Pub Date : 2023-12-12DOI: 10.1021/acsbiomedchemau.3c00030
Francesco Nai, Maria Paula Flores Espinoza, Annalisa Invernizzi, Pablo Andrés Vargas-Rosales, Olga Bobileva, Marcin Herok and Amedeo Caflisch*,
We discovered the first inhibitors of the m7G-RNA writer METTL1 by high-throughput docking and an enzymatic assay based on luminescence. Eleven compounds, which belong to three different chemotypes, show inhibitory activity in the range 40–300 μM. Two adenine derivatives identified by docking have very favorable ligand efficiency of 0.34 and 0.31 kcal/mol per non-hydrogen atom, respectively. Molecular dynamics simulations provide evidence that the inhibitors compete with the binding of the cosubstrate S-adenosyl methionine to METTL1. We also present a soakable crystal form that was used to determine the structure of the complex of METTL1 with sinefungin at a resolution of 1.85 Å.
{"title":"Small-Molecule Inhibitors of the m7G-RNA Writer METTL1","authors":"Francesco Nai, Maria Paula Flores Espinoza, Annalisa Invernizzi, Pablo Andrés Vargas-Rosales, Olga Bobileva, Marcin Herok and Amedeo Caflisch*, ","doi":"10.1021/acsbiomedchemau.3c00030","DOIUrl":"10.1021/acsbiomedchemau.3c00030","url":null,"abstract":"<p >We discovered the first inhibitors of the m7G-RNA writer METTL1 by high-throughput docking and an enzymatic assay based on luminescence. Eleven compounds, which belong to three different chemotypes, show inhibitory activity in the range 40–300 μM. Two adenine derivatives identified by docking have very favorable ligand efficiency of 0.34 and 0.31 kcal/mol per non-hydrogen atom, respectively. Molecular dynamics simulations provide evidence that the inhibitors compete with the binding of the cosubstrate <i>S</i>-adenosyl methionine to METTL1. We also present a soakable crystal form that was used to determine the structure of the complex of METTL1 with sinefungin at a resolution of 1.85 Å.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.3c00030","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138581705","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-12-11DOI: 10.1021/acsbiomedchemau.3c00060
Alexander J. Lander, Yifu Kong, Yi Jin*, Chuanliu Wu* and Louis Y. P. Luk*,
Many cell-surface receptors are promising targets for chemical synthesis because of their critical roles in disease development. This synthetic approach enables investigations by racemic protein crystallography and ligand discovery by mirror-image methodologies. However, due to their complex nature, the chemical synthesis of a receptor can be a significant challenge. Here, we describe the chemical synthesis and folding of a central, cysteine-rich domain of the cell-surface receptor tumor necrosis factor 1 which is integral to binding of the cytokine TNF-α, namely, TNFR-1 CRD2. Racemic protein crystallography at 1.4 Å confirmed that the native binding conformation was preserved, and TNFR-1 CRD2 maintained its capacity to bind to TNF-α (KD ≈ 7 nM). Encouraged by this discovery, we carried out mirror-image phage display using the enantiomeric receptor mimic and identified a d-peptide ligand for TNFR-1 CRD2 (KD = 1 μM). This work demonstrated that cysteine-rich domains, including the central domains, can be chemically synthesized and used as mimics for investigations.
