BACKGROUND
the precedence for tumor necrosis factor alpha  as a target has been well established by the anti-tnf biological therapeutics currently on the market  <cit> .
while the biologic therapies available are targeted at tnf directly, tnf production can be regulated at intracellular several points as well, including transcription, translation and shedding from its membrane-anchored precursor on the cell surface, all of which have been pursued as drug targets by various companies  <cit> .
p <dig> mapk was originally identified as the target of a compound that regulated the production of multiple pro-inflammatory cytokines, including tnf.
p38's regulation of tnf production is largely thought to be mediated via mk <dig>  one of its many substrates.
active mk <dig> serves to stabilize tnf mrna, thereby positively contributing to tnf production  <cit> .
dozens of small molecule p <dig> inhibitors have been put into the clinic for the treatment of chronic inflammatory diseases such as ra  <cit> .
these compounds represent a diverse chemical space  <cit>  and in spite of being highly selective  <cit> , none has yet made it to the market, with many failing due to adverse events, most notably liver enzyme elevation and skin rashes  <cit> .
it has been hypothesized that the adverse events may be mechanistically linked to p <dig>  <cit> , possibly due to the disruption of the normal p <dig> function within the cell, beyond regulation of tnf production.
thus, drug discovery teams have sought means to provide a more selective inhibition of tnf production.
it is very attractive to attempt to selectively block tnf by targeting intracellular signaling mechanisms regulating its production.
further, it has been surmised that intervening proximal to tnf  will help avoid unwanted effects.
to this end, mk <dig> has been investigated as a potential target  <cit> .
however, mk <dig> itself has proven to be a challenging molecule to selectively target with small molecules  <cit> .
consequently, attention has reverted to p <dig>  based on the established druggability of p <dig> as a target, its diverse role in cellular function and the specific role for mk <dig> in tnf production it has been hypothesized that specific modulation of this interaction would lead to an improved safety profile over previous p <dig> inhibitors.
this is the basis for the development of so-called 'substrate-selective' inhibitors as described by davidson, et al.
<cit> .
in davidson, et al.
a 'substrate-selective' a p38-alpha inhibitor was described that prevented p38-alpha-dependent mk <dig> phosphorylation  but did not prevent phosphorylation of another p <dig> substrate, atf- <dig>   <cit> .
the structural details of how this molecule is able to elicit differential inhibition of mk <dig> and atf <dig> were not disclosed or not known.
atf <dig> was used as a representative nuclear localized transcription factor in this assay, even though it is known to be phosphorylated by other kinases, such as jnk  <cit> .
atf <dig> is a histone acteyltransferase that binds dna in a sequence-specific manner  <cit> .
it activates a variety of gene targets including cyclin a, cyclin d, and c-jun, which are involved in oncogenesis  <cit> .
p <dig> phosphorylates atf <dig> on thr  <dig> and thr  <dig>  <cit> .
compounds satisfying the 'substrate-selective' criteria can be discovered through high-throughput screening approaches.
two screens are set up: one looking for p38-alpha mediated mk- <dig> phosphorylation and one for p38alpha mediated atf <dig> phosphorylation.
compounds are selected such that their potency in the mk <dig> assay >> atf <dig> assay.
thus, by the construction of the screening campaign, such compounds are said to be 'substrate-selective'.
in this work, we investigate the degree to which 'substrate-selectivity' holds as these classes of compounds are tested under conditions with multiple competing substrates.
using a combination of biochemical experiments and kinetic modeling we explore the contributions of mechanism and stoichiometry in determining the feasibility of the 'substrate-selective' mechanism under more complex, multi-substrate conditions.
RESULTS
previous work has defined a 'substrate-selective' p <dig> inhibitor as a compound that has a lower ic <dig> for one of its substrates than another, as assessed in independent assays  <cit> .
this behavior has been demonstrated for a p <dig> inhibitor described in davidson, et al  that exhibited a lower ic <dig> for mk <dig> than for atf- <dig>  both well-known substrates of p <dig>  <cit> .
in order to verify this behavior two assays were developed on the meso-scale discovery platform, one for the phosphorylated form of the transcription factor, atf <dig> and one for phosphorylated form of the kinase mk <dig>  as described in methods.
in each case, the degree of phosphorylation serves as a readout of the activity of p <dig> for its respective substrate.
we chose to evaluate the compound from the original davidson paper, cmpd <dig>  <cit>  with  <dig> traditional p <dig> inhibitors: sd- <dig>  <cit>  and birb  <dig>  <cit> .
using these assays, ic50's of the  <dig> compounds were measured against either mk <dig> or atf <dig>  shown in figure  <dig>  traditional p <dig> inhibitors, sd- <dig> and birb  <dig>  inhibited mk <dig> phosphorylation and atf <dig> phosphorylation in a dose-dependent manner with ic50s within 10-fold of each other .
by contrast, the phospho-atf <dig> dose-response curve for the cmpd <dig> are significantly right-shifted relative to the phospho-mk <dig> dose-response curve.
similar results were obtained for an in-house substrate-selective compound .
ic50's are shown ± standard error.
*substrate selectivity is defined as a relative measure between inhibiting phospho-atf <dig> and phospho-mk <dig> = /.
this quantity is distinct and separable from a compound's potency.
**cmpd <dig> is unable to completely inhibit phospho-atf <dig> within its solubility limits.
atf2/mk <dig> dual substrate assay
we next sought to determine how substrate-selective compounds would behave in a context where multiple competing substrates were present.
to this end, we designed a dual-substrate assay in which p <dig> could simultaneously phosphorylate mk <dig> and/or atf <dig>  the assay conditions chosen were  <dig>  nm p <dig>   <dig> nm atf <dig>   <dig> nm mk <dig>   <dig> um atp.
as with the single substrate assay, mk <dig> phosphorylation was assayed at  <dig> min and atf <dig> phosphorylation was assayed at  <dig> min to ensure that each measurement was within the linear range of the assay.
atp levels were measured at the end of the assay, to confirm that it was not being depleted.
the dual substrate assay was run in the absence of compound to examine the effect of the second substrate.
in the absence of compound, the addition of atf <dig> had no discernable effect on mk <dig> phosphorylation .
