Pub Date : 2024-08-30DOI: 10.1021/acsptsci.4c00383
Kathrin Schorr, Xinyu Chen, Takanori Sasaki, Anahi Paula Arias-Loza, Johannes Lang, Takahiro Higuchi, Achim Goepferich
Thin-layer chromatography (TLC) is commonly employed to screen technetium-99m labeled polymer nanoparticle batches for unreduced pertechnetate and radio-colloidal impurities. Although this method is widely accepted, our findings applying radiolabeled PLGA/PLA–PEG nanoparticles underscore its lack of transferability between different settings and its limitations as a standalone quality control tool. While TLC profiles may appear similar for purified and radiocolloid containing nanoparticle formulations, their in vivo behavior can vary significantly, as demonstrated by discrepancies between TLC results and single-photon emission computed tomography (SPECT) and biodistribution data. This highlights the urgent need for a case-by-case evaluation of TLC methods for each specific nanoparticle type. Our study revealed that polymeric nanoparticles cannot be considered analytically uniform entities in the context of TLC analysis, emphasizing the complex interplay between nanoparticle composition, radiolabeling conditions, and subsequent biological behavior.
{"title":"Rethinking Thin-Layer Chromatography for Screening Technetium-99m Radiolabeled Polymer Nanoparticles","authors":"Kathrin Schorr, Xinyu Chen, Takanori Sasaki, Anahi Paula Arias-Loza, Johannes Lang, Takahiro Higuchi, Achim Goepferich","doi":"10.1021/acsptsci.4c00383","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00383","url":null,"abstract":"Thin-layer chromatography (TLC) is commonly employed to screen technetium-99m labeled polymer nanoparticle batches for unreduced pertechnetate and radio-colloidal impurities. Although this method is widely accepted, our findings applying radiolabeled PLGA/PLA–PEG nanoparticles underscore its lack of transferability between different settings and its limitations as a standalone quality control tool. While TLC profiles may appear similar for purified and radiocolloid containing nanoparticle formulations, their in vivo behavior can vary significantly, as demonstrated by discrepancies between TLC results and single-photon emission computed tomography (SPECT) and biodistribution data. This highlights the urgent need for a case-by-case evaluation of TLC methods for each specific nanoparticle type. Our study revealed that polymeric nanoparticles cannot be considered analytically uniform entities in the context of TLC analysis, emphasizing the complex interplay between nanoparticle composition, radiolabeling conditions, and subsequent biological behavior.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"61 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142218146","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 : 2024-08-30DOI: 10.1021/acsptsci.4c00102
Sadhana Sharma, Vincenzo S. Gilberto, Cassandra L. Levens, Anushree Chatterjee, Kristine A. Kuhn, Prashant Nagpal
Autoimmune and autoinflammatory diseases account for more than 80 chronic conditions affecting more than 24 million people in the US. Among these autoinflammatory diseases, noninfectious chronic inflammation of the gastrointestinal (GI) tract causes inflammatory bowel diseases (IBDs), primarily Crohn’s and ulcerative colitis (UC). IBD is a complex disease, and one hypothesis is that these are either caused or worsened by compounds produced by bacteria in the gut. While traditional approaches have focused on pan immunosuppressive techniques (e.g., steroids), low remission rates, prolonged illnesses, and an increased frequency of surgical procedures have prompted the search for more targeted and precision therapeutic approaches. IBD is a complex disease resulting from both genetic and environmental factors, but several recent studies have highlighted the potential pivotal contribution of gut microbiota dysbiosis. Gut microbiota are known to modulate the immune status of the gut by producing metabolites that are encoded in biosynthetic gene clusters (BGCs) of the bacterial genome. Here, we show a targeted and high-throughput screening of more than 90 biosynthetic genes in 41 gut anaerobes, through downselection using available bioinformatics tools, targeted gene manipulation in these genetically intractable organisms using the Nanoligomer platform, and identification and synthesis of top microbiome targets as a Nanoligomer BGC cocktail (SB_BGC_CK1, abbreviated as CK1) as a feasible precision therapeutic approach. Further, we used a host-directed immune target screening to identify the NF-κB and NLRP3 cocktail SB_NI_112 (or NI112 for short) as a targeted inflammasome inhibitor. We used these top two microbe- and host-targeted Nanoligomer cocktails in acute and chronic dextran sulfate sodium (DSS) mouse colitis and in TNF<sup>ΔARE/+</sup> transgenic mice that develop spontaneous Crohn’s like ileitis. The mouse microbiome was humanized to replicate that in human IBD through antibiotic treatment, followed by mixed fecal gavage from 10 human donors and spiked with IBD-inducing microbial species. Following colonization, colitis was induced in mice using 1 week of 3% DSS (acute) or 6 weeks of 3 rounds of 2.5% DSS induction for a week followed by 1 week of no DSS (chronic colitis model). Both Nanoligomer cocktails (CK1 and NI112) showed a strong reduction in disease severity, significant improvement in disease histopathology, and profound downregulation of disease biomarkers in colon tissue, as assessed by multiplexed ELISA. Further, we used two different formulations of intraperitoneal injections (IP) and Nanoligomer pills in the chronic DSS colitis model. Although both formulations were highly effective, the oral pill formulation demonstrated a greater reduction in biochemical markers compared to IP. A similar therapeutic effect was observed in the TNF<sup>ΔARE/+</sup> model. Overall, these results point to the potential for further development and testin
