Pub Date : 2024-09-17DOI: 10.1124/molpharm.124.000912
Carrie O'Connor, Mathew Schneider, Jade M Katinas, Md Junayed Nayeen, Khushbu Shah, Tejashree Magdum, Abhishekh Sharma, Seongho Kim, Xun Bao, Jing Li, Charles E Dann, Aleem Gangjee, Larry H Matherly, Zhanjun Hou
Folate-dependent one-carbon (C1) metabolism encompasses distinct cytosolic and mitochondrial pathways connected by an interchange among serine, glycine, and formate. In both the cytosol and mitochondria, folates exist as polyglutamates, with polyglutamylation catalyzed by folylpolyglutamate synthetase (FPGS), including cytosolic and mitochondrial isoforms. Serine is metabolized by serine hydroxymethyltransferase (SHMT)2 in the mitochondria and generates glycine and C1 units for cellular biosynthesis in the cytosol. AGF347 is a novel pyrrolo[3,2-day]pyrimidine antifolate that targets SHMT2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. FPGS is expressed in primary pancreatic cancer specimens, and FPGS levels correlate with in vitro efficacies of AGF347 toward human pancreatic cancer cells. MIA PaCa-2 pancreatic cancer cells with CRISPR knockout of FPGS were engineered to express doxycycline-inducible FPGS exclusively in the cytosol (cFPGS) or in both the cytosol and mitochondria (mFPGS). Folate and AGF347 accumulations increased in both the cytosol and mitochondria with increased mFPGS but were restricted to the cytosol with cFPGS. AGF347-Glu5 inhibited SHMT2 ∼19-fold greater than AGF347 By metabolomics analysis, mFPGS stimulated the C1 flux from serine in the mitochondria and de novo purine and dTTP synthesis far greater than cFPGS. mFPGS enhanced in vitro inhibition of MIA PaCa-2 cell proliferation by AGF347 (∼30-fold) more than cFPGS (∼4.9-fold). Similar results were seen with other pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320); however, elevated mFPGS adversely impacted inhibition by the nonclassical SHMT2/SHMT1 inhibitor SHIN1. These results suggest a critical role of mFPGS levels in determining antitumor efficacies of mitochondrial-targeted pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer. SIGNIFICANCE STATEMENT: AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets serine hydroxymethyltransferase (SHMT)2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. AGF347 accumulation increases with folylpolyglutamate synthetase (FPGS) levels in both the cytosol and mitochondria. Increased mitochondrial FPGS stimulated one-carbon metabolic fluxes in the cytosol and mitochondria and substantially enhanced in vitro inhibition of pancreatic cancer cells by AGF347. Mitochondrial FPGS levels play important roles in determining the antitumor efficacies of pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer.
{"title":"Role of Mitochondrial and Cytosolic Folylpolyglutamate Synthetase in One-Carbon Metabolism and Antitumor Efficacy of Mitochondrial-Targeted Antifolates.","authors":"Carrie O'Connor, Mathew Schneider, Jade M Katinas, Md Junayed Nayeen, Khushbu Shah, Tejashree Magdum, Abhishekh Sharma, Seongho Kim, Xun Bao, Jing Li, Charles E Dann, Aleem Gangjee, Larry H Matherly, Zhanjun Hou","doi":"10.1124/molpharm.124.000912","DOIUrl":"10.1124/molpharm.124.000912","url":null,"abstract":"<p><p>Folate-dependent one-carbon (C1) metabolism encompasses distinct cytosolic and mitochondrial pathways connected by an interchange among serine, glycine, and formate. In both the cytosol and mitochondria, folates exist as polyglutamates, with polyglutamylation catalyzed by folylpolyglutamate synthetase (FPGS), including cytosolic and mitochondrial isoforms. Serine is metabolized by serine hydroxymethyltransferase (SHMT)2 in the mitochondria and generates glycine and C1 units for cellular biosynthesis in the cytosol. <b>AGF347</b> is a novel pyrrolo[3,<i>2-day</i>]pyrimidine antifolate that targets SHMT2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. FPGS is expressed in primary pancreatic cancer specimens, and FPGS levels correlate with in vitro efficacies of <b>AGF347</b> toward human pancreatic cancer cells. MIA PaCa-2 pancreatic cancer cells with CRISPR knockout of FPGS were engineered to express doxycycline-inducible FPGS exclusively in the cytosol (cFPGS) or in both the cytosol and mitochondria (mFPGS). Folate and <b>AGF347</b> accumulations increased in both the cytosol and mitochondria with increased mFPGS but were restricted to the cytosol with cFPGS. <b>AGF347-Glu<sub>5</sub></b> inhibited SHMT2 ∼19-fold greater than <b>AGF347</b> By metabolomics analysis, mFPGS stimulated the C1 flux from serine in the mitochondria and de novo purine and dTTP synthesis far greater than cFPGS. mFPGS enhanced in vitro inhibition of MIA PaCa-2 cell proliferation by <b>AGF347</b> (∼30-fold) more than cFPGS (∼4.9-fold). Similar results were seen with other pyrrolo[3,<i>2-d</i>]pyrimidine antifolates (<b>AGF291, AGF320</b>); however, elevated mFPGS adversely impacted inhibition by the nonclassical SHMT2/SHMT1 inhibitor SHIN1. These results suggest a critical role of mFPGS levels in determining antitumor efficacies of mitochondrial-targeted pyrrolo[3,<i>2-d</i>]pyrimidine antifolates for pancreatic cancer. SIGNIFICANCE STATEMENT: <b>AGF347</b> is a novel pyrrolo[3,<i>2-d</i>]pyrimidine antifolate that targets serine hydroxymethyltransferase (SHMT)2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. <b>AGF347</b> accumulation increases with folylpolyglutamate synthetase (FPGS) levels in both the cytosol and mitochondria. Increased mitochondrial FPGS stimulated one-carbon metabolic fluxes in the cytosol and mitochondria and substantially enhanced in vitro inhibition of pancreatic cancer cells by <b>AGF347</b>. Mitochondrial FPGS levels play important roles in determining the antitumor efficacies of pyrrolo[3,<i>2-d</i>]pyrimidine antifolates for pancreatic cancer.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11413923/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141759764","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-17DOI: 10.1124/molpharm.124.000932
