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1.
Toxicology ; 505: 153830, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38754619

ABSTRACT

The use of tyrosine kinase inhibitors (TKIs) has resulted in significant occurrence of arrhythmias. However, the precise mechanism of the proarrhythmic effect is not fully understood. In this study, we found that nilotinib (NIL), vandetanib (VAN), and mobocertinib (MOB) induced the development of "cellrhythmia" (arrhythmia-like events) in a concentration-dependent manner in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Continuous administration of NIL, VAN, or MOB in animals significantly prolonged the action potential durations (APD) and increased susceptibility to arrhythmias. Using phosphoproteomic analysis, we identified proteins with altered phosphorylation levels after treatment with 3 µM NIL, VAN, and MOB for 1.5 h. Using these identified proteins as substrates, we performed kinase-substrate enrichment analysis to identify the kinases driving the changes in phosphorylation levels of these proteins. MAPK and WNK were both inhibited by NIL, VAN, and MOB. A selective inhibitor of WNK1, WNK-IN-11, induced concentration- and time-dependent cellrhythmias and prolonged field potential duration (FPD) in hiPSC-CMs in vitro; furthermore, administration in guinea pigs confirmed that WNK-IN-11 prolonged ventricular repolarization and increased susceptibility to arrhythmias. Fingding indicated that WNK1 inhibition had an in vivo and in vitro arrhythmogenic phenotype similar to TKIs. Additionally,three of TKIs reduced hERG and KCNQ1 expression at protein level, not at transcription level. Similarly, the knockdown of WNK1 decreased hERG and KCNQ1 protein expression in hiPSC-CMs. Collectively, our data suggest that the proarrhythmic effects of NIL, VAN, and MOB occur through a kinase inhibition mechanism. NIL, VAN, and MOB inhibit WNK1 kinase, leading to a decrease in hERG and KCNQ1 protein expression, thereby prolonging action potential repolarization and consequently cause arrhythmias.


Subject(s)
Action Potentials , Arrhythmias, Cardiac , Myocytes, Cardiac , Piperidines , Proteomics , Pyrimidines , Quinazolines , Humans , Arrhythmias, Cardiac/chemically induced , Animals , Proteomics/methods , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Piperidines/pharmacology , Piperidines/toxicity , Pyrimidines/toxicity , Pyrimidines/pharmacology , Quinazolines/toxicity , Quinazolines/pharmacology , Action Potentials/drug effects , Protein Kinase Inhibitors/toxicity , Protein Kinase Inhibitors/pharmacology , Phosphorylation , ERG1 Potassium Channel/metabolism , ERG1 Potassium Channel/antagonists & inhibitors , ERG1 Potassium Channel/genetics , Guinea Pigs , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Male , KCNQ1 Potassium Channel/metabolism , KCNQ1 Potassium Channel/genetics , KCNQ1 Potassium Channel/drug effects , Phosphoproteins/metabolism , Dose-Response Relationship, Drug
2.
Environ Toxicol ; 39(7): 3833-3845, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38546377

ABSTRACT

Depleted uranium (DU) retains the radiological toxicities, which accumulates preferentially in the kidneys. Hedgehog (Hh) pathway plays a critical role in tissue injury. However, the role of Hh in DU-induced nephrotoxicity was still unclear. This study was carried out to investigate the effect of Gli2, which was an important transcription effector of Hh signaling, on DU induced nephrotoxicity. To clarify it, CK19 positive tubular epithelial cells specific Gli2 conditional knockout (KO) mice model was exposed to DU, and then histopathological damage and Hh signaling pathway activation was analyzed. Moreover, HEK-293 T cells were exposed to DU with Gant61 or Gli2 overexpression, and cytotoxicity of DU as analyzed. Results showed that DU caused nephrotoxicity accompanied by activation of Hh signaling pathway. Meanwhile, genetic KO of Gli2 reduced DU-induced nephrotoxicity by normalizing biochemical indicators and reducing Hh pathway activation. Pharmacologic inhibition of Gli1/2 by Gant61 reduced DU induced cytotoxicity by inhibiting apoptosis, ROS formation and Hh pathway activation. However, overexpression of Gli2 aggravated DU-induced cytotoxicity by increasing the levels of apoptosis and ROS formation. Taken together, these results revealed that Hh signaling negatively regulated DU-inducted nephrotoxicity, and that inhibition of Gli2 might serve as a promising nephroprotective target for DU-induced kidney injury.