conversely, we found that addition of mk <dig> markedly inhibited atf <dig> phosphorylation, as one might expect given that mk <dig> has a much higher affinity for p <dig> than atf <dig> .
in order to aid in the interpretation of the dual substrate assay, we developed a simple kinetic model of p38-mediated phosphorylation of two substrates: atf <dig> and mk <dig> .
since p <dig> has a single kinase domain and atp binding site, we assumed that p <dig> can only act on a single substrate at a time.
for each substrate, a random-order bi-substrate  reaction mechanism was assumed.
active p <dig> can reversibly bind atp, with affinity kd, atp and atp binding is independent of further complex formation.
active p <dig>  can reversibly bind to atf <dig> or mk <dig> to form complexes p38-atf <dig> or p38-mk <dig>  respectively.
each complex undergoes an irreversible catalysis step to form products phospho-atf <dig>  or phospho-mk <dig> .
the model equations are a set of ordinary differential equations written in terms of mass-action kinetics.
binding interactions are characterized by affinities kd, atf <dig> or kd, mk <dig>  respectively, as listed in table  <dig>  catalysis rates kcat, atf <dig> and kcat, mk <dig> are also listed in table  <dig>  for the case where only one substrate is present, this model reduces to the single substrate assay.
using this simple competitive model, we simulated the single and dual-substrate assays .
the simulation results over a  <dig> min time scale indicate very subtle differences in atf <dig> phosphorylation between the single and dual substrate assays.
however, the experimental results from the dual substrate assay indicate a far more pronounced inhibition of atf <dig> phosphorylation in the dual substrate assay than seen in the simulation results.
thus, this basic competitive mechanism was not quantitatively consistent with the experimental data and prompted us to examine the basic mechanism further.
we next experimentally measured the effect of mk <dig> levels on the degree of atf <dig> phosphorylation for a fixed concentration of p <dig>  this demonstrated that the inhibition of atf <dig> phosphorylation by mk <dig> was dose-dependent.
.
secondly, we questioned whether the inhibition effect was due to mk <dig> specifically, or simply required any second p <dig> substrate.
for this we chose to use another known p <dig> substrate, 'peptide 4'  <cit> .
in our assays, the true km of this peptide was determined to be roughly  <dig> um .
the inhibition of atf <dig> phosphorylation was measured in the presence of peptide  <dig> at  <dig>   <dig>   <dig> and  <dig> um, and shown to have no effect on phospho-atf <dig>  independent of p <dig> levels used .
in order to explain the mk2-induced inhibition of atf <dig> phosphorylation seen in the experimental data, we hypothesized five alternate mechanisms:  <cit>  phospho-mk <dig> was inhibiting p <dig> via substrate inhibition;  <cit>  phospho-mk <dig> was binding atf <dig> preventing its interaction with p38; or  <cit>  p <dig> itself is modified after phosphorylating mk <dig> either by  <cit>  altering its affinity for atp,  <cit>  altering its affinity for atf <dig> or  <cit>  altering its catalytically activity for atf <dig>  each model was coded into the corresponding biochemical reaction scheme .
for each reaction scheme the single and dual substrate assays were simulated , as well as the dose-dependence on mk <dig> levels .
mechanism #1
in this mechanism, substrates are permitted to rebind to p <dig> after getting phosphorylated.
while this mechanism successfully impedes atf <dig> phosphorylation, it can nonetheless be ruled out because it is predicted to reduce overall mk <dig> phosphorylation.
further, this substrate inhibition mechanism fails to reproduce the observed dose-dependence on mk <dig> concentration .
if phospho-mk <dig> stays associated with p <dig> it will affect p38's ability to further phosphorylate other mk <dig> molecules resulting in the decrease in overall p-mk <dig>  finally, this mechanism also did not show sufficient sensitivity to mk <dig> levels as seen in the mk <dig> dose-response curve.
intuitively this occurs because mk <dig> levels already exceed p <dig> level in the assay, consequently, p <dig> is quickly saturated by phospho-mk <dig>
mechanism #2
in this mechanism, mk <dig> is allowed to bind atf <dig> and prevent its interaction with p <dig>  intuitively, this mechanism is limited by the stoichiometry of the assay, in which mk <dig> is at a 10-fold lower concentration than atf <dig>  consequently, this mechanism can be ruled out because no effect was seen on mk <dig> in addition to an insufficient magnitude of effect on the mk <dig> dose-response curve at high mk <dig> concentrations.
mechanisms #3-5
the remaining mechanisms investigate  <dig> ways in which the activity of the p <dig> kinase might be altered following interaction with mk <dig>  in mechanism # <dig>  mk <dig> alters the affinity of p <dig> for atp.
in this case, phosphorylation of atf <dig> is significantly inhibited.
by contrast, phospho-mk <dig> is relatively unaffected due to its high affinity for p <dig>  however, this mechanism shows little to no sensitivity to mk <dig> concentration and can consequently be ruled out as an independent mechanism.
mechanisms # <dig> and # <dig> posit that mk <dig> alters the affinity for atf <dig>  and the catalytic activity  of p <dig>  respectively.
each parameter was assumed to be affected 10-fold.
in both cases, these mechanisms are qualitatively consistent with the observed data.
they have no discernable effect on phosphorylation of mk <dig>  while dramatically inhibiting phosphorylation of atf <dig>  further, each shows a dose-dependence with total mk <dig> concentration.
model validation
in order to validate the model, we aimed to predict and measure the behavior of a perfectly non-substrate selective p <dig> inhibitor.
since we cannot be guaranteed that any of the compounds exhibit this idealized behavior we devised a 'virtual p <dig> compound' that could be tested experimentally.
conceptually, an ideal non-substrate-selective inhibitor of p <dig> would bind p <dig> and prevent its activity, effectively titrating out the p <dig>  experimentally and computationally, this could be performed by simply lowering the p <dig> level in concordance with a simple inhibitor-p <dig> binding isotherm .
the resulting relationship between 'virtual compound' and free p <dig> is shown in figure 7a.
using the model, the virtual inhibitor is simulated.
mechanisms # <dig> & # <dig> predict a discernable left-shift in ic <dig> for the dual-substrate assay and no effect on the phospho-mk <dig> assay .
the magnitude of the shift in each case is dependent on how much the corresponding parameter is affected following mk <dig> interaction.
