Comparison of LanthaScreen Eu kinase binding assay and surface plasmon resonance method in elucidating the binding kinetics of focal adhesion kinase inhibitors.

An understanding of the dynamics of drug-target interactions is important in the drug discovery process. Information related to the binding kinetics of a drug toward its target or off-target aids in determining the efficacy or toxicity of a drug. Biophysical techniques such as surface plasmon resonance (SPR) have been available for over 20 years, but have been predominantly utilized to characterize protein-protein interactions. With improvements in instrument sensitivity and data analysis software, interactions between proteins (such as kinases) and small molecules have been successfully evaluated. More recently, the LanthaScreen Eu kinase binding assay for characterizing kinase inhibitors has been described. This assay monitors displacement of an Alexa Fluor 647-labeled tracer from the ATP-binding site of an epitope-tagged kinase by a test compound. Such behavior results in a decrease in time-resolved fluorescence energy transfer signal. In this report, a side-by-side comparison of the LanthaScreen Eu kinase binding assay and the SPR method was performed using inhibitors of focal adhesion kinase. The two methods yielded comparable results and identified compounds with time-dependent inhibition and relatively slow dissociation.

A selective, orally bioavailable 1,2,4-triazolo[1,5-a]pyridine-based inhibitor of Janus kinase 2 for use in anticancer therapy: discovery of CEP-33779.

Members of the JAK family of nonreceptor tyrosine kinases play a critical role in the growth and progression of many cancers and in inflammatory diseases. JAK2 has emerged as a leading therapeutic target for oncology, providing a rationale for the development of a selective JAK2 inhibitor. A program to optimize selective JAK2 inhibitors to combat cancer while reducing the risk of immune suppression associated with JAK3 inhibition was undertaken. The structure-activity relationships and biological evaluation of a novel series of compounds based on a 1,2,4-triazolo[1,5-a]pyridine scaffold are reported. Para substitution on the aryl at the C8 position of the core was optimum for JAK2 potency (17). Substitution at the C2 nitrogen position was required for cell potency (21). Interestingly, meta substitution of C2-NH-aryl moiety provided exceptional selectivity for JAK2 over JAK3 (23). These efforts led to the discovery of CEP-33779 (29), a novel, selective, and orally bioavailable inhibitor of JAK2.

Improvement of inhibitor identification for heat shock protein 90α by utilizing a red-shifted fluorescence polarization probe.

Heat shock protein-90 (HSP90) is an ATP-dependent molecular chaperone with intrinsic ATPase activity. HSP90 is required for the stability and function of client proteins, many of which are involved in oncogenesis. Thus, identification of HSP90 inhibitors would potentially lead to the discovery of cancer therapeutics. Here, we present a high-throughput screening campaign utilizing two geldanamycin (GM)-labeled probes in a fluorescence polarization (FP) assay. For the primary screen, a previously reported green BODIPY-labeled GM (GM-BODIPY) was used to evaluate a library collection of about 400,000 compounds. From this screen, 3058 compounds showed >30% inhibition. To distinguish true positives from compound interference, a confirmatory screen was deemed necessary. Accordingly, a red-shifted FP binding assay was developed using GM labeled with red BODIPY. This tool enabled reliable identification of promising HSP90α inhibitors.

Time-resolved fluorescence resonance energy transfer as a versatile tool in the development of homogeneous cellular kinase assays.

Homogeneous cellular assays can streamline product detection in the drug discovery process. One commercially available assay employing time-resolved fluorescence resonance energy transfer (TR-FRET) that detects phosphorylated products was used to evaluate inhibitors of the receptor tyrosine kinase AXL in a cell line expressing an AXL-green fluorescent protein fusion protein. This TR-FRET assay was modified to evaluate the phosphorylation state of the AXL family member MER in a cell line expressing MER with a V5 tag by adding a fluorescein-labeled anti-V5 antibody. This homogeneous cellular assay was further modified to evaluate the nonreceptor tyrosine kinase focal adhesion kinase (FAK) in cell lines that expressed an untagged kinase by the inclusion of a commercially available anti-FAK antibody conjugated with an acceptor dye. The methods described here can be further adapted for TR-FRET detection of other cellular kinase activities.