Subject(s)
Hedgehog Proteins , Kidney , Mice, Knockout , Signal Transduction , Zinc Finger Protein Gli2 , Animals , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Humans , HEK293 Cells , Signal Transduction/drug effects , Zinc Finger Protein Gli2/metabolism , Zinc Finger Protein Gli2/genetics , Kidney/drug effects , Kidney/pathology , Kidney/metabolism , Mice , Uranium/toxicity , Apoptosis/drug effects , Pyridines/pharmacology , Pyridines/toxicity , Male , Kidney Diseases/chemically induced , Kidney Diseases/pathology , Kidney Diseases/metabolism , Pyrimidines/pharmacology , Pyrimidines/toxicity , Mice, Inbred C57BL , Reactive Oxygen Species/metabolism
3.
J Med Chem ; 67(6): 5032-5052, 2024 Mar 28.
Article in English | MEDLINE | ID: mdl-38482820

ABSTRACT

Gout and hyperuricemia are metabolic diseases characterized with high serum uric acid (SUA) levels that significantly impact human health. Lesinurad, a uricosuric agent, is limited to concurrent use with xanthine oxidase inhibitors (XOIs) in clinical practice due to its restricted efficacy and potential nephrotoxicity. Herein, extensive structural modifications of lesinurad were conducted through scaffold hopping and substituent modification strategies, affording 54 novel derivatives containing pyrimidine-fused cyclic structures. Notably, the thienopyrimidine compound 29 demonstrated a remarkable 2-fold increase in SUA-lowering in vivo activity compared to lesinurad, while exhibiting potent inhibitory activity against the urate transporter 1 (URAT1, IC50 = 2.01 µM) and glucose transporter 9 (GLUT9, IC50 = 18.21 µM). Furthermore, it possessed a lower effective dosage of 0.5 mg/kg, favorable safety profile without any apparent acute toxicity at doses of 1000 mg/kg, and improved pharmacokinetic properties. Overall, we have discovered an efficacious URAT1/GLUT9 dual inhibitor for inhibiting urate reabsorption with favorable pharmacokinetic profiles.


Subject(s)
Gout , Hyperuricemia , Organic Anion Transporters , Thioglycolates , Triazoles , Humans , Uric Acid/therapeutic use , Gout/drug therapy , Hyperuricemia/drug therapy , Uricosuric Agents/therapeutic use , Pyrimidines/toxicity , Pyrimidines/therapeutic use , Glucose Transport Proteins, Facilitative , Organic Cation Transport Proteins
4.
Sci Rep ; 14(1): 1516, 2024 01 17.
Article in English | MEDLINE | ID: mdl-38233515

ABSTRACT

The exponential development of resistance to conventional chemical insecticides adds another important motive for the creation of novel insecticidal active agents. One of the keys to meeting this challenge is the exploration of novel classes of insecticidal molecules with different modes of action. Herein, a novel series of spiro pyrimidine derivatives was prepared using some green synthetic methodologies such as microwave irradiation, and sonication under ultrasound waves. Spiro pyrimidine aminonitrile 1 is a key starting material for the synthesis of targets 2-9 by reaction with different carbon electrophiles and nitrogen nucleophiles. The structures of all the newly synthesized compounds were approved using spectral data. The toxicological efficiency and biological impacts of the synthesized spiro pyrimidine derivatives were assessed against Culex pipiens L. larvae. The toxicity of synthesized compounds showed remarkable variations against the C. pipiens larvae. Where, 3, 4 and 2 were the most efficient compounds with LC50 values of 12.43, 16.29 and 21.73 µg/mL, respectively. While 1 was the least potent compound with an LC50 value of 95.18 µg/mL. As well, other compounds were arranged according to LC50 values as follows 5 > 7 > 6 > 9 > 8. In addition, 3 and 4 exhibited significant prolongation of the developmental duration and greatly inhibited adult emergence. Moreover, many morphological deformities were observed in all developmental stages. Furthermore, cytotoxicity of the most effective compounds was assessed against the normal human cells (WI-38) as non-target organisms, where compounds 2, 4 and 3 showed weak to non-toxic effects. The study of binding affinity and correlation between chemical structure and reactivity was carried out using molecular docking study and DFT calculations to investigate their mode of action. This study shed light on promising compounds with larvicidal activity and biological impacts on the C. pipiens life cycle.


Subject(s)
Culex , Insecticides , Animals , Humans , Molecular Docking Simulation , Insecticides/pharmacology , Insecticides/chemistry , Larva , Pyrimidines/toxicity
5.
Toxicol In Vitro ; 95: 105754, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38061604

ABSTRACT

In this study, we aimed to analyze the effects of first and second-generation Bcr-Abl tyrosine kinase inhibitors, imatinib and nilotinib on LPS/IFN gamma activated RAW 264.7 macrophages. Our data revealed that imatinib was less effective on nitrite levels and more toxic on macrophages compared to nilotinib. Therefore, we further analysed the effect of nilotinib on various inflammatory markers including iNOS, COX-2, NFkB, IL-6, p-ERK, p-p38 and p-JNK in LPS/IFN gamma activated RAW264.7 macrophages. Spectrophotometric viability test and Griess assay,western blot, RT-PCR and luciferase reporter assays were used to analyze the biological activity of nilotinib. Our findings revealed that nilotinib decreases nitrite levels, iNOS mRNA, iNOS and p-p38 protein expressions significantly whereas induces IL-6 mRNA and p-JNK protein expressions at particular doses. We did not find significant effect of nilotinib on COX-2, p-ERK and nuclear p65 proteins and NFkB transcriptional activity. In addition, the binding mode of nilotinib to iNOS protein was predicted by molecular docking. According to the docking analyses, nilotinib exhibited hydrophobic interactions between MET349, ALA191, VAL346, PHE363, TYR367, MET368, CYS194, TRP366 residues at the binding pocket and the molecule as well as van der Waals interactions at specific residues. In conclusion, our results reveal that, in addition to its anticancer activity, nilotinib can exhibit immune modulatory effects on macrophages through its effects on iNOS, IL-6, p-p38 and p-JNK.


