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1
Neal, Frances, Joanne Arnold, Christine J. Rossant, et al. "Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays." Journal of Biomolecular Screening 21, no. 1 (2015): 24–34. http://dx.doi.org/10.1177/1087057115610070.
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Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets.
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Benicchi, Tiziana, Sara Iozzi, Andreas Svahn, et al. "A Homogeneous HTRF Assay for the Identification of Inhibitors of the TWEAK-Fn14 Protein Interaction." Journal of Biomolecular Screening 17, no. 7 (2012): 933–45. http://dx.doi.org/10.1177/1087057112447873.
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The TWEAK-Fn14 pathway is upregulated in models of inflammation, autoimmune diseases, and cancer. Both TWEAK and Fn14 show increased expression also in the CNS in response to different stimuli, particularly astrocytes, microglia, and neurons, leading to activation of NF-κB and release of proinflammatory cytokines. Although neutralizing antibodies against these proteins have been shown to have therapeutic efficacy in animal models of inflammation, no small-molecule therapeutics are yet available. Here, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF)–based screening assay together with several counterassays for the identification of small-molecule inhibitors of this protein-protein interaction. Recombinant HIS-TWEAK and Fn14-Fc proteins as well as FLAG-TWEAK and Fn14-FLAG proteins and an anti-Fn14 antibody were used to establish and validate these assays and to screen a library of 60 000 compounds. Two HTRF counterassays with unrelated proteins in the same assay format, an antiaggregation assay and a redox assay, were applied to filter out potential false-positive compounds. The novel assay and associated screening cascade should be useful for the discovery of small-molecule inhibitors of the TWEAK-Fn14 protein interaction.
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Jordan, Allan, Philip Addis, Toby Allen, et al. "Abstract LB030: Building a platform of validated high throughput screening approaches for molecular glue degrader identification." Cancer Research 84, no. 7_Supplement (2024): LB030. http://dx.doi.org/10.1158/1538-7445.am2024-lb030.
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Abstract Over recent years, Induced proximity therapeutics (IPT) and targeted protein degradation (TPD) approaches have become increasingly popular. Whilst significant initial efforts focused upon heterobifunctional degraders, molecular glue degraders (MGDs) represent the next generation proximity-based drugs, but their rational discovery remains challenging. Here, we describe unbiased and broadly applicable approaches for molecular glue hit ID through the development of a suite of orthogonal and highly complementary screening approaches, relying on Homogeneous Time-Resolved Fluorescence (HTRF), Surface Plasmon Resonance (SPR) and High Content Imaging (HCI). To demonstrate the utility of these assays, we have focussed on the therapeutically relevant protein SOS1 as a model target. SOS1 is the major guanine nucleotide exchange factor (GEF) for KRAS, where inhibition of the SOS1/KRAS interaction reduces tumor growth in preclinical settings, and small molecule inhibitors of the SOS1/KRAS interaction are currently undergoing clinical testing for KRAS driven cancers. Our HTRF approach measures the induced interaction between the ubiquitin ligase CRBN/DDB1 and the SOS1 protein. The assay was successfully developed and validated in a miniaturized format utilizing a bifunctional tool compound as a positive control. Complementing our HTRF based approach, we also developed a highly sensitive SPR assay for detecting compounds that generate a ternary complex with CRBN/DDB1. These assay formats are amenable for large scale HTS screening, and as proof-of-concept we have established workflows and screened compound collections from the Sygnature LeadFinder and fragment libraries, in each of these assay platforms. Our combined biochemical/biophysical approach to glue identification using HTRF and SPR demonstrates the successful and scalable use of these methodologies for HTS, with the exciting potential for identification of novel MGD candidates for SOS1. To further complement these strategies for glue identification, we have additionally developed a functional cell-based approach utilizing HCI. HCI offers a medium-high throughput approach to monitor endogenous protein abundance and localization in complex cellular models. One major advantage of imaging assays is that they are amenable to multiplexing, allowing simultaneous detection of target engagement, biomarker analysis and compound toxicity. Here, we showcase HCI as a robust, high throughput method of screening for induced protein degradation at the endogenous level, again focusing on the KRAS pathway. Using this approach we quantified endogenous protein abundance in response to compound treatment, as well as downstream effects in the RAS pathway. Together, these approaches demonstrate multiple, complementary options for identifying molecular glue degraders. Further, the described assays are also applicable for screening and characterization of bifunctional degrader compounds. This platform is poised to identify high quality lead compounds across a range of oncology targets in areas of unmet clinical need. Citation Format: Allan Jordan, Philip Addis, Toby Allen, Tarun Ayra, Sarah Beck, Roslyn Brant, Roland Hjerpe, Benoit Gourdet, Martin Jennings, Rachel Lawrence, Sigrun Campbell-Maurer, Pei Cing Ng, Lyn Parkinson, Eva Rejnowicz, Gonzalo Robles, Elizabeth Rosethorne, Joshua Shaw, Duncan Smith, Denise Swift, Daniel Tait, Stuart Thomson, Chris Tomlinson, Ailsa Townley, Stephanie Ward, Clare Wilson. Building a platform of validated high throughput screening approaches for molecular glue degrader identification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB030.
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Thomas-Fowlkes, Brande, Steven Cifelli, Sarah Souza, et al. "Cell-Based In Vitro Assay Automation: Balancing Technology and Data Reproducibility/Predictability." SLAS TECHNOLOGY: Translating Life Sciences Innovation 25, no. 3 (2020): 276–85. http://dx.doi.org/10.1177/2472630320902095.
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G-protein-coupled receptors (GPCRs) are modulated by many marketed drugs, and as such, they continue to be key targets for drug discovery and development. Many GPCR targets at Merck Research Laboratories (MRL) are profiled using homogenous time-resolved fluorescence (HTRF) inositol monophosphate (IP-1) cell-based functional assays using adherent cells in 384-well microplates. Due to discrepancies observed across several in vitro assays supporting lead optimization structure–activity relationship (SAR) efforts, different assay paradigms were evaluated for removing growth medium from the assay plates prior to compound addition and determination of IP-1 accumulation. Remarkably, employing the noncontact centrifugation BlueWasher method leads to left-shifted potencies across multiple structural classes and rescues “false negatives” relative to the traditional manual evacuation method. Further, assay performance is improved, with the minimum significant ratio of challenging chemotypes dropping from ~5–6 to <3. While the impact of BlueWasher on a broad range of our GPCR targets remains to be determined, for highly protein-bound small molecules, it provides a path toward improving assay reproducibility across scientists and sites as well as reducing replicates in SAR assay support.
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Ding, Mei, Anders Cavallin, Nils-Olov Hermansson, Pia Berntsson, Lisa Jinton, and Sandra Rodrigo Blomqvist. "Comparing Flow Cytometry QBeads PlexScreen Assays with Other Immunoassays for Determining Multiple Analytes." SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, no. 7 (2018): 676–86. http://dx.doi.org/10.1177/2472555218771610.
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Immunoassays, utilizing the affinity of antibodies to their antigens, are powerful techniques and have been widely used for quantifying analytes, such as cytokines, in biological samples in the clinic and in drug discovery. Various immunoassays have been developed to fit for different purposes. Recently, bead-based flow cytometry assays have emerged as interesting options for multiplex quantification of analytes. In this study, we compared high-throughput flow cytometry multiplex iQue QBeads PlexScreen assays with several other commonly used immunoassays, including MSD, Luminex, ELISA, HTRF, and AlphaLISA assays. Head-to-head comparisons of quantification data of the following cytokines were made: (1) IL-2, IL-4, IL-6, IL-13, IL-17A, IFNγ, KC/GRO, RANTES, and TNFα in mouse bronchoalveolar lavage fluid samples; (2) IL-10 and TNFα in supernatants from a THP-1 cell assay; (3) IL-6, IL-10, IL-12p70, and TNFα in supernatants from a human monocyte-derived dendritic cell assay; and (4) IL-2 in supernatants from a human CD4+ cell assay. The results demonstrated a good assay correlation between the iQue and the compared assays for the cytokine studied. Although overall good assay correlations were observed, our results showed that the iQue assay generated different absolute cytokine values for some cytokines in the same sample sets compared with other assays.
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Kimos, Martha, Maggi Burton, David Urbain, et al. "Development of an HTRF Assay for the Detection and Characterization of Inhibitors of Catechol-O-Methyltransferase." Journal of Biomolecular Screening 21, no. 5 (2015): 490–95. http://dx.doi.org/10.1177/1087057115616793.
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Catechol-O-methyltransferase (COMT) plays an important role in the deactivation of catecholamine neurotransmitters and hormones. Inhibitors of COMT, such as tolcapone and entacapone, are used clinically in the treatment of Parkinson’s disease. Discovery of novel inhibitors has been hampered by a lack of suitable assays for high-throughput screening (HTS). Although assays using esculetin have been developed, these are affected by fluorescence, a common property of catechol-type compounds. We have therefore evaluated a new homogenous time-resolved fluorescence (HTRF)–based assay from CisBio (Codolet, France), which measures the production of S-adenosyl-L-homocysteine (SAH). The assay has been run in both HTS and medium-throughput screening (MTS) modes. The assay was established using membranes expressing human membrane-bound COMT and was optimized for protein and time to give an acceptable signal window, good potency for tolcapone, and a high degree of translation between data in fluorescence ratio and data in terms of [SAH] produced. pIC50 values for the hits from the HTS mode were determined in the MTS mode. The assay also proved suitable for kinetic studies such as Km,app determination.
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Surmiak, Ewa, Katarzyna Magiera-Mularz, Bogdan Musielak, et al. "PD-L1 Inhibitors: Different Classes, Activities, and Mechanisms of Action." International Journal of Molecular Sciences 22, no. 21 (2021): 11797. http://dx.doi.org/10.3390/ijms222111797.
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Targeting the programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) interaction has become an established strategy for cancer immunotherapy. Although hundreds of small-molecule, peptide, and peptidomimetic inhibitors have been proposed in recent years, only a limited number of drug candidates show good PD-1/PD-L1 blocking activity in cell-based assays. In this article, we compare representative molecules from different classes in terms of their PD-1/PD-L1 dissociation capacity measured by HTRF and in vitro bioactivity determined by the immune checkpoint blockade (ICB) co-culture assay. We point to recent discoveries that underscore important differences in the mechanisms of action of these molecules and also indicate one principal feature that needs to be considered, which is the eventual human PD-L1 specificity.
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Wewer Albrechtsen, Nicolai J., Simon Veedfald, Astrid Plamboeck, et al. "Inability of Some Commercial Assays to Measure Suppression of Glucagon Secretion." Journal of Diabetes Research 2016 (2016): 1–5. http://dx.doi.org/10.1155/2016/8352957.
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Glucagon levels are increasingly being included as endpoints in clinical study design and more than 400 current diabetes-related clinical trials have glucagon as an outcome measure. The reliability of immune-based technologies used to measure endogenous glucagon concentrations is, therefore, important. We studied the ability of immunoassays based on four different technologies to detect changes in levels of glucagon under conditions where glucagon levels are strongly suppressed. To our surprise, the most advanced technological methods, employing electrochemiluminescence or homogeneous time resolved fluorescence (HTRF) detection, were not capable of detecting the suppression induced by a glucose clamp (6 mmol/L) with or without atropine in five healthy male participants, whereas a radioimmunoassay and a spectrophotometry-based ELISA were. In summary, measurement of glucagon is challenging even when state-of-the-art immune-based technologies are used. Clinical researchers using glucagon as outcome measures may need to reconsider the validity of their chosen glucagon assay. The current study demonstrates that the most advanced approach is not necessarily the best when measuring a low-abundant peptide such as glucagon in humans.
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Musielak, Bogdan, Justyna Kocik, Lukasz Skalniak, et al. "CA-170 – A Potent Small-Molecule PD-L1 Inhibitor or Not?" Molecules 24, no. 15 (2019): 2804. http://dx.doi.org/10.3390/molecules24152804.
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CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins – important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 – a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57.
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Palaniappan, Murugesan, Kurt M. Bohren, Yong Wang, Damian W. Young, Suzanne A. Fuqua, and Martin M. Matzuk. "Abstract P6-10-12: Discovery and Development of Next-Generation Estrogen Receptor Mutant Inhibitors using DNA-Encoded Chemical Library Screening." Cancer Research 83, no. 5_Supplement (2023): P6–10–12—P6–10–12. http://dx.doi.org/10.1158/1538-7445.sabcs22-p6-10-12.
