ABSTRACT
Background: Velcrins are molecular glues that kill cells by inducing the formation of a protein complex between the RNase SLFN12 and the phosphodiesterase PDE3A. Formation of the complex activates SLFN12, which cleaves tRNALeu(TAA) and induces apoptosis. Velcrins such as the clinical investigational compound, BAY 2666605, were found to have activity across multiple solid tumor cell lines from the cancer cell line encyclopedia, including glioblastoma cell lines. We therefore aim to characterize velcrins as novel therapeutic agents in glioblastoma. Materials and Methods: PDE3A and SLFN12 expression levels were measured in glioblastoma cell lines, the Cancer Genome Atlas (TCGA) tumor samples, and tumor neurospheres. Velcrin-treated cells were assayed for viability, induction of apoptosis, cell cycle phases, and global changes in translation. Transcriptional profiling of the cells was obtained. Xenograft-harboring mice treated with velcrins were also monitored for survival. Results: We identified several velcrin-sensitive glioblastoma cell lines and 4 velcrin-sensitive glioblastoma patient-derived models. We determined that BAY 2666605 crosses the blood-brain barrier and elicits full tumor regression in an orthotopic xenograft model of GB1 cells. We also determined that the velcrins BAY 2666605 and BRD3800 induce tumor regression in subcutaneous glioblastoma PDX models. Conclusions: Velcrins have antitumor activity in preclinical models of glioblastoma, warranting further investigation as potential therapeutic agents.
ABSTRACT
This study describes the identification and target deconvolution of small molecule inhibitors of oncogenic Yes-associated protein (YAP1)/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex as the direct target of YAP1/TAZ pathway inhibitors. The small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling in vivo.
Subject(s)
Adaptor Proteins, Signal Transducing , Antineoplastic Agents , Cell Proliferation , High-Throughput Screening Assays , Signal Transduction , Transcription Factors , YAP-Signaling Proteins , Humans , Transcription Factors/metabolism , Transcription Factors/antagonists & inhibitors , YAP-Signaling Proteins/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Signal Transduction/drug effects , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Animals , Cell Proliferation/drug effects , Mice , rho GTP-Binding Proteins/metabolism , rho GTP-Binding Proteins/antagonists & inhibitors , Cell Line, Tumor , Phosphoproteins/metabolism , Phosphoproteins/antagonists & inhibitors , Drug Screening Assays, Antitumor , Alkyl and Aryl Transferases/antagonists & inhibitors , Alkyl and Aryl Transferases/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Drug Discovery , Mice, Nude , Acyltransferases/antagonists & inhibitors , Acyltransferases/metabolism , Phenotype , Structure-Activity Relationship , Transcriptional Coactivator with PDZ-Binding Motif ProteinsABSTRACT
USP21 belongs to the ubiquitin-specific protease (USP) subfamily of deubiquitinating enzymes (DUBs). Due to its relevance in tumor development and growth, USP21 has been reported as a promising novel therapeutic target for cancer treatment. Herein, we present the discovery of the first highly potent and selective USP21 inhibitor. Following high-throughput screening and subsequent structure-based optimization, we identified BAY-805 to be a non-covalent inhibitor with low nanomolar affinity for USP21 and high selectivity over other DUB targets as well as kinases, proteases, and other common off-targets. Furthermore, surface plasmon resonance (SPR) and cellular thermal shift assays (CETSA) demonstrated high-affinity target engagement of BAY-805, resulting in strong NF-κB activation in a cell-based reporter assay. To the best of our knowledge, BAY-805 is the first potent and selective USP21 inhibitor and represents a valuable high-quality in vitro chemical probe to further explore the complex biology of USP21.
