Activity-Based Profiling of Proteases

Abstract

Proteolytic enzymes are key signaling molecules in both normal physiological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the application of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers.

Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells

“…We note that this approach is distinct from activity-based protein profiling with chemoselective probes (23)(24)(25), where the objective is functional protein classification based on probe reactivity. The use of click analog molecular probes to assess target selectivity of candidate proteasome inhibitors has been described (53,54), but the assessment of global target specificity is a novel, but important extension of this approach.…”

“…The integration of alkynyl probes and cleavable click reagents for the purification of adducted proteins into the MS workflow has greatly broadened the utility of this technique. This approach has proven valuable for functional proteomics studies by activity-based proteome profiling (23)(24)(25) and to explore cellular protein targets of oxidants and electrophiles (26 -31).…”

Metal assisted peptide bond hydrolysis: Chemistry, biotechnology and toxicological implications

“…The replacement of Ce(IV) with Zr(IV) or Hf(IV) gave the same pattern of hydrolysis, but lowered the reaction rates [32]. A more detailed study regarding the nature of the interaction of proteins and metal-substituted POMs was conducted using HSA and different types of Zr(IV)-substituted POMs: (i) Wells-Dawsontype POMs, 1:2 Zr(IV)-WD POM and 4:2 Zr(IV)-WD POM, previously described in Section 2.1.4; (ii) Keggin type POMs, the previously described 2:2 Zr(IV)-Kg POM and its analog 1:2 Zr(IV)-Kg POM (Et 2 NH 2 ) 10 The most effective Zr(IV) compounds were Wells-Dawson POMs, 1:2 Zr(IV)-WD POM and 4:2 Zr(IV)-WD POM, with 75 and 50% conversion degrees after 48 h, at pH 7.4 and 60 • C. Less than 35% of HSA was hydrolyzed under the same conditions by other POMs. The significant role of the incorporation of Zr(IV) into POM for metal-supported hydrolysis was proved by the absence of reactivity or a very low reaction yield for ZrCl 4 or a POM without Zr(IV).…”

“…The N-terminal part of the substrate is released simultaneously. In the second step of this reaction type the acyl-enzyme intermediate is hydrolyzed finally by an activated water molecule [10,11].…”

Tailored Ahp‐cyclodepsipeptides as Potent Non‐covalent Serine Protease Inhibitors

“…In this work, we demonstrate that Ahp-cyclodepsipeptides represent as uitable scaffold for generating target-tailored inhibitors of serine proteases.F or efficient synthetic access,wedeveloped apractical mixed solidand solution-phase synthesis that we validated through performing the first chemical synthesis of the two natural products Tasipeptin Aand B. The suitability of the Ahp-cyclodepsipeptide scaffold for tailored inhibitor synthesis is showcased by the generation of the most potent human HTRA protease inhibitors to date.W ea nticipate that our approach maya lso be applied to other serine proteases,thus opening new avenues for asystematic discovery of serine protease inhibitors.Serine proteases of the S1 family (trypsin/chymotrypsinlike) are one of the largest and biomedically relevant protease families.[1] In contrast to the design of covalent inhibitors or activity-based probes for this enzyme class, [2] generic approaches for designing potent, enzyme-class-specific,c ellactive,and non-covalent small-molecule inhibitors are highly limited and have been achieved only for distinct serine proteases.[3] Consequently,a lternative approaches for the systematic generation of such chemical probes are urgently required.Ahp-cyclodepsipeptides (also termed cyanopeptolins or peptolides) are aclass of over 100 peptidic natural products of non-ribosomal origin that display potent inhibitory properties against serine proteases.[4] All Ahp-cyclodepsipeptides are containing an Ahp (3-amino-6-hydroxy-2-piperidone) unit and an N-methyl aromatic amino acid at conserved positions in their 19-membered ring structure,while other residues are much less conserved (Figure 1a). Crystal structures of Ahpcyclodepsipeptides in complex with serine proteases indicate that inhibition is based on as ubstrate-like binding mode in which distinct amino acid residues occupy the S-and S'-pockets,h owever,p roteolytic cleavage does not occur (Figure 1b,S chechter and Berger nomenclature is used).…”