Fluorophosphonate probes

Many gene products are uncharacterized enzymes that lack a specific class assignment. As discussed in Sect. 2.3, ABPP probes can be used to identify structurally disparate members of enzyme families based on their reactivity with mechanism-based inhibitors. To date, many uncharacterized enzymes have been classified upon their identification via ABPP. For example, the use of a fluorophosphonate probe by Jessani et al. led to the characterization of sialyl acetylesterase - expressed... 

Figure 9 Serine hydrolases as targets of ABPP. (a) Catalytic mechanism of peptide substrate cleavage via the action of a catalytic triad, (b) Aryl phosphonate and fluorophosphonate probe scaffolds.

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

A competitive version of ABPP identifies the target(s) and assesses the selectivity of an enzyme inhibitor in biological systems by gauging how well the inhibitor slows the enzyme s reaction with an ABP. For example, fluorophosphonate ABP 3 was used to profile the selectivity of fatty acid amide hydrolase (FAAH) inhibitors within the serine hydrolase superfamily [27] (FAAH hydrolyzes endocannabinoids such as anandamide). Serine hydrolases that exhibited reduced labeling by the probe in the presence of inhibitor were scored as targets of the inhibitor. Urea FAAH inhibitors exemplified by PF-3845 (5) that covalently modify the active-site serine nucleophile of FAAH were found to be exquisitely selective for FAAH in brain and liver... 

S.G. Withers, i.P. Street, M.D. Percivai, Fiuorinated carbohydrates as probes of enzyme specificity and mechanism, ACS Symposium Series 374 (1988) 59-77. W.G. Stirtan, S.G. Withers, Phosphonate and aipha-fluorophosphonate analogue probes of the ionization state of pyridoxai 5 -phosphate (PLP) in glycogen phosphor-ylase. Biochemistry 35 (1996) 15057-15064. 

The greatly increased nucleophilicity of the catalytic serine distinguishes it from all other serine residues and makes it an ideal candidate for modification via activity-based probes [58]. Of the electrophilic probe types to profile serine hydrolases, the fluorophosphonate (FP)-based probes are the most extensively used and were first introduced by Cravatt and coworkers [38, 39]. FPs have been well-known inhibitors of serine hydrolases for over 80 years and were first applied as chemical weapons as potent acetylcholine esterase inhibitors. As FPs do not resemble a peptide or ester substrate, they are nonselective towards a particular serine hydrolase, thus allowing the entire family to be profiled. FPs also show minimal cross-reactivity with other classes of hydrolases such as cysteine-, metallo-, and aspartylhydrolases [59]. Furthermore, FP-based probes react only with the active serine hydrolase, and not the inactive zymogen, allowing these probes to interact only with functional species within the proteome [59]. Extensive use of this probe family has demonstrated their remarkable selectivity for serine hydrolases and resulted in the identification of over 100 distinct serine hydrolases... 

Figure 3 Design of an ABPP probe. The design is driven by the choice of the active site-directed group (e.g., fluorophosphonate (FP), shown in green) and a fluorescent tag (rhodamine, shown in red) separated by a spacer group. FPs react covalently with the active site serine residue of the serine hydrolase enzyme family (see also Figure 9). B, base. Reproduced by permission of The Royal Society of Chemistry.

Figure 2.3 Serine protease and hydrolase ABPs. (A) Reaction of a general serine hydrolase probe containing a fluorophosphonate (FP) reactive electrophile. This class of probes has been used extensively to label various classes of serine hydrolases including proteases, esterases, lipases and others. (B) The peptide diphenyl phosphonate (DPP) reacts with the serine nucleophile in the active site of serine proteases. This probe is much less reactive than the FP class of probes but is more selective towards serine proteases over other types of serine hydrolases.(C) The natural product epoxomicin contains a keto-epoxide that selectively reacts with the catalytic N-terminal threonine of the proteasome P-subunit. This reaction results in the formation of a stable six-membered ring. This class of electrophile has been used in probes of the proteasome.

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