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Abstract Background: Activating somatic ESR1 mutations Y537S and D538G occur more frequently in endocrine therapy-resistant metastatic breast cancer, which is associated with an aggressive phenotype and poor survival in breast cancer patients. These gain of function mutant receptors are constitutively active and allow resistance to first-line endocrine therapies. Therefore, the development of next-generation small molecule drugs targeting mutant estrogen receptor (ER) is an important priority. Here, we searched the small molecule inhibitors for Y537S and D538D ER mutants using DNA-encoded chemical library screening. Methods: Wild type (WT) and mutant ER ligand binding domain (LBD) proteins were expressed in E. coli. The soluble proteins were purified by Ni-NTA chromatography followed by anion exchange and size exclusion chromatography. Homogeneous time-resolved fluorescence (HTRF) and fluorescent polarization (FP) assays were performed in these purified proteins. We employed a DNA-encoded chemical library affinity selection using our in-house collection of 6 billion compounds. Hit compounds were resynthesized and validated in biochemical assays. Finally, we have performed functional studies in CRISPR-Cas9 knock-in of Y537S and D538G mutant MCF-7 breast cancer cells. Results: We have successfully purified microgram amounts of ERα LBD of WT, Y537S, and D538G proteins. To test whether the purified WT and mutant proteins are active, HTRF and FP assays were performed in the presence and absence of estradiol and 4OH tamoxifen. Steroid receptor coactivator 3 (SRC3) peptide binding to the WT ER protein occurred only in the presence of estradiol. However, Y537S and D538G proteins are recruited by the SRC3 peptide in the absence of estradiol, indicating that these mutants are constitutively active and bind to SRC3. Furthermore, an in vitro biochemical FP assay was also established for WT and mutants in the presence of estradiol and 4OH tamoxifen. The screen of our multibillion small molecule collection of DNA-encoded chemical libraries identified several hits in WT and mutant ER. To confirm the selection output, we synthesized off-DNA compounds and validated these in biochemical and cell-based studies. We have identified that the compounds, CDD-1272 and CDD-1274, are active in HTRF and FP assays. Furthermore, these compounds inhibit WT and mutant cell growth in the presence of estradiol. More importantly, CDD-1274 degrades ER mutant and cyclin D1 proteins. In addition, CDD-1274 induced p21 protein expression in WT and mutant cells. Conclusions: We have identified potent novel ER mutant binders by using our DNA-encoded chemical library platform. Our compounds are active in biochemical and ER mutant cell lines, suggesting these molecules are potential chemical probes to explore in in vivo models of breast cancer. Support: NIH/NCI R03 CA259664 and CPRIT RP220524 to MP. Citation Format: Murugesan Palaniappan, Kurt M. Bohren, Yong Wang, Damian W. Young, Suzanne A. Fuqua, Martin M. Matzuk. Discovery and Development of Next-Generation Estrogen Receptor Mutant Inhibitors using DNA-Encoded Chemical Library Screening [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-12.
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Katsuya, Ken, Yuji Hori, Daisuke Oikawa, et al. "High-Throughput Screening for Linear Ubiquitin Chain Assembly Complex (LUBAC) Selective Inhibitors Using Homogenous Time-Resolved Fluorescence (HTRF)-Based Assay System." SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, no. 10 (2018): 1018–29. http://dx.doi.org/10.1177/2472555218793066.
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The nuclear factor κB (NF-κB) pathway is critical for regulating immune and inflammatory responses, and uncontrolled NF-κB activation is closely associated with various inflammatory diseases and malignant tumors. The Met1-linked linear ubiquitin chain, which is generated by linear ubiquitin chain assembly complex (LUBAC), is important for regulating NF-κB activation. This process occurs through the linear ubiquitination of NF-κB essential modulator, a regulatory subunit of the canonical inhibitor of the NF-κB kinase complex. In this study, we have established a robust and efficient high-throughput screening (HTS) platform to explore LUBAC inhibitors, which may be used as tool compounds to elucidate the pathophysiological role of LUBAC. The HTS platform consisted of both cell-free and cell-based assays: (1) cell-free LUBAC-mediated linear ubiquitination assay using homogenous time-resolved fluorescence technology and (2) cell-based LUBAC assay using the NF-κB luciferase reporter gene assay. By using the HTS platform, we performed a high-throughput chemical library screen and identified several hit compounds with selectivity against a counterassay. Liquid chromatography–mass spectrometry analysis revealed that these compounds contain a chemically reactive lactone structure, which is transformed to give reactive α,β-unsaturated carbonyl compounds. Further investigation revealed that the reactive group of these compounds is essential for the inhibition of LUBAC activity.
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Dobritsa, Svetlana V., Iok Teng Kuok, Hai Nguyen, et al. "Development of a High-Throughput Cell-Based Assay for Identification of IL-17 Inhibitors." Journal of Biomolecular Screening 18, no. 1 (2012): 75–84. http://dx.doi.org/10.1177/1087057112459350.
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Human interleukin 17 (IL-17) is a proinflammatory cytokine derived mainly from activated T cells. Extensive evidence points to a significant role of IL-17 in many autoimmune and infectious diseases, as well as tumorigenesis and transplant rejection, and suggests that targeting IL-17 could be a promising therapeutic strategy. Robust cell-based assays would thus be essential for lead identification and the optimization of therapeutic candidates. Herein, we report a well-characterized two-step assay, consisting of (a) in vitro activation and stimulation of CD4+ T lymphocytes by a defined complex of antibodies and cytokines, leading to T helper 17 (Th17) cell differentiation and IL-17 production, and (b) IL-17 quantification in cell supernatants using a homogeneous time-resolved fluorescence (HTRF) assay. The system was optimized for and shown to be reliable in high-throughput compatible 96- and 384-well plate formats. The assay is robust (Z′ > 0.5) and simple to perform, yields a stable response, and allows for sufficient discrimination of positive (IL-17–producing cells) and negative controls (uninduced cells). The assay was validated by performing dose-response testing of rapamycin and cyclosporine A, which had previously been reported to inhibit IL-17, and determining, for the first time, their in vitro potencies (IC50s of 80 ± 23 pM and 223 ± 52 nM, respectively). Also, IKK 16, a selective small-molecule inhibitor of IκB kinase, was found to inhibit IL-17 production, with an IC50 of 315 ± 79 nM.
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Douayry, Najim, Milind Rajopadhye, and Olivia J. Kelada. "Abstract 4133: Cyclin-dependent kinase signaling in oncology: assessing the effectiveness of a CDK9-targeting using no-wash immunoassays." Cancer Research 85, no. 8_Supplement_1 (2025): 4133. https://doi.org/10.1158/1538-7445.am2025-4133.
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Abstract Cyclins are regulatory subunits that bind to a cyclin dependent kinase (CDK), thus activating the kinase and controlling its activity. Most CDKs form a complex with a particular cyclin and are involved in the regulation of either cell cycle or cell transcription. Dysregulation of CDKs has been found to be a primary driver in tumorigenesis. Thus, CDK9 is an attractive target for cancer therapeutics due to its crucial role in transcription regulation, particularly of short-lived anti-apoptotic proteins such as MCL-1 and XIAP. Among the investigated CDK9-based therapeutic strategies, the degradation of CDK9 using specific compounds like proteolysis targeting chimera (PROTAC) is of high interest. The purpose of our study was to determine whether homogeneous time-resolved fluorescence (HTRF) and a bead-based homogeneous immunoassay platform (AlphaLISA Surefire Ultra, ALSU) assays are effective solutions for identifying and characterizing PROTAC degradation of CDK9. Both assays were used to monitor the expression levels of CDK9 or GAPDH in cell lysates from HeLa or MCF-7 cells. Assessing CDK9 levels allowed the identification of a PROTAC degradation of CDK9 while the determination of GADPH levels was used as an internal control for potential changes to global protein levels in drug treatment. Cells were treated for 4h either with 1µM of a reference CDK9 degrader, Thal-SNS-032 or with the same concentration of its two individual chemical components, Thalidomide and SNS-032 (as negative controls). Cells were then lysed, and the corresponding lysates were assessed in either CDK9 or GAPDH assays. Thal-SNS6032 induced a robust degradation of CDK9 (80 to 95%) while Thalidomide and SNS-032 did not affect the expression level of CDK9. Moreover, none of the tested compounds affected the GAPDH expression level suggesting that the changes of CDK9 levels is not due to changes in the global protein concentration. To further characterize the effect of Thal-SNS-032, a dose-response treatment was carried out on HeLa and MCF-7 cells. CDK9 and GAPDH levels were assessed in the corresponding cell lysates and a dose-dependent degradation of CDK9 was observed with half-maximal degradation values (DC50) ranging from 60 to 166nM while GADPH levels remained unchanged. These data suggest that HTRF and ALSU assays are efficient solutions to identify and characterize PROTAC targeting of CDK9. Results obtained for CDK9 can be translated to other CDK targets of interest in oncology research as a broad portfolio of total CDK detection kits is available using the two assay platforms. Citation Format: Najim Douayry, Milind Rajopadhye, Olivia J. Kelada. Cyclin-dependent kinase signaling in oncology: assessing the effectiveness of a CDK9-targeting using no-wash immunoassays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4133.
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Wu, Jianghong, Yong Wan, Shuguang Liang, et al. "Abstract 4704: Biochemical and cell-based assay platforms for development of RAF inhibitors against human cancers." Cancer Research 84, no. 6_Supplement (2024): 4704. http://dx.doi.org/10.1158/1538-7445.am2024-4704.
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Abstract The RAF protein kinases are key intermediates in cellular signal transduction, functioning as direct effectors of the RAS GTPases and as the initiating kinases in the ERK cascade. In human cancer, RAF activity is frequently dysregulated due to mutations in the RAF family members (ARAF, BRAF, and CRAF) or to alterations in upstream RAF regulators, including RAS and receptor tyrosine kinases. The first and second generations of RAF inhibitors have yielded dramatic responses in malignant melanomas containing BRAF mutations; however, their overall usefulness has been limited by both intrinsic and acquired drug resistance. In addition, cancers with hyperactive RAS exhibit intrinsic resistance to these drugs. In particular, issues related to the dimerization of the RAF kinases can impact the efficacy of these compounds and are a primary cause of drug resistance. We have established biochemical HotSpotTM kinase assay, NanoBRETTM and NanoBITTM cell assay platforms for High Through Screening of kinase inhibitors. Here, we demonstrate that the 3rd generation of pan-RAF inhibitors LY3009120, LXH254, and Belvarafenib inhibit ARAF, BRAF, CRAF, BRAF(V600E), and CRAF(R391W) kinase activity in biochemical HotSpotTM assay. Our NanoBRETTM target engagement and NanoBITTM cellular assay data show that LY3009120, LXH254, and Belvarafenib bind to KRAS(G12C) primed BRAF and CRAF, and block BRAF and CRAF dimerization. Furthermore, our results show these inhibitors can block the downstream ERK phosphorylation in cellular HTRF assay and induce caspase-3/7 activation in Western blot assay in the triple negative breast cancer MDA-MB-231 cells. Taken together, our results indicate the biochemical HotSpotTM kinase activity assay, and NanoBRETTM target engagement and NanoBITTM cellular assays can serve as great platforms to facilitate RAF drug discovery against human cancers. Citation Format: Jianghong Wu, Yong Wan, Shuguang Liang, Peter Gallagher, Li Liang, Christian Loch, Haiching Ma. Biochemical and cell-based assay platforms for development of RAF inhibitors against human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4704.
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AbdulSalam, Safnas F., Sung Won Oh, Brenna P. Lee, Joseph J. Ferry, John R. Ries, and Kurumi Y. Horiuchi. "Abstract 4417: Development of biochemical screening assays to facilitate drug discovery in RNA m6A modification regulators." Cancer Research 84, no. 6_Supplement (2024): 4417. http://dx.doi.org/10.1158/1538-7445.am2024-4417.
