Pub Date : 2024-08-16DOI: 10.1101/2024.08.13.607728
Stefanie Kickinger, Anna Seiler, Daniela Digles, Gerhard F Ecker
Proteochemometric modeling (PCM) combines ligand information as well as target information in order to predict an output variable of interest (e.g. activity of a compound). The big advantage of PCM compared to conventional Quantitative Structure-Activity Relationship (QSAR) modeling is, that by creating a single model one can not only predict the affinity of a diverse set of compounds to a diverse set of targets, but also extrapolate the specific ligand-protein interactions that might be relevant for activity. In this study, we compiled a dataset of 323 compounds and their bioactivity data regarding the inhibition of the four GABA-transporter (GAT1/BGT1/GAT2/GAT3) subtypes, which are potential new drug targets for treating epilepsy. Proteochemometric modeling using partial least squares and random forest provided models which performed equally well than conventional QSAR models for each individual transporter. However, by analyzing the importance of the protein descriptors used in the PCM models, we identified the amino acid Leu300/Q299/L294/L314/ in GAT1/BGT1/GAT2/GAT3 to be relevant for binding and subtype selectivity.
{"title":"Proteochemometric modeling strengthens the role of Q299 for GABA transporter subtype selectivity","authors":"Stefanie Kickinger, Anna Seiler, Daniela Digles, Gerhard F Ecker","doi":"10.1101/2024.08.13.607728","DOIUrl":"https://doi.org/10.1101/2024.08.13.607728","url":null,"abstract":"Proteochemometric modeling (PCM) combines ligand information as well as target information in order to predict an output variable of interest (e.g. activity of a compound). The big advantage of PCM compared to conventional Quantitative Structure-Activity Relationship (QSAR) modeling is, that by creating a single model one can not only predict the affinity of a diverse set of compounds to a diverse set of targets, but also extrapolate the specific ligand-protein interactions that might be relevant for activity. In this study, we compiled a dataset of 323 compounds and their bioactivity data regarding the inhibition of the four GABA-transporter (GAT1/BGT1/GAT2/GAT3) subtypes, which are potential new drug targets for treating epilepsy. Proteochemometric modeling using partial least squares and random forest provided models which performed equally well than conventional QSAR models for each individual transporter. However, by analyzing the importance of the protein descriptors used in the PCM models, we identified the amino acid Leu300/Q299/L294/L314/ in GAT1/BGT1/GAT2/GAT3 to be relevant for binding and subtype selectivity.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"58 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142184301","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The Fusarium mycotoxin Deoxynivalenol (DON) represents a significant threat to both human and animal health. It can cross the blood-brain barrier and disrupt cerebral functions. One of the main mechanisms underlying DON toxicity involves the activation of mitogen-activated protein kinases (MAPKs). Gonadotropin-Releasing Hormone (GnRH) stimulation of pituitary gonadotroph cells to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH) triggers the MAPKs pathway too. Yet, no research investigated the endocrine-disrupting effects of DON on pituitary gonadotropins. To address this gap, our study investigated the effects of DON on gonadotroph cell viability, and its impact on the GnRH-stimulated secretion of FSH and LH, using the murine gonadotroph LβT2 cells. Our results uncovered that low-dose exposure to DON (1nM) significantly impairs viability in gonadotroph cells at both 24 and 48 h. Moreover, exposure to DON shows to induce membrane phosphatidylserine translocation without loss of membrane integrity, supporting a DON-induced cytotoxicity through apoptosis initiation. Furthermore, DON specifically inhibits GnRH-induced Erk phosphorylation, while leaving p38 unaffected. Subsequent experiments with DON-treated LβT2 cells stimulated with GnRH showed a dose-dependent reduction in gene expression associated Gonadotropin-Releasing Hormone receptor (GnRHr), Luteinizing Hormone subunit beta (LHβ), and Glycoprotein Hormones, alpha subunit (Cgα), along with a reduction in LH production. Our findings underscore the induction of DON cytotoxicity through active apoptosis and its impact on LH secretion by inhibiting Erk phosphorylation within the MAPKs pathway. This research contributes to a better understanding of the neurotoxic effects of DON and establishes a foundation for further studies exploring the neuroendocrine impact of mycotoxins.
