Synthesis and Application of Activity-Based Probes for Proteases

Abstract

The detection, visualization, and identification of active proteases can be facilitated by activity-based probes, which covalently bind to a catalytic residue of the target protease. The synthesis of activity-based probes can be challenging. We here outline a simple protocol for probe synthesis based on standard solid phase peptide synthesis followed by capping of the N-terminus with a reactive electrophile as a warhead. The applicability of the probes is illustrated by labeling cysteine proteases in cell and tissue lysates with Western blotting or fluorescence scanning as a readout.

Azanitrile Inhibitors of the SmCB1 Protease Target Are Lethal to Schistosoma mansoni: Structural and Mechanistic Insights into Chemotype Reactivity

“…Melting points were determined on a Buchi (Essen, Germany) 510 oil bath apparatus. 1 H NMR (500 MHz) and 13 C NMR (125 MHz) spectra were recorded on a Bruker Avance DRX 500 spectrometer and 1 H NMR (600 MHz) and 13 C NMR (150 MHz) spectra, on a Bruker Avance III 600 NMR spectrometer. Chemical shifts δ are given in ppm referring to the signal center using the solvent peaks for reference: DMSO-d 6 2.49/ 39.7 ppm.…”

“…Azapeptides, peptides in which the CαH of at least one amino acid has been replaced with nitrogen, have emerged as particularly important peptidomimetic structures (Figure ). Compared to their parent carbapeptide analogs, bioactive azapeptides can possess improved potency and target selectivity as well as superior pharmacokinetics. − Azadipeptide nitriles were introduced as a class of efficient inhibitors of human cysteine cathepsins. − This chemotype supported the successful development of activity-based probes, modified for organelle-specific delivery to lysosomal cysteine proteases and applied as PET-imaging agents for tumor-associated cathepsin activity. − These reports have highlighted that azadipeptide nitriles can enrich the portfolio of inhibitors of cysteine proteases suitable as activity-based probes , and potential therapeutics against parasitic and protozoal infections. − Similar to the well-established dipeptide nitriles, azadipeptide nitriles are thought to undergo a covalent, reversible interaction with the target proteases by forming a stabilized isothiosemicarbazide adduct (Figure ). − …”

“…6−9 This chemotype supported the successful development of activity-based probes, modified for organelle-specific delivery to lysosomal cysteine proteases and applied as PET-imaging agents for tumor-associated cathepsin activity. 10−12 These reports have highlighted that azadipeptide nitriles can enrich the portfolio of inhibitors of cysteine proteases suitable as activity-based probes 13,14 and potential therapeutics against parasitic and protozoal infections. 15−18 Similar to the well-established dipeptide nitriles, azadipeptide nitriles are thought to undergo a covalent, reversible interaction with the target proteases by forming a stabilized isothiosemicarbazide adduct (Figure 1).…”

Synthesis and Configurational Assignment of Vinyl Sulfoximines and Sulfonimidamides

“…The majority of the previously established covalent warheads do not possess convenient chemical handles for attachment of alkyne tags . As such, the design of fully functionalized probes often requires preinstallation of the alkyne handle into the pharmacophore, which can be synthetically challenging . Therefore, a general method for late-stage propargylation of small molecules would present significant benefits and opportunities to chemical biologists.…”