Pub Date : 2024-09-13DOI: 10.1101/2024.09.09.612002
Anastasia Kremer, Tatyana Ryaykenen, Xavier Segarra-Visent, Melanie Sauer, Qi Tang, David A Cooper, Dimas Echeverria, Clothilde Philouze, Emilie Bayon, Dan Georgess, Reka A Haraszti
RNA interference (RNAi)-based medicines offer precise targeting of virtually any transcript, making them an appealing new drug class for addressing unmet needs in immune-oncological applications. While RNAi therapies show exceptional duration of effect in non-dividing cells, their efficacy in rapidly dividing cells, crucial for immune-oncology, remains largely unexplored. Unlike in non-dividing cells, full chemical modification in rapidly dividing cells has not consistently extended silencing duration, according to limited data available. In this study, we investigated key factors affecting the duration of effect for three main types of RNAi-based therapeutics (siRNA, miRNA mimics, and miRNA inhibitors) in rapidly dividing cancer and immune cells. Saturation of intracellular depots by multiple loading doses, a common strategy to prolong silencing duration in non-dividing hepatocytes, had minimal impact on siRNA duration of effect in rapidly dividing cells. However, modifying the antisense strand with a 5'-(E)-vinylphosphonate (5'-VP) to protect siRNAs from exonucleases and enhance AGO2 binding significantly extended siRNA silencing duration to over 30 days both in vitro and in vivo. For miRNA mimics, extensive stabilization of the antisense strand with phosphorothioates was not effective and led to reduced potency and silencing duration. Interestingly, a shorter duplex region commonly seen in therapeutic siRNAs partially rescued duration of silencing in miRNA mimics with extended phosphorothioate modifications. On the other hand, miRNA inhibitors demonstrated robust reversal of miRNA activity for an impressive 25 days in cancer cell lines. Our findings enable the rational design of the chemical architecture and administration regimens of RNAi-based therapies in oncology and immunology.
{"title":"Structure - silencing duration relationships in RNAi medicines in rapidly dividing cells","authors":"Anastasia Kremer, Tatyana Ryaykenen, Xavier Segarra-Visent, Melanie Sauer, Qi Tang, David A Cooper, Dimas Echeverria, Clothilde Philouze, Emilie Bayon, Dan Georgess, Reka A Haraszti","doi":"10.1101/2024.09.09.612002","DOIUrl":"https://doi.org/10.1101/2024.09.09.612002","url":null,"abstract":"RNA interference (RNAi)-based medicines offer precise targeting of virtually any transcript, making them an appealing new drug class for addressing unmet needs in immune-oncological applications. While RNAi therapies show exceptional duration of effect in non-dividing cells, their efficacy in rapidly dividing cells, crucial for immune-oncology, remains largely unexplored. Unlike in non-dividing cells, full chemical modification in rapidly dividing cells has not consistently extended silencing duration, according to limited data available. In this study, we investigated key factors affecting the duration of effect for three main types of RNAi-based therapeutics (siRNA, miRNA mimics, and miRNA inhibitors) in rapidly dividing cancer and immune cells. Saturation of intracellular depots by multiple loading doses, a common strategy to prolong silencing duration in non-dividing hepatocytes, had minimal impact on siRNA duration of effect in rapidly dividing cells. However, modifying the antisense strand with a 5'-(E)-vinylphosphonate (5'-VP) to protect siRNAs from exonucleases and enhance AGO2 binding significantly extended siRNA silencing duration to over 30 days both in vitro and in vivo. For miRNA mimics, extensive stabilization of the antisense strand with phosphorothioates was not effective and led to reduced potency and silencing duration. Interestingly, a shorter duplex region commonly seen in therapeutic siRNAs partially rescued duration of silencing in miRNA mimics with extended phosphorothioate modifications. On the other hand, miRNA inhibitors demonstrated robust reversal of miRNA activity for an impressive 25 days in cancer cell lines. Our findings enable the rational design of the chemical architecture and administration regimens of RNAi-based therapies in oncology and immunology.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"41 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259622","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 : 2024-09-13DOI: 10.1101/2024.09.07.611751
Yuanbao Ai, Siyu Xu, Yan Zhang, Zhaoxiang Liu, Sen Liu
Targeted covalent inhibitors (TCIs) form covalent bonds with targets following initial non-covalent binding. The advantages of TCIs have driven a resurgence in rational TCI design over the past decade, resulting in the approval of several blockbuster covalent drugs. To support TCI discovery, various computational methods have been developed. However, accurately predicting TCI reactivity remains challenging due to interference between non-covalent scaffolds and reactive warheads, leading to inefficiencies in computational screening and high experimental costs. In this study, we enhanced the SCARdock protocol, a validated computational screening tool developed by our lab, by incorporating quantum chemistry-based warhead reactivity calculations. By integrating these calculations with non-covalent docking scores, docking ranks, and bonding-atom distances, non-covalent and covalent inhibitors of S-adenosylmethionine decarboxylase (AdoMetDC) were correctly classified. Using the optimized SCARdock, we successfully identified twelve new AdoMetDC covalent inhibitors from 17 compounds, achieving a 70.6% hit rate. From these novel inhibitors, we analyzed the contributions of non-covalent interactions and covalent bonding, enabling a structure-activity relationship (SAR) analysis for AdoMetDC covalent inhibitors, which was previously unexplored with substrate-based inhibitors. Overall, this work presents an efficient computational protocol for TCI discovery and offers new insights into AdoMetDC inhibitor design. We anticipate that this approach will stimulate TCI development by improving computational screening efficiency and reducing experimental costs.
