X66, a novel N-terminal heat shock protein 90 inhibitor, exerts antitumor effects without induction of heat shock response

Heat shock protein 90 (HSP90) is essential for cancer cells to assist the function of various oncoproteins, and it has been recognized as a promising target in cancer therapy. Although the HSP90 inhibitors in clinical trials have shown encouraging clinical efficacy, these agents induce heat shock response (HSR), which undermines their therapeutic effects. In this report, we detailed the pharmacologic properties of 4-(2-((1H-indol-3-yl)methylene)hydrazinyl)-N-(4-bromophenyl)-6-(3,5- dimethyl-1H -pyrazol-1-yl)-1,3,5-triazin-2-amine (X66), a novel and potent HSP90 inhibitor. X66 binds to the N-terminal domain in a different manner from the classic HSP90 inhibitors. Cellular study showed that X66 depleted HSP90 client proteins, resulted in cell cycle arrest and apoptosis, and inhibition of proliferation in cancer cell lines. X66 did not activate heat shock factor-1 (HSF-1) or stimulate transcription of HSPs. Moreover, the combination of X66 with HSP90 and proteasome inhibitors yielded synergistic cytotoxicity which was involved in X66-mediated abrogation of HSR through inhibition of HSF-1 activity. The intraperitoneal administration of X66 alone depleted client protein and inhibited tumor growth, and led to enhanced activity when combined with celastrol as compared to either agent alone in BT-474 xenograft model. Collectively, the HSP90 inhibitory action and the potent antitumor activity, with the anti-HSR action, promise X66 a novel HSP90-targeted agent, which merits further research and development.


Synthesis and characterization of X66 and Biotin-X66
General: All starting materials and solvents were purchased from commercial suppliers and used without further purification. Reactions were monitored by thin layer chromatography (TLC) of silica gel (HSGF254) at 254 nm wavelength. The LRMS and HRMS were recorded on Finnigan LCQ/DECA and Micromass Ultra Q-TOF (ESI) spectrometer, respectively.
The 1 H and 13 C spectra were taken on Bruker Avance -400 and 500 NMR spectrometer operating at 400 MHz for 1 H and 126 MHz for 13 CNMR, respectively, using TMS as the internal standard and CDCl 3 or DMSO-d 6 as the solvent. Chemical shifts are given in δ values of ppm. The abbreviations s is singlet, d is doublet, t is triplet and m is multiplet. Coupling constants (J) were measured in hertz (Hz). 1 H and 13 C spectrums of X-66 and Biotin-X66 exhibited special signals while using CDCl 3 or DMSO-d 6 as solvent, which may be due to the structural resonance. The special signals are indicated by major or minor.

Plasmids construction
The His-HSP90α expression vector (pET28a-HSP90α) was constructed by first amplifying the fulllength human HSP90 cDNA from pUC-HSP90α by PCR, and subcloned into pET28a using SalI and NotI. Subsequently, the regions coding of amino acids 1-236 and 629-732 which correspond to the N-terminal domain and C-terminal domain of the full-length protein, respectively, was amplified by PCR, and subcloned back into pET28a using EcoRI and XhoI to produce pET28a-HSP90NT and pET28a-HSP90CT.

Protein purification
The BL21-CondonPlus (DE3)-RIL-X strain was used as the host strain for expression of the full-length human HSP90α, fragment of N-terminal domain and fragment of C-terminal domain. An overnight culture of E.coli carrying HSP90α, N-terminal fragment, C-terminal fragment or yeast HSP90 was used to inoculate LB containing 50 μg/ml kanamycin or 100 μg/ml ampicillin. The culture was grown to an OD 600 of 0.8, induced with 1mM IPTG and grown for 3 h before collecting by centrifugation. Cells were re-suspended in buffer A (20 mM HEPES-HCl pH 8.0, 0.5 M NaCl), 8 mM imidazole and 15% glycerol. Cells were disrupted by sonication in the presence of protease inhibitors (Roche life science, Indianapolis, IN, USA) and the lysate spun at 18000 × g at 4°C for 60 min. The supernatant was loaded onto a NI-NTA column (QIAGEN) which was equilibrated with buffer A containing 8 mM imidazole. The bound His-tagged protein was subsequently eluted with buffer A containing 100-300 mM imidazole, and concentrated using Amicon Ultra-15 centrifugal filter (Millipore). The concentrated protein was diluted with buffer B (20 mM HEPES-HCl pH 7.4) and loaded onto Q-sepharose column (GE healthcare). A linear gradient of NaCl (0-1 M) in buffer B was applied to the column for protein elution. The fraction was dialyzed in 20 mM HEPES-HCl pH 7.4 containing 150 mM NaCl and concentrated using Amicon Ultra-15 centrifugal filter. All proteins were store at -80°C.

Cell cycle analysis
Cells were collected, washed with PBS twice, and fixed with 70% ethanol overnight at -20°C. After RNaseA incubation and propidium iodide staining, cells were analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA, US) with CellQuest and ModiFit LT3.0 software.

LC-MS/MS-based bioanalytical assays
Validated bioanalytical methods were used to measure test compounds in plasma and tumor tissue using a API4000 instrument (Applied Biosystems, Foster, CA, USA) interfaced via an electrospray ionization probe with a liquid chromatography (Agilent Technologies, Waldbronn, Germany). Chromatographic separations for measurement of X66 were achieved on a Phenomenex Gemini 5-μm C 18 column (50 × 2.0 mm, Torrance, CA, USA). Mobile phases for measurement of X66 were acetonitrile (containing 0.1% formic acid; solvent A) and water (containing 0.1% formic acid and 5 mM ammonium acetate; solvent B). A binary escalation gradient elusion was performed, which consisted of three isocratic segments, i.e., 0 to 0.5 min at 70% B, 0.9 to 1.9 min at 10% B, and 2 to 3.5 min at 70% B. MS/MS was performed in the positive ion mode using the precursor-to-product ion pairs m/z 502.8→407.1 and 502.8→386.4 for X66 in plasma and tumor tissue, respectively, and m/z 324 →127 for the internal standard (IS) gliclazide in both plasma and tumor tissue.
The LC effluent flow was introduced into the electrospray ionization probe at a flow rate of 0.5 ml·min −1 . To a 25 μl aliquot of plasma sample, 25 μl of internal standard (50.0 ng/ml, gliclazide), and 100 μl of acetonitrile were added. To a 80 μl aliquot of tumor tissue sample, 20 μl of IS, and 100 μl of acetonitrile were added. After votex-mixing for 1 min and centrifugation at 14000 rpm for 5 min, the supernatant (20 μl for plasma and 10μl for tumor tissue, respectively) was applied to LC-MS/MS-based analysis. Matrix-matched calibration curves were constructed for the X66 using weighted (1/X) linear regressions of the compound response (peak area; Y) against the corresponding nominal concentrations.

Molecular docking
The X-ray crystal structure of the N-terminus domain of HSP90 (PDB code: 2YK9) [1] was retrieved from the Protein Data Bank. Molecular docking was performed in standard procedure by Glide v5.6 in its SP mode [2,3]. LigPrep v2.4 was utilized to pre-process the compound using default parameters.