{"title":"Microbiome- and Host Inflammasome-Targeting Inhibitor Nanoligomers Are Therapeutic in the Murine Colitis Model","authors":"Sadhana Sharma, Vincenzo S. Gilberto, Cassandra L. Levens, Anushree Chatterjee, Kristine A. Kuhn, Prashant Nagpal","doi":"10.1021/acsptsci.4c00102","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00102","url":null,"abstract":"Autoimmune and autoinflammatory diseases account for more than 80 chronic conditions affecting more than 24 million people in the US. Among these autoinflammatory diseases, noninfectious chronic inflammation of the gastrointestinal (GI) tract causes inflammatory bowel diseases (IBDs), primarily Crohn’s and ulcerative colitis (UC). IBD is a complex disease, and one hypothesis is that these are either caused or worsened by compounds produced by bacteria in the gut. While traditional approaches have focused on pan immunosuppressive techniques (e.g., steroids), low remission rates, prolonged illnesses, and an increased frequency of surgical procedures have prompted the search for more targeted and precision therapeutic approaches. IBD is a complex disease resulting from both genetic and environmental factors, but several recent studies have highlighted the potential pivotal contribution of gut microbiota dysbiosis. Gut microbiota are known to modulate the immune status of the gut by producing metabolites that are encoded in biosynthetic gene clusters (BGCs) of the bacterial genome. Here, we show a targeted and high-throughput screening of more than 90 biosynthetic genes in 41 gut anaerobes, through downselection using available bioinformatics tools, targeted gene manipulation in these genetically intractable organisms using the Nanoligomer platform, and identification and synthesis of top microbiome targets as a Nanoligomer BGC cocktail (SB_BGC_CK1, abbreviated as CK1) as a feasible precision therapeutic approach. Further, we used a host-directed immune target screening to identify the NF-κB and NLRP3 cocktail SB_NI_112 (or NI112 for short) as a targeted inflammasome inhibitor. We used these top two microbe- and host-targeted Nanoligomer cocktails in acute and chronic dextran sulfate sodium (DSS) mouse colitis and in TNF<sup>ΔARE/+</sup> transgenic mice that develop spontaneous Crohn’s like ileitis. The mouse microbiome was humanized to replicate that in human IBD through antibiotic treatment, followed by mixed fecal gavage from 10 human donors and spiked with IBD-inducing microbial species. Following colonization, colitis was induced in mice using 1 week of 3% DSS (acute) or 6 weeks of 3 rounds of 2.5% DSS induction for a week followed by 1 week of no DSS (chronic colitis model). Both Nanoligomer cocktails (CK1 and NI112) showed a strong reduction in disease severity, significant improvement in disease histopathology, and profound downregulation of disease biomarkers in colon tissue, as assessed by multiplexed ELISA. Further, we used two different formulations of intraperitoneal injections (IP) and Nanoligomer pills in the chronic DSS colitis model. Although both formulations were highly effective, the oral pill formulation demonstrated a greater reduction in biochemical markers compared to IP. A similar therapeutic effect was observed in the TNF<sup>ΔARE/+</sup> model. Overall, these results point to the potential for further development and testin","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142218145","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 : 2024-08-26DOI: 10.1021/acsptsci.4c00129
Nicholas A. Weirath, Carrie Haskell-Luevano
The melanocortin receptors are a centrally and peripherally expressed family of Class A GPCRs with physiological roles, including pigmentation, steroidogenesis, energy homeostasis, and others yet to be fully characterized. There are five melanocortin receptor subtypes that, apart from the melanocortin-2 receptor (MC2R), are stimulated by a shared set of endogenous agonists. Until 2020, X-ray crystallographic and cryo-electron microscopic (cryo-EM) structures of these receptors were unavailable, and the investigation of their mechanisms of action and putative ligand–receptor interactions was driven by site-directed mutagenesis studies of the receptors and targeted structure–activity relationship (SAR) studies of the endogenous and derivative synthetic ligands. Synthetic derivatives of the endogenous agonist ligand α-MSH have evolved into a suite of powerful ligands such as NDP-MSH (melanotan I), melanotan II (MTII), and SHU9119. This suite of tool compounds now enables the study of the melanocortin receptors and serves as scaffolds for FDA-approved drugs, means of validating stably expressing melanocortin receptor cell lines, core ligands in assessing cryo-EM structures of active and inactive receptor complexes, and essential references for high-throughput discovery and mechanism of action studies. Herein, we review the history and significance of a finite set of these essential tool compounds and discuss how they are being utilized to further the field’s understanding of melanocortin receptor physiology and greater druggability.