Kelly A Manthei, Grace E Tremonti, Louise Chang, Akseli Niemelä, Laura Giorgi, Artturi Koivuniemi, John Joseph Grubb Tesmer
Lecithin:cholesterol acyltransferase (LCAT) deficiencies represent severe disorders characterized by aberrant cholesterol esterification in plasma, leading to life-threatening conditions. This study investigates the efficacy of Compound 2, a piperidinyl pyrazolopyridine allosteric activator that binds the membrane-binding domain of LCAT, in rescuing the activity of LCAT variants associated with disease. The variants K218N, N228K, and G230R, all located in the cap and lid domains of LCAT, demonstrated notable activity restoration in response to Compound 2. Molecular dynamics simulations and structural modeling indicate that these mutations disrupt the lid and membrane binding domain, with Compound 2 potentially dampening these structural alterations. Conversely, variants such as M252K and F382V in the cap and α/β-hydrolase domain, respectively, exhibited limited or no rescue by Compound 2. Future research should prioritize in vivo investigations that would validate the therapeutic potential of Compound 2 and related activators in familial LCAT deficiency patients with mutations in the cap and lid of the enzyme. SIGNIFICANCE STATEMENT: Lecithin:cholesterol acyltranferase (LCAT) catalyzes the first step of reverse cholesterol transport, namely the esterification of cholesterol in high density lipoprotein particles. Somatic mutations in LCAT lead to excess cholesterol in blood plasma and, in severe cases, kidney failure. In this study, we show that recently discovered small molecule activators can rescue function in LCAT-deficient variants when the mutations occur in the lid and cap domains of the enzyme.
{"title":"Rescue of Familial Lecithin:Cholesterol Acyltranferase Deficiency Mutations with an Allosteric Activator.","authors":"Kelly A Manthei, Grace E Tremonti, Louise Chang, Akseli Niemelä, Laura Giorgi, Artturi Koivuniemi, John Joseph Grubb Tesmer","doi":"10.1124/molpharm.124.000932","DOIUrl":"10.1124/molpharm.124.000932","url":null,"abstract":"<p><p>Lecithin:cholesterol acyltransferase (LCAT) deficiencies represent severe disorders characterized by aberrant cholesterol esterification in plasma, leading to life-threatening conditions. This study investigates the efficacy of Compound 2, a piperidinyl pyrazolopyridine allosteric activator that binds the membrane-binding domain of LCAT, in rescuing the activity of LCAT variants associated with disease. The variants K218N, N228K, and G230R, all located in the cap and lid domains of LCAT, demonstrated notable activity restoration in response to Compound 2. Molecular dynamics simulations and structural modeling indicate that these mutations disrupt the lid and membrane binding domain, with Compound 2 potentially dampening these structural alterations. Conversely, variants such as M252K and F382V in the cap and <i>α</i>/<i>β</i>-hydrolase domain, respectively, exhibited limited or no rescue by Compound 2. Future research should prioritize in vivo investigations that would validate the therapeutic potential of Compound 2 and related activators in familial LCAT deficiency patients with mutations in the cap and lid of the enzyme. SIGNIFICANCE STATEMENT: Lecithin:cholesterol acyltranferase (LCAT) catalyzes the first step of reverse cholesterol transport, namely the esterification of cholesterol in high density lipoprotein particles. Somatic mutations in LCAT lead to excess cholesterol in blood plasma and, in severe cases, kidney failure. In this study, we show that recently discovered small molecule activators can rescue function in LCAT-deficient variants when the mutations occur in the lid and cap domains of the enzyme.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11413911/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141996146","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-17DOI: 10.1124/molpharm.124.000974
David Salom, Arum Wu, Chang C Liu, Krzysztof Palczewski
The family of human G protein-coupled receptors (GPCRs) comprises about 800 different members, with about 35% of current pharmaceutical drugs targeting GPCRs. However, GPCR structural biology, necessary for structure-guided drug design, has lagged behind that of other membrane proteins, and it was not until the year 2000 when the first crystal structure of a GPCR (rhodopsin) was solved. Starting in 2007, the determination of additional GPCR structures was facilitated by protein engineering, new crystallization techniques, complexation with antibody fragments, and other strategies. More recently, the use of camelid heavy-chain-only antibody fragments (nanobodies) as crystallographic chaperones has revolutionized the field of GPCR structural biology, aiding in the determination of more than 340 GPCR structures to date. In most cases, the GPCR structures solved as complexes with nanobodies (Nbs) have revealed the binding mode of cognate or non-natural ligands; in a few cases, the same Nb has acted as an orthosteric or allosteric modulator of GPCR signaling. In this review, we summarize the multiple ingenious strategies that have been conceived and implemented in the last decade to capitalize on the discovery of nanobodies to study GPCRs from a structural perspective. SIGNIFICANCE STATEMENT: G protein-coupled receptors (GPCRs) are major pharmacological targets, and the determination of their structures at high resolution has been essential for structure-guided drug design and for insights about their functions. Single-domain antibodies (nanobodies) have greatly facilitated the structural determination of GPCRs by forming complexes directly with the receptors or indirectly through protein partners.