{"title":"Deciphering the Synthetic and Refolding Strategy of a Cysteine-Rich Domain in the Tumor Necrosis Factor Receptor (TNF-R) for Racemic Crystallography Analysis and d-Peptide Ligand Discovery","authors":"Alexander J. Lander, Yifu Kong, Yi Jin*, Chuanliu Wu* and Louis Y. P. Luk*, ","doi":"10.1021/acsbiomedchemau.3c00060","DOIUrl":"10.1021/acsbiomedchemau.3c00060","url":null,"abstract":"<p >Many cell-surface receptors are promising targets for chemical synthesis because of their critical roles in disease development. This synthetic approach enables investigations by racemic protein crystallography and ligand discovery by mirror-image methodologies. However, due to their complex nature, the chemical synthesis of a receptor can be a significant challenge. Here, we describe the chemical synthesis and folding of a central, cysteine-rich domain of the cell-surface receptor tumor necrosis factor 1 which is integral to binding of the cytokine TNF-α, namely, TNFR-1 CRD2. Racemic protein crystallography at 1.4 Å confirmed that the native binding conformation was preserved, and TNFR-1 CRD2 maintained its capacity to bind to TNF-α (<i>K</i><sub>D</sub> ≈ 7 nM). Encouraged by this discovery, we carried out mirror-image phage display using the enantiomeric receptor mimic and identified a <span>d</span>-peptide ligand for TNFR-1 CRD2 (<i>K</i><sub>D</sub> = 1 μM). This work demonstrated that cysteine-rich domains, including the central domains, can be chemically synthesized and used as mimics for investigations.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.3c00060","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138567038","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-20DOI: 10.1021/acsbiomedchemau.3c00067
Photis Rotsides, Paula J. Lee, Nakoa Webber, Kimberly C. Grasty, Joris Beld and Patrick J. Loll*,
Vancomycin’s interactions with cellular targets drive its antimicrobial activity and also trigger expression of resistance against the antibiotic. Interaction partners for vancomycin have previously been identified using photoaffinity probes, which have proven to be useful tools for exploring vancomycin’s interactome. This work seeks to develop diazirine-based vancomycin photoprobes that display enhanced specificity and bear fewer chemical modifications as compared to previous photoprobes. Using proteins fused to vancomycin’s main cell-wall target, d-alanyl-d-alanine, we used mass spectrometry to show that these photoprobes specifically label known vancomycin-binding partners within minutes. In a complementary approach, we developed a Western-blot strategy targeting the vancomycin adduct of the photoprobes, eliminating the need for affinity tags and simplifying the analysis of photolabeling reactions. Together, the probes and identification strategy provide a novel and streamlined pipeline for identifying vancomycin-binding proteins.
{"title":"Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins","authors":"Photis Rotsides, Paula J. Lee, Nakoa Webber, Kimberly C. Grasty, Joris Beld and Patrick J. Loll*, ","doi":"10.1021/acsbiomedchemau.3c00067","DOIUrl":"10.1021/acsbiomedchemau.3c00067","url":null,"abstract":"<p >Vancomycin’s interactions with cellular targets drive its antimicrobial activity and also trigger expression of resistance against the antibiotic. Interaction partners for vancomycin have previously been identified using photoaffinity probes, which have proven to be useful tools for exploring vancomycin’s interactome. This work seeks to develop diazirine-based vancomycin photoprobes that display enhanced specificity and bear fewer chemical modifications as compared to previous photoprobes. Using proteins fused to vancomycin’s main cell-wall target, <span>d</span>-alanyl-<span>d</span>-alanine, we used mass spectrometry to show that these photoprobes specifically label known vancomycin-binding partners within minutes. In a complementary approach, we developed a Western-blot strategy targeting the vancomycin adduct of the photoprobes, eliminating the need for affinity tags and simplifying the analysis of photolabeling reactions. Together, the probes and identification strategy provide a novel and streamlined pipeline for identifying vancomycin-binding proteins.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.3c00067","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138528976","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-15DOI: 10.1021/acsbiomedchemau.3c00039
Elena Arutyunova, Alexandr Belovodskiy*, Pu Chen, Muhammad Bashir Khan, Michael Joyce, Holly Saffran, Jimmy Lu, Zoe Turner, Bing Bai, Tess Lamer, Howard S. Young, John C. Vederas, D. Lorne Tyrrell, M. Joanne Lemieux* and James A. Nieman,
This study explores the relationship between structural alterations of nirmatrelvir, such as homologation and deuteration, and metabolic stability of newly synthesized derivatives. We developed a reliable synthetic protocol toward dideutero-nirmatrelvir and its homologated analogues with high isotopic incorporation. Deuteration of the primary metabolic site of nirmatrelvir provides a 3-fold improvement of its human microsomal stability but is accompanied by an increased metabolism rate at secondary sites. Homologation of the lactam ring allows the capping group modification to decrease and delocalize the molecule’s lipophilicity, reducing the metabolic rate at secondary sites. The effect of deuteration was less pronounced for the 6-membered lactam than for its 5-membered analogue in human microsomes, but the trend is reversed in the case of mouse microsomes. X-ray data revealed that the homologation of the lactam ring favors the orientation of the drug’s nitrile warhead for interaction with the catalytic sulfur of the SARS-CoV-2 Mpro, improving its binding. Comparable potency against SARS-CoV-2 Mpro from several variants of concern and selectivity over human cysteine proteases cathepsin B, L, and S was observed for the novel deuterated/homologated derivative and nirmatrelvir. Synthesized compounds displayed a large interspecies variability in hamster, rat, and human hepatocyte stability assays. Overall, we aimed to apply a rational approach in changing the physicochemical properties of the drug to refine its biochemical and biological parameters.