the 'virtual compound' was tested in the single and dual substrate assays, shown in figure 7c.
as predicted with the kinetic model, there was no effect on the mk <dig> ic <dig> and a significant left-shift in the atf <dig> ic <dig>  thus, the simple presence of mk <dig> reduces the ability of p <dig> to phosphorylate atf <dig>  even in the absence of compound.
further, this effect is qualitatively and quantitatively consistent with mk <dig> altering p38's affinity and/or catalytic activity for atf <dig>
if the p38-mk <dig> interaction affected p38's subsequent ability to phosphorylate atf <dig>  we predict that p <dig> should have a markedly different ability to phosphorylate atf <dig> with and without pre-incubation with mk <dig>  to test this, p <dig> was incubated with different amounts of mk <dig> for  <dig> min, under the same reaction conditions as in the single substrate assay.
following incubation, p <dig> was immunoprecipitated to remove it from mk <dig>  the immunoprecipitates were analyzed by western blot to demonstrate there was no mk <dig> contamination .
finally, the immunoprecipitated p <dig> was then used in a subsequent reaction with atf <dig> and phospho-atf <dig> measured after  <dig> min of incubation time.
as shown in figure  <dig>  pre-incubation with mk <dig> dramatically inhibited the ability of p <dig> to phosphorylate atf <dig>
having validated the model and developed a grasp of the potential mechanisms underlying the behavior of the dual substrate assay, we then sought to simulate the effects of both classical and substrate-selective p <dig> inhibitors.
a classical, non-substrate-selective p <dig> inhibitor was simulated in a standard fashion by adding to the model an inhibitor that could interact with all forms of p <dig> with equal affinity, characterized by ki.
in order to simulate the presence of a 'substrate-selective' compound we added a compound to the kinetic model that binds the p38-mk <dig> complex with affinity ki.
the compound was allowed to bind to free p <dig> and p38-atf <dig> with a reduced affinity kd, i/ where f is the selectivity .
this compound serves to stabilize the p38-mk <dig> complex and thus the mk <dig> dissociation rate constant must be multiplied by  to satisfy thermodynamic constraints of microscopic reversibility.
for our purposes, we assumed a highly potent and selective compound with ki =  <dig> nm and f =  <dig> .
the details of kinetic mechanisms are cartooned in figure  <dig>  both atp-competitive and atp non-competitive compounds were simulated.
dose-response curves were simulated for classical and substrate-selective inhibitors, predicting the effects on phospho-atf <dig> and phospho-mk <dig> for both the single and dual substrate assays .
inhibitor dose-response curves for the phospho-atf <dig> and phospho-mk <dig> measurements were generated at the  <dig> and  <dig> min time points, respectively.
the model simulations predict that as one moves from the single substrate assay to the dual substrate assay, there will be a left-shift in phospho-atf <dig> ic <dig> for the classical p <dig> inhibitors and an even greater left-shift for the substrate-selective inhibitors.
meanwhile, the simulations predicted no change in the phospho-mk <dig> ic <dig> between the single and dual substrate assay for either inhibitor.
simulation results are shown in figure  <dig>  both atp-competitive and atp non-competitive compounds had qualitatively indistinguishable results.
compound evaluation
we next tested our set of compounds in the dual substrate assay.
all compounds were run in the single and dual substrate assays and ic50's determined for phospho-atf <dig> and phospho-mk <dig> .
as predicted by the model, there was no discernable effect on mk <dig> ic <dig>  further, the model correctly predicted a left-shift in atf <dig> ic <dig>  regardless of whether or not the compound was 'substrate-selective'.
there were two novel findings that were correctly predicted via the modeling effort:  the substrate-selective inhibitor showed a loss of substrate-selective behavior in the dual substrate assays and  the classical p <dig> inhibitors also showed a decrease in the atf <dig> ic <dig> in the dual substrate assay.
the loss of substrate-selectivity in the dual substrate assay was also replicated with a proprietary substrate-selective compound .
ic50's are shown ± standard error.
substrate selectivity = /
the modest left-shift predicted and experimentally observed in phospho-atf <dig> ic <dig> in the classical inhibitors is due to the effect of mk <dig> on p38's ability to phosphorylate atf <dig>  the greater left-shift seen in substrate-selective inhibitors can be broken down into two parts:  the mk2-mediated effect on p <dig> and a second effect : mechanistically, a substrate-selective compound is designed to stabilize the p38-mk <dig> complex .
when compound is added, mass-action drives the formation of the p38-mk2-compound complex.
since  >  in our assay design, all of the active p <dig> will be sequestered into the p38-mk2-inhibitor complex, reducing the pool of active p <dig> that is free to phosphorylate atf <dig>  in spite of the apparent 'substrate-selective' behavior seen in the single assays.
cell characterization
thus far, we have demonstrated both in silico and biochemically, that the presence of additional substrates results in the loss of substrate selectivity.
due to the sequestration effect of substrate selective compounds, we have found that a necessary condition for this to take place in vivo is that  < .
in order to see if this holds in relevant cell types, we measured protein expression levels of p <dig> and mk <dig> in the pma-activated u <dig> and thp- <dig> monocytic cell lines as well as in primary peripheral blood mononuclear cells .
protein expression was measured via western blot on a per cell basis using recombinant standards and quantifying bands via densitometry, shown in figure  <dig>  in both u <dig> and thp- <dig> cells, there are comparable protein levels of p <dig> and mk <dig> and in pbmcs the total protein level of mk <dig> significantly exceeds the total protein level of p <dig>  further, given that not all p <dig> in a cell is in the active conformation, our data strongly suggests that in situ  < .
this is also consistent with other reports of protein expression levels seen in mapk signaling cascades  <cit> .
under these conditions one would expect the sequestration effect of substrate-selective compounds to take place.
discussion
the concept of a 'substrate-selective' inhibitor as a means to avoid unwanted side-effects is a very attractive one.
the use of multiple screening assays to identify such compounds is a convenient and efficient method for identifying chemical entities with specific effects.
however, great care should be taken to understand the cellular target to determine the feasibility of such strategies particularly in more complex environments.
from this work we specifically sought to explore the parameters governing the effectiveness of the substrate-selective inhibitor strategy in general and in particular for the p38-mk <dig> system.
