Comparative kinase and cancer cell panel profiling of kinase inhibitors approved for clinical use from 2018 to 2020.

During the last two decades, kinase inhibitors have become the major drug class for targeted cancer therapy. Although the number of approved kinase inhibitors increases rapidly, comprehensive in vitro profiling and comparison of inhibitor activities is often lacking in the public domain. Here we report the extensive profiling and comparison of 21 kinase inhibitors approved by the FDA for oncology indications since June 2018 and 13 previously approved comparators on panels of 255 biochemical kinase assays and 134 cancer cell line viability assays. Comparison of the cellular inhibition profiles of the EGFR inhibitors gefitinib, dacomitinib, and osimertinib identified the uncommon EGFR p.G719S mutation as a common response marker for EGFR inhibitors. Additionally, the FGFR inhibitors erdafitinib, infigratinib, and pemigatinib potently inhibited the viability of cell lines which harbored oncogenic alterations in FGFR1-3, irrespective of the specific clinical indications of the FGFR inhibitors. These results underscore the utility of in vitro kinase inhibitor profiling in cells for identifying new potential stratification markers for patient selection. Furthermore, comparison of the in vitro inhibition profiles of the RET inhibitors pralsetinib and selpercatinib revealed they had very similar biochemical and cellular selectivity. As an exception, an NTRK3 fusion-positive cell line was potently inhibited by pralsetinib but not by selpercatinib, which could be explained by the targeting of TRK kinases in biochemical assays by pralsetinib but not selpercatinib. This illustrates that unexpected differences in cellular activities between inhibitors that act through the same primary target can be explained by subtle differences in biochemical targeting. Lastly, FLT3-mutant cell lines were responsive to both FLT3 inhibitors gilteritinib and midostaurin, and the PI3K inhibitor duvelisib. Biochemical profiling revealed that the FLT3 and PI3K inhibitors targeted distinct kinases, indicating that unique dependencies can be identified by combined biochemical and cellular profiling of kinase inhibitors. This study provides the first large scale kinase assay or cell panel profiling study for newly approved kinase inhibitors, and shows that comprehensive in vitro profiling of kinase inhibitors can provide rationales for therapy selection and indication expansion of approved kinase inhibitors.

Pharmacological validation of TDO as a target for Parkinson's disease.

Parkinson's disease patients suffer from both motor and nonmotor impairments. There is currently no cure for Parkinson's disease, and the most commonly used treatment, levodopa, only functions as a temporary relief of motor symptoms. Inhibition of the expression of the L-tryptophan-catabolizing enzyme tryptophan 2,3-dioxygenase (TDO) has been shown to inhibit aging-related α-synuclein toxicity in Caenorhabditis elegans. To evaluate TDO inhibition as a potential therapeutic strategy for Parkinson's disease, a brain-penetrable, small molecule TDO inhibitor was developed, referred to as NTRC 3531-0. This compound potently inhibits human and mouse TDO in biochemical and cell-based assays and is selective over IDO1, an evolutionary unrelated enzyme that catalyzes the same reaction. In mice, NTRC 3531-0 increased plasma and brain L-tryptophan levels after oral administration, demonstrating inhibition of TDO activity in vivo. The effect on Parkinson's disease symptoms was evaluated in a rotenone-induced Parkinson's disease mouse model. A structurally dissimilar TDO inhibitor, LM10, was evaluated in parallel. Both inhibitors had beneficial effects on rotenone-induced motor and cognitive dysfunction as well as rotenone-induced dopaminergic cell loss and neuroinflammation in the substantia nigra. Moreover, both inhibitors improved intestinal transit and enhanced colon length, which indicates a reduction of the rotenone-induced intestinal dysfunction. Consistent with this, mice treated with TDO inhibitor showed decreased expression of rotenone-induced glial fibrillary acidic protein, which is a marker of enteric glial cells, and decreased α-synuclein accumulation in the enteric plexus. Our data support TDO inhibition as a potential therapeutic strategy to decrease motor, cognitive, and gastrointestinal symptoms in Parkinson's disease.