{"title":"Low doses of the food contaminant Deoxynivalenol trigger apoptosis and alter GnRH stimulation of gonadotroph cells","authors":"Guodong Cai, Lingchen Yang, Francis Marien-Bourgeois, Derek Boerboom, Gustavo Zamberlam, Imourana Alassane-Kpembi","doi":"10.1101/2024.08.13.607800","DOIUrl":"https://doi.org/10.1101/2024.08.13.607800","url":null,"abstract":"The Fusarium mycotoxin Deoxynivalenol (DON) represents a significant threat to both human and animal health. It can cross the blood-brain barrier and disrupt cerebral functions. One of the main mechanisms underlying DON toxicity involves the activation of mitogen-activated protein kinases (MAPKs). Gonadotropin-Releasing Hormone (GnRH) stimulation of pituitary gonadotroph cells to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH) triggers the MAPKs pathway too. Yet, no research investigated the endocrine-disrupting effects of DON on pituitary gonadotropins. To address this gap, our study investigated the effects of DON on gonadotroph cell viability, and its impact on the GnRH-stimulated secretion of FSH and LH, using the murine gonadotroph LβT2 cells. Our results uncovered that low-dose exposure to DON (1nM) significantly impairs viability in gonadotroph cells at both 24 and 48 h. Moreover, exposure to DON shows to induce membrane phosphatidylserine translocation without loss of membrane integrity, supporting a DON-induced cytotoxicity through apoptosis initiation. Furthermore, DON specifically inhibits GnRH-induced Erk phosphorylation, while leaving p38 unaffected. Subsequent experiments with DON-treated LβT2 cells stimulated with GnRH showed a dose-dependent reduction in gene expression associated Gonadotropin-Releasing Hormone receptor (GnRHr), Luteinizing Hormone subunit beta (LHβ), and Glycoprotein Hormones, alpha subunit (Cgα), along with a reduction in LH production. Our findings underscore the induction of DON cytotoxicity through active apoptosis and its impact on LH secretion by inhibiting Erk phosphorylation within the MAPKs pathway. This research contributes to a better understanding of the neurotoxic effects of DON and establishes a foundation for further studies exploring the neuroendocrine impact of mycotoxins.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142184302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-16DOI: 10.1101/2024.08.13.607806
Jeffrey Amoako Mensah, Kyle E. Thomson, Jennifer L. Huff, Tia Freeman, Christopher A Reilly, Joseph E. Rower, Cameron S Metcalf, Karen S Wilcox
Background: Patients with drug-resistant epilepsy, including Dravet syndrome (DS), are frequently prescribed multiple antiseizure medications (ASMs). Nevertheless, people with DS often have inadequate seizure control, and there is an ongoing unmet clinical need to identify novel therapeutics. As a proof-of-principle study to further validate and characterize the Scn1aA1783V/WT mouse model and identify a drug screening paradigm with face, construct, and predictive value, we assessed the efficacy of subchronic administration of stiripentol add-on to clobazam and valproic acid at clinically relevant doses using the Scn1aA1783V/WT mouse model. Methods: We evaluated the efficacy of STP add-on to CLB and VPA using hyperthermia-induced and video-EEG monitoring of spontaneous seizure tests following a 14-day treatment. VPA was delivered via osmotic minipump, while STP and CLB were administered via food pellets delivered through automatic feeders. Bioanalytical assays were performed to evaluate drug concentrations in plasma and brain using liquid chromatography-tandem mass spectrometry. Results: STP, CLB, N-desmethylclobazam, and VPA all yielded plasma concentrations within the human therapeutic plasma concentrations range. STP added to CLB and VPA significantly elevated the seizing temperatures in the hyperthermia-induced seizure assay. CLB, VPA, and STP coadministration significantly reduced spontaneous seizure frequency compared to CLB and VPA combined. Significance: This research lays the groundwork for exploring effective add-on compounds to CLB and VPA in treating DS. The study further highlights the utility of the Scn1aA1783V/WT mice in discovering therapies for DS-associated pharmacoresistant seizures.
