Virtual screening and molecular docking characterization of Isoxazole-based molecules as potential Hsp90 inhibitors: In silico insight

doi:10.21203/rs.3.rs-4542530/v1

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Virtual screening and molecular docking characterization of Isoxazole-based molecules as potential Hsp90 inhibitors: In silico insight

https://doi.org/10.21203/rs.3.rs-4542530/v1

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The heat shock protein 90 (Hsp90) is chaperone machinery involved in the activity of hundreds of client proteins. Numerous Hsp90 clients have been implicated in cancer initiation, progression, and metastasis. Hsp90 becomes more prone to binding to drug-like small molecules in the cancer milieu. Hence, modulating the Hsp90 activity by such molecules is a promising and growing approach for cancer treatment. The isoxazole-based molecules like Luminespib have shown potent inhibitory effects against Hsp90 activity in myriad human tumor cell lines. In this work, the chemical structure of Luminespib was employed to identify new potential Hsp90 inhibitors using a collection of in silico methods. Screening the ZINC database displayed that thirty-six isoxazole-based molecules can function as Hsp90 inhibitors. The molecular docking simulation analysis demonstrated that eleven of these ZINC-compounds have binding energies ranging from -8.00 to -8.42 Kcal/mol, which implies a high binding affinity compared to Luminespib, with a binding energy of -7.95 Kcal/mol. These compounds bind to Hsp90 via hydrogen bonds and hydrophobic interactions with crucial residues like Gly97, Asn51, and Lys58. Further optimization of these ZINC compounds could result in the discovery of potent anticancer agents targeting Hsp90.

Structural Biology

Applied Biochemistry

Biochemical Research Methods

Bioactive small molecules

Cancer

Hsp90

Molecular docking

Isoxazole.

Cancer is an infamous group of diseases that are classified as the second cause of death worldwide. Cancer is characterized by irregular and uncontrolled cell proliferation that extends to impact the normal ones¹. The severity of this pathological state has triggered the investigation to specify druggable protein targets for this health problem². In this regard, numerous proteins have been implicated in cancer initiation, progression, and metastasis. The ATP-dependent heat shock proteins (Hsps) have been determined in a broad spectrum of human cancers and were involved in all stages of carcinogenesis, from tumor initiation to invasion and metastasis³. The most abundant Hsps is a 90 kDa molecular chaperone known as Hsp90^2–4. Hsp90 plays a vital role in the activity of hundreds of client proteins in normal and pathological conditions⁴. In this respect, Hsp90 utilizes the energy of ATP hydrolysis to assist the proper folding of the nascent proteins and to sustain the stability and the conformation of the folded ones⁵. Many Hsp90-dependent proteins are involved in essential cellular processes, including DNA repair and signal transduction pathways⁶. On the other side, the dysregulation of the Hsp90 clients has been correlated to the development of several pathological conditions, including neurodegenerative diseases and cancer^7–9. In cancer, Hsp90 secures the stability of several oncogenic proteins, whose activities result in tumor cell survival and metastasis⁷. Notably, the overexpression of Hsp90 in tumor cells, 2-10 times compared to normal cells, renders elevated levels of the client proteins and, thereby, tumor cell growth⁸. Accordingly, Hsp90 is considered a valuable target for cancer treatment⁵.

Hsp90 is a homodimer protein, the monomer of which is constructed of three structural domains: the N-terminal domain (N-Hsp90), the C-terminal domain (C-Hsp90), and the middle domain (M-Hsp90)^6,10. The N-Hsp90 incorporates the ATP-binding pocket, which makes it a target for inhibitors that target Hsp90’s ATPase activity¹¹. Thus far, more than eighteen Hsp90 inhibitors have entered the clinical trials stage^7,12. However, none of them have reached the market as approved drugs. The emerging studies and the preclinical exploration demonstrated that these inhibitors bind to the N-Hsp90 deeply and impact the multiplication of cancer cells by inducing G1-G2 arrest and apoptosis¹³.