Chemotherapy sensitivity testing on ovarian cancer cells isolated from malignant ascites.

BACKGROUND: In epithelial ovarian cancer (EOC), 15-20% of the tumors do not respond to first-line chemotherapy (paclitaxel with platinum-based therapy), and in recurrences this number increases. Our aim is to determine the feasibility of cell proliferation assays of tumor cells isolated from malignant ascites to predict in vitro chemotherapy sensitivity, and to correlate these results with clinical outcome. MATERIALS AND METHODS: Ascites was collected from twenty women with advanced EOC. Cell samples were enriched for tumor cells and EOC origin was confirmed by intracellular staining of CK7, surface staining of CA125 and EpCAM, and HE4 gene expression. In vitro sensitivity to chemotherapy was determined in cell proliferation assays using intracellular ATP content as an indirect measure of cell number. In vitro drug response was quantified by calculation of the drug concentration at which cell growth was inhibited with 50%. Clinical outcome was determined using post-treatment CA125 level. RESULTS: Cell samples of twenty patients were collected, of which three samples that failed to proliferate were excluded in the analysis (15%). Three other samples were excluded, because clinical outcome could not be determined correctly. In twelve of the fourteen remaining cases (86%) in vitro drug sensitivity and clinical outcome corresponded, while in two samples (14%) there was no correspondence. CONCLUSIONS: Our study demonstrates the feasibility of drug sensitivity tests using tumor cells isolated from ascites of advanced EOC patients. Larger observational studies are required to confirm the correlation between the in vitro sensitivity and clinical outcome.

Targeting Indoleamine 2,3-Dioxygenase in Cancer Models Using the Novel Small Molecule Inhibitor NTRC 3883-0.

Indoleamine 2,3-dioxygenase (IDO1) is a key regulator of immune suppression by catalyzing the oxidation of L-tryptophan. IDO1 expression has been related to poor prognosis in several cancers and to resistance to checkpoint immunotherapies. We describe the characterization of a novel small molecule IDO1 inhibitor, NTRC 3883-0, in a panel of biochemical and cell-based assays, and various cancer models. NTRC 3883-0 released the inhibitory effect of IDO1 on CD8-positive T cell proliferation in co-cultures of IDO1-overexpressing cells with healthy donor lymphocytes, demonstrating its immune modulatory activity. In a syngeneic mouse model using IDO1-overexpressing B16F10 melanoma cells, NTRC 3883-0 effectively counteracted the IDO1-induced modulation of L-tryptophan and L-kynurenine levels, demonstrating its in vivo target modulation. Finally, we studied the expression and activity of IDO1 in primary cell cultures established from the malignant ascites of ovarian cancer patients. In these cultures, IDO1 expression was induced upon stimulation with IFNγ, and its activity could be inhibited by NTRC 3883-0. Based on these results, we propose the use of ascites cell-based functional assays for future patient stratification. Our results are discussed in light of the recent discontinuation of clinical trials of more advanced IDO1 inhibitors and the reconsideration of IDO1 as a valid drug target.

Cell Panel Profiling Reveals Conserved Therapeutic Clusters and Differentiates the Mechanism of Action of Different PI3K/mTOR, Aurora Kinase and EZH2 Inhibitors.