{"title":"Development of a preclinical screening platform for clinically relevant therapy of Dravet syndrome","authors":"Jeffrey Amoako Mensah, Kyle E. Thomson, Jennifer L. Huff, Tia Freeman, Christopher A Reilly, Joseph E. Rower, Cameron S Metcalf, Karen S Wilcox","doi":"10.1101/2024.08.13.607806","DOIUrl":"https://doi.org/10.1101/2024.08.13.607806","url":null,"abstract":"Background: Patients with drug-resistant epilepsy, including Dravet syndrome (DS), are frequently prescribed multiple antiseizure medications (ASMs). Nevertheless, people with DS often have inadequate seizure control, and there is an ongoing unmet clinical need to identify novel therapeutics. As a proof-of-principle study to further validate and characterize the Scn1aA1783V/WT mouse model and identify a drug screening paradigm with face, construct, and predictive value, we assessed the efficacy of subchronic administration of stiripentol add-on to clobazam and valproic acid at clinically relevant doses using the Scn1aA1783V/WT mouse model. Methods: We evaluated the efficacy of STP add-on to CLB and VPA using hyperthermia-induced and video-EEG monitoring of spontaneous seizure tests following a 14-day treatment. VPA was delivered via osmotic minipump, while STP and CLB were administered via food pellets delivered through automatic feeders. Bioanalytical assays were performed to evaluate drug concentrations in plasma and brain using liquid chromatography-tandem mass spectrometry. Results: STP, CLB, N-desmethylclobazam, and VPA all yielded plasma concentrations within the human therapeutic plasma concentrations range. STP added to CLB and VPA significantly elevated the seizing temperatures in the hyperthermia-induced seizure assay. CLB, VPA, and STP coadministration significantly reduced spontaneous seizure frequency compared to CLB and VPA combined. Significance: This research lays the groundwork for exploring effective add-on compounds to CLB and VPA in treating DS. The study further highlights the utility of the Scn1aA1783V/WT mice in discovering therapies for DS-associated pharmacoresistant seizures.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142184300","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-15DOI: 10.1101/2024.08.12.607677
Justin Shiau, Patti Engel, Mark Olsen, Gwendolyn Pais, Jack Chang, Marc H. Scheetz
Introduction: Vancomycin causes kidney injury by accumulating in the proximal tubule, likely mediated by megalin uptake. Protamine is a putative megalin inhibitor that shares binding sites with heparin and is approved for heparin overdose in patients. Methods: We employed a well characterized Sprague Dawley rat model to assess kidney injury and function in animals that received vancomycin, protamine alone, or vancomycin plus protamine over 5 days. Urinary KIM-1 was used as the primary measure for kidney injury while iohexol clearance was calculated to assess kidney function. Animals had samples drawn pre-treatment to serve as their own controls. Additionally, since protamine is not a known nephrotoxin, the protamine group also served as a control. Cellular inhibition studies were performed to assess the ability of protamine to inhibit OAT1, OAT3, and OCT2. Results: Rats that received vancomycin had significantly increased urinary KIM-1 on day 2 (24.9 ng/24h, 95% CI 1.87 to 48.0) compared to the protamine alone group. By day 4, animals that received protamine with vancomycin had urinary KIM-1 amounts that were elevated compared to protamine alone (KIM-1 29.0 ng/24h, 95% CI 5.0 to 53.0). No statistically significant differences were identified for iohexol clearance changes between drug groups or when comparing clearance change from baseline (P>0.05). No substantial inhibition of OAT1, OAT3, or OCT2 was observed with protamine. IC50 values for protamine were 1e-4 M for OAT1 and OAT3 and 4.3e-5 M for OCT2. Conclusion: Protamine, when added to vancomycin therapy, delays vancomycin induced kidney injury as defined by urinary KIM-1 in the rat model by one to three days. Protamine putatively acts through blockade of megalin and does not appear to have significant inhibition on OAT1, OAT3, or OCT2. Since protamine is an approved FDA medication, it has clinical potential as a therapeutic to reduce vancomycin related kidney injury; however, greater utility may be found by pursuing compounds with fewer adverse event liabilities.