Subject(s)
Signal Transduction , Ubiquitin-Specific Proteases , Gene Expression Regulation , EndopeptidasesABSTRACT
PPAR gamma (PPARG) is a ligand activated transcription factor that regulates genes involved in inflammation, bone biology, lipid homeostasis, as well as a master regulator of adipogenesis and a potential lineage driver of luminal bladder cancer. While PPARG agonists lead to transcriptional activation of canonical target genes, inverse agonists have the opposite effect through inducing a transcriptionally repressive complex leading to repression of canonical target gene expression. While many agonists have been described and tested clinically, inverse agonists offer an underexplored avenue to modulate PPARG biology in vivo. Current inverse agonists lack favorable in vivo properties; herein we describe the discovery and characterization of a series of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists, BAY-5516, BAY-5094, and BAY-9683. Structural studies of this series revealed distinct pre- and post-covalent binding positions, which led to the hypothesis that interactions in the pre-covalent conformation are primarily responsible for driving affinity, while interactions in the post-covalent conformation are more responsible for cellular functional effects by enhancing PPARG interactions with its corepressors. The need to simultaneously optimize for two distinct states may partially explain the steep SAR observed. Exquisite selectivity was achieved over related nuclear receptors in the subfamily due in part to a covalent warhead with low reactivity through an SNAr mechanism in addition to the specificity gained through covalent binding to a reactive cysteine uniquely positioned within the PPARG LBD. BAY-5516, BAY-5094, and BAY-9683 lead to pharmacodynamic regulation of PPARG target gene expression in vivo comparable to known inverse agonist SR10221 and represent new tools for future in vivo studies to explore their potential utility for treatment of disorders of hyperactivated PPARG including luminal bladder cancer and other disorders.
Subject(s)
PPAR gamma , Urinary Bladder Neoplasms , Humans , PPAR gamma/agonists , Drug Inverse Agonism , PPAR-gamma Agonists , Gene Expression RegulationABSTRACT
The ligand-activated nuclear receptor peroxisome-proliferator-activated receptor-γ (PPARG or PPARγ) represents a potential target for a new generation of cancer therapeutics, especially in muscle-invasive luminal bladder cancer where PPARγ is a critical lineage driver. Here we disclose the discovery of a series of chloro-nitro-arene covalent inverse-agonists of PPARγ that exploit a benzoxazole core to improve interactions with corepressors NCOR1 and NCOR2. In vitro treatment of sensitive cell lines with these compounds results in the robust regulation of PPARγ target genes and antiproliferative effects. Despite their imperfect physicochemical properties, the compounds showed modest pharmacodynamic target regulation in vivo. Improvements to the in vitro potency and efficacy of BAY-4931 and BAY-0069 compared to those of previously described PPARγ inverse-agonists show that these compounds are novel tools for probing the in vitro biology of PPARγ inverse-agonism.
Subject(s)
PPAR gamma , PPAR gamma/metabolism , LigandsABSTRACT
PIP4K2A is an insufficiently studied type II lipid kinase that catalyzes the conversion of phosphatidylinositol-5-phosphate (PI5P) into phosphatidylinositol 4,5-bisphosphate (PI4,5P2). The involvement of PIP4K2A/B in cancer has been suggested, particularly in the context of p53 mutant/null tumors. PIP4K2A/B depletion has been shown to induce tumor growth inhibition, possibly due to hyperactivation of AKT and reactive oxygen species-mediated apoptosis. Herein, we report the identification of the novel potent and highly selective inhibitors BAY-091 and BAY-297 of the kinase PIP4K2A by high-throughput screening and subsequent structure-based optimization. Cellular target engagement of BAY-091 and BAY-297 was demonstrated using cellular thermal shift assay technology. However, inhibition of PIP4K2A with BAY-091 or BAY-297 did not translate into the hypothesized mode of action and antiproliferative activity in p53-deficient tumor cells. Therefore, BAY-091 and BAY-297 serve as valuable chemical probes to study PIP4K2A signaling and its involvement in pathophysiological conditions such as cancer.
Subject(s)
Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Naphthyridines/chemistry , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , High-Throughput Screening Assays , Humans , Mice , Mice, Knockout , Mitochondria/drug effects , Mitochondria/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Structure-Activity RelationshipABSTRACT
Eukaryotes have evolved two major pathways to repair potentially lethal DNA double-strand breaks. Homologous recombination represents a precise, DNA-template-based mechanism available during the S and G2 cell cycle phase, whereas non-homologous end joining, which requires DNA-dependent protein kinase (DNA-PK), allows for fast, cell cycle-independent but less accurate DNA repair. Here, we report the discovery of BAY-8400, a novel selective inhibitor of DNA-PK. Starting from a triazoloquinoxaline, which had been identified as a hit from a screen for ataxia telangiectasia and Rad3-related protein (ATR) inhibitors with inhibitory activity against ATR, ATM, and DNA-PK, lead optimization efforts focusing on potency and selectivity led to the discovery of BAY-8400. In in vitro studies, BAY-8400 showed synergistic activity of DNA-PK inhibition with DNA damage-inducing targeted alpha therapy. Combination of PSMA-targeted thorium-227 conjugate BAY 2315497 treatment of human prostate tumor-bearing mice with BAY-8400 oral treatment increased antitumor efficacy, as compared to PSMA-targeted thorium-227 conjugate monotherapy.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , DNA-Activated Protein Kinase/metabolism , Gene Expression Regulation/drug effects , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation , DNA-Activated Protein Kinase/genetics , Drug Synergism , Drug Therapy, Combination , Hepatocytes/drug effects , Humans , Mice , Molecular Structure , Phosphatidylinositol 3-Kinases/genetics , Rats , Structure-Activity Relationship , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Selective inhibition of exclusively transcription-regulating positive transcription elongation factor b/CDK9 is a promising new approach in cancer therapy. Starting from atuveciclib, the first selective CDK9 inhibitor to enter clinical development, lead optimization efforts aimed at identifying intravenously (iv) applicable CDK9 inhibitors with an improved therapeutic index led to the discovery of the highly potent and selective clinical candidate VIP152. The evaluation of various scaffold hops was instrumental in the identification of VIP152, which is characterized by the underexplored benzyl sulfoximine group. VIP152 exhibited the best preclinical overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats upon once weekly iv administration. VIP152 has entered clinical trials for the treatment of cancer with promising longterm, durable monotherapy activity in double-hit diffuse large B-cell lymphoma patients.
Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Drug Discovery , Leukemia, Myeloid, Acute/drug therapy , Protein Kinase Inhibitors/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 9/metabolism , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Humans , Injections, Intravenous , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Nude , Molecular Structure , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Rats , Structure-Activity RelationshipABSTRACT
PURPOSE: 5' adenosine monophosphate-activated kinase (AMPK) is an essential regulator of cellular energy homeostasis and has been associated with different pathologies, including cancer. Precisely defining the biological role of AMPK necessitates the availability of a potent and selective inhibitor. METHODS: High-throughput screening and chemical optimization were performed to identify a novel AMPK inhibitor. Cell proliferation and mechanistic assays, as well as gene expression analysis and chromatin immunoprecipitation were used to investigate the cellular impact as well as the crosstalk between lipid metabolism and androgen signaling in prostate cancer models. Also, fatty acid turnover was determined by examining lipid droplet formation. RESULTS: We identified BAY-3827 as a novel and potent AMPK inhibitor with additional activity against ribosomal 6 kinase (RSK) family members. It displays strong anti-proliferative effects in androgen-dependent prostate cancer cell lines. Analysis of genes involved in AMPK signaling revealed that the expression of those encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), fatty acid synthase (FASN) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), all of which are involved in lipid metabolism, was strongly upregulated by androgen in responsive models. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) analysis identified several androgen receptor (AR) binding peaks in the HMGCR and PFKFB2 genes. BAY-3827 strongly down-regulated the expression of lipase E (LIPE), cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) and serine-threonine kinase AKT3 in responsive prostate cancer cell lines. Also, the expression of members of the carnitine palmitoyl-transferase 1 (CPT1) family was inhibited by BAY-3827, and this was paralleled by impaired lipid flux. CONCLUSIONS: The availability of the potent inhibitor BAY-3827 will contribute to a better understanding of the role of AMPK signaling in cancer, especially in prostate cancer.
Subject(s)
AMP-Activated Protein Kinases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Prostatic Neoplasms , Cell Line, Tumor , Humans , Male , Signal Transduction/drug effectsABSTRACT
Physiologically based pharmacokinetic modeling has become a standard tool to predict drug distribution in early stages of drug discovery; however, this does not currently encompass lysosomal trapping. For basic lipophilic compounds, lysosomal sequestration is known to potentially influence intracellular as well as tissue distribution. The aim of our research was to reliably predict the lysosomal drug content and ultimately integrate this mechanism into pharmacokinetic prediction models. First, we further validated our previously presented method to predict the lysosomal drug content (Schmitt et al., 2019) for a larger set of compounds (n = 41) showing a very good predictivity. Using the lysosomal marker lipid bis(monoacylglycero)phosphate, we estimated the lysosomal volume fraction for all major tissues in the rat, ranging from 0.03% for adipose up to 5.3% for spleen. The pH-driven lysosomal trapping was then estimated and fully integrated into the mechanistic distribution model published by Rodgers et al. (2005) Predictions of Kpu improved for all lysosome-rich tissues. For instance, Kpu increased for nicotine 4-fold (spleen) and 2-fold (lung and kidney) and for quinidine 1.8-fold (brain), although for most other drugs the effects were much less (≤7%). Overall, the effect was strongest for basic compounds with a lower lipophilicity, such as nicotine, for which the unbound volume of distribution at steady-state prediction changed from 1.34 to 1.58 l/kg. For more lipophilic (basic) compounds or those that already show strong interactions with acidic phospholipids, the additional contribution of lysosomal trapping was less pronounced. Nevertheless, lysosomal trapping will also affect intracellular distribution of such compounds. SIGNIFICANCE STATEMENT: The estimation of the lysosomal content in all body tissues facilitated the incorporation of lysosomal sequestration into a general physiologically based pharmacokinetic model, leading to improved predictions as well as elucidating its influence on tissue and subcellular distribution in the rat.