{"title":"The Impact of Nanobodies on G Protein-Coupled Receptor Structural Biology and Their Potential as Therapeutic Agents.","authors":"David Salom, Arum Wu, Chang C Liu, Krzysztof Palczewski","doi":"10.1124/molpharm.124.000974","DOIUrl":"10.1124/molpharm.124.000974","url":null,"abstract":"<p><p>The family of human G protein-coupled receptors (GPCRs) comprises about 800 different members, with about 35% of current pharmaceutical drugs targeting GPCRs. However, GPCR structural biology, necessary for structure-guided drug design, has lagged behind that of other membrane proteins, and it was not until the year 2000 when the first crystal structure of a GPCR (rhodopsin) was solved. Starting in 2007, the determination of additional GPCR structures was facilitated by protein engineering, new crystallization techniques, complexation with antibody fragments, and other strategies. More recently, the use of camelid heavy-chain-only antibody fragments (nanobodies) as crystallographic chaperones has revolutionized the field of GPCR structural biology, aiding in the determination of more than 340 GPCR structures to date. In most cases, the GPCR structures solved as complexes with nanobodies (Nbs) have revealed the binding mode of cognate or non-natural ligands; in a few cases, the same Nb has acted as an orthosteric or allosteric modulator of GPCR signaling. In this review, we summarize the multiple ingenious strategies that have been conceived and implemented in the last decade to capitalize on the discovery of nanobodies to study GPCRs from a structural perspective. SIGNIFICANCE STATEMENT: G protein-coupled receptors (GPCRs) are major pharmacological targets, and the determination of their structures at high resolution has been essential for structure-guided drug design and for insights about their functions. Single-domain antibodies (nanobodies) have greatly facilitated the structural determination of GPCRs by forming complexes directly with the receptors or indirectly through protein partners.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11413913/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141897829","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-17DOI: 10.1124/molpharm.124.000889
McKenna Losby, Matthew Hayes, Aurore Valfort, Danesh H Sopariwala, Ryan Sanders, John K Walker, Weiyi Xu, Vihang A Narkar, Lilei Zhang, Cyrielle Billon, Thomas P Burris
Autophagy is an essential self-degradative and recycling mechanism that maintains cellular homeostasis. Estrogen receptor-related orphan receptors (ERRs) are fundamental in regulating cardiac metabolism and function. Previously, we showed that ERR agonists improve cardiac function in models of heart failure and induce autophagy. Here, we characterized a mechanism by which ERRs induce the autophagy pathway in cardiomyocytes. Transcription factor EB (TFEB) is a master regulator of the autophagy-lysosome pathway and has been shown to be crucial regulator of genes that control autophagy. We discovered that TFEB is a direct ERR target gene whose expression is induced by ERR agonists. Activation of ERR results in increased TFEB expression in both neonatal rat ventricular myocytes and C2C12 myoblasts. An ERR-dependent increase in TFEB expression results in increased expression of an array of TFEB target genes, which are critical for the stimulation of autophagy. Pharmacologically targeting ERR is a promising potential method for the treatment of many diseases where stimulation of autophagy may be therapeutic, including heart failure. SIGNIFICANCE STATEMENT: Estrogen receptor-related receptor agonists function as exercise mimetics and also display efficacy in animal models of metabolic disease, obesity, and heart failure.
{"title":"The Estrogen Receptor-Related Orphan Receptors Regulate Autophagy through TFEB.","authors":"McKenna Losby, Matthew Hayes, Aurore Valfort, Danesh H Sopariwala, Ryan Sanders, John K Walker, Weiyi Xu, Vihang A Narkar, Lilei Zhang, Cyrielle Billon, Thomas P Burris","doi":"10.1124/molpharm.124.000889","DOIUrl":"10.1124/molpharm.124.000889","url":null,"abstract":"<p><p>Autophagy is an essential self-degradative and recycling mechanism that maintains cellular homeostasis. Estrogen receptor-related orphan receptors (ERRs) are fundamental in regulating cardiac metabolism and function. Previously, we showed that ERR agonists improve cardiac function in models of heart failure and induce autophagy. Here, we characterized a mechanism by which ERRs induce the autophagy pathway in cardiomyocytes. Transcription factor EB (TFEB) is a master regulator of the autophagy-lysosome pathway and has been shown to be crucial regulator of genes that control autophagy. We discovered that TFEB is a direct ERR target gene whose expression is induced by ERR agonists. Activation of ERR results in increased TFEB expression in both neonatal rat ventricular myocytes and C<sub>2</sub>C<sub>12</sub> myoblasts. An ERR-dependent increase in TFEB expression results in increased expression of an array of TFEB target genes, which are critical for the stimulation of autophagy. Pharmacologically targeting ERR is a promising potential method for the treatment of many diseases where stimulation of autophagy may be therapeutic, including heart failure. SIGNIFICANCE STATEMENT: Estrogen receptor-related receptor agonists function as exercise mimetics and also display efficacy in animal models of metabolic disease, obesity, and heart failure.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11413914/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142018047","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-16DOI: 10.1124/molpharm.124.000884
Michael Kurz, Michaela Ulrich, Sina B Kirchhofer, Alwina Bittner, Michael Daude, Wibke E Diederich, Kim Pauk, Holger Garn, Moritz Bünemann
Aberrant type 2 inflammatory responses are the underlying cause of the pathophysiology of allergic asthma, allergic rhinitis and other atopic diseases with an alarming prevalence in relevant parts of the western world. A bulk of evidence points out the important role of the DP2 receptor in this inflammation processes. A screening of different polyunsaturated fatty acids (PUFAs) at a fluorescence resonance energy transfer (FRET)-based DP2 receptor conformation sensor expressed in HEK cells revealed an agonistic effect of the prostaglandin (PG) D2 precursor arachidonic acid (AA) on DP2 receptor activity of about 80% of the effect induced by PGD2. In a combination of experiments at the conformation sensor and using a BRET-based G protein activation sensor expressed together with DP2 receptor-wt in HEK cells, we found that arachidonic acid act as a direct activator of the DP2 receptor but not DP1 receptor, in a concentration range considered physiologically relevant. Pharmacological inhibition of cyclooxygenases and lipoxygenases as well as cytochrome P450 did not lead to a diminished arachidonic acid response on the DP2 receptor, confirming a direct action of arachidonic acid on the receptor.