{"title":"The Effect of Deuteration and Homologation of the Lactam Ring of Nirmatrelvir on Its Biochemical Properties and Oxidative Metabolism","authors":"Elena Arutyunova, Alexandr Belovodskiy*, Pu Chen, Muhammad Bashir Khan, Michael Joyce, Holly Saffran, Jimmy Lu, Zoe Turner, Bing Bai, Tess Lamer, Howard S. Young, John C. Vederas, D. Lorne Tyrrell, M. Joanne Lemieux* and James A. Nieman, ","doi":"10.1021/acsbiomedchemau.3c00039","DOIUrl":"10.1021/acsbiomedchemau.3c00039","url":null,"abstract":"<p >This study explores the relationship between structural alterations of nirmatrelvir, such as homologation and deuteration, and metabolic stability of newly synthesized derivatives. We developed a reliable synthetic protocol toward dideutero-nirmatrelvir and its homologated analogues with high isotopic incorporation. Deuteration of the primary metabolic site of nirmatrelvir provides a 3-fold improvement of its human microsomal stability but is accompanied by an increased metabolism rate at secondary sites. Homologation of the lactam ring allows the capping group modification to decrease and delocalize the molecule’s lipophilicity, reducing the metabolic rate at secondary sites. The effect of deuteration was less pronounced for the 6-membered lactam than for its 5-membered analogue in human microsomes, but the trend is reversed in the case of mouse microsomes. X-ray data revealed that the homologation of the lactam ring favors the orientation of the drug’s nitrile warhead for interaction with the catalytic sulfur of the SARS-CoV-2 M<sup>pro</sup>, improving its binding. Comparable potency against SARS-CoV-2 M<sup>pro</sup> from several variants of concern and selectivity over human cysteine proteases cathepsin B, L, and S was observed for the novel deuterated/homologated derivative and nirmatrelvir. Synthesized compounds displayed a large interspecies variability in hamster, rat, and human hepatocyte stability assays. Overall, we aimed to apply a rational approach in changing the physicochemical properties of the drug to refine its biochemical and biological parameters.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.3c00039","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138528979","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-07DOI: 10.1021/acsbiomedchemau.3c00047
Adithi Kannan, Dhruv Kumar Chaurasiya and Athi N. Naganathan*,
The extent and molecular basis of interdomain communication in multidomain proteins, central to understanding allostery and function, is an open question. One simple evolutionary strategy could involve the selection of either conflicting or favorable electrostatic interactions across the interface of two closely spaced domains to tune the magnitude of interdomain connectivity. Here, we study a bilobed domain FF34 from the eukaryotic p190A RhoGAP protein to explore one such design principle that mediates interdomain communication. We find that while the individual structural units in wild-type FF34 are marginally coupled, they exhibit distinct intrinsic stabilities and low cooperativity, manifesting as slow folding. The FF3-FF4 interface harbors a frustrated network of highly conserved electrostatic interactions─a charge troika─that promotes the population of multiple, decoupled, and non-native structural modes on a rugged native landscape. Perturbing this network via a charge-reversal mutation not only enhances stability and cooperativity but also dampens the fluctuations globally and speeds up the folding rate by at least an order of magnitude. Our work highlights how a conserved but nonoptimal network of interfacial electrostatic interactions shapes the native ensemble of a bilobed protein, a feature that could be exploited in designing molecular systems with long-range connectivity and enhanced cooperativity.
