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.[1][2] 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.[1]
These labels are not limited to enzymes but may also be designed to react
with antibodies or ribozymes although this usage is less common. Although proteins
such as hemoglobin do not have an active site, binding pockets can be exploited for their
affinity and thus be labeled.
Classifications
Affinity labels can be broken down into three distinct categories based on their reactive
groups and mode of delivery.[3]
Classical affinity labels
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.[1][3]
Quiescent affinity labels
Quiescent affinity labels represent a promising approach for inhibiting enzymes using
‘masked’ reactive functionalities that are only uncovered within the active site. This
approach differs from mechanism-based inactivators in that the catalysis must be "offpathway". One of the best examples to explain this form of catalysis is in the inactivation
dimethylargine dimethylaminohydrolase (DDAH) by 4-halopyridines. At physiological pH,
the 4-halo group has near negligible reactivity with thiolates but upon protonation of the
nitrogen, the reactivity increases ~4500-fold. This protonation occurs off-pathway by an
aspartate residue that is not normally involved in catalysis. Following attack by the active
site cysteine and loss of the halide, the enzyme is irreversibly modified. This requirement
of catalysis tunes the selectivity of modification.[3] This class is not limited to
halopyridines and functional groups including epoxides and peptidyl acyloxymethyl
ketones have been used. The kinetic signature of this class resembles that of classical
affinity labels. This term has been previously used to describe affinity labels that contain
weakly reactive groups but recent literature has commenced on the requirement of offpathway catalysis.[4]
Photoaffinity labels
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.[2][3]
Uses of affinity labeling
When characterizing an enzyme, it is essential to identify the amino acid residues
responsible for catalysis. While it is clear that X-ray crystallography will provide more
detailed 3-D information about the active site, only a static picture is returned and
difficulties can be encountered with co-crystallization of the substrate or mimics due to
enzymatic turnover.
The classic example of the use of affinity labels for this purpose is in mapping the
topography of the active site of chymotrypsin. Through the use of three different affinity
labels that placed reactive groups (halomethyl ketones or phosphofluorides) on different
regions of the natural substrate core, the relative positions and identity of three different
amino acids could be determined.[1] Another notable example of using affinity labeling to
determine the active site of an enzyme is the work carried out by Grachev et al. which
resulted in characterization of the β-subunit of the core RNA polymerase as the sub-unit
responsible for phosphodiester-bond formation in the process of prokaryotic
transcription.[5]
Activity-based protein profiling (ABPP)
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).