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Nucleic Acids as Drug Targets - Influence of Platinum Complexes on Duplex Stability and Binding Kinetics

Polonyi, Christopher LU (2013)
Abstract (Swedish)
Popular Abstract in Swedish

Att få en övergripande förståelse för hur mänskliga celler påverkas av

metallbaserade läkemedel är viktigt, eftersom de har visat sig vara väldigt

effektiva för behandling av exempelvis testikel- och äggstockscancer. För att

utforska bakomliggande orsaker har forskningsprojektets utgångspunkt varit

att studera kliniskt välkarakteriserade metallbaserade läkemedel som

exempelvis cisplatin, och dess interaktioner med nukleinsyror. Kartläggning av

grundläggande mekanismer avseende vilken effekt strukturella skillnader i

RNA har på en specifik molekyls reaktivitet, bidrar till en djupare förståelse av

läkemedlets inverkan. Ny... (More)
Popular Abstract in Swedish

Att få en övergripande förståelse för hur mänskliga celler påverkas av

metallbaserade läkemedel är viktigt, eftersom de har visat sig vara väldigt

effektiva för behandling av exempelvis testikel- och äggstockscancer. För att

utforska bakomliggande orsaker har forskningsprojektets utgångspunkt varit

att studera kliniskt välkarakteriserade metallbaserade läkemedel som

exempelvis cisplatin, och dess interaktioner med nukleinsyror. Kartläggning av

grundläggande mekanismer avseende vilken effekt strukturella skillnader i

RNA har på en specifik molekyls reaktivitet, bidrar till en djupare förståelse av

läkemedlets inverkan. Ny kunskap angående molekylära mekanismer bidrar till

ökad förståelse på cellulär nivå, vilket ur ett patientperspektiv betyder mer

individbaserade behandlingsmetoder.

En metod har utvecklats för att kunna följa hur cisplatinets reaktivitet med

olika RNA strukturer ändrar sig med tiden och vid olika fysiologiska

betingelser. Det är möjligt att observera hur lång tid det tar för inbindningen

att äga rum och mäta den resulterande förändringen som inbindningen ger

upphov till. Läkemedelseffekten på biologiskt viktiga strukturer kan på så vis

undersökas, med målet att kunna beskriva och förutsäga hur inbindningen till

ett specifikt RNA inbindningssäte kommer att påverka nödvändiga funktioner i mänskliga celler. Metoden möjliggör även screening av miRNA reaktivitet,

vilket ger en bättre förståelse av hur olika celltyper med annorlunda miRNA

profiler kommer att reagera på en läkemedelsbehandling.

Ett annat viktigt mål är att utforska hur proteinuttrycksnivåer förändras i cellen, då en specifik miRNA familj har blivit utsatt för ett

läkemedel eller en miRNA mimic vars uppgift är att härma de naturligt

förekommande molekylerna innuti cellen. Responsen på proteinnivå kommer

att vara avgörande för cellens fortlevnad. Det är möjligt att cellen till slut blir

tvungen att genomgå programerad celldöd, ifall följden av behandlingen blir ett

haveri av cellens maskineri. Manipulering av proteinuttryck har studerats med

syfte att kunna påverka initiering, etabliering och metastasering av cancer. Ett

modelsystem har utvecklats för att med hjälp av cellförsök kunna mäta

effekten av intressanta läkemedelskandidater, som påverkar uttrycksnivåerna av

de proteiner som de viktiga cancerskyddsgenerna BRCA1 och BRCA2 kodar

för.

I pågående projekt genomförs en mer omfattande studie, med målet att uppnå

en detaljerad bild av hur olika miRNA familjer och biologiskt aktiva RNA

strukturer påverkas av metallbaserade läkemedel. Den omfattande studien

tillämpar den nyutvecklade kinetiska metoden, för att rangordna

inbindningsställen på RNA strukturer baserat på reaktivitet. Det bedrivs även

fortsatta studier på cellulär nivå, med målet att kartlägga mekanismer bakom

förändrade proteinuttrycksnivåer. Effekten av punktmutationer på RNA nivå

undersöks även. (Less)
Abstract
Gaining a detailed understanding of how human cells are affected by metal-based drugs is crucial, since these drugs have shown great potential in cancer treatment. Cisplatin is used to treat many different types of cancer, e.g. testicular, ovarian, melanoma, bladder, non-small cell lung cancer, small cell lung cancer, myelomas and lymphomas. The miRNA expression profiles in different human cancers differ from healthy tissue and can be used for classification. This difference makes it important to put every drug that is going to be used in a certain cancer treatment regime, in the context of the expression profile of the specific cancer type. The strong correlation between germline mutations in BRCA1 and cancer development makes it an... (More)
Gaining a detailed understanding of how human cells are affected by metal-based drugs is crucial, since these drugs have shown great potential in cancer treatment. Cisplatin is used to treat many different types of cancer, e.g. testicular, ovarian, melanoma, bladder, non-small cell lung cancer, small cell lung cancer, myelomas and lymphomas. The miRNA expression profiles in different human cancers differ from healthy tissue and can be used for classification. This difference makes it important to put every drug that is going to be used in a certain cancer treatment regime, in the context of the expression profile of the specific cancer type. The strong correlation between germline mutations in BRCA1 and cancer development makes it an essential target to study for creating novel treatment regimes.

