Chemicals and reagents

KNK437 (purity > 98%) was purchased from Energy Chemical and compounds 1–20 were synthesized before [14]. All the compounds were dissolved in dimethyl sulfoxide (DMSO). Paraformaldehyde, NaCl, KOH, KCl, Mg(OAc)2 and glycerol were purchased from Sigma-Aldrich (Santa Clara, CA, USA); Hepes, DTT and IPTG were purchased from Solarbio (Beijing, China); CCK-8 kit was purchased from Topscience (Shanghai, China); protein lysis buffer and chemiluminescent plus reagents were obtained from Beyotime Biotechnology (Shanghai, China); transfection reagent Lipo3000 were purchased from Thermo Fisher Scientific (Waltham, MA, USA); Green fluorescent protein (GFP)-tagged lentivirus (LV-HSPH1) and negative control lentivirus (CON522) were purchased from Genechem (Shanghai, China). All other reagents and chemicals were obtained from available commercial sources.

Cell lines and culture

Human PAECs, PASMCs, HLF-1 and HCT116 cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). HPAECs were cultured in DMEM/F-12 (1:1) supplemented with 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin mixture (Gibco, Grand Island, NY, USA). HPASMCs were cultured in SMCM supplemented with 2% FBS (ScienCell, USA), 1% SMCGS (ScienCell, USA) and 1% penicillin/streptomycin mixture (ScienCell, USA). HLF-1 cells were cultured in DMEM/F-12 K (1:1) supplemented with 10% FBS (Gibco, Grand Island, NY, USA) and penicillin/streptomycin mixture (Gibco, Grand Island, NY, USA). HCT116 cells were cultured in DMEM supplemented with 10% FBS (Gibco, Grand Island, NY, USA) and penicillin/streptomycin mixture (Gibco, Grand Island, NY, USA).

Elisa assay

The plasma samples from 10 patients, initially diagnosed with idiopathic PAH, (2 males and 8 females) aged 20–58 years old with a median of 44 years, healthy individuals (3 males and 7 females) aged 25–50 years old with a median of 40 years. All the patients received proper treatment including surgery or targeted drug therapy in Xiangya Hospital after plasma collection. This study was approved by the Medical Ethics Committee of Xiangya Hospital of Central South University and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient. The concentration of Hsp110 in human plasma was measured using a Hsp110 ELISA kit (Jingmei Biotechnology Co. Ltd, Jiangsu, China) according to the manufacturer’s instructions as previously described [31].

Immunohistochemistry and histology staining

These staining measurements were performed according to previously report [31]. In short, rat lung were fixed with 4% paraformaldehyde and embedded in paraffin, which were cut into 5 μm sections. For immunohistochemisty staining, serial above sections were digested with 3% H2O2 for 20 min at room temperature, and then preincubated with 10% non-immunized serum. Sections were incubated with rabbit anti-Hsp110 antibody (1:100, Abcam, UK) overnight at 4 °C. The sections were incubated with biotinylated goat anti-rabbit secondary antibody (1:500, Santa Cruz) after washing the unbound antibodies, and then incubated with streptavidin-HRP. Subsequently, diaminobenzidine was used for color reaction to detect a positive signal according to routine procedure. Slides were photographed by a cell imaging multi-mode reader (Biotek Cytation5). Brown and yellow staining indicated positive results. For histology staining, the sections were stained with hematoxylin and eosin, and snapshots of histology were captured by the cell imaging multi-mode reader.

Immunofluorescence

The method was performed as previously described [32]. In brief, the lung tissue sections were put onto gelatin-coated glass slides and permeabilized with 0.2% Triton X-100 for 20 min and blocked with 2% bovine serum albumin for 30 min. Then the tissues were incubated with Hsp110 antibody (1:100, Abcam, UK), α-SMA antibody (1:500, CST, USA) and CD31 antibody (1:800, CST, USA) overnight at 4 °C. Subsequently, the sections were washed three times with 1×PBS and then incubated with fluorescentlabeled secondary antibodies for 2 h. After washing with 1×PBS three times, the sections were stained nuclear with DAPI for 3 min at room temperature. The slides were covered with gelvatol and the immunofluorescence was visualized using a cell imaging multi-mode reader (Biotek Cytation5).