Heterocyclic compounds are continually found in the structure of bioactive molecules, including antibacterial, antiviral, antifungal, and anti-inflammatory agents¹⁴. Likewise, the manufactured analogs of these compounds have shown effective inhibition activities against a broad spectrum of enzymes. In the case of Hsp90, several heterocyclic-based compounds were constructed to modulate the functions of this chaperone machinery^15,16. These compounds include purine-, pyrazole-, triazine-, quinolines-, coumarin-, and isoxazole-based inhibitors¹⁴. Isoxazole is a five-membered ring compound made of three carbon atoms in addition to an oxygen atom and a nitrogen atom at positions 1 and 2, respectively¹⁷. As Hsp90 inhibitors, the isoxazole-based molecules have exhibited high efficacy, good pharmacokinetics profiles, and low toxicity¹⁸. For example, the N-(isoxazole-5-yl) amides containing Hsp90 inhibitors have shown distinct pharmacokinetic properties and delivered potent antitumor activities in both in vitro and in vivo studies¹⁹. Sharp et al. have reported the discovery of isoxazole-containing compounds, VER-50589, using high-throughput screening and the structure-based design²⁰. VER-50589 displayed a rugged inhibitory activity against Hsp90, comparable to the clinically approved drug 17-AAG¹⁹. Additionally, VER-50589 displayed an improved cellular uptake and good Pharmacokinetic and pharmacodynamic profiles likened to the lead Hsp90 inhibitor, CCT018159^14,19. Luminespib (AUY922) is another isoxazole-derived compound with high selectivity and potent inhibitory activity against Hsp90 in various human tumor cell lines²¹.

Computer-aided drug design (CADD) is widely utilized to determine drug-like small molecules and to explore their possible biological activities^22–24. In the present study, various in silico methods were employed to identify potential inhibitors that target Hsp90, as the inhibition of Hsp90 in the cancer milieu has arisen as an optimistic approach in cancer-targeted therapies. The ZINC database was screened for isoxazole-comprising compounds using the EXPASY Swiss Similarity platform and the chemical structure of the potent Hsp90 inhibitor Luminespib as a control molecule. Shortlisting the top-ranked 400 molecule candidates based on their drug-likeness, pharmacokinetics, and physiochemical properties resulted in a final list of thirty-six compounds. The binding possibility of the selected ZINC compounds to Hsp90 was sampled using online prediction servers such as SwissTargetPredction, Way2drug, SuperPred, and the SEA online tool.

Further, molecular docking simulation was executed using the SwissDock software to investigate the compounds' binding affinity and analyze their interactions with Hsp90. The findings of this study displayed that the selected molecules have shown potential binding to Hsp90. Moreover, eleven of the selected compounds exhibited higher binding affinity toward Hsp90, with binding energy ranging from -8.00 to -8.42 Kcal/mol, compared with the Luminespib, with a binding energy of -7.95 Kcal/mol. The potential binding is driven by diverse modes of interactions, including essential hydrogen bonds and hydrophobic contacts with crucial residues like Gly97, Asn51, and Lys58. Further optimization of these ZINC compounds could result in the discovery of powerful anticancer agents targeting Hsp90.

Ligands Screening and Selection

The virtual screening of the target molecules was performed using the Expasy SwissSimilarity platform and the chemical structure of the clinically verified Hsp90 inhibitor, Luminespib, as a control molecule. SwissSimilarity is an online service that provides a valuable tool for conducting virtual screening for bioactive molecules in chemical libraries^25,26. First, the Simplified Molecular Input Line Entry (SMILES) format of the control compound was retrieved from the PubChem database to be entered into the SwissSimilarity text box. The drug-like option was selected as the class of compound libraries to screen for the target molecules, and the 2D path-based FP2 fingerprint screening method was selected to search for the target compounds in the ZINC 20 database²⁵. Then, the top 400 ranked compounds were filtered based on Lipinski’s “Rule of five” (RO5) and the drug- and lead-likeness using the Expasy SwissADME web service. RO5 included the following criteria: a molecular mass < 500 Da, H-bond donors (HBD) equal or less than 5, H-bond acceptors (HBA) equal or less than 10, rotatable bonds (RB) no more than 10, and octanol-water partition coefficient (CLogP) equal or less than 5²⁷. The drug- and lead-likeness of the selected compounds were defined by medicinal chemistry rules, including Veber, Ghose, Egan, and Lipinski, using the Expasy SwissADME web service²⁸.

Ligands Binding to Hsp90 Prediction

The potential binding of Hsp90 to the isoxazoles compounds was predicted using online tools, including the EXPASY SwissTargetPrediction²⁹, SuperPred 3.0³⁰, prediction of activity spectra for substances (PASS)³¹, and Similarity Ensemble Approach (SEA)³². These platforms can accurately predict the targets of bioactive molecules based on their similarity to known ligands. The prediction process was executed by entering the selected molecules' SMILES files into the prediction webserver toolbox.

