Short Peptides with Uncleavable Peptide Bond Mimetics as Photoactivatable Caspase-3 Inhibitors

Abstract

Chemical probes that covalently interact with proteases have found increasing use for the study of protease function and localization. The design and synthesis of such probes is still a bottleneck, as the strategies to target different families are highly diverse. We set out to design and synthesize chemical probes based on protease substrate specificity with inclusion of an uncleavable peptide bond mimic and a photocrosslinker for covalent modification of the protease target. With caspase-3 as a model target protease, we designed reduced amide and triazolo peptides as substrate mimetics, whose sequences can be conveniently constructed by modified solid phase peptide synthesis. We found that these probes inhibited the caspase-3 activity, but did not form a covalent bond. It turned out that the reduced amide mimics, upon irradiation with a benzophenone as photosensitizer, are oxidized and form low concentrations of peptide aldehydes, which then act as inhibitors of caspase-3. This type of photoactivation may be utilized in future photopharmacology experiments to form protease inhibitors at a precise time and location.

Keywords: chemical probes; click chemistry; inhibitors; photoactivation; photocrosslinkers; proteases; triazoles.

Figure 1

Molecular design of general protease-targeting photocrosslinking probes. A protease substrate (scissile bond in red, also indicated with scissors) is surrounded by amino acids in the non-primed site (N-terminally; P1, P2, etc.) and the primed site (P1′, P2′, etc.) that accommodate recognition by certain proteases. The scissile bond is replaced by a non-cleavable analogue, and a photocrosslinker and a tag are incorporated.

Scheme 1

Construction of building blocks for solid phase peptide synthesis (SPPS) synthesis of ψ(CH₂NH) and triazole uncleavable peptide bond mimetics. Reagents and conditions: (i) N-,O-dimethyl-hydroxylamine, HBTU, DIEA, dichloromethane (DCM). (ii) LiAlH₄, THF. (iii) Bestmann–Ohira reagent, K₂CO₃, MeOH/MeCN. (iv) Imidazole-1-sulfonyl azide, K₂CO₃, CuSO₄, MeOH.

Scheme 2

(A) Synthesis of ψ(CH₂NH) probes containing a benzophenone photocrosslinker. (B) Synthesis of triazole peptide mimetics containing a benzophenone photocrosslinker Reagents and conditions: (i) Elongation: 20% piperidine in DMF, then coupling of amino acid with HBTU/DIEA. (ii) 20% piperidine in DMF, (iii) building block 3, NaBH₃CN, DMF/DCM/MeOH, AcOH, (iv) Boc₂O, pyridine, DIEA. (v) TFA/H₂O/TIS 95/2.5/2.5, (vi) CuBr, sodium ascorbate, 2,6-lutidine, DIEA, DMF/MeCN, (vii) PhSiH₃, Pd(Ph₃P)₄, DCM.

Figure 2

Inhibition of caspase-3. (A) Competitive activity-based protein profiling (ABPP) experiments using ψ(CH₂NH) or triazolo peptide derivatives 9–12 with or without irradiation. Compounds 9a–12a were used at 10 μM final concentration, 9b, 9c, 10b and 10c at 100 μM. Inh = caspase inhibitor biotin-DEVD-AOMK, used at 100 nM concentration. (B) Representative progress curves of fluorescence produced by Ac-DEVD-AMC (50 μM) cleavage by caspase-3, irradiated with or without 10 μM of the indicated compounds. (C) Bar graph of residual caspase-3 activity of replicate experiments (n = 2) of those in (B). (D) Inhibition of executioner’s caspase-3 and -7 in lysates of HEK293 cells, treated with cytochrome c/dATP to induce apoptosis.

Figure 3

Non-covalent inhibition of caspase-3 by compounds 9 and 10. (A) Fluorescent labeling of caspase-3 by ψ(CH₂NH) peptides 9–10 and triazolo peptides 11–12. Note that positive control probe 13 leads to strong, fluorescent labeling, but photocrosslinking peptides 9–12 do not. (B) Deconvoluted ESI-MS of caspase-3. (C) Deconvoluted ESI-MS of caspase-3 after incubation with covalent activity-based probe (ABP) 13. Note that the active site, located on the large subunit, is modified. (D) Deconvoluted ESI-MS of caspase-3 after irradiation in the presence of benzophenone ψ(CH₂NH) 10a, not resulting in covalent modification. (E) Competitive ABPP shows inhibition of caspase labeling by “UV preactivation.” Note that inhibition is seen for ψ(CH₂NH) peptides 9a–10b, but not for triazole peptides 11–12. (F) Structures of ψ(CH₂NH) peptides 14 and 15 without photocrosslinker. (G) UV irradiation of ψ(CH₂NH) peptides 14 and 15 in the presence of benzophenone leads to inhibition of caspase-3, as monitored by competitive ABPP. (H) Inhibition of caspase-3 by ψ(CH₂NH) peptide 14 needs both BP and UV irradiation as shown by competitive ABPP. (I) Competitive ABPP shows trapping of active aldehyde species by creating a sodium bisulfite adduct using 10 mM NaHSO₃. Ac-DEVD-aldehyde (100 nM) was used as a control compound, whereas 9a and 10a (10 μM) were irradiated for 10 min and reacted with sodium bisulfite for an additional 20 min before 30 min incubation with caspase-3 and read-out with a fluorescent caspase ABP.

Scheme 3

Proposed mechanism for photoinduced inhibition by ψ(CH₂NH) compounds. Upon irradiation with UV light in the presence of benzophenone, a small amount of secondary amine (A) is oxidized to imine (B). Upon hydrolysis of this imine, peptide aldehyde (C) is formed, which is a potent inhibitor of cysteine proteases.