Photoaffinity

While none of these neutrophil receptors has yet been sequenced, partial purification of the formyl peptide receptors has been reported (16). The formyl peptide receptor is particularly attractive because of the extensive structure-activity studies on peptide ligands by Freer and coworkers (17,18). The receptor is a trans-membrane glycoprotein with apparent molecular weight of 60,000 based on proteolysis (19) and photoaffinity (20) labeling studies. 

Dorman G (2000) Photoaffinity Labeling in Biological Signal Transduction. 211 169-225 Drabowicz J, Mikolajczyk M (2000) Selenium at Higher Oxidation States. 208 143-176 Eder B, see Wrodnigg TM (2001) The Amadori and Heyns Rearrangements Landmarks in the History of Carbohydrate Chemistry or Unrecognized Synthetic Opportunities 215 115-175... 

A photoaffinity label is a molecule that forms a highly reactive excited state when illuminated with light of an appropriate wavelength. While in this excited state the photoaffinity label can covalently modify groups on the enzyme molecule that are in close proximity to the label. Hence one can mix the compound and enzyme... 

Figure 8.19 Examples of photoaffinity labels. (A) An aryl azide and (B) a benzophenone.

Photoaffinity labeling can be particularly useful when dealing with noncompetitive inhibitors, where the site of binding cannot be inferred from competition with specific substrate or cofactor molecules. 

Figure 8.20 (A) Generic chemical structure of the y-secretase inhibitors described by Seiffert et al. (2000). (B) y-Secretase inhibitor incorporating a benzophenone photoaffinity label for crosslinking studies.

In addition to its interaction with central BZD receptors, pharmacological concentrations of melatonin can also bind to the peripheral-type BZ receptors (PBRs), which are involved in neurosteroidogenesis (Garcia-Ovejero et al. 2005). PBRs are primarily localized on the outer mitochondrial membrane these sites are therefore also referred to as mitochondrial BZD receptors. Using the isoquinoline carboxamide, PK14105, for photoaffinity labelling, an 18 kDa isoquinoline... 

Wieland, T., et al. Identity of hepatic membrane transport systems for bile salts, phalloidin, and antamanide by photoaffinity labeling. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 5232-5236. 

ABPP is only applicable to targets that possess a nucleophilic active-site residue (Ser, Cys, Lys) susceptible to covalent labeling by an electrophile. When this is lacking, an alternative is to add a photoaffinity group to an inhibitor scaffold so that a covalent adduct with the target can be created by exposure to UV light. 

The sustained attractiveness of photolabeling is apparent from its prominence in studies of y-secretase, an intramembrane protease that contributes to forming amyloid-p peptides and is a major target in Alzheimer s disease [60-62]. y-Secretase is a complex of at least four different polypeptides, and is difficult to engage with high-resolution structural methods. However, in a case of this kind that involves a known target, immunodetection of proteins can often specify the target of y-secretase inhibitor photoaffinity probes such as 19, and proteomic mass spectrometry is not needed. 

Figure 2 Double-stranded oligonucleotide photoprobes that simulate modified DNA and intended to cross-link to DNA-binding proteins. (A) Probe modeling interstrand cross-linking by cisplatin Source From Ref. [63], with permission from the American Chemical Society via the Rightslink service (license number 2458870278307 granted June 30, 2010). The benzophenone probe prior to reaction with DNA is shown in the lower part of the panel. (B) Photoaffinity probe for bacterial DNA repair proteins. TT is a simulated thymine dimer intended to be recognized as a site of damage in DNA, and T (two instances) is the diazirine thymine derivative T Source From Ref. [64], with permission from Wiley.

With apologies to authors whose relevant work was excluded because of space constraints, we conclude that the use of photoaffinity/ proteomic strategies in connection with drug function has gained significant momentum from the availability of proteomic analytical methods. 

Several papers in the last two years attempted to separate the photoreceptor-flavin (and/or the associated b-type cytochrome) from the overwhelming amount of bulk-flavin which is involved in metabolism rather than photoreception. However, a clear-cut assay for the bluelight receptor has not yet been found. Specific photoaffinity labeling and the considerable selectivity in the photoreduction of a b-type cytochrome, as mediated by suitable dyes, are the most promising attempts. 

---