on the path to understanding the behavior of substrate-selective inhibitors, an additional mechanism was uncovered: following interaction with mk <dig>  the activity of p <dig> with regard to atf <dig> is substantially reduced.
from our analysis, there are multiple mechanisms that could give rise to this, including alteration of the affinity for atf <dig> or the catalytic rate constant.
further determination of kinetic mechanism and molecular details was beyond the scope of this work.
one might hypothesize that mk <dig> may, in some way, be eliciting an inhibitory phosphorylation on p <dig>  however, this remains to be demonstrated.
given that mk <dig> already has a much higher affinity for p <dig> than atf <dig>  one may ask how atf <dig> would get phosphorylated at all within the cell .
in this case, one must recall that these are competing kinetic processes, rather than static events.
our dual substrate assay time course  confirms that atf <dig> phosphorylation continues at a measurable pace, albeit on a slower time-scale than mk <dig> .
thus, abundant mk <dig> does not prevent p <dig> from phosphorylating atf <dig> or other substrates, but merely slows it down.
simulations  further demonstrate that marked atf <dig> phosphorylation is also quantitatively consistent with reported affinities of p <dig> for atf <dig> and mk <dig>  even though p <dig> has markedly different affinities for atf <dig> and mk <dig>  we have demonstrated experimentally and computationally, that both substrates may get phosphorylated in a biochemical system with the key difference being the time scale over which they occur.
further, this work demonstrates that when atf <dig> and mk <dig> are both present, a so-called 'p <dig> substrate-selective' inhibitor will inhibit the p38-mediated phosphorylation of both substrates comparably as a consequence of a sequestration phenomenon driven by an excess of mk <dig> relative to active p <dig>
we have used our computational model to predict that the introduction of multiple substrates would result in the loss of substrate selectivity and experimentally validated this finding in a biochemical assay.
alternately stated, the addition of mk <dig> to the p38-atf <dig> reaction was able to make cmpd <dig> a potent inhibitor of atf <dig> phosphorylation.
through the construction of a kinetic model of the proposed mechanism of action we demonstrate that these findings are a general result and not a compound-specific finding.
our analysis demonstrated that relative p <dig> and mk <dig> levels play a defining role in determining that the substrate-selective mechanism is not likely to work as intended in vivo.
additionally, this mechanism of sequestration-mediated inhibition of secondary substrates would extend to other substrates than atf <dig> as well.
it is worth noting that the presence of scaffolding proteins and higher-order interactions taking place in the cell that may locally alter protein concentration and drive interaction that would otherwise not take place in free solution.
one cannot explicitly model such effects, however, given that their purpose is to locally increase protein concentration it is unlikely to change the outcome of our analysis.
in our kinetic model, we modeled each phosphorylation event as a one-hit reaction, even though p <dig> is known to phosphorylate mk <dig> and atf <dig> at multiple sites .
this was done to restrict the number of kinetic parameters in the model to as few as needed to adequately recapture the overall system behavior and be consistent with the experimentally measurable quantities, namely single phosphorylation sites.
conceptually, one could consider p38-mediated phosphorylation or activation as the process by which p <dig> comes into contact with one of its substrates and performs the necessary modifications, either via one single-step reaction, or multiple dissociation and rebinding events.
in order to perturb this process a compound needs to interfere with only one of these events.
our experimental data confirm that our compounds are able to interfere with mk <dig> and/or atf <dig> phosphorylation, even though only single site was measured.
if a substrate-selective inhibitor stabilizes the p38-mk <dig> interaction  it will effectively sequester and prevent p <dig> from performing atf <dig> phosphorylation, regardless of the number of phosphorylation sites.
consequently, the single phosphorylation site kinetic model was sufficient to describe and predict the observed behavior of both classical and substrate-selective model.
to further address the question of how multiple phosphorylation sites may affect a substrate-selective inhibitor we constructed a simplified model incorporating varying numbers of atf <dig> and mk <dig> phosphorylation sites .
it was found that increasing the number of atf <dig> phosphorylation sites resulted in an increased potency against maximal atf <dig> phosphorylation and this was independent of the number of mk <dig> phosphorylation sites.
similarly, increasing mk <dig> phosphorylation sites resulted in an increased potency against maximal mk <dig> phosphorylation .
importantly, these effects were independent of compound type.
thus, by having more mk <dig> phosphorylation sites than atf <dig> phosphorylation sites, cells may naturally achieve substrate-selectivity.
it is unclear what functional role, if any, this plays.
importantly, the incorporation of multiple phosphorylation sites did not affect the conclusion that under the condition where  > , a 'substrate-selective' inhibitor will inhibit the phosphorylation of both mk <dig> and atf <dig>
in three monocytic cell systems we have shown that total mk <dig> protein expression does not exceed that of p <dig> in resting cells.
in a resting cell inactive mk <dig> is reported to reside in the nucleus and the active form of p <dig> undergoes nuclear import  <cit> .
thus, when considering this compartmentalization, the level of mk <dig> may even further exceed the level of active p <dig>  consequently, it is unlikely that substrate selectivity can be achieved for any nuclear substrates of p <dig>  however, due to the compartmentalization and the nuclear/cytoplasmic shuttling of the active forms, it may be feasible to obtain substrate-selectivity for cytosolic substrates.
in these cases, however, substrate selectivity is not a general property and will vary based on substrate levels and substrate-dependent kinetic interaction rates.
our analysis has centered on a simplified model and experimental system and may differ from in vivo situations in a number of ways.
for example, this work did not take into account the mechanism by which p <dig> gets activated, whether it be via upstream kinases or via autophosphorylation  <cit> .
it is possible that various inhibitors may bind differently to active and inactive forms of p <dig>  further, many p <dig> compounds have been observed to inhibit p <dig> activation as well  <cit> .
neither of these phenomena would be expected to affect the stoichiometric effect by which mk <dig> levels would soak up all available active p <dig> when treated with 'substrate-selective' inhibitors.
when one considers that there are multiple converging pathways on atf <dig>  mk <dig> , as well positive and negative feedback loops that regulate activity and expression levels of enzymes and substrates, accurately predicting overall behavior becomes considerably more complex.
nonetheless, our kinetic model provides initial guidance for how a sub-system of the signaling network may operate and serves as a building block for adding in additional complexity.