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Abstract Over the recent years, knowledge of RNA modifications and their regulation has expanded extensively, providing novel insights and strategies to explore potential therapeutics for pathogenesis of various diseases, including cancer. Several chemical modifications in RNA have been identified so far, among these N6-methyladenosine (m6A) modification is the most abundant and well-studied epitranscriptomic marker found in mRNA and long noncoding RNA. Abnormal m6A expression is proven to be associated with tumorigenesis, cancer stemness and drug resistance of cancers. Methylation in 6-Adenosin of RNA is a dynamic and reversible process tightly regulated by its writer, m6A methyltransferase complex and erasers, FTO and ALKBH5. m6A methyltransferase complex contains METTL3-METTL14-WTAP as core components. METTL3 is the catalytic component, which is activated by heterodimer formation with METTL14. High METTL3 expression is found in several cancers such as breast, lung, liver, gastric, colorectal, AML and a METTL3 catalytic inhibitor is found to delay AML progression in mouse models. Protein that binds RNA m6A modification to execute it signaling are known as m6A readers. Readers are categorized into three main classes, YTH domain proteins, IGF2 mRNA-binding proteins and heterogeneous nuclear ribonucleoproteins. YTH family readers are found to have oncogenic roles in several cancers including AML, breast, lung, CRC and glioblastoma. Recent studies suggest inhibition of m6A binding of individual YTH family proteins is a promising therapeutic strategy however, potent inhibitors are yet to be identified. This poster summarizes current assays we have developed to facilitate cancer therapeutic discovery in m6A related protein targets. We present development and validation of an assay suitable identify in METTL3 catalytic inhibitors using our proprietary hotspot technology. We also show development of HTRF based biochemical assays for all five YTH family protein YTHDF1-3 and YTHDC1-2, to screen for molecules that disrupt protein-m6A interaction. We further study the selectivity of an FDA approved drug Tegaserod, a reported YTHDF1 inhibitor by a structure based virtual screening, between YTH family proteins. Citation Format: Safnas F. AbdulSalam, Sung Won Oh, Brenna P. Lee, Joseph J. Ferry, John R. Ries, Kurumi Y. Horiuchi. Development of biochemical screening assays to facilitate drug discovery in RNA m6A modification regulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4417.
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Patil, Sachin P., Elena Fattakhova, Jeremy Hofer, et al. "Machine-Learning Guided Discovery of Bioactive Inhibitors of PD1-PDL1 Interaction." Pharmaceuticals 15, no. 5 (2022): 613. http://dx.doi.org/10.3390/ph15050613.
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The selective activation of the innate immune system through blockade of immune checkpoint PD1-PDL1 interaction has proven effective against a variety of cancers. In contrast to six antibody therapies approved and several under clinical investigation, the development of small-molecule PD1-PDL1 inhibitors is still in its infancy with no such drugs approved yet. Nevertheless, a promising series of small molecules inducing PDL1 dimerization has revealed important spatio-chemical features required for effective PD1-PDL1 inhibition through PDL1 sequestration. In the present study, we utilized these features for developing machine-learning (ML) classifiers by fitting Random Forest models to six 2D fingerprint descriptors. A focused database of ~16 K bioactive molecules, including approved and experimental drugs, was screened using these ML models, leading to classification of 361 molecules as potentially active. These ML hits were subjected to molecular docking studies to further shortlist them based on their binding interactions within the PDL1 dimer pocket. The top 20 molecules with favorable interactions were experimentally tested using HTRF human PD1-PDL1 binding assays, leading to the identification of two active molecules, CRT5 and P053, with the IC50 values of 22.35 and 33.65 µM, respectively. Owing to their bioactive nature, our newly discovered molecules may prove suitable for further medicinal chemistry optimization, leading to more potent and selective PD1-PDL1 inhibitors. Finally, our ML models and the integrated screening protocol may prove useful for screening larger libraries for novel PD1-PDL1 inhibitors.
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Liang, Amy M., Emmanuel Claret, Josy Ouled-Diaf, et al. "Development of a Homogeneous Time-Resolved Fluorescence Leukotriene B4 Assay for Determining the Activity of Leukotriene A4 Hydrolase." Journal of Biomolecular Screening 12, no. 4 (2007): 536–45. http://dx.doi.org/10.1177/1087057107299873.
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Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotatic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA 4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF® assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB 4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay ( R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable4 assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase. ( Journal of Biomolecular Screening 2007:536-545)
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Qiu, Yuhan, Barbara Corkey, Jude Deeney, and Catherine Li. "Acyl-CoA Synthetase Inhibition Protects Clonal Pancreatic Beta-cell from Effects of Chronic Excess Nutrients." Current Developments in Nutrition 4, Supplement_2 (2020): 652. http://dx.doi.org/10.1093/cdn/nzaa049_045.
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Abstract Objectives A single nucleotide polymorphism (SNP) in the transcription factor 7 like 2 (TCF7L2) gene is strongly associated with Type 2 Diabetes (T2D), and deletion of this SNP has been shown to reduce long chain acyl-CoA synthetase 5 (ACSL5) mRNA level. Previous research in our lab has shown chronic exposure to excess nutrients (glucose and fatty acid (FA)) increases lipid droplets in beta-cells and causes basal insulin hypersecretion, left-shifted glucose-stimulated insulin secretion (GSIS), blunted maximal GSIS and reduced insulin content. We tested the efficacy of a known phenylpyrazole-carboxamide compound (herein named ADIPO C) to reduce accumulation of intracellular lipid droplets and reverse the left-shift of GSIS in beta-cells. Methods INS-1832/13 cells were cultured in RPMI media containing 10% FBS (source of FA) and either 4 or 11 mM glucose. Insulin was measured by HTRF assay (CisBio). Intracellular lipid was detected by fluorescence microscopy using Nile red. Fluorescent Bodipy-FA was used as a surrogate FA in both ACS activity assays and lipid incorporation into cells. Oxygen consumption rate was measured using the Seahorse ion flux analyzer and intracellular calcium was measured in fura 2 loaded cells mounted on an Olympus confocal microscope. Results Adipo C (10–25 µM) acutely inhibited acyl-CoA synthetase activity by up to 40% in cell homogenates and similarly reduced FA incorporation into neutral lipids in INS-1 (832/13) cells measured using thin layer chromatography. Longer exposure (72 hrs) to ADIPO C significantly decreased intracellular lipid droplets, right-shifted GSIS and increased insulin content in cells cultured in excess nutrients. Cells cultured in excess nutrients exhibited both increased basal oxygen consumption rate and intracellular calcium oscillations, which were both reduced with 72 hrs ADIPO C incubation. Conclusions Based on these results, we conclude that Adipo C has a protective effect on beta-cells exposed to a glucolipotoxic environment and thus may prove to have therapeutic potential in the prevention/treatment of T2D. Funding Sources No funding sources to report.
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Clemo, Nadine, Monica Roman-Trufero, Holger Auner, et al. "Efficacy of GCN2 inhibition by a novel small molecule AP030 in acute leukemia." Journal of Clinical Oncology 42, no. 16_suppl (2024): 6532. http://dx.doi.org/10.1200/jco.2024.42.16_suppl.6532.
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6532 Background: GCN2 is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR) that is activated in response to amino acid scarcity. Active GCN2 phosphorylates translation initiation factor eIF2α resulting in the attenuation of global protein synthesis. ISR signaling primarily promotes cell survival but may trigger cell death, dependent on the cellular context. In hematological tumors GCN2 promotes tumor cell survival under conditions of nutrient scarcity. Several GCN2 inhibitors are in clinical development for treatment of both solid and hematological tumors. Here, we describe the preclinical results of a novel, selective ATP-competitive inhibitor of GCN2. Methods: GCN2 inhibition by AP030 was determined using a biochemical Lanthascreen assay and a cell based HTRF assay measuring eIF2a phosphorylation following stimulation with Borrelidin. Kinase selectivity was determined using KinomeScan, and bespoke biochemical and cell-based assays. Interaction of AP030 with GCN2 was determined using X-ray crystallography. AP030 activity in disease-relevant tumor cell lines was determined using cell viability, caspase activation, protein expression and qPCR gene expression endpoints. Inhibition of hematological tumor growth and induction of cell death was investigated in patient samples, and ALL and AML in vivo animal models. Results: Potent inhibition of GCN2 in the Lanthascreen assay was demonstrated with AP030 (Ki of 4.4nM). Following stimulation with Borrelidin, AP030 inhibited eIF2a phosphorylation with an IC50 of 50.8nM and inhibited downstream targets CHAC1 and DDIT3 measured by qPCR. X-ray crystallography confirmed that AP030 binds to the ATP-binding site of GCN2. The KinomeScan confirmed that AP030 was highly selective against the human kinome. AP030 led to partial or complete reduction of AML cell line viability as a single agent and acted synergistically with asparaginase in ALL cell lines. Caspase 3/7 induction was observed in the AML and ALL cell lines evaluated and AP030 increased the proportion of cells undergoing apoptosis in AML patient primary samples. In the CCRF-CEM ALL systemic in vivo tumor model AP030 inhibited tumor growth in combination with asparaginase (TGI 79.33%). Treatment with 0.5-5mg/kg (q.d.) of AP030 resulted in dose-dependent tumor growth inhibition and regression of the MOLM-16 AML in vivo model (TGI 97.37% 5mg/kg). RNA sequencing of residual tumor cells revealed disruption of amino acid and protein metabolism, consistent with GCN2 inhibition. Conclusions: Targeting hematological tumor reliance on the GCN2 arm of the ISR during nutrient scarcity is a novel approach to preventing tumor cell survival. AP030 a novel, potent, selective, ATP-competitive kinase inhibitor has been shown to lead to impressive efficacy in acute leukemia. Based on these preclinical results, a phase 1/2 study has been initiated in AML.
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Perner, Florian, Sheng F. Cai, Daniela V. Wenge, et al. "Abstract 3457: Characterization of acquired resistance mutations to menin inhibitors." Cancer Research 83, no. 7_Supplement (2023): 3457. http://dx.doi.org/10.1158/1538-7445.am2023-3457.
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Abstract The menin-MLL1 interaction is critical for development of acute leukemias driven by MLL1 rearrangements (MLLr) or mutations in the Nucleophosmin 1 gene (NPM1c). Inhibition of the menin-MLL1 interaction by SNDX-5613 (revumenib) has demonstrated robust clinical responses in the current AUGMENT clinical trial (NCT04065399). During the trial, some responders relapsed during treatment due to acquired resistance in MEN1. Somatic MEN1 mutations were found at residues M327, G331 or T349 which diminished SDNX-5613 binding affinity and mediated therapeutic resistance. The presence of acquired resistance validates MEN1 as a therapeutic target in MLLr and NPM1c AML patients. Here we characterize the effects of these mutations on the activity of 6 menin inhibitor chemotypes currently in clinical trials (NCT04065399/Syndax, NCT04067336/Kura, NCT04811560/JNJ, NCT04988555/Sumitomo, NCT04752163/Daiichi, NCT05153330/Biomea). In vitro activity and binding modes for these compounds were evaluated in wild-type (WT) and mutant menins using (i) competition binding assays, (ii) cell-based proliferation assays and (iii) X-ray co-crystallography. For binding, His6-tagged MEN1 mutant proteins (G331R, M327I, M327V, T349M) were expressed and purified. Binding affinities were measured in competition binding format. The menin-MLL interaction was monitored by HTRF using Terbium labeled anti-His6 antibody and FITC labeled MLL peptide (4-43). Acquired mutations affected binding affinities (Ki) to varying degrees. Notably, M327I/V mutations reduced binding for all menin-MLL inhibitors ranging from ~30-300, indicating a class effect for this mutation. An irreversible Biomea chemotype did not inhibit menin-MLL binding in our assays. The decreased binding affinity to M327I was reflected in cell-based proliferation assays. Menin mutations were introduced into MV4;11 cells using CRISPR-Cas9 in conjunction with a homology directed repair template to edit the endogenous MEN1 coding sequence. Clonal lines were established harboring homozygous (homo) and heterozygous (het) M327I mutations. The M327I (het) MV4;11 cells experienced 15-50-fold shifts in IC50 vs WT cells, consistent with the reduced binding affinities. The molecular basis for sensitivity to M327 acquired resistance was examined by X-ray co-crystallography of inhibitors bound to M327I and WT menin. Both KO-539 and SNDX-5613 show notable changes in binding in M327I vs WT menin. The isoleucine creates a steric clash, displacing their position in the pocket as previously noted. The Janssen chemotype shows a novel binding mode. Although it has 30-fold lower affinity for M327I, it shows little change in its bound position to M327I menin. Given the clinical validation of menin inhibition in AML, the design of next generation compounds that block MLL1 binding while avoiding acquired MEN1 mutations may be a strategy to overcome acquired resistance to first generation menin inhibitors. Citation Format: Florian Perner, Sheng F. Cai, Daniela V. Wenge, Jeonghyeon Kim, Jevon Cutler, Radosław P. Nowak, Joel Cassel, Shivendra Singh, Shipra Bijpuria, William H. Miller, Eytan M. Stein, Ross L. Levine, Eric S. Fischer, Gerard M. McGeehan, Scott A. Armstrong. Characterization of acquired resistance mutations to menin inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3457.
