Phosphonate ester probes for proteolytic antibodies.

Area of Science:

• Biochemistry
• Chemical Biology
• Immunology

Background:

• Antibodies (Abs) possess inherent catalytic activity, particularly in enzyme-mimicking functions.
• Phosphonate esters are known inhibitors of serine proteases, targeting their active sites.

Purpose of the Study:

• To characterize the reactivity of phosphonate ester probes with antibody fragments.
• To investigate the potential of phosphonate esters in selecting and enriching catalytic antibody repertoires.

Main Methods:

• Incubation of antibody fragments with phosphonate diester and monoester probes.
• Analysis of adduct formation using column chromatography and denaturing electrophoresis.
• Chemical capture of phage particles displaying antibody fragments using biotinylated phosphonate esters.

Main Results:

• Irreversible, active site-directed inhibition of peptidase activity by phosphonate esters.
• Formation of stable phosphonate diester-antibody adducts.
• Selection of antibody fragments with enriched catalytic activity inhibitable by the selection reagent.
• Unexpected formation of stable adducts with phosphonate monoesters, leading to improved catalytic activity in selected phage antibodies.

Conclusions:

• Antibody fragments exhibit covalent reactivity with phosphonate esters, similar to serine proteases.
• Phosphonate esters can be utilized as effective reagents for selecting catalytic antibodies.
• The findings suggest antibodies can recapitulate the catalytic mechanisms of serine proteases.