Fluorogenic Substrates for In Situ Monitoring of Caspase-3 Activity in Live Cells

Abstract

The in situ detection of caspase-3 activity has applications in the imaging and monitoring of multiple pathologies, notably cancer. A series of cell penetrating FRET-based fluorogenic substrates were designed and synthesised for the detection of caspase-3 in live cells. A variety of modifications of the classical caspase-3 and caspase-7 substrate sequence Asp-Glu-Val-Asp were carried out in order to increase caspase-3 affinity and eliminate caspase-7 cross-reactivity. To allow cellular uptake and good solubility, the substrates were conjugated to a cationic peptoid. The most selective fluorogenic substrate 27, FAM-Ahx-Asp-Leu-Pro-Asp-Lys(MR)-Ahx, conjugated to the cell penetrating peptoid at the C-terminus, was able to detect and quantify caspase-3 activity in apoptotic cells without cross-reactivity by caspase-7.

Fig 1. The design of the FRET-based fluorogenic tetrapeptide substrates for caspase-3 detection in live cells.

The substrates bear 5(6)-carboxyfluorescein (λ_Ex/Em 488/528 nm) at the amino-terminus and a quencher coupled via a Lys side-chain. The recognition sequence is a tetrapeptide (Asp-X₃-X₂-Asp) with two variable positions (see Table 1). The C-terminus bears a “lysine-like” nonaresidue peptoid to enable cellular uptake.

Fig 2. Relative increase in fluorescence intensity of the FRET-based peptides, bearing different quenchers (MR, TAMRA or BHQ1) on the Lys side chain, after incubation with caspase-3 and 7.

The FRET-based peptides 1, 2 and 3 (6 μM) were incubated with caspase-3 and 7 (20 nM) and fluorescence recorded at 10, 20 and 40 min (n = 3, normalised to zero).

Fig 3. Structural modifications to the fluorogenic substrates with the aim of eliminating caspase-7 cross-reactivity.

Substrate 25 has a D-Lys residue, 26 an N-Methyl-Lys, and in substrate 27 the Ahx spacer has been moved between the Lys and the peptoid moiety. Caspase-3 selectivity was achieved with 27.

Fig 4. Flow cytometry analysis of healthy and apoptotic HEK293T cells treated with substrate 27.

The cells were incubated 5 h with fluorogenic substrate 27 (10 μM), detached, and analysed by flow cytometry (λ_Ex/Em 488/530 nm, x-axis = fluorescence intensity). (A) Healthy, non-apoptotic cells. (B) Apoptotic cells (induced by 1 μM staurosporine).

Fig 5. Confocal microscopy images of HEK293T and MCF-7 cells treated with substrate 27.

Confocal microscopy images (objective HCX PL APO ×63/1.40–0.6 Oil CS) of HEK273T cells with substrate 27 (10 μM) without staurosporine (STS) (A) and with staurosporine (1 μM) (B) induced apoptosis (green fluorescence is from fluorescein “turned on” by caspase-3 cleavage of the substrate, blue is DAPI nuclear stain). (C) Staurosporine treated MCF-7 cells with substrate 27.