The loss of peroxisome proliferator-activated receptor gamma (PPARγ) exacerbates pulmonary arterial hypertension (PAH), while its upregulation reduces cell proliferation and vascular remodeling, thereby decreasing PAH severity. SGLT2 inhibitors, developed for type 2 diabetes, might also affect signal transduction in addition to modulating sodium-glucose cotransporters. Pulmonary arterial smooth muscle cells (PASMCs) isolated from patients with idiopathic pulmonary arterial hypertension (IPAH) were treated with three SGLT2 inhibitors, canagliflozin (Cana), dapagliflozin (Dapa), and empagliflozin (Empa), to investigate their antiproliferative effects. To assess the impact of Empa on PPARγ, luciferase reporter assays and siRNA-mediated PPARγ knockdown were employed to examine regulation of the γ-secretase complex and its downstream target Notch3. Therapy involving daily administration of Empa was initiated 21 days after inducing hypoxia-induced PAH in mice. Empa exhibited significant antiproliferative effects on fast-growing IPAH PASMCs. Empa activated PPARγ to prevent formation of the γ-secretase complex, with specific impacts on presenilin enhancer 2 (PEN2), which plays a crucial role in maintaining γ-secretase complex stability, thereby inhibiting Notch3. Similar results were obtained in lung tissue of chronically hypoxic mice. Empa attenuated pulmonary arterial remodeling and right ventricle hypertrophy in a hypoxic PAH mouse model. Moreover, PPARγ expression was significantly decreased and PEN2, and Notch3 levels were increased in lung tissue from PAH patients compared with non-PAH lung tissue. Empa reverses vascular remodeling by activating PPARγ to suppress the γ-secretase-Notch3 axis. We propose Empa as a PPARγ activator and potential therapeutic for PAH.
{"title":"Empagliflozin Attenuates Pulmonary Arterial Remodeling Through Peroxisome Proliferator-Activated Receptor Gamma Activation","authors":"Ying-Ju Lai, Yung-Hsin Yeh, Yen-Lin Huang, Celina De Almeida, Gwo-Jyh Chang, Wei-Jan Chen, Hsao-Hsun Hsu","doi":"10.1021/acsptsci.4c00127","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00127","url":null,"abstract":"The loss of peroxisome proliferator-activated receptor gamma (PPARγ) exacerbates pulmonary arterial hypertension (PAH), while its upregulation reduces cell proliferation and vascular remodeling, thereby decreasing PAH severity. SGLT2 inhibitors, developed for type 2 diabetes, might also affect signal transduction in addition to modulating sodium-glucose cotransporters. Pulmonary arterial smooth muscle cells (PASMCs) isolated from patients with idiopathic pulmonary arterial hypertension (IPAH) were treated with three SGLT2 inhibitors, canagliflozin (Cana), dapagliflozin (Dapa), and empagliflozin (Empa), to investigate their antiproliferative effects. To assess the impact of Empa on PPARγ, luciferase reporter assays and siRNA-mediated PPARγ knockdown were employed to examine regulation of the γ-secretase complex and its downstream target Notch3. Therapy involving daily administration of Empa was initiated 21 days after inducing hypoxia-induced PAH in mice. Empa exhibited significant antiproliferative effects on fast-growing IPAH PASMCs. Empa activated PPARγ to prevent formation of the γ-secretase complex, with specific impacts on presenilin enhancer 2 (PEN2), which plays a crucial role in maintaining γ-secretase complex stability, thereby inhibiting Notch3. Similar results were obtained in lung tissue of chronically hypoxic mice. Empa attenuated pulmonary arterial remodeling and right ventricle hypertrophy in a hypoxic PAH mouse model. Moreover, PPARγ expression was significantly decreased and PEN2, and Notch3 levels were increased in lung tissue from PAH patients compared with non-PAH lung tissue. Empa reverses vascular remodeling by activating PPARγ to suppress the γ-secretase-Notch3 axis. We propose Empa as a PPARγ activator and potential therapeutic for PAH.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"201 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141866131","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 : 2024-08-01DOI: 10.1021/acsptsci.4c00326
Liang Xue, Daniel van Kalken, Erika M. James, Giulia Giammo, Matthew T. Labenski, Susan Cantin, Kelly Fahnoe, Karin Worm, Zhigang Wang, Alan F. Corin
Establishing target engagement is fundamental to effective target-based drug development. It paves the way for efficient medicinal chemistry design and definitive answers about target validation in the clinic. For irreversible targeted covalent inhibitor (TCI) drugs, there is a unique opportunity to establish and quantify the target engagement or occupancy. This is typically accomplished by using a covalent molecular probe, often a TCI analogue, derivatized to allow unoccupied target sites to be tracked; the difference of total sites minus unoccupied sites yields the occupied sites. When such probes are not available or the target is not readily accessible to covalent probes, another approach is needed. Receptor tyrosine-protein kinase erbB-2 (HER2) occupancy by afatinib presents such a case. Available HER2 covalent probes were unable to consistently modify HER2 after sample preparation, resulting in inadequate data. We demonstrate an alternative quantitative probe-free occupancy (PFO) method. It employs the immunoprecipitation of HER2 and direct mass spectrometer analysis of the cysteine-containing peptide that is targeted and covalently occupied by afatinib. Nontarget HER2 peptides provide normalization to the total protein. We show that HER2 occupancy by afatinib correlates directly to the inhibition of the receptor tyrosine kinase activity in NCI-N87 cells in culture and in vivo using those cells in a mouse tumor xenograft mode.
