhibitor for the specified time. Data are represented as percent control, i.e. the maximum velocity of the reaction with inhibitor divided by the maximum velocity of the reaction without inhibitor. The curves represent the fit of the data at each inhibitor concentration over several experiments to EQN 10. A, indomethacin/PGHS1, n = 2; B, indomethacin/PGHS2, n = 5; C, flurbiprofen/PGHS1, n = 5; D, flurbiprofen/PGHS2, n = 5; where n = the number of experiments.
The mathematical interpretation of the theoretical kinetic model in terms of the experimental protocol is subject to two phases, a preincubation phase in which inhibitor is incubated with enzyme in the absence of substrate, and a reaction phase, which begins when the substrate is added to the inhibitor enzyme mix. The following dynamics have been assumed to be associated with the preincubation phase.
Let E = E(t), I = I(t), EI = EI(t), and EI* = EI*(t) describe the concentration of free enzyme, free inhibitor, enzyme-inhibitor complex, and isomerized enzyme-inhibitor complex at time t, respectively. Applying the laws of mass action, we describe the dynamics in with the following system of differential equations:
We assume that (i) inhibitor is in excess of enzyme and (ii) the
reaction E + I &cjs0635; EI is in
quasiequilibrium (k
k
). As a result of assumption (ii), the E + I &cjs0635; EI equilibrium is essentially
achieved by t
0
. During all of the
preincubation then, we have the quasi-steady-state identity E = (K
/I)EI, where K
= k
/k
. Substituting this
identity into the conservation equation E
= E + EI + EI*, where E
is the initial enzyme concentration, then
substituting the resulting expression for EI into the
differential equation for EI* in , we have
The solution of this differential equation for EI*(t) with initial condition EI*(0) = 0 is
Now let =
(t) = E(t) + EI(t). Then since
= E
- EI*, from we have the final concentration of free enzyme and
enzyme-inhibitor complex (E + EI) at the end of T seconds of preincubation is approximately
This equation will be used for the initial enzyme concentration for the subsequent reaction.
The reaction phase of product formation is initiated by adding substrate after T seconds of preincubation, and the following dynamics are assumed.
In this diagram S, P, and E denote
substrate, product, and inactivated enzyme, respectively. Together with
the two previous assumptions made, we assume (iii) that substrate is in
excess of enzyme, so that Sk
and Ik
can be considered constants, (iv) that the rates k
are slow relative to the rates k
, k
, k
, and (v) that the rate k
is
slow relative to the rates k
, k
, k
. As a result of
assumptions (iv) and (v), the rates k
and k
are negligible in the initial stages of the
reaction. With these assumptions, the initial dynamics of the reaction
can be described with the following scheme.
Letting S = S(t), ES = ES(t) and P = P(t) describe the concentration of substrate, enzyme-substrate complex, and product at time t, respectively, and applying the law of mass action, we describe the dynamics in with the following system of differential equations:
The steady-state solution for can be computed and
depends on the length of the preincubation time T, giving the
steady-state concentration for ES after T seconds of
preincubation (ES(T)) as
where K = (k
+ k
)/k
and
= EI + E + ES, the conservation equation for the reaction phase. From and dP/dt in , we have the
maximum velocity of product formation after T seconds of
preincubation as
If there is no inhibitor (control), v is
computed by setting
= E
and I = 0 (in this case v
is
independent of T). Dividing by the control v
and using the expression for
in , we obtain the equation for the maximum velocity
after T seconds of preincubation as a percent of control.
Nonlinear regression as described under ``Experimental
Methods'' was used to fit the time-velocity curves to f(T) in . This estimates three parameters k, k
, and K
, using the known K
/S and
several inhibitor concentrations [I]. The overall inhibition
is defined by the following equation.
The estimated rate constants are shown in Table 1.
Comparison of the two inhibitors with two enzymes shows that all three
kinetic parameters must be examined in order to understand which
factors contribute to the overall affinities. For example, the K* values for flurbiprofen and indomethacin with
PGHS1 are similar, 0.017 and 0.032 µM, respectively.
However, the individual rate constants are vastly different; K
is 20-fold lower for flurbiprofen and k
is 30-fold slower for indomethacin.
