Design and high-throughput implementation of MALDI-TOF/MS-based assays for Parkin E3 ligase activity.

Parkinson's disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Loss-of-function mutations in the gene encoding the ubiquitin E3 ligase Parkin are causal for familial and juvenile PD. Among several therapeutic approaches being explored to treat or improve the prognosis of patients with PD, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy. A major barrier is the lack of high-throughput platforms for the robust and accurate quantification of Parkin activity in vitro. Here, we present two different and complementary Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF/MS)-based approaches for the quantification of Parkin E3 ligase activity in vitro. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators.

Generation of a chemical genetic model for JAK3.

Janus Kinases (JAKs) have emerged as an important drug target for the treatment of a number of immune disorders due to the central role that they play in cytokine signalling. 4 isoforms of JAKs exist in mammalian cells and the ideal isoform profile of a JAK inhibitor has been the subject of much debate. JAK3 has been proposed as an ideal target due to its expression being largely restricted to the immune system and its requirement for signalling by cytokine receptors using the common γ-chain. Unlike other JAKs, JAK3 possesses a cysteine in its ATP binding pocket and this has allowed the design of isoform selective covalent JAK3 inhibitors targeting this residue. We report here that mutating this cysteine to serine does not prevent JAK3 catalytic activity but does greatly increase the IC50 for covalent JAK3 inhibitors. Mice with a Cys905Ser knockin mutation in the endogenous JAK3 gene are viable and show no apparent welfare issues. Cells from these mice show normal STAT phosphorylation in response to JAK3 dependent cytokines but are resistant to the effects of covalent JAK3 inhibitors. These mice therefore provide a chemical-genetic model to study JAK3 function.

High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry-based deubiquitylating enzyme assay for drug discovery.

Deubiquitylating enzymes (DUBs) play a vital role in the ubiquitin pathway by editing or removing ubiquitin from their substrate. As breakthroughs within the ubiquitin field continue to highlight the potential of deubiquitylating enzymes as drug targets, there is increasing demand for versatile high-throughput (HT) tools for the identification of potent and selective DUB modulators. Here we present the HT adaptation of the previously published MALDI-TOF-based DUB assay method. In a MALDI-TOF DUB assay, we quantitate the amount of mono-ubiquitin generated by the in vitro cleavage of ubiquitin chains by DUBs. The method has been specifically developed for use with nanoliter-dispensing robotics to meet drug discovery requirements for the screening of large and diverse compound libraries. Contrary to the most common DUB screening technologies currently available, the MALDI-TOF DUB assay combines the use of physiological substrates with the sensitivity and reliability of the mass spectrometry-based readout.

Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases.

The related NUAK1 and NUAK2 are members of the AMPK (AMP-activated protein kinase) family of protein kinases that are activated by the LKB1 (liver kinase B1) tumour suppressor kinase. Recent work suggests they play important roles in regulating key biological processes including Myc-driven tumorigenesis, senescence, cell adhesion and neuronal polarity. In the present paper we describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms (IC50 for NUAK1 is 20 nM and for NUAK2 is 100 nM), whereas HTH-01-015 inhibits only NUAK1 (IC50 is 100 nM). These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases that were tested including ten AMPK family members. In all cell lines tested, WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterized substrate, MYPT1 (myosin phosphate-targeting subunit 1) that is phosphorylated by NUAK1 at Ser(445). We also identify a mutation (A195T) that does not affect basal NUAK1 activity, but renders it ~50-fold resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 mediating the phosphorylation of MYPT1 we find that in cells overexpressing drug-resistant NUAK1[A195T], but not wild-type NUAK1, phosphorylation of MYPT1 at Ser(445) is no longer suppressed by WZ4003 or HTH-01-015. We also demonstrate that administration of WZ4003 and HTH-01-015 to MEFs (mouse embryonic fibroblasts) significantly inhibits migration in a wound-healing assay to a similar extent as NUAK1-knockout. WZ4003 and HTH-01-015 also inhibit proliferation of MEFs to the same extent as NUAK1 knockout and U2OS cells to the same extent as NUAK1 shRNA knockdown. We find that WZ4003 and HTH-01-015 impaired the invasive potential of U2OS cells in a 3D cell invasion assay to the same extent as NUAK1 knockdown. The results of the present study indicate that WZ4003 and HTH-01-015 will serve as useful chemical probes to delineate the biological roles of the NUAK kinases.

Kinome-wide selectivity profiling of ATP-competitive mammalian target of rapamycin (mTOR) inhibitors and characterization of their binding kinetics.

