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Reagents and Methods for Protein Target Identification
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Reagents and Methods for Protein Target Identification

A special issue of Molecules (ISSN 1420-3049).

Deadline for manuscript submissions: closed (31 July 2013) | Viewed by 81319

Special Issue Editor

Dr. Nicholas James Westwood

E-Mail Website
Guest Editor
School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK
Interests: chemical biology; natural product synthesis

Special Issue Information

Dear Colleagues,

Two main factors are driving the resurgence in the use of protein target identification strategies. First, there is an increased interest in the identification of off-target effects of know drugs and second, improvements in phenotypic screening methods have led to a significant increase in the number of compounds that have been assigned as inducing an important biological phenotype through an unknown mechanism. In both cases, researchers at the chemistry-biology interface have been revisiting and in many cases inventing strategies to identify and subsequently validated protein targets. A number of target identification strategies use genomic and proteomic techniques. Typically these approaches, including the selection of resistant mutants followed by full genome sequencing, do not require the chemical synthesis of specific reagents. In other approaches, however, the preparation of specific reagents (radiolabeled, photoaffinity labelled, tagged etc) is essential. Structure activity relationship studies are also required to inform reagent synthesis programmes. Once the reagent has been prepared a range of methods for using it can then be envisaged. Overall, target identification is testing the creativity and skills of chemical biologists to the limit. In this special issue of Molecules, we aim to bring together papers that cover all aspects of the protein target identification challenge.

Dr. Nicholas James Westwood
Guest Editor

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Keywords

  • protein target identification
  • reagent synthesis
  • affinity chromatography
  • yeast-3-hybrid
  • photoaffinity labelling
  • radiolabelling
  • off target effects
  • genome-wide approaches
  • proteomic approaches
  • chemical proteomics
  • forward chemical genetics
  • protein target validation

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Published Papers (7 papers)