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Paprcka, Susan Lee, Armon Goshayeshi, Suan Liu, et al. "Abstract 518: AB801 is a highly potent and selective AXL kinase inhibitor that demonstrates significant anti-tumor activity." Cancer Research 83, no. 7_Supplement (2023): 518. http://dx.doi.org/10.1158/1538-7445.am2023-518.
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Abstract Background: AXL receptor tyrosine kinase (AXL) is overexpressed in a variety of tumors and correlates with poor prognosis in cancer patients. AXL is expressed in cancer, stromal, and select immune cells, and has been implicated in the development of resistance to chemotherapy, targeted therapies & immunotherapies. Activation of AXL can be mediated by its ligand, growth arrest specific protein 6 (GAS6), or via ligand-independent homo/hetero-dimerization, both of which facilitate AXL phosphorylation, initiation of signaling cascades that promote cancer cell proliferation, survival, and an immunosuppressive microenvironment. Here we present the discovery and characterization of a novel, highly potent and selective AXL inhibitor, AB801. Materials and Methods: The potency and selectivity of AB801 against AXL and other kinases were determined using a panel of HTRF KinEASE-TK assays and via a competition binding assay utilizing DNA-tagged kinases. The effects of AB801 were further assessed by a cell-based phospho-AXL ELISA. The pharmacokinetic (PK) profile of the molecule was evaluated in preclinical species. AB801 was characterized in routine in vitro safety assays, including hERG inhibition. Downstream signaling of AXL was evaluated by phospho-array, Western blot, and qPCR. Pharmacodynamics (PD) and anti-tumor efficacy in combination with standard of care (SOC) therapies were assessed in murine cancer models. Results: The novel AXL inhibitor AB801 is potent, reversible, and selective. AB801 exhibits cellular activity at low nanomolar concentrations and retains significant activity in 100% human serum. Excellent selectivity was observed against MERTK (860x), TYRO3 (1,400x), and the overall kinome. Importantly, AB801 does not show significant CYP450 or hERG inhibition. Favorable preclinical PK is consistent with projected once-a-day oral administration in humans. AB801 increases sensitivity to SOC therapeutics such as chemotherapy, and results in increased DNA damage. Moreover, AB801 treatment sensitizes tumors to checkpoint blockade by increasing immune cell activation. Significant anti-tumor efficacy is observed in combination SOC therapies in multiple in vivo models. Conclusions: AXL inhibition is a promising therapeutic mechanism for impairing the growth of tumors resistant to SOC therapeutics. AB801 exhibits improved potency, selectivity, and safety profiles compared to other AXL inhibitors currently advancing into clinical development. Citation Format: Susan Lee Paprcka, Armon Goshayeshi, Suan Liu, Ruben Flores, Lauren Rocha, Jhansi L. Leslie, Dillon H. Miles, Corinne N. Foley, Shiwei Qu, Manjunath Lamani, Srinivas Paladugu, Hsin-Ting Huang, Nidhi Tribewal, Ada Chen, Joseph Kulusich, Stefan Garrido-Shaqfeh, Patricia Fabila, Salema Jafri, Anuja Devarajan, Ester Fernandez-Salas. AB801 is a highly potent and selective AXL kinase inhibitor that demonstrates significant anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 518.
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Goedken, Eric R., Andrew I. Gagnon, Gary T. Overmeyer, et al. "HTRF-Based Assay for Microsomal Prostaglandin E2 Synthase-1 Activity." Journal of Biomolecular Screening 13, no. 7 (2008): 619–25. http://dx.doi.org/10.1177/1087057108321145.
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Microsomal prostaglandin E2 synthase-1 (mPGES-1) catalyzes the formation of prostaglandin E2 (PGE2) from the endoperoxide prostaglandin H 2 (PGH2). Expression of this enzyme is induced during the inflammatory response, and mouse knockout experiments suggest it may be an attractive target for antiarthritic therapies. Assaying the activity of this enzyme in vitro is challenging because of the unstable nature of the PGH 2 substrate. Here, the authors present an mPGES-1 activity assay suitable for characterization of enzyme preparations and for determining the potency of inhibitor compounds. This plate-based competition assay uses homogenous time-resolved fluorescence to measure PGE2 produced by the enzyme. The assay is insensitive to DMSO concentration up to 10% and does not require extensive washes after the initial enzyme reaction is concluded, making it a simple and convenient way to assess mPGES-1 inhibition. ( Journal of Biomolecular Screening 2008:619-625)
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Nakamura, Keiji, Chikashi Tokuda, Hideyuki Arimitsu, et al. "Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli." PeerJ 9 (July 28, 2021): e11871. http://dx.doi.org/10.7717/peerj.11871.
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Shiga toxin-producing Escherichia coli (STEC) is a major intestinal pathogen and causes serious gastrointestinal illness, which includes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which belong to the AB-type toxin family. Among several subtypes of Stx1 and Stx2, the production of Stx2a is thought to be a risk factor for severe STEC infections, but Stx2a production levels vary markedly between STEC strains, even strains with the same serotype. Therefore, quantitative analyses of Stx2 production by STEC strains are important to understand the virulence potential of specific lineages or sublineages. In this study, we developed a novel Stx2 quantification method by utilizing homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technology. To determine suitable “sandwich” assay conditions, we tested 6 combinations of fluorescence-labeled monoclonal antibodies (mAbs) specific to Stx2 and compared the HTRF signal intensities obtained at various incubation times. Through this analysis, we selected the most suitable mAb pair, one recognizing the A subunit and the other recognizing the B subunit, thus together detecting Stx holotoxins. The optimal incubation time was also determined (18 h). Then, we optimized the concentrations of the two mAbs based on the range for linearity. The established HTRF assay detected 0.5 ng/ml of the highly purified recombinant Stx2a and Stx2e proteins and the working range was 1–64 ng/ml for both Stx2a and Stx2e. Through the quantification analysis of Stx proteins in STEC cell lysates, we confirmed that other Stx2 subtypes (Stx2b, Stx2c, Stx2d and Stx2g) can also be quantified at a certain level of accuracy, while this assay system does not detect Stx2f, which is highly divergent in sequence from other Stx2 subtypes, and Stx1. As the HTRF protocol we established is simple, this assay system should prove useful for the quantitative analysis of Stx2 production levels of a large number of STEC strains.
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Dell, E. J., Franka Ganske, and Laurence Jacquemart. "GPCR activation is measured with Cisbio's cAMP and IP1 HTRF HTplex cell-based assay." BioTechniques 49, no. 4 (2010): 759. http://dx.doi.org/10.2144/000113517.
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Yoneyama-Hirozane, Mariko, Kohei Deguchi, Takeshi Hirakawa, et al. "Identification and Characterization of a New Series of Ghrelin O-Acyl Transferase Inhibitors." SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, no. 2 (2017): 154–63. http://dx.doi.org/10.1177/2472555217727097.
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Ghrelin O-acyl transferase (GOAT; MBOAT4) catalyzes O-acylation at serine-3 of des-acyl ghrelin. Acyl ghrelin is secreted by stomach X/A-like cells and plays a role in appetite and metabolism. Therefore, GOAT has been expected to be a novel antiobesity target because it is responsible for acyl ghrelin production. Here, we report homogeneous time-resolved fluorescence (HTRF) and enzyme-linked immunosorbent assay (ELISA) methods utilizing human GOAT-expressing microsomes as a novel high-throughput assay system for the discovery of hit compounds and optimization of lead compounds. Hit compounds exemplified by compound A (2-[(2,4-dichlorobenzyl)sulfanyl]-1,3-benzoxazole-5-carboxylic acid) were identified by high-throughput screening using the HTRF assay and confirmed to have GOAT inhibitory activity using the ELISA. Based on the hit compound information, the novel lead compound (compound B, (4-chloro-6-{[2-methyl-6-(trifluoromethyl)pyridin-3-yl]methoxy}-1-benzothiophen-3-yl)acetic acid) was synthesized and exhibited potent GOAT inhibition with oral bioavailability. Both the hit compound and lead compound showed octanoyl-CoA competitive inhibitory activity. Moreover, these two compounds decreased acyl ghrelin production in the stomach of mice after their oral administration. These novel findings demonstrate that GOAT is a druggable target, and its inhibitors are promising antiobesity drugs.
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Paprcka, Susan, Akshata Udyavar, Subhasree Sridhar, et al. "509 Potent and selective inhibition Of AXL receptor tyrosine kinase for the treatment of cancer." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (2020): A545. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0509.
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BackgroundAXL receptor tyrosine kinase (AXL) is a transmembrane protein that is over-expressed in a variety of cancer and immune cells. AXL signaling has been implicated in creating an immunosuppressive tumor microenvironment (TME) through both tumor-intrinsic and immunomodulatory mechanisms1,2,3,4,5 promoting resistance to various therapies.6,7,8,9MethodsCompound inhibition potency against the kinase activity of AXL and other kinases was determined by detecting phosphorylated substrate using homogeneous time-resolved fluorescence (HTRF). Binding affinity of inhibitor to intracellular AXL kinase was determined by monitoring displacement of a competitive fluorescent tracer using an AXL NanoBRET assay. Recombinant Gas6, cancer cell lines, whole blood or isolated cells from healthy donors were used to determine the reduction in AXL-mediated signaling in-vitro. PK/PD and anti-tumor effects of selected AXL inhibitors were evaluated in murine models.ResultsAXL is highly expressed on a subset of immune cells, including DC’s, NK cells and M2 macrophages as well as fibroblasts, which contribute to a blunted anti-tumor response. Consistent with these observations, AXL is strongly associated with increased infiltration of macrophages, exhausted NK and T-cells, as well as significantly increased CD73 expression in multiple cancer types in TCGA. Additionally, AXL expression is strongly and significantly correlated with epithelial-mesenchymal transition (EMT), which further generates an immunosuppressive TME and promotes resistance to immune, targeted and chemotherapies. High expression of AXL is also strongly associated with poor survival in NSCLC, pancreatic, breast, head & neck, stomach, colorectal, ovarian & prostate adenocarcinomas, especially in the metastatic setting. AXL inhibitors that exhibit high potency in both biochemical (IC <5nM) and cell-based (IC <25nM) assays in addition to good selectivity against closely related kinases MER and TYRO3 (>90x and >25x fold selectivity, respectively) as well as other kinases involved in downstream signaling such as PI3K have been developed. Initial studies in animal models indicate a favorable pharmacokinetic profile and anti-tumor efficacy.ConclusionsAXL is a promising therapeutic target involving both immunomodulatory and tumor-intrinsic mechanisms. AXL inhibition reduces the immunosuppressive TME, enables activation of an anti-tumor immune response and renders tumors more susceptible to previously resistant therapies. Highly potent and selective AXL inhibitors have been designed, displaying biological profiles superior to those of less-selective molecules currently advancing through clinical development.ReferencesGjerdrum C, Tiron C, Hoiby T, et al. Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival. PNAS 2010; 107(3):1124–1129.Ying X, Chen J, Huang X, Huang P, Yan S. Effect of AXL on the epithelial-to-mesenchymal transition in non-small cell lung cancer. Exp Ther Med 2017; 14:785–790.Tsukita Y, Fujino N, Miyauchi E, et al. Axl Kinase drives immune checkpoint and chemokine signalling pathways in lung adenocarcinomas. Molecular Cancer 2019;18:24.Terry S, Abdou A, Engelsen AST, et al. AXL targeting overcomes human lung cancer cell resistance to NK- and CTL-Mediated Cytotoxicity. Cancer Immunol Res 2019;7(11):1789–1802.Scutera S, Fraone T, Musso T, et al. Survival and migration of human dendritic cells are regulated by and IFN-alpha-Inducible Axl/Gas6 Pathway. J Immunol 2009; 183:3004–3013.Wilson C, Ye X, Pham T, et al. AXL Inhibition sensitizes mesenchymal cancer cells to antimitotic drugs. Cancer Res 2014;74(20):5878–5890.Ludwig KF, Du W, Sorrelle NB, et al. Small-Molecule Inhibition of Axl targets tumor immune suppression and enhances chemotherapy in pancreatic cancer. Cancer Res 2018; 78(1):246–255.Brand TM, Iida M, Stein AP, et al. AXL mediates resistance to cetuximab therapy. Cancer Res 2015;74(18):5152–5164.Guo Z, Li Y, Zhang D, Ma J. Axl Inhibition induces the antitumor response which can be further potentiated by PD-1 blockade in the mouse cancer models. Oncotarget 2017; 8(52):89761–89774
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Galcheva-Gargova, Zoya, Chia Lin Chu, Alison Long, Jay Duffner, Kimberly Holte, and Birgit Corinna Schultes. "Role of M402, a novel heparan sulfate mimetic, in pancreatic cancer cell invasion and metastasis: Inhibition of the Sonic Hedgehog pathway and heparanase activity." Journal of Clinical Oncology 30, no. 30_suppl (2012): 25. http://dx.doi.org/10.1200/jco.2012.30.30_suppl.25.