{"title":"Arachidonic acid directly activates the human DP2 receptor","authors":"Michael Kurz, Michaela Ulrich, Sina B Kirchhofer, Alwina Bittner, Michael Daude, Wibke E Diederich, Kim Pauk, Holger Garn, Moritz Bünemann","doi":"10.1124/molpharm.124.000884","DOIUrl":"https://doi.org/10.1124/molpharm.124.000884","url":null,"abstract":"Aberrant type 2 inflammatory responses are the underlying cause of the pathophysiology of allergic asthma, allergic rhinitis and other atopic diseases with an alarming prevalence in relevant parts of the western world. A bulk of evidence points out the important role of the DP2 receptor in this inflammation processes. A screening of different polyunsaturated fatty acids (PUFAs) at a fluorescence resonance energy transfer (FRET)-based DP2 receptor conformation sensor expressed in HEK cells revealed an agonistic effect of the prostaglandin (PG) D<sub>2</sub> precursor arachidonic acid (AA) on DP2 receptor activity of about 80% of the effect induced by PGD<sub>2</sub>. In a combination of experiments at the conformation sensor and using a BRET-based G protein activation sensor expressed together with DP2 receptor-wt in HEK cells, we found that arachidonic acid act as a direct activator of the DP2 receptor but not DP1 receptor, in a concentration range considered physiologically relevant. Pharmacological inhibition of cyclooxygenases and lipoxygenases as well as cytochrome P450 did not lead to a diminished arachidonic acid response on the DP2 receptor, confirming a direct action of arachidonic acid on the receptor.","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142263323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-30DOI: 10.1124/molpharm.124.000960
Spencer R Pierce, Allison L Germann, Douglas F Covey, Alex S Evers, Joe Henry Steinbach, Gustav Akk
The γ-aminobutyric acid type A (GABAA) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3β-hydroxy steroids inhibit while 3α-hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the α1β3γ2L GABAA receptor by the endogenous neurosteroid 3α-hydroxy-5β-pregnan-20-one (3α5βP) and the synthetic neuroactive steroid 3α-hydroxy-5α-androstane-17β-carbonitrile (ACN). The receptors were expressed in Xenopus oocytes. All experiments were done using two-electrode voltage-clamp electrophysiology. In the presence of low concentrations of GABA, 3α5βP and ACN potentiate the GABAA receptor. To reveal inhibition, we conducted the experiments on receptors activated by the combination of a saturating concentration of GABA and propofol to fully activate the receptors and mask potentiation, or on mutant receptors in which potentiation is ablated. Under these conditions, both steroids inhibited the receptor with IC50s of ~13 µM and maximal inhibitory effects of 70-90%. Receptor inhibition by 3α5βP was sensitive to substitution of the α1TM2-2' residue, previously shown to ablate inhibition by pregnenolone sulfate. However, results of coapplication studies and the apparent lack of state dependence suggest that pregnenolone sulfate and 3α5βP inhibit the GABAA receptor independently and through distinct mechanisms. Mutations to the neurosteroid binding sites in the α1 and β3 subunits significantly, albeit weakly and incompletely, reduced inhibition by 3α5βP and ACN. Significance Statement The heteromeric GABAA receptor is inhibited by sulfated steroids and 3β-hydroxy steroids while 3α-hydroxy steroids are considered to potentiate the receptor. We show here that 3α-hydroxy steroids have inhibitory effects on the α1β3γ2L receptor, which are observed in specific experimental settings and are expected to manifest under different physiological conditions.