{"title":"High-efficiency discovery and structure-activity-relationship analysis of non-substrate-based covalent inhibitors of S-adenosylmethionine decarboxylase","authors":"Yuanbao Ai, Siyu Xu, Yan Zhang, Zhaoxiang Liu, Sen Liu","doi":"10.1101/2024.09.07.611751","DOIUrl":"https://doi.org/10.1101/2024.09.07.611751","url":null,"abstract":"Targeted covalent inhibitors (TCIs) form covalent bonds with targets following initial non-covalent binding. The advantages of TCIs have driven a resurgence in rational TCI design over the past decade, resulting in the approval of several blockbuster covalent drugs. To support TCI discovery, various computational methods have been developed. However, accurately predicting TCI reactivity remains challenging due to interference between non-covalent scaffolds and reactive warheads, leading to inefficiencies in computational screening and high experimental costs. In this study, we enhanced the SCARdock protocol, a validated computational screening tool developed by our lab, by incorporating quantum chemistry-based warhead reactivity calculations. By integrating these calculations with non-covalent docking scores, docking ranks, and bonding-atom distances, non-covalent and covalent inhibitors of S-adenosylmethionine decarboxylase (AdoMetDC) were correctly classified. Using the optimized SCARdock, we successfully identified twelve new AdoMetDC covalent inhibitors from 17 compounds, achieving a 70.6% hit rate. From these novel inhibitors, we analyzed the contributions of non-covalent interactions and covalent bonding, enabling a structure-activity relationship (SAR) analysis for AdoMetDC covalent inhibitors, which was previously unexplored with substrate-based inhibitors. Overall, this work presents an efficient computational protocol for TCI discovery and offers new insights into AdoMetDC inhibitor design. We anticipate that this approach will stimulate TCI development by improving computational screening efficiency and reducing experimental costs.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"14 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259623","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 : 2024-09-12DOI: 10.1101/2024.09.06.611748
Rafael Sobrano Fais, Simon Gabriel Comerma Steffensen, Estéfano Pinilla, Vladimir V. Matchkov, Rita C Tostes, Fernando S. Carneiro, Ulf Simonsen
Membrane depolarization is implicated in the activation of the NLRP3 inflammasome. Downregulation of endothelial Ca2+-activated K+ channels type 2.3 (KCa 2.3) and endothelin-1 (ET-1) upregulation in the corpus cavernosum (CC) are associated with erectile dysfunction. We hypothesized that opening KCa 2.2/2.3 channels could reverse erectile dysfunction and NLRP3 activation in hypertensive DOCA/salt mice. Methods: Hypertension was induced in mice using a DOCA/salt model, with unilaterally nephrectomized mice as controls; blood pressure was measured by tail-cuff. Intracavernous pressure (ICP) and CC reactivity were assessed. Western blot analysis for KCa 2.3, caspase-1, and interleukin-1β (IL-1β) was performed. Endothelial cells from erectile tissue were isolated and stimulated with ET-1, and KCa 2.2/2.3-dependent currents were evaluated via voltage-clamp electrophysiology. Results: DOCA/salt mice exhibited impaired erectile function, increased pro-caspase-1 and caspase-1 expression, and reduced relaxations induced by acetylcholine (ACh), sodium nitroprusside (SNP), and electrical field stimulation (EFS). Treatment with either the endothelin receptor antagonist bosentan or the KCa 2.2/2.3 channel opener NS13001 reversed these dysfunctions and reduced ET-1-induced NLRP3 activation in DOCA/salt mice. NS13001 also restored the decreased current observed in primary endothelial cells exposed to ET-1. Apamin, a KCa 2.2/2.3 channel blocker, inhibited erectile responses in unilaterally nephrectomized mice and restored erectile responses in DOCA mice. Apamin did not affect EFS-, ACh-, or SNP-induced relaxation in CC from hypertensive DOCA/salt mice. Conclusion: NS13001 reversed ET-1-induced NLRP3 activation and erectile dysfunction in DOCA/salt mice, suggesting that KCa 2.2/2.3 channel modulation may restore erectile function in hypertension-related diseases.
