A senolysis-based theragnostic prodrug strategy towards chronic renal failure

Abstract

Selective elimination of senescent cells (senolysis) has become a promising therapeutic strategy for the management of chronic renal failure (CRF), but the senolytic molecular pathways towards CRF therapy are limited. Here, we present for the first time a senescence-associated β-galactosidase (SA-β-gal) activatable theragnostic prodrug strategy to pertinently and effectively treat CRF in mice with the aid of fluorescence-guided senolysis. The signs of premature senescence, including the overexpression of β-gal, have been found in kidneys of mice with CRF, making this enzyme particularly suitable as a trigger of prodrugs for CRF therapy. With this unique design, our pioneering prodrug TSPD achieved the activation of a fluorophore for tracking and the specific release of the parent drug, gemcitabine, in β-gal-enriched cells after activation with SA-β-gal. In mice with CRF, abdominal administration of TSPD was effective for improvement of the kidney functions, supporting the feasibility of the SA-β-gal-dependent senolysis therapy towards CRF.

Scheme 1. Synthetic route for TSPD. Reagents and conditions: (a) C₂H₅CO₂Na, (C₂H₅CO)₂O, triethylamine, 170 °C, and 10 h. (b) (1) NBS, AIBN, CCl₄, 85 °C, and 12 h; (2) NaOAc, CH₃COOH, 120 °C, and 12 h; (3) HCl, r.t., and 12 h. (c) NaBH₄, MeOH, 0 °C, and 2 h. (d) 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl bromide, Ag₂CO₃, HMTTA, dry CH₃CN, r.t., and 4 h. (e) 4-Nitrophenyl chloroformate, DIPEA, dry pyridine, THF, N₂, 0 °C, and 1 h. (f) (1) LiHMDS, N₂, dry THF, −78 °C, and 30 min; (2) dry THF, r.t., and 30 min. (g) (1) TBAF, dry THF, r.t., and 30 min; (2) CH₃ONa, dry CH₃OH, 0 °C, and 10 min.

Fig. 1. (A) Absorption spectra of TSPD (10 μM), compound 3 (10 μM) and TSPD (10 μM) after incubation with E. coli β-gal (1 U mL⁻¹) at 37 °C for 15 min. (B) Fluorescence spectra of TSPD (10 μM), compound 3 (10 μM) and TSPD (10 μM) after incubation with E. coli β-gal (1 U mL⁻¹) at 37 °C for 15 min. (C) Time dependent fluorescence spectra of TSPD (10 μM) after incubation with E. coli β-gal (1 U mL⁻¹) at 37 °C. Inset: changes in fluorescence intensity at 456 nm as a function of time after E. coli β-gal (1 U mL⁻¹) treatment. (D) Fluorescence spectra of TSPD (10 μM) after incubation with E. coli β-gal (0–5 U mL⁻¹) at 37 °C for 15 min. Inset: changes in fluorescence intensity at 456 nm as a function of the E. coli β-gal concentration. (E) Fluorescence spectra and (F) fluorescence intensity at 456 nm of TSPD (10 μM) upon incubation with E. coli β-gal and other various biological analytes. (λ_ex = 360 nm, slit: 5 nm/5 nm, PMT: 500 V) (n = 3).

Fig. 2. (A) Proposed activation mechanism of TSPD. (B) HPLC analysis of TSPD (50 μM) in the presence of E. coli β-gal (0.1 U mL⁻¹) at 37 °C for 0–30 min (eluent A, MeOH; eluent B, water; 0–30 min, A/B = 10/90–80/20; flow rate = 1.0 mL min⁻¹). (C) Release of gemcitabine from TSPD in the presence of different concentrations of E. coli β-gal. (D) Fluorescence imaging (left) and Coomassie blue staining (right) of SDS-PAGE gel. TSPD (20 μM) was incubated with proteins at 37 °C for 0.5 h. 1, TSPD + E. coli β-gal (1 U mL⁻¹) + BSA (1 mg mL⁻¹); 2, TSPD + E. coli β-gal (1 U mL⁻¹); 3, TSPD; λ_ex = 365 nm.

Fig. 3. (A) Fluorescence images of uninduced NRK-52E cells (control) and MitoC-induced senescent (sct) NRK-52E cells after incubation with TSPD (20 μM) for 4 h. Quantification of relative fluorescence intensity of (A) is on the right (n = 3). Scale bar = 20 μm. (B) X-gal staining images of uninduced NRK-52E cells and MitoC-induced senescent NRK-52E cells. The average percentage of SA-β-gal positive cells of (B) is on the right (n = 3). Scale bar = 20 μm. (C) Quantification of cell viability of uninduced NRK-52E cells and MitoC-induced senescent NRK-52E cells incubated with increasing concentrations of TSPD for 2 days (n = 3). (D) Western blotting analysis of p53 and γH2AX in uninduced NRK-52E cells and MitoC-induced senescent NRK-52E cells. GAPDH was chosen as an internal reference (n = 2). (E) Quantification of cell viability of uninduced NRK-52E cells and MitoC-induced senescent NRK-52E cells incubated with increasing concentrations of gemcitabine for 2 days (n = 3). Significant differences (*P < 0.05, **P < 0.01, and ***P < 0.001) are analyzed with the t-test.

Fig. 4. (A) Experimental design for renal unilateral ischemia reperfusion injury (UIRI). Mice (7 weeks-old) were subjected to renal unilateral ischemia reperfusion injury surgery or sham surgery 7 days before drug treatment. UIRI mice were intraperitoneally injected with gemcitabine (1 mg kg⁻¹ and 5 mg kg⁻¹), TSPD (1 mg kg⁻¹ and 5 mg kg⁻¹) or vehicle (2% DMSO, 90% PBS, 4% Tween-80, and 4% polyethylene glycol) twice a week for three weeks; sham surgery mice were treated with vehicle in the same way. (B) Representative images (left) and quantification (right) of lamin B1 staining of kidneys from sham or UIRI mice after vehicle, gemcitabine or TSPD treatment (n = 9 for each group). (C) Representative images of kidneys from sham or UIRI mice after vehicle, gemcitabine or TSPD treatment (n = 5 for each group). (D and E) Serum biochemical test. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) in sham or UIRI mice after vehicle, gemcitabine or TSPD treatment as shown (n = 5 for each group). (F) Representative images (left) and quantification (right) of SA-β-gal staining of kidneys from sham or UIRI mice after vehicle, gemcitabine or TSPD treatment (n = 4 for each group). (G) PSR (n = 7 for each group). (H) Ki67 (n = 9 for each group). (I) KIM-1 (n = 3 for each group). Scale bar = 20 μm. Significant differences (^##P < 0.01 and ^###P < 0.001, compared with the Sham group, *P < 0.05 and ***P < 0.001, compared with the UIRI group, and ⁺P < 0.05, ⁺⁺P < 0.01, and ⁺⁺⁺P < 0.001, compared with the UIRI + Gem group) are analyzed with the t-test.