Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometry

Autores
Boback, Nico; López, Jacob Gorenflos; Hackenberger, Christian P. R.; Di Lella, Santiago; Lauster, Daniel C.
Año de publicación
2025
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) 2-epimerase (GNE)/N-acetylmannosamine kinase is the rate-limiting enzyme in sialic acid biosynthesis and a promising therapeutic target. We applied mass photometry (MP) to investigate GNE oligomerization and its modulation by three small-molecule inhibitors (C5, C13, and C15). Substrate-binding (UDP-GlcNAc) stabilized tetramer formation by increasing dimer–dimer affinity 98-fold. All inhibitors destabilized tetramers in a concentration-dependent manner, with IC 50 values in the low micromolar range. Using a modified Cheng–Prusoffuation, IC 50 values were converted into KB,app values. Schild analysis and Operational Model of Allosterically Modulated Agonism were applied to estimate an apparent KB,app value and assess cooperative inhibition effects. Molecular docking confirmed competitive binding for all inhibitors and helped rationalize observed potency trends. While MP has previously been used to study protein assembly, our work demonstrates its applicability for the label-free, quantitative characterization of small-molecule inhibitors affecting protein oligomerization. These findings provide a foundation for further mechanistic studies and underscore the potential of MP in drug-target interaction profiling.
Fil: Boback, Nico. Institut Für Biochemie Und Chemie; Alemania
Fil: López, Jacob Gorenflos. Leibniz Forschungsinstitut Für Molekulare Pharmakologie; Alemania
Fil: Hackenberger, Christian P. R.. Leibniz Forschungsinstitut Für Molekulare Pharmakologie; Alemania
Fil: Di Lella, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina
Fil: Lauster, Daniel C.. Institut Für Biochemie Und Chemie; Alemania
Materia
ASSEMBLY INHIBITION
MASS PHOTOMETRY
MOLECULAR DOCKING
PROTEIN ASSEMBLY
SIALIC ACID BIOSYNTHESIS
UDP-GLCNAC-2-EPIMERASE
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/279328
Acceder
id CONICETDig_44aa92946430317ad0a3e2bc20d06e5e
oai_identifier_str oai:ri.conicet.gov.ar:11336/279328
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometryBoback, NicoLópez, Jacob GorenflosHackenberger, Christian P. R.Di Lella, SantiagoLauster, Daniel C.ASSEMBLY INHIBITIONMASS PHOTOMETRYMOLECULAR DOCKINGPROTEIN ASSEMBLYSIALIC ACID BIOSYNTHESISUDP-GLCNAC-2-EPIMERASEhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) 2-epimerase (GNE)/N-acetylmannosamine kinase is the rate-limiting enzyme in sialic acid biosynthesis and a promising therapeutic target. We applied mass photometry (MP) to investigate GNE oligomerization and its modulation by three small-molecule inhibitors (C5, C13, and C15). Substrate-binding (UDP-GlcNAc) stabilized tetramer formation by increasing dimer–dimer affinity 98-fold. All inhibitors destabilized tetramers in a concentration-dependent manner, with IC 50 values in the low micromolar range. Using a modified Cheng–Prusoffuation, IC 50 values were converted into KB,app values. Schild analysis and Operational Model of Allosterically Modulated Agonism were applied to estimate an apparent KB,app value and assess cooperative inhibition effects. Molecular docking confirmed competitive binding for all inhibitors and helped rationalize observed potency trends. While MP has previously been used to study protein assembly, our work demonstrates its applicability for the label-free, quantitative characterization of small-molecule inhibitors affecting protein oligomerization. These findings provide a foundation for further mechanistic studies and underscore the potential of MP in drug-target interaction profiling.Fil: Boback, Nico. Institut Für Biochemie Und Chemie; AlemaniaFil: López, Jacob Gorenflos. Leibniz Forschungsinstitut Für Molekulare Pharmakologie; AlemaniaFil: Hackenberger, Christian P. R.. Leibniz Forschungsinstitut Für Molekulare Pharmakologie; AlemaniaFil: Di Lella, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Lauster, Daniel C.. Institut Für Biochemie Und Chemie; AlemaniaJohn Wiley & Sons2025-10info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/279328Boback, Nico; López, Jacob Gorenflos; Hackenberger, Christian P. R.; Di Lella, Santiago; Lauster, Daniel C.; Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometry; John Wiley & Sons; Protein Science; 34; 11; 10-2025; 1-140961-8368CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://onlinelibrary.wiley.com/doi/10.1002/pro.70335info:eu-repo/semantics/altIdentifier/doi/10.1002/pro.70335info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2026-02-06T12:56:43Zoai:ri.conicet.gov.ar:11336/279328instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982026-02-06 12:56:43.87CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometry
title Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometry
spellingShingle Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometry
Boback, Nico
ASSEMBLY INHIBITION
MASS PHOTOMETRY
MOLECULAR DOCKING
PROTEIN ASSEMBLY
SIALIC ACID BIOSYNTHESIS
UDP-GLCNAC-2-EPIMERASE
title_short Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometry
title_full Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometry
title_fullStr Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometry
title_full_unstemmed Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometry
title_sort Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometry
dc.creator.none.fl_str_mv Boback, Nico
López, Jacob Gorenflos
Hackenberger, Christian P. R.