黑皮质素受体是一个在中心和外周表达的 A 类 GPCR 家族，其生理作用包括色素沉着、类固醇生成、能量平衡和其他尚未完全确定的作用。黑皮质素受体有五种亚型，除了黑皮质素-2 受体（MC2R）外，它们还受到一组共同的内源性激动剂的刺激。直到 2020 年，这些受体的 X 射线晶体学和低温电子显微镜（cryo-EM）结构仍无法获得，而对其作用机制和配体与受体之间假定相互作用的研究则是通过对受体进行定点突变研究以及对内源性配体和衍生合成配体进行有针对性的结构-活性关系（SAR）研究来推动的。内源性激动剂配体 α-MSH 的合成衍生物已发展成一套强大的配体，如 NDP-MSH（美拉罗坦 I）、美拉罗坦 II（MTII）和 SHU9119。目前，这一系列工具化合物可用于研究黑皮质素受体，并可作为 FDA 批准药物的支架、验证稳定表达黑皮质素受体细胞系的方法、评估活性和非活性受体复合物低温电子显微镜结构的核心配体，以及高通量发现和作用机制研究的重要参考。在此，我们将回顾这些重要工具化合物的历史和意义，并讨论如何利用它们来进一步加深本领域对黑色素皮质素受体生理学的理解，并提高其可药用性。
{"title":"Recommended Tool Compounds for the Melanocortin Receptor (MCR) G Protein-Coupled Receptors (GPCRs)","authors":"Nicholas A. Weirath, Carrie Haskell-Luevano","doi":"10.1021/acsptsci.4c00129","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00129","url":null,"abstract":"The melanocortin receptors are a centrally and peripherally expressed family of Class A GPCRs with physiological roles, including pigmentation, steroidogenesis, energy homeostasis, and others yet to be fully characterized. There are five melanocortin receptor subtypes that, apart from the melanocortin-2 receptor (MC2R), are stimulated by a shared set of endogenous agonists. Until 2020, X-ray crystallographic and cryo-electron microscopic (cryo-EM) structures of these receptors were unavailable, and the investigation of their mechanisms of action and putative ligand–receptor interactions was driven by site-directed mutagenesis studies of the receptors and targeted structure–activity relationship (SAR) studies of the endogenous and derivative synthetic ligands. Synthetic derivatives of the endogenous agonist ligand α-MSH have evolved into a suite of powerful ligands such as NDP-MSH (melanotan I), melanotan II (MTII), and SHU9119. This suite of tool compounds now enables the study of the melanocortin receptors and serves as scaffolds for FDA-approved drugs, means of validating stably expressing melanocortin receptor cell lines, core ligands in assessing cryo-EM structures of active and inactive receptor complexes, and essential references for high-throughput discovery and mechanism of action studies. Herein, we review the history and significance of a finite set of these essential tool compounds and discuss how they are being utilized to further the field’s understanding of melanocortin receptor physiology and greater druggability.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"14 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142218173","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 : 2024-08-21DOI: 10.1021/acsptsci.4c00485
Ilaria D’Acquarica, Israel Agranat
Page 209, bottom of left column. Dr. Sheila DeWitt (DeuteRx, LLC, Bedford, NH, USA; email: sdewitt@deuterx.com) has recently drawn our attention to a mistake in the following sentence: “The deuterated S-enantiomer of lenalidomide DP-053 (formerly CTP-221, Figure 2) was shown to be ‘greatly stabilized to epimerization and results in a more desirable pharmacokinetic profile than racemic lenalidomide’ 115 (see also ref 108, ref 56 therein).” There was also a mistake in ref 115 that was cited in that text, which is corrected in ref (1) herein. DP-053 was not formerly CTP-221. They are two different compounds that were pursued separately by Deuteria Pharmaceuticals, Inc. and Concert Pharmaceuticals, Inc., respectively. DP-053 is the structure shown in Figure 2 (D1-S-lenalidomide) which was pursued by Deuteria Pharmaceuticals, Inc. Celgene Corporation (now Bristol-Myers Squibb Company) acquired Deuteria and selected assets, including DP-053, in December 2012. The remaining assets were spun-out into DeuteRx, LLC. CTP-221 is D5-S-lenalidomide, which was pursued by Concert Pharmaceuticals, Inc. and then part of a development and license agreement with Celgene Corporation (now Bristol-Myers Squibb Company) in May 2013. The text should have stated that “CTP-221 was shown to be ‘greatly stabilized to epimerization and results in a more desirable pharmacokinetic profile than racemic lenalidomide’ 115 (see also ref 108, ref 56 therein).” We apologize for the above-mentioned mistakes. Other data shown in the published Perspective are correct, and this modification does not affect the overall conclusions of our study. We thank Dr. Sheila DeWitt for drawing our attention to these mistakes and for giving us the opportunity to correct them. This article references 1 other publications. Corrected ref 115 from the published Perspective: This article has not yet been cited by other publications.
