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Literature summary for 5.6.2.1 extracted from

  • Das, B.B.; Sen, N.; Roy, A.; Dasgupta, S.B.; Ganguly, A.; Mohanta, B.C.; Dinda, B.; Majumder, H.K.
    Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I (2006), Nucleic Acids Res., 34, 1121-1132.
    View publication on PubMedView publication on EuropePMC
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Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3)pLysS cells Leishmania donovani

Protein Variants

Protein Variants Comment Organism
delta 39-635 in contrast to camptothecin, baicalein and luteolin fail to inhibit the religation step when the drugs are added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and camptothecin result in investigations of the effect of baicalein and luteolin on camptothecin-resistant mutant enzyme LdTOP1D39LS lacking 1–39 amino acids of the large subunit Leishmania donovani
additional information Leishmania donovani strain Ld160CPTR camptothecin-resistant Leishmania donovani strain. The camptothecin-resistant strain reached a resistance level of 32fold over the wild-type Leishmania donovani AG83 strain. Baicalein and luteolin show inhibition of relaxation activity up to 60% at 30 mM concentration compared with that of camptothecin, where no inhibition of relaxationactivity is observed Leishmania donovani

Inhibitors

Inhibitors Comment Organism Structure
baicalein (5,6,7-trihydroxyflavone), naturally occurring flavone, isolated from the stem-bark of Oroxylum indicum, binds to the free enzyme and also intercalates into the DNA at a very high concentration (300 mM) without binding to the minor grove Leishmania donovani
camptothecin the best characterized topoisomerase IB inhibitor. Ability of the naturally occurring flavones to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT) Leishmania donovani
dihydrobetulinic acid DHBA, pentacyclic triterpenoid and catalytic inhibitor of both topoisomerases I and II that antagonizes camptothecin-induced cleavage, do not induce the formation of SDS-K+ precipitable complex, when Leishmania donovani cells incubated for 3 h with the compound Leishmania donovani
luteolin naturally occurring flavone, binds to the free enzyme and also intercalates into the DNA at a very high concentration (300 mM) without binding to the minor grove Leishmania donovani
additional information Comparisons of topoisomerase I-mediated DNA cleavage induced by flavones and camptothecin. The rates of stabilization of cleavable complex by the flavones are 2-to 3fold reduced compared with that of camptothecin. Analysis of drug induced covalent topoisomerase I–DNA complex formation in Leishmania donovani promastigotes by KCl-SDS precipitation assay. Treatment of the cells with baicalein, luteolin and quercetin for 5 h significantly increases the SDS-K+ precipitable complex compared with the untreated control cells. Relief of inhibition on dilution suggests that the selected flavones are acting reversibly against topoisomerase I. Fluorescence spectroscopy used to check directly the binding of luteolin, baicalein and quercetin to the enzyme (LdTOP1LS). Fluorescence titration data reveal that these selected flavones interact with free enzyme and perhaps explain for stronger inhibition in the enzyme–drug pre-incubation experiments. Ability of the selected flavones to displace ethidium bromide from DNA is determined by a fluorescence emission assay. Effect of flavones on single turnover cleavage and religation activity measured, interaction of these flavones with enzyme during trans-esterification reaction with DNA is a pre-requirement for the stabilization of topoisomerase I-cleavable complex and subsequently inhibiting the religation step Leishmania donovani
quercetin naturally occurring flavone, binds to the free enzyme and also intercalates into the DNA at a very high concentration (300 mM) without binding to the minor grove Leishmania donovani

Organism

Organism UniProt Comment Textmining
Leishmania donovani
-
-
-

Purification (Commentary)

Purification (Comment) Organism
Ni2+-NTA agarose column followed by phosphocellulose column. Concentrations of purified proteins quantified by Bradford reaction Leishmania donovani

Subunits

Subunits Comment Organism
heterodimer Leishmania donovani topoisomerase I is an unusual bi-subunit enzyme where the core DNA binding domain and the catalytic domain harboring the consensus SKXXY motif lie in separate subunits. The two subunits (LdTOP1L and LdTOP1S) are synthesized by two different genes, which associate with each other through protein–protein interaction to form an active heterodimeric topoisomerase I within the parasite Leishmania donovani

Synonyms

Synonyms Comment Organism
bi-subunit topoisomerase I
-
 Leishmania donovani
LdTOP1LS
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 Leishmania donovani
Topoisomerase I
-
 Leishmania donovani
Type I DNA topoisomerase
-
 Leishmania donovani

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.0015
-
 LdTOP1LS pre-incubated separately with different concentrations for 5 min at 37°C in relaxation buffer followed by addition of pBluescript(SK+) DNA and further incubated for 15 min at 37°C Leishmania donovani baicalein
0.0025
-
 LdTOP1LS pre-incubated separately with different concentrations for 5 min at 37°C in relaxation buffer followed by addition of pBluescript(SK+) DNA and further incubated for 15 min at 37°C Leishmania donovani luteolin
0.0055
-
 LdTOP1LS pre-incubated separately with different concentrations for 5 min at 37°C in relaxation buffer followed by addition of pBluescript(SK+) DNA and further incubated for 15 min at 37°C Leishmania donovani quercetin
0.009
-
 Quantitative representation of enzyme inhibition in the presence of flavones in relaxation experiments Leishmania donovani baicalein
0.014
-
 Quantitative representation of enzyme inhibition in the presence of flavones in relaxation experiments Leishmania donovani luteolin
0.017
-
 Quantitative representation of enzyme inhibition in the presence of flavones in relaxation experiments Leishmania donovani quercetin