A new ELISA plate based microtiter well assay for mycobacterial topoisomerase I for the direct screening of enzyme inhibitory monoclonal antibody supernatants

Leelaram, Majety Naga; Nunna, Suneetha; Nagaraja, Valakunja; Manjunath, Ramanathapuram

doi:10.1016/j.jim.2010.03.008

Cited by 5 publications

(6 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, in these experiments, the covalent reaction intermediates formed between wild‐type enzyme and DNA would also be precipitated, interfering with the analysis. To avoid this problem, a cleavage‐deficient active site mutant of MstopoI, Y339F (30) was used in these immunoprecipitation experiments, as described in Materials and Methods. DNA was retained in the pellet of high‐salt wash when 1E4F5 and 17B1IB4 were used in immunoprecipitation assays but not with a different anti‐MstopoI mAb, 2B1G5 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…DNA was retained in the pellet of high‐salt wash when 1E4F5 and 17B1IB4 were used in immunoprecipitation assays but not with a different anti‐MstopoI mAb, 2B1G5 (Fig. 4 B , top panel) that binds to the enzyme but does not inhibit the activity (30). DNA was not detectable in the samples treated with SDS, showing that the precipitation of DNA was protein‐mediated (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The inhibition is a specific property of the mAbs as mouse IgG did not show any effect (lane 3). A few other mAbs raised against MstopoI bound to the enzyme but did not inhibit the activity [not shown, (30)]. Importantly, both 1E4F5 and 17B1IB4 inhibited M. tuberculosis topoI (MttopoI), and the extent of inhibition was comparable to MstopoI inhibition ( Fig.…”

Section: Inhibition Of Dna Relaxation Activitymentioning

confidence: 99%

See 2 more Smart Citations

Type IA topoisomerase inhibition by clamp closure

Leelaram¹,

Bhat²,

Godbole³

et al. 2013

FASEB j.

Self Cite

View full text Add to dashboard Cite

Bacterial DNA topoisomerase I (topoI) catalyzes relaxation of negatively supercoiled DNA. The enzyme alters DNA topology through protein-operated DNA gate, switching between open and closed conformations during its reaction. We describe the mechanism of inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis topoI by monoclonal antibodies (mAbs) that bind with high affinity and inhibit at 10-50 nM concentration. Unlike other inhibitors of topoisomerases, the mAbs inhibited several steps of relaxation reaction, namely DNA binding, cleavage, strand passage, and enzyme-DNA dissociation. The enhanced religation of the cleaved DNA in presence of the mAb indicated closing of the enzyme DNA gate. The formation of enzyme-DNA heterocatenane in the presence of the mAbs as a result of closing the gate could be inferred by the salt resistance of the complex, visualized by atomic force microscopy and confirmed by fluorescence measurements. Locking the enzyme-DNA complex as a closed clamp restricted the movements of the DNA gate, affecting all of the major steps of the relaxation reaction. Enzyme trapped on DNA in closed clamp conformation formed roadblock for the elongating DNA polymerase. The unusual multistep inhibition of mycobacterial topoisomerases may facilitate lead molecule development, and the mAbs would also serve as valuable tools to probe the enzyme mechanism.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Inhibition Of Dna Relaxation Activitymentioning

confidence: 99%

See 1 more Smart Citation

Type IA topoisomerase inhibition by clamp closure

Leelaram¹,

Bhat²,

Godbole³

et al. 2013

FASEB j.

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, with new synthesis strategies and lowered monomer costs the interest in developing peptide drugs has markedly increased [24–27]. One of the very promising strategies was first presented by Nagaraja's research group, who had taken advantage of antibodies raised by the natural immune response of mice injected with the desired target, in the reported cases, Mycobacteria DNA gyrase or TopoI [28–30]. As a result different antibodies with specific inhibitory effects on either target have been identified.…”

Section: Discussionmentioning

confidence: 99%

“…The potential for synthetic peptides as efficient species-specific inhibitors even of discrete steps of Topo catalysis is highlighted in several studies by Nagaraja and co-workers describing the identification and characterization of species-specific antibodies with inhibitory activities against particular steps of Mycobacteria DNA gyrase or TopoI catalysis [28–30]. Peptides with similar inhibitory activities and potential in future antituberculosis treatment [29] are likely to be derived from such antibodies [31–34].…”

Section: Introductionmentioning

confidence: 99%

Peptide Inhibition of Topoisomerase IB fromPlasmodium falciparum

Roy

D’Annessa

Nielsen

et al. 2011

Molecular Biology International

View full text Add to dashboard Cite

Control of diseases inflicted by protozoan parasites such as Leishmania, Trypanosoma, and Plasmodium, which pose a serious threat to human health worldwide, depends on a rather small number of antiparasite drugs, of which many are toxic and/or inefficient. Moreover, the increasing occurrence of drug-resistant parasites emphasizes the need for new and effective antiprotozoan drugs. In the current study, we describe a synthetic peptide, WRWYCRCK, with inhibitory effect on the essential enzyme topoisomerase I from the malaria-causing parasite Plasmodium falciparum. The peptide inhibits specifically the transition from noncovalent to covalent DNA binding of P. falciparum topoisomerase I, while it does not affect the ligation step of catalysis. A mechanistic explanation for this inhibition is provided by molecular docking analyses. Taken together the presented results suggest that synthetic peptides may represent a new class of potential antiprotozoan drugs.

show abstract