{"title":"Conflicting Interfacial Electrostatic Interactions as a Design Principle to Modulate Long-Range Interdomain Communication","authors":"Adithi Kannan, Dhruv Kumar Chaurasiya and Athi N. Naganathan*, ","doi":"10.1021/acsbiomedchemau.3c00047","DOIUrl":"10.1021/acsbiomedchemau.3c00047","url":null,"abstract":"<p >The extent and molecular basis of interdomain communication in multidomain proteins, central to understanding allostery and function, is an open question. One simple evolutionary strategy could involve the selection of either conflicting or favorable electrostatic interactions across the interface of two closely spaced domains to tune the magnitude of interdomain connectivity. Here, we study a bilobed domain FF34 from the eukaryotic p190A RhoGAP protein to explore one such design principle that mediates interdomain communication. We find that while the individual structural units in wild-type FF34 are marginally coupled, they exhibit distinct intrinsic stabilities and low cooperativity, manifesting as slow folding. The FF3-FF4 interface harbors a frustrated network of highly conserved electrostatic interactions─a charge <i>troika</i>─that promotes the population of multiple, decoupled, and non-native structural modes on a rugged native landscape. Perturbing this network via a charge-reversal mutation not only enhances stability and cooperativity but also dampens the fluctuations globally and speeds up the folding rate by at least an order of magnitude. Our work highlights how a conserved but nonoptimal network of interfacial electrostatic interactions shapes the native ensemble of a bilobed protein, a feature that could be exploited in designing molecular systems with long-range connectivity and enhanced cooperativity.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.3c00047","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135539894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-10-31DOI: 10.1021/acsbiomedchemau.3c00032
Biswanath Maity, Hariharan Moorthy and Thimmaiah Govindaraju*,
Efficient delivery of bioactive ingredients into cells is a major challenge. Cell-penetrating peptides (CPPs) have emerged as promising vehicles for this purpose. We have developed novel CPPs derived from the flexible and disordered tail extensions of DNA-binding Ku proteins. Ku-P4, the lead CPP identified in this study, is biocompatible and displays high internalization efficacy. Biophysical studies show that the proline residue is crucial for preserving the intrinsically disordered state and biocompatibility. DNA binding studies showed effective DNA condensation to form a positively charged polyplex. The polyplex exhibited effective penetration through the cell membrane and delivered the plasmid DNA inside the cell. These novel CPPs have the potential to enhance the cellular uptake and therapeutic efficacy of peptide-drug or gene conjugates.