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25 Background: M402 is a novel heparin sulfate mimetic that binds to multiples growth factors, adhesion molecules, and cytokines to inhibit tumor angiogenesis, progression, and metastasis in nonclinical studies. We investigated if M402 could modulate tumor-stroma interactions in pancreatic cancer by inhibiting the Sonic Hedgehog (Shh) pathway as well as inhibit the activity of the extracellular matrix degrading enzyme, heparanase. Methods: Surface plasmon resonance (SPR) was used for analysis of M402 binding to Shh in vitro. A cell based Gli-1 reporter assay was implemented to assess the effect of M402 on Shh signaling. Immunohistochemistry and RT-qPCR were utilized to investigate M402’s effect on Shh activity in an orthotopic Capan-2 model in nude mice. The effect of M402 on heparanase activity in vitro and on Capan-2 tumor samples isolated from treated and untreated mice was measured using an HTRF-FRET assay. Results: There was specific binding of M402 to Shh in vitro. Additionally, Shh signaling was inhibited in the presence of M402. Immunohistochemistry and RT-qPCR of Capan-2 tumor samples from animals treated with M402 also demonstrated reduction of Shh signaling via Gli, its targeted transcription factor. The degree of inhibition of heparanase activity, as measured in the HTRF assay, was affected by the size, structure, and sulfation pattern of the different heparin sulfate mimetics evaluated. M402 was the most potent inhibitor of heparanse activity in vitro from all compounds tested. In addition, treatment with M402 inhibited heparanase activity in the pancreatic tumor lysates in a dose-dependent manner. Conclusions: M402 was shown in nonclinical studies to modulate tumor-stroma interactions involved in the metastatic, invasive, and desmoplastic pathways by simultaneously inhibiting two distinct pathways: Shh signaling and the activity of heparanase. M402 regulates a variety of polysaccharide-based binding proteins, which provides a rationale for the clinical investigation of M402 in a range of cancers.
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Newton, Philip, Desmond O’Shea, Edward Wells, et al. "Development of a Homogeneous High-Throughput Screening Assay for Biological Inhibitors of Human Rhinovirus Infection." Journal of Biomolecular Screening 18, no. 3 (2012): 237–46. http://dx.doi.org/10.1177/1087057112469047.
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Infection with human rhinovirus (HRV) is thought to result in acute respiratory exacerbations of chronic obstructive pulmonary disorder (COPD). Consequently, prevention of HRV infection may provide therapeutic benefit to these patients. As all major group HRV serotypes infect cells via an interaction between viral coat proteins and intercellular adhesion molecule–1 (ICAM-1), it is likely that inhibitors of this interaction would prevent or reduce infections. Our objective was to use phage display technology in conjunction with naive human antibody libraries to identify anti–ICAM-1 antibodies capable of functional blockade of HRV infection. Key to success was the development of a robust, functionally relevant high-throughput screen (HTS) compatible with the specific challenges of antibody screening. In this article, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF) assay based on the inhibition of soluble ICAM-1 binding to live HRV16. We describe the implementation of the method in an antibody screening campaign and demonstrate the biological relevance of the assay by confirming the activity of resultant antibodies in a cell-based in vitro HRV infection assay.
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Auriau, Johanna, Clara Roujeau, Zakia Belaid Choucair, et al. "Gain of affinity for VEGF165 binding within the VEGFR2/NRP1 cellular complex detected by an HTRF-based binding assay." Biochemical Pharmacology 158 (December 2018): 45–59. http://dx.doi.org/10.1016/j.bcp.2018.09.014.
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Weber, Elisabeth, Ina Rothenaigner, Stefanie Brandner, Kamyar Hadian, and Kenji Schorpp. "A High-Throughput Screening Strategy for Development of RNF8-Ubc13 Protein–Protein Interaction Inhibitors." SLAS DISCOVERY: Advancing the Science of Drug Discovery 22, no. 3 (2016): 316–23. http://dx.doi.org/10.1177/1087057116681408.
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The ubiquitin-proteasome system plays an essential role in a broad range of cellular signaling pathways. Ubiquitination is a posttranslational protein modification that involves the action of an enzymatic cascade (E1, E2, and E3 enzymes) for the covalent attachment of ubiquitin to target proteins. The emerging knowledge of the molecular mechanisms and correlation of deregulation of the ubiquitin system in human diseases is uncovering new opportunities for therapeutics development. The E3 ligase RNF8 acts in cooperation with the heterodimeric E2 enzyme Ubc13/Uev1a to generate ubiquitin conjugates at the sides of DNA double-strand breaks, and recent findings suggest RNF8 as a potential therapeutic target for the treatment of breast cancer. Here, we present a novel high-throughput screening (HTS)–compatible assay based on the AlphaScreen technology to identify inhibitors of the RNF8-Ubc13 protein–protein interaction, along with a follow-up strategy for subsequent validation. We have adapted the AlphaScreen assay to a 384-well format and demonstrate its reliability, reproducibility, and suitability for automated HTS campaigns. In addition, we have established a biochemical orthogonal homogeneous time-resolved fluorescence (HTRF) assay in HTS format and a cellular microscopy-based assay allowing verification of the primary hits. This strategy will be useful for drug screening programs aimed at RNF8-Ubc13 modulation.
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Shaabani, Shabnam, Louis Gadina, Ewa Surmiak, et al. "Biphenyl Ether Analogs Containing Pomalidomide as Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction." Molecules 27, no. 11 (2022): 3454. http://dx.doi.org/10.3390/molecules27113454.
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New biphenyl-based chimeric compounds containing pomalidomide were developed and evaluated for their activity to inhibit and degrade the programmed cell death-1/programmed cell death- ligand 1 (PD-1/PD-L1) complex. Most of the compounds displayed excellent inhibitory activity against PD-1/PD-L1, as assessed by the homogenous time-resolved fluorescence (HTRF) binding assay. Among them, compound 3 is one of the best with an IC50 value of 60 nM. Using an ex vivo PD-1/PD-L1 blockade cell line bioassay that expresses human PD-1 and PD-L1, we show that compounds 4 and 5 significantly restore the repressed immunity in this co-culture model. Western blot data, however, demonstrated that these anti-PD-L1/pomalidomide chimeras could not reduce the protein levels of PD-L1.
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Chen, Catherine Z., Noel Southall, Jingbo Xiao, et al. "Identification of Small-Molecule Agonists of Human Relaxin Family Receptor 1 (RXFP1) by Using a Homogenous Cell-Based cAMP Assay." Journal of Biomolecular Screening 18, no. 6 (2012): 670–77. http://dx.doi.org/10.1177/1087057112469406.
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The relaxin hormone is involved in a variety of biological functions, including female reproduction and parturition, as well as regulation of cardiovascular, renal, pulmonary, and hepatic functions. It regulates extracellular matrix remodeling, cell invasiveness, proliferation, differentiation, and overall tissue homeostasis. The G protein–coupled receptor (GPCR) relaxin family receptor 1 (RXFP1) is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. Although agonists of the receptor could have a wide range of pharmacologic utility, until now there have been no reported small-molecule agonists for relaxin receptors. Here, we report the development of a quantitative high-throughput platform for an RXFP1 agonist screen based on homogenous cell-based HTRF cyclic AMP (cAMP) assay technology. Two small molecules of similar structure were independently identified from a screen of more than 365 677 compounds. Neither compound showed activity in a counterscreen with HEK293T cells transfected with an unrelated GPCR vasopressin 1b receptor. These small-molecule agonists also demonstrated selectivity against the RXFP2 receptor, providing a basis for future medicinal chemistry optimization of selective relaxin receptor agonists.
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Klimek, Joanna, Oskar Kruc, Joanna Ceklarz, et al. "C2-Symmetrical Terphenyl Derivatives as Small Molecule Inhibitors of Programmed Cell Death 1/Programmed Death Ligand 1 Protein–Protein Interaction." Molecules 29, no. 11 (2024): 2646. http://dx.doi.org/10.3390/molecules29112646.
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The PD-1/PD-L1 complex is an immune checkpoint responsible for regulating the natural immune response, but also allows tumors to escape immune surveillance. Inhibition of the PD-1/PD-L1 axis positively contributes to the efficacy of cancer treatment. The only available therapeutics targeting PD-1/PD-L1 are monoclonal antibody-based drugs, which have several limitations. Therefore, small molecule compounds are emerging as an attractive alternative that can potentially overcome the drawbacks of mAb-based therapy. In this article, we present a novel class of small molecule compounds based on the terphenyl scaffold that bind to PD-L1. The general architecture of the presented structures is characterized by axial symmetry and consists of three elements: an m-terphenyl core, an additional aromatic ring, and a solubilizing agent. Using molecular docking, we designed a series of final compounds, which were subsequently synthesized and tested in HTRF assay and NMR binding assay to evaluate their activity. In addition, we performed an in-depth analysis of the mutual arrangement of the phenyl rings of the terphenyl core within the binding pocket of PD-L1 and found several correlations between the plane angle values and the affinity of the compounds towards the protein.
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Zhang, Da-Wei, Rong-Hua Luo, Lei Xu, et al. "A HTRF based competitive binding assay for screening specific inhibitors of HIV-1 capsid assembly targeting the C-Terminal domain of capsid." Antiviral Research 169 (September 2019): 104544. http://dx.doi.org/10.1016/j.antiviral.2019.104544.
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Enomoto, Koji, Yuko Aono, Takashi Mitsugi, et al. "High Throughput Screening for Human Interferon-y Production Inhibitor Using Homogenous Time-Resolved Fluorescence." Journal of Biomolecular Screening 5, no. 4 (2000): 263–68. http://dx.doi.org/10.1177/108705710000500409.
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An immunoassay for interferon-γ (IFN-γ) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IFN-γ can be detected by simply adding a mixture of three reagents-biotinylated polyclonal antibody, europium cryptate (fluorescence donor, EuK)-labeled monoclonal antibody, and crosslinked allophycocyanin (fluorescence acceptor, XL665) conjugated with streptavidin-and then measuring the time-resolved fluorescence. The detection limit of IFN-γ by the proposed method is about 625 pg/ml. We applied the method to the detection of IFN-γ secreted from NK3.3 cells and employed it in high throughput screening for IFN-γ production inhibitors. With this screening format, IFN-γ can be measured by directly adding the above reagents to microplate wells where NK3.3 cells are being cultured and stimulated with interleukin-12. This "in situ" immunoassay requires only pipetting reagents, with no need to transfer the culture supernatant to another microplate or wash the plate. Therefore, this screening format makes possible full automation of cell-based immunoassay, thus reducing cost and experimental time while increasing accuracy and throughput.
