scholarly journals Biolayer interferometry provides a robust method for detecting DNA binding small molecules in microbial extracts

Author(s):  
Ross D. Overacker ◽  
Birte Plitzko ◽  
Sandra Loesgen
2020 ◽  
Author(s):  
Ross D. Overacker ◽  
Birte Plitzko ◽  
Sandra Loesgen

DNA replication is an exceptional point of therapeutic intervention for many cancer types and several small molecules targeting DNA have been developed into clinically used antitumor agents. Many of these molecules are naturally occurring metabolites from plants and microorganisms, such as the widely used chemotherapeutic doxorubicin. While natural product sources contain a vast number of DNA binding small molecules, isolating and identifying these molecules is challenging. Typical screening campaigns utilize time-consuming bioactivity-guided fractionation approaches, which use sequential rounds of cell-based assays to guide the isolation of active compounds. In this study, we explore the use of Biolayer Interferometry (BLI) as a tool for rapidly screening natural products sources for DNA targeting small molecules. We first verified that BLI robustly detected DNA binding using designed GC and AT rich DNA oligonucleotides with known DNA intercalating, groove-, and covalent-binding agents including actinomycin D (<b>1</b>), doxorubicin (<b>2</b>), ethidium bromide (<b>3</b>), propidium iodide (<b>4</b>), Hoechst 33342 (<b>5</b>), netropsin (<b>6</b>), and cisplatin (<b>7</b>). Although binding varied with the properties of the oligonucleotides, measured binding affinities agreed with previously reported values. We next utilized BLI to screen over 100 bacterial extracts from our microbial library for DNA binding activity and found three highly active extracts. Binding-guided isolation was used to isolate the active principle component from each extract, which were identified as echinomycin (<b>8</b>), actinomycin V (<b>9</b>), and chartreusin (<b>10</b>). This biosensor-based DNA binding screen is a novel, low cost, easy to use, and sensitive approach for medium-throughput screening of complex chemical libraries.


2020 ◽  
Author(s):  
Ross D. Overacker ◽  
Birte Plitzko ◽  
Sandra Loesgen

DNA replication is an exceptional point of therapeutic intervention for many cancer types and several small molecules targeting DNA have been developed into clinically used antitumor agents. Many of these molecules are naturally occurring metabolites from plants and microorganisms, such as the widely used chemotherapeutic doxorubicin. While natural product sources contain a vast number of DNA binding small molecules, isolating and identifying these molecules is challenging. Typical screening campaigns utilize time-consuming bioactivity-guided fractionation approaches, which use sequential rounds of cell-based assays to guide the isolation of active compounds. In this study, we explore the use of Biolayer Interferometry (BLI) as a tool for rapidly screening natural products sources for DNA targeting small molecules. We first verified that BLI robustly detected DNA binding using designed GC and AT rich DNA oligonucleotides with known DNA intercalating, groove-, and covalent-binding agents including actinomycin D (<b>1</b>), doxorubicin (<b>2</b>), ethidium bromide (<b>3</b>), propidium iodide (<b>4</b>), Hoechst 33342 (<b>5</b>), netropsin (<b>6</b>), and cisplatin (<b>7</b>). Although binding varied with the properties of the oligonucleotides, measured binding affinities agreed with previously reported values. We next utilized BLI to screen over 100 bacterial extracts from our microbial library for DNA binding activity and found three highly active extracts. Binding-guided isolation was used to isolate the active principle component from each extract, which were identified as echinomycin (<b>8</b>), actinomycin V (<b>9</b>), and chartreusin (<b>10</b>). This biosensor-based DNA binding screen is a novel, low cost, easy to use, and sensitive approach for medium-throughput screening of complex chemical libraries.


BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Wangming Zhang ◽  
Shuang Yang ◽  
Jinhe Liu ◽  
Linchun Bao ◽  
He Lu ◽  
...  

Abstract Background The high expression of BLM (Bloom syndrome) helicase in tumors involves its strong association with cell expansion. Bisbenzylisoquinoline alkaloids own an antitumor property and have developed as candidates for anticancer drugs. This paper aimed to screen potential antiproliferative small molecules from 12 small molecules (the derivatives of bisbenzylisoquinoline alkaloids tetrandrine and fangchinoline) by targeting BLM642–1290 helicase. Then we explore the inhibitory mechanism of those small molecules on proliferation of MDA-MB-435 breast cancer cells. Methods Fluorescence polarization technique was used to screen small molecules which inhibited the DNA binding and unwinding of BLM642–1290 helicase. The effects of positive small molecules on the ATPase and conformation of BLM642–1290 helicase were studied by the malachite green-phosphate ammonium molybdate colorimetry and ultraviolet spectral scanning, respectively. The effects of positive small molecules on growth of MDA-MB-435 cells were studied by MTT method, colony formation and cell counting method. The mRNA and protein levels of BLM helicase in the MDA-MB-435 cells after positive small molecule treatments were examined by RT-PCR and ELISA, respectively. Results The compound HJNO (a tetrandrine derivative) was screened out which inhibited the DNA binding, unwinding and ATPase of BLM642–1290 helicase. That HJNO could bind BLM642–1290helicase to change its conformationcontribute to inhibiting the DNA binding, ATPase and DNA unwinding of BLM642–1290 helicase. In addition, HJNO showed its inhibiting the growth of MDA-MB-435 cells. The values of IC50 after drug treatments for 24 h, 48 h and 72 h were 19.9 μmol/L, 4.1 μmol/L and 10.9 μmol/L, respectively. The mRNA and protein levels of BLM helicase in MDA-MB-435 cells increased after HJNO treatment. Those showed a significant difference (P < 0.05) compared with negative control when the concentrations of HJNO were 5 μmol/L and 10 μmol/L, which might contribute to HJNO inhibiting the DNA binding, ATPase and DNA unwinding of BLM helicase. Conclusion The small molecule HJNO was screened out by targeting BLM642–1290 helicase. And it showed an inhibition on MDA-MB-435 breast cancer cells expansion.


Author(s):  
Miriam S. Butler ◽  
Mani Roshan-Moniri ◽  
Michael Hsing ◽  
Desmond Lau ◽  
Ari Kim ◽  
...  

2017 ◽  
Vol 114 (4) ◽  
pp. 681-686 ◽  
Author(s):  
Glen P. Liszczak ◽  
Zachary Z. Brown ◽  
Samuel H. Kim ◽  
Rob C. Oslund ◽  
Yael David ◽  
...  

Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small-molecule bromodomain and extra-terminal family bromodomain inhibitor JQ1 or a peptide-based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials.


2006 ◽  
Vol 100 (11) ◽  
pp. 1744-1754 ◽  
Author(s):  
Toshihiro Ihara ◽  
Takashi Ikegami ◽  
Tomohiro Fujii ◽  
Yusuke Kitamura ◽  
Shinji Sueda ◽  
...  

Author(s):  
Ritesh Pal ◽  
Jeet Chakraborty ◽  
Titas Kumar Mukhopadhyay ◽  
Ajay Kanungo ◽  
Rimita Saha ◽  
...  

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