Detection of allosteric kinase inhibitors by displacement of active site probes.

Non-adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)-based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes ("tracers"). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC(50) values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.

Development and applications of a broad-coverage, TR-FRET-based kinase binding assay platform.

The expansion of kinase assay technologies over the past decade has mirrored the growing interest in kinases as drug targets. As a result, there is no shortage of convenient, fluorescence-based methods available to assay targets that span the kinome. The authors recently reported on the development of a non-activity-based assay to characterize kinase inhibitors that depended on displacement of an Alexa Fluor 647 conjugate of staurosporine (a "tracer") from a particular kinase. Kinase inhibitors were characterized by a change in fluorescence lifetime of the tracer when it was bound to a kinase relative to when it was displaced by an inhibitor. Here, the authors report on improvements to this strategy by reconfiguring the assay in a time-resolved fluorescence resonance energy transfer (TR-FRET) format that simplifies instrumentation requirements and allows for the use of a substantially lower concentration of kinase than was required in the fluorescence-lifetime-based format. The authors use this new assay to demonstrate several aspects of the binding assay format that are advantageous relative to traditional activity-based assays. The TR-FRET binding format facilitates the assay of compounds against low-activity kinases, allows for the characterization of type II kinase inhibitors either using nonactivated kinases or by monitoring compound potency over time, and ensures that the signal being detected is specific to the kinase of interest and not a contaminating kinase.

Leucine-rich repeat kinase 2 mutants I2020T and G2019S exhibit altered kinase inhibitor sensitivity.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a frequent cause of late-onset autosomal dominant Parkinson's disease (PD). Some disease-associated mutations directly affect LRRK2 kinase activity and inhibition of LRRK2 is viewed as a potential therapeutic treatment for PD. We demonstrate by both binding and enzymatic assays that alterations in the kinase activity of the PD-associated mutants I2020T and G2019S are due in part to altered ATP affinity. In binding assays, G2019S and I2020T have approximately 2-fold lower and 6-fold higher ATP affinity, respectively, than wild-type LRRK2. Furthermore, using an in vitro kinase activity assay, we demonstrate that at ATP concentrations close to cellular levels (1 mM) I2020T is approximately 10-fold more resistant to ATP-competitive kinase inhibitors than wild-type whereas G2019S is 1.6-fold more sensitive. These results predict that LRRK2 status may impact kinase inhibitor potencies in vivo or in cellular models.

An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1.

The mammalian target of rapamycin (mTOR) kinase is the catalytic subunit of two functionally distinct complexes, mTORC1 and mTORC2, that coordinately promote cell growth, proliferation, and survival. Rapamycin is a potent allosteric mTORC1 inhibitor with clinical applications as an immunosuppressant and anti-cancer agent. Here we find that Torin1, a highly potent and selective ATP-competitive mTOR inhibitor that directly inhibits both complexes, impairs cell growth and proliferation to a far greater degree than rapamycin. Surprisingly, these effects are independent of mTORC2 inhibition and are instead because of suppression of rapamycin-resistant functions of mTORC1 that are necessary for cap-dependent translation and suppression of autophagy. These effects are at least partly mediated by mTORC1-dependent and rapamycin-resistant phosphorylation of 4E-BP1. Our findings challenge the assumption that rapamycin completely inhibits mTORC1 and indicate that direct inhibitors of mTORC1 kinase activity may be more successful than rapamycin at inhibiting tumors that depend on mTORC1.

Investigation of leucine-rich repeat kinase 2 : enzymological properties and novel assays.

Mutations in leucine-rich repeat kinase 2 (LRRK2) comprise the leading cause of autosomal dominant Parkinson's disease, with age of onset and symptoms identical to those of idiopathic forms of the disorder. Several of these pathogenic mutations are thought to affect its kinase activity, so understanding the roles of LRRK2, and modulation of its kinase activity,may lead to novel therapeutic strategies for treating Parkinson's disease. In this study, highly purified, baculovirus-expressed proteins have been used,for the first time providing large amounts of protein that enable a thorough enzymatic characterization of the kinase activity of LRRK2.Although LRRK2 undergoes weak autophosphorylation, it exhibits high activity towards the peptidic substrate LRRKtide, suggesting that it is a catalytically efficient kinase. We have also utilized a time-resolved fluorescence resonance energy transfer (TR-FRET) assay format (Lantha-ScreenTM) to characterize LRRK2 and test the effects of nonselective kinase inhibitors. Finally, we have used both radiometric and TR-FRETassays to assess the role of clinical mutations affecting LRRK2's kinase activity. Our results suggest that only the most prevalent clinical mutation,G2019S, results in a robust enhancement of kinase activity with LRRKtideas the substrate. This mutation also affects binding of ATP to LRRK2,with wild-type binding being tighter (Km,app of 57 lm) than with theG2019S mutant (Km,app of 134 lm). Overall, these studies delineate the catalytic efficiency of LRRK2 as a kinase and provide strategies by which a therapeutic agent for Parkinson's disease may be identified.

Pharmacological characterization of purified recombinant mTOR FRB-kinase domain using fluorescence-based assays.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in nutrient sensing and cell growth and is a validated target for oncology and immunosuppression. Two modes of direct small-molecule inhibition of mTOR activity are known: targeting of the kinase active site and a unique mode in which the small molecule rapamycin, in complex with FKBP12 (the 12-kDa FK506 binding protein), binds to the FRB (FKBP12/rapamycin binding) domain of mTOR and inhibits kinase activity through a poorly defined mechanism. To facilitate the study of these processes, the authors have expressed and purified a truncated version of mTOR that contains the FRB and kinase domains and have developed homogeneous fluorescence-based assays to study mTOR activity. They demonstrate the utility of these assays in studies of active site-directed and FRB domain-directed mTOR inhibition. The results suggest that these assays can replace traditional radiometric or Western blot-based assays.

Homogenous fluorescent assays for characterizing small-molecule activators of AMP-activated protein kinase (AMPK).

AMP activated protein kinase (AMPK) is a key regulator of cellular metabolism. AMPK activity is modulated in part by binding of AMP to the gamma-subunit of the kinase, which increases the activity of the catalytic alpha-subunit. Because increased AMPK activity in the liver and in skeletal muscle leads to increased fatty acid oxidation and decreased cholesterol and fatty acid biosynthesis, activators of AMPK are being sought for treatment of type-2 diabetes and other metabolic disorders. The unique mechanism of AMPK activation offers an opportunity to develop small molecules that directly upregulate AMPK activity, and there exists a need for simplified methods to identify and characterize small-molecules that show isoform-specific effects on AMPK. We have developed a suite of fluorescence-based assays to identify and characterize such compounds, and have used these to characterize and compare activity of recombinant AMPK alpha(1)beta(1)gamma(1) and alpha(2)beta(1)gamma(1) isoforms in response to small molecule activators and inhibitors.