{"title":"A KCa 2.2/2.3 opener reverses ET-1 induced NLRP3 activation in hypertensive mice","authors":"Rafael Sobrano Fais, Simon Gabriel Comerma Steffensen, Estéfano Pinilla, Vladimir V. Matchkov, Rita C Tostes, Fernando S. Carneiro, Ulf Simonsen","doi":"10.1101/2024.09.06.611748","DOIUrl":"https://doi.org/10.1101/2024.09.06.611748","url":null,"abstract":"Membrane depolarization is implicated in the activation of the NLRP3 inflammasome. Downregulation of endothelial Ca<sup>2+</sup>-activated K<sup>+</sup> channels type 2.3 (K<sub>Ca</sub> 2.3) and endothelin-1 (ET-1) upregulation in the corpus cavernosum (CC) are associated with erectile dysfunction. We hypothesized that opening K<sub>Ca</sub> 2.2/2.3 channels could reverse erectile dysfunction and NLRP3 activation in hypertensive DOCA/salt mice. Methods: Hypertension was induced in mice using a DOCA/salt model, with unilaterally nephrectomized mice as controls; blood pressure was measured by tail-cuff. Intracavernous pressure (ICP) and CC reactivity were assessed. Western blot analysis for K<sub>Ca</sub> 2.3, caspase-1, and interleukin-1β (IL-1β) was performed. Endothelial cells from erectile tissue were isolated and stimulated with ET-1, and K<sub>Ca</sub> 2.2/2.3-dependent currents were evaluated via voltage-clamp electrophysiology. Results: DOCA/salt mice exhibited impaired erectile function, increased pro-caspase-1 and caspase-1 expression, and reduced relaxations induced by acetylcholine (ACh), sodium nitroprusside (SNP), and electrical field stimulation (EFS). Treatment with either the endothelin receptor antagonist bosentan or the K<sub>Ca</sub> 2.2/2.3 channel opener NS13001 reversed these dysfunctions and reduced ET-1-induced NLRP3 activation in DOCA/salt mice. NS13001 also restored the decreased current observed in primary endothelial cells exposed to ET-1. Apamin, a K<sub>Ca</sub> 2.2/2.3 channel blocker, inhibited erectile responses in unilaterally nephrectomized mice and restored erectile responses in DOCA mice. Apamin did not affect EFS-, ACh-, or SNP-induced relaxation in CC from hypertensive DOCA/salt mice. Conclusion: NS13001 reversed ET-1-induced NLRP3 activation and erectile dysfunction in DOCA/salt mice, suggesting that K<sub>Ca</sub> 2.2/2.3 channel modulation may restore erectile function in hypertension-related diseases.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"62 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142223909","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 : 2024-09-11DOI: 10.1101/2024.09.06.611612
Maryam Latarani, Perla Pucci, Mark Eccleston, Massimiliano Manzo, Priyadarsini Gangadharannambiar, Ilaria Alborelli, Vera Mongiardini, Namra Mahmood, Mario Paolo Colombo, Benedetto Grimaldi, Sushila Rigas, Shusuke Akamatsu, Cheryl Hawkes, Yuzhuo Wang, Elena Jachetti, Francesco Crea
Background. EZH2 promotes aggressive-variant prostate cancer (AVPC) progression via histone H3-Lysine-27 tri-methylation (H3K27me3). We hypothesize that epigenetic reprogramming via EZH2 inhibitors (EZH2i) improves the efficacy of chemotherapy in AVPC. Methods. We studied the expression of EZH2 in clinical prostate cancer cohorts (bioinformatics). We determined the effect of EZH2i on both cellular and cell free-H3K27me3 levels. We measured effects of carboplatin with/without EZH2i on AVPC cell viability (IC50). We studied how EZH2i modulate gene expression (RNA Seq). Results. EZH2 was significantly up-regulated in AVPC vs other prostate cancer types. EZH2i reduced both cellular and cell free-H3K27me3 levels. EZH2i significantly reduced carboplatin IC50. EZH2i reduced the expression of DNA repair and increased the expression of pro-apoptotic genes.