Cancer cell line panels are important tools to characterize the in vitro activity of new investigational drugs. Here, we present the inhibition profiles of 122 anticancer agents in proliferation assays with 44 or 66 genetically characterized cancer cell lines from diverse tumor tissues (Oncolines). The library includes 29 cytotoxics, 68 kinase inhibitors, and 11 epigenetic modulators. For 38 compounds this is the first comparative profiling in a cell line panel. By strictly maintaining optimized assay protocols, biological variation was kept to a minimum. Replicate profiles of 16 agents over three years show a high average Pearson correlation of 0.8 using IC50 values and 0.9 using GI50 values. Good correlations were observed with other panels. Curve fitting appears a large source of variation. Hierarchical clustering revealed 44 basic clusters, of which 26 contain compounds with common mechanisms of action, of which 9 were not reported before, including TTK, BET and two clusters of EZH2 inhibitors. To investigate unexpected clusterings, sets of BTK, Aurora and PI3K inhibitors were profiled in biochemical enzyme activity assays and surface plasmon resonance binding assays. The BTK inhibitor ibrutinib clusters with EGFR inhibitors, because it cross-reacts with EGFR. Aurora kinase inhibitors separate into two clusters, related to Aurora A or pan-Aurora selectivity. Similarly, 12 inhibitors in the PI3K/AKT/mTOR pathway separated into different clusters, reflecting biochemical selectivity (pan-PI3K, PI3Kßγδ-isoform selective or mTOR-selective). Of these, only allosteric mTOR inhibitors preferentially targeted PTEN-mutated cell lines. This shows that cell line profiling is an excellent tool for the unbiased classification of antiproliferative compounds. Mol Cancer Ther; 15(12); 3097-109. ©2016 AACR.

Selective Targeting of CTNBB1-, KRAS- or MYC-Driven Cell Growth by Combinations of Existing Drugs.

The aim of combination drug treatment in cancer therapy is to improve response rate and to decrease the probability of the development of drug resistance. Preferably, drug combinations are synergistic rather than additive, and, ideally, drug combinations work synergistically only in cancer cells and not in non-malignant cells. We have developed a workflow to identify such targeted synergies, and applied this approach to selectively inhibit the proliferation of cell lines with mutations in genes that are difficult to modulate with small molecules. The approach is based on curve shift analysis, which we demonstrate is a more robust method of determining synergy than combination matrix screening with Bliss-scoring. We show that the MEK inhibitor trametinib is more synergistic in combination with the BRAF inhibitor dabrafenib than with vemurafenib, another BRAF inhibitor. In addition, we show that the combination of MEK and BRAF inhibitors is synergistic in BRAF-mutant melanoma cells, and additive or antagonistic in, respectively, BRAF-wild type melanoma cells and non-malignant fibroblasts. This combination exemplifies that synergistic action of drugs can depend on cancer genotype. Next, we used curve shift analysis to identify new drug combinations that specifically inhibit cancer cell proliferation driven by difficult-to-drug cancer genes. Combination studies were performed with compounds that as single agents showed preference for inhibition of cancer cells with mutations in either the CTNNB1 gene (coding for ß-catenin), KRAS, or cancer cells expressing increased copy numbers of MYC. We demonstrate that the Wnt-pathway inhibitor ICG-001 and trametinib acted synergistically in Wnt-pathway-mutant cell lines. The ERBB2 inhibitor TAK-165 was synergistic with trametinib in KRAS-mutant cell lines. The EGFR/ERBB2 inhibitor neratinib acted synergistically with the spindle poison docetaxel and with the Aurora kinase inhibitor GSK-1070916 in cell lines with MYC amplification. Our approach can therefore efficiently discover novel drug combinations that selectively target cancer genes.

High-throughput screening with immobilized metal ion affinity-based fluorescence polarization detection, a homogeneous assay for protein kinases.