Western blot analysis

The assays were conducted according to previously report [33]. Briefly, the concentrations of proteins extracted with RIPA buffer (Beyotime Biotechnology, China) including protease inhibitors and phosphatase inhibitors (Selleck, USA) were measured via the BCA Protein Assay Kit (KeyGEN, China). Protein samples were separated in SDS polyacrylamide gel and transferred to poly (vinylidene fluoride) membranes, which were blocked with 5% nonfat milk powder for 2 h at room temperature in Tris-buffered saline supplemented with 0.1% Tween 20. The membranes were incubated with primary antibodies overnight at 4 °C against the following proteins: anti-Hsp110 ( Abcam, UK); anti-Hsp70 (CST, USA); anti-Hsp90 (CST, USA); anti-STAT3 (CST, USA); anti-p-STAT3 (CST, USA); anti-c-Myc (CST, USA); anti-Cyclin D1(CST, USA); anti-PCNA (CST, USA); anti-β-actin (Proteintech, USA). Subsequently, the membranes were washed and incubated with secondary antibody for 2 h at room temperature. Finally, the bands were detected by a western fluorescent detection reagent (No. WBKLS0100, Millipore) and imaged within the ChemiDoc XRS + c imaging system (Bio-Rad).

Knockdown assay

The knockdown assay was performed as previously described [31, 32]. The siRNA targeting Hsp110 was purchased from Ribobio (Guangzhou, China). HPAECs were seeded in 6-well plates and cultured to ~ 30% confluency. For the transfection, diluting the siRNA (20 µM) to a final concentration of 50 nM with transfected medium (Opti-MEM, Gibco, Grand Island, NY, USA) and Lipo3000 (Invtrogen, USA), which was gently mixed and incubated for 15 min at room temperature. Then cell culture medium was removed, the mixture and DMEM/F-12 (1:1) supplemented with 10% FBS (Gibco, Grand Island, NY, USA) were added into wells and incubated for 48 h. Sequence of siRNA targeting Hsp110 was as follows:

5’- GCTAGAAGCTTTCTATTCT-3’Real-time quantitative PCR (RT-qPCR)

Total RNA was extracted from HPAECs using TRIZOL reagent (Invitrogen, Carlsbad, CA). Reverse transcription was performed according to the manufacturer’s protocol using the Takara RT kit (Takara, Dalian, China) with a reaction system containing 500 ng of total RNA in a volume of 10 µL. Real-time quantitative PCR (RT-qPCR) was performed using SYBR Premix EX TaqTM (TaKaRa). The expression level of Hsp110 was determined by RT-qPCR using GAPDH as a reference gene for the 2−∆∆Ct method. Primers for RT-qPCR are listed in Table S1.

Cell viability assay

For EdU assay, the method has been described before [34]. Briefly, HPAECs were plated into 96-well plates at a density appropriate about 4,000 cells/well. After cell adhesion, cells were treated with siRNA or compounds, and then cultured in incubators for 24 h at 37 °C. Then culture medium was replaced with fresh medium containing 10 µM EdU (Beyotime, China). After incubation for 3 h in incubators, the staining treatment was carried out on a cell imaging multi-mode reader (Biotek Cytation5). For CCK-8 assay, cells were seeded into 96-well plates at a density appropriate about 3,000 cells/well. After cell adhesion, HPAECs were treated with a range of concentrations of compounds for 48 h. Fresh CCK-8 (10 µL, 5 mg/mL, Biosharp) was added to each well and incubated at 37 °C for 2 h. The spectrophotometric absorbance of each well was measured at a wavelength of 450 nm. Three replicates were used for each treatment. The IC50 was calculated by GraphPad Prism 8 statistical software.