{"title":"Correction to “The Quest for Secondary Pharmaceuticals: Drug Repurposing/Chiral-Switches Combination Strategy”","authors":"Ilaria D’Acquarica, Israel Agranat","doi":"10.1021/acsptsci.4c00485","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00485","url":null,"abstract":"Page 209, bottom of left column. Dr. Sheila DeWitt (DeuteRx, LLC, Bedford, NH, USA; email: sdewitt@deuterx.com) has recently drawn our attention to a mistake in the following sentence: “The deuterated <i>S</i>-enantiomer of lenalidomide DP-053 (formerly CTP-221, Figure 2) was shown to be ‘greatly stabilized to epimerization and results in a more desirable pharmacokinetic profile than racemic lenalidomide’ <sup>115</sup> (see also ref 108, ref 56 therein).” There was also a mistake in ref 115 that was cited in that text, which is corrected in ref (1) herein. DP-053 was not formerly CTP-221. They are two different compounds that were pursued separately by Deuteria Pharmaceuticals, Inc. and Concert Pharmaceuticals, Inc., respectively. DP-053 is the structure shown in Figure 2 (D1-<i>S</i>-lenalidomide) which was pursued by Deuteria Pharmaceuticals, Inc. Celgene Corporation (now Bristol-Myers Squibb Company) acquired Deuteria and selected assets, including DP-053, in December 2012. The remaining assets were spun-out into DeuteRx, LLC. CTP-221 is D5-<i>S</i>-lenalidomide, which was pursued by Concert Pharmaceuticals, Inc. and then part of a development and license agreement with Celgene Corporation (now Bristol-Myers Squibb Company) in May 2013. The text should have stated that “CTP-221 was shown to be ‘greatly stabilized to epimerization and results in a more desirable pharmacokinetic profile than racemic lenalidomide’ <sup>115</sup> (see also ref 108, ref 56 therein).”<img alt=\"\" src=\"/cms/10.1021/acsptsci.4c00485/asset/images/medium/pt4c00485_0001.gif\"/> We apologize for the above-mentioned mistakes. Other data shown in the published Perspective are correct, and this modification does not affect the overall conclusions of our study. We thank Dr. Sheila DeWitt for drawing our attention to these mistakes and for giving us the opportunity to correct them. This article references 1 other publications. Corrected ref 115 from the published Perspective: This article has not yet been cited by other publications.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"303 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142218176","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 : 2024-08-20DOI: 10.1021/acsptsci.4c00462
Ahmed Kh. Abosalha, Paromita Islam, Jacqueline L. Boyajian, Rahul Thareja, Sabrina Schaly, Amal Kassab, Stephanie Makhlouf, Sarah Alali, Satya Prakash
Colorectal cancer (CRC) is the third most common cancer worldwide, acting as a significant public health problem. 5-Fluorouracil (5-FU) is a key chemotherapy for various types of cancer, due to its broad anticancer activity. However, the emergence of drug resistance is a considerable limitation in the clinical application of 5-FU. Quercetin (QC) is proposed as an adjuvant therapy to minimize drug resistance to chemotherapeutics and enhance their pharmacological efficacy. The oral delivery of 5-FU and QC is challenged by poor aqueous solubility of QC and poor cellular permeability of 5-FU. To solve this issue, novel polylactide-co-glycolide (PLGA) combinatorial nanoparticles loading 5-FU and QC were prepared to deliver them directly to the colon. These sustained-release combinatorial nanoparticles recorded a significant decrease in cancer cell proliferation, C-reactive protein (CRP) level, and Interleukin-8 (IL-8) expression by 30.08%, 40.7%, and 46.6%, respectively. The results revealed that this combination therapy may offer a new strategy for the targeted delivery of chemotherapeutics to the colon.
{"title":"Colon-Targeted Sustained-Release Combinatorial 5-Fluorouracil and Quercetin poly(lactic-co-glycolic) Acid (PLGA) Nanoparticles Show Enhanced Apoptosis and Minimal Tumor Drug Resistance for Their Potential Use in Colon Cancer","authors":"Ahmed Kh. Abosalha, Paromita Islam, Jacqueline L. Boyajian, Rahul Thareja, Sabrina Schaly, Amal Kassab, Stephanie Makhlouf, Sarah Alali, Satya Prakash","doi":"10.1021/acsptsci.4c00462","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00462","url":null,"abstract":"Colorectal cancer (CRC) is the third most common cancer worldwide, acting as a significant public health problem. 5-Fluorouracil (5-FU) is a key chemotherapy for various types of cancer, due to its broad anticancer activity. However, the emergence of drug resistance is a considerable limitation in the clinical application of 5-FU. Quercetin (QC) is proposed as an adjuvant therapy to minimize drug resistance to chemotherapeutics and enhance their pharmacological efficacy. The oral delivery of 5-FU and QC is challenged by poor aqueous solubility of QC and poor cellular permeability of 5-FU. To solve this issue, novel polylactide-<i>co</i>-glycolide (PLGA) combinatorial nanoparticles loading 5-FU and QC were prepared to deliver them directly to the colon. These sustained-release combinatorial nanoparticles recorded a significant decrease in cancer cell proliferation, C-reactive protein (CRP) level, and Interleukin-8 (IL-8) expression by 30.08%, 40.7%, and 46.6%, respectively. The results revealed that this combination therapy may offer a new strategy for the targeted delivery of chemotherapeutics to the colon.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"70 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142218174","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}
Obesity and osteoporosis are two prevalent conditions that are becoming increasingly common worldwide, primarily due to aging populations, imbalanced energy intake, and sedentary lifestyles. Obesity, characterized by excessive fat accumulation, and osteoporosis, marked by reduced bone density and increased fracture risk, are often interconnected. High-fat diets (HFDs) can exacerbate both conditions by promoting bone marrow adiposity and bone loss. The effect of WFA on the osteogenesis and adipogenesis was studied on the C3H10T1/2 cell line and bone marrow mesenchymal stem cells (BM-MSCs) isolated from mice. We used oil red O and alkaline phosphatase (ALP) staining to observe adipogenesis and osteogenesis, respectively, in MSCs. Real-time PCR and Western blot analyses were used to study the molecular effects of WFA on MSCs. We employed micro-CT to analyze the bone microarchitecture, bone mineral density (BMD), and abdominal fat mass in male mice. We have used osmium tetroxide (OsO4) staining to study the bone marrow fat. WFA induced the C3H10T1/2 cell line and BM-MSCs toward osteogenic lineage as evidenced by the higher ALP activity. WFA also downregulated the lipid droplet formation and adipocyte specific genes in MSCs. In the in vivo study, WFA also suppressed the bone catabolic effects of the HFD and maintained the bone microarchitecture and BMD in WFA-treated animals. The bone marrow adipose tissue was reduced in the tibia of WFA-treated groups in comparison with only HFD-fed animals. Withaferin A was able to improve the bone microarchitecture and BMD by committing BM-MSCs toward osteogenic differentiation and reducing marrow adiposity. The findings of this study could provide valuable insights into the therapeutic potential of Withaferin A for combating bone marrow obesity and osteoporosis, particularly in the context of diet-induced metabolic disturbances.