{"title":"A Probe-Free Occupancy Assay to Assess a Targeted Covalent Inhibitor of Receptor Tyrosine-Protein Kinase erbB-2","authors":"Liang Xue, Daniel van Kalken, Erika M. James, Giulia Giammo, Matthew T. Labenski, Susan Cantin, Kelly Fahnoe, Karin Worm, Zhigang Wang, Alan F. Corin","doi":"10.1021/acsptsci.4c00326","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00326","url":null,"abstract":"Establishing target engagement is fundamental to effective target-based drug development. It paves the way for efficient medicinal chemistry design and definitive answers about target validation in the clinic. For irreversible targeted covalent inhibitor (TCI) drugs, there is a unique opportunity to establish and quantify the target engagement or occupancy. This is typically accomplished by using a covalent molecular probe, often a TCI analogue, derivatized to allow unoccupied target sites to be tracked; the difference of total sites minus unoccupied sites yields the occupied sites. When such probes are not available or the target is not readily accessible to covalent probes, another approach is needed. Receptor tyrosine-protein kinase erbB-2 (HER2) occupancy by afatinib presents such a case. Available HER2 covalent probes were unable to consistently modify HER2 after sample preparation, resulting in inadequate data. We demonstrate an alternative quantitative probe-free occupancy (PFO) method. It employs the immunoprecipitation of HER2 and direct mass spectrometer analysis of the cysteine-containing peptide that is targeted and covalently occupied by afatinib. Nontarget HER2 peptides provide normalization to the total protein. We show that HER2 occupancy by afatinib correlates directly to the inhibition of the receptor tyrosine kinase activity in NCI-N87 cells in culture and <i>in vivo</i> using those cells in a mouse tumor xenograft mode.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"202 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141865965","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 : 2024-07-30DOI: 10.1021/acsptsci.4c00295
Nico Bückreiß, Marie Schulz-Fincke, Philipp König, Marco Maccarana, Toin H. van Kuppevelt, Jin-ping Li, Martin Götte, Gerd Bendas
The deregulation of cell surface heparan sulfate proteoglycans (HSPGs) is a main issue of cancer cells for increasing their malignancy. In these terms, the sulfation pattern of HS, created by an orchestrated activity of enzymes balancing a site-specific sulfation, is of key importance. These enzymes are often deregulated by epigenetic processes in cancer, e.g., being silenced by DNA hypermethylation. Here, we address this issue in human breast cancer cell lines aiming to target epigenetic processes to reactivate HS sulfation, shifting HS into an antithrombotic phenotype for which 3-O-sulfation is particularly important. Treatment of MCF-7 and MDA-MB-231 cells with nontoxic concentrations of 5-azacytidine (azacytidine) and 5-fluoro-2′-deoxycytidine (FdCyd) as DNMT inhibitors or vorinostat for targeting HDAC increased HS3-O-sulfation remarkably, as confirmed by fluorescence microscopy, by upregulating HS3-O-sulfotransferases, detected by quantitative real-time polymerase chain reaction and Western blot. Flow cytometry and microscopic approaches confirm that upon inhibitor treatment, increased HS3-O-sulfation improves cell binding to antithrombin, leading to an antithrombotic activity. Nevertheless, only azacytidine- and vorinostat-treated cells display anticoagulative properties, represented by attenuated thrombin formation, a lower activation of human platelet aggregation, or ATP release. In contrast, FdCyd additionally upregulated tissue factor expression in both cell lines, overshadowing the anticoagulant effects of HS, leading to an overall prothrombotic phenotype. Our data provide evidence for the first time that targeting epigenetic processes in HS sulfation is a valuable means to foster anticoagulative cell properties for decreasing malignancy and metastatic potency. These data warrant further investigations to fine-tune epigenetic targeting and to search for potential biomarkers attributed to these activities.