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Palomba, Tommaso, Giusy Tassone, Carmine Vacca, et al. "Exploiting ELIOT for Scaffold-Repurposing Opportunities: TRIM33 a Possible Novel E3 Ligase to Expand the Toolbox for PROTAC Design." International Journal of Molecular Sciences 23, no. 22 (2022): 14218. http://dx.doi.org/10.3390/ijms232214218.
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The field of targeted protein degradation, through the control of the ubiquitin–proteasome system (UPS), is progressing considerably; to exploit this new therapeutic modality, the proteolysis targeting chimera (PROTAC) technology was born. The opportunity to use PROTACs engaging of new E3 ligases that can hijack and control the UPS system could greatly extend the applicability of degrading molecules. To this end, here we show a potential application of the ELIOT (E3 LIgase pocketOme navigaTor) platform, previously published by this group, for a scaffold-repurposing strategy to identify new ligands for a novel E3 ligase, such as TRIM33. Starting from ELIOT, a case study of the cross-relationship using GRID Molecular Interaction Field (MIF) similarities between TRIM24 and TRIM33 binding sites was selected. Based on the assumption that similar pockets could bind similar ligands and considering that TRIM24 has 12 known co-crystalised ligands, we applied a scaffold-repurposing strategy for the identification of TRIM33 ligands exploiting the scaffold of TRIM24 ligands. We performed a deeper computational analysis to identify pocket similarities and differences, followed by docking and water analysis; selected ligands were synthesised and subsequently tested against TRIM33 via HTRF binding assay, and we obtained the first-ever X-ray crystallographic complexes of TRIM33α with three of the selected compounds.
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Wang, Yiqian, Xiangyan Jing, Hong Chen, et al. "Abstract 5444: BPI-371153, an orally bioavailable small molecule PD-L1 inhibitor." Cancer Research 82, no. 12_Supplement (2022): 5444. http://dx.doi.org/10.1158/1538-7445.am2022-5444.
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Abstract PD-1/PD-L1 antibodies have entered mainstream of cancer treatment, but they came with limitations such as weak tumor penetration, immunogenicity, immune-related risks with a long half-life, and high cost. Small molecule inhibitors of PD-L1 are expected to overcome these limitations. We hereby present BPI-371153, an orally bioavailable small molecule that disrupts PD-1/PD-L1 interaction similar to PD-1/PD-L1 antibodies. BPI-371153 demonstrates high binding affinity to human PD-L1, induces PD-L1 dimeralization and disrupts PD-L1:PD-1 interaction (IC50 &lt;1 nM by HTRF assay). BPI-371153 induces PD-L1 internalization with an IC50 value of 4.2 nM as detected by flow cytometry, activates nuclear factor of activated T cell (NFAT) signaling in cell-based reporter assay, and effectively stimulates IFN-γ release in human peripheral blood monocyte-mediated tumor cell killing assay. In a syngeneic mouse model with murine MC38 colon tumor cells expressing human PD-L1 (MC38-hPD-L1 model), BPI-371153 suppressed tumor growth to an extent comparable to an anti-PD-L1 antibody. In immunocompromised mice grafted with the same cell line, no growth inhibition was observed, confirming that the pharmacologic effect of BPI-371153 indeed came from the immune system. In addition, this compound exhibits favorable ADME properties, with high oral exposure across multiple pre-clinical species. Phase I clinical trial of BPI-371153 is planned for early 2022. Citation Format: Yiqian Wang, Xiangyan Jing, Hong Chen, Huijuan Zhang, Tianyi Ma, Yao Zhang, Chunhui Zhang, Guangzhi Zhang, Xiangyong Liu, Dan Yan, Jing Guo, Hong Lan, Jiabing Wang, Lieming Ding. BPI-371153, an orally bioavailable small molecule PD-L1 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5444.
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Günther, Madeline, Peter Schnierle, Thorsten Rose, et al. "AMPK Activation by Cimicifuga racemosa Extract Ze 450 Is Associated with Metabolic Effects and Cellular Resilience against Age-Related Pathologies in Different Tissue Cell Types." Pharmaceutics 16, no. 3 (2024): 393. http://dx.doi.org/10.3390/pharmaceutics16030393.
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Cimicifuga racemosa extracts (CREs) have gained well-established use for the treatment of menopausal symptoms such as hot flushes and excessive sweating, and weight gain. While the clinical effects of CREs have been well documented, the mechanisms underlying these effects are largely unknown. More recently, the metabolic effects of the CRE Ze 450 were demonstrated in cultured cells in vitro and in mouse models of obesity in vivo. At the molecular level, metabolic regulation, enhanced insulin sensitivity, and increased glucose uptake were linked to the activation of AMP-activated protein kinase (AMPK). Therefore, we tested the effects of Ze 450 on AMPK phosphorylation and thus activation in cells from different tissues, i.e., murine C2C12 myoblast cells, human HEPG2 liver cells, mouse HT22 neuronal cells, and in murine 3T3L1 adipocytes. Using a FRET-based HTRF-assay, we found that Ze 450 induced AMPK phosphorylation and the activation of this key enzyme of metabolic regulation in cells from various different tissues including C2C12 (muscle), HEPG2 (liver), HT22 (hippocampal), and 3T3-L1 (adipocyte) cells. In C2C12 muscle cells, enhanced AMPK activation was accompanied by reduced mitochondrial respiration and enhanced glucose uptake. Further, Ze 450 enhanced the resilience of the cells against oxidative death induced by ferroptosis inducers erastin or RSL3. Our findings suggest a general effect of Cimicifuga racemosa on AMPK activation in different tissues and across species. This may have a significant impact on expanded therapeutic applications of Ze 450, since AMPK activation and the related metabolic effects have been previously associated with anti-aging effects and the prevention of the metabolic syndrome.
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Deng, Changchun, Peter Sportelli, Richard Rodriguez, et al. "Novel PI3K Inhibitors Demonstrated Marked Cytotoxicity in T Cell Lymphoma Models, Caused Apoptosis and Were Synergistic with A Novel Anti-CD20 Monoclonal Antibody Ublituximab in B Cell Lymphoma Models." Blood 120, no. 21 (2012): 3725. http://dx.doi.org/10.1182/blood.v120.21.3725.3725.
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Abstract Abstract 3725 Background: Activation of PI3K has been shown to be required for the proliferation and survival of cancer cells. A selective PI3K-delta inhibitor, GS-1101/CAL-101, has produced promising results in the treatment of lymphoid malignancies. More recently, new chemical entities targeting PI3K have been developed, with pharmacologic and pharmacodynamic features distinct from CAL-101. We sought to determine the activities of two structurally related PI3K inhibitors, TGR-1202 and TGR-5237, in B- and T cell lymphoma models. Methods: The activity of TGR-1202 on individual PI3K isoforms was determined in a cell free system using the PI3K HTRF Assay Kit, and in a cell based assay using the Flow2CAST kit that measures the induction of CD63 surface expression on human whole blood basophils as a marker for PI3K-delta signaling. The cytotoxicity of TGR-1202 and TGR-5237 was studied in 4 mantle cell lymphoma (MCL) cell lines, 1 T-cell acute lymphoblastic leukemia (T-ALL) cell line (P12), and 1 cutaneous T cell lymphoma (CTCL) cell line (H9). Growth inhibition was determined using the ATP-based Cell Titer Glo assay, and apoptosis was determined by flow cytometry using the Alexa Fluo kit. The potency of ublituximab was determined in antigen recognition studies using the Human Whole Blood B-cell depletion assay. Cell cycle progression was evaluated using a Guava cell cycle assay kit in 4 lymphoma cell lines, including Daudi, Raji, U266B1, and DB. Results: In the enzyme based assay, TGR-1202 demonstrated marked potency against PI3K-delta, with a half maximal effective concentration (EC50) at 22 nM. TGR-1202 was 48- to 10,000-fold more selective for the PI3K-delta relative to the alpha, beta, and gamma isoforms. In the cell based assay, the EC50 of TGR-1202 for PI3K-delta was 67 nM, compared with 92 nM with CAL-101. In the cytotoxicity assay of TGR-5237, the concentration required to inhibit growth by 50% (IC50) ranged from 10 uM to 50 uM for the 4 MCL cells and the T-ALL cell P12. Surprisingly, the CTCL cell line H9, was exquisitely sensitive to TGR-5237, with IC50 below 0.1 uM (Figure 1). TGR-5237 induced concentration-dependent apoptosis in WSU-NHL, with its potency comparable to CAL-101 (Figure 2). TGR-1202 caused a concentration-dependent accumulation of cells in the G2-M phase in Raji, Daudi, U266B1, and DB. While TGR-1202 was not cytotoxic to CD20+ B-cells at the 1 uM concentration, combination of 1 uM TGR-1202 with ublituximab increased CD20+ cell depletion by 20% at the 0.1–10 ng/ml concentrations. Last, the combination of TGR-1202 and ublitiximab markedly increased the percentage of cells in sub-G0 phase in Daudi and Raji cells, indicative of their synergy in causing apoptosis (Figure 3). Conclusion: Two novel PI3K-delta inhibitors, TGR-1202 and TGR-5237, disrupted cell cycle progression, induced apoptosis, and inhibited cell growth and proliferation in B- and T cell lymphoma models. TGR-1202 enhanced the activity of a novel CD20 monoclonal antibody, ublituximab, in CD20 positive lymphoma cells. Disclosures: Sportelli: TG Therapeutics, Inc.: Employment, Equity Ownership. Miskin:TG Therapeutics, Inc.: Employment, Equity Ownership. Vakkalanka:Rhizen Pharmaceuticals: Employment, Equity Ownership. Viswanadha:Incozen Therapeutics: Employment. O'Connor:Millenium Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics, Inc: Consultancy; Seattle Genetics, Inc: Membership on an entity's Board of Directors or advisory committees; Allos Therapeutics, Inc: Consultancy.
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Bhavar, Prashant Kashinath, and Uday Kumar Surampudi. "Abstract C081: Novel and selective inhibitors of KRASG12V." Molecular Cancer Therapeutics 22, no. 12_Supplement (2023): C081. http://dx.doi.org/10.1158/1535-7163.targ-23-c081.
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Abstract KRAS is one the most mutated proteins in cancers and are commonly identified in pancreatic adenocarcinoma, colorectal and non-small cell lung cancer. Owing to its role in a different human cancer, KRAS has long been considered as an important therapeutic target in precision oncology. Although KRAS was considered undruggable due to the protein’s structural complexity, recent allele specific inhibitors that covalently bind and trap KRAS in its inactive GDP bound state have demonstrated promising approach for generating anti RAS therapies and is validated by the accelerated approval of Sotorasib and Adagrasib by the FDA for patients with lung cancer. Success of targeting mutant KRASG12C has opened opportunities to tackle other oncogenic KRAS variants such as KRASG12V. KRASG12V mutations occur frequently in multiple tumour histotypes such as adenocarcinoma of Pancreas (~32%), colorectal adenocarcinoma (~20%) and Lung (~19%) and represents a significant unmet medical need. Here we describe preclinical profile of a potent and selective KRASG12V inhibitor VRTX171. VRTX171 is a tool compound that was potent across a KRASG12V mutated cancer cell line and found to be several fold less active in non-KRASG12V mutant cell lines. Using a de novo drug design approach the compound was optimized to offer desired potency with high selectivity over WT KRAS and other KRAS mutant isoforms. Affinity for target protein, was assessed by HTRF based target engagement assay and inhibition of Raf/Mitogen-activated protein kinase (MAPK) pathway was determined using Western blot analysis in multiple cancer cell lines harbouring KRASG12V mutation. In-vivo studies of VRTX171 in Xenograft models harbouring KRASG12V mutations, as single agent as well as in combination with other targeted therapies are under consideration. Citation Format: Prashant Kashinath Bhavar, Uday Kumar Surampudi. Novel and selective inhibitors of KRASG12V [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C081.
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Ketcham, John M., David M. Briere, Aaron C. Burns, et al. "Abstract LB505: Design and discovery of MRTX0902, a potent, selective, and orally bioavailable SOS1 inhibitor." Cancer Research 82, no. 12_Supplement (2022): LB505. http://dx.doi.org/10.1158/1538-7445.am2022-lb505.