Subject(s)
Drug Development/methods , Lysosomes , Pharmaceutical Preparations/metabolism , Tissue Distribution/physiology , Animals , Lysosomes/chemistry , Lysosomes/drug effects , Lysosomes/physiology , Lysosomes/ultrastructure , Models, Biological , Pharmacokinetics , Rats , SolubilityABSTRACT
Despite extensive research on small molecule thrombin inhibitors for oral application in the past decades, only a single double prodrug with very modest oral bioavailability has reached human therapy as a marketed drug. We have undertaken major efforts to identify neutral, non-prodrug inhibitors. Using a holistic analysis of all available internal data, we were able to build computational models and apply these for the selection of a lead series with the highest possibility of achieving oral bioavailability. In our design, we relied on protein structure knowledge to address potency and identified a small window of favorable physicochemical properties to balance absorption and metabolic stability. Protein structure information on the pregnane X receptor helped in overcoming a persistent cytochrome P450 3A4 induction problem. The selected compound series was optimized to a highly potent, neutral, non-prodrug thrombin inhibitor by designing, synthesizing, and testing derivatives. The resulting optimized compound, BAY1217224, has reached first clinical trials, which have confirmed the desired pharmacokinetic properties.
Subject(s)
Anticoagulants/chemical synthesis , Drug Design , Thrombin/antagonists & inhibitors , Administration, Oral , Animals , Anticoagulants/chemistry , Anticoagulants/pharmacokinetics , Anticoagulants/pharmacology , Benzoxazoles/chemistry , Benzoxazoles/metabolism , Benzoxazoles/pharmacology , Binding Sites , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , Half-Life , Humans , Imidazoles/chemistry , Imidazoles/metabolism , Imidazoles/pharmacology , Inhibitory Concentration 50 , Male , Molecular Docking Simulation , Oxazolidinones/chemistry , Oxazolidinones/metabolism , Oxazolidinones/pharmacology , Pregnane X Receptor/genetics , Pregnane X Receptor/metabolism , Rats , Rats, Wistar , Structure-Activity Relationship , Thrombin/metabolism , Transcriptional Activation/drug effectsABSTRACT
The ATR kinase plays a key role in the DNA damage response by activating essential signaling pathways of DNA damage repair, especially in response to replication stress. Because DNA damage and replication stress are major sources of genomic instability, selective ATR inhibition has been recognized as a promising new approach in cancer therapy. We now report the identification and preclinical evaluation of the novel, clinical ATR inhibitor BAY 1895344. Starting from quinoline 2 with weak ATR inhibitory activity, lead optimization efforts focusing on potency, selectivity, and oral bioavailability led to the discovery of the potent, highly selective, orally available ATR inhibitor BAY 1895344, which exhibited strong monotherapy efficacy in cancer xenograft models that carry certain DNA damage repair deficiencies. Moreover, combination treatment of BAY 1895344 with certain DNA damage inducing chemotherapy resulted in synergistic antitumor activity. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965).
Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Morpholines/administration & dosage , Morpholines/pharmacokinetics , Pyrazoles/administration & dosage , Pyrazoles/pharmacokinetics , Administration, Oral , Animals , Antineoplastic Agents/chemistry , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Ataxia Telangiectasia Mutated Proteins/chemistry , Ataxia Telangiectasia Mutated Proteins/metabolism , Biological Availability , Carboplatin/administration & dosage , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Cytochrome P-450 CYP2C8 Inhibitors/chemistry , Cytochrome P-450 CYP2C8 Inhibitors/pharmacology , DNA Repair/drug effects , Dogs , Drug Discovery , Drug Screening Assays, Antitumor , Drug Stability , Female , Humans , Mice, SCID , Microsomes, Liver/drug effects , Morpholines/chemistry , Pyrazoles/chemistry , Rats, Wistar , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Inhibition of monopolar spindle 1 (MPS1) kinase represents a novel approach to cancer treatment: instead of arresting the cell cycle in tumor cells, cells are driven into mitosis irrespective of DNA damage and unattached/misattached chromosomes, resulting in aneuploidy and cell death. Starting points for our optimization efforts with the goal to identify MPS1 inhibitors were two HTS hits from the distinct chemical series "triazolopyridines" and "imidazopyrazines". The major initial issue of the triazolopyridine series was the moderate potency of the HTS hits. The imidazopyrazine series displayed more than 10-fold higher potencies; however, in the early project phase, this series suffered from poor metabolic stability. Here, we outline the evolution of the two hit series to clinical candidates BAY 1161909 and BAY 1217389 and reveal how both clinical candidates bind to the ATP site of MPS1 kinase, while addressing different pockets utilizing different binding interactions, along with their synthesis and preclinical characterization in selected in vivo efficacy models.