细胞表面硫酸肝素蛋白聚糖（HSPGs）的失调是癌细胞增加其恶性程度的一个主要问题。因此，由酶的协调活动平衡特定位点硫酸化作用而形成的硫酸化模式至关重要。在癌症中，这些酶通常会因表观遗传过程而失调，例如因 DNA 超甲基化而沉默。在此，我们在人类乳腺癌细胞系中解决了这一问题，目的是针对表观遗传过程重新激活HS硫酸化，将HS转变为抗血栓表型，其中3-O-硫酸化尤为重要。用无毒浓度的 5-氮杂胞苷（azacytidine）和 5-氟-2′-脱氧胞苷（FdCyd）（作为 DNMT 抑制剂）或伏立诺他（vorinostat）（用于靶向 HDAC）处理 MCF-7 和 MDA-MB-231 细胞，通过上调 HS3-O 磺化转移酶（通过实时定量聚合酶链式反应和 Western 印迹检测到），显著增加了 HS3-O 的硫酸化，荧光显微镜证实了这一点。流式细胞仪和显微镜方法证实，在抑制剂处理后，HS3-O-硫酸化的增加会改善细胞与抗凝血酶的结合，从而产生抗血栓活性。然而，只有阿扎胞苷（azacytidine）和伏立诺司他（vorinostat）处理过的细胞才具有抗凝特性，表现为凝血酶形成减弱、人血小板聚集活化程度降低或 ATP 释放减少。与此相反，FdCyd 会额外上调这两种细胞系中组织因子的表达，从而掩盖了 HS 的抗凝作用，导致整体的促血栓形成表型。我们的数据首次证明，针对 HS 硫酸化过程中的表观遗传过程是促进细胞抗凝特性以降低恶性程度和转移能力的重要手段。这些数据值得进一步研究，以微调表观遗传学靶向，并寻找这些活动的潜在生物标志物。
{"title":"Epigenetic Targeting of Heparan Sulfate 3-O- and 6-O-Sulfation in Breast Cancer Cells: Prospects for Attenuating Prothrombotic Tumor Cell Activities","authors":"Nico Bückreiß, Marie Schulz-Fincke, Philipp König, Marco Maccarana, Toin H. van Kuppevelt, Jin-ping Li, Martin Götte, Gerd Bendas","doi":"10.1021/acsptsci.4c00295","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00295","url":null,"abstract":"The deregulation of cell surface heparan sulfate proteoglycans (HSPGs) is a main issue of cancer cells for increasing their malignancy. In these terms, the sulfation pattern of HS, created by an orchestrated activity of enzymes balancing a site-specific sulfation, is of key importance. These enzymes are often deregulated by epigenetic processes in cancer, e.g., being silenced by DNA hypermethylation. Here, we address this issue in human breast cancer cell lines aiming to target epigenetic processes to reactivate HS sulfation, shifting HS into an antithrombotic phenotype for which 3-<i>O</i>-sulfation is particularly important. Treatment of MCF-7 and MDA-MB-231 cells with nontoxic concentrations of 5-azacytidine (azacytidine) and 5-fluoro-2′-deoxycytidine (FdCyd) as DNMT inhibitors or vorinostat for targeting HDAC increased HS3-<i>O</i>-sulfation remarkably, as confirmed by fluorescence microscopy, by upregulating HS3-<i>O</i>-sulfotransferases, detected by quantitative real-time polymerase chain reaction and Western blot. Flow cytometry and microscopic approaches confirm that upon inhibitor treatment, increased HS3-<i>O</i>-sulfation improves cell binding to antithrombin, leading to an antithrombotic activity. Nevertheless, only azacytidine- and vorinostat-treated cells display anticoagulative properties, represented by attenuated thrombin formation, a lower activation of human platelet aggregation, or ATP release. In contrast, FdCyd additionally upregulated tissue factor expression in both cell lines, overshadowing the anticoagulant effects of HS, leading to an overall prothrombotic phenotype. Our data provide evidence for the first time that targeting epigenetic processes in HS sulfation is a valuable means to foster anticoagulative cell properties for decreasing malignancy and metastatic potency. These data warrant further investigations to fine-tune epigenetic targeting and to search for potential biomarkers attributed to these activities.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141866135","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 : 2024-07-30DOI: 10.1021/acsptsci.4c00353
Saja Baraghithy, Asaad Gammal, Anna Permyakova, Sharleen Hamad, Radka Kočvarová, Yael Calles, Joseph Tam
The escalating prevalence of obesity and its related disorders represents a daunting global health challenge. Unfortunately, current pharmacological interventions for obesity remain limited and are often associated with debilitating side effects. Against this backdrop, the psychoactive aminoindane derivative 5-methoxy-2-aminoindane (MEAI) has gained considerable attention for its ability to induce a pleasurable, alcohol-like sensation while curbing alcohol consumption. Given the potential impact of MEAI on food addiction and energy homeostasis, we examined its metabolic efficacy on appetite regulation, obesity, and related comorbidities under acute and chronic settings, utilizing a mouse model of diet-induced obesity (DIO). Our results demonstrated that MEAI treatment significantly reduced DIO-induced overweight and adiposity by preserving lean mass and decreasing fat mass. Additionally, MEAI treatment exhibited positive effects on glycemic control by attenuating DIO-induced hyperglycemia, glucose intolerance, and hyperinsulinemia. Furthermore, MEAI reduced DIO-induced hepatic steatosis by decreasing hepatic lipid accumulation and lowering liver triglyceride and cholesterol levels, primarily by inhibiting de novo lipid synthesis. Metabolic phenotyping revealed that MEAI increased energy expenditure and fat utilization while maintaining food consumption similar to that of the vehicle-treated group. Lastly, MEAI normalized voluntary locomotion actions without any overstimulatory effects. These findings provide compelling evidence for the antiobesity effects of MEAI treatment and call for further preclinical testing. In conclusion, our study highlights the potential of MEAI as a novel therapeutic approach for treating obesity and its associated metabolic disorders, offering hope for the development of new treatment options for this global health challenge.