{"title":"EHZ2 inhibition enhances the activity of platinum chemotherapy in aggressive variant prostate cancer","authors":"Maryam Latarani, Perla Pucci, Mark Eccleston, Massimiliano Manzo, Priyadarsini Gangadharannambiar, Ilaria Alborelli, Vera Mongiardini, Namra Mahmood, Mario Paolo Colombo, Benedetto Grimaldi, Sushila Rigas, Shusuke Akamatsu, Cheryl Hawkes, Yuzhuo Wang, Elena Jachetti, Francesco Crea","doi":"10.1101/2024.09.06.611612","DOIUrl":"https://doi.org/10.1101/2024.09.06.611612","url":null,"abstract":"Background. EZH2 promotes aggressive-variant prostate cancer (AVPC) progression via histone H3-Lysine-27 tri-methylation (H3K27me3). We hypothesize that epigenetic reprogramming via EZH2 inhibitors (EZH2i) improves the efficacy of chemotherapy in AVPC.\u0000Methods. We studied the expression of EZH2 in clinical prostate cancer cohorts (bioinformatics). We determined the effect of EZH2i on both cellular and cell free-H3K27me3 levels. We measured effects of carboplatin with/without EZH2i on AVPC cell viability (IC50). We studied how EZH2i modulate gene expression (RNA Seq).\u0000Results. EZH2 was significantly up-regulated in AVPC vs other prostate cancer types. EZH2i reduced both cellular and cell free-H3K27me3 levels. EZH2i significantly reduced carboplatin IC50. EZH2i reduced the expression of DNA repair and increased the expression of pro-apoptotic genes.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"17 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142184230","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 : 2024-09-11DOI: 10.1101/2024.09.05.611567
Nada J. HABEICHI, Ghadir Amin, Solene E. Boitard, Cynthia Jean Tannous, Rana Ghali, Iman MOMKEN, Reine Diab, George W. Booz, Mathias Mericskay, Fouad A. Zouein
Background: Cardiorenal syndrome (CRS) type 1 is characterized by the development of acute kidney injury (AKI) following acute cardiac illness and notably acute myocardial infarction (MI). AKI is considered an independent risk factor increasing mortality rate substantially. Nicotinamide dinucleotide (NAD) is an important coenzyme in energy metabolism and oxidative phosphorylation and in its oxidized form, a substrate for multiple NAD+-dependent enzymes such as Sirtuins and poly-ADP ribose polymerases. Decreased cardiac NAD levels along with a down-regulation of the nicotinamide phosphoribosyl transferase (NAMPT) have been reported following MI. A compensatory upregulation in nicotinamide riboside kinase (NMRK) 2, an NAD+ biosynthetic enzyme that uses nicotinamide riboside (NR) to generate NAD+ takes place in the heart after MI but the impact on kidney NAD metabolism and function has not been addressed before. Methods: MI was induced by ligating the left anterior descending coronary artery in 2 months old C57BL6/J mice, followed by the administration of NR (IP injection, 400mg/kg/day) for four and seven days. We hypothesized that NR treatment could be a potential promising therapy for MI-induced AKI. Results: Our findings showed no significant improvement in cardiac ejection fraction following NR treatment at days 4 and 7 post-MI, whereas kidney functions were enhanced and morphological alterations and cell death decreased. The observed renal protection seems to be mediated by an up-regulation of NAMPT-mediated increase in renal NAD levels, notably in distal tubules. Conclusion: Our findings indicate that NR could be a potential promising therapy for AKI following an early stage of MI.
背景：1 型心肾综合征（CRS）的特点是在急性心脏病，尤其是急性心肌梗死（MI）之后发生急性肾损伤（AKI）。急性肾损伤被认为是一个独立的风险因素，会大大增加死亡率。烟酰胺二核苷酸（NAD）是能量代谢和氧化磷酸化过程中的一种重要辅酶，其氧化形式是多种依赖 NAD+ 的酶的底物，如 Sirtuins 和多-ADP 核糖聚合酶。有报道称，心肌梗死后，心脏中的 NAD 水平下降，烟酰胺磷酸核糖转移酶（NAMPT）下调。心肌梗死后，心脏中的烟酰胺核苷酸激酶（NMRK）2（一种利用烟酰胺核苷酸（NR）生成 NAD+ 的 NAD+ 生物合成酶）会出现代偿性上调，但其对肾脏 NAD 代谢和功能的影响尚未得到研究。研究方法：通过结扎 2 个月大的 C57BL6/J 小鼠的左前降支冠状动脉诱发心肌梗死，然后服用 NR（IP 注射，400 毫克/千克/天）4 天和 7 天。我们假设 NR 治疗可能是治疗 MI 引起的 AKI 的一种潜在疗法：结果：我们的研究结果表明，在心肌梗死后第 4 天和第 7 天服用 NR 后，心脏射血分数没有明显改善，而肾功能却有所增强，形态学改变和细胞死亡减少。观察到的肾脏保护似乎是通过上调 NAMPT 介导的肾脏 NAD 水平的增加而实现的，尤其是在远端肾小管。结论我们的研究结果表明，NR 可能是治疗早期心肌梗死后 AKI 的一种有潜力的疗法。
{"title":"Nicotinamide riboside: A promising therapy for MI-induced acute kidney injury by upregulating nicotinamide phosphoribosyltransferase-mediated NAD levels","authors":"Nada J. HABEICHI, Ghadir Amin, Solene E. Boitard, Cynthia Jean Tannous, Rana Ghali, Iman MOMKEN, Reine Diab, George W. Booz, Mathias Mericskay, Fouad A. Zouein","doi":"10.1101/2024.09.05.611567","DOIUrl":"https://doi.org/10.1101/2024.09.05.611567","url":null,"abstract":"Background: Cardiorenal syndrome (CRS) type 1 is characterized by the development of acute kidney injury (AKI) following acute cardiac illness and notably acute myocardial infarction (MI). AKI is considered an independent risk factor increasing mortality rate substantially. Nicotinamide dinucleotide (NAD) is an important coenzyme in energy metabolism and oxidative phosphorylation and in its oxidized form, a substrate for multiple NAD+-dependent enzymes such as Sirtuins and poly-ADP ribose polymerases. Decreased cardiac NAD levels along with a down-regulation of the nicotinamide phosphoribosyl transferase (NAMPT) have been reported following MI. A compensatory upregulation in nicotinamide riboside kinase (NMRK) 2, an NAD+ biosynthetic enzyme that uses nicotinamide riboside (NR) to generate NAD+ takes place in the heart after MI but the impact on kidney NAD metabolism and function has not been addressed before. Methods: MI was induced by ligating the left anterior descending coronary artery in 2 months old C57BL6/J mice, followed by the administration of NR (IP injection, 400mg/kg/day) for four and seven days. We hypothesized that NR treatment could be a potential promising therapy for MI-induced AKI.\u0000Results: Our findings showed no significant improvement in cardiac ejection fraction following NR treatment at days 4 and 7 post-MI, whereas kidney functions were enhanced and morphological alterations and cell death decreased. The observed renal protection seems to be mediated by an up-regulation of NAMPT-mediated increase in renal NAD levels, notably in distal tubules. Conclusion: Our findings indicate that NR could be a potential promising therapy for AKI following an early stage of MI.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"34 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142184229","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 : 2024-09-10DOI: 10.1101/2024.09.05.611349