{"title":"Protamine Protects Against Vancomycin Induced Kidney Injury","authors":"Justin Shiau, Patti Engel, Mark Olsen, Gwendolyn Pais, Jack Chang, Marc H. Scheetz","doi":"10.1101/2024.08.12.607677","DOIUrl":"https://doi.org/10.1101/2024.08.12.607677","url":null,"abstract":"Introduction: Vancomycin causes kidney injury by accumulating in the proximal tubule, likely mediated by megalin uptake. Protamine is a putative megalin inhibitor that shares binding sites with heparin and is approved for heparin overdose in patients. Methods: We employed a well characterized Sprague Dawley rat model to assess kidney injury and function in animals that received vancomycin, protamine alone, or vancomycin plus protamine over 5 days. Urinary KIM-1 was used as the primary measure for kidney injury while iohexol clearance was calculated to assess kidney function. Animals had samples drawn pre-treatment to serve as their own controls. Additionally, since protamine is not a known nephrotoxin, the protamine group also served as a control. Cellular inhibition studies were performed to assess the ability of protamine to inhibit OAT1, OAT3, and OCT2. Results: Rats that received vancomycin had significantly increased urinary KIM-1 on day 2 (24.9 ng/24h, 95% CI 1.87 to 48.0) compared to the protamine alone group. By day 4, animals that received protamine with vancomycin had urinary KIM-1 amounts that were elevated compared to protamine alone (KIM-1 29.0 ng/24h, 95% CI 5.0 to 53.0). No statistically significant differences were identified for iohexol clearance changes between drug groups or when comparing clearance change from baseline (P>0.05). No substantial inhibition of OAT1, OAT3, or OCT2 was observed with protamine. IC50 values for protamine were 1e-4 M for OAT1 and OAT3 and 4.3e-5 M for OCT2. Conclusion: Protamine, when added to vancomycin therapy, delays vancomycin induced kidney injury as defined by urinary KIM-1 in the rat model by one to three days. Protamine putatively acts through blockade of megalin and does not appear to have significant inhibition on OAT1, OAT3, or OCT2. Since protamine is an approved FDA medication, it has clinical potential as a therapeutic to reduce vancomycin related kidney injury; however, greater utility may be found by pursuing compounds with fewer adverse event liabilities.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"34 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142184334","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-15DOI: 10.1101/2024.08.12.607504
Adam Myszczyszyn, Anna Muench, Vivian Lehmann, Theo Sinnige, Frank G. van Steenbeek, Manon Bouwmeester, Roos-Anne Samsom, Marit Keuper-Navis, Thomas K. van der Made, Daniel Kogan, Sarah Braem, Luc J. W. van der Laan, Hossein Eslami Amirabadi, Evita van de Steeg, Rosalinde Masereeuw, Bart Spee
Liver-on-a-chip models predictive for both metabolism as well as canalicular and blood transport of drug candidates in humans are lacking. Here, we established an advanced, bioengineered and animal component-free hepatocyte-like millifluidic system based on 3D hollow fiber membranes (HFMs), recombinant human laminin 332 coating and adult human stem cell-derived organoids. Organoid fragments formed polarized and tight monolayers on HFMs with improved hepatocyte-like maturation, as compared to standard 3D organoid cultures in Matrigel from matched donors. Gene expression profiling and immunofluorescence revealed that hepatocyte-like monolayers expressed a broad panel of phase I (e.g., CYP3A4, CYP2D6) and II (UGTs, SULTs) drug-metabolizing enzymes and drug transporters (e.g., OATP1B3, MDR1 and MRP3). Moreover, statically cultured monolayers displayed phase I and II metabolism of a cocktail of six relevant compounds, including midazolam and 7-hydroxycoumarin. We also demonstrated the disposition of midazolam in the basal/blood-like circulation and apical/canalicular-like compartment of the millifluidic chip. Finally, we connected the system to the other two PK/ADME-most relevant organ systems, i.e. small intestine- and kidney proximal tubule-like to study the bioavailability of midazolam and coumarin, and excretion of metformin. In conclusion, we generated a proof-of-concept liver organoid-on-a-chip model for examining metabolism and transport of drugs, which can be further developed to predict PK/ADME profiles in humans.