Molecular Docking Simulation

The binding of potential inhibitors to Hsp90 at the atomic level resolution was carried out using molecular docking simulation. Molecular docking is a valuable technique for gaining in-depth insight into the mode of interaction between ligands and the target biomolecule³³. In this computer-aided method, specific ligands' predominant position and binding affinity are determined based on the solved structure of the biomolecule under investigation³⁴. In this study, the EXPASY SwissDock online server and the crystal structure of the Human Hsp90 protein (PDB ID: 6LTI) were employed to conduct molecular docking studies for the chosen ZINC-molecules, with Luminespib as a control for comparative analysis. The crystal structure of the Hsp90-Luminespib bound state was obtained from the Protein Data Bank (PDB ID: 6LTI, Fig. 1) and utilized as the protein target, while Luminespib was determined at the center of the ATP binding site using SwissDock³⁵. The protein preparation for the docking simulations was carried out using Chimera software³⁶. Initially, the protein crystal structure was loaded into Chimera and water molecules, and all the nonstandard residues, including the ligand, were removed. The protein's structure was minimized, hydrogens were added, and charges were assigned to improve the accuracy of the protein representation. No preparation step was conducted for the target ligands as they were acquired separately from the ZINC database in ready-to-dock, 3D formats³⁷. Finally, the actual docking studies were performed using SwissDock predefined parameters: WANTEDCONFS: 5000, NBFACTSEVAL: 5000, NBSEEDS: 250, SDSTEPS: 100, ABNRSTEPS: 250, CLUSTERINGRADIUS: 2.0, and MAXCLUSTERSIZE: 8³⁵. Henceforward, the docking results were visualized using UCSF Chimera package. The docking procedure was validated by re-docking Luminespib back to the ATP-binding site of N-Hsp90.

Computer-aided drug discovery is an emerging and evolving research domain that seeks to determine and characterize molecules of optimistic biological activity³⁸. The breakthroughs in machine-learning technologies have made this approach a valuable and reliable instrument to predict and validate the biological functions of chemical compounds²². In this work, in-silico methods were used to identify isoxazole-containing molecules as potential inhibitors for Hsp90 activity. Using drug-like small molecules as protein inhibitors is a promising protocol in cancer-based therapies³⁹. Compared to large-molecule drugs such as monoclonal antibodies and polypeptides, small-molecule drugs are inexpensive, compliant, and have good pharmacokinetic properties⁴⁰. Moreover, it covers numerous targets, including kinases, proteasomes, and several regulatory proteins⁴¹.

Isoxazoles Screening and Selection

To identify small-molecule candidates of potential Hsp90 inhibition activity, the ZINC database was screened using the EXPASY SwissSimilarity platform utilizing the chemical structure of Luminespib as a control molecule. Luminespib is an isoxazole-based molecule that inhibits Hsp90 ATPase activity by binding to the ATP binding pocket in the N-Hsp90⁴². Based on in vitro and in vivo studies, Luminespib has shown significant inhibitory activity, high selectivity, and good pharmacokinetics physiochemical properties compared to the prominent Hsp90 inhibitors such as 17-AAG^42,43. Additionally, it is one of the most investigated Hsp90 inhibitors in numerous human tumor cell lines⁴². Luminespib-induced inhibition impacts cancer cell proliferation by triggering cell cycle arrest, apoptosis, and depletion of client protein expression^42,44. Choosing Luminespib as a template molecule for screening processes is guided by the Similarity principle, which states that molecules with similar structures are likely to have identical biological influence^38,41.

The ZINC database comprises hundreds of millions of commercially available synthesized organic molecules ranging in size from 50 to 1000 Da³⁷. Screening the ZINC database for Luminespib analogs has revealed 400 potential candidates as plausible Hsp90 inhibitors. The selected molecules were shortlisted by applying Lipinski's RO5 and assessing their pharmacokinetics, physicochemical properties, drug-likeness, and medicinal chemistry using the EXPASY SwissADME website. The shortlisting processes have resulted in thirty-six potential Hsp90 inhibitors, Table 1, and Figure 1. The chemical structures of these molecules were drawn using ChemDraw 3D-Ultra version 19.0.0.22.

Table. 1 The selected ZINC compounds and Luminespib, molecular formula, molecule weight, and SMILE files.

