Fragment screening using WAC towards new SMARCA4 inhibitors
(2020) KASM05 20201Centre for Analysis and Synthesis
- Abstract
- The drug discovery and development process has changed dramatically during the last decades and the growth of more efficient techniques as high throughput screening combined with combinatorial synthesis using amino acids and carboxylic acids on solid support makes it possible to investigate the affinity of molecules towards a biological target. Fragment based drug discovery (FBDD) has been implemented into different techniques with purpose of discovering new drugs. In this study, a high throughput technique, weak affinity chromatography (WAC) has been used to screen small aromatics ( <400 Da) with purpose of finding a new starting point for a SMARCA4 inhibitor. It is known that the protein is involved in the transcription and repair of... (More)
- The drug discovery and development process has changed dramatically during the last decades and the growth of more efficient techniques as high throughput screening combined with combinatorial synthesis using amino acids and carboxylic acids on solid support makes it possible to investigate the affinity of molecules towards a biological target. Fragment based drug discovery (FBDD) has been implemented into different techniques with purpose of discovering new drugs. In this study, a high throughput technique, weak affinity chromatography (WAC) has been used to screen small aromatics ( <400 Da) with purpose of finding a new starting point for a SMARCA4 inhibitor. It is known that the protein is involved in the transcription and repair of DNA, hence the discovery of a new inhibitor is of great interest in medicinal chemistry. To find new inhibitors with high quality data, the need for a more neutral reference column is crucial. It has been observed that the current reference column which consists of ethanolamine have a charge impact on the results, where positively charged molecules being repelled and negatively charged molecules more attracted to the column, consequently the molecules elutes faster and slower respectively. Thus, a reference peptide column was developed by mimicking the isoelectrical point and the amino acid sequence of the protein with the purpose of having a reference column that better mimics the protein. During this study, a total of 216 molecules where synthesized, they were distributed in 13 different libraries and each mix was examined and evaluated after the screenings on WAC. WAC has been proven to be an efficient, sensitive, and a robust method, which also provides the possibility to calculate the dissociation constant (KD). Due to its smoothness, flexibility, and productivity, the technique has very high potential to contribute and to establish more reliable FBDD research. (Less)
- Popular Abstract (Swedish)
- Syftet med avhandlingen var att undersöka och utvärdera bindningen av molekyler mot ett målprotein som är involverad i tillväxten av cancerceller. Undersökning genomfördes på Red Glead Discovery med WAC (weak affinity chromotography) som är en kromatografisk separationsmetod, där molekyler med olika vikt pumpas genom olika kolonner och retentionstiden för molekylerna i kolonnen detekteras. Molekyler som elueras senare från proteinkolonen än referenskolonnen, anses inneha bindning med proteinet. Större skillnad i eluerings tid tyder på starkare bindning. För att kunna erhålla mer precis och konkret bindningsdata bör referenskolonnen vara så lik proteinkolonnen som möjligt. Den nuvarande referenskolonnen påverkar resultatet genom att... (More)
- Syftet med avhandlingen var att undersöka och utvärdera bindningen av molekyler mot ett målprotein som är involverad i tillväxten av cancerceller. Undersökning genomfördes på Red Glead Discovery med WAC (weak affinity chromotography) som är en kromatografisk separationsmetod, där molekyler med olika vikt pumpas genom olika kolonner och retentionstiden för molekylerna i kolonnen detekteras. Molekyler som elueras senare från proteinkolonen än referenskolonnen, anses inneha bindning med proteinet. Större skillnad i eluerings tid tyder på starkare bindning. För att kunna erhålla mer precis och konkret bindningsdata bör referenskolonnen vara så lik proteinkolonnen som möjligt. Den nuvarande referenskolonnen påverkar resultatet genom att kolonnen främjar repulsion av molekyler med positiv laddning och tvärtom för negativt laddade molekyler. Baserat på det uppstådda problemet designades en peptid baserat på proteinets isoelektriska punkt och dess aminosyra sekvens. Peptiden immobiliserades i en kolonn och användes som en referenskolonn i undersökningen av olika molekyler.
Det unika med metoden framhävs av förmågan att kunna detektera upp till 25 olika molekyler som finns i ett bibliotek. Antalet molekyler i ett bibliotek är endast begränsade av vikten på molekylerna och startmaterialens reaktivitet vid fastfas syntesen. Totalt syntetiserades 216 olika molekyler som var distribuerade i tretton olika bibliotek och varje bibliotek innehöll mellan sex och 25 olika molekyler.
Proteinet som har kodnamnet SMARCA4 har ofta upptäckts vara muterad hos cancerpatienter. Genom att inhibera proteinet förhindras okontrollerat tillväxt av cancerceller, eftersom proteinet är involverad i transkriptionen av DNA. Därför valdes det proteinet till denna studie.
Resultaten visade minskad påverkan av laddning med referenspeptid kolonnen, där negativt laddade molekyler hade kortare retentionstid på referenskolonnen än den nuvarande kolonnen. Detta visar att sökandet efter en ny referenskolonn material borde fortsättas. Under utvärderingen av molekyler som upptäcktes ett par stycken som visade affinitet för proteinet, även dessa borde bli fortsatt studerade. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9026986
- author
- Sahakjan, Artur LU
- supervisor
- organization
- course
- KASM05 20201
- year
- 2020
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- WAC, Fragments, SMARCA4, Library, Fragment Based Drug Discovery (FBDD), Peptide Synthesis, Affinity, Organic chemistry, Organisk kemi
- language
- English
- id
- 9026986
- date added to LUP
- 2020-09-10 13:12:33
- date last changed
- 2020-09-10 13:12:33
@misc{9026986,
abstract = {{The drug discovery and development process has changed dramatically during the last decades and the growth of more efficient techniques as high throughput screening combined with combinatorial synthesis using amino acids and carboxylic acids on solid support makes it possible to investigate the affinity of molecules towards a biological target. Fragment based drug discovery (FBDD) has been implemented into different techniques with purpose of discovering new drugs. In this study, a high throughput technique, weak affinity chromatography (WAC) has been used to screen small aromatics ( <400 Da) with purpose of finding a new starting point for a SMARCA4 inhibitor. It is known that the protein is involved in the transcription and repair of DNA, hence the discovery of a new inhibitor is of great interest in medicinal chemistry. To find new inhibitors with high quality data, the need for a more neutral reference column is crucial. It has been observed that the current reference column which consists of ethanolamine have a charge impact on the results, where positively charged molecules being repelled and negatively charged molecules more attracted to the column, consequently the molecules elutes faster and slower respectively. Thus, a reference peptide column was developed by mimicking the isoelectrical point and the amino acid sequence of the protein with the purpose of having a reference column that better mimics the protein. During this study, a total of 216 molecules where synthesized, they were distributed in 13 different libraries and each mix was examined and evaluated after the screenings on WAC. WAC has been proven to be an efficient, sensitive, and a robust method, which also provides the possibility to calculate the dissociation constant (KD). Due to its smoothness, flexibility, and productivity, the technique has very high potential to contribute and to establish more reliable FBDD research.}},
author = {{Sahakjan, Artur}},
language = {{eng}},
note = {{Student Paper}},
title = {{Fragment screening using WAC towards new SMARCA4 inhibitors}},
year = {{2020}},
}