{"title":"Inhibitory actions of potentiating neuroactive steroids in the human α1β3γ2L GABA<sub>A</sub> receptor.","authors":"Spencer R Pierce, Allison L Germann, Douglas F Covey, Alex S Evers, Joe Henry Steinbach, Gustav Akk","doi":"10.1124/molpharm.124.000960","DOIUrl":"https://doi.org/10.1124/molpharm.124.000960","url":null,"abstract":"<p><p>The γ-aminobutyric acid type A (GABA<sub>A</sub>) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3β-hydroxy steroids inhibit while 3α-hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the α1β3γ2L GABA<sub>A</sub> receptor by the endogenous neurosteroid 3α-hydroxy-5β-pregnan-20-one (3α5βP) and the synthetic neuroactive steroid 3α-hydroxy-5α-androstane-17β-carbonitrile (ACN). The receptors were expressed in <i>Xenopus</i> oocytes. All experiments were done using two-electrode voltage-clamp electrophysiology. In the presence of low concentrations of GABA, 3α5βP and ACN potentiate the GABA<sub>A</sub> receptor. To reveal inhibition, we conducted the experiments on receptors activated by the combination of a saturating concentration of GABA and propofol to fully activate the receptors and mask potentiation, or on mutant receptors in which potentiation is ablated. Under these conditions, both steroids inhibited the receptor with IC<sub>50</sub>s of ~13 µM and maximal inhibitory effects of 70-90%. Receptor inhibition by 3α5βP was sensitive to substitution of the α1TM2-2' residue, previously shown to ablate inhibition by pregnenolone sulfate. However, results of coapplication studies and the apparent lack of state dependence suggest that pregnenolone sulfate and 3α5βP inhibit the GABA<sub>A</sub> receptor independently and through distinct mechanisms. Mutations to the neurosteroid binding sites in the α1 and β3 subunits significantly, albeit weakly and incompletely, reduced inhibition by 3α5βP and ACN. <b>Significance Statement</b> The heteromeric GABA<sub>A</sub> receptor is inhibited by sulfated steroids and 3β-hydroxy steroids while 3α-hydroxy steroids are considered to potentiate the receptor. We show here that 3α-hydroxy steroids have inhibitory effects on the α1β3γ2L receptor, which are observed in specific experimental settings and are expected to manifest under different physiological conditions.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142109596","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-26DOI: 10.1124/molpharm.124.000743
Morgan B Dwyer, Jenna L Aumiller, Philip B Wedegaertner
G protein-coupled receptors (GPCRs) couple to heterotrimeric G proteins, comprised of α and βγ subunits, to convert extracellular signals into activation of intracellular signaling pathways. Canonically, GPCR-mediated activation results in the exchange of GDP for GTP on Gα and the dissociation of Gα-GTP and Gβγ, both of which can regulate a variety of signaling pathways. Hydrolysis of bound GTP by Gα returns the protein to Gα-GDP and allows reassociation with Gβγ to re-form the inactive heterotrimer. Naturally occurring mutations in Gα have been found at conserved glutamine and arginine amino acids that disrupt the canonical G protein cycle by inhibiting GTP hydrolysis, rendering these mutants constitutively active. Interestingly, these dysregulated Gα mutants are found in many different cancers due to their ability to sustain aberrant signaling without a need for activation by GPCRs. This review will highlight an increased recognition of the prevalence of such constitutively activating Gα mutations in cancers and the signaling pathways activated. In addition, we will discuss new knowledge regarding how these constitutively active Gα are regulated, how different mutations are biochemically distinct, and how mutationally activated Gα are unique compared to GPCR-activated Gα. Lastly, we will discuss recent progress in developing inhibitors directly targeting constitutively active Gα mutants. Significance Statement Constitutively activating mutations in G protein α subunits (Gα) widely occur in and contribute to the development of many human cancers. To develop ways to inhibit dysregulated, oncogenic signaling by these mutant Gα, it is crucial to better understand mechanisms that lead to constitutive Gα activation and unique mechanisms that regulate mutationally activated Gα in cells. The prevalence of activating mutations in Gα in various cancers make Gα proteins compelling targets for the development of therapeutics.
{"title":"<b><b>Going rogue: mechanisms, regulation, and roles of mutationally activated Gα in human cancer</b></b>.","authors":"Morgan B Dwyer, Jenna L Aumiller, Philip B Wedegaertner","doi":"10.1124/molpharm.124.000743","DOIUrl":"https://doi.org/10.1124/molpharm.124.000743","url":null,"abstract":"<p><p>G protein-coupled receptors (GPCRs) couple to heterotrimeric G proteins, comprised of α and βγ subunits, to convert extracellular signals into activation of intracellular signaling pathways. Canonically, GPCR-mediated activation results in the exchange of GDP for GTP on Gα and the dissociation of Gα-GTP and Gβγ, both of which can regulate a variety of signaling pathways. Hydrolysis of bound GTP by Gα returns the protein to Gα-GDP and allows reassociation with Gβγ to re-form the inactive heterotrimer. Naturally occurring mutations in Gα have been found at conserved glutamine and arginine amino acids that disrupt the canonical G protein cycle by inhibiting GTP hydrolysis, rendering these mutants constitutively active. Interestingly, these dysregulated Gα mutants are found in many different cancers due to their ability to sustain aberrant signaling without a need for activation by GPCRs. This review will highlight an increased recognition of the prevalence of such constitutively activating Gα mutations in cancers and the signaling pathways activated. In addition, we will discuss new knowledge regarding how these constitutively active Gα are regulated, how different mutations are biochemically distinct, and how mutationally activated Gα are unique compared to GPCR-activated Gα. Lastly, we will discuss recent progress in developing inhibitors directly targeting constitutively active Gα mutants. <b>Significance Statement</b> Constitutively activating mutations in G protein α subunits (Gα) widely occur in and contribute to the development of many human cancers. To develop ways to inhibit dysregulated, oncogenic signaling by these mutant Gα, it is crucial to better understand mechanisms that lead to constitutive Gα activation and unique mechanisms that regulate mutationally activated Gα in cells. The prevalence of activating mutations in Gα in various cancers make Gα proteins compelling targets for the development of therapeutics.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142073318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-26DOI: 10.1124/molpharm.124.000947
Ivone Gomes, Achla Gupta, Elyssa B Margolis, Lloyd D Fricker, Lakshmi A Devi
Ketamine is a glutamate receptor antagonist that was developed over 50 years ago as an anesthetic agent. At subanesthetic doses, ketamine and some metabolites are analgesics and fast-acting antidepressants, presumably through targets other than glutamate receptors. We tested ketamine and its metabolites for activity as allosteric modulators of opioid receptors expressed in recombinant receptors in heterologous systems and native receptors in rodent brain; signaling was examined by measuring GTP binding, b-arrestin recruitment, MAPK activation and neurotransmitter release. While micromolar concentrations of ketamine alone had weak agonist activity at mu opioid receptors, the combination of submicromolar concentrations of ketamine with endogenous opioid peptides produced robust synergistic responses with statistically significant increases in efficacies. All three opioid receptors (mu, delta, and kappa) showed synergism with submicromolar concentrations of ketamine and either Met-enkephalin, Leu-enkephalin, and/or dynorphin A17, albeit the extent of synergy was variable between receptors and peptides. S-ketamine exhibited higher modulatory effect compared to R-ketamine or racemic ketamine with nearly ~100% increase in efficacy. Importantly, the ketamine metabolite 6-hydroxynorketamine showed robust allosteric modulatory activity at mu opioid receptors; this metabolite is known to have analgesic and antidepressant activity but does not bind to glutamate receptors. Ketamine enhanced potency and efficacy of Met-enkephalin signaling both in mouse midbrain membranes and in rat ventral tegmental area neurons, as determined by electrophysiology recordings in brain slices. Taken together, these findings support the hypothesis that some of the therapeutic effects of ketamine and its metabolites are mediated by directly engaging the endogenous opioid system. Significance Statement We found that ketamine and its major biologically-active metabolites function as potent allosteric modulators of mu, delta, and kappa opioid receptors, with submicromolar concentrations of these compounds synergizing with endogenous opioid peptides such as enkephalin and dynorphin. This allosteric activity may contribute to ketamine's therapeutic effectiveness for treating acute and chronic pain and as a fast-acting antidepressant drug.
