General experimental procedures
Specific rotations were obtained by using a JASCO P-2200 digital polarimeter (l = 5 cm). ECD spectroscopy was recorded using a JASCO J-1500 spectrometer. FAB-MS and HR-FAB-MS were recorded by using a JEOL JMS-SX 102 A mass spectrometer. 1 H NMR spectroscopy was recorded on JEOL ECS400 (400 MHz) and JNM-ECA 600 (600 MHz) spectrometers. 13 C NMR spectroscopy was recorded on a JNM-ECA 600 (150 MHz) spectrometer. 2D-NMR experiments were carried out on a JEOL JNM-ECA 600 (600 MHz) spectrometer.
Normal phase silica gel column chromatography was carried out using Silica gel 60 (Kanto Chemical Co., Inc. 63–210 mesh), and reversed phase silica gel column chromatography was carried out using C18-OPN (Nacalai Tesque Co., Inc. 140 μm). High-performance liquid chromatography (HPLC) was performed using a Shimadzu SPD-M20A UV–vis detector, Shimadzu LC-20AD pump, and Shimadzu SIL-20 A auto-injector. COSMOSIL 5C18-MS-II (Nacalai Tesque Co., Inc. 250 × 4.6 mm i.d., 250 × 10 mm i.d., and 250 × 20 mm i.d.) columns were used for analytical and preparative work.
Collection and identification of the JKYM-AK1 strain
Airborne particles (PM10, aerodynamic diameter ≤ 10 μm) were collected in the city of Kyoto (135.81°E, 34.99°N) using a high-volume air sampler (HV1000R, Shibata Scientific Technology, Soka, Japan) equipped with an impactor (Shibata Scientific Technology) in September 2021. The collected airborne particles were immediately suspended in distilled H2O. The suspension was inoculated with potato dextrose agar (PDA) and chloramphenicol (0.1 g/L) for incubating at 28 °C for 3 days. The JKYM-AK1 strain was isolated as colonies and stored in 10% glycerin at − 80 °C. Using internal transcribed spacer 1 (ITS1, 5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) as primers, the ITS1-5.8 S-ITS2 sequence region (511 base pairs, GenBank accession number ON150838) of JKYM-AK1 was obtained. BLAST analysis (NCBI database) showed that the sequence has a 99.60% identity of Penicillium maximae (Accession number: EU427298).
Fermentation and extraction
The seed culture of the fungus was prepared in a potato dextrose medium and incubated at 28 °C for 5 days at 200 rpm. The seed culture was added to 2 L × 20 Erlenmeyer flasks containing potato dextrose medium (1 L). The cultures were further incubated at 28 °C for 20 days followed by filtration. Mycelia was extracted three times with methanol by refluxing for 1 h. The solvent was evaporated to obtain a methanol extract. The methanol extract was combined with the supernatant and partitioned using EtOAc–water mixture.
Isolation of the compounds produced by the JKYM-AK1 strain
The EtOAc soluble fraction (8.31 g) was purified by normal phase silica gel column chromatography [n-hexane–CHCl3 (1:1 → 1:4→ 0:1, v/v) → CHCl3–MeOH (50:1 → 30:1 → 20:1 → 10:1 → 5:1 → 1:1, v/v)] and ten fractions were collected. Fraction 4 (2.3 g) was further separated using reversed phase silica gel column chromatography and eight fractions were collected. Fraction 4 − 3 (73.3 mg) was purified using HPLC {H2O–CH3CN (30:70, v/v)} to obtain 1 (3.2 mg), 2 (5.1 mg), 3 (8.4 mg), and 4 (7.1 mg). Fraction 4–4 (118.5 mg) was purified using HPLC {H2O–CH3CN (40:60, v/v)} to obtain 5 (48.9 mg). Fraction 5 (1.1 g) was separated using reversed phase silica gel column chromatography and ten fractions were obtained. Fraction 5 − 4 (22.2 mg) was purified using HPLC {H2O–CH3CN (60:40, v/v)} to obtain 7 (2.3 mg). Fraction 5–5 (23.3 mg) was purified using HPLC {H2O–CH3CN (65:35, v/v)} to obtain 6 (2.7 mg).
Maximazaphilone I (1)
Yellow amorphous solid; [α]25
D +44.2 (c 0.16, MeOH); 1 H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz), see Table 1; positive-ion FAB-MS m/z 385 [M + H]+; HR-FAB-MS m/z 385.2018 [M + H]+ (calcd for C23H29O5, 385.2015).
Maximazaphilone II (2)
Yellow amorphous solid; [α]25
D +100.3 (c 0.27, MeOH); 1 H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz), see Table 1; positive-ion FAB-MS m/z 319 [M + H]+; HR-FAB-MS m/z 319.1928 [M + H]+ (calcd for C19H27O4, 319.1909).
Maximazaphilone III (3)
Yellow amorphous solid; [α]25
D +54.4 (c 0.21, MeOH); 1 H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz), see Table 1; positive-ion FAB-MS m/z 361 [M + H]+; HR-FAB-MS m/z 361.2025 [M + H]+ (calcd for C21H29O5, 361.2015).