将生物活性成分高效地输送到细胞中是一项重大挑战。为此,细胞穿透肽(CPPs)已成为一种很有前景的载体。我们从 DNA 结合 Ku 蛋白的柔性无序尾端延伸部分开发出了新型 CPPs。Ku-P4是本研究中发现的主要CPP,它具有生物相容性,并显示出很高的内化效力。生物物理研究表明,脯氨酸残基对于保持固有无序状态和生物相容性至关重要。DNA 结合研究表明,DNA 能有效缩合,形成带正电荷的多聚体。这种多聚体能有效穿透细胞膜,将质粒 DNA 送入细胞内。这些新型 CPPs 有潜力提高细胞对多肽-药物或基因共轭物的吸收和治疗效果。
{"title":"Intrinsically Disordered Ku Protein-Derived Cell-Penetrating Peptides","authors":"Biswanath Maity, Hariharan Moorthy and Thimmaiah Govindaraju*, ","doi":"10.1021/acsbiomedchemau.3c00032","DOIUrl":"10.1021/acsbiomedchemau.3c00032","url":null,"abstract":"<p >Efficient delivery of bioactive ingredients into cells is a major challenge. Cell-penetrating peptides (CPPs) have emerged as promising vehicles for this purpose. We have developed novel CPPs derived from the flexible and disordered tail extensions of DNA-binding Ku proteins. Ku-P4, the lead CPP identified in this study, is biocompatible and displays high internalization efficacy. Biophysical studies show that the proline residue is crucial for preserving the intrinsically disordered state and biocompatibility. DNA binding studies showed effective DNA condensation to form a positively charged polyplex. The polyplex exhibited effective penetration through the cell membrane and delivered the plasmid DNA inside the cell. These novel CPPs have the potential to enhance the cellular uptake and therapeutic efficacy of peptide-drug or gene conjugates.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.3c00032","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135872377","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-10-30DOI: 10.1021/acsbiomedchemau.3c00055
Dibakar Sarkar, and , Anirban Bhunia*,
The pursuit of a novel structural motif that can shed light on the key functional attributes is a primary focus in the study of protein folding disorders. Decades of research on Alzheimer’s disease (AD) have centered on the Amyloid β (Aβ) pathway, highlighting its significance in understanding the disorder. The diversity in the Aβ pathway and the possible silent tracks which are yet to discover, makes it exceedingly intimidating to the interdisciplinary scientific community. Over the course of AD research, Aβ has consistently been at the forefront of scientific inquiry and discussion. In this review, we epitomize the role of a potential structural motif (GxxxG motif) that may provide a new horizon to the Aβ conflict. We emphasize on how comprehensive understanding of this motif from a structure–function perspective may pave the way for designing novel therapeutics intervention in AD and related diseases.
{"title":"Delineating the Role of GxxxG Motif in Amyloidogenesis: A New Perspective in Targeting Amyloid-Beta Mediated AD Pathogenesis","authors":"Dibakar Sarkar, and , Anirban Bhunia*, ","doi":"10.1021/acsbiomedchemau.3c00055","DOIUrl":"10.1021/acsbiomedchemau.3c00055","url":null,"abstract":"<p >The pursuit of a novel structural motif that can shed light on the key functional attributes is a primary focus in the study of protein folding disorders. Decades of research on Alzheimer’s disease (AD) have centered on the Amyloid β (Aβ) pathway, highlighting its significance in understanding the disorder. The diversity in the Aβ pathway and the possible silent tracks which are yet to discover, makes it exceedingly intimidating to the interdisciplinary scientific community. Over the course of AD research, Aβ has consistently been at the forefront of scientific inquiry and discussion. In this review, we epitomize the role of a potential structural motif (GxxxG motif) that may provide a new horizon to the Aβ conflict. We emphasize on how comprehensive understanding of this motif from a structure–function perspective may pave the way for designing novel therapeutics intervention in AD and related diseases.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.3c00055","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"136070990","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-10-13DOI: 10.1021/acsbiomedchemau.3c00031
Katharine M. Wright, Hanjie Jiang, Wendy Xia, Michael B. Murphy, Tatiana N. Boronina, Justin N. Nwafor, HyoJeon Kim, Akunna M. Iheanacho, P. Aitana Azurmendi, Robert N. Cole, Philip A. Cole and Sandra B. Gabelli*,
NaV1.7, the neuronal voltage-gated sodium channel isoform, plays an important role in the human body’s ability to feel pain. Mutations within NaV1.7 have been linked to pain-related syndromes, such as insensitivity to pain. To date, the regulation and internalization mechanisms of the NaV1.7 channel are not well known at a biochemical level. In this study, we perform biochemical and biophysical analyses that establish that the HECT-type E3 ligase, NEDD4L, ubiquitinates the cytoplasmic C-terminal (CT) region of NaV1.7. Through in vitro ubiquitination and mass spectrometry experiments, we identify, for the first time, the lysine residues of NaV1.7 within the CT region that get ubiquitinated. Furthermore, binding studies with an NEDD4L E3 ligase modulator (ubiquitin variant) highlight the dynamic partnership between NEDD4L and NaV1.7. These investigations provide a framework for understanding how NEDD4L-dependent regulation of the channel can influence the NaV1.7 function.
