Faculty of Natural Sciences

TY - JOUR

T1 - Simple and fast DNA based sensor system for screening of small-molecule compounds targeting eukaryotic topoisomerase 1

AU - Petersen, Kamilla Vandsø

AU - Selas, Asier

AU - Hymøller, Kirstine Mejlstrup

AU - Mizielinski, Karol

AU - Thorsager, Maria

AU - Stougaard, Magnus

AU - Alonso, Concepcion

AU - Palacios, Francisco

AU - Pérez-Pertejo, Yolanda

AU - Reguera, Rosa M.

AU - Balaña-Fouce, Rafael

AU - Knudsen, Birgitta R.

AU - Tesauro, Cinzia

PY - 2021/8

Y1 - 2021/8

N2 - Background: Eukaryotic topoisomerase 1 is a potential target of anti-parasitic and anti-cancer drugs. Parasites require topoisomerase 1 activity for survival and, consequently, compounds that inhibit topoisomerase 1 activity may be of interest. All effective topoisomerase 1 drugs with anti-cancer activity act by inhibiting the ligation reaction of the enzyme. Screening for topoisomer-ase 1 targeting drugs, therefore, should involve the possibility of dissecting which step of topoiso-merase 1 activity is affected. Methods: Here we present a novel DNA-based assay that allows for screening of the effect of small-molecule compounds targeting the binding/cleavage or the ligation steps of topoisomerase 1 catalysis. This novel assay is based on the detection of a rolling circle amplification product generated from a DNA circle resulting from topoisomerase 1 activity. Results: We show that the binding/cleavage and ligation reactions of topoisomerase 1 can be investigated separately in the presented assay termed REEAD (C|L) and demonstrate that the assay can be used to investigate, which of the individual steps of topoisomerase 1 catalysis are affected by small-mol-ecule compounds. The assay is gel-free and the results can be detected by a simple colorimetric readout method using silver-on-gold precipitation rendering large equipment unnecessary. Con-clusion: REEAD (C|L) allows for easy and quantitative investigations of topoisomerase 1 targeting compounds and can be performed in non-specialized laboratories.

AB - Background: Eukaryotic topoisomerase 1 is a potential target of anti-parasitic and anti-cancer drugs. Parasites require topoisomerase 1 activity for survival and, consequently, compounds that inhibit topoisomerase 1 activity may be of interest. All effective topoisomerase 1 drugs with anti-cancer activity act by inhibiting the ligation reaction of the enzyme. Screening for topoisomer-ase 1 targeting drugs, therefore, should involve the possibility of dissecting which step of topoiso-merase 1 activity is affected. Methods: Here we present a novel DNA-based assay that allows for screening of the effect of small-molecule compounds targeting the binding/cleavage or the ligation steps of topoisomerase 1 catalysis. This novel assay is based on the detection of a rolling circle amplification product generated from a DNA circle resulting from topoisomerase 1 activity. Results: We show that the binding/cleavage and ligation reactions of topoisomerase 1 can be investigated separately in the presented assay termed REEAD (C|L) and demonstrate that the assay can be used to investigate, which of the individual steps of topoisomerase 1 catalysis are affected by small-mol-ecule compounds. The assay is gel-free and the results can be detected by a simple colorimetric readout method using silver-on-gold precipitation rendering large equipment unnecessary. Con-clusion: REEAD (C|L) allows for easy and quantitative investigations of topoisomerase 1 targeting compounds and can be performed in non-specialized laboratories.

KW - Colorimetric readout

KW - Drug screening

KW - Enzyme activity

KW - REEAD

KW - Rolling circle amplification

KW - Small-molecule compounds

KW - Topoisomerase 1

UR - http://www.scopus.com/inward/record.url?scp=85113320400&partnerID=8YFLogxK

U2 - 10.3390/pharmaceutics13081255

DO - 10.3390/pharmaceutics13081255

M3 - Journal article

C2 - 34452216

AN - SCOPUS:85113320400

VL - 13

JO - Pharmaceutics

JF - Pharmaceutics

SN - 1999-4923

IS - 8

M1 - 1255

ER -