Beverly P. Holskin

Inhibition of mixed lineage kinase 3 attenuates MPP+-induced neurotoxicity in SH-SY5Y cells

The neuropathology of Parkinsons Disease has been modeled in experimental animals following MPTP treatment and in dopaminergic cells in culture treated with the MPTP neurotoxic metabolite, MPP(+). MPTP through MPP(+) activates the stress-activated c-Jun N-terminal kinase (JNK) pathway in mice and SH-SY5Y neuroblastoma cells. Recently, it was demonstrated that CEP-1347/KT7515 attenuated MPTP-induced nigrostriatal dopaminergic neuron degeneration in mice, as well as MPTP-induced JNK phosphorylation. Presumably, CEP-1347 acts through inhibition of at least one upstream kinase within the mixed lineage kinase (MLK) family since it has been shown to inhibit MLK 1, 2 and 3 in vitro. Activation of the MLK family leads to JNK activation. In this study, the potential role of MLK and the JNK pathway was examined in MPP(+)-induced cell death of differentiated SH-SY5Y cells using CEP-1347 as a pharmacological probe and dominant negative adenoviral constructs to MLKs. CEP-1347 inhibited MPP(+)-induced cell death and the morphological features of apoptosis. CEP-1347 also prevented MPP(+)-induced JNK activation in SH-SY5Y cells. Endogenous MLK 3 expression was demonstrated in SH-SY5Y cells through protein levels and RT-PCR. Adenoviral infection of SH-SY5Y cells with a dominant negative MLK 3 construct attenuated the MPP(+)-mediated increase in activated JNK levels and inhibited neuronal death following MPP(+) addition compared to cultures infected with a control construct. Adenoviral dominant negative constructs of two other MLK family members (MLK 2 and DLK) did not protect against MPP(+)-induced cell death. These studies show that inhibition of the MLK 3/JNK pathway attenuates MPP(+)-mediated SH-SY5Y cell death in culture and supports the mechanism of action of CEP-1347 as an MLK family inhibitor.

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.

Discovery of 7-arylsulfonyl-1,2,3,4, 4a,9a-hexahydro-benzo[4,5]furo[2,3-c]pyridines: Identification of a potent and selective 5-HT6 receptor antagonist showing activity in rat social recognition test

Serotoninergic neurotransmission has been implicated in modulation of learning and memory. It has been demonstrated that 5-hydroxytryptamine(6) (5-HT(6)) receptor antagonists show beneficial effect on cognition in several animal models. Based on a pharmacophore model reported in the literature, we have designed and successfully identified a 7-benzenesulfonyl-1,2,3,4-tetrahydro-benzo[4,5]furo[2,3-c]pyridine (3a) scaffold as a novel class of 5-HT(6) receptor antagonists. Despite good activity against 5-HT(6) receptor, 3a exhibited poor liver microsome stability in mouse, rat and dog. It was demonstrated that the saturation of the double bond of the tetrahydropyridine ring of 3a enhanced metabolic stability. However the resulting compound, 4a (7-phenylsulfonyl-1,2,3,4,4a,9a-hexahydro-benzo[4,5]furo[2,3-c] pyridine-HCl salt) exhibited ∼30-fold loss in potency along with introduction of two chiral centers. In our optimization process for this series, we found that substituents at the 2 or 3 positions on the distal aryl group are important for enhancing activity against 5-HT(6). Separation of enantiomers and subsequent optimization and SAR with bis substituted phenyl sulfone provided potent 5-HT(6) antagonists with improved PK profiles in rat. A potent, selective 5-HT(6)R antagonist (15k) was identified from this study which showed good oral bioavailability (F=39%) in rat with brain penetration (B/P=2.76) and in vivo activity in a rat social recognition test.

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.

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 Invitrogens 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.

Novel brain penetrant benzofuropiperidine 5-HT6 receptor antagonists

7-Arylsulfonyl substituted benzofuropiperidine was discovered as a novel scaffold for 5HT(6) receptor antagonists. Optimization by substitution at C-1 position led to identification of selective, orally bioavailable, brain penetrant antagonists with reduced hERG liability. An advanced analog tested in rat social recognition model showed significant activity suggesting potential utility in the enhancement of short-term memory.

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 Invitrogens 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.

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.