Gao Zhang, Jiaxin Liang, Gang Wen, Mingli Yao, Yuqing Jia, Bo Feng, Jishun Li, Zunsheng Han, Qingxin Liu, Tianlei Li, Wenxuan Zhang, Hongwei Jin, Jie Xia, Liang Peng, Song Wu
Antibiotic resistance has become a serious threat to public health, thus novel antibiotics are urgently needed to combat drug-resistant bacteria including MRSA (methicillin-resistant S. aureus). The 1,4-dicarbonylthiosemicarbazide is an interesting chemotype that could exhibit antibacterial activity. However, the currently available compounds are not as potent as clinical antibiotics. Herein, we adopted the computer-aided drug design strategy, substructure search, to retrieve antibacterial 1,4-dicarbonylthiosemicarbazide derivatives, and identified compound B5 (Specs ID: AG-690/15432331) from the Specs chemical library that exhibited moderate activity (minimum inhibitory concentration (MIC): 6.25 μg/mL) against S. aureus (ATCC 29213). Based on that compound, we further designed and synthesized 45 derivatives, and evaluated their antibacterial activity. Eight derivatives were more potent than or equivalent to vancomycin (MIC: 1.56 μg/mL). We compared the three most potent ones for their cytotoxicity to HepG2 and HUVEC cells and selected compound 1b as our lead compound for comprehensive biological evaluation. As a result, compound 1b exhibited a bacteriostatic mode, and was active against a panel of gram-positive bacteria strains, metabolically stable, and effective to protect the mice from MRSA infection. More importantly, we applied 2D similarity calculation and reverse docking to predict potential targets of compound 1b. Through experimental validation and molecular dynamics simulation, we were able to confirm that compound 1b inhibited S. aureus DNA gyrase (IC50: 1.81 μM) and DNA supercoiling, potentially by binding to the ATPase domain, where ASP81, GLU58 and GLN91 formed key hydrogen bonds. Taken together, we have discovered a new class of DNA gyrase inhibitors represented by compound 1b for the treatment of MRSA infection, through the design, synthesis, and biological evaluation of novel 1,4-dicarbonylthiosemicarbazides.
{"title":"Discovery of novel 1,4-dicarbonylthiosemicarbazides as DNA gyrase inhibitors for the treatment of MRSA infection","authors":"Gao Zhang, Jiaxin Liang, Gang Wen, Mingli Yao, Yuqing Jia, Bo Feng, Jishun Li, Zunsheng Han, Qingxin Liu, Tianlei Li, Wenxuan Zhang, Hongwei Jin, Jie Xia, Liang Peng, Song Wu","doi":"10.1101/2024.09.05.611349","DOIUrl":"https://doi.org/10.1101/2024.09.05.611349","url":null,"abstract":"Antibiotic resistance has become a serious threat to public health, thus novel antibiotics are urgently needed to combat drug-resistant bacteria including MRSA (methicillin-resistant S. aureus). The 1,4-dicarbonylthiosemicarbazide is an interesting chemotype that could exhibit antibacterial activity. However, the currently available compounds are not as potent as clinical antibiotics. Herein, we adopted the computer-aided drug design strategy, substructure search, to retrieve antibacterial 1,4-dicarbonylthiosemicarbazide derivatives, and identified compound B5 (Specs ID: AG-690/15432331) from the Specs chemical library that exhibited moderate activity (minimum inhibitory concentration (MIC): 6.25 μg/mL) against S. aureus (ATCC 29213). Based on that compound, we further designed and synthesized 45 derivatives, and evaluated their antibacterial activity. Eight derivatives were more potent than or equivalent to vancomycin (MIC: 1.56 μg/mL). We compared the three most potent ones for their cytotoxicity to HepG2 and HUVEC cells and selected compound 1b as our lead compound for comprehensive biological evaluation. As a result, compound 1b exhibited a bacteriostatic mode, and was active against a panel of gram-positive bacteria strains, metabolically stable, and effective to protect the mice from MRSA infection. More importantly, we applied 2D similarity calculation and reverse docking to predict potential targets of compound 1b. Through experimental validation and molecular dynamics simulation, we were able to confirm that compound 1b inhibited S. aureus DNA gyrase (IC50: 1.81 μM) and DNA supercoiling, potentially by binding to the ATPase domain, where ASP81, GLU58 and GLN91 formed key hydrogen bonds. Taken together, we have discovered a new class of DNA gyrase inhibitors represented by compound 1b for the treatment of MRSA infection, through the design, synthesis, and biological evaluation of novel 1,4-dicarbonylthiosemicarbazides.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142184251","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 : 2024-09-09DOI: 10.1101/2024.09.07.611781
Yang Hu, Ping Wang, Jian Xiang, Lu Han, Biyun Zhang, Xiaohua Liu, Hua Nie, Guobing Chen, Weibing Qin