Cell migration assay

The methods to test HPAECs migration via cell scratch and transwell chamber (with a pore diameter of 8 μm) experiments were reported previously [33]. For scratch assay, cells were cultured in 6-well plates, in which 2 × 106 cells were seeded per well. After cell adhesion, straight scratches lines for each well were made using a sterile 200 µL pipette tip and washed with PBS three times. After 24 h under hypoxia (1% O2) treatment with siRNA or compounds in DMEM/F12 medium containing 1% FBS, pictures were captured from the same areas as those recorded at 0 h time points. Then the migration distances were analyzed by Image J software. For transwell assay, 1 × 104 HPAECs treated with siRNA or compounds were seeded into the upper chamber containing the culture medium with 1% FBS per well, and 600 µL of complete medium was added to the lower chamber. After incubation for 24 h at 37 °C with 1% O2 and 5% CO2, the cells that migrated to the lower membrane were fixed with 4% paraformaldehyde and stained with crystal violet (Solarbio, China). Quantification was performed by observing and photographing the stained cells using a cell imaging multi-mode reader (Biotek Cytation5).

Lentivirus construction and cell transfection

Green fluorescent protein (GFP)-tagged lentivirus (LV-Hsp110) and negative control lentivirus (CON522) were constructed and purified by Genechem (Shanghai, China). After 24 h, when the cell density was approximately 30%, the original medium was discarded and starved with serum-free medium for 12 h. Polybrene was diluted to 5 µg/mL with infection enhancer, then Hsp110 or blank lentivirus was added to make a lentiviral infection solution with MOI = 20 and mixed well. The plate was washed three times with PBS, then 1 mL of virus infection solution was added to each well, and after 12 h of infection, the plate was replaced with normal medium, and after 48 h, the transfection efficiency was observed by fluorescence. Finally, the samples were collected.

Protein expression and purification

The human Hsp110 proteins were expressed using the pET-28a plasmid in the Novagen’s Rosetta2(DE3)pLysS strain (MilliporeSigma) and purified as previously described with some modifications [35, 36]. Briefly, Luria-Bertani (LB) medium containing kanamycin (50 µg/mL) was used to inoculate the fresh transformants untill the O.D. 600 reached around 0.6–0.8 at 37 °C after transformation. Then, induction was performed by adding IPTG to medium with the final concentration of 0.5 mM. Cells were harvested after induction for 12 h or overnight at 16 °C. After centrifugation, the pellet was resuspended in lysis buffer (25 mM Hepes-KOH, pH 7.5, 300 mM NaCl, 10% glycerol, and 1 mM DTT). The expressed His6-SUMO-Hsp110 fusion proteins were first purified on a HisTrap column. After eluting with a linear gradient of imidazole in lysis buffer,the fractions including pure His6-SUMO-Hsp110 fusion protein were pooled. The His6-SUMO tag was cleaved by Ulp1 protease and removed by a second HisTrap column. Then Hsp110 was further purified on a HiTrap Q column and eluted with gradient concentrations of NaCl. The peak fractions was concentrated to more than 10 mg/mL in buffer containing 25 mM Hepes-KOH, pH 7.5, 50 mM KCl, and 1 mM DTT. Finally, the purified Hsp110 protein was flash frozen in liquid nitrogen and stored in -80 °C freezer.

