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

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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

Schematic representation of chemical proteomics and reverse chemical proteomics. In chemical proteomics (A), one uses a small molecule (e.g. marine natural product) to construct an affinity or activity probe to isolate specific proteins or a family of proteins. In reverse chemical proteomics (B), we start with the transcriptome, which is cloned into an amplifiable vector (e.g. a virus) that expresses a single protein from the proteome on its surface. A tagged natural product can be used to probe the tagged proteome in an iterative manner.
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f1-marinedrugs-03-00036: Schematic representation of chemical proteomics and reverse chemical proteomics. In chemical proteomics (A), one uses a small molecule (e.g. marine natural product) to construct an affinity or activity probe to isolate specific proteins or a family of proteins. In reverse chemical proteomics (B), we start with the transcriptome, which is cloned into an amplifiable vector (e.g. a virus) that expresses a single protein from the proteome on its surface. A tagged natural product can be used to probe the tagged proteome in an iterative manner.

Mentions: In reverse chemical proteomics (Fig 1B), the starting point is a transcriptome of a phenotype of interest. A cDNA library is cloned into an expression system and the resulting tagged proteome is screened against a small molecule. To date, only phage display has been used in a reverse chemical proteomics context, although other methods, such bacterial [25] and yeast [26] cell-surface display, are also possible, but need to be developed further. If an entire cDNA library is cloned into a phage display vector, each phage particle will contain a different gene insert and will express the protein encoded by that gene on its surface. The displayed proteins often behave as if they were free in solution, so phage displaying the target protein can be rescued from an entire library using an immobilised natural product, as per standard chemical proteomics experiments. However, the real power of phage display comes from the fact that rescued phages can be amplified by transfection into E. coli and then subjected to another round of affinity selection with the immobilised natural product. This cycle can be repeated numerous times, allowing the most avid binders to be identified, even if they are only present in very small amounts in the original cDNA library. This is a significant advantage over chemical proteomics where the most abundant binding protein is usually isolated at the expense of the most avid binders [27]. A significant disadvantage with phage display is that the proteins are not expressed in their native environments, so they will not contain any post-translational modifications and may not fold correctly. In addition, membrane-bound proteins and proteins comprised of multiple subunits are unlikely to be displayed correctly on the surface of a phage particle.


Quality not Quantity: The Role of Marine Natural Products in Drug Discovery and Reverse Chemical Proteomics
Schematic representation of chemical proteomics and reverse chemical proteomics. In chemical proteomics (A), one uses a small molecule (e.g. marine natural product) to construct an affinity or activity probe to isolate specific proteins or a family of proteins. In reverse chemical proteomics (B), we start with the transcriptome, which is cloned into an amplifiable vector (e.g. a virus) that expresses a single protein from the proteome on its surface. A tagged natural product can be used to probe the tagged proteome in an iterative manner.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756327&req=5

f1-marinedrugs-03-00036: Schematic representation of chemical proteomics and reverse chemical proteomics. In chemical proteomics (A), one uses a small molecule (e.g. marine natural product) to construct an affinity or activity probe to isolate specific proteins or a family of proteins. In reverse chemical proteomics (B), we start with the transcriptome, which is cloned into an amplifiable vector (e.g. a virus) that expresses a single protein from the proteome on its surface. A tagged natural product can be used to probe the tagged proteome in an iterative manner.
Mentions: In reverse chemical proteomics (Fig 1B), the starting point is a transcriptome of a phenotype of interest. A cDNA library is cloned into an expression system and the resulting tagged proteome is screened against a small molecule. To date, only phage display has been used in a reverse chemical proteomics context, although other methods, such bacterial [25] and yeast [26] cell-surface display, are also possible, but need to be developed further. If an entire cDNA library is cloned into a phage display vector, each phage particle will contain a different gene insert and will express the protein encoded by that gene on its surface. The displayed proteins often behave as if they were free in solution, so phage displaying the target protein can be rescued from an entire library using an immobilised natural product, as per standard chemical proteomics experiments. However, the real power of phage display comes from the fact that rescued phages can be amplified by transfection into E. coli and then subjected to another round of affinity selection with the immobilised natural product. This cycle can be repeated numerous times, allowing the most avid binders to be identified, even if they are only present in very small amounts in the original cDNA library. This is a significant advantage over chemical proteomics where the most abundant binding protein is usually isolated at the expense of the most avid binders [27]. A significant disadvantage with phage display is that the proteins are not expressed in their native environments, so they will not contain any post-translational modifications and may not fold correctly. In addition, membrane-bound proteins and proteins comprised of multiple subunits are unlikely to be displayed correctly on the surface of a phage particle.

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