Resistance to platinum is the main challenge in the chemotherapy of ovarian cancer (OV). Therefore, developing a response signature to platinum is essential for the precision therapy of OV. Existing quantitative signatures of platinum are susceptible to batch effects and sequencing platform variations. To address this, we developed a transcriptome-based platinum signature, named PRSM, consisting of 15 genes, based on within-sample prognostic and relative expression ordering of genes, to predict individual responses to platinum in OV. The PRSM model demonstrated superior classification accuracy compared to previous quantitative signatures. Resistant samples classified by PRSM exhibited poorer overall survival, lower SNV neoantigen load, tumor mutational burden, and distinct methylation patterns compared to sensitive samples. Pathway analysis revealed the activation of MYC targets V2 and oxidative phosphorylation in resistant tumors. Single-cell analysis highlighted the roles of NK and epithelial cells in resistance. Among the 15 core genes, five (TFAP2B, KRT81, PAGE1, CRNN, UGT2B17) were linked to poor prognosis, with TFAP2B having the highest contribution to PRSM. Overexpression of TFAP2B in A2780 cells enhanced cisplatin sensitivity, while in A2780cis cells, it inhibited growth. In brief, our findings provide a multi-dimensional view of platinum resistance in ovarian cancer, introducing a robust predictive model and identifying potential therapeutic targets.
{"title":"A 15-Gene prognostic signature with TFAP2B functioning in Platinum Resistance of Ovarian Carcinoma","authors":"Yang Hu, Ping Wang, Jian Xiang, Lu Han, Biyun Zhang, Xiaohua Liu, Hua Nie, Guobing Chen, Weibing Qin","doi":"10.1101/2024.09.07.611781","DOIUrl":"https://doi.org/10.1101/2024.09.07.611781","url":null,"abstract":"Resistance to platinum is the main challenge in the chemotherapy of ovarian cancer (OV). Therefore, developing a response signature to platinum is essential for the precision therapy of OV. Existing quantitative signatures of platinum are susceptible to batch effects and sequencing platform variations. To address this, we developed a transcriptome-based platinum signature, named PRSM, consisting of 15 genes, based on within-sample prognostic and relative expression ordering of genes, to predict individual responses to platinum in OV. The PRSM model demonstrated superior classification accuracy compared to previous quantitative signatures. Resistant samples classified by PRSM exhibited poorer overall survival, lower SNV neoantigen load, tumor mutational burden, and distinct methylation patterns compared to sensitive samples. Pathway analysis revealed the activation of MYC targets V2 and oxidative phosphorylation in resistant tumors. Single-cell analysis highlighted the roles of NK and epithelial cells in resistance. Among the 15 core genes, five (TFAP2B, KRT81, PAGE1, CRNN, UGT2B17) were linked to poor prognosis, with TFAP2B having the highest contribution to PRSM. Overexpression of TFAP2B in A2780 cells enhanced cisplatin sensitivity, while in A2780cis cells, it inhibited growth. In brief, our findings provide a multi-dimensional view of platinum resistance in ovarian cancer, introducing a robust predictive model and identifying potential therapeutic targets.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"178 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142184239","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 : 2024-09-09DOI: 10.1101/2024.09.05.611570
Faizan Ahmad, Anik Karan, Richard L Jayaraj
Methylsulfonylmethane (MSM) is a common dietary supplement mainly used for inflammatory disorders as well as MSM had shown anti-tumor effects on different types of cancers. However, the glioma cell line has not been tested against MSM, and we are reporting it in our study for the first time. This research used an in silico study in which sphingosine kinase 1(SphK1) is used as a therapeutic target which is associated with Glioblastoma multiforme(GBM). SphK1 is pivotal enzyme for sphingolipid metabolism whose high expression level is thought to be associated with cancer alongside other inflammatory diseases and it is a potential drug target for various types of cancer. First, in silico analysis was executed to evaluate the inhibitory effect of MSM on SphK1.Then we further observed the anti-tumor activities of MSM on the C6 glioma cell line. During in silico investigation at the initial stage, we performed molecular docking with Auto Dock Vina followed by molecular dynamics simulation at 100ns with Gromacs Software Package. MSM binds with SphK1 with a docked score of -2.1 kcal mol1. During molecular dynamics simulation complex maintain stability at 10ns but we ran simulation till 100ns to confirm the stability. We performed in depth analysis which includes post trajectory analysis like free energy landscape (FEL), principal constant analysis (PCA) with kernel density (KDE)estimation plots as well as probability distribution plots. Even molecular dynamics simulation shows stability, compactness and interaction of MSM with Sphk1, we calculated MMPBSA binding energy calculation is -13.922 +/- 19.518 kJ/mol- The viability and cellular metabolic activity of the C6 glioma in the presence of MSM showed 393.459 mM /ml of MSM reduced cell viability by 50% (CTC50) value in dose dependent manner. Further analysis like DNA fragmentation assay and Acridine orange and ethidium bromide (AO/EB) staining were carried out, which clearly depicts MSM inducing apoptosis in C6 gliomas. Based on in silico and in vitro results, for the first time, we are reporting it in our study, and we conclude that MSM acts as a potential inhibitor for SphK1 as well as inhibits the growth of glioma cells and acts as a potential dietary supplement for the management of GBM which can cross the blood-brain barrier (BBB) and not toxic to cells even at high dose.