Protein kinases are one of the most important target classes in high-throughput screening today. The use of generic assay technologies facilitates assay development for new targets and decreases the time needed for implementation of assays in robotic screening. For tyrosine kinases, several generic assay technology platforms are available. These technologies make use of high-affinity antibodies that discriminate between phosphorylated tyrosines and non-phosphorylated tyrosines. Similar generic antibodies specific for phosphoserine or phosphothreonine are lacking. Recently, a non-antibody-based fluorescence polarization assay for protein kinases has become available, called IMAP (Molecular Devices, Sunnyvale, CA). In this assay, a fluorescently labeled peptide substrate that is phosphorylated by kinase is captured on metal-derivatized nanoparticles. We have evaluated IMAP in high-throughput screening, and compared this technology with a competition fluorescence polarization immunoassay based on an antibody specific for a phosphorylated peptide substrate. A random collection of >250000 compounds was screened with the two assays. Fluorescent library compounds were identified by calculation of fluorescence intensity values from the screening data, and by assaying in the absence of fluorescent reagents. Fluorescence polarization artifacts were filtered out further by testing in an ELISA-based kinase assay. Our data show that IMAP is a robust technology for high-throughput screening of kinase targets, and suggest that it is less susceptible to fluorescence polarization artifacts than the competition fluorescence polarization immunoassay.

High-throughput fluorescence-based screening assays for tryptophan-catabolizing enzymes.

Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecule with an excitation wavelength of 400 nm and an emission wavelength of 510 nm. We provide the first side-by-side comparison of a number of published inhibitors of IDO1 and TDO and reveal that the preclinical IDO1 inhibitor Compound 5l shows significant cross-reactivity with TDO, while the relative selectivity of other published inhibitors was confirmed. The suitability for high-throughput screening of the assays was demonstrated by screening a library of 87,000 chemical substances in 384- or 1536-well format. Finally, we demonstrate that the assay can also be used to measure the capacity of cells to metabolize tryptophan and to measure the cellular potency of IDO1 and TDO inhibitors.

Comparison of the cancer gene targeting and biochemical selectivities of all targeted kinase inhibitors approved for clinical use.

The anti-proliferative activities of all twenty-five targeted kinase inhibitor drugs that are in clinical use were measured in two large assay panels: (1) a panel of proliferation assays of forty-four human cancer cell lines from diverse tumour tissue origins; and (2) a panel of more than 300 kinase enzyme activity assays. This study provides a head-on comparison of all kinase inhibitor drugs in use (status Nov. 2013), and for six of these drugs, the first kinome profiling data in the public domain. Correlation of drug activities with cancer gene mutations revealed novel drug sensitivity markers, suggesting that cancers dependent on mutant CTNNB1 will respond to trametinib and other MEK inhibitors, and cancers dependent on SMAD4 to small molecule EGFR inhibitor drugs. Comparison of cellular targeting efficacies reveals the most targeted inhibitors for EGFR, ABL1 and BRAF(V600E)-driven cell growth, and demonstrates that the best targeted agents combine high biochemical potency with good selectivity. For ABL1 inhibitors, we computationally deduce optimized kinase profiles for use in a next generation of drugs. Our study shows the power of combining biochemical and cellular profiling data in the evaluation of kinase inhibitor drug action.

Multidimensional profiling of CSF1R screening hits and inhibitors: assessing cellular activity, target residence time, and selectivity in a higher throughput way.

Over the past years, improvements in high-throughput screening (HTS) technology and compound libraries have resulted in a dramatic increase in the amounts of good-quality screening hits, and there is a growing need for follow-on hit profiling assays with medium throughput to further triage hits. Here the authors present such assays for the colony-stimulating factor 1 receptor (CSF1R, Fms), including tests for cellular activity and a homogeneous assay to measure affinity for inactive CSF1R. They also present a high-throughput assay to measure target residence time, which is based on competitive binding kinetics. To better fit k(off) rates, they present a modified mathematical model for competitive kinetics. In all assays, they profiled eight reference inhibitors (imatinib, sorafenib, sunitinib, tandutinib, dasatinib, GW2580, Ki20227, and J&J's pyrido[2,3-d]pyrimidin-5-one). Using the known biochemical selectivities of these inhibitors, which can be quantified using metrics such as the selectivity entropy, the authors have determined which assay readout best predicts hit selectivity. Their profiling shows surprisingly that imatinib has a preference for the active form of CSF1R and that Ki20227 has an unusually slow target dissociation rate. This confirms that follow-on hit profiling is essential to ensure that the best hits are selected for lead optimization.