{"title":"5-Methoxy-2-aminoindane Reverses Diet-Induced Obesity and Improves Metabolic Parameters in Mice: A Potential New Class of Antiobesity Therapeutics","authors":"Saja Baraghithy, Asaad Gammal, Anna Permyakova, Sharleen Hamad, Radka Kočvarová, Yael Calles, Joseph Tam","doi":"10.1021/acsptsci.4c00353","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00353","url":null,"abstract":"The escalating prevalence of obesity and its related disorders represents a daunting global health challenge. Unfortunately, current pharmacological interventions for obesity remain limited and are often associated with debilitating side effects. Against this backdrop, the psychoactive aminoindane derivative 5-methoxy-2-aminoindane (MEAI) has gained considerable attention for its ability to induce a pleasurable, alcohol-like sensation while curbing alcohol consumption. Given the potential impact of MEAI on food addiction and energy homeostasis, we examined its metabolic efficacy on appetite regulation, obesity, and related comorbidities under acute and chronic settings, utilizing a mouse model of diet-induced obesity (DIO). Our results demonstrated that MEAI treatment significantly reduced DIO-induced overweight and adiposity by preserving lean mass and decreasing fat mass. Additionally, MEAI treatment exhibited positive effects on glycemic control by attenuating DIO-induced hyperglycemia, glucose intolerance, and hyperinsulinemia. Furthermore, MEAI reduced DIO-induced hepatic steatosis by decreasing hepatic lipid accumulation and lowering liver triglyceride and cholesterol levels, primarily by inhibiting <i>de novo</i> lipid synthesis. Metabolic phenotyping revealed that MEAI increased energy expenditure and fat utilization while maintaining food consumption similar to that of the vehicle-treated group. Lastly, MEAI normalized voluntary locomotion actions without any overstimulatory effects. These findings provide compelling evidence for the antiobesity effects of MEAI treatment and call for further preclinical testing. In conclusion, our study highlights the potential of MEAI as a novel therapeutic approach for treating obesity and its associated metabolic disorders, offering hope for the development of new treatment options for this global health challenge.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"50 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141866132","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 : 2024-07-26DOI: 10.1021/acsptsci.4c00119
Ana Semeano, Rian Garland, Alessandro Bonifazi, Kuo Hao Lee, John Famiglietti, Wenqi Zhang, Yoon Jae Jo, Francisco O. Battiti, Lei Shi, Amy Hauck Newman, Hideaki Yano
Bitopic ligands bind both orthosteric and allosteric or secondary binding sites within the same receptor, often resulting in an improvement of receptor selectivity, potency, and efficacy. In particular, for both agonists and antagonists of the dopamine D2 and D3 receptors (D2R and D3R), the primary therapeutic targets for several neurological and neuropsychiatric disorders, bitopic ligand design has proved advantageous in achieving better pharmacological profiles in vitro. Although the two pharmacophores within a bitopic ligand are typically considered the main drivers of conformational change for a receptor, the role of the linker that connects the two has not yet been systematically studied for its relevance in receptor activity profiles. Here, we present a comprehensive analysis of sumanirole and PF592,379-based indole-containing bitopic compounds in agonist activity at D2R and D3R, with a focus on linker chemical space and stereochemistry through testing six distinct chirally resolved linkers and a simple aliphatic linker. The structure activity relationships (SARs) of these linkers are examined extensively, beyond the conventional level, by characterizing the activation of all putative transducers over a 44 min time course. Our multiparametric analysis reveals previously unappreciated specific linker-dependent effects on primary pharmacophores, receptors, transducer activation kinetics, and bias, highlighting the utility of this comprehensive approach and the significance of the linker type in shaping transducer bias profiles.
{"title":"Linkers in Bitopic Agonists Shape Bias Profile among Transducers for the Dopamine D2 and D3 Receptors","authors":"Ana Semeano, Rian Garland, Alessandro Bonifazi, Kuo Hao Lee, John Famiglietti, Wenqi Zhang, Yoon Jae Jo, Francisco O. Battiti, Lei Shi, Amy Hauck Newman, Hideaki Yano","doi":"10.1021/acsptsci.4c00119","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00119","url":null,"abstract":"Bitopic ligands bind both orthosteric and allosteric or secondary binding sites within the same receptor, often resulting in an improvement of receptor selectivity, potency, and efficacy. In particular, for both agonists and antagonists of the dopamine D2 and D3 receptors (D2R and D3R), the primary therapeutic targets for several neurological and neuropsychiatric disorders, bitopic ligand design has proved advantageous in achieving better pharmacological profiles <i>in vitro</i>. Although the two pharmacophores within a bitopic ligand are typically considered the main drivers of conformational change for a receptor, the role of the linker that connects the two has not yet been systematically studied for its relevance in receptor activity profiles. Here, we present a comprehensive analysis of sumanirole and PF592,379-based indole-containing bitopic compounds in agonist activity at D2R and D3R, with a focus on linker chemical space and stereochemistry through testing six distinct chirally resolved linkers and a simple aliphatic linker. The structure activity relationships (SARs) of these linkers are examined extensively, beyond the conventional level, by characterizing the activation of all putative transducers over a 44 min time course. Our multiparametric analysis reveals previously unappreciated specific linker-dependent effects on primary pharmacophores, receptors, transducer activation kinetics, and bias, highlighting the utility of this comprehensive approach and the significance of the linker type in shaping transducer bias profiles.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"28 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141772073","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}
