A PIKfyve modulator combined with an integrated stress response inhibitor to treat lysosomal storage diseases

Abstract

Lysosomal degradation pathways coordinate the clearance of superfluous and damaged cellular components. Compromised lysosomal degradation is a hallmark of many degenerative diseases, including lysosomal storage diseases (LSDs), which are caused by loss-of-function mutations within both alleles of a lysosomal hydrolase, leading to lysosomal substrate accumulation. Gaucher's disease, characterized by <15% of normal glucocerebrosidase function, is the most common LSD and is a prominent risk factor for developing Parkinson's disease. Here, we show that either of two structurally distinct small molecules that modulate PIKfyve activity, identified in a high-throughput cellular lipid droplet clearance screen, can improve glucocerebrosidase function in Gaucher patient-derived fibroblasts through an MiT/TFE transcription factor that promotes lysosomal gene translation. An integrated stress response (ISR) antagonist used in combination with a PIKfyve modulator further improves cellular glucocerebrosidase activity, likely because ISR signaling appears to also be slightly activated by treatment by either small molecule at the higher doses employed. This strategy of combining a PIKfyve modulator with an ISR inhibitor improves mutant lysosomal hydrolase function in cellular models of additional LSD.

Keywords: Gaucher’s disease; PIKfyve; Parkinson’s disease; integrated stress response; lysosomal storage disease.

Fig. 1.

Small molecules identified in previous HTS increase GCase activity. (A) Chemical structures of A16 and A18. (B) 72 h treatment of A16 or A18 increased GCase activity in non-Gaucher patient–derived fibroblasts, as measured by 4-MUG substrate hydrolysis in cell lysate. (C) Immunofluorescence confocal microscopy of HeLa cells after 20 h treatment of A16 and A18 showed TFEB nuclear localization; (D) quantification. (Scale bar, 40 µm.) (E) Non-Gaucher patient fibroblasts that were transduced with shRNA targeting TFEB, TFE3, or nontargeting shRNA were treated with A16 and A18 for 48 h. (F) Immunofluorescence confocal microscopy of fibroblasts after 24 h treatment of A16 showed TFE3 nuclear localization; (G) quantification. (Scale bar, 60 µm.) Bar graphs show mean ± 95% CI, and statistics were performed using the Kruskal–Wallis test with multiple comparisons (D and E) or Mann–Whitney T test (G). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Experiments were done with at least three replicates (N ≥ 3).

Fig. 2.

A16 and A18 improve GCase activity in Gaucher patient–derived fibroblasts. (A) 72 h treatment of A16 or A18 improved GCase activity in Gaucher fibroblasts with L444P/L444P or N370S/84GG mutations on GBA1, as measured by 4-MUG hydrolysis in cell lysate. (B) Treatment of L444P/L444P Gaucher fibroblasts with A16 or A18 for 24 h increased GCase mRNA levels, as determined by qPCR. (C) N370S/84GG Gaucher fibroblasts that were treated with A16 or A18 for 72 h had their lysate subjected to EndoH digestion. Western blots of lysate showed increased EndoH-resistant GCase bands in small molecule–treated groups. See SI Appendix, Fig. S5 for quantification of EndoH-sensitive GCase. (D) L444P/L444P Gaucher fibroblasts that had been treated with A16 or A18 for 72 h were labeled in situ with fluorescent GCase activity-based probe EW644. Lysates were run on SDS-PAGE, showing higher amounts of ABP-conjugated GCase in small molecule–treated groups. (E) Lysates conjugated with A16 and A18 showed shifted band density to higher molecular weights on ABP-conjugated GCase. Bar graphs show mean ± 95% CI, and statistics were performed using the Kruskal–Wallis test with multiple comparisons. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Experiments were done with at least three replicates (N ≥ 3).

Fig. 3.