Maximazaphilone IV (4)
Yellow amorphous solid; [α]25
D +173.0 (c 0.15, MeOH); 1 H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz), see Table 1; positive-ion FAB-MS m/z 319 [M + H]+; HR-FAB-MS m/z 319.1919 [M + H]+ (calcd for C19H27O4, 319.1909).
Calculation of theoretical ECD spectra for maximazaphilone I, II, and IV (1, 2, and 4)
A preliminary molecular mechanics (MM) conformational search was carried out on 1, 2, and 4 in vacuum by using the Merck molecular force field (MMFF) as implemented in Spartan’16 program [22]. The stable energy conformers of each compound with Boltzmann distributions over 1% were further optimized at the B3LYP/def2-TZVP level of density functional theory (DFT). The normal mode analysis was done at the same level to confirm none of the conformers showed imaginary frequencies and to obtain the Gibbs free-energies [23]. The low free-energy conformers for each compound with Boltzmann distributions over 1% (Fig. S 4) were subjected to the ECD calculations using time-dependent density functional theory (TD-DFT) at the B3LYP/def2-TZVPP level. Both for the DFT and TD-DFT calculations were performed using an integral equation formalism polarizable continuum model (IEFPCM) in MeOH using Gaussian 16 [24]. The resultant rotatory strengths of the lowest 30 excited states for each conformer were converted into Gaussian-type curves with half-bands (0.30 eV) using SpecDis v1.71 [25]. The calculated ECD spectra of each of 1, 2, and 4 were composed after correction based on the Boltzmann distribution of conformers and their relative free-energy.
Cells
Human cervical carcinoma (HeLa) cells and mouse pGL105/C3H cells [26] were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with low glucose (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 5% fetal bovine serum (Merck, Darmstadt, Germany) under a 5% CO2 atmosphere at 37 ℃.
Measurement of Luc activity
Stable hsp105 promoter-luciferase reporter cell lines (pGL105/C3H cells) were cultured in a flat-bottomed 96-well plate (Coster 3596; Corning, NY, USA) and incubated to reach 70 − 80% confluence. The cells were pretreated with test compounds for 30 min and exposed to heat shock at 41℃ for 3 h using a water bath. After washing the cells with PBS (-) (Wako Pure Chemical Industries) twice, 150 µL of 1X Glo Lysis Buffer (Promega, Madison, WI, USA) was added to each well and mixed by shaking for 20 min at room temperature. Luciferase activity and cell viability were measured on the 96-well white plate (136,101; Thermo Fisher Scientific, Waltham, MA, USA) by using a luminometer (GloMax® Discover System; Promega). Luciferase assay reagent (Promega) and CellTiter-Glo® 3D reagent (Promega) were used for the measurement of luminescence and cell viability, respectively.
Time-lapse imaging
Time-lapse imaging was performed using an Operetta high-content imaging system (PerkinElmer, Waltham, MA) as described previously [27]. The cells were cultured in a flat-bottomed 24-well plate (Coaster 3526; Corning) to reach 70 − 80% confluence. The cells were treated with test compounds or Adriamycin prior to the time-lapse cell imaging. Images were captured at 10 min intervals for 24 h under a 5% CO2 atmosphere at 37 ℃.
Evaluation of Hsps expression by western blot analysis
HeLa cells (1.0 × 105) were seeded in 35-mm dishes and cultured. After 24 h of incubation, the cells were treated with KRIBB11 or 6 for 30 min. Then, cells were exposed to 42 °C for 1 h for Hsps induction and were recovered at 37 °C for 5 h. Western blot analysis was performed as described previously [10]. Briefly, cells were lysed with SDS sample buffer and boiled at 100 °C for 5 min. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, USA). Blots were incubated with Blocking One reagent (Nacalai Tesque, Kyoto, Japan) and sequentially incubated with appropriate primary and secondary antibodies. Chemiluminescence was detected with an LAS-4000 mini-image analysis system (Fujifilm, Tokyo, Japan) using Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA). Antibodies used in this study were as follows: mouse monoclonal anti-Hsp105 (1:1000–2000; clone B-7, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Hsp90 (1:2000; clone AC88, Enzo Life Sciences, Farmingdale, NY, USA), anti-Hsp70 (1:2000; clone C92F3A-5, Enzo Life Sciences) and rabbit monoclonal anti-a-tubulin (1:2000; clone DM1A, Sigma-Aldrich, St. Louis, MO, USA) antibodies. HRP-conjugated donkey anti-mouse (1:4000; 712-035-151, Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) and donkey anti-rabbit (1:4000; 712-035-152, Jackson Immuno Research Laboratories Inc.) IgG antibodies.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.21 software. Statistical analysis was conducted using a one-way analysis of variance (ANOVA) followed by a Dunnett’s test or a Tukey–Kramer test to analyze the differences between the treatment groups. The significance level used for statistical analysis with two-tailed testing was 5%.