CONCLUSIONS
the analysis presented herein suggests that the substrate-selective mechanism of inhibiting is not an effective strategy for p38/mk2/atf <dig> system in cases where the concentration of mk <dig> exceeds the concentration of p <dig>  this work may point to general properties regarding the substrate-selective inhibitor concept.
with regard to other secondary substrates, beyond atf <dig>  we believe they will also be inhibited due to the stoichiometric excess of mk <dig> relative to active p <dig>  further, we believe this work contributes to determining biological conditions that are required for the substrate-selective inhibitor strategy to be effective.
there may be other kinase-substrate pairs in which the stoichiometry is not limiting for the substrate-selective mechanism and our computational model is easily adapted for their evaluation.
however, moving closer to the receptor level may broaden the downstream effects by virtue of being farther from transcriptional and translational endpoints.
overall, targeting kinase-substrate complexes may offer a general approach and new avenue for achieving selectivity for kinases with high structural similarity to other proteins.
the different requirements of targeting the p38-mk <dig> complex vs. free p <dig> may lead us into new chemical space that could still find alternate avenues to differentiate from previous p <dig> inhibitors.

 the precedence for tumor necrosis factor alpha as a target has been well established by the anti-tnf biological therapeutics currently on the market. while the biologic therapies available are targeted at tnf directly, tnf production can be regulated at intracellular several points as well, including transcription, translation and shedding from its membrane-anchored precursor on the cell surface, all of which have been pursued as drug targets by various companies. p mapk was originally identified as the target of a compound that regulated the production of multiple pro-inflammatory cytokines, including tnf. p38's regulation of tnf production is largely thought to be mediated via mk one of its many substrates. active mk serves to stabilize tnf mrna, thereby positively contributing to tnf production. dozens of small molecule p inhibitors have been put into the clinic for the treatment of chronic inflammatory diseases such as ra. these compounds represent a diverse chemical space and in spite of being highly selective, none has yet made it to the market, with many failing due to adverse events, most notably liver enzyme elevation and skin rashes. it has been hypothesized that the adverse events may be mechanistically linked to p, possibly due to the disruption of the normal p function within the cell, beyond regulation of tnf production. thus, drug discovery teams have sought means to provide a more selective inhibition of tnf production. it is very attractive to attempt to selectively block tnf by targeting intracellular signaling mechanisms regulating its production. further, it has been surmised that intervening proximal to tnf will help avoid unwanted effects. to this end, mk has been investigated as a potential target. however, mk itself has proven to be a challenging molecule to selectively target with small molecules. consequently, attention has reverted to p based on the established druggability of p as a target, its diverse role in cellular function and the specific role for mk in tnf production it has been hypothesized that specific modulation of this interaction would lead to an improved safety profile over previous p inhibitors. this is the basis for the development of so-called'substrate-selective' inhibitors as described by davidson, et al.. in davidson, et al. a'substrate-selective' a p38-alpha inhibitor was described that prevented p38-alpha-dependent mk phosphorylation but did not prevent phosphorylation of another p substrate, atf-. the structural details of how this molecule is able to elicit differential inhibition of mk and atf were not disclosed or not known. atf was used as a representative nuclear localized transcription factor in this assay, even though it is known to be phosphorylated by other kinases, such as jnk. atf is a histone acteyltransferase that binds dna in a sequence-specific manner. it activates a variety of gene targets including cyclin a, cyclin d, and c-jun, which are involved in oncogenesis. p phosphorylates atf on thr and thr. compounds satisfying the'substrate-selective' criteria can be discovered through high-throughput screening approaches. two screens are set up: one looking for p38-alpha mediated mk- phosphorylation and one for p38alpha mediated atf phosphorylation. compounds are selected such that their potency in the mk assay >> atf assay. thus, by the construction of the screening campaign, such compounds are said to be'substrate-selective'. in this work, we investigate the degree to which'substrate-selectivity' holds as these classes of compounds are tested under conditions with multiple competing substrates. using a combination of biochemical experiments and kinetic modeling we explore the contributions of mechanism and stoichiometry in determining the feasibility of the'substrate-selective' mechanism under more complex, multi-substrate conditions.  previous work has defined a'substrate-selective' p inhibitor as a compound that has a lower ic for one of its substrates than another, as assessed in independent assays. this behavior has been demonstrated for a p inhibitor described in davidson, et al that exhibited a lower ic for mk than for atf- both well-known substrates of p. in order to verify this behavior two assays were developed on the meso-scale discovery platform, one for the phosphorylated form of the transcription factor, atf and one for phosphorylated form of the kinase mk as described in methods. in each case, the degree of phosphorylation serves as a readout of the activity of p for its respective substrate. we chose to evaluate the compound from the original davidson paper, cmpd with traditional p inhibitors: sd- and birb. using these assays, ic50's of the compounds were measured against either mk or atf shown in figure traditional p inhibitors, sd- and birb inhibited mk phosphorylation and atf phosphorylation in a dose-dependent manner with ic50s within 10-fold of each other. by contrast, the phospho-atf dose-response curve for the cmpd are significantly right-shifted relative to the phospho-mk dose-response curve. similar results were obtained for an in-house substrate-selective compound. ic50's are shown ± standard error. *substrate selectivity is defined as a relative measure between inhibiting phospho-atf and phospho-mk = /. this quantity is distinct and separable from a compound's potency. **cmpd is unable to completely inhibit phospho-atf within its solubility limits. atf2/mk dual substrate assay we next sought to determine how substrate-selective compounds would behave in a context where multiple competing substrates were present. to this end, we designed a dual-substrate assay in which p could simultaneously phosphorylate mk and/or atf the assay conditions chosen were nm p nm atf nm mk um atp. as with the single substrate assay, mk phosphorylation was assayed at min and atf phosphorylation was assayed at min to ensure that each measurement was within the linear range of the assay. atp levels were measured at the end of the assay, to confirm that it was not being depleted. the dual substrate assay was run in the absence of compound to examine the effect of the second substrate. in the absence of compound, the addition of atf had no discernable effect on mk phosphorylation. conversely, we found that addition of mk markedly inhibited atf phosphorylation, as one might expect given that mk has a much higher affinity for p than atf. in order to aid in the interpretation of the dual substrate assay, we developed a simple kinetic model of p38-mediated phosphorylation of two substrates: atf and mk. since p has a single kinase domain