目前还缺乏可预测候选药物在人体内代谢以及管腔和血液转运的肝脏芯片模型。在这里，我们基于三维中空纤维膜（HFMs）、重组人层粘蛋白 332 涂层和成人干细胞衍生的类器官，建立了一种先进的、生物工程的、不含动物成分的类肝细胞毫流体系统。与在匹配供体的 Matrigel 中进行的标准三维类器官培养相比，类器官片段在 HFM 上形成了极化和紧密的单层，并改善了肝细胞样成熟度。基因表达谱分析和免疫荧光显示，肝细胞样单层表达了一系列I期（如CYP3A4、CYP2D6）和II期（UGTs、SULTs）药物代谢酶和药物转运体（如OATP1B3、MDR1和MRP3）。此外，静态培养的单层细胞显示了包括咪达唑仑和 7-羟基香豆素在内的六种相关化合物的 I 期和 II 期代谢。我们还证明了咪达唑仑在毫流体芯片的基底/类血液循环和顶端/类毛细血管腔中的分布。最后，我们将该系统与另外两个与 PK/ADME 最相关的器官系统（即小肠和肾近曲小管）连接起来，研究了咪达唑仑和香豆素的生物利用度以及二甲双胍的排泄情况。总之，我们建立了一个概念验证型肝脏类器官芯片模型，用于研究药物的代谢和转运，该模型可进一步开发用于预测人体的PK/ADME情况。
{"title":"A hollow fiber membrane-based liver organoid-on-a-chip model for examining drug metabolism and transport","authors":"Adam Myszczyszyn, Anna Muench, Vivian Lehmann, Theo Sinnige, Frank G. van Steenbeek, Manon Bouwmeester, Roos-Anne Samsom, Marit Keuper-Navis, Thomas K. van der Made, Daniel Kogan, Sarah Braem, Luc J. W. van der Laan, Hossein Eslami Amirabadi, Evita van de Steeg, Rosalinde Masereeuw, Bart Spee","doi":"10.1101/2024.08.12.607504","DOIUrl":"https://doi.org/10.1101/2024.08.12.607504","url":null,"abstract":"Liver-on-a-chip models predictive for both metabolism as well as canalicular and blood transport of drug candidates in humans are lacking. Here, we established an advanced, bioengineered and animal component-free hepatocyte-like millifluidic system based on 3D hollow fiber membranes (HFMs), recombinant human laminin 332 coating and adult human stem cell-derived organoids. Organoid fragments formed polarized and tight monolayers on HFMs with improved hepatocyte-like maturation, as compared to standard 3D organoid cultures in Matrigel from matched donors. Gene expression profiling and immunofluorescence revealed that hepatocyte-like monolayers expressed a broad panel of phase I (e.g., CYP3A4, CYP2D6) and II (UGTs, SULTs) drug-metabolizing enzymes and drug transporters (e.g., OATP1B3, MDR1 and MRP3). Moreover, statically cultured monolayers displayed phase I and II metabolism of a cocktail of six relevant compounds, including midazolam and 7-hydroxycoumarin. We also demonstrated the disposition of midazolam in the basal/blood-like circulation and apical/canalicular-like compartment of the millifluidic chip. Finally, we connected the system to the other two PK/ADME-most relevant organ systems, i.e. small intestine- and kidney proximal tubule-like to study the bioavailability of midazolam and coumarin, and excretion of metformin. In conclusion, we generated a proof-of-concept liver organoid-on-a-chip model for examining metabolism and transport of drugs, which can be further developed to predict PK/ADME profiles in humans.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142184303","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Many plant extracts such as celastrol (CLT) have excellent pharmacological effects. However, exploring their drug targets is challenging. Here we show that CLT imitates ATP and binds to its pocket on pyruvate kinase PKM (PKM) while inhibiting the enzyme activity. Quantum calculation predicts hydrogen bond formation between CLT and asparagine. Liquid chromatography-mass spectrometry further identifies PKM as CLT's target. The inhibition of PKM is stronger and longer for CLT while weak and short for ATP. Notably, CLT-PKM interaction perfectly underlies the hypoglycemic effects by forming glucose carbon flux U turn before PKM. Besides, the PKM inhibition induces tricarboxylic acid (TCA) vortex which could promote amino acid and lipid degradation as the energy compensation, leading to a significant weight-loss. Additionally, CLT exerts efficient antioxidant effects by altering the glucose flux to strengthen the pentose phosphate pathway. Consequently, the CLT-PKM interaction vividly reproduces ATP-PKM interaction named "ATP resistance" in the diabetes pathogenesis.
