Novel broad-spectrum activity-based probes to profile malarial cysteine proteases

Abstract

Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prevent its transmission. In order to study the biological function of these proteases and to chemically validate some of them as viable drug targets, highly specific inhibitors need to be developed. This is especially challenging given the large number of clan CA proteases present in Plasmodium species (ten in Plasmodium falciparum), and the difficulty of designing selective inhibitors that do not cross-react with other members of the same family. Additionally, any efforts to develop antimalarial drugs targeting these proteases will also have to take into account potential off-target effects against the 11 human cysteine cathepsins. Activity-based protein profiling has been a very useful tool to determine the specificity of inhibitors against all members of an enzyme family. However, current clan CA proteases broad-spectrum activity-based probes either target endopeptidases or dipeptidyl aminopeptidases, but not both subfamilies efficiently. In this study, we present a new series of dipeptydic vinyl sulfone probes containing a free N-terminal tryptophan and a fluorophore at the P1 position that are able to label both subfamilies efficiently, both in Plasmodium falciparum and in mammalian cells, thus making them better broad-spectrum activity-based probes. We also show that some of these probes are cell permeable and can therefore be used to determine the specificity of inhibitors in living cells. Interestingly, we show that the choice of fluorophore greatly influences the specificity of the probes as well as their cell permeability.

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

“… 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 ).…”

“…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 ). − …”

The Impact of Activity‐Based Protein Profiling in Malaria Drug Discovery

“…However, FY01 has a more selective labeling profile. [23] Tan et al [24] recently reported a new series of broadspectrum vinyl sulfone ABPs that efficiently labeled both subfamilies in lysates and intact cells. An alkyne handle was included, and different fluorophores were appended by click chemistry.…”

“…Tan et al [24] . recently reported a new series of broad‐spectrum vinyl sulfone ABPs that efficiently labeled both subfamilies in lysates and intact cells.…”

Chemoproteomics for Plasmodium Parasite Drug Target Discovery

“…Subsequent studies have largely expanded the scope of activity‐based probes to target Plasmodium serine hydrolases, [38] metallo‐aminopeptidases [39] and the proteasome [40–43] . The expansion of ABPP probes to a larger number of diverse proteins could provide a platform to screen different enzymes as candidate targets of antimalarial inhibitors [44] …”