Riboflavin transporter 3 (RFVT3) represents a potential cardioprotective biotarget in energetic metabolism reprogramming after myocardial infarction/reperfusion (MI/R). This study investigated the feasibility of noninvasive real-time quantification of RFVT3 expression after MI/R with an radiolabeled probe 18F-RFTA in a preclinical rat model of MI/R. The tracer 18F-RFTA was radio-synthesized manually and characterized on the subjects of radiolabeling yield, radiochemical purity, and stability in vivo. MI/R and sham-operated rat models were confirmed by cardiac magnetic resonance imaging (cMRI) and single-photon-emission computed tomography (SPECT) myocardial perfusion imaging (MPI) with technetium-99m sestamibi (99mTc-MIBI). Positron emission tomography (PET) imaging of MI/R and sham-operated rat models were conducted with 18F-RFTA. Ex vivo autoradiography and RFVT3 immunohistochemical (IHC) staining were conducted to verify the RFVT3 expression in infarcted and normal myocardium. 18F-RFTA injection was prepared with high radiochemical purity (>95%) and kept stable in vitro and in vivo. 18F-RFTA PET revealed significant uptake in the infarcted myocardium at 8 h after reperfusion, as confirmed by lower 99mTc-MIBI perfusion and decreased intensity of cMRI. Conversely, there were only the tiniest uptakes in the normal myocardium and blocked infarcted myocardium, which was further corroborated by ex vivo autoradiography. The RFVT3 expression was further confirmed by IHC staining in the infarcted and normal myocardium. We first demonstrate the feasibility of imaging RFVT3 in infarcted myocardium. 18F-RFTA is an encouraging PET probe for imaging cardioprotective biotarget RFVT3 in mitochondrial energetic metabolism reprogramming after myocardial infarction. Noninvasive imaging of cardioprotective biotarget RFVT3 has potential value in the diagnosis and therapy of patients with MI.
{"title":"MicroPET Imaging of Riboflavin Transporter 3 Expression in Myocardial Infarction/Reperfusion Rat Models with Radiofluorinated Riboflavin","authors":"Jindian Li, Xingfang Hong, Yingxi Chen, Bin Yin, Hongzhang Yang, Changrong Shi, Xinying Zeng, Deliang Zhang, Zhide Guo, Xianzhong Zhang","doi":"10.1021/acsptsci.4c00175","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00175","url":null,"abstract":"Riboflavin transporter 3 (RFVT3) represents a potential cardioprotective biotarget in energetic metabolism reprogramming after myocardial infarction/reperfusion (MI/R). This study investigated the feasibility of noninvasive real-time quantification of RFVT3 expression after MI/R with an radiolabeled probe <sup>18</sup>F-RFTA in a preclinical rat model of MI/R. The tracer <sup>18</sup>F-RFTA was radio-synthesized manually and characterized on the subjects of radiolabeling yield, radiochemical purity, and stability <i>in vivo</i>. MI/R and sham-operated rat models were confirmed by cardiac magnetic resonance imaging (cMRI) and single-photon-emission computed tomography (SPECT) myocardial perfusion imaging (MPI) with technetium-99m sestamibi (<sup>99m</sup>Tc-MIBI). Positron emission tomography (PET) imaging of MI/R and sham-operated rat models were conducted with <sup>18</sup>F-RFTA. <i>Ex vivo</i> autoradiography and RFVT3 immunohistochemical (IHC) staining were conducted to verify the RFVT3 expression in infarcted and normal myocardium. <sup>18</sup>F-RFTA injection was prepared with high radiochemical purity (>95%) and kept stable <i>in vitro</i> and <i>in vivo</i>. <sup>18</sup>F-RFTA PET revealed significant uptake in the infarcted myocardium at 8 h after reperfusion, as confirmed by lower <sup>99m</sup>Tc-MIBI perfusion and decreased intensity of cMRI. Conversely, there were only the tiniest uptakes in the normal myocardium and blocked infarcted myocardium, which was further corroborated by <i>ex vivo</i> autoradiography. The RFVT3 expression was further confirmed by IHC staining in the infarcted and normal myocardium. We first demonstrate the feasibility of imaging RFVT3 in infarcted myocardium. <sup>18</sup>F-RFTA is an encouraging PET probe for imaging cardioprotective biotarget RFVT3 in mitochondrial energetic metabolism reprogramming after myocardial infarction. Noninvasive imaging of cardioprotective biotarget RFVT3 has potential value in the diagnosis and therapy of patients with MI.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"17 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141772189","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 : 2024-07-25DOI: 10.1021/acsptsci.4c00277
Yi-Hua Chiang, Erin C. Berthold, Michelle A. Kuntz, Siva Rama Raju Kanumuri, Alexandria S. Senetra, Sushobhan Mukhopadhyay, Aidan J. Hampson, Christopher R. McCurdy, Abhisheak Sharma
This study reports the steady-state pharmacokinetic parameters for mitragynine and characterizes its elimination in male and female rats. Four male and female rats were dosed q12h with 40 mg/kg, and orally administered mitragynine for 5 and 6 days, respectively. Using a validated ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method, the plasma concentrations of mitragynine, its metabolites (7-hydroxymitragynine, 9-hydroxycorynantheidine, and mitragynine acid), and a non-CYP oxidation product (3-dehydromitragynine) were determined at various time points. Sex differences in pharmacokinetics were observed, with females demonstrating significantly higher systemic exposure of mitragynine than males. The mitragynine area under the curve normalized by the dose interval (AUC/τ) was 6741.6 ± 869.5 h*ng/mL in female rats and 1808.9 ± 191.3 h*ng/mL in males (p < 0.05). Both sexes produced similar metabolite profiles; the major metabolites were mitragynine acid and 9-hydroxycorynantheidine. 7-Hydroxymitragynine was a minor metabolite. However, higher exposure (AUCs) and the maximum plasma concentrations (Cmax) of active metabolites, 7-hydroxymitragynine and 9-hydroxycorynantheidine, were observed in female rats and exhibited substantial sex differences. Renal clearance of mitragynine (CLr) was low (0.64 ± 0.3 mL/h in males and 0.98 ± 0.4 mL/h in females), and unchanged mitragynine accounted for <1% of the dose excreted in feces (both sexes). The clinical chemistry, complete blood count, and hematological test results reported no abnormal hematological findings after multiple dosing in either sex.