{"title":"The C-Terminal of NaV1.7 Is Ubiquitinated by NEDD4L","authors":"Katharine M. Wright, Hanjie Jiang, Wendy Xia, Michael B. Murphy, Tatiana N. Boronina, Justin N. Nwafor, HyoJeon Kim, Akunna M. Iheanacho, P. Aitana Azurmendi, Robert N. Cole, Philip A. Cole and Sandra B. Gabelli*, ","doi":"10.1021/acsbiomedchemau.3c00031","DOIUrl":"10.1021/acsbiomedchemau.3c00031","url":null,"abstract":"<p >Na<sub>V</sub>1.7, the neuronal voltage-gated sodium channel isoform, plays an important role in the human body’s ability to feel pain. Mutations within Na<sub>V</sub>1.7 have been linked to pain-related syndromes, such as insensitivity to pain. To date, the regulation and internalization mechanisms of the Na<sub>V</sub>1.7 channel are not well known at a biochemical level. In this study, we perform biochemical and biophysical analyses that establish that the HECT-type E3 ligase, NEDD4L, ubiquitinates the cytoplasmic C-terminal (CT) region of Na<sub>V</sub>1.7. Through in vitro ubiquitination and mass spectrometry experiments, we identify, for the first time, the lysine residues of Na<sub>V</sub>1.7 within the CT region that get ubiquitinated. Furthermore, binding studies with an NEDD4L E3 ligase modulator (ubiquitin variant) highlight the dynamic partnership between NEDD4L and Na<sub>V</sub>1.7. These investigations provide a framework for understanding how NEDD4L-dependent regulation of the channel can influence the Na<sub>V</sub>1.7 function.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.3c00031","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135857986","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-09-08DOI: 10.1021/acsbiomedchemau.3c00046
Mayako Michino*, Alexandre Beautrait, Nicholas A. Boyles, Aparna Nadupalli, Alexey Dementiev, Shan Sun, John Ginn, Leigh Baxt, Robert Suto, Ruslana Bryk, Steven V. Jerome, David J. Huggins* and Jeremie Vendome*,
Lpd (lipoamide dehydrogenase) in Mycobacterium tuberculosis (Mtb) is required for virulence and is a genetically validated tuberculosis (TB) target. Numerous screens have been performed over the last decade, yet only two inhibitor series have been identified. Recent advances in large-scale virtual screening methods combined with make-on-demand compound libraries have shown the potential for finding novel hits. In this study, the Enamine REAL library consisting of ∼1.12 billion compounds was efficiently screened using the GPU Shape screen method against Mtb Lpd to find additional chemical matter that would expand on the known sulfonamide inhibitor series. We identified six new inhibitors with IC50 in the range of 5–100 μM. While these compounds remained chemically close to the already known sulfonamide series inhibitors, some diversity was found in the cores of the hits. The two most potent hits were further validated by one-step potency optimization to submicromolar levels. The co-crystal structure of optimized analogue TDI-13537 provided new insights into the potency determinants of the series.
