Ilona Kariv

MK-2461, a Novel Multitargeted Kinase Inhibitor, Preferentially Inhibits the Activated c-Met Receptor

The receptor tyrosine kinase c-Met is an attractive target for therapeutic blockade in cancer. Here, we describe MK-2461, a novel ATP-competitive multitargeted inhibitor of activated c-Met. MK-2461 inhibited in vitro phosphorylation of a peptide substrate recognized by wild-type or oncogenic c-Met kinases (N1100Y, Y1230C, Y1230H, Y1235D, and M1250T) with IC(50) values of 0.4 to 2.5 nmol/L. In contrast, MK-2461 was several hundredfold less potent as an inhibitor of c-Met autophosphorylation at the kinase activation loop. In tumor cells, MK-2461 effectively suppressed constitutive or ligand-induced phosphorylation of the juxtamembrane domain and COOH-terminal docking site of c-Met, and its downstream signaling to the phosphoinositide 3-kinase-AKT and Ras-extracellular signal-regulated kinase pathways, without inhibiting autophosphorylation of the c-Met activation loop. BIAcore studies indicated 6-fold tighter binding to c-Met when it was phosphorylated, suggesting that MK-2461 binds preferentially to activated c-Met. MK-2461 displayed significant inhibitory activities against fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor, and other receptor tyrosine kinases. In cell culture, MK-2461 inhibited hepatocyte growth factor/c-Met-dependent mitogenesis, migration, cell scatter, and tubulogenesis. Seven of 10 MK-2461-sensitive tumor cell lines identified from a large panel harbored genomic amplification of MET or FGFR2. In a murine xenograft model of c-Met-dependent gastric cancer, a well-tolerated oral regimen of MK-2461 administered at 100 mg/kg twice daily effectively suppressed c-Met signaling and tumor growth. Similarly, MK-2461 inhibited the growth of tumors formed by s.c. injection of mouse NIH-3T3 cells expressing oncogenic c-Met mutants. Taken together, our findings support further preclinical development of MK-2461 for cancer therapy.

Discovery of PDK1 Kinase Inhibitors with a Novel Mechanism of Action by Ultrahigh Throughput Screening

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen® screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, Km values were determined as 5.6 μm for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1× and 10× Km concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.

Quality Control Procedures for Dose-Response Curve Generation Using Nanoliter Dispense Technologies

With the advancement of high-throughput biomolecular screening techniques to the lead optimization stage, there is a critical need to quality control (QC) dose-response curves generated by robotic liquid handlers to ensure accurate affinity determinations. One challenge in evaluating the performance of liquid handlers is identifying and validating a robust method for testing dispense volumes across different instruments. Although traditional automated liquid handlers are still considered the standard platform in many laboratories, nanoliter dispensers are becoming more common and pose new challenges for routine quality control procedures. For example, standard gravimetric measurements are unreliable for testing the accuracy of nanoliter liquid dispenses. However, nanoliter dispensing technology allows for the conservation of compound, reduces compound carryover from well to well through discrete dispenses, and eliminates the need for intermediate compound dilution steps to achieve a low final DMSO assay concentration. Moreover, an intermediate dilution step in aqueous solution might result in compound precipitation at high concentrations. This study compared representative automation procedures done on a variety of liquid dispensers, including manual, traditional, and nanodispense volumes. The data confirmed the importance of establishing robust QC procedures for dose-response generation in addition to accuracy and precision determinations for each instrument, and they validated the use of nanoliter pipettors for dose-response testing. The results of this study also support the requirement for thorough mixing during serial compound dilutions prepared for high-throughput lead optimization strategies using traditional liquid handlers. (Journal of Biomolecular Screening 2007:891-899)

Development of High-Throughput TR-FRET and AlphaScreen® Assays for Identification of Potent Inhibitors of PDK1

The PI3K/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a Ser/Thr protein kinase, which catalyzes the phosphorylation of a conserved residue in the activation loop of a number of AGC kinases, including proto-oncogenes Akt, p70S6K, and RSK kinases. To find new small-molecule inhibitors of this important regulator kinase, the authors have developed PDK1-specific high-throughput enzymatic assays in time-resolved fluorescence resonance energy transfer (TR-FRET) and AlphaScreen® formats, monitoring phosphorylation of a biotinylated peptide substrate derived from the activation loop of Akt. Development of homogeneous assays enabled screening of a focused kinase library of ~21,500 compounds in 1536-well TR-FRET format in duplicate. Upon validation of hits in an alternative 384-well AlphaScreen® assay, several classes of structurally diverse PDK1 inhibitors, including tetracyclics, tricyclics, azaindoles, indazoles, and indenylpyrazoles, were identified, thus confirming the utility and sensitivity of the developed assays. Further testing in PC3 prostate cancer cells confirmed that representatives of the tetracyclic series showed intracellular modulation of the PDK1 activity, as evident from decreased phosphorylation levels of AKT, RSK, and S6-ribosomal protein.