A16 and A18 operate through PIKfyve modulation in order to improve GCase activation. (A) Structures of A18 with established PIKfyve modulators Apilimod and YM201636, highlighting structural similarities in red. (B) A16, A18, and Apilimod bound PIKfyve as shown by DiscoveRX KINOMEScan for PIKfyve. (C and D) Treatment of Gaucher fibroblasts with (C) Apilimod and (D) YM201636 for 72 h improved GCase activity, as measured by 4-MUG hydrolysis in cell lysate. (E) L444P/L444P Gaucher fibroblasts that were transduced with nontargeting and PIKfyve-targeting shRNA, followed by 72 h treatment with A16 or A18. A16 showed reduced efficacy in improving GCase for the shPIKfyve-transduced cells than the shNonTargeting transduced cells, as measured by 4-MUG hydrolysis in cell lysate. Bar graphs show mean ± 95% CI, and statistics were performed using the Kruskal–Wallis test with multiple comparisons. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Experiments were done with at least three replicates (N ≥ 3).

Fig. 4.

Medicinal chemistry efforts on A18 gave rise to multiple chemical analogues with different effects on GCase improvement and PIKfyve binding. (A) GCase activity in L444P/L444P Gaucher fibroblasts after 72 h treatment with A18 analogues LM-2-34 and LM-2-27A, as determined by 4-MUG hydrolysis in cell lysate. (B) Structure of A18 analogues LM-2-34 and LM-2-27A, with differences from A18 highlighted in red. (C) A18 analogues bound to PIKfyve as shown by DiscoveRX KINOMEScan for PIKfyve. (D) Western blots of EndoH treated/nontreated cell lysate from Gaucher fibroblasts that have been treated with A18 or LM-2-27A for 72 h. See SI Appendix, Fig. S8F for quantification of EndoH-sensitive GCase. (E) Treatment of Gaucher fibroblasts with LM-2-27A for 24 h increased GCase mRNA levels, as determined by qPCR. The bar graph shows mean ± 95% CI, and statistics were performed using the Kruskal–Wallis test with multiple comparisons. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Experiments were done with at least three replicates (N ≥ 3).

Fig. 5.

Inhibition of the ISR via ISRIB improves GCase activity and can amplify the effect of PIKfyve modulators, as determined by 4-MUG hydrolysis in cell lysate. (A) 72 h treatment of ISRIB improved GCase activity in L444P/L444P Gaucher fibroblasts. (B) 72 h cotreatment of ISRIB with A16, A18, or Apilimod further improved GCase activity in L444P/L444P Gaucher fibroblasts over individual treatments. (C) Ratiometric GCase activity of (B), comparing the improvement of ISRIB when cotreated with PIKfyve modulators. (D) 72 h cotreatment of ISRIB with LM-2-34, LM-2-27A, or YM201636 further improved GCase activity in L444P/L444P Gaucher fibroblasts over individual treatments. (E) L444P/L444P Gaucher fibroblasts that had been treated with Apilimod and/or ISRIB for 72 h were labeled in situ with fluorescent GCase activity-based probe EW644. Lysates were run on SDS-PAGE, showing higher amounts of ABP-conjugated GCase in Apilimod-treated cells, and further increase with ISRIB cotreatment. (F) 24 h treatment of L444P/L444P Gaucher fibroblasts with Apilimod increased GCase transcription, but ISRIB did not alter GCase mRNA levels, nor did it make any contribution to GCase mRNA levels when cotreated with Apilimod, as determined by qPCR. (A–D) was performed by measuring 4-MUG hydrolysis in cell lysate. Bar graphs show mean ± 95% CI, and statistics were performed using the Kruskal–Wallis test with multiple comparisons. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Experiments were done with at least three replicates (N ≥ 3).

Fig. 6.

PIKfyve modulators improve multiple lysosomal enzyme activities in various LSD fibroblasts. (A) Fabry, (B) Pompe, (C and D) mucopolysaccharidosis IV, (E and F) α-mannosidosis fibroblasts that were treated with PIKfyve modulators for 72 h had increased mutant enzyme activity, as measured by respective 4-MU-based fluorescent substrate assays in cell lysate. See SI Appendix, Fig. S14 for corresponding ratiometric data. Bar graphs show mean ± 95% CI, and statistics were performed using the Kruskal–Wallis test with multiple comparisons. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Experiments were done with at least three replicates (N ≥ 3).