Whereas K
and k
were
the critical kinetic parameters in explaining the similarities in
affinities for flurbiprofen and indomethacin for PGHS1, the 30-fold
selectivity of indomethacin for PGHS1 versus PGHS2 is
accounted for by the difference in k
. The K
and k
are similar,
while there is a 10-fold difference in k
for the
two enzymes, 0.334 s
for PGHS1 and 0.034
s
for PGHS2.
The plateau phase of the curves upon
casual inspection appears to approach saturation (Fig. 2).
Indeed, this is consistent with the predictions of the model. The
plateau represents the amount of inhibitor in the EI* complex
after equilibrium has been achieved in the preincubation phase.
Saturation of the enzyme with inhibitor results in the saturation of
the EI* complex and accordingly, the plateau. Computing the
limit of in as I and T
, the percentage of enzyme in EI* at saturation, termed
% EI
, can be expressed as:
The % EI for flurbiprofen was
determine to be 97 and 92.7% for PGHS1 and PGHS2, respectively.
Therefore, the amount of activity remaining at saturation is 3 and
7.3%, respectively. The % EI
for indomethacin
was 99.7 and 91.6% for PGHS1 and PGHS2, respectively. These data
indicate that even when the enzyme is fully saturated with inhibitor,
not all of the enzyme is in the EI* complex. This is
consistent with an equilibrium between EI and EI*
that is not affected directly by inhibitor concentration. This is
inconsistent with a single-step reaction in which a rapid equilibrium
is not established prior to the formation of EI* ().
The K and forward isomerization rates were
determined by Kulmacz and Lands (7) for the inhibition of ram
PGHS1 by flurbiprofen and indomethacin assuming irreversible
inhibition. The results they reported were similar to those reported
here for human PGHS1. For flurbiprofen and indomethacin, Kulmacz and
Lands (7) reported K
values of 0.2 and 1.7
µM, respectively, and forward isomerization rates of 0.27
and 0.25 s
, respectively. The similarity in the data
is surprising given the difference in methods and enzymes. These data
suggest that the interactions that promote the slow binding kinetics
may be similar for the human and ram PGHS1, although no definitive
conclusions can be made without determining the off rates for the ram
enzyme. More recently, Quellet and Percival (11) used the same
type analysis to determine the rate constants associated with
inhibition of human PGHS2 by indomethacin and flurbiprofen. The K
values were 114 and 0.17 µM,
respectively, and the rate of k
(k
) was 0.035 and 0.018
s
, respectively.
Calculation of the binding
energies associated with the kinetic constants using the Eyring
equation gives further insight into the factors that affect the
selectivity. The calculated free energies, G, are plotted
against the reaction coordinate in Fig. 3. The transition state
energy in going from EI to EI* is lower for PGHS1 by
approximately 1.4 kcal/mol for both inhibitors. The ground state energy
for EI* is 2.07 kcal/mol lower for PGHS1 with indomethacin and
0.61 kcal/mol lower with flurbiprofen. Accordingly, indomethacin
selectivity results from a combination of interactions from the
transition state between EI and EI* and the EI* ground state. The selectivity observed with flurbiprofen
appears to be exclusively the result of transition state interactions.
Figure 3: Reaction coordinate associated with the inhibition of PGHS1 and PGHS2 by indomethacin (A) and flurbiprofen (B).
One obvious question from this analysis is what structurally is the difference between EI* and EI and what interactions influence the transition state structure? The recently described crystal structure by the Garavito and co-workers (13) of flurbiprofen bound to ram PGHS1 is most likely the EI* complex. The identity of EI is unknown. The difference between EI and EI* could be the displacement of a water molecule, as has been proposed for slow binding inhibition of stromelysin, thermolysin, and pepsin (14, 15, 16) or a larger protein-inhibitor conformation change. Further work will be needed to define the structural factors that contribute to these kinetic observations.
Characterizing and quantitating the factors important to the
inhibition of PGHSs for use in drug design has been a challenging
undertaking. It has not been resolved which in vitro parameters are most predictive of in vivo efficacy, and
in addition, it has been difficult to correlate in vitro enzyme data with cell-based data. An example of this is seen when
comparing the IC values determined for the inhibition of
human PGHS1 and PGHS2 by indomethacin under a variety of different
conditions (Table 2). The variability in the results is not
surprising when one considers that there are at least five dynamic
processes involved in the slow binding inhibition of PGHS and the
results are but a single snapshot of the dynamics. The five dynamic
processes are (i) the association and (ii) dissociation rates of
inhibitor with EI*, (iii) the rate of enzyme autoinactivation,
(iv) the rate of substrate turnover, and (v) the rate of catalytic
activation of the enzyme. Instead of a single snapshot, our analysis of
the reactions has led to the quantitation of the effect of the rates of
association and dissociation of the inhibitor upon the overall
inhibition of substrate turnover by minimizing the impact of the
autoinactivation of the enzyme.
