Complex Crystal Structure Determination and in vitro Anti–non–small Cell Lung Cancer Activity of Hsp90N Inhibitor SNX-2112

SNX-2112, as a promising anticancer lead compound targeting heat shock protein 90 (Hsp90), absence of complex crystal structure of Hsp90N-SNX-2112 hindered further structural optimization and understanding on molecular interaction mechanism. Herein, a high-resolution complex crystal structure of Hsp90N-SNX-2112 was successfully determined by X-ray diffraction, resolution limit, 2.14 Å, PDB ID 6LTK, and their molecular interaction was analyzed in detail, which suggested that SNX-2112 was well accommodated in the ATP-binding pocket to disable molecular chaperone activity of Hsp90, therefore exhibiting favorable inhibiting activity on three non–small cell lung cancer (NSCLC) cell lines (IC50, 0.50 ± 0.01 μM for A549, 1.14 ± 1.11 μM for H1299, 2.36 ± 0.82 μM for H1975) by inhibited proliferation, induced cell cycle arrest, and aggravated cell apoptosis. SNX-2112 exhibited high affinity and beneficial thermodynamic changes during the binding process with its target Hsp90N confirmed by thermal shift assay (TSA, ΔTm, and −9.51 ± 1.00°C) and isothermal titration calorimetry (Kd, 14.10 ± 1.60 nM). Based on the complex crystal structure and molecular interaction analysis, 32 novel SNX-2112 derivatives were designed, and 25 new ones displayed increased binding force with the target Hsp90N verified by molecular docking evaluation. The results would provide new references and guides for anti-NSCLC new drug development based on the lead compound SNX-2112.


INTRODUCTION
Lung cancer, as the leading cause of cancer death worldwide, accounts for 18% of total cancer deaths each year (Petrek and Yu, 2019). According to histological types and therapeutic aims, lung cancer can be traditionally classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC); NSCLC accounts for 80-85% (Ettinger et al., 2017). NSCLC is increasingly thought as an oncogene addicted tumor (Molina et al., 2008); therefore, targeted drugs may be an effective way for NSCLC treatment (Tomasini et al., 2016;Xiong et al., 2019).
Heat shock protein 90 (Hsp90), as an evolutionarily conserved chaperone in eukaryotes, plays a vital role in cell differentiation, proliferation, and apoptosis by assisting more than 300 client protein proper folding or conformational maturation (Li et al., 2012a;Vahid et al., 2017). Among the client proteins, 48 proteins play an important, even decisive role in tumorigenesis and development, such as transmembrane tyrosine kinase, steroid receptors, and cell cycle regulators, etc. (Jhaveri et al., 2014;Hyun et al., 2018). Hsp90 N inhibitors lead to multiple misfolded or immature client proteins to be degraded by the ubiquitin protease complex pathway (Chen et al., 2014), which involves simultaneous intervention of multiple potential antitumor target proteins and a "multipoint attack" on the tumor to achieve favorable therapeutic efficacy (Chehab et al., 2015;Dutta Gupta et al., 2019). Hsp90 inhibitor development, therefore, has become a promising strategy in cancer therapy (Barrott and Haystead, 2013;Soga et al., 2013;Hu et al., 2019).
So far, there is rare report concerning anti-NSCLC activity of SNX-2112 (Hendriks and Dingemans, 2017). Our preliminary experimental results indicated that SNX-2112 exhibited favorable inhibition activity on NSCLC cell lines (A549, H1299, and H1975). However, absence of the complex crystal structure of Hsp90 N -SNX-2112 hampered further structural optimization of SNX-2112 and understanding of binding mode and molecular interaction of Hsp90 N -SNX-2112. Herein, the complex crystal structure of Hsp90 N -SNX-2112 was determined by X-ray diffraction (XRD); molecular interaction analysis based on complex crystal structure, thermal shift assay (TSA), and isothermal titration calorimetry (ITC) was performed. Meanwhile, in vitro anti-NSCLC activity of SNX-2112 was evaluated, which would provide new references for anti-NSCLC new drug development based on the lead compound SNX-2112.

Materials and Reagents
A549, H1299, and H1975 NSCLC cell lines were obtained from the AOLUKEJI (Shanghai, China). Fetal bovine serum and RPMI 1640 medium were purchased from Gibco-BRL (Gaithersburg, MD, United States).