{"title":"<b>Ketamine and major ketamine metabolites function as allosteric modulators of opioid receptors</b>.","authors":"Ivone Gomes, Achla Gupta, Elyssa B Margolis, Lloyd D Fricker, Lakshmi A Devi","doi":"10.1124/molpharm.124.000947","DOIUrl":"https://doi.org/10.1124/molpharm.124.000947","url":null,"abstract":"<p><p>Ketamine is a glutamate receptor antagonist that was developed over 50 years ago as an anesthetic agent. At subanesthetic doses, ketamine and some metabolites are analgesics and fast-acting antidepressants, presumably through targets other than glutamate receptors. We tested ketamine and its metabolites for activity as allosteric modulators of opioid receptors expressed in recombinant receptors in heterologous systems and native receptors in rodent brain; signaling was examined by measuring GTP binding, b-arrestin recruitment, MAPK activation and neurotransmitter release. While micromolar concentrations of ketamine alone had weak agonist activity at mu opioid receptors, the combination of submicromolar concentrations of ketamine with endogenous opioid peptides produced robust synergistic responses with statistically significant increases in efficacies. All three opioid receptors (mu, delta, and kappa) showed synergism with submicromolar concentrations of ketamine and either Met-enkephalin, Leu-enkephalin, and/or dynorphin A17, albeit the extent of synergy was variable between receptors and peptides. S-ketamine exhibited higher modulatory effect compared to R-ketamine or racemic ketamine with nearly ~100% increase in efficacy. Importantly, the ketamine metabolite 6-hydroxynorketamine showed robust allosteric modulatory activity at mu opioid receptors; this metabolite is known to have analgesic and antidepressant activity but does not bind to glutamate receptors. Ketamine enhanced potency and efficacy of Met-enkephalin signaling both in mouse midbrain membranes and in rat ventral tegmental area neurons, as determined by electrophysiology recordings in brain slices. Taken together, these findings support the hypothesis that some of the therapeutic effects of ketamine and its metabolites are mediated by directly engaging the endogenous opioid system. <b>Significance Statement</b> We found that ketamine and its major biologically-active metabolites function as potent allosteric modulators of mu, delta, and kappa opioid receptors, with submicromolar concentrations of these compounds synergizing with endogenous opioid peptides such as enkephalin and dynorphin. This allosteric activity may contribute to ketamine's therapeutic effectiveness for treating acute and chronic pain and as a fast-acting antidepressant drug.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142073261","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-26DOI: 10.1124/molpharm.124.000957
Hannah J Goudsward, Victor Ruiz-Velasco, Salvatore L Stella, Paul B Herold, Gregory M Holmes
The orexigenic gut peptide ghrelin is an endogenous ligand for the growth hormone secretagogue receptor type 1a (GHSR1a). Systemic ghrelin administration has previously been shown to increase gastric motility and emptying. While these effects are known to be mediated by the vagus nerve, the cellular mechanism underlying these effects remains unclear. Therefore, the purpose of the present study was to investigate the signaling mechanism by which GHSR1a inhibits voltage-gated Ca2+ channels in isolated rat gastric vagal afferent neurons using whole-cell patch-clamp electrophysiology. The ghrelin pharmacological profile indicated that Ca2+ currents were inhibited with a log (Ic50)=-2.10 {plus minus} 0.44 and a maximal inhibition of 42.8 {plus minus} 5.0%. Exposure to the GHSR1a receptor antagonist (D-Lys3)-GHRP-6 reduced ghrelin-mediated Ca2+ channel inhibition (29.4 {plus minus} 16.7% vs 1.9 {plus minus} 2.5%, n=6, p=0.0064). Interestingly, we observed that activation of GHSR1a inhibited Ca2+ currents through both voltage-dependent and voltage-independent pathways. We also treated the gastric neurons with either pertussis toxin (PTX) or YM-254890 to examine whether the Ca2+ current inhibition was mediated by Gαi/o or Gαq/11 family of subunits. Treatment with both PTX (Ca2+ current inhibition=15.7 {plus minus} 10.6%, n=8, p=0.0327) and YM-254890 (15.2 {plus minus} 11.9%, n=8, p=0.0269) blocked ghrelin's effects on Ca2+ currents, as compared to control neurons (34.3 {plus minus} 18.9%, n=8). These results indicate GHSR1a can couple to both Gαi/o and Gαq/11 in gastric vagal afferent neurons. Overall, our findings suggest GHSR1a-mediated inhibition of Ca2+ currents occurs through two distinct pathways, offering necessary insights into the cellular mechanisms underlying ghrelin's regulation of gastric vagal afferents. Significance Statement This study demonstrated that in gastric vagal afferent neurons, activation of GHSR1a by ghrelin inhibits voltage-gated Ca2+ channels through both voltage-dependent and voltage-independent signaling pathways. These results provide necessary insight into the cellular mechanism underlying ghrelin regulation of gastric vagal afferent activity, which may benefit future studies investigating ghrelin mimetics to treat gastric motility disorders.