肥胖症和骨质疏松症是两种在全球范围内日益普遍的疾病，其主要原因是人口老龄化、能量摄入失衡和久坐不动的生活方式。以脂肪过度堆积为特征的肥胖症和以骨密度降低和骨折风险增加为特征的骨质疏松症往往相互关联。高脂饮食（HFDs）会促进骨髓肥胖和骨质流失，从而加重这两种情况。我们研究了 WFA 对 C3H10T1/2 细胞系和小鼠骨髓间充质干细胞（BM-MSCs）成骨和成脂的影响。我们使用油红 O 和碱性磷酸酶（ALP）染色法分别观察间充质干细胞的脂肪生成和骨生成。实时 PCR 和 Western 印迹分析用于研究 WFA 对间充质干细胞的分子影响。我们采用显微 CT 分析了雄性小鼠的骨微结构、骨矿物质密度（BMD）和腹部脂肪量。我们使用四氧化锇（OsO4）染色来研究骨髓脂肪。WFA诱导C3H10T1/2细胞系和骨髓间充质干细胞向成骨细胞系生长，这一点可以从较高的ALP活性得到证明。WFA 还能下调间充质干细胞的脂滴形成和脂肪细胞特异性基因。在体内研究中，WFA 还能抑制 HFD 对骨代谢的影响，并维持 WFA 处理动物的骨微结构和 BMD。与仅摄入高纤维食物的动物相比，WFA 处理组动物胫骨中的骨髓脂肪组织有所减少。Withaferin A能使骨髓间充质干细胞向成骨分化方向发展并减少骨髓脂肪，从而改善骨的微结构和BMD。这项研究的结果为研究威化沙棘素 A 对抗骨髓肥胖和骨质疏松症的治疗潜力提供了有价值的见解，尤其是在饮食引起的代谢紊乱的情况下。
{"title":"Withaferin A Ameliorated the Bone Marrow Fat Content in Obese Male Mice by Favoring Osteogenesis in Bone Marrow Mesenchymal Stem Cells and Preserving the Bone Mineral Density","authors":"Ashish Kumar Tripathi, Anirban Sardar, Nikhil Rai, Divya Rai, Aboli Girme, Shradha Sinha, Kunal Chutani, Lal Hingorani, Prabhat Ranjan Mishra, Ritu Trivedi","doi":"10.1021/acsptsci.3c00356","DOIUrl":"https://doi.org/10.1021/acsptsci.3c00356","url":null,"abstract":"Obesity and osteoporosis are two prevalent conditions that are becoming increasingly common worldwide, primarily due to aging populations, imbalanced energy intake, and sedentary lifestyles. Obesity, characterized by excessive fat accumulation, and osteoporosis, marked by reduced bone density and increased fracture risk, are often interconnected. High-fat diets (HFDs) can exacerbate both conditions by promoting bone marrow adiposity and bone loss. The effect of WFA on the osteogenesis and adipogenesis was studied on the C3H10T1/2 cell line and bone marrow mesenchymal stem cells (BM-MSCs) isolated from mice. We used oil red O and alkaline phosphatase (ALP) staining to observe adipogenesis and osteogenesis, respectively, in MSCs. Real-time PCR and Western blot analyses were used to study the molecular effects of WFA on MSCs. We employed micro-CT to analyze the bone microarchitecture, bone mineral density (BMD), and abdominal fat mass in male mice. We have used osmium tetroxide (OsO<sub>4</sub>) staining to study the bone marrow fat. WFA induced the C3H10T1/2 cell line and BM-MSCs toward osteogenic lineage as evidenced by the higher ALP activity. WFA also downregulated the lipid droplet formation and adipocyte specific genes in MSCs. In the <i>in vivo</i> study, WFA also suppressed the bone catabolic effects of the HFD and maintained the bone microarchitecture and BMD in WFA-treated animals. The bone marrow adipose tissue was reduced in the tibia of WFA-treated groups in comparison with only HFD-fed animals. Withaferin A was able to improve the bone microarchitecture and BMD by committing BM-MSCs toward osteogenic differentiation and reducing marrow adiposity. The findings of this study could provide valuable insights into the therapeutic potential of Withaferin A for combating bone marrow obesity and osteoporosis, particularly in the context of diet-induced metabolic disturbances.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"164 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142218175","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 : 2024-08-19DOI: 10.1021/acsptsci.4c00236
Jolien De Neve, Émile Breault, Santo Previti, Esaü Vangeloven, Bobbi Loranger, Magali Chartier, Rebecca Brouillette, Annik Lanoie, Brian J. Holleran, Jean-Michel Longpré, Louis Gendron, Dirk Tourwé, Philippe Sarret, Steven Ballet
Linking an opioid to a nonopioid pharmacophore represents a promising approach for reducing opioid-induced side effects during pain management. Herein, we describe the optimization of the previously reported opioid-neurotensin hybrids (OPNT-hybrids), SBL-OPNT-05 & -10, containing the μ-/δ-opioid agonist H-Dmt-d-Arg-Aba-β-Ala-NH2 and NT(8–13) analogs optimized for NTS2 affinity. In the present work, the constrained dipeptide Aba-β-Ala was modified to investigate the optimal linker length between the two pharmacophores, as well as the effect of expanding the aromatic moiety within constrained dipeptide analogs, via the inclusion of a naphthyl moiety. Additionally, the N-terminal Arg residue of the NT(8–13) pharmacophore was substituted with β3hArg. For all analogs, affinity was determined at the MOP, DOP, NTS1, and NTS2 receptors. Several of the hybrid ligands showed a subnanomolar affinity for MOP, improved binding for DOP compared to SBL-OPNT-05 & -10, as well as an excellent NTS2-affinity with high selectivity over NTS1. Subsequently, the Gαi1 and β-arrestin-2 pathways were evaluated for all hybrids, along with their stability in rat plasma. Upon MOP activation, SBL-OPNT-13 and -18 were the least effective at recruiting β-arrestin-2 (Emax = 17 and 12%, respectively), while both compounds were also found to be partial agonists at the Gαi1 pathway, despite improved potency compared to DAMGO. Importantly, these analogs also showed a half-life in rat plasma in excess of 48 h, making them valuable tools for future in vivo investigations.