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Abstract KRAS mutations are the most common activating mutations in human cancer that ultimately lead to hyperactivation of the MAPK pathway and uncontrolled growth. KRAS functions as a small GTPase that cycles through its GTP-loaded “on” state and its GDP-loaded “off” state, a highly regulated process that is crucial for normal cell proliferation and survival. The guanine nucleotide exchange factor (GEF) SOS1 plays a critical role in this process by regulating the “on/off” state of KRAS. The protein-protein interaction between SOS1 and KRAS facilitates turnover of KRAS from the GDP-loaded inactive state to its activated and GTP-loaded state, a critical step to enable productive KRAS effector binding and activation of downstream signaling. The KRASG12C inhibitor, adagrasib (MRTX849), irreversibly binds to the GDP-loaded inactive conformation of KRASG12C and has recently shown encouraging clinical activity across several cancer types. As adagrasib binds preferentially to the inactive state of KRAS, blockade of SOS1 is anticipated to shift KRASG12C into the adagrasib-susceptible GDP-loaded state. Furthermore, this combination strategy could be used to target other mutant-driven cancers within the MAPK pathway using the appropriate KRASmut inhibitors and/or inhibitors of other targets within the MAPK pathway including MEK or EGFR. MRTX0902 was identified using iterative structure-based design as a selective inhibitor of SOS1 that demonstrates an IC50 value of 2 nM in a SOS1 HTRF binding assay and 30 nM in an MKN1 cellular assay. In pharmacokinetic evaluation across species, MRTX0902 demonstrated low extraction ratios and moderate to high bioavailability in mice, rats, and dogs. In preclinical models, MRTX0902 augmented the antitumor activity of adagrasib and other selected therapies. The design, discovery, and preclinical characterization of the potential best-in-class candidate MRTX0902 will be described. Citation Format: John M. Ketcham, David M. Briere, Aaron C. Burns, James G. Christensen, Robin J. Gunn, Jacob Haling, Anthony Ivetac, Shilpi Khare, Jon Kuehler, Svitlana Kulyk, Jade Laguer, John D. Lawson, Krystal Moya, Natalie Nguyen, Peter Olson, Lisa Rahbaek, Christopher R. Smith, Niranjan Sudhakar, Nicole C. Thomas, Darin Vanderpool, Xiaolun Wang, Matthew A. Marx. Design and discovery of MRTX0902, a potent, selective, and orally bioavailable SOS1 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB505.
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Timiraos, Ana Belen Fraga, Caterina Facchin, Nadia Babaa, Anne-Laure Larroque-Lombard, and Bertrand Jean-Claude. "Abstract 3104: Synthesis and optimization of small molecules designed to stimulate the immune system by inducing DNA damage and blocking PD-1/PD-L1." Cancer Research 83, no. 7_Supplement (2023): 3104. http://dx.doi.org/10.1158/1538-7445.am2023-3104.
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Abstract Introduction. Activating the immune system against cancer is becoming an increasingly effective therapy option that can result in dramatic and durable responses in several cancer types. One approach to achieve the reactivation of endogenous antitumor T cells is by blocking PD-1/PD-L1 immune checkpoints expressed on T cells and other leukocytes. However, only limited cancer patients (15-25%) respond to anti-PD-1/PD-L1 immunotherapy. One of the most pressing current clinical challenges is to convert nonresponsive, “cold” tumors to responsive, “hot” tumors. Interestingly, after DNA-damaging chemotherapy, the immune environment may be changed from “cold” tumors to “hot” tumors by increasing the tumor mutation burden and the generation of neoantigens on the surface of cancer cells. Therefore, we surmised that a molecule capable of inducing promutagenic DNA and block PD-1/PD-L1 could not only induce neoantigens but also synergistically enhance immune response against the targeted tumor. Using an approach developed in our laboratory termed the “combi-targeting” strategy, we designed and synthesized a series of “combi-molecules” programmed to generate the promutagenic species and a small molecule capable of blocking PD-1/PD-L1. Material and methods. Melanoma cell line B16-F10 was used to determine IC50 of the new molecules with SRB assay. Homogenous time-resolved fluorescence (HTRF) binding assay was used to determine the IC50 inhibition of PD-1/PD-L1. Drug metabolism in extracted cells was measured by LC-MS. Results. We discovered a structure activity relationship of the combi-molecules based on the substitution of the side chain of the alkylating agent and the PD-1/PD-L1 scaffold. By altering the scaffolds of the combi-molecules from sulfonamides to biphenyl derivatives, we optimized binding to PD-1/PD-L1 from millimolar to micromolar levels. In vitro growth inhibitory analysis showed that the combi-molecules with biphenyl scaffold were 24-fold more potent in B16-F10. Importantly, analysis of intracellular metabolites of the combi-molecules revealed three main metabolites that can only result from the release of the shortlived promutagenic species. Conclusions. The biphenyl scaffold is optimal for maintaining strong PD-1/PD-L1 binding potency and enough potent to contribute to cell death. Hydrolytic cleavage of the alkylating agent is an indirect evidence of the formation of the alkylating species required to induce promutagenic lesions. Citation Format: Ana Belen Fraga Timiraos, Caterina Facchin, Nadia Babaa, Anne-Laure Larroque-Lombard, Bertrand Jean-Claude. Synthesis and optimization of small molecules designed to stimulate the immune system by inducing DNA damage and blocking PD-1/PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3104.
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Shang, Erchang, Boyu Zhong, Tony Zhang, et al. "Abstract 3315: Preclinical studies of TSN1611, a potent, selective, and orally bioavailable KRASG12D inhibitor." Cancer Research 84, no. 6_Supplement (2024): 3315. http://dx.doi.org/10.1158/1538-7445.am2024-3315.
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Abstract Background: KRAS mutations are the most frequently encountered driver oncogene, involved in ~25% of all human cancers [1,2]. KRASG12D is the predominant KRAS mutation isoform, detected in approximately 35% of pancreatic cancer, 13% of colorectal cancer, and 5% of NSCLC [3]. Compared to KRASG12C, targeting KRASG12D has proven to be more challenging since the target protein lacks a reactive amino acid residue for irreversible inhibitory modification by a ligand. Herein, we disclose TSN1611, a potent and selective KRASG12D inhibitor, which possesses favorable oral PK profiles and demonstrates significant in vitro and in vivo anti-tumor activity in various KRASG12D-mutant models. Method: Biochemical HTRF assay was used to measure the inhibition of TSN1611 to both GDP-bound and GTP-bound state of KRASG12D. Biophysical SPR method was used to directly measure the binding of TSN1611 to GDP-bound KRASG12D and KRASWT. Cell based activities were evaluated in a series of in vitro cell proliferation assay utilizing Ba/F3 cells engineered with KRASG12D or non-KRASG12D mutations and tumor cell lines harboring KRASG12D mutation. Human cancer cell-derived xenograft models of HPAC (pancreatic) and GP2D (colorectal) were used to evaluate its in vivo antitumor effect. in vitro and in vivo PK studies were performed in mouse, rat, and dog. Systematic nonclinical safety evaluations, including safety panel screen testing, safety pharmacology studies, and repeat-dose toxicity studies were carried out to assess its preliminary toxicity profile. Results: TSN1611 inhibited both active (GTP-bound) and inactive (GDP-bound) forms of KRASG12D protein at IC50 1.23 and 1.49 nM, respectively; the KD value of its direct binding to KRASG12D protein is 1.93 pM in SPR assay. TSN1611 demonstrated potent anti-proliferation activity against several tumor cell lines harboring KRASG12D mutation, and excellent selectivity over cells of NRAS, HRAS, and other KRAS isoforms. It also showed dose-dependent anti-tumor efficacy in GP2D and HPAC models. Mechanism of action studies concluded that the antitumor effect of TSN1611 is resulted from its effective inhibition of KRAS signaling pathway. Oral bioavailability and safety profile across multiple species supported its further development. Conclusion: TSN1611 is a selective KRASG12D inhibitor. It exhibited excellent selectivity and activity both in vitro and in vivo; it demonstrated favorable physicochemical properties, oral PK profiles, and brain penetration potential; it also showed acceptable margins of safety. The preclinical data supports further development. Pending regulatory submission and review, the phase I/II study is planned to start in H1 of 2024. References: [1] Cox, A.D. et al. Nat. Rev. Drug. Discov. 2014, 13, 828. [2] Indini, A. et al. Pharmaceutics 2021, 13, 653. [3] Moore, A. R. et al. Nat. Rev. Drug. Discov. 2020, 19, 533. Citation Format: Erchang Shang, Boyu Zhong, Tony Zhang, Chunlan Dong, Shengtang Ma, Anjiang Yang, Ziyang Jia, Renjuan Zheng, Jing Li, Han Fu, Liangbao Lai. Preclinical studies of TSN1611, a potent, selective, and orally bioavailable KRASG12D inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3315.
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Yang, Chao-Yie, Xinrui Yuan, Mona Kazemi Sabzvar, et al. "Small-Molecule U2 Auxiliary Factor Homology Motif (UHM) Domain Inhibitors Cause Splicing Pattern Changes in U2AF1 Mutant Leukemia Cells and Induce Sub-G1 Cell Cycle Arrest." Blood 142, Supplement 1 (2023): 115. http://dx.doi.org/10.1182/blood-2023-190365.
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Mutations in factors involved in the spliceosomal pathway have emerged to be an important contributor to the development of myeloid neoplasms including MDS and secondary AML (sAML). A set of frequently mutated genes encoding RNA splicing factors including SF3B1, U2AF1, SRSF2, and ZRSR2 or haploinsufficiency of LUC7L2 have beenreported, but pharmacological intervention targeting these genes remains limited and under-developed. Combination of mutations of these splicing factors are rarely found in patients indicating that multiple defects in a spliceosome pathway may be deleterious. Heterozygous mutation in U2AF1 found in MDS leads to the acquisition of the neomorphic mutant U2AF1 (U2AF1 wt) that relies on wild-type U2AF1 (U2AF1 wt) to survive and proliferate. To support our hypothesis, we undertook a discovery campaign to identify small-molecule compounds targeting U2AF1. By recognizing the challenge of discovering compounds selectively targeting the mutant zinc-finger domains in U2AF1 found clinically, we devised a tractable intervention strategy to inhibit the UHM domain of U2AF1 (U2AF1-UHM). U2AF1-UHM binds with U2AF2 to form a protein complex (U2AF1/U2AF2) that recognizes the 3' splice site in mRNA processing. U2AF1-UHM inhibitors may decrease the binding between U2AF1 and U2AF2 and abrogate U2AF1/U2AF2 functions to change expression patterns of protein isoforms. To identify hit compounds for development, we performed screening of ~3000 fragment molecules against U2AF1-UHM using the thermal shift assay. Two of the top hits were derivatives of the same chemical scaffold. We verified that both hits inhibited the binding of U2AF1-UHM and U2AF2-ULM (U2AF ligand motifs) in our Homogenous Time-Resolved Fluorescence (HTRF) assay. We next evaluated the top hit, SF-1-8, in our K562-U2AF1 S34F mutant cell line and obtained an IC50 at the micromolar range. In contrast, SF-1-8 had no activity in K562-U2AF1 wt cell line and bone marrow cells obtained from healthy individuals. Based on SF-1-8, we performed chemical modifications to develop structure-activity relationship (SAR) of SF-1-8 and obtained SF-1-50 that was two-fold more potent than SF-1-8. To assess the selectivity of SF-1-8 and analogs to other UHM containing proteins, we further determined the IC50 values of our SF-1-8 analogs to RBM39-UHM, SPF45-UHM, and PUF60-UHM to construct the selectivity profiles of our compounds. The selectivity profiles of SF-1-8 and SF-1-50 showed they were selectively more effective to U2AF1-UHM than RBM39-UHM, SPF45-UHM, and PUF60-UHM. We then studied pathways impacted by these inhibitors in K562-U2AF1 S34F cells by performing RNA-seq in K562-U2AF1 S34F cells treated with SF-1-8 at 5 uM and control. We found that 36 and 63 genes were significantly (p &lt;0.002) up- and down-regulated respectively by SF-1-8 in K562-U2AF1 S34F cells. Downregulated genes included CBL, CBLL1, and a subset of collagen genes ( COL1, COL3, COL5) and upregulated genes included ATF3 and BCL2. When analyzing the transcript changes affected by SF-1-8, we identified substantial protein isoform changes in genes involved with proteosome, endocytosis, apoptosis, extracellular matrix/cell adhesion, histones, and stress response. Primary effects from changes of protein isoform patterns ameliorated the restoration of the trans-Golgi network ( ERGIC3, COPB2), secretory pathway ( RUSC1, AP4E1) and impairment of clathrin mediated endocytosis and endosome-lysosome transport ( CBL, PICALM, VAMP7, ASAP1). NKM-1 cell line was previously characterized to carry U2AF1 mutation. We also found SF-1-50 caused accumulation of sub-G1 cells in NKM-1 cells in a dose-dependent manner. In summary, SF-1-8 caused disruption of extra- and intracellular protein transport in K562-U2AF1 S34F cells and represented a new class of small-molecule inhibitors to target U2AF1-UHM. Further optimization of this class of compounds will allow us to develop effective chemical probes for study in the U2af1 murine models and assess the potential of U2AF1 as a therapeutic target in MDS and sAML.