An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 µM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 µM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 µM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.

Structural features underlying the selectivity of the kinase inhibitors NBC and dNBC: role of a nitro group that discriminates between CK2 and DYRK1A.

8-hydroxy-4-methyl-9-nitrobenzo(g)chromen-2-one (NBC) has been found to be a fairly potent ATP site-directed inhibitor of protein kinase CK2 (Ki = 0.22 µM). Here, we show that NBC also inhibits PIM kinases, especially PIM1 and PIM3, the latter as potently as CK2. Upon removal of the nitro group, to give 8-hydroxy-4-methyl-benzo(g)chromen-2-one (here referred to as "denitro NBC", dNBC), the inhibitory power toward CK2 is almost entirely lost (IC(50) > 30 µM) whereas that toward PIM1 and PIM3 is maintained; in addition, dNBC is a potent inhibitor of a number of other kinases that are weakly inhibited or unaffected by NBC, with special reference to DYRK1A whose IC(50) values with NBC and dNBC are 15 and 0.60 µM, respectively. Therefore, the observation that NBC, unlike dNBC, is a potent inducer of apoptosis is consistent with the notion that this effect is mediated by inhibition of endogenous CK2. The structural features underlying NBC selectivity have been revealed by inspecting its 3D structure in complex with the catalytic subunit of Z. mays CK2. The crucial role of the nitro group is exerted both through a direct electrostatic interaction with the side chain of Lys68 and, indirectly, by enhancing the acidic dissociation constant of the adjacent hydroxyl group which interacts with a conserved water molecule in the deepest part of the cavity. By contrast, the very same nitro group is deleterious for the binding to the active site of DYRK1A, as disclosed by molecular docking. This provides the rationale for preferential inhibition of DYRK1A by dNBC.

The specificities of small molecule inhibitors of the TGFß and BMP pathways.

Small molecule inhibitors of type 1 receptor serine threonine kinases (ALKs1-7), the mediators of TGFß and BMP signals, have been employed extensively to assess their physiological roles in cells and organisms. While all of these inhibitors have been reported as "selective" inhibitors of specific ALKs, extensive specificity tests against a wide array of protein kinases have not been performed. In this study, we examine the specificities and potencies of the most frequently used small molecule inhibitors of the TGFß pathway (SB-431542, SB-505124, LY-364947 and A-83-01) and the BMP pathway (Dorsomorphin and LDN-193189) against a panel of up to 123 protein kinases covering a broad spectrum of the human kinome. We demonstrate that the inhibitors of the TGFß pathway are relatively more selective than the inhibitors of the BMP pathway. Based on our specificity and potency profile and published data, we recommend SB-505124 as the most suitable molecule for use as an inhibitor of ALKs 4, 5 and 7 and the TGFß pathway. We do not recommend Dorsomorphin, also called Compound C, for use as an inhibitor of the BMP pathway. Although LDN-193189, a Dorsomorphin derivative, is a very potent inhibitor of ALK2/3 and the BMP-pathway, we found that it potently inhibited a number of other protein kinases at concentrations sufficient to inhibit ALK2/3 and its use as a selective BMP-pathway inhibitor has to be considered cautiously. Our observations have highlighted the need for caution when using these small molecule inhibitors to assess the physiological roles of BMP and TGFß pathways.

Comparison of the specificity of Trk inhibitors in recombinant and neuronal assays.

Neurotrophins are important mediators of neuronal development, survival and plasticity. They act via binding to Trk receptors, which results in the stimulation of the intracellular tyrosine kinase domain of the receptor leading to autophosphorylation of this domain. This in turn creates a scaffold that recruits various adapter proteins allowing the activation of intracellular signaling cascades including the PLCγ, MAPK and PI3K pathways. Compounds that specifically block the activity of the tyrosine kinase domain of Trk receptors would provide a powerful tool to study the role of these receptors in cells. K252a has previously been used for this purpose, however we show here that it can inhibit many tyrosine and serine/threonine kinases in vitro. Profiling of 3 newer inhibitors, referred to here as SHN-753, SHN-722 and GSK-Trk, demonstrate that they have significantly improved specificity for the kinase activity of TrkA in vitro compared to K252a. In addition these compounds were found to block the TrkB mediated activation of ERK1/2 by BDNF, but did not affect NMDA induced ERK1/2 activation. These compounds, while still not completely specific for Trk receptor kinase activity, do represent a considerable improvement over K252a and should prove valuable in the study of neurotrophin-mediated actions in the nervous system.