{"title":"Celastrol binds to pyruvate kinase and imitates the metabolic reprogramming of ATP","authors":"Xuan Zhang, Xudong Jiang, Xiaobin Wu, Youhao Yang, Jintian Wang, Junfeng Zheng, Miao Zhou, Chen Qian","doi":"10.1101/2024.08.09.607407","DOIUrl":"https://doi.org/10.1101/2024.08.09.607407","url":null,"abstract":"Many plant extracts such as celastrol (CLT) have excellent pharmacological effects. However, exploring their drug targets is challenging. Here we show that CLT imitates ATP and binds to its pocket on pyruvate kinase PKM (PKM) while inhibiting the enzyme activity. Quantum calculation predicts hydrogen bond formation between CLT and asparagine. Liquid chromatography-mass spectrometry further identifies PKM as CLT's target. The inhibition of PKM is stronger and longer for CLT while weak and short for ATP. Notably, CLT-PKM interaction perfectly underlies the hypoglycemic effects by forming glucose carbon flux U turn before PKM. Besides, the PKM inhibition induces tricarboxylic acid (TCA) vortex which could promote amino acid and lipid degradation as the energy compensation, leading to a significant weight-loss. Additionally, CLT exerts efficient antioxidant effects by altering the glucose flux to strengthen the pentose phosphate pathway. Consequently, the CLT-PKM interaction vividly reproduces ATP-PKM interaction named \"ATP resistance\" in the diabetes pathogenesis.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"159 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141933275","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.01.606254
jian zuo, Zhe Yang
Objective: Qing-Luo-Yin (QLY) is an anti-rheumatic herbal formula with potentials activating PPARγ. This study investigated if its anti-angiogenesis effects are related to immune modulation. Method: Adjuvant-induced arthritis (AIA) rats were orally treated by QLY or rosiglitazone (a PPARγ agonist) for 30 days. Their immune and metabolism statues were investigated afterward. Isolated monocytes and lymphocytes were co-cultured reciprocally, and treated by different serums. Healthy rats received blood transfusion from QLY-treated or AIA model rats. Two days ahead of sacrifice, a matrigel plug was planted. The plug and some blood immune indicators were examined. AIA rat serum-incubated THP-1 and Jurkat cells were treated by sinomenine, berberine and palmatine. The medium and T0070907 (a PPARγ inhibitor) were used to stimulate HUVEC cells. Results: QLY showed similar therapeutic effects on AIA to rosiglitazone, alleviating joint injuries, synovial angiogenesis, and metabolic disorders. Although QLY impaired inflammatory phenotype of AIA monocytes in vivo, the effect was hardly achieved or sustained in vitro. T cells from QLY-treated AIA rats showed the weakened inflammatory phenotype, and were unable to induce monocytes inflammatory polarization. AIA rat lymphocytes induced angiogenesis in the matrigel plug in healthy recipients. In lymphocytes enrichment site, QLY reduced the secretion of IL-17A, IFNγ, and many angiogenesis-related cytokines. QLY-related components affected Jurkat but not THP-1 cells. Jurkat T cells induced angiogenesis of HUVEC cells when cultured by AIA rat serum. Inhibitory effects of the compounds on it were abolished by T0070907. Conclusion: PPARγ activation in T cells is a foundation for the anti-angiogenesis property of QLY.
{"title":"Qing-Luo-Yin eases angiogenesis in adjuvant-induced arthritis rats by activating PPARγ","authors":"jian zuo, Zhe Yang","doi":"10.1101/2024.08.01.606254","DOIUrl":"https://doi.org/10.1101/2024.08.01.606254","url":null,"abstract":"Objective: Qing-Luo-Yin (QLY) is an anti-rheumatic herbal formula with potentials activating PPARγ. This study investigated if its anti-angiogenesis effects are related to immune modulation. Method: Adjuvant-induced arthritis (AIA) rats were orally treated by QLY or rosiglitazone (a PPARγ agonist) for 30 days. Their immune and metabolism statues were investigated afterward. Isolated monocytes and lymphocytes were co-cultured reciprocally, and treated by different serums. Healthy rats received blood transfusion from QLY-treated or AIA model rats. Two days ahead of sacrifice, a matrigel plug was planted. The plug and some blood immune indicators were examined. AIA rat serum-incubated THP-1 and Jurkat cells were treated by sinomenine, berberine and palmatine. The medium and T0070907 (a PPARγ inhibitor) were used to stimulate HUVEC cells. Results: QLY showed similar therapeutic effects on AIA to rosiglitazone, alleviating joint injuries, synovial angiogenesis, and metabolic disorders. Although QLY impaired inflammatory phenotype of AIA monocytes in vivo, the effect was hardly achieved or sustained in vitro. T cells from QLY-treated AIA rats showed the weakened inflammatory phenotype, and were unable to induce monocytes inflammatory polarization. AIA rat lymphocytes induced angiogenesis in the matrigel plug in healthy recipients. In lymphocytes enrichment site, QLY reduced the secretion of IL-17A, IFNγ, and many angiogenesis-related cytokines. QLY-related components affected Jurkat but not THP-1 cells. Jurkat T cells induced angiogenesis of HUVEC cells when cultured by AIA rat serum. Inhibitory effects of the compounds on it were abolished by T0070907. Conclusion: PPARγ activation in T cells is a foundation for the anti-angiogenesis property of QLY.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"85 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141933278","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.07.606978
Alice Valentini, Bethany Dibnah, Marija Ciba, Trond Ulven, Brian D Hudson, Elisabeth Rexen Ulven
G protein coupled receptors (GPCRs) are the largest family of signalling proteins and highly successful drug targets. To date, most GPCR drugs interact with the binding pocket for the natural ligand, typically near the extracellular part of the transmembrane region. Recent advancements in structural biology have identified additional allosteric binding sites in other parts of these receptors. Allosteric sites provide several theoretical advantages, including the ability to modulate natural ligand function, and there is a need for better ways to study how ligands bind and interact with diVerent GPCR binding sites. We have developed an approach to study multiple ligands binding to the same receptor based on sequential resonance energy transfer between two fluorescent ligands bound simultaneously to a GPCR. We use this approach to gain insight into allosteric ligand interactions for a clinically relevant receptor. This method will provide important information to aid development of new GPCR drugs.