Subject(s)
Antineoplastic Agents/metabolism , Cell Cycle Proteins/metabolism , Drug Delivery Systems/methods , Drug Discovery/methods , M Phase Cell Cycle Checkpoints/drug effects , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Spindle Apparatus/drug effects , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Cell Line, Tumor , Dogs , Female , HT29 Cells , HeLa Cells , Humans , M Phase Cell Cycle Checkpoints/physiology , Male , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Structure, Tertiary , Protein-Tyrosine Kinases/antagonists & inhibitors , Rats , Rats, Wistar , Spindle Apparatus/metabolism , Treatment OutcomeABSTRACT
A short synthetic approach with broad scope to access five- to seven-membered cyclic sulfoximines in only two to three steps from readily available thiophenols is reported. Thus, simple building blocks were converted to complex molecular structures by a sequence of S-alkylation and one-pot sulfoximine formation, followed by intramolecular cyclization. Seventeen structurally diverse cyclic sulfoximines were prepared in high overall yields. In vitro evaluation of these underrepresented, three-dimensional, cyclic sulfoximines with respect to properties relevant to medicinal chemistry did not reveal any intrinsic flaw for application in drug discovery.
Subject(s)
Drug Discovery/methods , Methionine Sulfoximine/chemical synthesis , Alkylation , Chemistry, Pharmaceutical , Cyclization , Methionine Sulfoximine/chemistry , Molecular StructureABSTRACT
The DNA damage response (DDR) secures the integrity of the genome of eukaryotic cells. DDR deficiencies can promote tumorigenesis but concurrently may increase dependence on alternative repair pathways. The ataxia telangiectasia and Rad3-related (ATR) kinase plays a central role in the DDR by activating essential signaling pathways of DNA damage repair. Here, we studied the effect of the novel selective ATR kinase inhibitor BAY 1895344 on tumor cell growth and viability. Potent antiproliferative activity was demonstrated in a broad spectrum of human tumor cell lines. BAY 1895344 exhibited strong monotherapy efficacy in cancer xenograft models that carry DNA damage repair deficiencies. The combination of BAY 1895344 with DNA damage-inducing chemotherapy or external beam radiotherapy (EBRT) showed synergistic antitumor activity. Combination treatment with BAY 1895344 and DDR inhibitors achieved strong synergistic antiproliferative activity in vitro, and combined inhibition of ATR and PARP signaling using olaparib demonstrated synergistic antitumor activity in vivo Furthermore, the combination of BAY 1895344 with the novel, nonsteroidal androgen receptor antagonist darolutamide resulted in significantly improved antitumor efficacy compared with respective single-agent treatments in hormone-dependent prostate cancer, and addition of EBRT resulted in even further enhanced antitumor efficacy. Thus, the ATR inhibitor BAY 1895344 may provide new therapeutic options for the treatment of cancers with certain DDR deficiencies in monotherapy and in combination with DNA damage-inducing or DNA repair-compromising cancer therapies by improving their efficacy.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , DNA Damage/drug effects , Neoplasms/drug therapy , Animals , Female , Humans , MiceABSTRACT
6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, potently and selectively inhibits phosphodiesterases 3A and 3B (PDE3A and PDE3B) and kills cancer cells by inducing PDE3A/B interactions with SFLN12. The structure-activity relationship (SAR) of DNMDP analogs was evaluated using a phenotypic viability assay, resulting in several compounds with suitable pharmacokinetic properties for in vivo analysis. One of these compounds, BRD9500, was active in an SK-MEL-3 xenograft model of cancer.