Comparison of two homogeneous cell-based kinase assays for JAK2 V617F: SureFire pSTAT5 and GeneBLAzer fluorescence resonance energy transfer assays.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays an important role in cellular responses to cytokines and growth factors. Recent studies have identified a recurrent somatic activating mutation (JAK2 V617F) in majority of patients with myeloproliferative disorders (MPDs). Development of drugs that target JAK2 V617F is, therefore, of therapeutic relevance. To discover small molecule inhibitors for this target, robust and reliable cell-based assays are important. Here, we present a comparison of two homogeneous, 384-well plate-based cellular assays using Invitrogen's CellSensor® JAK2 V617F interferon regulatory factor-1 (irf1)-beta-lactamase (bla) human erythroleukemia line (HEL): (1) SureFire® pSTAT5 AlphaScreen® assay from PerkinElmer; and (2) GeneBLAzer® fluorescence resonance energy transfer assay from Invitrogen. HEL cells are growth factor-independent due to JAK2 V617F mutation that causes constitutive STAT5 activation. The SureFire assay measures levels of phosphorylated STAT5 downstream of JAKs, while the GeneBLAzer assay is a reporter assay that monitors bla activity further downstream of STAT5. Evaluation of a number of chemically diverse JAK2 inhibitors in the two cellular assays yielded comparable half-maximal inhibitory concentration (IC50) values, boding well for the utility of these assay formats in compound profiling.

Modification of CellSensor irf1-bla TF-1 and irf1-bla HEL assays for direct comparison of wild-type JAK2 and JAK2 V617F inhibition.

The Janus kinase (JAK)-signal transducer and activator of transcription pathway is an important therapeutic target because of its role in the regulation of cell growth. Aberrant, constitutive activation of JAK2 signaling has been implicated in myeloproliferative disorders with a single, activating somatic V617F mutation in the JH2 pseudokinase domain of JAK2 as the prevalent molecular lesion. Invitrogen has developed the CellSensor(®) cell lines interferon regulatory factor-1 (irf1)-beta-lactamase (bla) TF-1 and irf1-bla HEL for use in evaluating inhibitors of wild-type JAK2 and mutant JAK2 V617F, respectively. Both contain a bla reporter gene downstream of the irf1 response element stably integrated into either TF-1 or HEL cells. A fluorescence resonance energy transfer-based bla substrate is utilized to give a robust detection of JAK2 activity. Examination of Invitrogen's protocols for the two cell lines revealed significant differences that are not conducive to direct comparison of inhibitor activities against wild-type and mutant JAK2. Systematic changes to standardize the two assays were incorporated and evaluated for effects on assay response ratio, assay quality, and potency for a diverse series of inhibitors.

Amino-terminal isoforms of the human glycine transporter GlyT1 exhibit similar pharmacology.

Alternative promoter usage and mRNA precursor splicing produce three amino-terminal isoforms of the human glycine transporter type 1 (GlyT1). To enable discovery of pharmacological tools that might distinguish them, each of these isoforms was stably expressed in CHO-K1 cells and clonal isolates were generated by limiting dilution. Glycine uptake assays were validated for two lines for each isoform, one low and one high expressor. The data show a modest trend for lower potency against higher expressing lines. IC(50) values for reference GlyT1 inhibitors ALX-5407 (Allelix), (S)-13h (Merck), and SSR504734 (Sanofi-Synthelabo) were similar across isoforms. The greatest variation was observed for ALX-5407, and its IC(50) values across isoforms were still within one log unit of each other. Antipsychotics previously shown to be weak inhibitors of GlyT1 likewise had similar potency against all three isoforms. The cell lines validated here are tools for discovering inhibitors that might distinguish among GlyT1 isoforms.

8-THP-DHI analogs as potent Type I dual TIE-2/VEGF-R2 receptor tyrosine kinase inhibitors.

A novel series of 8-(2-tetrahydropyranyl)-12,13-dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazoles (THP-DHI) was synthesized and evaluated as dual TIE-2 and VEGF-R2 receptor tyrosine kinase inhibitors. Development of the structure-activity relationships (SAR) with the support of X-ray crystallography led to identification of 7f and 7g as potent, selective dual TIE-2/VEGF-R2 inhibitors with excellent cellular potency and acceptable pharmacokinetic properties. Compounds 7f and 7g were orally active in tumor models with no observed toxicity.

Aequorin functional assay for characterization of G-protein-coupled receptors: implementation with cryopreserved transiently transfected cells.

Assay technologies that measure intracellular Ca(2+) release are among the predominant methods for evaluation of GPCR function. These measurements have historically been performed using cell-permeable fluorescent dyes, although the use of the recombinant photoprotein aequorin (AEQ) as a Ca(2+) sensor has gained popularity with recent advances in instrumentation. The requirement of the AEQ system for cells expressing both the photoprotein and the GPCR target of interest has necessitated the labor-intensive development of cell lines stably expressing both proteins. With the goal of streamlining this process, transient transfections were used to either (1) introduce AEQ into cells stably expressing the GPCR of interest or (2) introduce the GPCR into cells stably expressing the AEQ protein, employing the human muscarinic M(1) receptor as a model system. Robust results were obtained from cryopreserved cells prepared by both strategies, yielding agonist and antagonist pharmacology in good agreement with literature values. Good reproducibility was observed between multiple transient transfection events. These results indicate that transient transfection is a viable and efficient method for production of cellular reagents for use in AEQ assays.