Fluorescence polarization assay for determining ATP binding to Hsp110

The method was performed as previously described [35,36,37]. Briefly, a fluorescence polarization (FP) assay was performed to determine the binding affinity of Hsp110 for ATP using the fluorescence-labeled ATP (N6-(6-amino) hexyl-ATP-5-FAM, ATP-FAM, Jena Bioscience, Jena, Germany). For compounds screening, Hsp110 was diluted in the buffer (25 mM Hepes, 150 mM KCl, 10 mM Mg(OAc)2, 10% glycerol, and1 mM DTT) to 0.2 µM. Then, the mixture of Hsp110 proteins and each compound (final concentration: 100 µM) was incubated at the room temperature for 1 h. The ATP-FAM was added to the mixture at a final concentration of 20 nM, then the reactions were incubated for another 1 h at the room temperature, allowing ATP-FAM to bind to Hsp110 efficiently. The fluorescence polarization measurements were performed on a cell imaging multi-mode reader (Biotek Cytation5), and the reaction with 1% DMSO was used as a positive control. In order to test the IC50 of compound 6 against Hsp110, serial dilutions of 6 were incubated with Hsp110 for 1 h at the room temperature before ATP-FAM adding. Inhibition rates of each compound were measured using GraphPad Prism 8 software.

Anti-aggregation activity of Hsp110

Hsp110 preventing firefly luciferase (Sigma-Aldrich, SRE0045) aggregation assay was carried out as previously described with minor modifications [35,36,37,38]. Before performing the assay, purified human Hsp110 was first treated with compounds at the room temperature for 2 h in buffer (25 mM Hepes-KOH, pH 7.5, 50 mM KCl, 5 mM Mg(OAc)2, 2 mM ATP, and 2 mM DTT). Then, the compounds-treated Hsp110 was mixed with luciferase in buffer, and the final concentrations of luciferase, Hsp110 and compounds were 2 µM, 1.5 µM and 100 µM, respectively. The mixture was plated 100 µL into per well in the 96-well plate, then cultured in incubators at 42 °C, and UV absorbance at 320 nm was monitored over time. Compounds were added in reaction buffer to achieve the indicated concentrations, with a held final concentration of 1% DMSO. Luciferase alone was used as a negative control, and luciferase with the addition of Hsp110 as a positive control. Anti-aggregation activities of Hsp110 and the UV absorbance at 320 nm were measured using GraphPad Prism 8 software.

Surface plasmon resonance (SPR) analysis

This assay was performed as our previously report [39]. In brief, the binding of compound 6 or KNK437 to the Hsp110 protein was monitored with the OpenSPRTM system. According to the OpenSPRTM standard operating procedures, installing acid-coupling chip, running PBS (pH 7.4) at a maximum flow rate of 150 µL/min, loading 200 µL of 80% isopropanol (IPA) to remove bubbles, washing with buffer to evacuate air. Before reaching the baseline, binding studies were performed at a constant flow rate of 20 µL/min. Subsequently, 200 µL solution of EDC/NHS was loaded to the sample loop, then washed with buffer and evacuated with air. Injecting Diluted Hsp110 with buffer and run for 4 min, followed by injection of 200 µL of blocking solution. Then washing the sample loop with buffer to evacuate air. SPR binding data were obtained througth an appropriate gradient dilution series of compounds, and the injections were consecutively performed for 120–140 s association and 160 s dissociation, respectively. Finally, the affinity parameters were evaluated in a steady-state one-to-one analysis model using the TraceDrawer (Ridgeview Instruments ab, Sweden).

Drug Affinity Responsive Target Stability (DARTS) analysis

Given the observation that some ligands can protect the target protein from degradation by proteases, DARTS assay has well been used to detect the interaction between a ligand and a protein when mixed in a solution [40, 41]. In short, about 2 × 107 HPAECs were grown to 90% confluency in 10 cm cell culture dishes and lysed with M-PER lysis buffer (Thermo Fisher, USA) containing protease and phosphatase inhibitor (Selleck, USA) for 10 min at 4 °C. After centrifugation, the cell lysates were removed to new tubes and assigned to different groups, which were added with 10 × TNC buffer to afford 1 × TNC buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM CaCl2). The mixture was treated with compound 6 (10 µM) and 1% DMSO respectively for 2 h at room temperature. Then each sample was treated with different concentrations of using pronase (Roche, Switzerland) for 30 min at 37 °C. The proteolysis was terminated by adding 5 × loading buffer and denaured for 10 min at 100 °C. Finally, the target protein Hsp110 was investigated by western blotting analysis.