Theoretically, under equilibrium
conditions, the IC values determined with preincubation
should approximate K
*. However, when the rate of
autoinactivation is faster than the other dynamic processes, an
equilibrium can never be established, and the IC
will not
approximate K
*. The methodology required to
accurately determine a meaningful IC
associated with the
slow binding inhibition of PGHS by indomethacin requires the
preincubation and/or incubation time to be long enough to allow for the
association of indomethacin into EI* (k
),
and the incubations must be long enough to allow for equilibrium
dissociation from EI* (k
).
Therefore, equilibrium will only be established after five half-lives
of the slowest process, (
)which for indomethacin is the
dissociation from EI*. The rates of dissociation from EI* were 0.0011 s
and 0.0031 s
for PGHS1 and PGHS2, respectively. The corresponding half-lives
are 630 and 224 s, respectively. Accordingly, it will take
approximately 50 and 20 min, respectively, for slow binding equilibrium
to be established with indomethacin. The reactions must proceed at
least this long for the IC
to be a good estimate of K
*. This is impossible in vitro because
of the rapid autoinactivation. Consequently, the IC
values
can only represent pre-equilibrium inhibition because the dissociation
rates are much slower than the rates of autoinactivation. This being
the case, the apparent IC
values will vary depending on
preincubation time, incubation time, and factors that affect the rate
of enzyme autoinactivation. In general, IC
determinations
in which preincubation times are less than 5 times the EI*
association half-life will underestimate the affinity. Reactions in
which the EI* dissociation rate is greater than the
inactivation rate or incubation time will overestimate the affinity
because the enzyme will be bound in EI* and unable to compete
with substrate. Considering the complexity of the system, it is not
unexpected that the IC
values determined with different
assay protocols will vary considerably (Table 2). The reported
values for inhibition of human PGHS1 by indomethacin following
preincubations from 5 to 30 min range from 13 nM to 1.7
µM, and those for inhibition of human PGHS2 range from 74
nM to 25 µM. The selectivity ranged from 2.3 to
17. We determined an intrinsic selectivity of 30.
The apparent K represents the affinity of inhibitor for the
first equilibrium complex, EI. IC
values
determined by inhibition of the initial velocities should be
independent of the reaction dynamics and approximate the apparent K
. Laneuville and co-workers (19) reported
IC
values associated with instantaneous inhibition
determined from the inhibition of the initial rate of oxygen
consumption(19) . A good correlation was observed between the
IC
reported by Laneuville et al.(19) and K
values reported here for PGHS1. However, there
was a large discrepancy between the two PGHS2 sets of data. The
IC
values for instantaneous inhibition by indomethacin and
flurbiprofen were nearly 100 and 5 times greater, respectively, than
the K
values. We used the method of Laneuville et al.(19) and calculated IC
values
nearly identical to those they had reported (Table 2).
Surprisingly, when we determined the IC
values by adding
the inhibitors immediately prior to addition of the substrate instead
of simultaneously (preincubation less than 5 s), the IC
values corresponded to the K
values (Table 2). The only dynamic process we are aware of that could
potentially influence these determinations is the rate of catalytic
activation of the enzyme. It is well documented that the activation of
PGHSs by hydroperoxides must proceed the cyclooxygenase activity and
oxygen consumption. This results in a lag in enzyme activity after the
addition of arachidonic acid as a result of the time required to
synthesize sufficient quantity of the hydroperoxide product,
prostaglandin G
, needed to activate all the PGHS in the
reaction mixture. When inhibitor is added to an incubation prior to
arachidonic acid, it will interact only with unactivated enzyme,
whereas when inhibitor and arachidonic acid are added simultaneously
the inhibitor will be competing with arachidonic acid for binding to a
dynamic mixture of activated and unactivated enzyme. We can envision
two possible scenarios to account for the differences in IC
values between the two methods: (i) preincubation of inhibitor
with enzyme, even for 5 s, delays the activation and increases the
apparent affinity and/or (ii) the inhibitors have a different affinity
for activated and unactivated enzyme. While we cannot rule out the
first scenario, our laboratory does have preliminary evidence from
other studies that supports the hypothesis of an inhibitor sensitive
allosteric activation of the PGHS cyclooxygenase activity. (
)Our data suggests that there is a separate SAR for the
activated and unactivated states of both PGHS1 and PGHS2. Therefore,
the selectivity will be determined by the state of enzyme activation
and the sensitivity of the inhibitors to the activation state of the
enzyme. Indomethacin and flurbiprofen appear to be sensitive to the
activation state of PGHS2, not PGHS1.