This thesis has a focus on studies of the clinically well-characterized platinum drugs cisplatin and oxaliplatin, and how these affect the stability and melting behaviour of similar sequence DNA and RNA duplexes. The studied RNA duplexes showed a more pronounced sensitivity towards platination compared to the DNA duplexes. A novel method was developed to monitor the binding kinetics of cisplatin towards the mature form of miR-146a and short RNAs containing biologically important sites with altered base complementarity. Spectral changes and binding kinetics were monitored over time at 38 °C and the observed rate constants were linearly dependent on the concentration of mono-aquated cisplatin. Binding of mono-aquated cisplatin to the studied RNA duplexes was shown to be salt dependent, with an observed variation in reactivity. Different mimics of miR-146a, which is known to be up-regulated in invasive cell lines, were constructed to study the influence on BRCA1 down-regulation in vitro. The mimics included several types of chemical modifications and modifications made to the duplex architecture. The degree of complementarity of the guide strand towards the target mRNA was gradually increased to study and evaluate the corresponding change in silencing capacity. The idea behind the construction of a fully complementary mimic, was to study the possibility of obtaining similar silencing levels as with siRNAs. The mimic having full complementarity towards the mRNA target did indeed show higher silencing activity compared to the less complementary mimics. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Chow, Christine S., Department of Chemistry, Wayne State University, Detroit, USA
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Anticancer drugs, platinum complexes, cisplatin, oxaliplatin, nucleic acid, RNA, DNA, duplex melting, thermodynamic stability, kinetics, non-coding RNA, miRNA, siRNA, wobble base pair, loop, BRCA1, mimic, luciferase assay
publisher
Lund University, dept. of Chemistry
defense location
Lecture Hall B, Centre for Chemistry and Chemical Engineering, Getingevägen 60, Lund
defense date
2013-12-20 10:15
ISBN
978-91-7422-336-1
language
English
LU publication?
yes
id
0ad2b95b-0d5a-41a9-bd47-f32be50771f6 (old id 4174913)
date added to LUP
2013-12-10 14:30:43
date last changed
2016-09-19 08:45:11
@phdthesis{0ad2b95b-0d5a-41a9-bd47-f32be50771f6,
  abstract     = {Gaining a detailed understanding of how human cells are affected by metal-based drugs is crucial, since these drugs have shown great potential in cancer treatment. Cisplatin is used to treat many different types of cancer, e.g. testicular, ovarian, melanoma, bladder, non-small cell lung cancer, small cell lung cancer, myelomas and lymphomas. The miRNA expression profiles in different human cancers differ from healthy tissue and can be used for classification. This difference makes it important to put every drug that is going to be used in a certain cancer treatment regime, in the context of the expression profile of the specific cancer type. The strong correlation between germline mutations in BRCA1 and cancer development makes it an essential target to study for creating novel treatment regimes.<br/><br>
This thesis has a focus on studies of the clinically well-characterized platinum drugs cisplatin and oxaliplatin, and how these affect the stability and melting behaviour of similar sequence DNA and RNA duplexes. The studied RNA duplexes showed a more pronounced sensitivity towards platination compared to the DNA duplexes. A novel method was developed to monitor the binding kinetics of cisplatin towards the mature form of miR-146a and short RNAs containing biologically important sites with altered base complementarity. Spectral changes and binding kinetics were monitored over time at 38 °C and the observed rate constants were linearly dependent on the concentration of mono-aquated cisplatin. Binding of mono-aquated cisplatin to the studied RNA duplexes was shown to be salt dependent, with an observed variation in reactivity. Different mimics of miR-146a, which is known to be up-regulated in invasive cell lines, were constructed to study the influence on BRCA1 down-regulation in vitro. The mimics included several types of chemical modifications and modifications made to the duplex architecture. The degree of complementarity of the guide strand towards the target mRNA was gradually increased to study and evaluate the corresponding change in silencing capacity. The idea behind the construction of a fully complementary mimic, was to study the possibility of obtaining similar silencing levels as with siRNAs. The mimic having full complementarity towards the mRNA target did indeed show higher silencing activity compared to the less complementary mimics.},
  author       = {Polonyi, Christopher},
  isbn         = {978-91-7422-336-1},
  keyword      = {Anticancer drugs,platinum complexes,cisplatin,oxaliplatin,nucleic acid,RNA,DNA,duplex melting,thermodynamic stability,kinetics,non-coding RNA,miRNA,siRNA,wobble base pair,loop,BRCA1,mimic,luciferase assay},
  language     = {eng},
  publisher    = {Lund University, dept. of Chemistry},
  school       = {Lund University},
  title        = {Nucleic Acids as Drug Targets - Influence of Platinum Complexes on Duplex Stability and Binding Kinetics},
  year         = {2013},
}