{"title":"Design, Synthesis, and In Vitro Characterization of Proteolytically-Stable Opioid-Neurotensin Hybrid Peptidomimetics","authors":"Jolien De Neve, Émile Breault, Santo Previti, Esaü Vangeloven, Bobbi Loranger, Magali Chartier, Rebecca Brouillette, Annik Lanoie, Brian J. Holleran, Jean-Michel Longpré, Louis Gendron, Dirk Tourwé, Philippe Sarret, Steven Ballet","doi":"10.1021/acsptsci.4c00236","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00236","url":null,"abstract":"Linking an opioid to a nonopioid pharmacophore represents a promising approach for reducing opioid-induced side effects during pain management. Herein, we describe the optimization of the previously reported opioid-neurotensin hybrids (OPNT-hybrids), <b>SBL-OPNT-05</b> & <b>-10</b>, containing the μ-/δ-opioid agonist H-Dmt-<span>d</span>-Arg-Aba-β-Ala-NH<sub>2</sub> and NT(8–13) analogs optimized for NTS2 affinity. In the present work, the constrained dipeptide Aba-β-Ala was modified to investigate the optimal linker length between the two pharmacophores, as well as the effect of expanding the aromatic moiety within constrained dipeptide analogs, via the inclusion of a naphthyl moiety. Additionally, the <i>N</i>-terminal Arg residue of the NT(8–13) pharmacophore was substituted with β<sup>3</sup><i>h</i>Arg. For all analogs, affinity was determined at the MOP, DOP, NTS1, and NTS2 receptors. Several of the hybrid ligands showed a subnanomolar affinity for MOP, improved binding for DOP compared to <b>SBL-OPNT-05</b> & <b>-10</b>, as well as an excellent NTS2-affinity with high selectivity over NTS1. Subsequently, the G<sub>αi1</sub> and β-arrestin-2 pathways were evaluated for all hybrids, along with their stability in rat plasma. Upon MOP activation, <b>SBL-OPNT-13</b> and <b>-18</b> were the least effective at recruiting β-arrestin-2 (<i>E</i><sub>max</sub> = 17 and 12%, respectively), while both compounds were also found to be partial agonists at the G<sub>αi1</sub> pathway, despite improved potency compared to DAMGO. Importantly, these analogs also showed a half-life in rat plasma in excess of 48 h, making them valuable tools for future <i>in vivo</i> investigations.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142218177","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: 10.1021/acsptsci.4c00331
Megan N. Doleschal, Jenna Miller, Sankalp Jain, Alexey V. Zakharov, Ganesha Rai, Anton Simeonov, Bolormaa Baljinnyam, Zhihao Zhuang
Ubiquitination is a post-translational modification that elicits a variety of cellular responses. Deubiquitinases (DUBs) remove ubiquitin moieties from proteins and modulate cellular processes by counteracting the ubiquitin ligase activities. Ubiquitination and deubiquitination processes are tightly regulated by different mechanisms and their dysregulation is associated with many diseases. Discovery of DUB inhibitors could not only lead to therapeutics but also facilitate the understanding of ubiquitination/deubiquitination processes and their regulatory mechanisms. To enable the inhibitor discovery against DUBs, we developed a cell-based DUB assay that utilizes a cell-permeable ubiquitin probe, Biotin-cR10-Ub-PA, to covalently label DUBs in their native cellular environment. Amplified luminescent proximity homogeneous assay (Alpha, specifically AlphaLISA) is utilized to quantitatively assess the capture of the target DUB by the Biotin-cR10-Ub-PA probe. We demonstrated that this new cell-based DUB assay is robust and amenable to high-throughput screening. Human USP15 was selected as a DUB of interest and screened against a library of protease inhibitors as a proof of concept. In addition to the widely adopted pan-DUB inhibitor PR-619, several other DUB inhibitors from the library were also identified as hits. This new DUB assay can be readily adapted for inhibitor discovery against many other human DUBs to identify potent and cell-permeable inhibitors.
