The p97 Inhibitor UPCDC-30245 Blocks Endo-Lysosomal Degradation

Abstract

The diverse modes of action of small molecule inhibitors provide versatile tools to investigate basic biology and develop therapeutics. However, it remains a challenging task to evaluate their exact mechanisms of action. We identified two classes of inhibitors for the p97 ATPase: ATP competitive and allosteric. We showed that the allosteric p97 inhibitor, UPCDC-30245, does not affect two well-known cellular functions of p97, endoplasmic-reticulum-associated protein degradation and the unfolded protein response pathway; instead, it strongly increases the lipidated form of microtubule-associated proteins 1A/1B light chain 3B (LC3-II), suggesting an alteration of autophagic pathways. To evaluate the molecular mechanism, we performed proteomic analysis of UPCDC-30245 treated cells. Our results revealed that UPCDC-30245 blocks endo-lysosomal degradation by inhibiting the formation of early endosome and reducing the acidity of the lysosome, an effect not observed with the potent p97 inhibitor CB-5083. This unique effect allows us to demonstrate UPCDC-30245 exhibits antiviral effects against coronavirus by blocking viral entry.

Keywords: coronavirus; endo-lysosomal degradation; lysomotropic agents; p97 inhibitor; proteomics.

Figure 1

Differential cellular effects between UPCDC-30245 and CB-5083 or NMS-873 in HCT116 cells. (A) qRT-PCR analysis of CHOP, ATF3, p97 and p62 RNAs following treatment with DMSO, 5 μM of UPCDC-30245, 5 μM of CB-5083 or 5 μM of NMS-874 for 6 h. N = 3. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.0005; ****, p < 0.0001; according to one-way ANOVA with multiple comparison tests. (B) The cellular viability curves for UPCDC-30245, CB-5083 and NMS-873. N = 4.

Figure 2

Proteomics reveals impairment of endo-lysosomal pathways in cells treated with UPCDC-30245 in HCT116 cells. (A) PCA revealed a separation between the treatment with DMSO and 5 of UPCDC-30245 for 6 h. (B) Volcano plot displaying the proteomic changes following UPCDC-30245 treatment in HCT116 cells, log₂ (fold change) indicates the logarithm to the base 2 of fold change, n = 3. (C) Functional enrichment analysis on proteins affected by UPCDC-30245. (D) Heatmap showing fold change in early endosome proteins, autophagy and lysosome related proteins which are significantly dysregulated by UPCDC-30245. (E) Enzymatic activity of HEXB and NAGLU. Cells indicates enzymatic activity was detected in HCT116 cells pre-treated with DMSO or 5 μM of UPCDC-30245 for 6 h. Lysate indicates enzymatic activity was detected using by treating HCT116 cell lysate with DMSO or 5 μM UPCDC-30245 for 30 min. An increase in enzyme activity was observed in the cells treated with UPCDC-30245, but not in the lysate, which indicates that UPCDC-30245 increased the amount of enzyme. N = 3. *, p < 0.05; **, p < 0.01; according to one-way ANOVA tests.

Figure 3

UPCDC-30245 inhibits the formation of early endosome and autophagy flux in H1299 and HeLa cells. (A,B) UPCDC-30245 (5 μM) inhibits the formation of EEA1 positive puncta after 1 h of treatment in H1299. 550 (DMSO), 528 (CB-5083) and 228 (UPCDC-30245) cells were counted from triplicates. Scale bar represents 25 μm. (C,D) The number of autophagosomes were increased and autolysosomes were reduced by 5 μM of UPCDC-30245, 50 μM of HCQ and 10 μM of Baf-A1, but not by 5 μM of CB-5083 in HeLa cells stably expressing mRFP-GFP-LC3 reporter after 2 h of treatment. Scale bar represents 50 μm. (E) UPCDC-30245 and HCQ induced the formation of large autophagosomes in HeLa cells after 2 h of treatment. The 210 (DMSO), 158 (CB-5083), 215 (UPCDC-30245), 225 (HCQ) and 291 (Baf-A1) cells were counted from four replicates. ns, not significant; ***, p < 0.0005; ****, p < 0.0001 according to one-way ANOVA with multiple comparison tests.

Figure 4

UPCDC-30245 decreases the acidity of lysosomes in H1299 and HeLa cells. (A) Structures of UPCDC-30245 and HCQ. (B,C) UPCDC-30245 (5 μM) decreases the LysoTracker puncta staining in both HeLa and H1299 cells. 220 to 290 (H1299) and 250 to 350 (HeLa) were counted from triplicates. Scale bar represents 50 μm. (D,E) Decreasing the acidity of lysosomes using Baf-A1 blocks the formation of large autophagosomes induced by UPCDC-30245 and HCQ in HeLa cells. HeLa cells were pretreated with DMSO, 10 μM of Baf-A1, 50 μM of HCQ or 5 μM of UPCDC-30245 for 30 min. Then 5 μM of UPCDC-30245 or 50 μM of HCQ were added. Images were taken at 2 h after the addition of UPCDC-30245 and HCQ. A total of 141 to 201 (HCQ) and 81 to 161 (UPCDC-30245) cells were counted from triplicates. Scale bar represents 50 μm. ns, not significant; ****, p < 0.0001 according to one-way ANOVA with multiple comparison tests.

Figure 5

UPCDC-30245 inhibits HCoV-229E infection via blocking virus entry in H1299 cells. (A) Schema of time-of-addition experiment. 1. Added compound for 5 min, added virus for 2 h, washed out, added fresh media with DMSO for 6 h. 2. Added compound and virus together for 2 h, washed out, added fresh media with compound for 2 h, washout, add fresh media and DMSO for 4 h. 3. Added compound and virus together for 2 h, washed out, added fresh media with compound for 6 h. 4. Added DMSO and virus together for 2 h, washed out, added fresh media with DMSO for 2 h, washed out, and added fresh media with compound for 4 h. (B) The HCoV-229E RNA levels in H1299 cells were significantly reduced by 5 μM of UPCDC-30245 and 50 μM of HCQ at 8 h post infection. Data were normalized to the RNA levels of human GAPDH. Numbers 1 to 4 represent the time-of-addition described in Figure 5A. N = 3. (C) The protection of CPE activity (black curve) and toxicity (TOX, red curve) curves for UPCDC-30245 and HCQ. N = 4. (D) TCID₅₀/mL measurements in a viral titer reduction assay for DMSO, UPCDC-30245 and 25 μM of HCQ after 24 h of HCoV-229E infection. N = 3. (E) TCID₅₀/mL measurements for remdesivir. DMSO or 2.5 μM of UPCDC-3245 was added 5 min before virus infection and washed out with virus after 4 h of infection, then remdesivir was added and incubated for 20 h. N = 3. (F) The protection of CPE activity curves for remdesivir (0–0.42 μM, 3-fold dilution) with or without the pretreatment of 2.5 μM of UPCDC-30245 during virus infection. N = 3. ns, not significant; *, p < 0.05; **, p < 0.01; ****, p < 0.0001 according to one-way ANOVA with multiple comparison tests.