ZINC Code	Molecular Formula	MW (g/mol)	SMILE
ZINC000020510095	C₂₀H₁₈N₂O₃	334.37	C[C@H](NC(=O)C1=NOC2=C1COC1=CC=C(C)C=C21)C1=CC=CC=C1
ZINC000035477380	C₁₅H₁₄N₂O₃	258.27	CCNC(=O)C1=NOC2=C1COC1=CC=C(C)C=C21
ZINC000020509814	C₁₆H₁₈N₂O₃	286.33	CC[C@@H](C)NC(=O)C1=NOC2=C1COC1=CC=C(C)C=C21
ZINC000020509816	C₁₆H₁₈N₂O₃	286.33	CC[C@H](C)NC(=O)C1=NOC2=C1COC1=CC=C(C)C=C21
ZINC000032920495	C₁₉H₁₆N₂O₃	320.34	CC1=CC=C2OCC3=C(ON=C3C(=O)NCC3=CC=CC=C3)C2=C1
ZINC000020509949	C₂₀H₁₈N₂O₃	334.37	CC1=CC=C(CNC(=O)C2=NOC3=C2COC2=CC=C(C)C=C32)C=C1
ZINC000035477309	C₁₅H₁₆N₂O₃	272.30	CC[C@@H](C)NC(=O)C1=NOC2=C1COC1=CC=CC=C21
ZINC000035477310	C₁₅H₁₆N₂O₃	272.30	CC[C@H](C)NC(=O)C1=NOC2=C1COC1=CC=CC=C21
ZINC000015673863	C₁₇H₂₀N₂O₃	300.35	CCCN(CCC)C(=O)C1=NOC2=C1COC1=CC=CC=C21
ZINC000035537827	C₁₆H₁₆N₂O₄	300.31	CC1=CC=C2OCC3=C(ON=C3C(=O)N3CCOCC3)C2=C1
ZINC000020484607	C₁₅H₁₆N₂O₃	272.30	CCCCNC(=O)C1=NOC2=C1COC1=CC=CC=C21
ZINC000020484604	C₁₄H₁₄N₂O₄	274.27	COCCNC(=O)C1=NOC2=C1COC1=CC=CC=C21
ZINC000020484847	C₁₈H₁₄N₂O₃	306.32	O=C(NCC1=CC=CC=C1)C1=NOC2=C1COC1=CC=CC=C21
ZINC000020509776	C₁₈H₂₀N₂O₃	312.36	CC1=CC=C2OCC3=C(ON=C3C(=O)NC3CCCCC3)C2=C1
ZINC000003908545	C₁₈H₁₆N₂O₃	308.33	C[C@H](NC(=O)C1=NOC(=C1)C1=CC=CC=C1O)C1=CC=CC=C1
ZINC000003908546	C₁₈H₁₆N₂O₃	308.33	C[C@@H](NC(=O)C1=NOC(=C1)C1=CC=CC=C1O)C1=CC=CC=C1
ZINC000020901917	C₁₅H₁₄N₂O₄	286.28	O=C(N1CCOCC1)C1=NOC2=C1COC1=CC=CC=C21
ZINC000020484748	C₁₈H₁₃ClN₂O₃	340.76	ClC1=CC=C(CNC(=O)C2=NOC3=C2COC2=CC=CC=C32)C=C1
ZINC000020510020	C₁₇H₁₈N₂O₅	330.34	CCOC(=O)[C@@H](C)NC(=O)C1=NOC2=C1COC1=CC=C(C)C=C21
ZINC000020510023	C₁₇H₁₈N₂O₅	330.34	CCOC(=O)[C@H](C)NC(=O)C1=NOC2=C1COC1=CC=C(C)C=C21
ZINC000003908532	C₁₇H₁₃FN₂O₃	312.30	OC1=CC=CC=C1C1=CC(=NO1)C(=O)NCC1=CC=C(F)C=C1
ZINC000003908564	C₁₄H₁₆N₂O₃	260.29	CCN(CC)C(=O)C1=NOC(=C1)C1=CC=CC=C1O
ZINC000003908535	C₁₈H₁₆N₂O₃	308.33	OC1=CC=CC=C1C1=CC(=NO1)C(=O)NCCC1=CC=CC=C1
ZINC000033028831	C₁₆H₁₆N₂O₅	316.31	CCOC(=O)[C@@H](C)NC(=O)C1=NOC2=C1COC1=CC=CC=C21
ZINC000033028832	C₁₆H₁₆N₂O₅	316.31	CCOC(=O)[C@H](C)NC(=O)C1=NOC2=C1COC1=CC=CC=C21
ZINC000033028795	C₁₇H₁₉N₃O₄	329.35	O=C(NCCN1CCOCC1)C1=NOC2=C1COC1=CC=CC=C21
ZINC000003908558	C₁₄H₁₆N₂O₃	260.29	CC(C)CNC(=O)C1=NOC(=C1)C1=CC=CC=C1O
ZINC000013119868	C₁₄H₁₆N₂O₃	260.29	CC[C@@H](C)NC(=O)C1=NOC(=C1)C1=CC=CC=C1O
ZINC000013119870	C₁₄H₁₆N₂O₃	260.29	CC[C@H](C)NC(=O)C1=NOC(=C1)C1=CC=CC=C1O
ZINC000032920422	C₁₈H₁₅N₃O₃	321.33	CC1=CC=C2OCC3=C(ON=C3C(=O)NCC3=CC=CN=C3)C2=C1
ZINC000003908529	C₁₈H₁₆N₂O₃	308.33	CN(CC1=CC=CC=C1)C(=O)C1=NOC(=C1)C1=CC=CC=C1O
ZINC000003908536	C₁₇H₁₄N₂O₃	294.30	OC1=CC=CC=C1C1=CC(=NO1)C(=O)NCC1=CC=CC=C1
ZINC000003908554	C₁₄H₁₆N₂O₃	260.29	CCCCNC(=O)C1=NOC(=C1)C1=CC=CC=C1O
ZINC000003908038	C₁₄H₁₆N₂O₃	260.29	CCCNC(=O)C1=NOC(=C1)C1=CC=CC=C1OC
ZINC000047528273	C₁₃H₁₁F₃N₂O₃	300.23	COC1=CC=CC=C1C1=CC(=NO1)C(=O)NCC(F)(F)F
ZINC000003908547	C₁₈H₁₆N₂O₄	324.33	COC1=CC=C(CNC(=O)C2=NOC(=C2)C2=CC=CC=C2O)C=C1
ZINC000014974852	C₁₈H₂₀N₂O₃	388.807	CCNC(=O)C1=NOC(=C1C2=CC=C(C=C2)CN3CCOCC3)C4=CC(=C(C=C4O)O)C(C)C

