Screening Approaches to Identify Allosteric Group I Pak Inhibitors

Many high-throughput assays have been developed for the identification of kinase inhibitors. The majority of these assays simply report on the phosphorylation of model substrates and therefore do not discriminate between compounds acting via a particular mechanism. The identification of compounds that target group I Paks via the allosteric mechanism of stabilization of the native autoinhibited conformation requires special considerations in assay design. For example, kinase inhibitor screens frequently utilize constitutively active, truncated fragments comprising the isolated kinase domain. These constructs may lack important domains that regulate catalytic activity that could be targeted by an allosteric inhibitor. Therefore, full-length Pak1 should be used to enable the detection of allosteric inhibitors. In addition, kinase activity screens are frequently performed in the presence of low (||M) ATP concentrations, thus rendering these assays highly sensitive to ATP-competitive inhibitors. One simple change that could bias such a screen towards non-ATP competitive inhibitors would be to monitor Pak1 kinase activity at high (mM) ATP concentrations. Subsequent mechanistic or enzymological follow-up studies could then be conducted to determine if inhibitory compounds are indeed non-competitive with ATP.

Another approach to address a Pak1 inhibitor's mechanism of action takes advantage of the ability to control the activation state of Pak1 in vitro. The interaction between purified autoregulatory and kinase domains of Pak1 is abolished when these fragments are autophosphorylated by Pak1 (Zenke et al. 1999; Buchwald et al. 2001), suggesting that once activated (and autophosphorylated), full length Pak1 does not reform the autoinhibited conformation until acted upon by protein phosphatases. Therefore, in an in vitro assay with no phosphatases present, it is possible to drive the regulatory equilibrium of Pak1 irreversibly towards the activated state by pre-incubating the kinase with activated Rac or Cdc42 and ATP. A small molecule that inhibits Pak1 activity by stabilizing the autoinhibited Pak1 complex should be ineffective at inhibiting this pre-active Pak1. This suggests that a simple "order of addition" experiment could be used to discriminate between inhibitors targeting the active site and those that target the Pak1 activation mechanism. In this approach, two separate kinase assays would be conducted in parallel in which the kinase reaction components are the same, but the order of their addition to the reaction is different. In the first assay, potential inhibitors would be added to autoinhibited Pak1 dimers prior to the addition of activator (activated Rac or Cdc42) and ATP. In the second assay, small molecules would be added to Pak1 after activation of the kinase via incubation with activator and ATP. Compounds that inhibit substrate phosphorylation by Pak1 in both assays likely do not depend on the formation of inactive Pak1 dimers. However, inhibition of Pak1 catalytic activity by a compound only when added prior to Pak1 activation would be consistent with a compound antagonizing the activation process. In this manner it may be possible to initially identify allosteric Pak1 inhibitors.

Alternatively, an assay that directly monitors the interaction between Pakl dimers could be performed so as to select for compounds that stabilize the Pak1 autoinhibited conformation. One such assay could involve a fluorescence resonance energy transfer (FRET)-based approach utilizing recombinant Pakl fragments comprising the catalytic domain and the autoregulatory domain individually tagged with either FRET donor or acceptor molecules. In the autoinhibited state, these tagged Pakl fragments would dimerize and undergo FRET. Upon addition of Cdc42, FRET would be abolished due to the dissociation of the autoregulatory domain from the kinase domain. In this assay, individual compounds would be added to inactive Pakl dimers and the maintenance of FRET monitored upon addition of Cdc42. Those reactions that maintain FRET in the presence of Cdc42 could act by stabilizing Pakl dimers. Subsequent assays could then be performed to confirm that compounds identified by this approach do indeed inhibit Pakl catalytic activity.

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