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Quality not Quantity: The Role of Marine Natural Products in Drug Discovery and Reverse Chemical Proteomics

View Article: PubMed Central

ABSTRACT

Reverse chemical proteomics combines affinity chromatography with phage display and promises to be a powerful new platform technology for the isolation of natural product receptors, facilitating the drug discovery process by rapidly linking biologically active small molecules to their cellular receptors and the receptors’ genes. In this paper we review chemical proteomics and reverse chemical proteomics and show how these techniques can add value to natural products research. We also report on techniques for the derivatisation of polystyrene microtitre plates with cleavable linkers and marine natural products that can be used in chemical proteomics or reverse chemical proteomics. Specifically, we have derivatised polystyrene with palau’amine and used reverse chemical proteomics to try and isolate the human receptors for this potent anticancer marine drug.

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Alignment of the C-terminal domain (aa 350–650 of 699) of hNopp140 (Swiss-Prot: Q14978) with clones rescued from human brain (HB) and human breast tumour (BrT) cDNA libraries using palau’amine immobilised on PS microtitre plate wells. Similar clones isolated from T7 phage-displayed libraries by Yu et al. [63] (using biotinylated doxorubicin immobilised on a streptavidin-coated plate) and Gearhart et al. [64] (using 2-methylnorharman adsorbed to nitrocellulose discs) are also shown. Black shading = 80% homology; Grey shading = 60% homology.
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f13-marinedrugs-03-00036: Alignment of the C-terminal domain (aa 350–650 of 699) of hNopp140 (Swiss-Prot: Q14978) with clones rescued from human brain (HB) and human breast tumour (BrT) cDNA libraries using palau’amine immobilised on PS microtitre plate wells. Similar clones isolated from T7 phage-displayed libraries by Yu et al. [63] (using biotinylated doxorubicin immobilised on a streptavidin-coated plate) and Gearhart et al. [64] (using 2-methylnorharman adsorbed to nitrocellulose discs) are also shown. Black shading = 80% homology; Grey shading = 60% homology.

Mentions: The DNA gels of the PCR products obtained from randomly selected plaques showed a range of different sized DNA inserts in all five libraries, indicating the selections had not reached a consensus after five rounds. Analysis of the DNA sequences obtained revealed the majority of clones contained DNA inserts that were either in backwards (25%), not in the correct reading frame (20%), not in the coding region of the encoded gene (10%) or expressed only short peptides (32%). However, two clones rescued from the T7Select10-3 human brain library (E9/F1) contained the C-terminal domain of nucleolar and coiled-body phosphoprotein 1 (hNopp140), and one clone rescued from the breast tumour library (G11) contained the C-terminal domain of the almost identical nucleolar phosphoprotein p130 (Fig. 14), both of which are involved in biogenesis of the nucleolus [62].


Quality not Quantity: The Role of Marine Natural Products in Drug Discovery and Reverse Chemical Proteomics
Alignment of the C-terminal domain (aa 350–650 of 699) of hNopp140 (Swiss-Prot: Q14978) with clones rescued from human brain (HB) and human breast tumour (BrT) cDNA libraries using palau’amine immobilised on PS microtitre plate wells. Similar clones isolated from T7 phage-displayed libraries by Yu et al. [63] (using biotinylated doxorubicin immobilised on a streptavidin-coated plate) and Gearhart et al. [64] (using 2-methylnorharman adsorbed to nitrocellulose discs) are also shown. Black shading = 80% homology; Grey shading = 60% homology.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756327&req=5

f13-marinedrugs-03-00036: Alignment of the C-terminal domain (aa 350–650 of 699) of hNopp140 (Swiss-Prot: Q14978) with clones rescued from human brain (HB) and human breast tumour (BrT) cDNA libraries using palau’amine immobilised on PS microtitre plate wells. Similar clones isolated from T7 phage-displayed libraries by Yu et al. [63] (using biotinylated doxorubicin immobilised on a streptavidin-coated plate) and Gearhart et al. [64] (using 2-methylnorharman adsorbed to nitrocellulose discs) are also shown. Black shading = 80% homology; Grey shading = 60% homology.
Mentions: The DNA gels of the PCR products obtained from randomly selected plaques showed a range of different sized DNA inserts in all five libraries, indicating the selections had not reached a consensus after five rounds. Analysis of the DNA sequences obtained revealed the majority of clones contained DNA inserts that were either in backwards (25%), not in the correct reading frame (20%), not in the coding region of the encoded gene (10%) or expressed only short peptides (32%). However, two clones rescued from the T7Select10-3 human brain library (E9/F1) contained the C-terminal domain of nucleolar and coiled-body phosphoprotein 1 (hNopp140), and one clone rescued from the breast tumour library (G11) contained the C-terminal domain of the almost identical nucleolar phosphoprotein p130 (Fig. 14), both of which are involved in biogenesis of the nucleolus [62].

View Article: PubMed Central

ABSTRACT

Reverse chemical proteomics combines affinity chromatography with phage display and promises to be a powerful new platform technology for the isolation of natural product receptors, facilitating the drug discovery process by rapidly linking biologically active small molecules to their cellular receptors and the receptors’ genes. In this paper we review chemical proteomics and reverse chemical proteomics and show how these techniques can add value to natural products research. We also report on techniques for the derivatisation of polystyrene microtitre plates with cleavable linkers and marine natural products that can be used in chemical proteomics or reverse chemical proteomics. Specifically, we have derivatised polystyrene with palau’amine and used reverse chemical proteomics to try and isolate the human receptors for this potent anticancer marine drug.

No MeSH data available.


Related in: MedlinePlus