Cell cycle assay
Non-small cell lung cancer cell lines A549, H1299, and H1975 were seeded in a 6-well culture plate at density 3 × 10 5 cells per well, which were treated with different concentrations of SNX-2112 or DMSO for 72 h (4 × 0.50 µM for A549, 4 × 1.14 µM for H1299, and 4 × 2.36 µM for H1975). Cells were then gathered and washed with cold phosphatebuffered saline (PBS). And then at 4 • C, 1 mL PBS and 2 mL 100% ethanol were was added. After centrifugation at 1,000 g for 5 min, they were washed with 2 mL PBS and resuspended with 400 µL PBS. In the end, 50 µL propidium iodide (PI) and 50 µL RNase were added, and cells were darkly cultivated for 30 min at 37 • C. The effects of SNX-2112 on cell cycle distribution were analyzed with flow cytometry (Becton Dickinson, Franklin Lakes, NJ, United States; Olszewska et al., 2020) Cell apoptosis assay Based on instructions from the manufacturer, the apoptosis rate was detected by the annexin V-fluorescein isothiocyanate (FITC)/PI Apoptosis Detection kit (556547, Becton Dickinson, United States). Cells were treated identically with mentioned as Cell Cycle Analysis. And then, cells were gathered and washed with 2 mL PBS and resuspended in 100 µL binding buffer. Lastly, 5 µL annexin V-FITC and 10 µL PI were added to the buffer and darkly cultivated at 4 • C for 30 min. Effects of SNX-2112 on cell apoptosis were determined by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, United States; Ye et al., 2017).

Molecular Interaction Analysis Thermal shift assay
Thermal shift assay was applied for evaluating molecular interaction between target Hsp90 N and its ligand SNX-2112 using real-time PCR (7500, ABI Corp., United States). A 20-µL reaction system consisted of Hsp90 N (1 mg/mL) 2 µL, ligand SNX-2112 (dissolved in DMSO, 100 mM) 0.5 µL, buffer (pH 7.5, 20 mM Tris-HCl, 150 mM NaCl, and 10% glycerol) 10 µL, Protein Thermal Shift Buffer (Applied Biosystems, United States) 5 µL, and TSA dye (Applied Biosystems, United States) 2.5 µL, eight replicates. The samples were centrifuged at 1,000 revolutions/min (rpm) for 1 min. The samples were run from 25 to 95 • C with a ramp rate of 1 • C/min. The protein unfolds when it is heated and exposes hydrophobic regions to bind the environmentally sensitive TSA dye and fluoresces. The melting temperature (Tm) and melting temperature differences ( Tm) were derived from the melting curve, which was related to the binding affinity of Hsp90 N -SNX-2112 (Andreotti et al., 2015).

ITC
Isothermal titration calorimetry was used for further detecting Hsp90 N -SNX-2112 molecular interactions using ITC (ITC-200, Malvern Instrument Ltd., United Kingdom). SNX-2112 in DMSO was diluted with buffer for protein purification (pH 7.5, 20 mM Tris-HCl and 150 mM NaCl) to 500 µM. Fresh-purified Hsp90 was extensively dialyzed against the same buffer and concentrated to 50 µM. After being centrifuged and degassed, 2 µL aliquots of SNX-2112 were injected into Hsp90 N solution in the cell with an interval of 200 s and 750-rpm stirring speed. With Microcal Origin software, the experimental data were fitted with a bimolecular binding model with stoichiometry (n), enthalpy ( H • ), and association constant (K a ) as adjustable parameters. The thermodynamic parameters G • (free energy) and S • (entropy) were derived from the equation -RT ln K a = G • = H • -T S (He et al., 2014b).