{"title":"Shape-Based Virtual Screening of a Billion-Compound Library Identifies Mycobacterial Lipoamide Dehydrogenase Inhibitors","authors":"Mayako Michino*, Alexandre Beautrait, Nicholas A. Boyles, Aparna Nadupalli, Alexey Dementiev, Shan Sun, John Ginn, Leigh Baxt, Robert Suto, Ruslana Bryk, Steven V. Jerome, David J. Huggins* and Jeremie Vendome*, ","doi":"10.1021/acsbiomedchemau.3c00046","DOIUrl":"10.1021/acsbiomedchemau.3c00046","url":null,"abstract":"<p >Lpd (lipoamide dehydrogenase) in <i>Mycobacterium tuberculosis</i> (Mtb) is required for virulence and is a genetically validated tuberculosis (TB) target. Numerous screens have been performed over the last decade, yet only two inhibitor series have been identified. Recent advances in large-scale virtual screening methods combined with make-on-demand compound libraries have shown the potential for finding novel hits. In this study, the Enamine REAL library consisting of ∼1.12 billion compounds was efficiently screened using the GPU Shape screen method against Mtb Lpd to find additional chemical matter that would expand on the known sulfonamide inhibitor series. We identified six new inhibitors with IC<sub>50</sub> in the range of 5–100 μM. While these compounds remained chemically close to the already known sulfonamide series inhibitors, some diversity was found in the cores of the hits. The two most potent hits were further validated by one-step potency optimization to submicromolar levels. The co-crystal structure of optimized analogue <b>TDI-13537</b> provided new insights into the potency determinants of the series.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.3c00046","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127259534","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-08-30DOI: 10.1021/acsbiomedchemau.3c00038
Ahmed M. Kamal El-sagheir, Ireny Abdelmesseh Nekhala, Mohammed K. Abd El-Gaber, Ahmed S. Aboraia, Jonatan Persson, Ann-Britt Schäfer, Michaela Wenzel* and Farghaly A. Omar*,
Fluoroquinolones are an important class of antibiotics with broad-spectrum antibacterial and antitubercular activity. Here, we describe the design and synthesis of a series of 38 N4-substituted piperazinyl norfloxacin derivatives. Their activity and mechanism of action were characterized using in silico, in vitro, and in vivo approaches. Several compounds displayed interesting activities against both Gram-negative and Gram-positive bacteria, and few displayed antimycobacterial activity, whereby some were as potent as norfloxacin and ciprofloxacin. Molecular docking experiments suggested that the new derivatives inhibit both DNA gyrase and DNA topoisomerase IV in a similar manner as norfloxacin. Selecting the most promising candidates for experimental mode of action analysis, we confirmed DNA gyrase and topoisomerase IV as targets of all tested compounds using enzymatic in vitro assays. Phenotypic analysis of both Escherichia coli and Bacillus subtilis confirmed a typical gyrase inhibition phenotype for all of the tested compounds. Assessment of possible additional targets revealed three compounds with unique effects on the B. subtilis cell wall synthesis machinery, suggesting that they may have an additional target in this pathway. Comparison with known cell wall synthesis inhibitors showed that the new compounds elicit a distinct and, so far, unique phenotype, suggesting that they act differently from known cell wall synthesis inhibitors. Interestingly, our phenotypic analysis revealed that both norfloxacin and ciprofloxacin displayed additional cellular effects as well, which may be indicative of the so far unknown additional mechanisms of fluoroquinolones.
氟喹诺酮类是一类重要的抗生素,具有广谱抗菌和抗结核活性。在此,我们介绍了一系列 38 个 N4 取代的哌嗪基诺氟沙星衍生物的设计与合成。我们采用硅学、体外和体内方法对它们的活性和作用机制进行了表征。一些化合物对革兰氏阴性菌和革兰氏阳性菌都显示出了有趣的活性,少数化合物显示出了抗霉菌活性,其中一些活性与诺氟沙星和环丙沙星相当。分子对接实验表明,新衍生物对 DNA 回旋酶和 DNA 拓扑异构酶 IV 的抑制作用与诺氟沙星相似。我们选择了最有希望的候选化合物进行实验性作用模式分析,并利用酶促体外实验证实了 DNA 回旋酶和拓扑异构酶 IV 是所有测试化合物的靶标。对大肠杆菌和枯草杆菌的表型分析证实,所有测试化合物都具有典型的回旋酶抑制表型。对可能的其他靶标进行评估后发现,有三种化合物对枯草芽孢杆菌细胞壁合成机制有独特的影响,这表明它们在这一途径中可能还有其他靶标。与已知的细胞壁合成抑制剂进行比较后发现,这些新化合物引发了独特的表型,而且迄今为止还是独一无二的,这表明它们的作用与已知的细胞壁合成抑制剂不同。有趣的是,我们的表型分析表明,诺氟沙星和环丙沙星也显示出了额外的细胞效应,这可能表明了氟喹诺酮类药物迄今未知的额外机制。
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