and atp binding site, we assumed that p can only act on a single substrate at a time. for each substrate, a random-order bi-substrate reaction mechanism was assumed. active p can reversibly bind atp, with affinity kd, atp and atp binding is independent of further complex formation. active p can reversibly bind to atf or mk to form complexes p38-atf or p38-mk respectively. each complex undergoes an irreversible catalysis step to form products phospho-atf or phospho-mk. the model equations are a set of ordinary differential equations written in terms of mass-action kinetics. binding interactions are characterized by affinities kd, atf or kd, mk respectively, as listed in table catalysis rates kcat, atf and kcat, mk are also listed in table for the case where only one substrate is present, this model reduces to the single substrate assay. using this simple competitive model, we simulated the single and dual-substrate assays. the simulation results over a min time scale indicate very subtle differences in atf phosphorylation between the single and dual substrate assays. however, the experimental results from the dual substrate assay indicate a far more pronounced inhibition of atf phosphorylation in the dual substrate assay than seen in the simulation results. thus, this basic competitive mechanism was not quantitatively consistent with the experimental data and prompted us to examine the basic mechanism further. we next experimentally measured the effect of mk levels on the degree of atf phosphorylation for a fixed concentration of p this demonstrated that the inhibition of atf phosphorylation by mk was dose-dependent.. secondly, we questioned whether the inhibition effect was due to mk specifically, or simply required any second p substrate. for this we chose to use another known p substrate,'peptide 4'. in our assays, the true km of this peptide was determined to be roughly um. the inhibition of atf phosphorylation was measured in the presence of peptide at and um, and shown to have no effect on phospho-atf independent of p levels used. in order to explain the mk2-induced inhibition of atf phosphorylation seen in the experimental data, we hypothesized five alternate mechanisms: phospho-mk was inhibiting p via substrate inhibition; phospho-mk was binding atf preventing its interaction with p38; or p itself is modified after phosphorylating mk either by altering its affinity for atp, altering its affinity for atf or altering its catalytically activity for atf each model was coded into the corresponding biochemical reaction scheme. for each reaction scheme the single and dual substrate assays were simulated, as well as the dose-dependence on mk levels. mechanism #1 in this mechanism, substrates are permitted to rebind to p after getting phosphorylated. while this mechanism successfully impedes atf phosphorylation, it can nonetheless be ruled out because it is predicted to reduce overall mk phosphorylation. further, this substrate inhibition mechanism fails to reproduce the observed dose-dependence on mk concentration. if phospho-mk stays associated with p it will affect p38's ability to further phosphorylate other mk molecules resulting in the decrease in overall p-mk finally, this mechanism also did not show sufficient sensitivity to mk levels as seen in the mk dose-response curve. intuitively this occurs because mk levels already exceed p level in the assay, consequently, p is quickly saturated by phospho-mk mechanism #2 in this mechanism, mk is allowed to bind atf and prevent its interaction with p intuitively, this mechanism is limited by the stoichiometry of the assay, in which mk is at a 10-fold lower concentration than atf consequently, this mechanism can be ruled out because no effect was seen on mk in addition to an insufficient magnitude of effect on the mk dose-response curve at high mk concentrations. mechanisms #3-5 the remaining mechanisms investigate ways in which the activity of the p kinase might be altered following interaction with mk in mechanism # mk alters the affinity of p for atp. in this case, phosphorylation of atf is significantly inhibited. by contrast, phospho-mk is relatively unaffected due to its high affinity for p however, this mechanism shows little to no sensitivity to mk concentration and can consequently be ruled out as an independent mechanism. mechanisms # and # posit that mk alters the affinity for atf and the catalytic activity of p respectively. each parameter was assumed to be affected 10-fold. in both cases, these mechanisms are qualitatively consistent with the observed data. they have no discernable effect on phosphorylation of mk while dramatically inhibiting phosphorylation of atf further, each shows a dose-dependence with total mk concentration. model validation in order to validate the model, we aimed to predict and measure the behavior of a perfectly non-substrate selective p inhibitor. since we cannot be guaranteed that any of the compounds exhibit this idealized behavior we devised a'virtual p compound' that could be tested experimentally. conceptually, an ideal non-substrate-selective inhibitor of p would bind p and prevent its activity, effectively titrating out the p experimentally and computationally, this could be performed by simply lowering the p level in concordance with a simple inhibitor-p binding isotherm. the resulting relationship between'virtual compound' and free p is shown in figure 7a. using the model, the virtual inhibitor is simulated. mechanisms # & # predict a discernable left-shift in ic for the dual-substrate assay and no effect on the phospho-mk assay. the magnitude of the shift in each case is dependent on how much the corresponding parameter is affected following mk interaction. the'virtual compound' was tested in the single and dual substrate assays, shown in figure 7c. as predicted with the kinetic model, there was no effect on the mk ic and a significant left-shift in the atf ic thus, the simple presence of mk reduces the ability of p to phosphorylate atf even in the absence of compound. further, this effect is qualitatively and quantitatively consistent with mk altering p38's affinity and/or catalytic activity for atf if the p38-mk interaction affected p38's subsequent ability to phosphorylate atf we predict that p should have a markedly different ability to phosphorylate atf with and without pre-incubation with mk to test this, p was incubated with different amounts of mk for min, under the same reaction conditions as in the single substrate assay. following incubation, p was immunoprecipitated to remove it from mk the immunoprecipitates were analyzed by western blot to demonstrate there was no mk contamination. finally, the immunoprecipitated p was then used in a subsequent reaction with atf and phospho-atf measured after min of incubation time. as shown in figure pre-incubation with mk dramatically inhibited the ability of p to phosphorylate atf having validated the model and developed a grasp of the potential mechanisms underlying the behavior of the dual substrate assay, we then sought to simulate the effects of both classical and substrate-selective p inhibitors. a classical, non-substrate-selective p inhibitor was simulated in a standard fashion by adding to the model an inhibitor that could interact with all forms of p with equal affinity, characterized by ki. in order to simulate the presence of a'substrate-selective' compound we added a compound to the kinetic model that binds the p38-mk complex with affinity ki. the compound was allowed to bind to free p and p38-atf with a reduced affinity kd, i/ where f is the selectivity. this compound serves to stabilize the p38-mk complex and thus the mk dissociation rate constant must be multiplied by to satisfy thermodynamic constraints of microscopic reversibility. for our purposes, we assumed a highly potent and selective compound with ki = nm and f =. the details of kinetic mechanisms are cartooned in figure both atp-competitive and atp non-competitive