{"title":"Methylsulfonylmethane: A Potential Dietary Supplement targeting sphingosine kinase 1 involved in Glioblastomamultiforme","authors":"Faizan Ahmad, Anik Karan, Richard L Jayaraj","doi":"10.1101/2024.09.05.611570","DOIUrl":"https://doi.org/10.1101/2024.09.05.611570","url":null,"abstract":"Methylsulfonylmethane (MSM) is a common dietary supplement mainly used for inflammatory disorders as well as MSM had shown anti-tumor effects on different types of cancers. However, the glioma cell line has not been tested against MSM, and we are reporting it in our study for the first time. This research used an in silico study in which sphingosine kinase 1(SphK1) is used as a therapeutic target which is associated with Glioblastoma multiforme(GBM). SphK1 is pivotal enzyme for sphingolipid metabolism whose high expression level is thought to be associated with cancer alongside other inflammatory diseases and it is a potential drug target for various types of cancer. First, in silico analysis was executed to evaluate the inhibitory effect of MSM on SphK1.Then we further observed the anti-tumor activities of MSM on the C6 glioma cell line. During in silico investigation at the initial stage, we performed molecular docking with Auto Dock Vina followed by molecular dynamics simulation at 100ns with Gromacs Software Package. MSM binds with SphK1 with a docked score of -2.1 kcal mol1. During molecular dynamics simulation complex maintain stability at 10ns but we ran simulation till 100ns to confirm the stability. We performed in depth analysis which includes post trajectory analysis like free energy landscape (FEL), principal constant analysis (PCA) with kernel density (KDE)estimation plots as well as probability distribution plots. Even molecular dynamics simulation shows stability, compactness and interaction of MSM with Sphk1, we calculated MMPBSA binding energy calculation is -13.922 +/- 19.518 kJ/mol- The viability and cellular metabolic activity of the C6 glioma in the presence of MSM showed 393.459 mM /ml of MSM reduced cell viability by 50% (CTC50) value in dose dependent manner. Further analysis like DNA fragmentation assay and Acridine orange and ethidium bromide (AO/EB) staining were carried out, which clearly depicts MSM inducing apoptosis in C6 gliomas. Based on in silico and in vitro results, for the first time, we are reporting it in our study, and we conclude that MSM acts as a potential inhibitor for SphK1 as well as inhibits the growth of glioma cells and acts as a potential dietary supplement for the management of GBM which can cross the blood-brain barrier (BBB) and not toxic to cells even at high dose.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142184250","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 : 2024-09-08DOI: 10.1101/2024.09.06.611550
Antonia C Darragh, Andrew M Hanna, Justin H Lipner, Alastair J King, Nicole B Servant, Mirza Jahic
Uncovering a drug's mechanism of action and possible adverse effects are critical components in drug discovery and development. Moreover, it provides evidence for why some drugs prove more effective than others, and how to design better drugs altogether. Here we demonstrate the utility of a high-throughput in vitro screening platform along with a comprehensive panel to aid in the characterization of fifteen BTK inhibitors that are either approved by the FDA or presently under clinical evaluation. To compare the potency of these drugs, we measured the binding affinity of each to wild-type BTK, as well as a clinically relevant resistance mutant of BTK (BTK C481S). In doing so, we discovered a considerable difference in the selectivity and potency of these BTK inhibitors to the wild-type and mutant proteins. Some of this potentially contributes to the adverse effects experienced by patients undergoing therapy using these drugs. Overall, non-covalent BTK inhibitors showed stronger potency for both the wild-type and mutant BTK when compared with that of covalent inhibitors, with the majority demonstrating a higher specificity and less off-target modulation. Additionally, we compared biological outcomes for four of these inhibitors in human cell-based models. As expected, we found different phenotypic profiles for each inhibitor. However, the two non-covalent inhibitors had fewer off-target biological effects when compared with the two covalent inhibitors. This and similar in-depth preclinical characterization of drug candidates can provide critical insights into the efficacy and mechanism of action of a compound that may affect its safety in a clinical setting.