Complex Crystal Structure Determination
Homo sapiens Hsp90 N gene containing residues 9 to 236 was artificially synthesized and cloned into plasmid pET-28a, which was transformed into Escherichia coli BL21 (DE3; TIANGEN Biotech Corp., Beijing, China) to express and purify Hsp90 N as reported by Cao (Cao et al., 2017).
With 5:1 molar ratio, SNX-2112 was added into Hsp90 N to incubate for 30 min at 4 • C. Then, the mixture was centrifuged for 10 min at 3,000 g, and the supernatant was taken to mix with the same amount of a crystal precipitant [pH 8.5, 100 mM Tris-HCl, 200 mM MgCl 2 , 25-30% PEG4000 (Stebbins et al., 1997)]. With the hanging drop vapor diffusion method, cocrystallization was carried out at 4 • C for 3-7 days in an incubator controlled by a bath circulator (PolyScience 9712, PolyScience, United States). Complex crystal image of Hsp90 N -SNX-2112 was captured by a stereomicroscope (M165, Leica Microsystems, Germany).
Complex crystals were mounted with cryo-loop (Hampton Research Corp., Aliso Viejo, CA, United States), and then quickly soaked in the cryoprotectant solution containing 20-25% glycerol and crystal reagent mentioned previously (Prodromou et al., 2000). Then, crystals were flash-frozen in liquid nitrogen for XRD. With an ADSC Quantum 315r CCD detector (Wang et al., 2015), all data sets were collected at 100 K on Macromolecular Crystallography Beamline17U1 (BL17U1) at Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China; Wang et al., 2018).
Aquarium pipeline was applied for automatic processing of diffraction data (Yu et al., 2019b). Using the crystal structure of Hsp90 N -FS23 (PDB ID 5CF0) as the research model (Li et al., 2015), the complex crystal structure was determined and refined by molecular replacement with PHENIX software (Adams et al., 2010;Yu et al., 2019a). The initial model was rebuilt by Coot software (Emsley et al., 2010), and CCP4MG software was applied for graphing and molecular interaction analysis (McNicholas et al., 2011;Winn et al., 2011).

Design of New SNX-2112 Derivatives and Molecular Docking Evaluation
A series of new SNX-2112 derivatives were designed based on the complex crystal structure and molecular interaction analysis, which were evaluated by molecular docking with software SYBYL-X 2.0 (Tripos Associates Inc., St. Louis, MO, United States). The crystal structure of Hsp90 N -FS23 (PDB ID 5CF0) was as the docking model. First, a compound library of newly designed SNX-2112 derivatives was built, and new derivatives were conducted with geometric and force field optimization for 10,000-100,000 times. Second, the target Hsp90 N was optimized by adding hydrogen and charges. Last, molecular docking was carried out using the Surflex-Dock module of SYBYL software. Total score and Cscore were chosen as parameters to evaluate the binding affinity between the target Hsp90 and new ligands. CCP4MG software was applied for simulated complex threedimensional (3D) structure reconstruction and molecular interaction analysis.

Statistical Analysis
Statistical analysis was conducted with SPSS 13.0 software (International Business Machines Corporation, United States) and GraphPad Prism 5.01 software (GraphPad Software, San Diego, United States). Data were presented as mean ± standard deviation (SD). Differences between two groups were performed according to unpaired Student t test. P < 0.05 was deemed as statistically significant.

Molecular Interaction Analysis
TSA Usually, every protein has a specific Tm value under certain conditions, which would change when binding a ligand. Greater Tm means higher affinity, and absolute Tm value greater than three is deemed as a favorable ligand (Lo et al., 2004;Andreotti et al., 2015). As can be seen from Figure 4, Tm value of Hsp90 N was 48.36 ± 0.82 • C (Figure 4A), and Tm was shifted to 38.84 ± 1.35 • C after Hsp90 N combining with SNX-2112 ( Figure 4B). As a result, Tm value was -9.51 ± 1.00 • C ( * * * p < 0.001 vs. control, Hsp90 N , n = 8; Figure 4C). It suggested that an intense binding force existed between the ligand SNX-2112 and its target Hsp90 N . Figure 4D, the upper panel manifested the raw titration data. The power was supplied to the system to maintain a constant temperature against time, and the area of each peak meant interaction heat for injection. The lower panel exhibited bimolecular fit of normalized interaction heats plotted against molar concentration. Hsp90 N binding with SNX-2112 was exothermic at 25 • C from the negative peaks in the raw ITC titration data. A fine S-shaped curve was obtained after nonlinear data fitting. The stoichiometry was determined to be 0.83 ± 0.01, indicating a 1:1 binding mode between target Hsp90 N and ligand SNX-2112. As shown in Table 1, the K d values here were 14.10 ± 1.60 nM, which indicated an intense binding force between the ligand SNX-2112 and its target Hsp90 N . The thermodynamic signature was dominated by both favorable enthalpy change ( H a , −6.80 ± 0.20 kcal/mol) and entropy change (T S a , 3.90 kcal/mol), indicating the establishment of favorable interactions during the Hsp90 N -SNX-2112 binding process (He et al., 2011(He et al., , 2014a.

