Discovery of Spirosnuolides A-D, Type I/III Hybrid Polyketide Spiro-Macrolides for a Chemotherapeutic Lead against Lung Cancer

Abstract

Four new macrolides, spirosnuolides A-D (1-4, respectively), were discovered from the termite nest-derived Kitasatospora sp. INHA29. Spirosnuolides A-D are 18-membered macrolides sharing an embedded [6,6]-spiroketal functionality inside the macrocycle and are conjugated with structurally uncommon side chains featuring cyclopentenone, 1,4-benzoquinone, hydroxyfuroic acid, or butenolide moieties. Structure elucidation was achieved using a combination of spectroscopic analyses, multiple chemical derivatizations (methylation, methanolysis, Luche reduction, and Mosher's reaction), X-ray diffraction analysis, and computational ECD calculations. Interestingly, genome sequencing analysis suggests that spirosnuolides were biosynthesized through a rare type I/III hybrid polyketide synthase. Importantly, spirosnuolide B displayed potent antiproliferative effects against various cancer cell lines at nanomolar concentrations, particularly against HCC827 cells, an EGFR mutant non-small-cell lung cancer (NSCLC) cell line, with a high safety index value. Based on in vitro studies, the antiproliferative mechanism of spirosnuolide B involved the activation of AMPK signaling, leading to cell cycle arrest and apoptosis in HCC827 cells. Its potent efficacy was also proven in vivo by the effective inhibition of tumor growth in mouse xenograft studies. Moreover, cotreatment with spirosnuolide B and gefitinib, synergistically enhanced the antiproliferative activity and apoptosis, suggesting a potential strategy to overcome gefitinib resistance in EGFR mutant NSCLC.

Figure 1

(a) Structures of spirosnuolides A–D (1–4, respectively). (b) Key COSY, HMBC, and LR-HSQMBC correlations for constructing the planar structure of spirosnuolide A (1) and its methylated derivative (5). (c) ¹³C NMR spectra of spirosnuolide A (1) in CD₃OD (upper) and 1:1 CD₃OD-CD₃OH mixture (lower). (d) J-based configurational analysis for the relative configuration of spirosnuolides at C-2 and C-3. (e) Coupling constants and key ROESY correlations for determination of relative configuration of the spiro-bis-tetrahydropyran ketal portion of spirosnuolides.

Scheme 1. Chemical Derivatization of Spirosnuolide A (1)

Figure 2

(a) ¹³C–¹³C COSY spectrum (bold line) of spirosnuolide B (2). (b) Methylation derivatization of spirosnuolide B with HMBC correlations (arrows). (c) Comparison of the experimental ECD spectrum with the averaged calculated ECD spectrum of spirosnuolide B (2). ORTEP drawings with 30% probability ellipsoids shown of the crystal structures of (d) spirosnuolide B (2), (e) spirosnuolide C (3), and (f) spirosnuolide D (4).

Figure 3

In vitro efficacy of spirosnuolide B (2). (a) HCC827 cells were seeded in 96-well plates and treated with spirosnuolide B (2) for 24–72 h. IC₅₀ values were determined by SRB assay. Data are presented as mean ± SD (n = 3). (b) Effects of spirosnuolide B (2) on colony formation in HCC827 cells. HCC827 cells were treated with 1 μM spirosnuolide B (2) for 24 h, and then cells were replaced with fresh medium every 3 days and incubated for additional 21 days. (c) HCC827 cells were treated with indicated concentrations of spirosnuolide B (2) for 24 h, and the expression of AMPKα and its downstream targets was evaluated by Western blot analysis. β-Actin was used as an internal control.

Figure 4

Effects of spirosnuolide B (2) on cell cycle distribution in HCC827 cells. (a) Cells were treated with spirosnuolide B (2) for 24 h, and then the cells were collected, fixed with 70% cold ethanol in PBS, and stained with propidium iodide (PI). Cell cycle distribution was measured by flow cytometry. *p < 0.05 and **p < 0.01 represent statistically significant differences compared to the vehicle-treated control group. (b) Cells were treated with indicated concentrations of spirosnuolide B (2) for 24 h, and then the expressions of proteins were analyzed by Western blotting. β-Actin was used as an internal control.

Figure 5

Effects of spirosnuolide B (2) on induction of apoptosis in HCC827 cells. (a) Cells were treated with spirosnuolide B (2) for 48 h, collected, stained with PI, and annexin V-FITC for 15 min, and apoptotic cell distribution was analyzed by flow cytometry. ***p < 0.001 represents a statistically significant difference compared to the vehicle-treated control group. (b) Cells were treated with spirosnuolide B (2) for 48 h, lysed, and then expressions of proteins were determined by Western blot analysis. β-Actin was used as an internal control.

Figure 6

In vivo efficacy of spirosnuolide B (2). (a) HCC827 cells were implanted subcutaneously into the flank of Balb/C-nude mice. After tumor volume reached approximately 100 mm³, gefitinib (10 mg/kg) was administered orally or the vehicle and spirosnuolide B (0.5 or 1 mg/kg) was administered intraperitoneally three times per week for 22 days. Tumor volume was measured every 2–3 days. Gefitinib was used as a positive control. *p < 0.05, **p < 0.01, and ***p < 0.001 represent statistically significant differences compared to the vehicle-treated control group. (b) Body weight was monitored twice per week. (c) Excised tumor section was fixed with formalin, embedded in paraffin, incubated with indicated antibodies, and photographed under an inverted phase-contrast microscope.

Figure 7

Combination index (CI) value with cotreatment of spirosnuolide B (2) and gefitinib in HCC827 cells. Cells were treated with spirosnuolide B (2) and gefitinib with indicated concentrations for 48 h, and CI values were calculated for the combination effects.