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Ishizawa, Jo, Kenji Nakamaru, Takahiko Seki, et al. "Discovery of Predictive Gene Signatures for Tumor Sensitivity to MDM2 Inhibition in Development of a Novel MDM2 Inhibitor DS-3032b." Blood 128, no. 22 (2016): 2893. http://dx.doi.org/10.1182/blood.v128.22.2893.2893.
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Abstract Development of MDM2 inhibitors enabled successful induction of p53-mediated apoptosis in tumor cells without a risk of DNA damage. Early clinical trials of MDM2 inhibitors demonstrated proof-of-concept (Andreeff et al., Clin Can Res, 2015). However, a clinical challenge is that not all the tumors bearing wild-type TP53 are sensitive to MDM2 inhibition. We here discovered novel gene profiling-based algorithms for predicting tumor sensitivity to MDM2 inhibition, using DS-3032b, a novel potent MDM2 inhibitor, which is currently in early clinical trials. In vitro inhibitory effects of DS-3032b on MDM2-p53 interaction was demonstrated using the homogeneous time resolved fluorescence (HTRF) assay (IC50 5.57 nM). DS-3032b treatment (30-1000 nM) indeed increased p53 protein in a dose-dependent manner, and also the p53 targets MDM2 and p21, in cancer cell lines with wild-type TP53 (SJSA-1, MOLM-13, DOHH-2, and WM-115), showing around 10-fold potent growth inhibition effects compared to Nutlin-3a (Table 1). The xenograft mouse models with SJSA-1 and MOLM-13 cells showed > 90% reduction in tumor growth with oral administrations of 25 and 50 mg/kg/day. For discovering predictive gene signatures, we performed two different approaches. In the first approach, 240 cell lines available as OncoPanel were treated with DS-3032b, another prototypic MDM2 inhibitor DS-5272, and Nutlin-3a, and determined 62 sensitive and 164 resistant lines, based on GI50s. Using gene expression profiling (GEP) publicly available for all the cell lines, we selected 175 top-ranked genes with highest expression in the 62 sensitive cell lines. We thus defined the average of Z-scores of the 175 gene expression as "sensitivity score". To validate the 175-gene signature, we evaluated in vivo anti-tumor activities of DS-3032b in 13 patient-derived tumor xenografts (melanoma, NSCLC, colorectal and pancreatic cancers). The prediction accuracy, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) were 85, 88, 88 and 80% respectively. As another validation set, 41 primary AML samples were treated with DS-3032b to define the top and bottom one-third most sensitive or resistant samples (14 each), and GEP was performed in every sample. TP53 mutations were detected in 8 specimens by next generation sequencing and confirmed by Sanger sequencing. The 175-gene signature was applied to the AML dataset, and the accuracy, sensitivity, PPV and NPV to predict the 14 sensitive or resistant samples were 79, 93, 72 and 90% respectively. Importantly, this signature was more predictive than the TP53 mutation status alone applied (68, 93, 62 and 86%). (Table 2A-B) In contrast to the cell line-based approach, the second approach defined an AML-specific gene signature. Specifically, we used the same dataset of 41 primary AML samples described above as training and validation set, by performing random forest methods with cross validation. Using a routine way in bioinformatics analysis of classifying gene signature, we first selected the 1500 top-ranked genes with highest expression variance among all the specimens. In addition, p53-related 32 genes that potentially have predictive values were also selected based on the previous reports. Classification was performed using the random forest method to identify a predictive algorithm with the 1500-gene set, 32-gene set or combined 1525-gene set (7 genes were overlapped), thus we found that the 1525-gene set had highest performance than each gene set alone. However, applying this method to all the 41 samples showed inferior predictive performance than applied only to the 33 wild-type TP53 samples (the prediction accuracy, sensitivity, PPV and NPV were 68, 72, 67 and 69%, vs. 77, 82, 75 and 80%).(Table 2C) Finally, we combined each of the two algorithms (Table 2B-C) with TP53 mutation status. Specifically, the samples with TP53 mutations were predicted as resistant, then either of gene signatures was applied to the rest of the samples with wild-type TP53. Predictive performance (Table 2D-E) was improved in both signatures compared to the others, especially showing the highest PPVs (80 and 82%, respectively). Taken together, gene signatures discovered in the present study, by combining with TP53 mutation status, provided new highly predictive algorithms for therapy of MDM2 inhibition. Our findings will be tested in ongoing clinical trials of DS-3032b. Disclosures Nakamaru: Daiichi Sankyo Co., Ltd: Employment. Seki:2Daiichi Sankyo Co., Ltd.: Employment. Tazaki:2Daiichi Sankyo Co., Ltd.: Employment. DiNardo:Celgene: Research Funding; Novartis: Other: advisory board, Research Funding; Abbvie: Research Funding; Agios: Other: advisory board, Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding. Tse:Daiichi Sankyo, Inc.: Employment.
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Wei, Zhengxi, Tuan Xu, Judy Strickland, et al. "Use of in vitro methods combined with in silico analysis to identify potential skin sensitizers in the Tox21 10K compound library." Frontiers in Toxicology 6 (February 28, 2024). http://dx.doi.org/10.3389/ftox.2024.1321857.
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Introduction: Skin sensitization, which leads to allergic contact dermatitis, is a key toxicological endpoint with high occupational and consumer prevalence. This study optimized several in vitro assays listed in OECD skin sensitization test guidelines for use on a quantitative high-throughput screening (qHTS) platform and performed in silico model predictions to assess the skin sensitization potential of prioritized compounds from the Tox21 10K compound library.Methods: First, we screened the entire Tox21 10K compound library using a qHTS KeratinoSensTM (KS) assay and built a quantitative structure–activity relationship (QSAR) model based on the KS results. From the qHTS KS screening results, we prioritized 288 compounds to cover a wide range of structural chemotypes and tested them in the solid phase extraction–tandem mass spectrometry (SPE–MS/MS) direct peptide reactivity assay (DPRA), IL-8 homogeneous time-resolved fluorescence (HTRF) assay, CD86 and CD54 surface expression in THP1 cells, and predicted in silico sensitization potential using the OECD QSAR Toolbox (v4.5).Results: Interpreting tiered qHTS datasets using a defined approach showed the effectiveness and efficiency of in vitro methods. We selected structural chemotypes to present this diverse chemical collection and to explore previously unidentified structural contributions to sensitization potential.Discussion: Here, we provide a skin sensitization dataset of unprecedented size, along with associated tools, and analysis designed to support chemical assessments.
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Ho, Jo‐Hao, Kimberly Lovell, Lei Zhou, Kevin Frankowski, Jeffrey Aubé, and Laura Bohn. "Characterization of Novel Biased KOR Agonist‐Mediated Cell Signaling." FASEB Journal 30, S1 (2016). http://dx.doi.org/10.1096/fasebj.30.1_supplement.931.5.
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Kappa opioid receptors (KORs) are widely expressed in the central nervous system and are involved in the regulation of pain perception. Therefore, KORs have been attractive drug targets for treating pain; however, KOR agonists also produce sedation and dysphoria. Recent evidence suggests that while G protein signaling may promote KOR‐mediated antinociception, βarrestin‐mediated signaling may lead to the dysphoric effects. In an effort to begin to determine whether KOR activation without βarrestin2 recruitment would maintain the therapeutic potency of KOR agonists while avoiding the aversive effects, we evaluated two classes of KOR agonists, triazole analogue and isoquinolinone analogue, both of which potently activate G protein coupling but weakly recruit βarrestin2. Canonically, KOR couples to inhibitory G proteins that suppress adenylyl cyclase. However, the biased KOR agonists have very weak potency for inhibiting adenylyl cyclase in cell‐based signaling assays. Therefore, we hypothesize that these novel KOR agonists preferentially transduce signaling through different types of Gα or Gβγ proteins. In this study, we compare these novel KOR agonists to the reference compound, U69,593, for their abilities to activate three different KOR‐mediated G protein signaling events: adenylyl cyclase activity by cAMP HTRF technology, G protein‐coupled inwardly‐rectifying potassium (GIRK) channel activity by FLIPR potassium technology, and intracellular calcium mobilization by FLIPR calcium technology. These novel KOR agonists display weak potency in regulating adenylyl cyclase activity and stimulating calcium response; however, they potently activate GIRK channels in cell‐based assays. Together, these results suggest that the novel KOR agonists display different signatures in KOR‐mediated G protein signaling. This study provides insights into the novel KOR agonist‐mediated signaling pathways which will inform future behavioral studies.Support or Funding InformationThis work was supported by NIH/NIDA R01 DA031927 to LMB and JA.
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Heinz, Beverly A., John M. Schaus, James P. Beck, et al. "In Vitro Characterization of a Novel Allosteric Potentiator of the Dopamine D1 Receptor." FASEB Journal 30, S1 (2016). http://dx.doi.org/10.1096/fasebj.30.1_supplement.708.1.
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Allosteric potentiators of the dopamine D1 receptor could be useful for treatment of Parkinson's disease, schizophrenia, depression, ADHD, and narcolepsy. We identified a novel D1 potentiator (“DETQ”)by high‐throughput screening followed by iterative chemical optimization. In a HEK293 cell line expressing the human D1 receptor, agonist‐stimulated cAMP production was measured in the presence of an EC20 concentration of dopamine using homogeneous time resolved fluorescence (HTRF) technology. DETQ potentiated the dopamine response with a mean EC50 of 5.8 nM and mean efficacy corresponding to 95% of the maximum response to dopamine. In the absence of exogenous dopamine (agonist mode), the maximum efficacy of DETQ was 11.6% with an EC50 of 30 nM; based on the initial slopes of the two curves (Ehlert R‐ratio), DETQ was thus 43‐fold less potent as an allosteric agonist than as a potentiator. When concentration‐response curves for dopamine were carried out at multiple DETQ concentrations in the cAMP assay, DETQ shifted the dopamine curve 21‐fold to the left with a KB of 26 nM. In experiments measuring binding of the D1 antagonist 3H‐SCH23390 to the human D1 receptor, 100 nM DETQ caused a 5‐fold leftward shift in the concentration‐inhibition curve for dopamine, indicating adirect effect of DETQ on the D1 receptor. The potency of DETQ in the cAMP assay was about 50‐fold lower at the mouse and rat D1 receptors than at human, rhesus, and dog D1 receptors. DETQ was inactive at concentrations up to atleast 10 uM when tested in agonist and potentiator modes at related receptors, including D2, D5, β1, β2, β3, and 5HT6. DETQ was also inactive in binding and/or functional assays at a largeset of unrelated targets.These results suggest that DETQ could be a useful tool to probe applications of dopamine D1 potentiators for CNS disorders.
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Annor-Gyamfi, Joel, Felix Nwogbo, Kun Qian, et al. "Fused tetrahydroquinolines (THQ): potential PAINS compounds in a recent HTS for moesin-CD44 pathway inhibitors." April 13, 2021. https://doi.org/10.5281/zenodo.4685122.
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Ildefeld, Niklas, Dieter Steinhilber, Ewgenij Proschak, and Jan Heering. "HTRF-based assay for detection of mono-ADP-ribosyl hydrolyzing macrodomains and inhibitor screening." iScience, June 2024, 110333. http://dx.doi.org/10.1016/j.isci.2024.110333.
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