Di Lella, Santiago
Lauster, Daniel C.
author Boback, Nico
author_facet Boback, Nico
López, Jacob Gorenflos
Hackenberger, Christian P. R.
Di Lella, Santiago
Lauster, Daniel C.
author_role author
author2 López, Jacob Gorenflos
Hackenberger, Christian P. R.
Di Lella, Santiago
Lauster, Daniel C.
author2_role author
author
author
author
dc.subject.none.fl_str_mv ASSEMBLY INHIBITION
MASS PHOTOMETRY
MOLECULAR DOCKING
PROTEIN ASSEMBLY
SIALIC ACID BIOSYNTHESIS
UDP-GLCNAC-2-EPIMERASE
topic ASSEMBLY INHIBITION
MASS PHOTOMETRY
MOLECULAR DOCKING
PROTEIN ASSEMBLY
SIALIC ACID BIOSYNTHESIS
UDP-GLCNAC-2-EPIMERASE
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) 2-epimerase (GNE)/N-acetylmannosamine kinase is the rate-limiting enzyme in sialic acid biosynthesis and a promising therapeutic target. We applied mass photometry (MP) to investigate GNE oligomerization and its modulation by three small-molecule inhibitors (C5, C13, and C15). Substrate-binding (UDP-GlcNAc) stabilized tetramer formation by increasing dimer–dimer affinity 98-fold. All inhibitors destabilized tetramers in a concentration-dependent manner, with IC 50 values in the low micromolar range. Using a modified Cheng–Prusoffuation, IC 50 values were converted into KB,app values. Schild analysis and Operational Model of Allosterically Modulated Agonism were applied to estimate an apparent KB,app value and assess cooperative inhibition effects. Molecular docking confirmed competitive binding for all inhibitors and helped rationalize observed potency trends. While MP has previously been used to study protein assembly, our work demonstrates its applicability for the label-free, quantitative characterization of small-molecule inhibitors affecting protein oligomerization. These findings provide a foundation for further mechanistic studies and underscore the potential of MP in drug-target interaction profiling.
Fil: Boback, Nico. Institut Für Biochemie Und Chemie; Alemania
Fil: López, Jacob Gorenflos. Leibniz Forschungsinstitut Für Molekulare Pharmakologie; Alemania
Fil: Hackenberger, Christian P. R.. Leibniz Forschungsinstitut Für Molekulare Pharmakologie; Alemania
Fil: Di Lella, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina
Fil: Lauster, Daniel C.. Institut Für Biochemie Und Chemie; Alemania
description Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) 2-epimerase (GNE)/N-acetylmannosamine kinase is the rate-limiting enzyme in sialic acid biosynthesis and a promising therapeutic target. We applied mass photometry (MP) to investigate GNE oligomerization and its modulation by three small-molecule inhibitors (C5, C13, and C15). Substrate-binding (UDP-GlcNAc) stabilized tetramer formation by increasing dimer–dimer affinity 98-fold. All inhibitors destabilized tetramers in a concentration-dependent manner, with IC 50 values in the low micromolar range. Using a modified Cheng–Prusoffuation, IC 50 values were converted into KB,app values. Schild analysis and Operational Model of Allosterically Modulated Agonism were applied to estimate an apparent KB,app value and assess cooperative inhibition effects. Molecular docking confirmed competitive binding for all inhibitors and helped rationalize observed potency trends. While MP has previously been used to study protein assembly, our work demonstrates its applicability for the label-free, quantitative characterization of small-molecule inhibitors affecting protein oligomerization. These findings provide a foundation for further mechanistic studies and underscore the potential of MP in drug-target interaction profiling.
publishDate 2025
dc.date.none.fl_str_mv 2025-10
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/279328
Boback, Nico; López, Jacob Gorenflos; Hackenberger, Christian P. R.; Di Lella, Santiago; Lauster, Daniel C.; Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometry; John Wiley & Sons; Protein Science; 34; 11; 10-2025; 1-14
0961-8368
CONICET Digital
CONICET
url http://hdl.handle.net/11336/279328
identifier_str_mv Boback, Nico; López, Jacob Gorenflos; Hackenberger, Christian P. R.; Di Lella, Santiago; Lauster, Daniel C.; Quantitative analysis of inhibitor‐induced assembly disruption in human UDP ‐ GlcNAc 2‐epimerase using mass photometry; John Wiley & Sons; Protein Science; 34; 11; 10-2025; 1-14
0961-8368
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://onlinelibrary.wiley.com/doi/10.1002/pro.70335
info:eu-repo/semantics/altIdentifier/doi/10.1002/pro.70335
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv John Wiley & Sons
publisher.none.fl_str_mv John Wiley & Sons
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1856403322578141184
score 13.11174

Publicaciones similares