Successful identification of glycine transporter inhibitors using an adaptation of a functional cell-based assay.

Glycine transporter (GlyT1) function is typically measured by radiolabeled glycine uptake using lysis methods or scintillation proximity assays (SPAs), which have limited throughput. This study shows the adaptation of the standard cell lysis method to a screening assay with improved throughput and assay characteristics. The assay takes advantage of the 384-well format, standard laboratory automation, and cryopreserved CHO-K1 cells stably overexpressing human GlyT1a transporter (CHO-K1/hGlyT1a) that were validated and banked in advance of screening. The assay was evaluated for the time course of glycine uptake, K(m), V(max), Z' factor analysis, and IC(50) value determination with reference GlyT1 inhibitors. Screening of 118,000 compounds at 10 microM identified 4556 compounds (3.9%) as inhibitors. Positive compounds (>50% inhibition) were retested in the assay at 4 inhibitor concentrations. Compounds demonstrating greater than 40% inhibition at 10 microM were considered as confirmed positives, yielding a 68% confirmation rate from the original screen. To eliminate compounds that nonspecifically inhibited glycine uptake, IC(50) values were determined in both GlyT1 and GlyT2 assays, and those compounds that inhibited GlyT2 were removed from consideration. The screening campaign identified 300 small molecules as selective GlyT1 inhibitors for lead optimization, demonstrating the utility of this cost-effective method.

ALK mutants in the kinase domain exhibit altered kinase activity and differential sensitivity to small molecule ALK inhibitors.

Abnormal expression of constitutively active anaplastic lymphoma kinase (ALK) chimeric proteins in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is well established. Recent studies with small molecule kinase inhibitors have provided solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK (+) ALCL cells. In this study, several missense mutants of ALK in the phosphate anchor and gatekeeper regions were generated and their kinase activity was measured. NPM-ALK L182M, L182V, and L256M mutants displayed kinase activity in cells comparable to or higher than that of NPM-ALK wild type (WT) and rendered BaF3 cells into IL-3-independent growth, while NPM-ALK L182R, L256R, L256V, L256P, and L256Q displayed much weaker or little kinase activity in cells. Similar kinase activities were obtained with corresponding GST-ALK mutants with in vitro kinase assays. With regard to inhibitor response, NPM-ALK L182M and L182V exhibited sensitivity to a fused pyrrolocarbazole (FP)-derived ALK inhibitor comparable to that of NPM-ALK WT but were dramatically less sensitive to a diaminopyrimidine (DAP)-derived ALK inhibitor. On the other hand, NPM-ALK L256M exhibited >30-fold lower sensitivity to both FP-derived and DAP-derived ALK inhibitors. The growth inhibition and cytotoxicity of BaF3/NPM-ALK mutant cells induced by ALK inhibitors were consistent with inhibition of cellular NPM-ALK autophosphorylation. In a mouse survival model, treatment with the orally bioavailable DAP-ALK inhibitor substantially extended the survival of the mice inoculated with BaF3/NPM-ALK WT cells but not those inoculated with BaF3/NPM-ALK L256M cells. Binding of ALK inhibitors to ALK WT and mutants was analyzed using ALK homology models. In summary, several potential active ALK mutants were identified, and our data indicate that some of these mutants are resistant to select small molecule ALK inhibitors. Further characterization of these mutants may help to identify and develop potent ALK inhibitors active against both WT and resistant mutants of ALK.

Mixed-lineage kinase 1 and mixed-lineage kinase 3 subtype-selective dihydronaphthyl[3,4-a]pyrrolo[3,4-c]carbazole-5-ones: optimization, mixed-lineage kinase 1 crystallography, and oral in vivo activity in 1-methyl-4-phenyltetrahydropyridine models.

The optimization of the dihydronaphthyl[3,4-a]pyrrolo[3,4-c]carbazole-5-one R(2) and R(12) positions led to the identification of the first MLK1 and MLK3 subtype-selective inhibitors within the MLK family. Compounds 14 (CEP-5104) and 16 (CEP-6331) displayed good potency for MLK1 and MLK3 inhibition with a greater than 30- to 100-fold selectivity for related family members MLK2 and DLK. Compounds 14 and 16 were orally active in vivo in a mouse MPTP biochemical efficacy model that was comparable to the first-generation pan-MLK inhibitor 1 (CEP-1347). The MLK1 structure-activity relationships were supported by the first-reported X-ray crystal structure of MLK1 bound with 16.

Anaplastic lymphoma kinase activity is essential for the proliferation and survival of anaplastic large-cell lymphoma cells.

