Betulinic acid, a potent inhibitor of eukaryotic topoisomerase I: identification of the inhibitory step, the major functional group responsible and development of more potent derivatives.
BACKGROUND
Betulinic acid, a naturally abundant, plant derived, pentacyclic triterpenoid possesses anti-HIV, anti-malarial and anti-inflammatory properties and has recently emerged as a potent anti-tumor compound. This study explores the mode of action of betulinic acid on eukaryotic topoisomerase I and identifies the major functional group responsible along with more potent derivatives.


METHODS
Topoisomerase I relaxation activity was electrophoretically measured by the decreased mobility of the relaxed monomers followed by ethidium bromide staining. DNA cleavage was studied by electrophoretic separation of the nicked monomers from the relaxed and supercoiled monomers in presence of ethidium bromide. In-vivo DNA cleavage was studied in blasted mouse splenocytes by the SDS-K+ trapping of 3H-DNA-topoisomerase I-camptothecin ternary complex.


RESULTS
Betulinic acid exerts its inhibitory effect by preventing topoisomerase I-DNA interaction as a result of which the 'cleavable complex' is not formed. In consequence, it also acts as an antagonist to camptothecin-mediated cleavage. A series of analogues modified at C-3, C-17 and C-20 positions of betulinic acid were subsequently assayed for inhibition of topoisomerase I catalytic activity. Replacement of the 17-carboxylic group reduces the inhibitory effect and decarboxylation leads to the complete loss of inhibitory effect.


CONCLUSIONS
This study is the first detail report of betulinic acid as a very potent inhibitior of eukaryotic topoisomerase I and highlights the necessity of the carboxylic functional group. Dihydro betulinic acid is the most potent (IC50=0.5 mM) pentacyclic triterpenoid to inhibit eukaryotic topoisomerase I till date and can be exploited as a strong candidate for anti-tumor drug designing.
Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, Kolkata, India.
Arnab Roy
AR
Chowdhury
Suparna
S
Mandal
Bidyottam
B
Mittra
Shalini
S
Sharma
Sibabrata
S
Mukhopadhyay
Hemanta K
HK
Majumder
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002166:Camptothecin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D015394:Molecular Structure; D053978:Pentacyclic Triterpenes; D010936:Plant Extracts / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D059004:Topoisomerase I Inhibitors; D014315:Triterpenes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000094062:Betulinic Acid
D000972:Antineoplastic Agents, Phytogenic; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D053978:Pentacyclic Triterpenes; D010936:Plant Extracts; D059004:Topoisomerase I Inhibitors; D014315:Triterpenes; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin; D000094062:Betulinic Acid