Subject(s)
Lipopolysaccharides , Nitrites , Imatinib Mesylate/pharmacology , Lipopolysaccharides/pharmacology , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Nitrites/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Molecular Docking Simulation , Macrophages , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , NF-kappa B/metabolism , Nuclear Proteins/metabolism , Pyrimidines/toxicity , RNA, Messenger/metabolism
6.
Toxicol In Vitro ; 93: 105699, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37751784

ABSTRACT

Cytotoxicity of some pesticides is a disadvantage for the Salmonella/microsome assay with regard to the equivalence assessment of pesticide technical grade active ingredients to the original products and detection of low-level impurities. The technical grade active ingredients (TGAIs) of pesticides from certain chemical classes were found to be toxic for Salmonella typhimurium strains. Among the highly cytotoxic compounds were sulfonylureas, which include 20 active ingredients. In addition, this class includes active pharmaceutical ingredients used for the manufacture of antidiabetics drugs. A traditional selection methodology was applied using the cultivation of S. typhimurium TA100 in the presence of high concentrations of thifensulfuronmethyl (TFSM) to obtain a resistant test strain insusceptible to sulfonylurea toxic effect. Two strains resistant not only to sulfonylureas (SFU) but also triazolepyrimidines were received. The first mutant strain (deposited as S. typhimurium VKPM B-14099 in the Russian National Collection of Industrial Microorganisms) demonstrated the TA100 phenotypic characteristics: hisG46, rfa, ΔuvrB-bio, pKM101. The second strain (deposited as S. typhimurium VKPM B-14359) showed the TA1535 phenotypic characteristics and probably lost the R-factor due to the selection using the poor Gm-media with TFSM. Positive controls caused pronounced mutagenic effects (±S9) in both strains, consequently the mutants did not lose the ability to respond to induction of the reverse gene mutations. The maximum non-cytotoxic concentrations of SFUs and triazole-pyrimidines for the Ames test strains did not exceed 0.05-0.125 mg/plate, while no evidence of cytotoxicity was observed for the mutants up to 5.0 mg/plate. Electron microscopy of the ultrathin sections of Salmonella cells grown with and without TFSM showed an obvious difference in the structure of the cell wall and cytoplasm in mutant and parental cultures. The concurrent resistance both to SFU and triazolepyrimidines was assumed to be mediated by the same mechanism of action of the pesticides from these classes - inhibition of acetohydroxyacid synthase. To confirm this hypothesis, the tests in the presence of branched-chain amino acids were carried out. The enrichment of agar with isoleucine prevented the toxic effects of SFU and triazolepyrimidines for all Ames test strains used in the study, while strong cytotoxicity was observed in the presence of valine and leucine. Considering the tolerance of strains both to SFU and triazolpyrimidines and the results with branched-chain amino acids, the modification of target acetohydroxyacid synthase was supposed the key to the acquired resistance. The new strains resistant to sulfonylureas and triazole-pyrimidines expands the possibilities to reveal mutagenic impurities that may occur in TGAIs in small amounts.


Subject(s)
Herbicides , Mutagenicity Tests/methods , Herbicides/toxicity , Mutagens/toxicity , Salmonella typhimurium/genetics , Amino Acids, Branched-Chain/genetics , Amino Acids, Branched-Chain/pharmacology , Pyrimidines/toxicity , Triazoles/pharmacology
7.
Int J Mol Sci ; 24(4)2023 Feb 15.
Article in English | MEDLINE | ID: mdl-36835302

ABSTRACT

Over the past 20 years, numerous tyrosine kinase inhibitors (TKIs) have been introduced for targeted therapy of various types of malignancies. Due to frequent and increasing use, leading to eventual excretion with body fluids, their residues have been found in hospital and household wastewaters as well as surface water. However, the effects of TKI residues in the environment on aquatic organisms are poorly described. In the present study, we investigated the cytotoxic and genotoxic effects of five selected TKIs, namely erlotinib (ERL), dasatinib (DAS), nilotinib (NIL), regorafenib (REG), and sorafenib (SOR), using the in vitro zebrafish liver cell (ZFL) model. Cytotoxicity was determined using the MTS assay and propidium iodide (PI) live/dead staining by flow cytometry. DAS, SOR, and REG decreased ZFL cell viability dose- and time-dependently, with DAS being the most cytotoxic TKI studied. ERL and NIL did not affect viability at concentrations up to their maximum solubility; however, NIL was the only TKI that significantly decreased the proportion of PI negative cells as determined by the flow cytometry. Cell cycle progression analyses showed that DAS, ERL, REG, and SOR caused the cell cycle arrest of ZFL cells in the G0/G1 phase, with a concomitant decrease of cells in the S-phase fraction. No data could be obtained for NIL due to severe DNA fragmentation. The genotoxic activity of the investigated TKIs was evaluated using comet and cytokinesis block micronucleus (CBMN) assays. The dose-dependent induction of DNA single strand breaks was induced by NIL (≥2 µM), DAS (≥0.006 µM), and REG (≥0.8 µM), with DAS being the most potent. None of the TKIs studied induced micronuclei formation. These results suggest that normal non-target fish liver cells are sensitive to the TKIs studied in a concentration range similar to those previously reported for human cancer cell lines. Although the TKI concentrations that induced adverse effects in exposed ZFL cells are several orders of magnitude higher than those currently expected in the aquatic environment, the observed DNA damage and cell cycle effects suggest that residues of TKIs in the environment may pose a hazard to non-intentionally exposed organisms living in environments contaminated with TKIs.