The roles of aberrant expression of constitutively active ALK chimeric proteins in the pathogenesis of anaplastic large-cell lymphoma (ALCL) have been well defined; nevertheless, the notion that ALK is a molecular target for the therapeutic modulation of ALK+ ALCL has not been validated thus far. Select fused pyrrolocarbazole (FP)-derived small molecules with ALK inhibitory activity were used as pharmacologic tools to evaluate whether functional ALK is essential for the proliferation and survival of ALK+ ALCL cells in culture. These compounds inhibited interleukin 3 (IL-3)-independent proliferation of BaF3/NPM-ALK cells in an ALK inhibition-dependent manner and significantly blocked colony formation in agar of mouse embryonic fibroblast (MEF) cells harboring NPM-ALK. Inhibition of NPM-ALK phosphorylation in the ALK+ ALCL-derived cell lines resulted in significant inhibition of cell proliferation and induction of apoptotic-cell death, while having marginal effects on the proliferation and survival of K562, an ALK- leukemia cell line. ALK inhibition resulted in cell-cycle G1 arrest and inactivation of ERK1/2, STAT3, and AKT signaling pathways. Potent and selective ALK inhibitors may have therapeutic application for ALK+ ALCL and possibly other solid and hematologic tumors in which ALK activation is implicated in their pathogenesis.

Phosphoregulation of mixed-lineage kinase 1 activity by multiple phosphorylation in the activation loop.

Mixed-lineage kinase 1 (MLK1) is a mitogen-activated protein kinase kinase kinase capable of activating the c-Jun NH(2)-terminal kinase (JNK) pathway. Full-length MLK1 has 1104 amino acids and a domain structure identical to MLK2 and MLK3. Immunoblot and mass spectrometry show that MLK1 is threonine (and possibly serine) phosphorylated in or near the activation loop. A kinase-dead mutant is not, consistent with autophosphorylation. Mutation to alanine of any of the four serine or threonine residues in the activation loop reduces both the activity of the recombinant kinase domain and JNK pathway activation driven by full-length MLK1 expressed in mammalian cells. Furthermore, the gel mobility of the mutant MLK1s is closer to that of the kinase-dead than wild type, consistent with reduced phosphorylation. Thr312 is the key residue: MLK1[T312A] retains only basal activity (about 1-2% of wild type), and its gel mobility is indistinguishable from kinase-dead. Thr312 does not suffice, however; phosphorylation of multiple sites is necessary for full activation of MLK1. An activation mechanism consistent with these data involves phosphorylation of multiple sites in the activation loop, with phosphorylation of Thr312 required for full phosphorylation. This mechanism is broadly similar to that previously reported for MLK3 [Leung, I. W., and Lassam, N. (2001) J. Biol. Chem. 276, 1961-1967], but the key residue differs.

A new class of potent vascular endothelial growth factor receptor tyrosine kinase inhibitors: structure-activity relationships for a series of 9-alkoxymethyl-12-(3-hydroxypropyl)indeno[2,1-a]pyrrolo[3,4-c]carbazole-5-ones and the identification of CEP-5214 and its dimethylglycine ester prodrug clinical candidate CEP-7055.

A series of potent vascular endothelial growth factor R2 (VEGF-R2) tyrosine kinase inhibitors from a new indenopyrrolocarbazole template is reported. The structure-activity relationships for a series of 9-alkoxymethyl-12-(3-hydroxypropyl)indeno[2,1-a]pyrrolo[3,4-c]carbazole-5-ones revealed an optimal R9 substitution with ethoxymethyl 19 (VEGF-R2 IC(50) = 4 nM) and isopropoxymethyl 21 (VEGF-R2 IC(50) = 8 nM) being the most potent inhibitors in the series. The VEGF-R2 activity was reduced appreciably by increasing the size of the R9 alkoxy group or by alpha-methyl branching adjacent to the ring. The combined R9 alkoxymethyl and N12 hydroxypropyl substitutions were required for potent VEGF-R2 activity, and the corresponding thioether analogues were weaker than their ether counterparts. Compound 21 (R9 isopropoxymethyl, CEP-5214) was identified as a potent, low-nanomolar pan inhibitor of human VEGF-R tyrosine kinases, displaying IC(50) values of 16, 8, and 4 nM for VEGF-R1/FLT-1, VEGF-R2/KDR, and VEGF-R3/FLT-4, respectively, with cellular activity equivalent to the isolated enzyme activity. Compound 21 exhibited good selectivity against numerous tyrosine and serine/threonine kinases including PKC, Tie2, TrkA, CDK1, p38, JNK, and IRK. To increase water solubility and oral bioavailability, the N,N-dimethylglycine ester 40 was prepared. In pharmacokinetic studies in mice and rats, increased plasma levels of 21 were observed after oral administration of 40. Compound 21 demonstrated significant in vivo antitumor activity in numerous tumor models and was advanced into phase I clinical trials as the water-soluble N,N-dimethylglycine ester prodrug 40 (CEP-7055).