Complex Crystal Structure Determination
With a molecular weight of 25 kDa, Hsp90 N was expressed in E. coli BL21 (DE3) strain. Purified Hsp90 N protein was obtained by immobilized Ni ++ affinity chromatography and molecular sieve chromatography. Complex crystals of Hsp90 N -SNX-2112 were obtained by the hanging-drop method at 4 • C for 3-5 days. As shown in Supplementary Image 1, the average dimension of rhombus crystals was approximately 230 µm × 130 µm × 50 µ m. Crystal structure of Hsp90 N -FS23 (PDB ID 5CF0) was reported as the research model; a complex crystal structure of Hsp90 N -SNX-2112 was successfully determined by molecular replacement. The complete data collection and refinement statistics are shown in Table 2. The coordinates and structure factors have been deposited in PDB (PDB ID 6LTK). Diffraction data were collected up to 2.14 Å resolution limit with R free = 0.24 and R work = 0.21. The space groups showed I222 (unit cell parameter, a = 69.70 Å, b = 88.87 Å, c = 96.55 Å; α = β = γ = 90.00 • ).
As shown in Figure 5, the asymmetric unit of the refined model included a protein monomer (residues 17-224) and 47 water molecules, as well as one SNX-2112 molecule in the ATPbinding pocket. No electron density was observed for residues 9-16 and residues 225-236 of N-and C-terminal protein, which was attributed to disordered electron density. The refinement results showed that continuous and strong electron density appears in the empty space of ATP-binding site of Hsp90 N with a shape to well accommodate SNX-2112 molecule. SNX-2112 was perfectly sitting at the ATP-binding pocket to disable molecular chaperone activity of Hsp90 by blocking ATP binding, therefore suppressing cancer cells.
As shown in Figure 5B, various interactions, including hydrogen bonds, π-π interactions, and hydrophobic interactions, contributed to the intense binding force between the ligand SNX-2112 and its target Hsp90 N . SNX-2112 formed three hydrogen bonds with residue Y139 (2.7 Å), residue D93 (2.9 Å), and residue K58 (3.0 Å) of Hsp90 N . Meanwhile, SNX-2112 also arranged a network of water-mediated hydrogen bonds with residues S52, D93, and T184. The special feature of SNX-2112 contained three fluorine atoms, and one of them formed a halogen bond (2.7 Å) with the carbonyl group on the main chain of residue G135. In addition, the side chain of residue F138 faced a parallel plan with the pyrazol ring of SNX-2112, for which π-π interaction contributed to their binding. SNX-2112 formed multiple hydrophobic interactions with residues M98, L103, L107, F138, Y139, W162, N51, A55, D93, and T184 of Hsp90 N , which was responsible for the intense binding force between the ligand SNX-2112 and its target Hsp90 N .

New SNX-2112 Derivatives Design
Based on the complex crystal structure and molecular interaction analysis, 32 new SNX-2112 derivatives were designed, as shown in Figure 6A. It was important to maintain the special molecular configuration to achieve further structural optimization of SNX-2112. Hydrogen bonds were favorable binding force, and thus, hydrophilic groups were adopted for structural modification of SNX-2112. For instance, the hydroxy group on cyclohexanol moiety was varied to an amino group or sulfhydryl group. The amino group on benzamide moiety was replaced by a hydroxy group. The carbonyl group of 6,6-dimethyl-1,5,6,7tetrahydro-indazol-4-one moiety was substituted by a nitro group. Hydrophobic interactions also contributed more, and introduction of hydrophobic functional groups would be a suitable scheme for structural modification. To be specific, the trifluoromethyl part was modified to a dimethylamino or tertbutyl group. Considering the π-π interactions formed, the 1 Hpyrazole ring of SNX-2112 was replaced by a 1 H-pyrrole ring.