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Research

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279 KiB  
Communication
Design and Synthesis of a Biotinylated Chemical Probe for Detecting the Molecular Targets of an Inhibitor of the Production of the Pseudomonas aeruginosa Virulence Factor Pyocyanin
by Ysobel R. Baker, Warren R. J. D. Galloway, James T. Hodgkinson and David R. Spring
Molecules 2013, 18(10), 11783-11796; https://doi.org/10.3390/molecules181011783 - 25 Sep 2013
Cited by 13 | Viewed by 8453
Abstract
Pseudomonas aeruginosa is a human pathogen associated with a variety of life-threatening nosocomial infections. This organism produces a range of virulence factors which actively cause damage to host tissues. One such virulence factor is pyocyanin, known to play a crucial role in the [...] Read more.
Pseudomonas aeruginosa is a human pathogen associated with a variety of life-threatening nosocomial infections. This organism produces a range of virulence factors which actively cause damage to host tissues. One such virulence factor is pyocyanin, known to play a crucial role in the pathogenesis of P. aeruginosa infections. Previous studies had identified a novel compound capable of strongly inhibiting the production of pyocyanin. It was postulated that this inhibition results from modulation of an intercellular communication system termed quorum sensing, via direct binding of the compound with the LasR protein receptor. This raised the possibility that the compound could be an antagonist of quorum sensing in P. aeruginosa, which could have important implications as this intercellular signaling mechanism is known to regulate many additional facets of P. aeruginosa pathogenicity. However, there was no direct evidence for the binding of the active compound to LasR (or any other targets). Herein we describe the design and synthesis of a biotin-tagged version of the active compound. This could potentially be used as an affinity-based chemical probe to ascertain, in a direct fashion, the active compound’s macromolecular biological targets, and thus better delineate the mechanism by which it reduces the level of pyocyanin production. Full article
(This article belongs to the Special Issue Reagents and Methods for Protein Target Identification)
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1196 KiB  
Article
A Modular Approach to Triazole-Containing Chemical Inducers of Dimerisation for Yeast Three-Hybrid Screening
by Fanny Tran, Anahi V. Odell, Gary E. Ward and Nicholas J. Westwood
Molecules 2013, 18(9), 11639-11657; https://doi.org/10.3390/molecules180911639 - 23 Sep 2013
Cited by 14 | Viewed by 10600
Abstract
The yeast three-hybrid (Y3H) approach shows considerable promise for the unbiased identification of novel small molecule-protein interactions. In recent years, it has been successfully used to link a number of bioactive molecules to novel protein binding partners. However despite its potential importance as [...] Read more.
The yeast three-hybrid (Y3H) approach shows considerable promise for the unbiased identification of novel small molecule-protein interactions. In recent years, it has been successfully used to link a number of bioactive molecules to novel protein binding partners. However despite its potential importance as a protein target identification method, the Y3H technique has not yet been widely adopted, in part due to the challenges associated with the synthesis of the complex chemical inducers of dimerisation (CIDs). The development of a modular approach using potentially “off the shelf” synthetic components was achieved and allowed the synthesis of a family of four triazole-containing CIDs, MTX-Cmpd2.2-2.5. These CIDs were then compared using the Y3H approach with three of them giving a strong positive interaction with a known target of compound 2, TgCDPK1. These results showed that the modular nature of our synthetic strategy may help to overcome the challenges currently encountered with CID synthesis and should contribute to the Y3H approach reaching its full potential as an unbiased target identification strategy. Full article
(This article belongs to the Special Issue Reagents and Methods for Protein Target Identification)
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4706 KiB  
Article
Combination of Small Molecule Microarray and Confocal Microscopy Techniques for Live Cell Staining Fluorescent Dye Discovery
by Eszter Molnár, Soujanya Kuntam, Pradeep Kumar Reddy Cingaram, Begüm Peksel, Bhavyashree Suresh, Gabriella Fábián, Liliána Z. Fehér, Attila Bokros, Ágnes Medgyesi, Ferhan Ayaydin and László G. Puskás
Molecules 2013, 18(8), 9999-10013; https://doi.org/10.3390/molecules18089999 - 20 Aug 2013
Cited by 10 | Viewed by 8821
Abstract
Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS) methods. In the present study a combination of small molecule microarray (SMM) prescreening and confocal laser scanning microscopy (CLSM) was developed in order to discover novel cell staining fluorescent dyes. Compounds with high [...] Read more.
Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS) methods. In the present study a combination of small molecule microarray (SMM) prescreening and confocal laser scanning microscopy (CLSM) was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment-specific, cell-permeable (or plasma membrane-targeted) fluorochromes were identified. Their cytotoxicity was tested and found that between 1–10 micromolar range, they were non-toxic even during long-term incubations. Full article
(This article belongs to the Special Issue Reagents and Methods for Protein Target Identification)
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244 KiB  
Article
Distinct Metabolites for Photoreactive l-Phenylalanine Derivatives in Klebsiella sp. CK6 Isolated from Rhizosphere of a Wild Dipterocarp Sapling
by Lei Wang, Wataru Hisano, Yuta Murai, Munenori Sakurai, Yasuyuki Muto, Haruka Ikemoto, Masashi Okamoto, Takashi Murotani, Reika Isoda, Dongyeop Kim, Yasuko Sakihama, Irnayuli R. Sitepu, Yasuyuki Hashidoko, Yasumaru Hatanaka and Makoto Hashimoto
Molecules 2013, 18(7), 8393-8401; https://doi.org/10.3390/molecules18078393 - 16 Jul 2013
Cited by 10 | Viewed by 10434
Abstract
Photoaffinity labeling is a reliable analytical method for biological functional analysis. Three major photophores—aryl azide, benzophenone and trifluoromethyldiazirine—are utilized in analysis. Photophore-bearing l-phenylalanine derivatives, which are used for biological functional analysis, were inoculated into a Klebsiella sp. isolated from the rhizosphere of a [...] Read more.
Photoaffinity labeling is a reliable analytical method for biological functional analysis. Three major photophores—aryl azide, benzophenone and trifluoromethyldiazirine—are utilized in analysis. Photophore-bearing l-phenylalanine derivatives, which are used for biological functional analysis, were inoculated into a Klebsiella sp. isolated from the rhizosphere of a wild dipterocarp sapling in Central Kalimantan, Indonesia, under nitrogen-limiting conditions. The proportions of metabolites were quite distinct for each photophore. These results indicated that photophores affected substrate recognition in rhizobacterial metabolic pathways, and differential photoaffinity labeling could be achieved using different photophore-containing l-phenylalanine derivatives. Full article
(This article belongs to the Special Issue Reagents and Methods for Protein Target Identification)
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Review