{"title":"Ghrelin modulates voltage-gated Ca<sup>2+</sup> channels through voltage-dependent and voltage-independent pathways in rat gastric vagal afferent neurons.","authors":"Hannah J Goudsward, Victor Ruiz-Velasco, Salvatore L Stella, Paul B Herold, Gregory M Holmes","doi":"10.1124/molpharm.124.000957","DOIUrl":"https://doi.org/10.1124/molpharm.124.000957","url":null,"abstract":"<p><p>The orexigenic gut peptide ghrelin is an endogenous ligand for the growth hormone secretagogue receptor type 1a (GHSR1a). Systemic ghrelin administration has previously been shown to increase gastric motility and emptying. While these effects are known to be mediated by the vagus nerve, the cellular mechanism underlying these effects remains unclear. Therefore, the purpose of the present study was to investigate the signaling mechanism by which GHSR1a inhibits voltage-gated Ca<sup>2+</sup> channels in isolated rat gastric vagal afferent neurons using whole-cell patch-clamp electrophysiology. The ghrelin pharmacological profile indicated that Ca<sup>2+</sup> currents were inhibited with a log (Ic<sub>50</sub>)=-2.10 {plus minus} 0.44 and a maximal inhibition of 42.8 {plus minus} 5.0%. Exposure to the GHSR1a receptor antagonist (D-Lys3)-GHRP-6 reduced ghrelin-mediated Ca<sup>2+</sup> channel inhibition (29.4 {plus minus} 16.7% vs 1.9 {plus minus} 2.5%, n=6, p=0.0064). Interestingly, we observed that activation of GHSR1a inhibited Ca<sup>2+</sup> currents through both voltage-dependent and voltage-independent pathways. We also treated the gastric neurons with either pertussis toxin (PTX) or YM-254890 to examine whether the Ca<sup>2+</sup> current inhibition was mediated by Gα<sub>i/o</sub> or Gα<sub>q/11</sub> family of subunits. Treatment with both PTX (Ca<sup>2+</sup> current inhibition=15.7 {plus minus} 10.6%, n=8, p=0.0327) and YM-254890 (15.2 {plus minus} 11.9%, n=8, p=0.0269) blocked ghrelin's effects on Ca<sup>2+</sup> currents, as compared to control neurons (34.3 {plus minus} 18.9%, n=8). These results indicate GHSR1a can couple to both Gα<sub>i/o</sub> and Gα<sub>q/11</sub> in gastric vagal afferent neurons. Overall, our findings suggest GHSR1a-mediated inhibition of Ca<sup>2+</sup> currents occurs through two distinct pathways, offering necessary insights into the cellular mechanisms underlying ghrelin's regulation of gastric vagal afferents. <b>Significance Statement</b> This study demonstrated that in gastric vagal afferent neurons, activation of GHSR1a by ghrelin inhibits voltage-gated Ca2+ channels through both voltage-dependent and voltage-independent signaling pathways. These results provide necessary insight into the cellular mechanism underlying ghrelin regulation of gastric vagal afferent activity, which may benefit future studies investigating ghrelin mimetics to treat gastric motility disorders.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142073262","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-26DOI: 10.1124/molpharm.124.000934
Pedro Azalim-Neto, François Noël, Simone C Silva, José A F P Villar, Leandro Barbosa, George A O'Doherty, Luis Eduardo M Quintas
The antitumor effect of cardiotonic steroids (CTS) has stimulated the search for new methods to evaluate both kinetic and thermodynamic aspects of their binding to Na+/K+-ATPase (NKA, EC 3.6.3.9). We propose a real-time assay based on a chromogenic substrate for phosphatase activity (pNPPase activity), using only two concentrations with an inhibitory progression curve, to obtain the association rate (kon), dissociation rate (koff) and equilibrium (Ki) constants of CTS for structure-kinetics relationship in drug screening. We show that changing conditions (from ATPase to pNPPase activity) resulted in an increase of Ki of the cardenolides digitoxigenin, essentially due to a reduction of kon In contrast, the Ki of the structurally related bufadienolide bufalin increased much less due to the reduction of its koff partially compensating the decrease of its kon When evaluating the kinetics of 15 natural and semi-synthetic CTS, we observed that both kon and koff correlated with Ki (Spearman test), suggesting that differences in potency depend on variations of both kon and koff A rhamnose in C3 of the steroidal nucleus enhanced the inhibitory potency by a reduction of koff rather than an increase of kon Rising the temperature did not alter the koff of digitoxin, generating a ∆H‡ (koff) of -10.4 {plus minus} 4.3 kJ/mol, suggesting a complex dissociation mechanism. Based on a simple and inexpensive methodology, we determined the values of kon, koff, and Ki of the CTS and provided original kinetics and thermodynamics differences between CTS that could help the design of new compounds. Significance Statement We described a fast, simple, and cost-effective method for the measurement of phosphatase pNPPase activity enabling structure-kinetics relationships of Na+/K+-ATPase inhibitors, which are important compounds due to their antitumor effect and endogenous role. Using 15 compounds, some of them original, we were able to delineate the kinetics and/or thermodynamics differences due to the type of sugar and lactone ring present in the steroid structure.