G 蛋白偶联受体（GPCR）是最大的信号蛋白家族，也是非常成功的药物靶点。迄今为止，大多数 GPCR 药物都与天然配体的结合袋相互作用，通常靠近跨膜区的细胞外部分。结构生物学的最新进展已经在这些受体的其他部位发现了更多的异生结合位点。异构位点具有多种理论优势，包括能够调节天然配体的功能，因此需要更好的方法来研究配体如何与不同的 GPCR 结合位点结合和相互作用。我们开发了一种研究多种配体与同一受体结合的方法，这种方法基于同时与 GPCR 结合的两种荧光配体之间的顺序共振能量转移。我们利用这种方法来深入了解一种临床相关受体的异构配体相互作用。这种方法将为帮助开发新的 GPCR 药物提供重要信息。
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Identifying novel, high-yield drug targets is challenging and often results in a high failure rate. However, recent data indicates that leveraging human genetic evidence to identify and validate these targets significantly increases the likelihood of success in drug development. Two recent papers from Open Targets claimed that around half of FDA-approved drugs had targets with direct human genetic evidence. By expanding target identification to include protein network partners—molecules in physical contact—the proportion of drug targets with genetic evidence support increased to two-thirds. However, the efficacy of using these network partners for target identification was not formally tested. To address this, we tested the approach on a list of robust positive control genes. We used the IntAct database to find molecular interacting proteins of genes identified by exome-wide association studies (ExWAS) and genome-wide association studies (GWAS) combined with a locus-to-gene mapping algorithm called the Effector Index (Ei). We assessed how accurately including interacting genes with the ExWAS and Effector Index selected genes identified positive controls, focusing on precision, sensitivity, and specificity. Our results indicated that although molecular interactions led to higher sensitivity in identifying positive control genes, their practical application is limited by low precision. Hence, expanding genetically identified targets to include network partners did not increase the chance of identifying drug targets, suggesting that such results should be interpreted with caution.
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Pub Date : 2024-08-09DOI: 10.1101/2024.08.08.607074
Wilson Peng, Kaitlin B Chung, B. Paige Lawrence, M. Kerry O'Banion, Robert T Dirksen, Andrew P Wojtovich, John O Onukwufor
Iron is critical for neuronal activity and metabolism, and iron dysregulation alters these functions in age-related neurodegenerative disorders, such as Alzheimers disease (AD). AD is a chronic neurodegenerative disease characterized by neuronal dysfunction, memory loss and decreased cognitive function. AD patients exhibit elevated iron levels in the brain compared to age-matched non-AD individuals. However, the degree to which iron overload contributes to AD pathogenesis is unclear. Here, we evaluated the involvement of ferroptosis, an iron-dependent cell death process, in mediating AD-like pathology in C. elegans. Results showed that iron accumulation occurred prior to the loss of neuronal function as worms age. In addition, energetic imbalance was an early event in iron-induced loss of neuronal function. Furthermore, the loss of neuronal function was, in part, due to increased mitochondrial reactive oxygen species mediated oxidative damage, ultimately resulting in ferroptotic cell death. Mitochondrial redox environment and ferroptosis were modulated by pharmacologic processes that exacerbate or abolish iron accumulation both in wild-type worms and worms with increased levels of neuronal amyloid beta. However, neuronal amyloid beta worms were more sensitive to ferroptosis-mediated neuronal loss, and this increased toxicity was ameliorated by limiting the uptake of ferrous iron through knockout of divalent metal transporter 1 (DMT1). In addition, the loss of DMT1 completely suppressed phenotypic measures of amyloid beta toxicity with age. Overall, our findings suggest that iron-induced ferroptosis alters the mitochondrial redox environment to drive oxidative damage when neuronal amyloid beta is overexpressed. DMT1 knockout abolishes neuronal amyloid beta pathology by reducing neuronal iron uptake