Subject(s)
Antineoplastic Agents , Hepatocytes , Animals , Humans , Antineoplastic Agents/toxicity , Hepatocytes/drug effects , Liver , Pyrimidines/toxicity , Sorafenib/toxicity , Zebrafish
8.
Reprod Toxicol ; 113: 35-41, 2022 10.
Article in English | MEDLINE | ID: mdl-35944741

ABSTRACT

Bone has recently emerged as a target organ for some Janus kinase (JAK) inhibitors in adult and/or juvenile animal toxicity studies. Oral administration of tofacitinib, a JAK inhibitor, was not associated with clinical or macroscopic effects on bone growth and development in a rat juvenile animal study (JAS) with tofacitinib dosing starting on postnatal day (PND) 21. However, given that previous JAS did not include a targeted evaluation of bone, inclusive of microscopic examination, an additional rat JAS was conducted to further assess this risk. In this subsequent JAS, administration of tofacitinib from PND 7-49 or from PND 21-49 did not result in any direct effects on bone, with no histologic effects on developing bone. The only bone effect in this JAS was nonadverse shorter femur length, which was not considered to be a direct effect of tofacitinib, but rather an indicator of growth delay, as this was associated with lower body weights. There were no effects on femur length or body weight after a 2-month recovery period. To further explore the relationship between body weight and femur length, historical control data were analyzed from control rats in other JAS. This analysis clearly demonstrated that shorter femur length can occur as an indirect effect that is highly associated with lower body weight, consistent with what was observed in the JAS with tofacitinib. These analyses provide a robust and valuable data set to support the interpretation of such data in JAS, and further support the lack of direct effects of tofacitinib on bone growth and development. As with the previously conducted juvenile studies with tofacitinib, the additional JAS did not identify any special JAS-based concerns for use in pediatric patients as young as 2 years of age.


Subject(s)
Janus Kinase Inhibitors , Animals , Body Weight , Femur , Janus Kinase Inhibitors/toxicity , Janus Kinases , Piperidines/toxicity , Pyrimidines/toxicity , Rats
9.
Toxicol Lett ; 367: 48-58, 2022 Aug 15.
Article in English | MEDLINE | ID: mdl-35868497

ABSTRACT

Sarin was used as a chemical weapon due to its high neurotoxicity and mortality. Subacute sarin induced cognitive and behavioral disorder. However, the underlying mechanism is still unclear. Here we offered a multi-omic approach for the analysis of altered metabolites, lipids, and proteins to explore the neurotoxicity of subacute sarin. Guinea pigs were administered between the shoulder blades 16.8 µg/kg of sarin in a volume of 1.0 ml/kg body weight by subcutaneous injection once daily for 14 days. At the end of the final injection, guinea pigs were sacrificed, and striatum were dissected for analysis. A total of 138 different metabolites were identified in the metabolome analysis. Lipids and lipid-like molecules is the largest group (38.41%). For lipidomic analysis, a total of 216 lipids were identified. In proteomic study, over 4300 proteins were identified and quantified. By integrating these enriched components, we demonstrated that the joint pathways disturbed by subacute sarin mainly involving lipid, purine and pyrimidine metabolism in guinea pig striatum. Overall, this study highlights the powerfulness of omics platforms to deepen the understanding of nerve agents caused neurotoxicity.


Subject(s)
Chemical Warfare Agents , Neurotoxicity Syndromes , Animals , Chemical Warfare Agents/toxicity , Cholinesterase Inhibitors/toxicity , Guinea Pigs , Homeostasis , Lethal Dose 50 , Lipidomics , Lipids , Neurotoxicity Syndromes/metabolism , Proteomics , Purines , Pyrimidines/toxicity , Sarin/toxicity
10.
Mar Drugs ; 20(3)2022 Mar 08.
Article in English | MEDLINE | ID: mdl-35323495

ABSTRACT

Five new alkaloids have been isolated from the lipophilic extract of the Antarctic tunicate Synoicum sp. Deep-sea specimens of Synoicum sp. were collected during a 2011 cruise of the R/V Nathanial B. Palmer to the southern Scotia Arc, Antarctica. Crude extracts from the invertebrates obtained during the cruise were screened in a zebrafish-based phenotypic assay. The Synoicum sp. extract induced embryonic dysmorphology characterized by axis truncation, leading to the isolation of aminopyrimidine substituted indolone (1-4) and indole (5-12) alkaloids. While the primary bioactivity tracked with previously reported meridianins A-G (5-11), further investigation resulted in the isolation and characterization of australindolones A-D (1-4) and the previously unreported meridianin H (12).


