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Enabling Fragment-Based Drug Discovery for Membrane Proteins: Development of a Nanodisc-Based Weak Affinity Chromatography Method

Kruse, Linnea LU and Degrér, Vilma LU (2025) KFKM05 20251
Biophysical Chemistry
Abstract
Fragment-based drug discovery (FBDD) has in recent years emerged as an interesting method to target challenging binding sites, such as allosteric sites or small pockets. Weak Affinity Chromatography (WAC™) is a FBDD technology where dissociation constants of injected compounds are determined by comparing retention times between a protein column to a reference column. The goal of this project was to facilitate the application of WAC™ technology with membrane proteins, primarily Leucine Transporter (LeuT), also Aquaporin 4 (AQP4). This was done through the use of nanodiscs, consisting of two Membrane Scaffold Proteins (MSP) encircled around a phospholipid bilayer. Both proteins were successfully inserted into nanodiscs. For LeuT, different... (More)
Fragment-based drug discovery (FBDD) has in recent years emerged as an interesting method to target challenging binding sites, such as allosteric sites or small pockets. Weak Affinity Chromatography (WAC™) is a FBDD technology where dissociation constants of injected compounds are determined by comparing retention times between a protein column to a reference column. The goal of this project was to facilitate the application of WAC™ technology with membrane proteins, primarily Leucine Transporter (LeuT), also Aquaporin 4 (AQP4). This was done through the use of nanodiscs, consisting of two Membrane Scaffold Proteins (MSP) encircled around a phospholipid bilayer. Both proteins were successfully inserted into nanodiscs. For LeuT, different molar ratios between LeuT:MSP:Lipids:Cholate was investigated to optimize the yield of complete nanodiscs. A slight increase in LeuT relative to MSP proved successful whereas increase in lipids to MSP ratio decreased the yield.

Furthermore, immobilization and packing of the LeuT nanodiscs on the column proved successful in regard of stable pressure and flow during all WAC™ runs. WAC™-UV experiments with the binder L-tryptophan in a salt-containing buffer showed retention time differences between LeuT and the empty nanodisc reference column, suggesting salt-dependent binding. This shift was not observed in salt-free buffer using WAC™-MS. Desipramine, another binder, exhibited a retention time difference in WAC™-MS. However, long retention on the reference column indicated non-specific interactions, likely due to the amphipathic properties of desipramine.

The results demonstrate that membrane protein nanodiscs can be used successfully in combination with WAC™ technology. However, further optimization is necessary for broader application. (Less)
Abstract (Swedish)
Fragmentbaserad läkemedelsutveckling (FBDD) har på senare tid utvecklats som en lovande alternativ metod för att identifiera svåråtkomliga bindningsställen, såsom små fickor eller allosteriska säten. Weak affinity chromatography (WAC™) är en FBDD-metod där dissociationskonstanten kan beräknas genom att jämföra retentionstiden på en proteinkolonn med en referenskolonn. Målet med projektet är att möjligöra kombinationen av WAC™ med membranproteiner, först och främst Leucintransportören (LeuT) men också Aquaporin 4 (AQP4). Detta gjordes genom att använda nanodiskar bestående två membrane scaffold proteiner (MSP) som innesluter målproteinet i ett dubbelskikt av fosfolipider. Båda proteinerna var framgångsrikt inmonterade i nanodiskarna. För... (More)
Fragmentbaserad läkemedelsutveckling (FBDD) har på senare tid utvecklats som en lovande alternativ metod för att identifiera svåråtkomliga bindningsställen, såsom små fickor eller allosteriska säten. Weak affinity chromatography (WAC™) är en FBDD-metod där dissociationskonstanten kan beräknas genom att jämföra retentionstiden på en proteinkolonn med en referenskolonn. Målet med projektet är att möjligöra kombinationen av WAC™ med membranproteiner, först och främst Leucintransportören (LeuT) men också Aquaporin 4 (AQP4). Detta gjordes genom att använda nanodiskar bestående två membrane scaffold proteiner (MSP) som innesluter målproteinet i ett dubbelskikt av fosfolipider. Båda proteinerna var framgångsrikt inmonterade i nanodiskarna. För att optimera inmonteringsprocessen av LeuT undersöktes förhållandet mellan komponenterna LeuT:Lipider:MSP:Cholat. Det visade att en liten ökning av LeuT relativt MSP var lyckat medan en ökning av lipider relativt MSP gav en minskning i utbyte.

I nästa steg utfördes immobilisering och packning av leuT-nanodiskarna på kolonnen och tomma nanodiskar på referenskolonnen. Processen resulterade i stabilt tryck och flöde under samtliga WAC™-försök. WAC™-UV experiment med saltbuffert och injektion av liganden
L-tryptofan gav signifikant skillnad i retentionstid mellan kolonnerna. Skiftet kunde dock inte observeras i frånvaro av salt under WAC™-MS körningen, vilket kan tyda på en saltberoende bindning till L-tryptopfan. En annan bindare, desipramin, påvisade en skillnad i retentionstid i frånvaro av salt vid WAC™-MS. Dock var retentionstiden också lång på referenskolonnen, vilket indikerar ospecifika interaktioner, troligen orsakade av desipramins amfipatiska egenskaper.