{"title":"Multiple-Dose Pharmacokinetics and Safety of Mitragynine, the Major Alkaloid of Kratom, in Rats","authors":"Yi-Hua Chiang, Erin C. Berthold, Michelle A. Kuntz, Siva Rama Raju Kanumuri, Alexandria S. Senetra, Sushobhan Mukhopadhyay, Aidan J. Hampson, Christopher R. McCurdy, Abhisheak Sharma","doi":"10.1021/acsptsci.4c00277","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00277","url":null,"abstract":"This study reports the steady-state pharmacokinetic parameters for mitragynine and characterizes its elimination in male and female rats. Four male and female rats were dosed q12h with 40 mg/kg, and orally administered mitragynine for 5 and 6 days, respectively. Using a validated ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method, the plasma concentrations of mitragynine, its metabolites (7-hydroxymitragynine, 9-hydroxycorynantheidine, and mitragynine acid), and a non-CYP oxidation product (3-dehydromitragynine) were determined at various time points. Sex differences in pharmacokinetics were observed, with females demonstrating significantly higher systemic exposure of mitragynine than males. The mitragynine area under the curve normalized by the dose interval (AUC/τ) was 6741.6 ± 869.5 h*ng/mL in female rats and 1808.9 ± 191.3 h*ng/mL in males (<i>p</i> < 0.05). Both sexes produced similar metabolite profiles; the major metabolites were mitragynine acid and 9-hydroxycorynantheidine. 7-Hydroxymitragynine was a minor metabolite. However, higher exposure (AUCs) and the maximum plasma concentrations (<i>C</i><sub>max</sub>) of active metabolites, 7-hydroxymitragynine and 9-hydroxycorynantheidine, were observed in female rats and exhibited substantial sex differences. Renal clearance of mitragynine (CL<sub>r</sub>) was low (0.64 ± 0.3 mL/h in males and 0.98 ± 0.4 mL/h in females), and unchanged mitragynine accounted for <1% of the dose excreted in feces (both sexes). The clinical chemistry, complete blood count, and hematological test results reported no abnormal hematological findings after multiple dosing in either sex.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"177 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141786027","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 : 2024-07-24DOI: 10.1021/acsptsci.4c00308
Jiaqi Huang, Aishik Chakraborty, Lakshmi Suchitra Tadepalli, Arghya Paul
DNA nanostructures have been widely researched in recent years as emerging biomedical materials for drug delivery, biosensing, and cancer therapy, in addition to their hereditary function. Multiple precisely designed single-strand DNAs can be fabricated into complex, three-dimensional DNA nanostructures through a simple self-assembly process. Among all of the synthetic DNA nanostructures, tetrahedral DNA nanostructures (TDNs) stand out as the most promising biomedical nanomaterial. TDNs possess the merits of structural stability, cell membrane permeability, and natural biocompatibility due to their compact structures and DNA origin. In addition to their inherent advantages, TDNs were shown to have great potential in delivering therapeutic agents through multiple functional modifications. As a multifunctional material, TDNs have enabled innovative pharmaceutical applications, including antimicrobial therapy, anticancer treatment, immune modulation, and cartilage regeneration. Given the rapid development of TDNs in the biomedical field, it is critical to understand how to successfully produce and fine-tune the properties of TDNs for specific therapeutic needs and clinical translation. This article provides insights into the synthesis and functionalization of TDNs and summarizes the approaches for TDN-based therapeutics delivery as well as their broad applications in the field of pharmaceutics and nanomedicine, challenges, and future directions.