compounds were simulated. dose-response curves were simulated for classical and substrate-selective inhibitors, predicting the effects on phospho-atf and phospho-mk for both the single and dual substrate assays. inhibitor dose-response curves for the phospho-atf and phospho-mk measurements were generated at the and min time points, respectively. the model simulations predict that as one moves from the single substrate assay to the dual substrate assay, there will be a left-shift in phospho-atf ic for the classical p inhibitors and an even greater left-shift for the substrate-selective inhibitors. meanwhile, the simulations predicted no change in the phospho-mk ic between the single and dual substrate assay for either inhibitor. simulation results are shown in figure both atp-competitive and atp non-competitive compounds had qualitatively indistinguishable results. compound evaluation we next tested our set of compounds in the dual substrate assay. all compounds were run in the single and dual substrate assays and ic50's determined for phospho-atf and phospho-mk. as predicted by the model, there was no discernable effect on mk ic further, the model correctly predicted a left-shift in atf ic regardless of whether or not the compound was'substrate-selective'. there were two novel findings that were correctly predicted via the modeling effort: the substrate-selective inhibitor showed a loss of substrate-selective behavior in the dual substrate assays and the classical p inhibitors also showed a decrease in the atf ic in the dual substrate assay. the loss of substrate-selectivity in the dual substrate assay was also replicated with a proprietary substrate-selective compound. ic50's are shown ± standard error. substrate selectivity = / the modest left-shift predicted and experimentally observed in phospho-atf ic in the classical inhibitors is due to the effect of mk on p38's ability to phosphorylate atf the greater left-shift seen in substrate-selective inhibitors can be broken down into two parts: the mk2-mediated effect on p and a second effect: mechanistically, a substrate-selective compound is designed to stabilize the p38-mk complex. when compound is added, mass-action drives the formation of the p38-mk2-compound complex. since > in our assay design, all of the active p will be sequestered into the p38-mk2-inhibitor complex, reducing the pool of active p that is free to phosphorylate atf in spite of the apparent'substrate-selective' behavior seen in the single assays. cell characterization thus far, we have demonstrated both in silico and biochemically, that the presence of additional substrates results in the loss of substrate selectivity. due to the sequestration effect of substrate selective compounds, we have found that a necessary condition for this to take place in vivo is that <. in order to see if this holds in relevant cell types, we measured protein expression levels of p and mk in the pma-activated u and thp- monocytic cell lines as well as in primary peripheral blood mononuclear cells. protein expression was measured via western blot on a per cell basis using recombinant standards and quantifying bands via densitometry, shown in figure in both u and thp- cells, there are comparable protein levels of p and mk and in pbmcs the total protein level of mk significantly exceeds the total protein level of p further, given that not all p in a cell is in the active conformation, our data strongly suggests that in situ <. this is also consistent with other reports of protein expression levels seen in mapk signaling cascades. under these conditions one would expect the sequestration effect of substrate-selective compounds to take place. discussion the concept of a'substrate-selective' inhibitor as a means to avoid unwanted side-effects is a very attractive one. the use of multiple screening assays to identify such compounds is a convenient and efficient method for identifying chemical entities with specific effects. however, great care should be taken to understand the cellular target to determine the feasibility of such strategies particularly in more complex environments. from this work we specifically sought to explore the parameters governing the effectiveness of the substrate-selective inhibitor strategy in general and in particular for the p38-mk system. on the path to understanding the behavior of substrate-selective inhibitors, an additional mechanism was uncovered: following interaction with mk the activity of p with regard to atf is substantially reduced. from our analysis, there are multiple mechanisms that could give rise to this, including alteration of the affinity for atf or the catalytic rate constant. further determination of kinetic mechanism and molecular details was beyond the scope of this work. one might hypothesize that mk may, in some way, be eliciting an inhibitory phosphorylation on p however, this remains to be demonstrated. given that mk already has a much higher affinity for p than atf one may ask how atf would get phosphorylated at all within the cell. in this case, one must recall that these are competing kinetic processes, rather than static events. our dual substrate assay time course confirms that atf phosphorylation continues at a measurable pace, albeit on a slower time-scale than mk. thus, abundant mk does not prevent p from phosphorylating atf or other substrates, but merely slows it down. simulations further demonstrate that marked atf phosphorylation is also quantitatively consistent with reported affinities of p for atf and mk even though p has markedly different affinities for atf and mk we have demonstrated experimentally and computationally, that both substrates may get phosphorylated in a biochemical system with the key difference being the time scale over which they occur. further, this work demonstrates that when atf and mk are both present, a so-called'p substrate-selective' inhibitor will inhibit the p38-mediated phosphorylation of both substrates comparably as a consequence of a sequestration phenomenon driven by an excess of mk relative to active p we have used our computational model to predict that the introduction of multiple substrates would result in the loss of substrate selectivity and experimentally validated this finding in a biochemical assay. alternately stated, the addition of mk to the p38-atf reaction was able to make cmpd a potent inhibitor of atf phosphorylation. through the construction of a kinetic model of the proposed mechanism of action we demonstrate that these findings are a general result and not a compound-specific finding. our analysis demonstrated that relative p and mk levels play a defining role in determining that the substrate-selective mechanism is not likely to work as intended in vivo. additionally, this mechanism of sequestration-mediated inhibition of secondary substrates would extend to other substrates than atf as well. it is worth noting that the presence of scaffolding proteins and higher-order interactions taking place in the cell that may locally alter protein concentration and drive interaction that would otherwise not take place in free solution. one cannot explicitly model such effects, however, given that their purpose is to locally increase protein concentration it is unlikely to change the outcome of our analysis. in our kinetic model, we modeled each phosphorylation event as a one-hit reaction, even though p is known to phosphorylate mk and atf at multiple sites. this was done to restrict the number of kinetic parameters in the model to as few as needed to adequately recapture the overall system behavior and be consistent with the experimentally measurable quantities, namely single phosphorylation sites. conceptually, one could consider p38-mediated phosphorylation or activation as the process by which p comes into contact with one of its substrates and performs the necessary modifications, either via one single-step reaction, or multiple dissociation and rebinding events. in