Sammanfattningsvis visade projektet att membranproteiner har potential att framgångsrikt kunna användas i kombination med WAC™-teknologin. Dock krävs ytterligare optimering för en trovärdig medicinsk tillämpning. (Less)
Popular Abstract
Drug Discovery Method Adapted to Target Membrane Proteins.
Membrane proteins are the target of a majority of medicine due to their vital role in the body. By adapting a drug discovery method to membrane proteins, this study contributes to an efficient way of developing new drugs.
This project focused on two specific membrane proteins. One is closely related to human proteins with connections to depression, schizophrenia and epilepsy. The other one has been linked to Alzheimer’s and Parkinson’s disease, which both are of significant interest in the field.
A technique for identifying small fragments that bind to a target protein has in this study been applied to membrane proteins. These weakly binding fragments can by themselves or... (More)
Drug Discovery Method Adapted to Target Membrane Proteins.
Membrane proteins are the target of a majority of medicine due to their vital role in the body. By adapting a drug discovery method to membrane proteins, this study contributes to an efficient way of developing new drugs.
This project focused on two specific membrane proteins. One is closely related to human proteins with connections to depression, schizophrenia and epilepsy. The other one has been linked to Alzheimer’s and Parkinson’s disease, which both are of significant interest in the field.
A technique for identifying small fragments that bind to a target protein has in this study been applied to membrane proteins. These weakly binding fragments can by themselves or combined with other fragments become drugs specific for the studied protein. Membrane proteins are complicated to study since they need the cell membrane to function normally. That is why they cannot be studied in the same way that water soluble proteins can. To overcome this problem, the project involved encapsulating the membrane proteins in artificial membrane discs, mimicking their natural environment. These membrane discs made the proteins functional outside of the cell membrane, making it possible to study them in a similar way as water soluble proteins.
This project successfully put two different membrane proteins into artificial membrane discs. It also succeeded in detecting an interaction between one of the studied proteins and two known binders, testifying that the technique works and keeps the proteins stable.
Hopefully, the study will lead to the technique being fully applied to membrane proteins. Artificial membrane discs, with adaptations to the target protein, can potentially be relevant to all important membrane proteins. This could lead to a cheaper and more efficient method for creating new medication for a wide range of diseases. (Less)
Please use this url to cite or link to this publication:
author
Kruse, Linnea LU and Degrér, Vilma LU
supervisor
organization
alternative title
Fragmentbaserad Läkemedelsutveckling för Membranproteiner: Nanodiskbaserad Metod för Weak Affinity-Kromatografi
course
KFKM05 20251
year
type
H3 - Professional qualifications (4 Years - )
subject
keywords
Weak Affinity Chromatography, WAC, Membrane proteins, Nanodiscs, Affinity-based screening, Membrane protein purification, Chromatographic methods, Protein-ligand interactions, Fragment-based Drug Discovery, Biophysical Chemistry
language
English
id
9197326
date added to LUP
2025-06-12 11:54:50
date last changed
2025-06-12 11:54:50
@misc{9197326,
  abstract     = {{Fragment-based drug discovery (FBDD) has in recent years emerged as an interesting method to target challenging binding sites, such as allosteric sites or small pockets. Weak Affinity Chromatography (WAC™) is a FBDD technology where dissociation constants of injected compounds are determined by comparing retention times between a protein column to a reference column. The goal of this project was to facilitate the application of WAC™ technology with membrane proteins, primarily Leucine Transporter (LeuT), also Aquaporin 4 (AQP4). This was done through the use of nanodiscs, consisting of two Membrane Scaffold Proteins (MSP) encircled around a phospholipid bilayer. Both proteins were successfully inserted into nanodiscs. For LeuT, different molar ratios between LeuT:MSP:Lipids:Cholate was investigated to optimize the yield of complete nanodiscs. A slight increase in LeuT relative to MSP proved successful whereas increase in lipids to MSP ratio decreased the yield. 

Furthermore, immobilization and packing of the LeuT nanodiscs on the column proved successful in regard of stable pressure and flow during all WAC™ runs. WAC™-UV experiments with the binder L-tryptophan in a salt-containing buffer showed retention time differences between LeuT and the empty nanodisc reference column, suggesting salt-dependent binding. This shift was not observed in salt-free buffer using WAC™-MS. Desipramine, another binder, exhibited a retention time difference in WAC™-MS. However, long retention on the reference column indicated non-specific interactions, likely due to the amphipathic properties of desipramine. 

The results demonstrate that membrane protein nanodiscs can be used successfully in combination with WAC™ technology. However, further optimization is necessary for broader application.}},
  author       = {{Kruse, Linnea and Degrér, Vilma}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Enabling Fragment-Based Drug Discovery for Membrane Proteins: Development of a Nanodisc-Based Weak Affinity Chromatography Method}},
  year         = {{2025}},
}