Subject(s)
Indole Alkaloids , Pyrimidines , Urochordata/chemistry , Animals , Antarctic Regions , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/drug effects , Indole Alkaloids/chemistry , Indole Alkaloids/toxicity , Pyrimidines/chemistry , Pyrimidines/toxicity , Zebrafish
11.
J Med Chem ; 65(4): 3229-3248, 2022 02 24.
Article in English | MEDLINE | ID: mdl-35138851

ABSTRACT

Herein, we report two promising compounds 30 and 36 possessing nanomolar FLT3 inhibitory activities (IC50 = 1.5-7.2 nM), high selectivity over c-KIT (>1000-fold), and excellent anti-AML activity (MV4-11 IC50 = 0.8-3.2 nM). Furthermore, these two compounds efficiently inhibited the growth of multiple mutant BaF3 cells expressing FLT3-ITD, FLT3-D835V/F, FLT3-F691L, FLT3-ITD-F691L, and FLT3-ITD-D835Y. Oral administration of 30 and 36 at 6 mg/kg/d could significantly suppress tumor growth in the MV4-11 cell-inoculated xenograft model, exhibiting tumor growth inhibitory rates of 83.5% and 95.1%, respectively. Importantly, 36 could prolong the mouse survival time in the FLT3-ITD-TKD dual mutation syngeneic mouse model (BaF3-FLT3-ITD-D835Y) at a dose of 6 mg/kg p.o. bid/4W. No clear myelosuppression was observed in the treated group of 36 in the MPO strain of zebrafish, even at 10 µM. In summary, our data demonstrated that 36 may represent a promising candidate for the treatment of FLT3 mutant AML.


Subject(s)
Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Animals , Cell Line, Tumor , Demyelinating Diseases/chemically induced , Demyelinating Diseases/pathology , Dose-Response Relationship, Drug , Humans , Leukemia, Myeloid, Acute/drug therapy , Mice , Mice, Inbred BALB C , Protein Kinase Inhibitors/toxicity , Pyrimidines/toxicity , Signal Transduction/drug effects , Substrate Specificity , Xenograft Model Antitumor Assays , Zebrafish
12.
J Med Chem ; 65(3): 2471-2496, 2022 02 10.
Article in English | MEDLINE | ID: mdl-35077178

ABSTRACT

Novel analogues of C-2-substituted thienopyrimidine-based bisphosphonates (C2-ThP-BPs) are described that are potent inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS). Members of this class of compounds induce target-selective apoptosis of multiple myeloma (MM) cells and exhibit antimyeloma activity in vivo. A key structural element of these inhibitors is a linker moiety that connects their (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)bisphosphonic acid core to various side chains. The structural diversity of this linker moiety, as well as the side chains attached to it, was investigated and found to significantly impact the toxicity of these compounds in MM cells. The most potent inhibitor identified was evaluated in mouse and rat for liver toxicity and systemic exposure, respectively, providing further optimism for the potential value of such compounds as human therapeutics.


Subject(s)
Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Geranylgeranyl-Diphosphate Geranylgeranyltransferase/antagonists & inhibitors , Multiple Myeloma/drug therapy , Pyrimidines/therapeutic use , Thiophenes/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Antineoplastic Agents/toxicity , Bone Marrow Cells/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/toxicity , Female , Fungal Proteins/antagonists & inhibitors , Fungal Proteins/metabolism , Geranylgeranyl-Diphosphate Geranylgeranyltransferase/metabolism , Humans , Liver/drug effects , Male , Mice, Inbred C57BL , Molecular Structure , Protein Binding , Pyrimidines/chemical synthesis , Pyrimidines/metabolism , Pyrimidines/toxicity , Rats , Saccharomyces cerevisiae/enzymology , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/metabolism , Thiophenes/toxicity
13.
J Pharmacol Exp Ther ; 380(2): 114-125, 2022 02.
Article in English | MEDLINE | ID: mdl-34794962