{"title":"Cell-Based Covalent-Capture Deubiquitinase Assay for Inhibitor Discovery","authors":"Megan N. Doleschal, Jenna Miller, Sankalp Jain, Alexey V. Zakharov, Ganesha Rai, Anton Simeonov, Bolormaa Baljinnyam, Zhihao Zhuang","doi":"10.1021/acsptsci.4c00331","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00331","url":null,"abstract":"Ubiquitination is a post-translational modification that elicits a variety of cellular responses. Deubiquitinases (DUBs) remove ubiquitin moieties from proteins and modulate cellular processes by counteracting the ubiquitin ligase activities. Ubiquitination and deubiquitination processes are tightly regulated by different mechanisms and their dysregulation is associated with many diseases. Discovery of DUB inhibitors could not only lead to therapeutics but also facilitate the understanding of ubiquitination/deubiquitination processes and their regulatory mechanisms. To enable the inhibitor discovery against DUBs, we developed a cell-based DUB assay that utilizes a cell-permeable ubiquitin probe, Biotin-cR<sub>10</sub>-Ub-PA, to covalently label DUBs in their native cellular environment. Amplified luminescent proximity homogeneous assay (Alpha, specifically AlphaLISA) is utilized to quantitatively assess the capture of the target DUB by the Biotin-cR<sub>10</sub>-Ub-PA probe. We demonstrated that this new cell-based DUB assay is robust and amenable to high-throughput screening. Human USP15 was selected as a DUB of interest and screened against a library of protease inhibitors as a proof of concept. In addition to the widely adopted pan-DUB inhibitor PR-619, several other DUB inhibitors from the library were also identified as hits. This new DUB assay can be readily adapted for inhibitor discovery against many other human DUBs to identify potent and cell-permeable inhibitors.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142218179","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: 10.1021/acsptsci.4c00336
Mohammad Nasrullah, Remant KC, Kyle Nickel, Kylie Parent, Cezary Kucharski, Daniel Nisakar Meenakshi Sundaram, Amarnath Praphakar Rajendran, Xiaoyan Jiang, Joseph Brandwein, Hasan Uludağ
The therapeutic potential of small interfering RNAs (siRNAs) in gene-targeted treatments is substantial, but their suboptimal delivery impedes widespread clinical applications. Critical among these is the inability of siRNAs to traverse the cell membranes due to their anionic nature and high molecular weight. This limitation is particularly pronounced in lymphocytes, which pose additional barriers due to their smaller size and scant cytoplasm. Addressing this, we introduce an innovative lipid-conjugated polyethylenimine lipopolymer platform, engineered for delivery of therapeutic siRNAs into lymphocytes. This system utilizes the cationic nature of the polyethylenimine for forming stable complexes with anionic siRNAs, while the lipid component facilitates cellular entry of siRNA. The resulting lipopolymer/siRNA complexes are termed lipopolymer nanoparticles (LPNPs). We comprehensively profiled the efficacy of this platform in human peripheral blood mononuclear cells (PBMCs) as well as in vitro and in vivo models of acute lymphoblastic leukemia (ALL), emphasizing the inhibition of the oncogenic signal transducer and activator of transcription 5A (STAT5A) gene. The lipopolymers demonstrated high efficiency in delivering siRNA to ALL cell lines (RS4;11 and SUP-B15) and primary patient cells, effectively silencing the STAT5A gene. The resultant gene silencing induced apoptosis and significantly reduced colony formation in vitro. Furthermore, in vivo studies showed a significant decrease in tumor volumes without causing substantial toxicity. The lipopolymers did not induce the secretion of proinflammatory cytokines (IL-6, TNF-α, and INF-γ) in PBMCs from healthy volunteers, underscoring their immune safety profile. Our observations indicate that LPNP-based siRNA delivery systems offer a promising therapeutic approach for ALL in terms of both safety and therapeutic efficacy.