A Simplified Scintillation Proximity Assay for Fatty Acid Synthase Activity: Development and Comparison with Other FAS Activity Assays

Fatty acid synthase (FAS), an essential enzyme for de novo lipogenesis, has been implicated in a number of disease states, including obesity, dyslipidemia, and cancer. To identify small-molecule inhibitors of FAS, the authors developed a bead-based scintillation proximity assay (SPA) to detect the fatty acid products of FAS enzymatic activity. This homogeneous SPA assay discriminates between a radiolabeled hydrophilic substrate of FAS (acetyl-coenzyme A) and the labeled lipophilic products of FAS (fatty acids), generating signal only when labeled fatty acids are present. The assay requires a single addition of unmodified polystyrene imaging SPA beads and can be miniaturized to 384- or 1536-well density with appropriate assay statistics for high-throughput screening. High-potency FAS inhibitors were used to compare the sensitivity of the SPA bead assay with previously described assays that measure FAS reaction intermediates (CoA-SH and NADP +). The advantages and disadvantages of these different FAS assays in small-molecule inhibitor discovery are discussed. (Journal of Biomolecular Screening 2009:636-642)

High-Throughput Analysis of HGF-Stimulated Cell Scattering:

Historically, only relatively low-throughput or expensive methods have been available to measure cell migration. Hepatocyte growth factor (HGF) is a ligand for the tyrosine kinase receptor Met that, in addition to mediating proliferation and survival, increases cell motility and metastasis. The authors have developed a high-throughput imaging assay for measuring inhibition of HGF-induced scattering in human HPAF-II pancreatic adenocarcinoma cells. Following treatment with test compounds and HGF for 24 h, cells are labeled with a nuclear stain and imaged at 10× magnification. The proximity of neighboring nuclei is measured, and the distribution of internuclear distances across each field of view is used to calculate the fraction of scattered cells. This method of analysis can be extended to other cell types and signaling pathways and, compared with other membrane-based migration assays currently available, the assay is significantly lower in cost, is less labor intensive, and provides higher throughput. (Journal of Biomolecular Screening 2008:847-854)

Lead optimization of 4,4-biaryl piperidine amides as γ-secretase inhibitors.

Alzheimers disease is a major unmet medical need with pathology characterized by extracellular proteinaceous plaques comprised primarily of β-amyloid. γ-Secretase is a critical enzyme in the cellular pathway responsible for the formation of a range of β-amyloid peptides; one of which, Aβ42, is believed to be responsible for the neuropathological features of the disease. Herein, we report 4,4 disubstituted piperidine γ-secretase inhibitors that were optimized for in vitro cellular potency and pharmacokinetic properties in vivo. Key agents were further characterized for their ability to lower cerebral Aβ42 production in an APP-YAC mouse model. This structural series generally suffered from sub-optimal pharmacokinetics but hypothesis driven lead optimization enabled the discovery of γ-secretase inhibitors capable of lowering cerebral Aβ42 production in mice.

Evaluation of JAK3 biology in autoimmune disease using a highly selective, irreversible JAK3 inhibitor

Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km value for each JAK isozyme ranged from 31.8 to 2.9 μM for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and >35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.

Integration of Affinity Selection-Mass Spectrometry and Functional Cell-Based Assays to Rapidly Triage Druggable Target Space within the NF-κB Pathway.

The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection–mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway.

Development of a Cell-Based Assay for Measurement of c-Met Phosphorylation Using AlphaScreenTM Technology and High-Content Imaging Analysis

c-Met is a receptor tyrosine kinase (RTK) with a critical role in many fundamental cellular processes, including cell proliferation and differentiation. Deregulated c-Met signaling has been implicated in both the initiation and progression of human cancers and therefore represents an attractive target for anticancer therapy. Monitoring the phosphorylation status of relevant tyrosine residues provides an important method of assessing c-Met kinase activity. This report describes a novel assay to monitor c-Met phosphorylation in cells using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen™) technology. Using AlphaScreen™, the authors were able to detect both global and site-specific phosphorylation of c-Met in transformed cell lines. Data obtained from the AlphaScreen™ assay were compared to data obtained from a high-content imaging (HCI) method developed in parallel to monitor c-Met phosphorylation at the single cell level. The AlphaScreen™ assay was miniaturized to a 384-well format with acceptable signal-to-background ratio (S/B) and Z′ statistics and was employed to measure c-Met kinase activity in situ after treatment with potent c-Met-specific kinase inhibitors. The authors discuss the utility of quantifying endogenous cellular c-Met phosphorylation in lead optimization and how the modular design of the AlphaScreen™ assay allows its adaptation to measure cellular activity of other kinases. (Journal of Biomolecular Screening 2009:404-411)