order to perturb this process a compound needs to interfere with only one of these events. our experimental data confirm that our compounds are able to interfere with mk and/or atf phosphorylation, even though only single site was measured. if a substrate-selective inhibitor stabilizes the p38-mk interaction it will effectively sequester and prevent p from performing atf phosphorylation, regardless of the number of phosphorylation sites. consequently, the single phosphorylation site kinetic model was sufficient to describe and predict the observed behavior of both classical and substrate-selective model. to further address the question of how multiple phosphorylation sites may affect a substrate-selective inhibitor we constructed a simplified model incorporating varying numbers of atf and mk phosphorylation sites. it was found that increasing the number of atf phosphorylation sites resulted in an increased potency against maximal atf phosphorylation and this was independent of the number of mk phosphorylation sites. similarly, increasing mk phosphorylation sites resulted in an increased potency against maximal mk phosphorylation. importantly, these effects were independent of compound type. thus, by having more mk phosphorylation sites than atf phosphorylation sites, cells may naturally achieve substrate-selectivity. it is unclear what functional role, if any, this plays. importantly, the incorporation of multiple phosphorylation sites did not affect the conclusion that under the condition where >, a'substrate-selective' inhibitor will inhibit the phosphorylation of both mk and atf in three monocytic cell systems we have shown that total mk protein expression does not exceed that of p in resting cells. in a resting cell inactive mk is reported to reside in the nucleus and the active form of p undergoes nuclear import. thus, when considering this compartmentalization, the level of mk may even further exceed the level of active p consequently, it is unlikely that substrate selectivity can be achieved for any nuclear substrates of p however, due to the compartmentalization and the nuclear/cytoplasmic shuttling of the active forms, it may be feasible to obtain substrate-selectivity for cytosolic substrates. in these cases, however, substrate selectivity is not a general property and will vary based on substrate levels and substrate-dependent kinetic interaction rates. our analysis has centered on a simplified model and experimental system and may differ from in vivo situations in a number of ways. for example, this work did not take into account the mechanism by which p gets activated, whether it be via upstream kinases or via autophosphorylation. it is possible that various inhibitors may bind differently to active and inactive forms of p further, many p compounds have been observed to inhibit p activation as well. neither of these phenomena would be expected to affect the stoichiometric effect by which mk levels would soak up all available active p when treated with'substrate-selective' inhibitors. when one considers that there are multiple converging pathways on atf mk, as well positive and negative feedback loops that regulate activity and expression levels of enzymes and substrates, accurately predicting overall behavior becomes considerably more complex. nonetheless, our kinetic model provides initial guidance for how a sub-system of the signaling network may operate and serves as a building block for adding in additional complexity.  the analysis presented herein suggests that the substrate-selective mechanism of inhibiting is not an effective strategy for p38/mk2/atf system in cases where the concentration of mk exceeds the concentration of p this work may point to general properties regarding the substrate-selective inhibitor concept. with regard to other secondary substrates, beyond atf we believe they will also be inhibited due to the stoichiometric excess of mk relative to active p further, we believe this work contributes to determining biological conditions that are required for the substrate-selective inhibitor strategy to be effective. there may be other kinase-substrate pairs in which the stoichiometry is not limiting for the substrate-selective mechanism and our computational model is easily adapted for their evaluation. however, moving closer to the receptor level may broaden the downstream effects by virtue of being farther from transcriptional and translational endpoints. overall, targeting kinase-substrate complexes may offer a general approach and new avenue for achieving selectivity for kinases with high structural similarity to other proteins. the different requirements of targeting the p38-mk complex vs. free p may lead us into new chemical space that could still find alternate avenues to differentiate from previous p inhibitors. 
we find that, given our current understanding, it is unlikely that'substrate-selective' inhibitors of p will work as originally intended when placed in the context of more complex cellular environments, largely due to a stoichiometric excess of mk relative to p the success of anti-tnf biologics for the treatment of rheumatoid arthritis has highlighted the importance of understanding the intracellular pathways that regulate tnf production in the quest for an orally-available small molecule inhibitor. p is known to strongly regulate tnf production via mk the failure of several p inhibitors in the clinic suggests the importance of other downstream pathways in normal cell function. importantly, it was found that excess mk results in a stoichiometric effect in which the formation of p38-mk2-inhibitor complex prevents the phosphorylation of atf despite the preference of the compound for the p38-mk complex over the p38-atf complex. we found that in a dual substrate assay, mk2-p interaction reduced the activity of p against atf we further constructed a detailed kinetic mechanistic model of p phosphorylation in the presence of multiple substrates and successfully predicted the performance of classical and so-called'substrate-selective' p inhibitors in the dual substrate assay.
 the success of anti-tnf biologics for the treatment of rheumatoid arthritis has highlighted the importance of understanding the intracellular pathways that regulate tnf production in the quest for an orally-available small molecule inhibitor. p is known to strongly regulate tnf production via mk the failure of several p inhibitors in the clinic suggests the importance of other downstream pathways in normal cell function. recent work has described a'substrate-selective' p inhibitor that is able to preferentially block the activity of p against one substrate versus another. using a combined experimental and computational approach, we have examined this mechanism in greater detail for two p substrates, mk and atf  we found that in a dual substrate assay, mk2-p interaction reduced the activity of p against atf we further constructed a detailed kinetic mechanistic model of p phosphorylation in the presence of multiple substrates and successfully predicted the performance of classical and so-called'substrate-selective' p inhibitors in the dual substrate assay. importantly, it was found that excess mk results in a stoichiometric effect in which the formation of p38-mk2-inhibitor complex prevents the phosphorylation of atf despite the preference of the compound for the p38-mk complex over the p38-atf complex. mk and p protein expression levels were quantified in u thp- and pbmcs and found that >.  our integrated mechanistic modeling and experimental validation provides an example of how systems biology approaches can be applied to drug discovery and provide a basis for decision-making with limited chemical matter. we find that, given our current understanding, it is unlikely that'substrate-selective' inhibitors of p will work as originally intended when placed in the context of more complex cellular environments, largely due to a stoichiometric excess of mk relative to p 