{"title":"Comprehensive Characterization of BTK Inhibitor Specificity, Potency, and Biological Effects: Insights into Covalent and Non-covalent Mechanistic Signatures","authors":"Antonia C Darragh, Andrew M Hanna, Justin H Lipner, Alastair J King, Nicole B Servant, Mirza Jahic","doi":"10.1101/2024.09.06.611550","DOIUrl":"https://doi.org/10.1101/2024.09.06.611550","url":null,"abstract":"Uncovering a drug's mechanism of action and possible adverse effects are critical components in drug discovery and development. Moreover, it provides evidence for why some drugs prove more effective than others, and how to design better drugs altogether. Here we demonstrate the utility of a high-throughput in vitro screening platform along with a comprehensive panel to aid in the characterization of fifteen BTK inhibitors that are either approved by the FDA or presently under clinical evaluation. To compare the potency of these drugs, we measured the binding affinity of each to wild-type BTK, as well as a clinically relevant resistance mutant of BTK (BTK C481S). In doing so, we discovered a considerable difference in the selectivity and potency of these BTK inhibitors to the wild-type and mutant proteins. Some of this potentially contributes to the adverse effects experienced by patients undergoing therapy using these drugs. Overall, non-covalent BTK inhibitors showed stronger potency for both the wild-type and mutant BTK when compared with that of covalent inhibitors, with the majority demonstrating a higher specificity and less off-target modulation. Additionally, we compared biological outcomes for four of these inhibitors in human cell-based models. As expected, we found different phenotypic profiles for each inhibitor. However, the two non-covalent inhibitors had fewer off-target biological effects when compared with the two covalent inhibitors. This and similar in-depth preclinical characterization of drug candidates can provide critical insights into the efficacy and mechanism of action of a compound that may affect its safety in a clinical setting.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142223911","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 : 2024-09-08DOI: 10.1101/2024.09.06.611731
Yingjie Sun, Changheng Li, Xiaofei Deng, Wenjie Li, Xiaoyi Deng, Weiqi Ge, Miaoyuan Shi, Ying Guo, Yanxun V. Yu, Hai-bing Zhou, Youngnam N Jin
Identifying target proteins for bioactive molecules is essential for understanding their mechanisms, developing improved derivatives, and minimizing off-target effects. Despite advances in target identification (target-ID) technologies, significant challenges remain, impeding drug development. Most target-ID methods use cell lysates, but maintaining an intact cellular context is vital for capturing specific drug-protein interactions, such as those with transient protein complexes and membrane-associated proteins. To address these limitations, we developed POST-IT (Pup-On-target for Small molecule Target Identification Technology), a non-diffusive proximity tagging system for live cells, orthogonal to the eukaryotic system. POST-IT utilizes an engineered fusion of proteasomal accessory factor A (PafA) and HaloTag to transfer Pup to proximal proteins upon directly binding to the small molecule. After significant optimization to eliminate self-pupylation and polypupylation, minimize depupylation, and optimize chemical linkers, POST-IT successfully identified known targets and discovered a new binder, SEPHS2, for dasatinib, and VPS37C as a new target for hydroxychloroquine, enhancing our understanding these drugs’ mechanisms of action. Furthermore, we demonstrated the application of POST-IT in live zebrafish embryos, highlighting its potential for broad biological research and drug development.
{"title":"Target protein identification in live cells and organisms with a non-diffusive proximity tagging system","authors":"Yingjie Sun, Changheng Li, Xiaofei Deng, Wenjie Li, Xiaoyi Deng, Weiqi Ge, Miaoyuan Shi, Ying Guo, Yanxun V. Yu, Hai-bing Zhou, Youngnam N Jin","doi":"10.1101/2024.09.06.611731","DOIUrl":"https://doi.org/10.1101/2024.09.06.611731","url":null,"abstract":"Identifying target proteins for bioactive molecules is essential for understanding their mechanisms, developing improved derivatives, and minimizing off-target effects. Despite advances in target identification (target-ID) technologies, significant challenges remain, impeding drug development. Most target-ID methods use cell lysates, but maintaining an intact cellular context is vital for capturing specific drug-protein interactions, such as those with transient protein complexes and membrane-associated proteins. To address these limitations, we developed POST-IT (Pup-On-target for Small molecule Target Identification Technology), a non-diffusive proximity tagging system for live cells, orthogonal to the eukaryotic system. POST-IT utilizes an engineered fusion of proteasomal accessory factor A (PafA) and HaloTag to transfer Pup to proximal proteins upon directly binding to the small molecule. After significant optimization to eliminate self-pupylation and polypupylation, minimize depupylation, and optimize chemical linkers, POST-IT successfully identified known targets and discovered a new binder, SEPHS2, for dasatinib, and VPS37C as a new target for hydroxychloroquine, enhancing our understanding these drugs’ mechanisms of action. Furthermore, we demonstrated the application of POST-IT in live zebrafish embryos, highlighting its potential for broad biological research and drug development.","PeriodicalId":501518,"journal":{"name":"bioRxiv - Pharmacology and Toxicology","volume":"1693 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142184253","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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