Molecular Docking Evaluation
Based on the design scheme, 32 new SNX-2112 derivatives were designed, and the binding capacity with the target Hsp90 N was evaluated by molecular docking from theoretical calculation aspect using the software SYBYL-X 2.0. Total score and CScore were chosen as key evaluation parameters, which were positively related to their binding force. Their chemical structures, docking results, and total score increment compared with SNX-2112 are shown in Supplementary Table 1. Among them, a total score of 25 new derivatives increased compared with SNX-2112, which suggested a favorable design scheme. The top 10 new derivatives with high total score increment compared with SNX-2112 and chemical structures were shown in Figure 6B. The total score of the optimal one S15 was as high as 9.5 (increment, 3.5 vs. SNX-2112), which suggested that there was an intense banding force between S15 and its target Hsp90 N , and S15 was a favorable ligand for target Hsp90 N . There were obvious differences in structures between S15 and SNX-2112. First, compared to SNX-2112, the hydroxy group on cyclohexanol moiety of SNX-2112 was varied to an amino group in S15. Second, the carbonyl group of 6,6dimethyl-1,5,6,7-tetrahydro-indazol-4-one moiety of SNX-2112 was substituted by a nitro group in S15. Last, the trifluoromethyl part of SNX-2112 was modified to a dimethylamino group in S15. The changes in the structure improved the Hsp90 N -S15 binding affinity.
In the optimal derivative S15 as a representative (Figure 6F), simulated complex 3D structure reconstruction and molecular interaction analysis of Hsp90 N -S15 were performed by SYBYL software and CCP4MG software, as shown in Figures 6C-E. S15 was suitably located in the ATP-binding pocket of Hsp90 N (Figures 6C,E). Hydrogen bonds were responsible for their intense binding force. S15 formed three hydrogen bonds with residues N51 (2.9 Å), F138 (2.7 Å), and T184 (3.0 Å) of Hsp90 N . Moreover, the hydrophobic effects apparently also played a vital role for binding of S15 to Hsp90 N . Specifically, the hydrophobic interactions appeared between the 4,5,6,7-tetrahydro-1 H-indazole moiety and residues L107, G135, and Y139 of Hsp90 N . In addition, the benzamide moiety together with cyclohexylamine moiety formed the hydrophobic interactions with residues A55, K58, and M98 ( Figure 6D).
In short, hydrogen bonds and hydrophobic interactions of Hsp90 N -S15 were responsible for their intense binding force, which would provide a potential lead compound for anti-NSCLC new drug development.