ABSTRACT

Drug-induced liver injury (DILI) is the leading cause of acute liver failure and a major concern in drug development. Altered bile acid homeostasis via inhibition of the bile salt export pump (BSEP) is one mechanism of DILI. Dasatinib, pazopanib, and sorafenib are tyrosine kinase inhibitors (TKIs) that competitively inhibit BSEP and increase serum biomarkers for hepatotoxicity in ∼25-50% of patients. However, the mechanism(s) of hepatotoxicity beyond competitive inhibition of BSEP are poorly understood. This study examined mechanisms of TKI-mediated hepatotoxicity associated with altered bile acid homeostasis. Dasatinib, pazopanib, and sorafenib showed bile acid-dependent toxicity at clinically relevant concentrations, based on the C-DILI assay using sandwich-cultured human hepatocytes (SCHH). Among several bile acid-relevant genes, cytochrome P450 (CYP) 7A1 mRNA was specifically upregulated by 6.2- to 7.8-fold (dasatinib) and 5.7- to 9.3-fold (pazopanib), compared with control, within 8 hours. This was consistent with increased total bile acid concentrations in culture medium up to 2.3-fold, and in SCHH up to 1.4-fold, compared with control, within 24 hours. Additionally, protein abundance of sodium taurocholate co-transporting polypeptide (NTCP) was increased up to 2.0-fold by these three TKIs. The increase in NTCP protein abundance correlated with increased function; dasatinib and pazopanib increased hepatocyte uptake clearance (CLuptake) of taurocholic acid, a probe bile acid substrate, up to 1.4-fold. In conclusion, upregulation of CYP7A1 and NTCP in SCHH constitute novel mechanisms of TKI-associated hepatotoxicity. SIGNIFICANCE STATEMENT: Understanding the mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors (TKIs) is fundamental to development of effective and safe intervention therapies for various cancers. Data generated in sandwich-cultured human hepatocytes, an in vitro model of drug-induced hepatotoxicity, revealed that TKIs upregulate bile acid synthesis and alter bile acid uptake and excretion. These findings provide novel insights into additional mechanisms of bile acid-mediated drug-induced liver injury, an adverse effect that limits the use and effectiveness of TKI treatment in some cancer patients.


Subject(s)
Antineoplastic Agents/toxicity , Bile Acids and Salts/metabolism , Chemical and Drug Induced Liver Injury/metabolism , Hepatocytes/drug effects , Protein Kinase Inhibitors/toxicity , ATP Binding Cassette Transporter, Subfamily B, Member 11/metabolism , Cells, Cultured , Cholesterol 7-alpha-Hydroxylase/genetics , Cholesterol 7-alpha-Hydroxylase/metabolism , Dasatinib/toxicity , Hepatocytes/metabolism , Humans , Indazoles/toxicity , Organic Anion Transporters, Sodium-Dependent/metabolism , Pyrimidines/toxicity , Sorafenib/toxicity , Sulfonamides/toxicity , Symporters/metabolism
14.
PLoS One ; 16(12): e0259383, 2021.
Article in English | MEDLINE | ID: mdl-34855766

ABSTRACT

Endocrine-disrupting compounds as pesticides affect the hormonal balance, and this can result in several diseases. Therefore, the analysis of representative hormones with acetamiprid (AC) and azoxystrobin (AZ) was a good strategy for the investigation of the endocrine-disrupting activity of pesticides. Hence, a sensitive and rapid analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The method was validated for the analysis of AC, AZ, estriol, estrone, progesterone, and testosterone in the serum, testis, and liver of rats. The correlation between the residues of pesticides and the disturbance of the endocrine system was evaluated. The different mass parameters, mobile phase types, analytical columns, injection volumes, and extraction solvents were compared to get the lowest limit of detection of the studied compounds. The detection limits of AC, AZ, estriol, estrone, progesterone, and testosterone were 0.05, 0.05, 1.0, 10, and 1.0 ng/ml, respectively. The method developed was applied to evaluate the changes in these hormones induced by the duration of exposure to AC and AZ in rat testis and serum. The hormones level in rat serum and testis had a significant decrease as they were oral gavage treated with different high concentrations of studied pesticides. Both pesticides were distributed in the body of rats by the multi-compartment model (liver, testis, and serum).


Subject(s)
Endocrine Disruptors/toxicity , Gonadal Steroid Hormones/analysis , Neonicotinoids/toxicity , Pyrimidines/toxicity , Strobilurins/toxicity , Animals , Calibration , Chromatography, Liquid/methods , Endocrine Disruptors/administration & dosage , Endocrine Disruptors/analysis , Endocrine Disruptors/pharmacokinetics , Estriol/analysis , Estrone/analysis , Limit of Detection , Male , Neonicotinoids/administration & dosage , Neonicotinoids/analysis , Neonicotinoids/pharmacokinetics , Pesticides/toxicity , Pyrimidines/administration & dosage , Pyrimidines/analysis , Pyrimidines/pharmacokinetics , Rats, Wistar , Reproducibility of Results , Strobilurins/administration & dosage , Strobilurins/analysis , Strobilurins/pharmacokinetics , Tandem Mass Spectrometry/methods , Testosterone/analysis , Tissue Distribution
15.
Sci Rep ; 11(1): 21653, 2021 11 05.
Article in English | MEDLINE | ID: mdl-34741036

ABSTRACT

Pollinators, particularly wild bees, are suffering declines across the globe, and pesticides are thought to be drivers of these declines. Research into, and regulation of pesticides has focused on the active ingredients, and their impact on bee health. In contrast, the additional components in pesticide formulations have been overlooked as potential threats. By testing an acute oral dose of the fungicide product Amistar, and equivalent doses of each individual co-formulant, we were able to measure the toxicity of the formulation and identify the ingredient responsible. We found that a co-formulant, alcohol ethoxylates, caused a range of damage to bumble bee health. Exposure to alcohol ethoxylates caused 30% mortality and a range of sublethal effects. Alcohol ethoxylates treated bees consumed half as much sucrose as negative control bees over the course of the experiment and lost weight. Alcohol ethoxylates treated bees had significant melanisation of their midguts, evidence of gut damage. We suggest that this gut damage explains the reduction in appetite, weight loss and mortality, with bees dying from energy depletion. Our results demonstrate that sublethal impacts of pesticide formulations need to be considered during regulatory consideration, and that co-formulants can be more toxic than active ingredients.


