Genetic approaches have demonstrated that the p110β isoform of PI3K is essential for the growth of PTEN-null tumors. Thus, it is desirable to develop p110β-specific inhibitors for cancer therapy. Using a panel of PI3K isoform-specific cellular assays, we screened a collection of compounds possessing activities against kinases in the PI3K superfamily and identified a potent and selective p110β inhibitor: KIN-193. We show that KIN-193 is efficacious specifically in blocking AKT signaling and tumor growth that are dependent on p110β activation or PTEN-loss. Broad profiling across a panel of 422 human tumor cell lines demonstrates that the PTEN mutation status of cancer cells strongly correlates with their response to KIN-193. Together, our data provide the first pharmacological evidence that PTEN-deficient tumors are dependent on p110β in animals, and suggest that KIN-193 can be pursued as a drug to treat tumors that are dependent on p110β, while sparing other PI3K isoforms.
Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (Roc R1441H ) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis.M utation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD) (1-5). LRRK2 is a large (2,527-aa) multidomain protein consisting of seven putative domains (2), including a Ras-like GTPase domain called Ras of complex proteins (Roc), followed by a domain called C-terminal of Roc (COR), which is then followed by a kinase domain (Kin). It remains unclear how perturbations of these activities result in disease; however, the most common mutation in LRRK2-associated PD, G2019S in the kinase domain, shows higher kinase activity than wild type; therefore, its overactivation might be associated with disease pathogenesis (6).The tandem Roc-COR-Kin arrangement suggests that their activities might be coupled such that the GTPase activity of Roc might modulate the kinase activity. Indeed, several studies have shown that GTP binding to the Roc domain regulates the activity of the Kin domain (7,8). Moreover, a PD-associated mutation in the Roc domain (R1441C) has been shown to have higher kinase activity (9), thus suggesting that mutations in the Roc domain, also up-regulate kinase activity.Understanding the function of Roc and its mechanism of action is important for understanding the mechanism of PD pathogenesis and therapeutic development. However, because of the lack sufficient quantity of protein samples amendable for detailed investigations, the biochemical properties and enzymatic activities of the Roc domain of LRRK2 are poorly understood.Here, we describe a stably folded construct of human Roc domain that enabled us to investigate quantitatively its biochemical and enzymatic properties. The results revealed that a PD-causing mutation R1441H in the Roc domain renders it less active at hydrolyzing GTP, as well as having higher affinity for GTP, than its wild-type counterpart, thereby increasing the residence time of its GTP-bound "active state," which is associated with PD pathogenesis (8). Results and DiscussionConstruction of a Stable Human LRR...
Missense mutations in the Leucine-Rich Repeat protein Kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson’s disease (PD) (Farrer et al., 2005; Skipper et al., 2005; Di Fonzo et al., 2006; Healy et al., 2008; Paisan-Ruiz et al., 2008; Lesage et al., 2010). LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955, and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955, and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro, and Tyr1699Cys, can positively enhance LRRK2 kinase activity, while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and Okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.
Background: Several new ATP-competitive mTOR inhibitors have been described, but their kinome-wide selectivity profiles have not been disclosed. Results: Four different profiling technologies revealed a different spectrum of targets for four recently described mTOR inhibitors. Conclusion: Diverse heterocyclic mTOR inhibitors have unique pharmacology.Significance: Profiling data guide choices of mTOR inhibitors for particular applications and provide new potential targets for medicinal chemistry efforts.