ABSTRACT
Bis(monoacylglycero)phosphate (BMP) and phosphatidylglycerol (PG) are structural isomeric phospholipids with very different properties and biological functions. Due to their isomeric nature, it has thus far been challenging to simultaneously quantify BMP and PG lipids in tissue samples by mass spectrometry. Therefore, we have developed a sensitive LC-MS/MS based approach with prior methylation derivatization that is able to handle large batches of samples. Using this high throughput platform, a simulated MS/MS database was established for confident lipid assignment. In this work, we have simultaneously identified and quantified BMP and PG lipid molecules in different body tissues of rats and mice. We report for the first time a quantitative molecular atlas of BMP and PG lipids for 14 different tissues and organs in Wistar rats, NMRI and CD1 mice. Organ- and species-specificity was analyzed and compared for both lipid molecule classes. A total of 34 BMP and 10â¯PG molecules were quantified, with PG concentrations being generally much higher across tissues than BMP, but BMP lipids showing a much higher molecular diversity between animal organs. The large diversity of the BMP lipids with regard to their abundance and molecular composition suggests distinct biological function(s) of the individual BMP molecules in different tissues and organs of body. Particularly high tissue levels of BMP were seen in spleen, lung, liver, kidney and small intestines, i.e. tissues that are known for their high abundance and/or activity level of lysosomes late and endosomes. Elevated BMP levels in brain tissue of APP/PSEN transgenic compared to age matched wild-type mice were also observed using this platform. This analytical methodology presented a high throughput LC-based approach incorporating simulated MS/MS database to identify and quantify BMP lipids as well as PG molecules.
Subject(s)
Lysophospholipids/analysis , Membrane Lipids/chemistry , Monoglycerides/analysis , Phosphatidylglycerols/analysis , Animals , Chromatography, Liquid , Male , Membrane Lipids/isolation & purification , Methylation , Mice , Mice, Inbred Strains , Rats , Rats, Wistar , Tandem Mass SpectrometryABSTRACT
Lysosomal sequestration may affect the pharmacokinetics, efficacy, and safety of new basic lipophilic drug candidates potentially impacting their intracellular concentrations and tissue distribution. It may also be involved in drug-drug interactions, drug resistance, and phospholipidosis. However, currently there are no assays to evaluate the lysosomotropic behavior of compounds in a setting fully meeting the needs of drug discovery. We have, therefore, integrated a set of methods to reliably rank order, quantify, and calculate the extent of lysosomal sequestration in rat hepatocytes. An indirect fluorescence-based assay monitors the displacement of the fluorescence probe LysoTracker Red by test compounds. Using a lysosomal-specific evaluation algorithm allows one to generate IC50 values at lower than previously reported concentrations. The concentration range directly agrees with the concentration dependency of the lysosomal drug content itself directly quantified by liquid chromatography-tandem mass spectrometry and thus permits a quantitative link between the indirect and the direct trapping assay. Furthermore, we have determined the full pH profile and corresponding volume fractions of the endo-/lysosomal system in plated rat hepatocytes, enabling a more accurate in silico prediction of the extent of lysosomal trapping based only on pK a values as input, allowing early predictions even prior to chemical synthesis. The concentration dependency-i.e., the saturability of the trapping-can then be determined by the IC50 values generated in vitro. Thereby, a more quantitative assessment of the susceptibility of basic lipophilic compounds for lysosomal trapping is possible.
Subject(s)
Biological Assay/methods , Drug Discovery/methods , Hepatocytes/metabolism , Lysosomes/metabolism , Pharmaceutical Preparations/analysis , Amines/chemistry , Animals , Cells, Cultured , Computer Simulation , Hepatocytes/chemistry , Hepatocytes/cytology , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Lysosomes/chemistry , Microscopy, Fluorescence , Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/metabolism , Primary Cell Culture , Rats , Rats, Wistar , Tandem Mass Spectrometry/methods , Tissue DistributionABSTRACT
An unprecedented set of structurally diverse sulfonimidamides (47â compounds) has been prepared by various N-functionalization reactions of tertiary =NH sulfonimidamide 2 aa. These N-functionalization reactions of model compoundâ 2 aa include arylation, alkylation, trifluoromethylation, cyanation, sulfonylation, alkoxycarbonylation (carbamate formation) and aminocarbonylation (urea formation). Small molecule X-ray analyses of selected N-functionalized products are reported. To gain further insight into the properties of sulfonimidamides relevant to medicinal chemistry, a variety of structurally diverse reaction products were tested in selected in vitro assays. The described N-functionalization reactions provide a short and efficient approach to structurally diverse sulfonimidamides which have been the subject of recent, growing interest in the life sciences.