The real value of any
simplified in vitro methodology is the ability to predict what
will occur in a more complex whole cell in vivo system. In the
preceding paragraphs, we presented an argument for why the kinetic
constants we have determined are a more accurate measure of the overall
inhibition, as defined by K*, than IC
values determined after preincubation. We have also discussed
factors that may effect K
and the IC
values associated with instantaneous inhibition. The next
question to answer is whether these kinetic parameters have any
relevance to inhibition in whole cells and in vivo.
Interestingly, the two reported studies of the inhibition human PGHSs
by indomethacin in whole cells had very different protocols and
results. Patrignani et al.(20) measured the
inhibition of platelet PGHS1 by indomethacin for 1 h and compared it
with the inhibition of lipopolysaccharide-induced monocyte PGHS2 for 4
h in human whole blood with no addition of exogenous arachidonic acid
and reported IC
values of 0.7 and 0.36 µM,
respectively. Chan et al.(21) measured the inhibition
of PGHS1 in U-937 cell and PGHS2 in osteosarcoma cells following a
15-min preincubation and a 10-min incubation with arachidonic acid and
determined IC
values of 5 and 10 nM,
respectively. One major difference between these two protocols is the
amount of time in which indomethacin is incubated with the enzymes. In
the experiments by Chan et al.(21) the reaction times
are much shorter than 5 times the half-life for dissociation, therefore
the enzyme will stay bound in the EI* complex during the
reaction and will not be able to equilibrate with the substrate. This
protocol should and does enhance the apparent affinity. In the
experiments by Patrignani et al.(20) the reaction
times are much longer than the time needed to dissociate from the
enzyme, therefore we would expect the IC
values to
approach the K
* values. This is the case for PGHS2
(IC
= 0.36 µM; K
*
= 1.02 µM) but not for PGHS1 (IC
= 0.7 µM; K
* =
0.03 µM).
Since we do not know the rates of
autoinactivation and catalytic activation in these cells, we cannot
determine if these dynamic processes play a role in the discrepancies
between the observed IC value and the K
*. Unfortunately it is these factors that may
play the most important role in determining the efficacy of inhibitors in vivo. First, if the rate of enzyme inactivation is less
than the dissociation rate, then the dissociation rates (k
), not the overall affinity (K
*) may be a better predictor of in vivo activity. And second, if the affinity of the inhibitor is
different for activated and unactivated enzyme, the in vivo efficacy will depend upon whether an inhibitor is introduced to a
cell whose PGHS system is activated or unactivated.
Not all time-dependent PGHS inhibitors are reversible slow binding inhibitors. Another class of time-dependent PGHS inhibitors, exemplified by NS-398 and DuP 697, was recently described by Copeland and co-workers (22) to be selective, irreversible inhibitors of PGHS2. Similar to the slow binding inhibitors, indomethacin, and flurbiprofen, these compounds could be recovered unaltered from the reaction mixture; however, the enzyme was not active after these compounds dissociated from the enzyme. These compounds appear to form an enzyme-inhibitor complex, which promotes inactivation of the enzyme in the absence of substrate. The advantage to be gained by inhibiting PGHS2 in vivo with a time-dependent irreversible inhibitor as compared to a time-dependent reversible inhibitor remains to be determined.
We
have presented a methodology for evaluation of intrinsic kinetic
parameters for slow binding inhibitors. This allows for the first time
the determination of the dissociation rates from the EI*
complex (k), the evaluation of the critical
enzyme-inhibitor structural interactions for K
, k
, and k
, the
determination of the overall affinity of a slow binding inhibitor (K
*), and the impact of these kinetic parameters
upon the cell-based and in vivo efficacy of PGHS inhibitors.
This should allow us to determine what factors affect the potency and
selectivity of slow binding inhibitors.