DISCUSSION
Non-small cell lung cancer accounts for more than 80% of lung cancer cases, which is the leading cause of cancer death worldwide (Petrek and Yu, 2019). Targeted drugs, for NSCLC as an oncogene addicted tumor, could be an effective way of treatment. However, drug resistance and toxicity confined their clinical applications (Tomasini et al., 2016;Xiong et al., 2019). Hsp90, as a chaperone, plays a vital and even decisive role in tumorigenesis and development by assisting 300 client proteins to obtain their correct folding and mature conformation (Jhaveri et al., 2014;Hyun et al., 2018). Hsp90 exists with an α-α or ββ homodimer in the cytoplasm, and each monomer consists of three domains. The C-terminal domain (Hsp90 C ) plays a key role for dimerization of Hsp90, whereas the central domain (Hsp90 M ) Top 10 optimal new SNX-2112 derivatives with high total score increment evaluated by molecular docking using software SYBYL-X 2.0. (C) Overall fold of complex crystal of Hsp90 N -S15. Hsp90 N and S15 are shown as cartoon and stick, respectively. (D) Molecular interaction between Hsp90 N and S15. The carbon, nitrogen, and oxygen atoms of S15 are colored in green, blue, and red, respectively. In addition, the carbon, nitrogen, oxygen, and sulfur atoms of Hsp90 N residues are shown in cyan, blue, red, and yellow, respectively. The hydrogen bonds are represented as red dashed lines. (E) Connolly surface of ATP-binding site of S15 in Hsp90 N . (F) Molecular structure of S15.
possesses a large hydrophobic surface to bind client proteins and facilitate their folding. The N-terminal domain (Hsp90 N ), with ATP-hydrolase activity, contains an ATP-binding pocket that is responsible for ATP binding and hydrolysis to provide energy for client protein correct folding or conformation maturation, which is indispensable for the chaperone function of Hsp90 (Donnelly and Blagg, 2008;Mayer et al., 2012).
Accordingly, Hsp90 inhibitors were divided as Hsp90 N inhibitors, Hsp90 M inhibitors and Hsp90 C inhibitors. Hsp90 N inhibitors could bind in the ATP-binding pocket to prevent ATP binding and then disable chaperone function of Hsp90 (Prodromou et al., 2000;Li et al., 2012b). Hsp90 N inhibitor SNX-2112 was perfectly sitting at the ATP-binding pocket of Hsp90 N , which prevented ATP binding to disable chaperone function of Hsp90. Therefore, immature or misfolding client proteins would be captured and degraded by proteasomes (Chen et al., 2014). With degraded proteins accumulated, multiple signaling pathways can be derailed simultaneously, which can hinder proliferation, and progression of cancer (Chehab et al., 2015;Dutta Gupta et al., 2019). Hsp90, therefore, was paid more attention to as a promising anticancer drug target (Barrott and Haystead, 2013;Soga et al., 2013;Hu et al., 2019).
Absence of the complex crystal structure of Hsp90 N -SNX-2112, however, prevented further structural optimization of SNX-2112 and understanding of binding mode and molecular interaction of Hsp90 N -SNX-2112. A high-resolution complex crystal structure of Hsp90 N -SNX-2112 was successfully determined by XRD (resolution limit, 2.10 Å, PDB ID 6LTK), and their molecular interaction was analyzed in detail. The complex crystal structure suggested that SNX-2112 was well accommodated in the ATP-binding pocket to disable molecular chaperone activity of Hsp90, therefore exhibiting favorable inhibiting activity on three NSCLC cell lines (IC 50 , 0.50 ± 0.01 µM for A549, 1.14 ± 1.11 µM for H1299, and 2.36 ± 0.82 µM for H1975) by inhibited proliferation, induced cell cycle arrest, and aggravated cell apoptosis.
The molecular binding mode and interaction mechanism of Hsp90 N -SNX-2112 were analyzed in detail by the complex crystal structure, which suggested that hydrogen bonds, hydrophobic interactions, and π-π interactions significantly contributed to the intense binding force between the ligand SNX-2112 and its target Hsp90 N , which was verified by TSA and ITC. TSA was a useful tool for target-ligand binding force valuation by determining melting temperature differences ( Tm) of the target with or without binding a ligand. Every protein has a definite melting temperature under certain conditions, and it changes after binding a ligand. Greater absolute Tm value means stronger binding force. It is deemed as a favorable ligand with absolute Tm value greater than 3. The TSA result ( Tm, -9.51 ± 1.00 • C) indicated an intense binding force between Hsp90 N and SNX-2112, which was verified again by ITC. ITC, another tool for binding force determination, measured heat absorption or release amount during target-ligand binding process. Smaller K d means stronger binding force and protein thermostability. The ITC results (K d , 14.10 ± 1.60 nM) suggested that the thermostability of Hsp90 N increased binding with its ligand SNX-2112.
Based on the complex crystal structure, molecular interaction analysis from TSA and ITC, the design scheme of new SNX-2112 derivatives focused on promoting the interactions by group replacements, such as hydrogen bonds, hydrophobic interactions, and π-π interactions, contributed to the intense binding force of Hsp90 N -SNX-2112. Thirty-two novel SNX-2112 derivatives were designed, and 25 exhibited increased binding force with the target Hsp90 N , which confirmed a favorable design scheme evaluated by molecular docking assay. The results would provide promising lead compounds for anti-NSCLC new drug development based on the lead compound SNX-2112.

CONCLUSION
SNX-2112 is a promising compound, but the absence of the complex crystal structure of Hsp90N-SNX-2112 restricted the structural optimization of SNX-2112. Herein, a complex crystal structure of Hsp90 N -SNX-2112 was determined, and anti-NSCLC activity in vitro of SNX-2112 was evaluated. The main results were as follows: (1) SNX-2112 displayed favorable anti-NSCLC activity (IC 50 , 0.50 ± 0.01 µM for A549, 1.14 ± 1.11 µM for H1299, and 2.36 ± 0.82 µM for H1975) by inhibiting cell viability, inducing cell cycle arrest, and aggravating cell apoptosis.
(2) A high-resolution complex crystal structure Hsp90 N -SNX-2112 was successfully determined by XRD (resolution limit, 2.14 Å, PDB ID 6LTK). It was indicated that SNX-2112 well accommodated in the ATP-binding pocket to disable molecular chaperone function of Hsp90, therefore suppressing cancer cells.
(3) The result from TSA disclosed that an intense binding force resulted in a more stable conformation of Hsp90 N after binding with SNX-2112 ( Tm, -9.51 ± 1.00 • C), which was verified from favorable thermodynamic changes by ITC (K d , 14.10 ± 1.60 nM). (4) Based on complex crystal structure and molecular interaction analysis of Hsp90 N -SNX-2112, 32 new SNX-2112 derivatives were successfully designed. Among them, 25 new derivatives exhibited increased binding force with Hsp90 N , which verified a favorable design scheme by molecular docking evaluation. (5) The results would provide new references and guides for anti-NSCLC new drug development basis on the lead compound SNX-2112.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.