Co-immunoprecipitation (co-IP) assay

This assay was conducted as follows: Firstly, HPAECs and HCT116 cells were seeded into 10 cm cell culture dishes and incubated for 12 h to adherence at 37 °C with 5% CO2. Then, cells were treated with compound 6 (10 µM) and KNK437 (20 µM) for 48 h, respectively. Cells were lysed with IP buffer (Beyotime Biotechnology, China) at 4 °C for 15 min in the tube. The lysate was centrifuged at 4 °C in a rate of 12,000 rpm for 20 min, and the supernatants were taken into the corresbonding new tubes. The concentration of collected protein samples was measured through BCA method. The collected protein samples were incubated with mouse STAT3 antibody overnight at 4 °C before adding protein A agarose beads. After incubation 3 h at 4 °C, tubes were centrifuged at 4 °C in a rate of 8000 rpm for 2 min. The pellets were washed with PBS two times and denatured with loading buffer. Finally, the precipitated proteins were subjected to western blotting analysis with the indicated antibodies, including rabbit anti-Hsp110 (Abcam, UK); mouse anti-STAT3 (CST, USA); rabbit anti-β-actin (Proteintech, USA).

Animal experiment

The specific scheme of animal experiment was carried out according to our previously reports [14, 33, 34]. Briefly, healthy male Sprague-Dawley (SD) rats (weighing 160–180 g) were purchased from Hunan SJA Laboratory Animal Co. Ltd. (NO: SYXK (Xiang) 2015-0017). All the experiments were approved by Hunan Normal University Experimental Animal Welfare Ethics Committee and Animal Management Committee (IRB approval number : D2021041). All rats were acclimated at 20–25 °C with 50-60% humidity for 7 days and then were randomly divided into 4 groups (n = 7 per group): (i) control group, rats were exposed to normobaric normoxia (21% O2) continuously for 4 weeks (ii) hypoxia group, rats were exposed to 10% O2 continuously for 4 weeks (iii) hypoxia plus KNK437 (15 mg/kg) treatment (3 weeks) group, and (iv) hypoxia plus compound 6 (15 mg/kg) treatment (3 weeks) group.

After PAH modeling, rats were anesthetized with 1% sodium pentobarbital (50 mg/kg, i.p.). Then, the mPAP was measured using a BL-420 F biological signal acquisition and analysis system (TECHMAN, Chengdu, China). A single lumen PE50 tubing (0.5 mm inner diameter, 0.9 mm outer diameter, SP0109, AD Instruments, Australia) filled with heparinized saline was inserted into the right external jugular vein, and the other end was connected to the measurement system. The mPAP trace curve was used to confirm the catheter position and representative shapes of the pressure was electronically measured. After that, the right ventricle (RV) and left ventricle (LV) plus interventricular septum (S) were dissected from the heart, which were weighed to evaluate RVH index through calculating the ratio of RV/(LV + S). Following hemodynamic measurements, lung tissues were fixed with paraffin and then stained with HE to measure morphology. The slices were examined under a cell imaging multi-mode reader (Biotek Cytation5) to visualize the morphology of pulmonary arterioles (diameter between 50 and 150 μm). The relative PAMT (%, distance between outer and inner elastic lamina) was calculated by 100 × PAMT/external diameter.

Statistical analysis

All statistical tests were performed using GraphPad Prism 8.0 software (San Diego, CA, USA), and representative data were selected to create the figures. Statistical significance was determined either by the two-tailed Student’s t test (for two groups) or a one-way analysis of variance (ANOVA) (for three or more groups). A significant difference was assumed at the P < 0.05 and error bars represent SD of three experiments unless stated otherwise.