Design of a Selective Substrate and Activity Based Probe for Human Neutrophil Serine Protease 4

Human neutrophil serine protease 4 (NSP4), also known as PRSS57, is a recently discovered fourth member of the neutrophil serine proteases family. Although its biological function is not precisely defined, it is suggested to regulate neutrophil response and innate immune reactions. To create optimal substrates and visualization probes for NSP4 that distinguish it from other NSPs we have employed a Hybrid Combinatorial Substrate Library approach that utilizes natural and unnatural amino acids to explore protease subsite preferences. Library results were validated by synthesizing individual substrates, leading to the identification of an optimal substrate peptide. This substrate was converted to a covalent diphenyl phosphonate probe with an embedded biotin tag. This probe demonstrated high inhibitory activity and stringent specificity and may be suitable for visualizing NSP4 in the background of other NSPs.


Library synthesis
Synthesis of P1 Arg (in Arg-HyCoSuL) was carried out in a similar manner as described previously [1][2][3]. The peptide chain was elongated with standard Fmocsynthesis procedure. 1eq of Rink Amide resin (13g, 9.1 mmol, 0.7mmol/g, mesh 200-300) was swollen in a glass reaction vessel in DCM, stirred gently once per 10 minutes for an hour. The resin was washed three times with DMF, the Fmoc protecting group was removed with 20% piperidine in DMF with three washes for 5, 5 and 25 minutes, followed by gently washing with DMF (6x). A ninhydrin test was performed to confirm amine group deprotection. In the next step 3eq of Fmoc-ACC-OH were preactivated with 3eq of HOBt and 3eq of DICI in DMF, and after 5 minutes the mixture was poured into the resin. The reaction was carried out by gentle stirring for 24 hours in room temperature. Next the resin was washed three times with DMF and Fmoc-ACC-OH coupling was repeated with 1.5 eq of reagents to increase loading efficiency. After 24 hours, the resin was washed with DMF and coupling was confirmed by a ninhydrin test. The Fmoc group was removed as described above, resin was washed gently and 3 eq of Fmoc-Arg(Pbf)-OH was preactivated with 3 eq of HATU and 3eq of collidine in DMF for 5 minutes, followed by addition to the resin.
The reaction was agitated for 24 hours in room temperature and then a second coupling was carried out with 1.5 eq of reagents to increase the loading efficiency.
To cap unreacted aminocoumarin reporter groups, the resin was treated with 10 eq of acetic acid, 10 eq of 3-nitro-1,2,4-triazole and 10 eq of DICI in DMF for 24 hours, followed by addition of 2 eq of DIPEA for 3 hours [3]. Next the amine group from Arg was deprotected with 20% piperidine in DMF and washed with DMF (6x), DCM (3x) and MeOH (3x) and dried over P 2 O 5 overnight. Resin was divided in 120 portions and 48 of these portions were added to 48 wells of solid phase peptide synthesis reactor [1,2,4]. The resin was swollen with DCM for an hour, shaking once every 10 minutes. In the next step, resin was washed with DMF and 3eq of each of 48 different amino acids (separately in Eppendorf tubes) were preactivated with 3eq of HOBt and 3eq of DICI in a minimal amount of DMF for 3 minutes and added to the glass SPPS reactor wells. The reaction was carried out for 3 hours, followed by washing with DMF (3x). The Fmoc-protecting group was removed, resin was washed DMF (6x) and 5eq of isokinetic mixture of natural amino acids (omitting Cys and substituting Nle for Met) was preactivated with 5eq of HOBt and 5eq of DICI for 5 minutes and then added to the resin. The Fmoc protecting group was removed with 20% PIP/DMF, resin washed with DMF (6x) and the isokinetic mixture was attached to the P4 position as described above. After Fmoc group removal, the free amine group was protected with 5eq of acetic acid, 5eq of HBTU and 5eq of DIPEA in DMF.
Finally, tetrapeptide derivatives were cleaved from the resin with a standard

