Affinity label

Affinity labels are a class of enzyme inhibitors that covalently bind to their target causing its inactivation. The hallmark of an affinity label is the use of a targeting moiety to specifically and reversibly deliver a weakly reactive group to the enzyme that irreversibly binds to an amino acid residue. The targeting portion of the label often resembles the enzyme's natural substrate so that a similar mode of noncovalent binding is used prior to the covalent linkage. Their usefulness in medicine can be limited by the specificity of the first noncovalent binding step whereas indiscriminate action can be utilized for purposes such as affinity labeling a technique for the validation of substrate specific binding of compounds.

This category encompasses the simplest approach of coupling an electrophile with low intrinsic reactivity to a noncovalent binding moiety which frequently mimics the natural substrate. Key to this designation is that the reactivity of the electrophile is not altered by the enzyme and that the noncovalent binding moiety serves to increase the presence and lifetime of the electrophile in the active site (effective molarity). The weakly reactive group may react with functional groups outside of the active site or on other proteins but the selectivity is conferred by the noncovalent binding moiety. Kinetic signatures of this type of inhibitor can be found in saturation because of the covalent reaction (kinact) becomes the rate limiting step at high concentrations of inhibitor. A handful of drugs such as afatinib have gained FDA approval through this approach. The inverse approach of using a weakly nucleophilic inhibitor to attack a protein bound electrophile has also been studied. This approach has received much less attention due to the lack of protein electrophiles and only those with suitable cofactors can be targeted.

Photoaffinity labels are characterized by nonenzymatic reactivity produced by exposure to light and a noncovalent targeting moiety to enhance the effective molarity of this reactive group in the active site. While this technique appears sound in theory, low degree of labeling is frequently observed primarily due to quenching of the reactive species by solvent or other species in solution. However, this quenching can be advantageous as it is such a fast process that once the reactive species is formed, it will not diffuse to any appreciable extent and will only react with molecules to which it is immediately adjacent. Photoaffinity labels do not show great promise for inhibition or in the use of drugs but are appropriately suited to identify ligand binding sites. Reactive groups such as nitrenes or 2 aryl 5 carboxytetrazoles are often employed to generate highly reactive, nonselective carbenes or moderately selective nitrile imine intermediates, respectively.

The basic unit of activity based proteomics is the probe, which typically consists of two elements: a reactive group (RG, sometimes called a "warhead") and a tag. Additionally, some probes may contain a binding group which enhances selectivity. The reactive group usually contains a specially designed electrophile that becomes covalently linked to a nucleophilic residue in the active site of an active enzyme. An enzyme that is inhibited or post translationally modified will not react with an activity based probe. The tag may be either a reporter such as a fluorophore or an affinity label such as biotin or an alkyne or azide for use with the Huisgen 1,3 dipolar cycloaddition (also known as click chemistry).