{"title":"Lipopolymer/siRNA Nanoparticles Targeting the Signal Transducer and Activator of Transcription 5A Disrupts Proliferation of Acute Lymphoblastic Leukemia","authors":"Mohammad Nasrullah, Remant KC, Kyle Nickel, Kylie Parent, Cezary Kucharski, Daniel Nisakar Meenakshi Sundaram, Amarnath Praphakar Rajendran, Xiaoyan Jiang, Joseph Brandwein, Hasan Uludağ","doi":"10.1021/acsptsci.4c00336","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00336","url":null,"abstract":"The therapeutic potential of small interfering RNAs (siRNAs) in gene-targeted treatments is substantial, but their suboptimal delivery impedes widespread clinical applications. Critical among these is the inability of siRNAs to traverse the cell membranes due to their anionic nature and high molecular weight. This limitation is particularly pronounced in lymphocytes, which pose additional barriers due to their smaller size and scant cytoplasm. Addressing this, we introduce an innovative lipid-conjugated polyethylenimine lipopolymer platform, engineered for delivery of therapeutic siRNAs into lymphocytes. This system utilizes the cationic nature of the polyethylenimine for forming stable complexes with anionic siRNAs, while the lipid component facilitates cellular entry of siRNA. The resulting lipopolymer/siRNA complexes are termed lipopolymer nanoparticles (LPNPs). We comprehensively profiled the efficacy of this platform in human peripheral blood mononuclear cells (PBMCs) as well as <i>in vitro</i> and <i>in vivo</i> models of acute lymphoblastic leukemia (ALL), emphasizing the inhibition of the oncogenic signal transducer and activator of transcription 5A (<i>STAT5A)</i> gene. The lipopolymers demonstrated high efficiency in delivering siRNA to ALL cell lines (RS4;11 and SUP-B15) and primary patient cells, effectively silencing the <i>STAT5A</i> gene. The resultant gene silencing induced apoptosis and significantly reduced colony formation <i>in vitro</i>. Furthermore, <i>in vivo</i> studies showed a significant decrease in tumor volumes without causing substantial toxicity. The lipopolymers did not induce the secretion of proinflammatory cytokines (IL-6, TNF-α, and INF-γ) in PBMCs from healthy volunteers, underscoring their immune safety profile. Our observations indicate that LPNP-based siRNA delivery systems offer a promising therapeutic approach for ALL in terms of both safety and therapeutic efficacy.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"37 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142218178","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 : 2024-08-14DOI: 10.1021/acsptsci.4c00002
Andras Bilkei-Gorzo, Britta Schurmann, Marion Schneider, Michael Kraemer, Prakash Nidadavolu, Eva C. Beins, Christa E. Müller, Mona Dvir-Ginzberg, Andreas Zimmer
Brain aging is associated with cognitive decline, reduced synaptic plasticity, and altered metabolism. The activity of mechanistic target of rapamycin (mTOR) has a major impact on aging by regulating cellular metabolism. Although reduced mTOR signaling has a general antiaging effect, it can negatively affect the aging brain by reducing synaptogenesis and thus cognitive functions. Increased mTOR activity facilitates aging and is responsible for the amnestic effect of the cannabinoid receptor 1 agonist Δ9-tetrahydrocannabinol (THC) in higher doses. Long-term low-dose Δ9-THC had an antiaging effect on the brain by restoring cognitive abilities and synapse densities in old mice. Whether changes in mTOR signaling and metabolome are associated with its positive effects on the aging brain is an open question. Here, we show that Δ9-THC treatment has a tissue-dependent and dual effect on mTOR signaling and the metabolome. In the brain, Δ9-THC treatment induced a transient increase in mTOR activity and in the levels of amino acids and metabolites involved in energy production, followed by an increased synthesis of synaptic proteins. Unexpectedly, we found a similar reduction in the mTOR activity in adipose tissue and in the level of amino acids and carbohydrate metabolites in blood plasma as in animals on a low-calorie diet. Thus, long-term Δ9-THC treatment first increases the level of energy and synaptic protein production in the brain, followed by a reduction in mTOR activity and metabolic processes in the periphery. Our study suggests that a dual effect on mTOR activity and the metabolome could be the basis for an effective antiaging and pro-cognitive medication.
{"title":"Bidirectional Effect of Long-Term Δ9-Tetrahydrocannabinol Treatment on mTOR Activity and Metabolome","authors":"Andras Bilkei-Gorzo, Britta Schurmann, Marion Schneider, Michael Kraemer, Prakash Nidadavolu, Eva C. Beins, Christa E. Müller, Mona Dvir-Ginzberg, Andreas Zimmer","doi":"10.1021/acsptsci.4c00002","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00002","url":null,"abstract":"Brain aging is associated with cognitive decline, reduced synaptic plasticity, and altered metabolism. The activity of mechanistic target of rapamycin (mTOR) has a major impact on aging by regulating cellular metabolism. Although reduced mTOR signaling has a general antiaging effect, it can negatively affect the aging brain by reducing synaptogenesis and thus cognitive functions. Increased mTOR activity facilitates aging and is responsible for the amnestic effect of the cannabinoid receptor 1 agonist Δ<sup>9</sup>-tetrahydrocannabinol (THC) in higher doses. Long-term low-dose Δ<sup>9</sup>-THC had an antiaging effect on the brain by restoring cognitive abilities and synapse densities in old mice. Whether changes in mTOR signaling and metabolome are associated with its positive effects on the aging brain is an open question. Here, we show that Δ<sup>9</sup>-THC treatment has a tissue-dependent and dual effect on mTOR signaling and the metabolome. In the brain, Δ<sup>9</sup>-THC treatment induced a transient increase in mTOR activity and in the levels of amino acids and metabolites involved in energy production, followed by an increased synthesis of synaptic proteins. Unexpectedly, we found a similar reduction in the mTOR activity in adipose tissue and in the level of amino acids and carbohydrate metabolites in blood plasma as in animals on a low-calorie diet. Thus, long-term Δ<sup>9</sup>-THC treatment first increases the level of energy and synaptic protein production in the brain, followed by a reduction in mTOR activity and metabolic processes in the periphery. Our study suggests that a dual effect on mTOR activity and the metabolome could be the basis for an effective antiaging and pro-cognitive medication.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142218180","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}
We apologize for the above-mentioned mistakes. Other data shown in the published Perspective are correct, and this modification does not affect the overall conclusions of our study. We thank Dr. Sheila DeWitt for drawing our attention to these mistakes and for giving us the opportunity to correct them. This article references 1 other publications. Corrected ref 115 from the published Perspective: This article has not yet been cited by other publications.
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