Individual substrates synthesis
In a glass peptide synthesis vessel, 1eq of Rink Amide resin (0.7 mmol/g, 200-300 mesh) was swollen in DCM for 1 hour stirring once per 10 minutes and then washed with DMF (3x). The Fmoc-protecting group was removed with 20% PIP in DMF for 5, 5 and 25 minutes. Resin was washed with portions of DMF (6x) and deprotection was confirmed by a ninhydrin test (resin beads were navy blue).
Afterwards, to the resin 3eq of Fmoc-ACC-OH was loaded with 3eq of HOBt and 3eq of DICI (preactivated for 3 minutes), followed by 24 hours agitation at room temperature. Resin was washed with DMF (3x) and the coupling procedure was repeated with 1.5 eq of Fmoc-ACC-OH, HOBt and DICI. The resin was washed with DMF (3x) and coupling was confirmed by ninhydrin test, followed by Fmoc protecting group removed with 20% PIP/DMF for 5, 5 and 25 minutes. Resin was washed carefully with DMF (6x) and 3eq of first amino acid (Fmoc-Arg(Pbf)-OH) was attached to the resin with 3eq of HATU and 3 eq of collidine as a coupling reagents, stirring gently for 24 hours. The resin was washed with DMF (3x) and loading was repeated with 1.5eq of reagents for additional 24 hours. The resin was washed with DMF (3x) and Fmoc protecting group was removed with 20% PIP/DMF (5, 5 and 25 minutes) and resin was washed with DMF (6x). To confirm deprotection, a ninhydrin test was performed.
Resin was stirred gently for 3 hours and loading was confirmed with a ninhydrin test.
The Fmoc protecting group was removed as described above.

hCha-Phe(guan)-Oic-OH)
2-chlorotrityl chloride resin (100mg, loading 1.6 mmol/g) dry DCM was added in a glass peptide synthesis vessel and resin was swollen for 1 hour shaking once each 10 minutes. The resin was washed three times with dry DCM, following by addition of

Biot-Ahx-hCha-Phe(guan)-Oic-Arg P (OPh) 2 synthesis (PK401)
To a round bottom flask fitted with a stirring bar, 1eq of Biot-Ahx-hCha-Phe(guan(Boc) 2 )-Oic-OH, 1 eq of H 2 N-R(Boc 2 ) P (OPh) 2 , 1 eq of HATU, and 3 eq of collidine (pH was adjusted to 7-8) in DMF were added and stirred for 2 hours at room temperature. The reaction was monitored by analytical HPLC. Crude compound was purified by HPLC (Discovery BIO Wide Pore C8-10, semi-preparative column) and lyophilized. Purity of the compound was confirmed by analytical HPLC (Discovery BIO Wide Pore C8, analytical column). Finally, Boc protecting groups were removed with TFA:DCM (1:1, v/v) within 30 minutes -the reaction progress was monitored once by analytical HPLC. Crude compound was then purified by HPLC (Discovery BIO Wide Pore C8-10, semi-preparative column) and lyophilized. Purity of the compound was confirmed by analytical HPLC and HRMS.

NSP's visualization by PK401
A standard SDS broad spectrum marker (Biorad) was used. 100nM of each NE< PR3, CatG and NSP4 were treated with PK401 at 100nM for 10 minutes at 37°C. Separately, 100nM of NE, PR3, CatG and NSP4 were incubated with buffer for 10 minutes in 37°C. Samples were heated for 5 minutes at 95 o C in 4xSDS buffer and 21µL of each was run at the Bolt 4-12% Bis-Tris Plus Gels, 10-well (165V, 36min), followed by transfer to nitrocellulose membrane (10V, 60min) in MES running buffer.
The membrane was blocked with 1.5% BSA in TBST overnight at 4°C and treated with IRDye800CW Streptavidin (dilution 1:10000 with 1,5% BSA in TBST) for 45