铁对神经元的活动和新陈代谢至关重要，铁失调会改变老年性神经退行性疾病（如阿尔茨海默病（AD））的这些功能。阿尔茨海默病是一种慢性神经退行性疾病，以神经元功能障碍、记忆力减退和认知功能下降为特征。与年龄匹配的非阿兹海默症患者相比，阿兹海默症患者大脑中的铁含量升高。然而，铁超载在多大程度上导致了AD发病机制尚不清楚。在这里，我们评估了铁跃迁（一种依赖铁的细胞死亡过程）在介导优雅小鼠 AD 类病理学中的参与情况。结果表明，随着蠕虫年龄的增长，铁积累发生在神经元功能丧失之前。此外，能量失衡是铁诱导神经元功能丧失的早期事件。此外，神经元功能丧失的部分原因是线粒体活性氧介导的氧化损伤增加，最终导致铁跃迁细胞死亡。在野生型蠕虫和神经元淀粉样蛋白 beta 水平升高的蠕虫中，线粒体氧化还原环境和铁凋亡可通过药物过程进行调节，从而加剧或消除铁积累。然而，神经元淀粉样 beta 蠕虫对铁突变介导的神经元损失更为敏感，而通过敲除二价金属转运体 1（DMT1）限制亚铁的吸收可改善毒性的增加。此外，随着年龄的增长，DMT1的缺失完全抑制了淀粉样β毒性的表型测量。总之，我们的研究结果表明，当神经元淀粉样蛋白β过度表达时，铁诱导的铁变态反应会改变线粒体氧化还原环境，从而驱动氧化损伤。DMT1基因敲除可通过减少神经元铁摄取而消除神经元淀粉样蛋白β病理变化
{"title":"DMT1 knockout abolishes ferroptosis induced mitochondrial dysfunction in C. elegans amyloid beta proteotoxicity","authors":"Wilson Peng, Kaitlin B Chung, B. Paige Lawrence, M. Kerry O'Banion, Robert T Dirksen, Andrew P Wojtovich, John O Onukwufor","doi":"10.1101/2024.08.08.607074","DOIUrl":"https://doi.org/10.1101/2024.08.08.607074","url":null,"abstract":"Iron is critical for neuronal activity and metabolism, and iron dysregulation alters these functions in age-related neurodegenerative disorders, such as Alzheimers disease (AD). AD is a chronic neurodegenerative disease characterized by neuronal dysfunction, memory loss and decreased cognitive function. AD patients exhibit elevated iron levels in the brain compared to age-matched non-AD individuals. However, the degree to which iron overload contributes to AD pathogenesis is unclear. Here, we evaluated the involvement of ferroptosis, an iron-dependent cell death process, in mediating AD-like pathology in C. elegans. Results showed that iron accumulation occurred prior to the loss of neuronal function as worms age. In addition, energetic imbalance was an early event in iron-induced loss of neuronal function. Furthermore, the loss of neuronal function was, in part, due to increased mitochondrial reactive oxygen species mediated oxidative damage, ultimately resulting in ferroptotic cell death. Mitochondrial redox environment and ferroptosis were modulated by pharmacologic processes that exacerbate or abolish iron accumulation both in wild-type worms and worms with increased levels of neuronal amyloid beta. However, neuronal amyloid beta worms were more sensitive to ferroptosis-mediated neuronal loss, and this increased toxicity was ameliorated by limiting the uptake of ferrous iron through knockout of divalent metal transporter 1 (DMT1). In addition, the loss of DMT1 completely suppressed phenotypic measures of amyloid beta toxicity with age. Overall, our findings suggest that iron-induced ferroptosis alters the mitochondrial redox environment to drive oxidative damage when neuronal amyloid beta is overexpressed. DMT1 knockout abolishes neuronal amyloid beta pathology by reducing neuronal iron uptake","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"22 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141933277","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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