DNA 纳米结构作为一种新兴的生物医学材料，除了具有遗传功能外，还可用于药物输送、生物传感和癌症治疗，近年来已被广泛研究。通过简单的自组装过程，可以将多条精确设计的单链 DNA 制成复杂的三维 DNA 纳米结构。在所有合成的 DNA 纳米结构中，四面体 DNA 纳米结构（TDNs）是最有前途的生物医学纳米材料。四面体 DNA 纳米结构因其紧凑的结构和 DNA 起源而具有结构稳定性、细胞膜渗透性和天然生物相容性等优点。除了其固有的优点外，TDNs 还通过多种功能修饰，在递送治疗药物方面具有巨大潜力。作为一种多功能材料，TDNs 实现了创新药物应用，包括抗菌治疗、抗癌治疗、免疫调节和软骨再生。鉴于 TDNs 在生物医学领域的快速发展，了解如何成功生产和微调 TDNs 的特性以满足特定的治疗需求和临床转化至关重要。本文深入探讨了 TDNs 的合成和功能化，总结了基于 TDN 的治疗递送方法及其在制药学和纳米医学领域的广泛应用、挑战和未来发展方向。
{"title":"Adoption of a Tetrahedral DNA Nanostructure as a Multifunctional Biomaterial for Drug Delivery","authors":"Jiaqi Huang, Aishik Chakraborty, Lakshmi Suchitra Tadepalli, Arghya Paul","doi":"10.1021/acsptsci.4c00308","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00308","url":null,"abstract":"DNA nanostructures have been widely researched in recent years as emerging biomedical materials for drug delivery, biosensing, and cancer therapy, in addition to their hereditary function. Multiple precisely designed single-strand DNAs can be fabricated into complex, three-dimensional DNA nanostructures through a simple self-assembly process. Among all of the synthetic DNA nanostructures, tetrahedral DNA nanostructures (TDNs) stand out as the most promising biomedical nanomaterial. TDNs possess the merits of structural stability, cell membrane permeability, and natural biocompatibility due to their compact structures and DNA origin. In addition to their inherent advantages, TDNs were shown to have great potential in delivering therapeutic agents through multiple functional modifications. As a multifunctional material, TDNs have enabled innovative pharmaceutical applications, including antimicrobial therapy, anticancer treatment, immune modulation, and cartilage regeneration. Given the rapid development of TDNs in the biomedical field, it is critical to understand how to successfully produce and fine-tune the properties of TDNs for specific therapeutic needs and clinical translation. This article provides insights into the synthesis and functionalization of TDNs and summarizes the approaches for TDN-based therapeutics delivery as well as their broad applications in the field of pharmaceutics and nanomedicine, challenges, and future directions.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141772190","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 : 2024-07-22DOI: 10.1021/acsptsci.4c00256
Xin Hu, Paul Shinn, Zina Itkin, Lin Ye, Ya-Qin Zhang, Min Shen, Stephanie Ford-Scheimer, Matthew D. Hall
As part of the NIH Helping to End Addiction Long-term (HEAL) Initiative, the National Center for Advancing Translational Sciences is dedicated to the development of new pharmacological tools and investigational drugs for managing and treating pain as well as the prevention and treatment of opioid misuse and addiction. In line with these objectives, we created a comprehensive, annotated small molecule library including drugs, probes, and tool compounds that act on published pain- and addiction-relevant targets. Nearly 3000 small molecules associated with approximately 200 known and hypothesized HEAL targets have been assembled, curated, and annotated in one collection. Physical samples of the library compounds have been acquired and plated in 1536-well format, enabling a rapid and efficient high-throughput screen against a wide range of assays. The creation of the HEAL Targets and Compounds Library, coupled with an integrated computational platform for AI-driven machine learning, structural modeling, and virtual screening, provides a valuable source for strategic drug repurposing, innovative profiling, and hypothesis testing of novel targets related to pain and opioid use disorder (OUD). The library is available to investigators for screening pain and OUD-relevant phenotypes.
作为美国国立卫生研究院（NIH）"帮助戒除毒瘾长期计划"（HEAL）的一部分，美国国家转化科学促进中心（National Center for Advancing Translational Sciences）致力于开发新的药理学工具和研究药物，用于控制和治疗疼痛以及预防和治疗阿片类药物滥用和成瘾。根据这些目标，我们创建了一个全面的、附有注释的小分子化合物库，其中包括作用于已公布的疼痛和成瘾相关靶点的药物、探针和工具化合物。与大约 200 个已知和假设的 HEAL 靶点相关的近 3000 种小分子化合物已被收集、整理和注释在一个集合中。该化合物库的物理样本已采集完毕，并以 1536 孔的格式进行了培养，从而能够针对各种检测方法进行快速、高效的高通量筛选。HEAL 靶点和化合物库的建立，加上人工智能驱动的机器学习、结构建模和虚拟筛选的集成计算平台，为疼痛和阿片类药物使用障碍 (OUD) 相关新靶点的战略药物再利用、创新性分析和假设检验提供了宝贵的资源。研究人员可利用该库筛选与疼痛和阿片类药物使用障碍相关的表型。
{"title":"A Comprehensive Collection of Pain and Opioid Use Disorder Compounds for High-Throughput Screening and Artificial Intelligence-Driven Drug Discovery","authors":"Xin Hu, Paul Shinn, Zina Itkin, Lin Ye, Ya-Qin Zhang, Min Shen, Stephanie Ford-Scheimer, Matthew D. Hall","doi":"10.1021/acsptsci.4c00256","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00256","url":null,"abstract":"As part of the NIH Helping to End Addiction Long-term (HEAL) Initiative, the National Center for Advancing Translational Sciences is dedicated to the development of new pharmacological tools and investigational drugs for managing and treating pain as well as the prevention and treatment of opioid misuse and addiction. In line with these objectives, we created a comprehensive, annotated small molecule library including drugs, probes, and tool compounds that act on published pain- and addiction-relevant targets. Nearly 3000 small molecules associated with approximately 200 known and hypothesized HEAL targets have been assembled, curated, and annotated in one collection. Physical samples of the library compounds have been acquired and plated in 1536-well format, enabling a rapid and efficient high-throughput screen against a wide range of assays. The creation of the HEAL Targets and Compounds Library, coupled with an integrated computational platform for AI-driven machine learning, structural modeling, and virtual screening, provides a valuable source for strategic drug repurposing, innovative profiling, and hypothesis testing of novel targets related to pain and opioid use disorder (OUD). The library is available to investigators for screening pain and OUD-relevant phenotypes.