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432 KiB  
Review
Radiolabeling Strategies for Tumor-Targeting Proteinaceous Drugs
by Grant Sugiura, Helen Kühn, Max Sauter, Uwe Haberkorn and Walter Mier
Molecules 2014, 19(2), 2135-2165; https://doi.org/10.3390/molecules19022135 - 18 Feb 2014
Cited by 52 | Viewed by 16017
Abstract
Owing to their large size proteinaceous drugs offer higher operative information content compared to the small molecules that correspond to the traditional understanding of druglikeness. As a consequence these drugs allow developing patient-specific therapies that provide the means to go beyond the possibilities [...] Read more.
Owing to their large size proteinaceous drugs offer higher operative information content compared to the small molecules that correspond to the traditional understanding of druglikeness. As a consequence these drugs allow developing patient-specific therapies that provide the means to go beyond the possibilities of current drug therapy. However, the efficacy of these strategies, in particular “personalized medicine”, depends on precise information about individual target expression rates. Molecular imaging combines non-invasive imaging methods with tools of molecular and cellular biology and thus bridges current knowledge to the clinical use. Moreover, nuclear medicine techniques provide therapeutic applications with tracers that behave like the diagnostic tracer. The advantages of radioiodination, still the most versatile radiolabeling strategy, and other labeled compounds comprising covalently attached radioisotopes are compared to the use of chelator-protein conjugates that are complexed with metallic radioisotopes. With the techniques using radioactive isotopes as a reporting unit or even the therapeutic principle, care has to be taken to avoid cleavage of the radionuclide from the protein it is linked to. The tracers used in molecular imaging require labeling techniques that provide site specific conjugation and metabolic stability. Appropriate choice of the radionuclide allows tailoring the properties of the labeled protein to the application required. Until the event of positron emission tomography the spectrum of nuclides used to visualize cellular and biochemical processes was largely restricted to iodine isotopes and 99m-technetium. Today, several nuclides such as 18-fluorine, 68-gallium and 86-yttrium have fundamentally extended the possibilities of tracer design and in turn caused the need for the development of chemical methods for their conjugation. Full article
(This article belongs to the Special Issue Reagents and Methods for Protein Target Identification)
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1974 KiB  
Review
Photoaffinity Labeling of Plasma Proteins
by Victor Tuan Giam Chuang and Masaki Otagiri
Molecules 2013, 18(11), 13831-13859; https://doi.org/10.3390/molecules181113831 - 8 Nov 2013
Cited by 16 | Viewed by 8902
Abstract
Photoaffinity labeling is a powerful technique for identifying a target protein. A high degree of labeling specificity can be achieved with this method in comparison to chemical labeling. Human serum albumin (HSA) and α1-acid glycoprotein (AGP) are two plasma proteins that [...] Read more.
Photoaffinity labeling is a powerful technique for identifying a target protein. A high degree of labeling specificity can be achieved with this method in comparison to chemical labeling. Human serum albumin (HSA) and α1-acid glycoprotein (AGP) are two plasma proteins that bind a variety of endogenous and exogenous substances. The ligand binding mechanism of these two proteins is complex. Fatty acids, which are known to be transported in plasma by HSA, cause conformational changes and participate in allosteric ligand binding to HSA. HSA undergoes an N-B transition, a conformational change at alkaline pH, that has been reported to result in increased ligand binding. Attempts have been made to investigate the impact of fatty acids and the N-B transition on ligand binding in HSA using ketoprofen and flunitrazepam as photolabeling agents. Meanwhile, plasma AGP is a mixture of genetic variants of the protein. The photolabeling of AGP with flunitrazepam has been utilized to shed light on the topology of the protein ligand binding site. Furthermore, a review of photoaffinity labeling performed on other major plasma proteins will also be discussed. Using a photoreactive natural ligand as a photolabeling agent to identify target protein in the plasma would reduce non-specific labeling. Full article
(This article belongs to the Special Issue Reagents and Methods for Protein Target Identification)
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1586 KiB  
Review
Recent Advances in Target Characterization and Identification by Photoaffinity Probes
by Jitapa Sumranjit and Sang J. Chung
Molecules 2013, 18(9), 10425-10451; https://doi.org/10.3390/molecules180910425 - 29 Aug 2013
Cited by 101 | Viewed by 16718
Abstract
Target identification of biologically active molecules such as natural products, synthetic small molecules, peptides, and oligonucleotides mainly relies on affinity chromatography, activity-based probes, or photoaffinity labeling (PAL). Amongst them, activity-based probes and PAL have offered great advantages in target identification technology due to [...] Read more.
Target identification of biologically active molecules such as natural products, synthetic small molecules, peptides, and oligonucleotides mainly relies on affinity chromatography, activity-based probes, or photoaffinity labeling (PAL). Amongst them, activity-based probes and PAL have offered great advantages in target identification technology due to their ability to form covalent bonds with the corresponding targets. Activity-based probe technology mainly relies on the chemical reactivity of the target proteins, thereby limiting the majority of the biological targets to enzymes or proteins which display reactive residues at the probe-binding site. In general, the probes should bear a reactive moiety such as an epoxide, a Michael acceptor, or a reactive alkyl halide in their structures. On the other hand, photoaffinity probes (PAPs) are composed of a target-specific ligand and a photoactivatable functional group. When bound to the corresponding target proteins and activated with wavelength-specific light, PAPs generate highly reactive chemical species that covalently cross-link proximal amino acid residues. This process is better known as PAL and is widely employed to identify cellular targets of biologically active molecules. This review highlights recent advances in target identification by PAL, with a focus on the structure and chemistry of the photoaffinity probes developed in the recent decade, coupled to the target proteins identified using these probes. Full article
(This article belongs to the Special Issue Reagents and Methods for Protein Target Identification)
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