A Mitochondria-Targeting SIRT3 Inhibitor with Activity against Diffuse Large B Cell Lymphoma

Abstract

Diffuse large B-cell lymphomas (DLBCLs) are heterogeneous cancers that still require better and less toxic treatments. SIRT3, a member of the sirtuin family of NAD⁺-dependent protein deacylase, is critical for DLBCL growth and survival. A mitochondria-targeted SIRT3 small-molecule inhibitor, YC8-02, exhibits promising activity against DLBCL. However, YC8-02 has several limitations including poor solubility. Here, we report our medicinal chemistry efforts that led to an improved mitochondria-targeted SIRT3 inhibitor, SJ-106C, achieved by using a triethylammonium group, which helps to increase both solubility and SIRT3 inhibition potency. SJ-106C, while still inhibiting SIRT1 and SIRT2, is enriched in the mitochondria to help with SIRT3 inhibition. It is more active against DLBCL than other solid tumor cells and effectively inhibits DLBCL xenograft tumor growth. The findings provide useful insights for the development of SIRT3 inhibitors and mitochondrial targeting agents and further support the notion that SIRT3 is a promising druggable target for DLBCL.

Figure 1

Structures of various SIRT3 inhibitors developed and in vitro IC₅₀ values against SIRT1/2/3. Enzymatic activity was detected by using an HPLC assay.

Figure 2

X-ray crystal structure of SIRT3 in the complex with NH6-10 explains the structure–activity trends observed for the inhibitors shown in Figure 1. (A) Structure of SIRT3 in a complex with NH6-10. The covalent intermediate formed between NH6-10 and NAD⁺ is shown in stick representation. The two key residues, P297 and E325, are also shown. (B,C) The structures of SIRT2 (PDB 6NR0) (B) and SIRT1 (PDB 4I5I) (C) were superimposed to that of SIRT3 in (A). The covalent intermediate formed between NH6-10 and NAD⁺ on SIRT3 is shown in stick representation to highlight the active sites. The residues corresponding to P297 and E325 of SIRT3 are also shown (S238 and Q267 in SIRT2, and N417 and R446 in SIRT1).

Figure 3

SJ-106C inhibits the viability of various cancer cells, particularly in DLBCLs. (A) Relative cell viability of the indicated cells was assessed after treatment with increasing concentrations of SJ-106C for 72 h. (B) Calculated IC₅₀ values (μM) of SJ-106C for 72 h in various cancer cells.

Figure 4

Negative control compound, SJ-155, lost its ability to inhibit SIRT1/2/3 (compared to SJ-106C). Enzymatic activity was detected using an HPLC assay.

Figure 5

SJ-106C as a mitochondria-targeting SIRT3 inhibitor. (A) 20 million of Karpas 422 cells were treated with SJ-106C, YC8-02, or JH-T4 at 5 μM for 6 h. Quantification of compound amount (in nmol) in whole cell lysate and mitochondrial fractions was detected by LC-MS; (B) the ratio of the inhibitor amount in mitochondria to that in whole cell lysate for each inhibitor was calculated based on the compound amount in each exacted portion normalized to the exact volume of extraction. Data represent three independent experiments and are presented as mean ± s.d. *p < 0.05, **p < 0.01.

Figure 6

SJ-106C inhibits SIRT3 in the cells. (A) Western blot and densitometry analysis of mitochondrial acetylation from Karpas 422 cells (1 million/mL) were treated with indicated compounds for 12 h. The mitochondria were isolated and blotted for acetyl-lysine. Western blots were quantified by densitometry, normalized to the intensity of the corresponding VDAC, and then further normalized to the DMSO group. (B) Western blot analysis of acetylated IDH2 (K413) after immunoprecipitation of Flag-tagged IDH2 in MDA-MB-231 cells treated with DMSO, SJ-106C, YC8-02, or SJ-155 at the indicated concentrations for 6 h. This cell line expressed Flag-IDH2 in a doxycycline-inducible manner. Western blots were quantified by densitometry, normalized to the intensity of the corresponding IP Flag-IDH2, and then further normalized to the DMSO group.

Figure 7

Pharmacokinetics of SJ-106C in NSG mice. SJ-106C was intraperitoneally (IP) injected at 100 mg/kg. Blood and various organs were collected at specified time points of 1, 3, 6, and 24 h after SJ-106C administration. Plasma isolation, tissue lysis, and extraction of SJ-106C with methanol were performed for LC-MS detection (n = 3 for each time point).

Figure 8

Effects of SIRT3 inhibitors on the OCI-LY7 tumor xenograft model. (A) Image of the OCI-LY7 xenograft tumors dissected from NSG mice treated with SJ-106C or vehicle control. (B) Tumor weights in different groups of mice were measured. (C) Tumor growth curves of the OCI-LY7 tumor xenografts with intraperitoneal administration of vehicle, 50 mg/kg SJ-106C five times per week for 24 days (n = 5 mice per group). (D) Average body weight of mice in different groups. Data are shown as mean ± sd of 5 mice per group. P-values were determined by Student’s t test. *p < 0.05, **p < 0.01.