Affinity-labeling agents

Chlornaltrexamine (/f-CNA, 15) another naltrexone derivative modified at C-6, is a nonequilibrium antagonist which blocks irreversibly the three major opioid receptor types (ji, k and 8). Portoghese and his collaborators have developed this compound as the first affinity labelling agent of its class [58-61]. Compound (15) has an alkylating function at C-6 (classic nitrogen mustards) able to bind covalently to opioid receptors. In the tail flick assay in mice, /f-CNA inhibited morphine-induced antinociception for 3-6... 

Affinity labeling agents are intrinsically reactive compounds that initially bind reversibly to the active site of the enzyme then undergo chemical reaction (generally an acylation or alkylation reaction) with a nucleophile on the enzyme (Scheme 8). To differentiate a reversible inhibitor from an irreversible one, often the dissociation constant is written with a capital i, K (65), instead of a small i, K, which is used for reversible inhibitors. The K denotes the concentration of an inactivator that produces half-maximal inactivation. Note that this kinetic Scheme is similar to that for substrate turnover except instead of the catalytic rate constant, kcat for product formation, kmact is used to denote the maximal rate constant for inactivation. 

Figure 16 (a) A plot showing time-dependent inactivation by affinity labeling agents and mechanism-based inactivators used for determination of kinetic constants, (b) Replot of the half-lives of inactivation from Fig. 16a versus the inverse of the inactivator concentration to determine the K and values for affinity labeling agents and mechanism-based inactivators. 

M. Ichikawa, Y. Ichikawa, A mechanism-based affinity-labeling agent for possible use in isolating N-acetylglucosaminidase, Bioorg. 

Wolfenden R. Transition-state analogues as potential affinity labeling agents. Methods Enzymol 1977 46 15-28. 

A number of opioid receptor selective affinity labeling agents (i.e., compounds that form an irreversible covalent bond with the receptor protein) have been developed (Fig. 24.7). These compounds have been important... 

Examples of important affinity labeling agents are P-CNA, which because of its highly reactive 2-chloroethylamine electrophilic group irreversibility binds to all three opioid receptor types (50). The structurally related compound p-FNA has a less reactive fumaramide electrophilic group and reacts irreversibly with only p receptors (51). Derivatives of the fentanyl series, FIT and... 

Apparently, when these agents are bound to p receptors, the electrophilic isothiocyanate group is not oriented in proper juxtaposition to a receptor nucleophile for covalent bond formation to occur. Incorporation of the electrophilic isothiocyanate into the structure of the highly k receptor-selective arylacetamides has provided affinity labeling agents (UPHIT and DIPPA) for k receptors (54,55). 

The possibility of combining the features of a transition state analog with those of an affinity labeling agent remains to be explored in detail, but it appears to have been realized unexpectedly in two cases. Bacterial /8-gaIactosidase is rather strongly inhibited by n-galactal. ... 

It had been known for some time > that FdUMP is an extremely potent inhibitor of thymidylate synthetase, but the nature of inhibition was the topic of considerable controversy. Since the 6-position of 1-sub-stituted 5-fluorouracils is quite susceptible toward nucleophilic attack, it was suspected that FdUMP might exert its inhibitory effect by reaction with the proposed nucleophilic catalyst of thymidylate synthetase. It is now well established that, in the presence of CH,-H4folate, 5-fluoro-2 -deoxyuridylate (FdUMP) behaves as a quasi-substrate for thymidylate synthetase " and is, in effect, an affinity labeling agent for the enzyme. Whereas FdUMP binds relatively poorly to free enzyme, in the presence of the cofactor, CHa-Hifolate, a covalent bond is formed between an amino acid residue of the enzyme and the 6-position of the nucleotide to give the complex depicted in Fig. 1. Although covalent bonds are involved in linking the components of the complex, the reaction is slowly reversible. Nevertheless, the complex is sufficiently stable Ka 10" M) to permit isolation and characterization. 

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