Subject(s)
Bees/drug effects , Feeding Behavior/drug effects , Fungicides, Industrial/toxicity , Pyrimidines/toxicity , Strobilurins/toxicity , Animals , Body Weight/drug effects , Gastrointestinal Tract/drug effects , Sucrose
16.
Int J Mol Sci ; 22(22)2021 Nov 19.
Article in English | MEDLINE | ID: mdl-34830376

ABSTRACT

Fungicides often cause genotoxic stress and neurodevelopmental disorders such as autism (ASD). Fungicide-azoxystrobin (AZOX) showed acute and chronic toxicity to various organisms, and remained a concern for ill effects in developing neurons. We evaluated the neurotoxicity of AZOX in developing mouse brains, and observed prenatal exposure to AZOX reduced neuronal viability, neurite outgrowth, and cortical migration process in developing brains. The 50% inhibitory concentration (IC50) of AZOX for acute (24 h) and chronic (7 days) exposures were 30 and 10 µM, respectively. Loss in viability was due to the accumulation of reactive oxygen species (ROS), and inhibited neurite outgrowth was due to the deactivation of mTORC1 kinase activity. Pretreatment with ROS scavenger- N-acetylcysteine (NAC) reserved the viability loss and forced activation of mTORC1 kinase revived the neurite outgrowth in AZOX treated neurons. Intra-amniotic injection of AZOX coupled with in utero electroporation of GFP-labelled plasmid in E15.5 mouse was performed and 20 mg/kg AZOX inhibited radial neuronal migration. Moreover, the accumulation of mitochondria was significantly reduced in AZOX treated primary neurons, indicative of mitochondrial deactivation and induction of apoptosis, which was quantified by Bcl2/Bax ratio and caspase 3 cleavage assay. This study elucidated the neurotoxicity of AZOX and explained the possible cure from it.


Subject(s)
Apoptosis/drug effects , Neurogenesis/drug effects , Neurons/drug effects , Pyrimidines/pharmacology , Strobilurins/pharmacology , Acetylcysteine/pharmacology , Animals , Autistic Disorder/chemically induced , Autistic Disorder/genetics , Autistic Disorder/pathology , Cell Movement/drug effects , Cell Survival/drug effects , Female , Fungicides, Industrial/toxicity , Humans , Mechanistic Target of Rapamycin Complex 1/genetics , Mice , Mitochondria/drug effects , Neuronal Outgrowth/drug effects , Neurons/pathology , Pregnancy , Prenatal Exposure Delayed Effects , Pyrimidines/toxicity , Reactive Oxygen Species/antagonists & inhibitors , Strobilurins/toxicity
17.
An Acad Bras Cienc ; 93(suppl 3): e20200860, 2021.
Article in English | MEDLINE | ID: mdl-34550199

ABSTRACT

The use of agrochemicals in agriculture may impact aquatic ecosystems, particularly influencing the stream insect communities. Among aquatic insects, the family Chironomidae is the most abundant and species-diverse insect group found in freshwater ecosystems. However, in the southern hemisphere, studies with Chironomidae are still sparse, compared to Europe and North America. The present study evaluates the responses of Chironomidae species (Insecta: Diptera) to pyrimethanil fungicide in a mesocosm experiment. Water contamination and chironomid community were monitored over 10 months. After five months of monitoring, the pyrimethanil fungicide was completely degraded and there was a statistically significant increase in the Margalef Richness and Shannon-Wiener Index (H') in the control units when compared with the contaminated mesocosms (p = 0.003). Our results point out that the utilization of agrochemicals can be a harmful factor influencing negatively the Chironomidae populations. This finding has key implications for insect conservation strategies and ecological management environments.


Subject(s)
Chironomidae , Fungicides, Industrial , Animals , Ecosystem , Fungicides, Industrial/toxicity , Pyrimidines/toxicity
18.
Sci Rep ; 11(1): 16856, 2021 08 19.
Article in English | MEDLINE | ID: mdl-34413359

ABSTRACT

P-gp-associated multidrug resistance is a major impediment to the success of chemotherapy. With the aim of finding non-toxic and effective P-gp inhibitors, we investigated a panel of quinolin-2-one-pyrimidine hybrids. Among the active compounds, two of them significantly increased intracellular doxorubicin and rhodamine 123 accumulation by inhibiting the efflux mediated by P-gp and restored doxorubicin toxicity at nanomolar range. Structure-activity relationships showed that the number of methoxy groups, an optimal length of the molecule in its extended conformation, and at least one flexible methylene group bridging the quinolinone to the moiety bearing the pyrimidine favored the inhibitory potency of P-gp. The best compounds showed a similar binding pattern and interactions to those of doxorubicin and tariquidar, as revealed by MD and hybrid QM/MM simulations performed with the recent experimental structure of P-gp co-crystallized with paclitaxel. Analysis of the molecular interactions stabilizing the different molecular complexes determined by MD and QTAIM showed that binding to key residues from TMH 4-7 and 12 is required for inhibition.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Drug Resistance, Multiple/drug effects , Pyrimidines/pharmacology , Quinolones/pharmacology , Cell Death/drug effects , Humans , K562 Cells , Molecular Dynamics Simulation , Protein Transport/drug effects , Pyrimidines/chemistry , Pyrimidines/toxicity , Quinolones/chemistry , Quinolones/toxicity , Rhodamine 123/metabolism , Structure-Activity Relationship , Thermodynamics
19.
J Pharmacol Exp Ther ; 379(2): 166-174, 2021 11.
Article in English | MEDLINE | ID: mdl-34373353