强心类固醇(CTS)的抗肿瘤作用促使人们寻找新的方法来评估其与 Na+/K+-ATP 酶(NKA,EC 3.6.3.9)结合的动力学和热力学方面。我们提出了一种基于磷酸酶活性(pNPPase 活性)显色底物的实时测定方法,只需使用两种浓度的抑制进展曲线,即可获得 CTS 的结合率(kon)、解离率(koff)和平衡常数(Ki),从而在药物筛选中建立结构-动力学关系。我们发现,改变条件(从 ATP 酶活性到 pNPP 酶活性)会导致红豆杉内酯地高辛的 Ki 增加,这主要是由于 kon 的降低、在评估 15 种天然和半合成 CTS 的动力学时,我们注意到 kon 和 koff 都与 Ki 相关(斯皮尔曼检验),这表明药效的差异取决于 kon 和 koff 的变化。4 {正负} 4.3 kJ/mol,这表明存在复杂的解离机制。基于一种简单而廉价的方法,我们确定了 CTS 的 kon、koff 和 Ki 值,并提供了 CTS 之间的原始动力学和热力学差异,这有助于新化合物的设计。意义声明 我们描述了一种快速、简单且经济有效的磷酸酶 pNPPase 活性测定方法,该方法可建立 Na+/K+-ATPase 抑制剂的结构-动力学关系,而 Na+/K+-ATPase 抑制剂因其抗肿瘤作用和内源性作用而成为重要化合物。通过使用 15 种化合物(其中一些是原创化合物),我们能够划定因甾体结构中存在的糖和内酯环类型而产生的动力学和/或热力学差异。
{"title":"<b><i>Simplified Method for Kinetic and Thermodynamic Screening of Cardiotonic Steroids Through the K</i></b> <b> <i> <sup><b><i>+</i></b> </sup> </i> </b> <b> <i><b><i>-Dependent Phosphatase Activity of Na</i></b> </i> </b> <b> <i> <sup><b><i>+</i></b> </sup> </i> </b> <b> <i><b><i>/K</i></b> </i> </b> <b> <i> <sup><b><i>+</i></b> </sup> </i> </b> <b> <i><b><i>-ATPase with Chromogenic pNPP Substrate</i></b> </i></b>.","authors":"Pedro Azalim-Neto, François Noël, Simone C Silva, José A F P Villar, Leandro Barbosa, George A O'Doherty, Luis Eduardo M Quintas","doi":"10.1124/molpharm.124.000934","DOIUrl":"https://doi.org/10.1124/molpharm.124.000934","url":null,"abstract":"<p><p>The antitumor effect of cardiotonic steroids (CTS) has stimulated the search for new methods to evaluate both kinetic and thermodynamic aspects of their binding to Na<sup>+</sup>/K<sup>+</sup>-ATPase (NKA, EC 3.6.3.9). We propose a real-time assay based on a chromogenic substrate for phosphatase activity (pNPPase activity), using only two concentrations with an inhibitory progression curve, to obtain the association rate (k<sub>on</sub>), dissociation rate (k<sub>off</sub>) and equilibrium (K<sub>i</sub>) constants of CTS for structure-kinetics relationship in drug screening. We show that changing conditions (from ATPase to pNPPase activity) resulted in an increase of K<sub>i</sub> of the cardenolides digitoxigenin, essentially due to a reduction of k<sub>on</sub> In contrast, the K<sub>i</sub> of the structurally related bufadienolide bufalin increased much less due to the reduction of its k<sub>off</sub> partially compensating the decrease of its k<sub>on</sub> When evaluating the kinetics of 15 natural and semi-synthetic CTS, we observed that both k<sub>on</sub> and k<sub>off</sub> correlated with K<sub>i</sub> (Spearman test), suggesting that differences in potency depend on variations of both k<sub>on</sub> and k<sub>off</sub> A rhamnose in C3 of the steroidal nucleus enhanced the inhibitory potency by a reduction of k<sub>off</sub> rather than an increase of k<sub>on</sub> Rising the temperature did not alter the k<sub>off</sub> of digitoxin, generating a ∆H<sup>‡</sup> (k<sub>off</sub>) of -10.4 {plus minus} 4.3 kJ/mol, suggesting a complex dissociation mechanism. Based on a simple and inexpensive methodology, we determined the values of k<sub>on</sub>, k<sub>off</sub>, and K<sub>i</sub> of the CTS and provided original kinetics and thermodynamics differences between CTS that could help the design of new compounds. <b>Significance Statement</b> We described a fast, simple, and cost-effective method for the measurement of phosphatase pNPPase activity enabling structure-kinetics relationships of Na<sup>+</sup>/K<sup>+</sup>-ATPase inhibitors, which are important compounds due to their antitumor effect and endogenous role. Using 15 compounds, some of them original, we were able to delineate the kinetics and/or thermodynamics differences due to the type of sugar and lactone ring present in the steroid structure.</p>","PeriodicalId":18767,"journal":{"name":"Molecular Pharmacology","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142073319","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}