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"93 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141754025","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}
Claudin18.2 (CLDN18.2) has emerged as a significant target in the treatment of advanced gastric cancer. The screening of patients positive for CLDN18.2 is crucial for the effective application of targeted therapies specific to CLND18.2. In this study, we developed a novel nanobody-based probe, [99mTc]Tc-PHG102, for use in nuclear medicine. We analyzed its radiochemical yield and stability to ensure accurate probe characterization. Additionally, we assessed the probe’s affinity and specificity toward the CLDN18.2 target and evaluated its efficacy in the BGC82318.2 xenograft model for SPECT/CT imaging of gastric cancer. The binding of [99mTc]Tc-PHG102 to HEK-293T18.2 and BGC82318.2 cells was notably higher than its binding to HEK-293T18.1, HEK-293T, and BGC823 cells, with bound values of 12.87 ± 1.46%, 6.16 ± 0.34%, 1.25 ± 0.22%, 1.14 ± 0.26%, and 1.32 ± 0.07% AD, respectively. The binding ability of [99mTc]Tc-PHG102 was significantly different between CLDN18.2-positive and negative cells (P < 0.001). Imaging results demonstrated a time-dependent tumor accumulation of the radiotracer. Notably, at 0.5 h postinjection, rapid accumulation was observed with an average tumor uptake of 4.63 ± 0.81% ID/cc (n = 3), resulting in clear tumor visualization. By 1 h postinjection, as [99mTc]Tc-PHG102 was rapidly metabolized, a decrease in uptake by other organs was noted. Preliminary clinical imaging trials further confirmed the safety and effectiveness of the probe, indicating specificity for lesions expressing CLDN18.2 in gastric cancer and favorable in vivo metabolic properties. In conclusion, the nanobody-based probe [99mTc]Tc-PHG102 proves to be a safe and effective tool for detecting CLDN18.2 expression levels in gastric cancer tumors and for screening CLDN18.2-positive patients.
{"title":"Claudin18.2-Targeted SPECT/CT Imaging for Gastric Cancer: Preclinical Evaluation and Clinical Translation of the 99mTc-Labeled Nanobody (PHG102) Radiotracer","authors":"Zhidong Bai, Xin Xie, Chenzhen Li, Yuchen Wang, Yuanbo Wang, Huijie Li, Rui Gao, Bing Jia","doi":"10.1021/acsptsci.4c00280","DOIUrl":"https://doi.org/10.1021/acsptsci.4c00280","url":null,"abstract":"Claudin18.2 (CLDN18.2) has emerged as a significant target in the treatment of advanced gastric cancer. The screening of patients positive for CLDN18.2 is crucial for the effective application of targeted therapies specific to CLND18.2. In this study, we developed a novel nanobody-based probe, [<sup>99m</sup>Tc]Tc-PHG102, for use in nuclear medicine. We analyzed its radiochemical yield and stability to ensure accurate probe characterization. Additionally, we assessed the probe’s affinity and specificity toward the CLDN18.2 target and evaluated its efficacy in the BGC823<sup>18.2</sup> xenograft model for SPECT/CT imaging of gastric cancer. The binding of [<sup>99m</sup>Tc]Tc-PHG102 to HEK-293T<sup>18.2</sup> and BGC823<sup>18.2</sup> cells was notably higher than its binding to HEK-293T<sup>18.1</sup>, HEK-293T, and BGC823 cells, with bound values of 12.87 ± 1.46%, 6.16 ± 0.34%, 1.25 ± 0.22%, 1.14 ± 0.26%, and 1.32 ± 0.07% AD, respectively. The binding ability of [<sup>99m</sup>Tc]Tc-PHG102 was significantly different between CLDN18.2-positive and negative cells (<i>P</i> < 0.001). Imaging results demonstrated a time-dependent tumor accumulation of the radiotracer. Notably, at 0.5 h postinjection, rapid accumulation was observed with an average tumor uptake of 4.63 ± 0.81% ID/cc (<i>n</i> = 3), resulting in clear tumor visualization. By 1 h postinjection, as [<sup>99m</sup>Tc]Tc-PHG102 was rapidly metabolized, a decrease in uptake by other organs was noted. Preliminary clinical imaging trials further confirmed the safety and effectiveness of the probe, indicating specificity for lesions expressing CLDN18.2 in gastric cancer and favorable in vivo metabolic properties. In conclusion, the nanobody-based probe [<sup>99m</sup>Tc]Tc-PHG102 proves to be a safe and effective tool for detecting CLDN18.2 expression levels in gastric cancer tumors and for screening CLDN18.2-positive patients.","PeriodicalId":501473,"journal":{"name":"ACS Pharmacology & Translational Science","volume":"43 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141754026","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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