ABSTRACT

During drug development, potential safety issues can occur at any time. Understanding the cause of a toxicity can help with deciding on how to advance the drug development program. Chemoproteomics provides a way to help understand the cause of a toxicity wherein the affected tissue is accessible and can be probed with a covalently binding compound that is analogous to the offending drug. In this case, N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292), a covalently binding Bruton's tyrosine kinase inhibitor, had produced testicular toxicity in rodents. Experiments were conducted using a CC-292 analog that could be chemically modified with biotin to probe rodent testes homogenates for potential binding sites that were subsequently recovered with avidin beads. These biotin-tagged proteins undergo trypsin digest on the avidin beads to yield peptides that are identified using mass spectrometry. Two proteins were identified from the testicular homogenates of both rats and mice, namely retinal dehydrogenase 1 (ALDH1A1) and retinal dehydrogenase 2 (ALDH1A2). Literature confirmed a link between inhibition of these enzymes and testicular toxicity. Subsequently, molecular modeling was used to demonstrate that CC-292 can be docked into both the nicotinamide adenine dinucleotide and retinal binding pockets of the analogous human ALDH1A2 enzyme. These data suggest that the off-target binding site for CC-292 on retinal dehydrogenase enzymes may provide a mechanistic explanation to the testicular toxicity observed in rodents and that there may be a potential concern for human male fertility. SIGNIFICANCE STATEMENT: Biotinylated covalently binding drug analogues are used to enrich bound proteins from tissue homogenates wherein toxicity was observed in rodents. Bound proteins were subsequently identified by mass spectroscopy. Competition of the analog binding with the parent inhibitor itself and three-dimensional molecular modeling were used to establish a likely link between the off-targets of CC-292, ALDH1A1, and ALDH1A2 with potential testicular toxicity.


Subject(s)
Acrylamides/toxicity , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Protein Kinase Inhibitors/toxicity , Proteomics/methods , Pyrimidines/toxicity , Testis/drug effects , Testis/enzymology , Agammaglobulinaemia Tyrosine Kinase/genetics , Agammaglobulinaemia Tyrosine Kinase/metabolism , Amino Acid Sequence , Animals , Dose-Response Relationship, Drug , Humans , Male , Mice , Mice, Inbred C57BL , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , Rats, Sprague-Dawley
20.
J Med Chem ; 64(16): 11857-11885, 2021 08 26.
Article in English | MEDLINE | ID: mdl-34374541

ABSTRACT

Cathepsin C (Cat C) participates in inflammation and immune regulation by affecting the activation of neutrophil serine proteases (NSPs). Therefore, cathepsin C is an attractive target for treatment of NSP-related inflammatory diseases. Here, the complete discovery process of the first potent "non-peptidyl non-covalent cathepsin C inhibitor" was described with hit finding, structure optimization, and lead discovery. Starting with hit 14, structure-based optimization and structure-activity relationship study were comprehensively carried out, and lead compound 54 was discovered as a potent drug-like cathepsin C inhibitor both in vivo and in vitro. Also, compound 54 (with cathepsin C Enz IC50 = 57.4 nM) exhibited effective anti-inflammatory activity in an animal model of chronic obstructive pulmonary disease. These results confirmed that the non-peptidyl and non-covalent derivative could be used as an effective cathepsin C inhibitor and encouraged us to continue further drug discovery on the basis of this finding.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Cathepsin C/antagonists & inhibitors , Inflammation/drug therapy , Protease Inhibitors/therapeutic use , Pulmonary Disease, Chronic Obstructive/drug therapy , Pyrimidines/therapeutic use , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/toxicity , Cathepsin C/metabolism , Cell Line, Tumor , Drug Discovery , Humans , Inflammation/etiology , Inflammation/pathology , Lung/drug effects , Lung/pathology , Male , Mice, Inbred ICR , Microsomes, Liver/metabolism , Molecular Docking Simulation , Molecular Structure , Protease Inhibitors/chemical synthesis , Protease Inhibitors/metabolism , Protease Inhibitors/toxicity , Protein Binding , Pulmonary Disease, Chronic Obstructive/complications , Pulmonary Disease, Chronic Obstructive/pathology , Pyrimidines/chemical synthesis , Pyrimidines/metabolism , Pyrimidines/toxicity , Rats, Sprague-Dawley , Structure-Activity Relationship
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