Receptor tyrosine kinases (RTKs) are important pharmacological targets in oncology. Current biochemical assays designed to assess compound efficacy utilize truncated forms of the kinase lacking the transmembrane or extracellular domains and fail to address how these domains might affect the observed pharmacology. Purification of the full-length recombinant receptor can be both costly and difficult to scale for large screening campaigns. Additionally, because of the generic nature of most substrates used in activity assays for RTKs, small amounts of contaminating kinases can interfere with assay results. Numerous cell-based assays for pathway analysis can also serve as readouts for these targets, but they are not a direct measurement of kinase activity. To address these limitations, we have employed a BacMam-mediated gene delivery system to express full-length receptor tyrosine kinases with C-terminal green fluorescent protein (GFP) fusions in a variety of cellular backgrounds. Expression of these full-length RTKs has enabled the development of two complimentary assay platforms. First, we present a competitive displacement assay that uses a europium (Eu)-labeled anti-GFP antibody and an AlexaFluor® 647-labeled active site probe to characterize compound binding to the RTK in a lysate-based format. Here, the GFP moiety serves as an epitope tag to allow for very specific and sensitive detection of compound binding, discriminating compounds with sub-nanomolar affinity. Using the same BacMam RTK-GFP constructs, we have also developed a simple, addition-only, cell-based method to detect auto-phosphorylation of the RTK via TR-FRET between a terbium (Tb)-labeled anti-phospho-tyrosine antibody and the GFP moiety. Together, these tools can be used in a high-throughput screening format with a fluorescence-based readout to more fully characterize compound efficacy against full-length RTKs expressed in native cellular contexts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3881. doi:1538-7445.AM2012-3881
There is great potential for the utilization of selective kinase inhibitors in the treatment of many clinical indications, especially in oncology and autoimmune conditions. Kinase inhibitor selectivity profiling has become an important tool during lead development to understand a compound's selectivity, potential off-target effects and possible new indications for the compound. We have utilized the LanthaScreen® Eu Kinase Binding Assay platform to develop a simple, robust means for profiling compounds across the kinome utilizing pre-plated reagents and controls. The assay is based on competitive displacement of a tracer (a fluorophore conjugated to a kinase inhibitor scaffold) from specific kinases of interest. Binding of the tracer to a kinase is detected by addition of a europium-labeled anti-tag antibody. Binding of the tracer and antibody to a kinase results in a high degree of FRET, whereas displacement of the tracer with a kinase inhibitor results in a loss of FRET. This platform is useful to explore activation-state binding selectivity, time-dependent binding, and binding to kinases for which substrates have not been identified. The assay detects both ATP-competitive as well as non-competitive, allosteric kinase inhibitors. We will present data demonstrating the utility of these reagents for kinase profiling efforts with a variety of kinase inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5499.
In addition to drug-target affinity, drug-target residence time is becoming recognized as a critical parameter influencing drug efficacy. Desired compound action can only take place when a drug is interacting with its target protein, which is governed both by the affinity of the compound for the target, as well as the dissociative off-rate of the compound from the drug-target complex. A compound with a slower dissociative off-rate may require reduced dosing schedule relative to a compound with a rapid off-rate, and effective target specificity may be increased if the off-rate for the desired drug-target complex is greater than that for off-target complexes. With respect to kinase inhibitors, a slow off-rate for Tykerb relative to the related EGFR inhibitors Tarceva and Iressa has been proposed to explained longer lasting effects in cell culture. Currently, off rates for kinases can be measured by surface plasmon resonance (SPR)-based instruments or by detailed enzyme kinetics experiments. While SPR yields highly quantitative data, it is low-throughput and requires costly instrumentation. Furthermore, it often involves covalent conjugation of protein targets to surfaces, which can lead to loss of protein function. Off-rate measurements by classical enzyme kinetics methods are established, but fairly labor-intensive. We have developed and will present data on a fully homogenous (in-solution) method for determining kinase-compound off-rates that is simple to perform in a standard fluorescence-based plate reader. In this method, a drug-kinase complex is first formed at supra-Kd concentrations of drug, and then rapidly diluted into a solution containing a fluorescent, ATP-site directed small molecule probe. As the compound dissociates from the kinase the fluorescent probe associates with the kinase, causing an increase in the fluorescence signal that can be monitored in real time. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5496.
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