Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity

Abstract

Many biologically and chemically based approaches have been developed to design highly active and selective protease substrates and probes. It is, however, difficult to find substrate sequences that are truly selective for any given protease, as different proteases can demonstrate a great deal of overlap in substrate specificities. In some cases, better enzyme selectivity can be achieved using peptide libraries containing unnatural amino acids such as the hybrid combinatorial substrate library (HyCoSuL), which uses both natural and unnatural amino acids. HyCoSuL is a combinatorial library of tetrapeptides containing amino acid mixtures at the P4-P2 positions, a fixed amino acid at the P1 position, and an ACC (7-amino-4-carbamoylmethylcoumarin) fluorescent tag occupying the P1' position. Once the peptide is recognized and cleaved by a protease, the ACC is released and produces a readable fluorescence signal. Here, we describe the synthesis and screening of HyCoSuL for human caspases and legumain. We also discuss possible modifications and adaptations of this approach that make it a useful tool for developing highly active and selective reagents for a wide variety of proteolytic enzymes. The protocol can be divided into three major parts: (i) solid-phase synthesis of the fluorescence-labeled HyCoSuL, (ii) screening of protease P4-P2 preferences, and (iii) synthesis of the optimized activity probes equipped with an AOMK (acyloxymethyl ketone) reactive group and a biotin label for easy detection. Beginning with the library design, the entire protocol can be completed in 4-8 weeks (HyCoSuL synthesis: 3-5 weeks; HyCoSuL screening per enzyme: 4-8 d; and activity-based probe synthesis: 1-2 weeks).

Activity profiling and structures of inhibitor-bound SARS-CoV-2-PLpro protease provides a framework for anti-COVID-19 drug design

“…These results suggest that more detailed mapping of binding pocket architecture should facilitate design of new, active substrates as well as optimal peptide sequences for inhibitor development efforts. To achieve this goal we developed a defined and combinatorial substrate library (HyCoSuL) containing wide variety of nonproteinogenic amino acids [24]. Since tetrapeptide fluorogenic substrates are not very efficiently hydrolyzed by enzymes exhibiting deubiquitinating activity, we designed and synthesized the P2 defined library with a general structure of Ac-LRXG-ACC (X -19 natural and 109 unnatural amino acids) and a hybrid combinatorial substrate library, where three positions were fixed and one position contains an equimolar mixture of 19 amino acids (Mix), (P3 sublibrary: Ac-Mix-P3-Gly-Gly-ACC, P4 sublibrary: Ac-P4-Mix-Gly-Gly-ACC; P3 and P4 -a natural or unnatural amino acid) [26].…”

“…Individual fluorogenic substrates were synthesized on a solid support using the SPPS method as previously described [29,30]. Each substrate was purified by HPLC and analyzed using analytical HPLC and HRMS.…”

“…We have developed recently a novel chemical approach, called HyCoSuL (Hybrid Combinatorial Substrate Library), to dissect a broad substrate specificity of proteolytic enzymes [24]. The use of natural and a wide range of unnatural amino acids in HyCoSuL structure allows for ultrafast discovery of highly active and selective substrates, inhibitors, and activity-based probes for multiple proteases.…”

Substrate specificity profiling of SARS-CoV-2 main protease enables design of activity-based probes for patient-sample imaging

“…ACC-labeled substrates were synthesized on the solid support according to the solid phase peptide synthesis method described elsewhere. [15] In brief, Fmoc-ACC-OH (2.5 eq) was attached to a Rink-amide resin using HOBt (2.5 eq) and DICI (2.5 eq) in DMF as coupling reagents. Then, the Fmoc protecting group was removed using 20% piperidine in DMF (three cycles: 5, 5, and 25 min).…”

Applications of Unnatural Amino Acids in Protease Probes

“…Usually, the amino acid residue at P1 must remain constant (Scheme ). The HyCoSuL protocol is described in detail elsewhere …”