Luminespib is a monocarboxylic acid amide in which the isoxazole ring is connected to two aryl groups at 4 and 5 positions⁴². Likewise, ZINC molecules are constructed on the isoxazole moiety, which is attached to diverse functionalities of the ring, Figure 1.

Per Lipinski's RO5, the selected molecules have molecular weights smaller than 500 Da (ranging from 258.27 to 340.76) Da, Table 1. The number of H-bond donors and H-bond acceptors does not violate RO5, which is crucial for adequate drug absorption and to preclude unnecessary interactions, Table 2. For flexibility purposes and to facilitate binding to the target protein, the number of rotatable bonds is less than ten, Table 2. Additionally, the ZINC molecules satisfy the drug- and lead likeness standards with no violations, Table. 3. The water solubility of the drug is essential for a sufficient pharmacological response dosage⁴⁵. Generally, based on the solubility scale, drug solubility ranges from insoluble to very and highly soluble. The SwissADME data indicated that all selected molecules are classified as soluble to moderately soluble. The topological polar surface area (TPSA) estimates the degree of ligand polarity that arises from the molecule's polar atoms, such as sulfur, nitrogen, and oxygen²⁴. Also, the TPSA value is critical to determining the degree of molecule solubility in lipids and provides insight into ligand-protein interactions. Most of the selected candidates have TPSA values within the optimal TPSA value range, 60 to 140 Å², which makes them well-suited for oral absorption. Lipophilicity is an essential drug property that affects drug uptake and metabolism⁴⁶. The ideal value for drug lipophilicity (LogP) is less than 5⁴⁷. The LogP values of the chosen compounds are less than five, which gives them a good lipophilicity profile.

Table. 2 The physicochemical properties of selected ZINC compounds and Luminespib

Zinc Code	HBD	HBA	NRB	Log P	TPSA (Å²)	Log S	Solubility (mg/mL)	Water Solubility
ZINC000020510095	1	4	4	3.43	64.36	-4.32	1.6E-02	Moderately soluble
ZINC000035477380	1	4	3	2.23	64.36	-2.88	3.39E-01	Soluble
ZINC000020509814	1	4	4	2.87	64.36	-3.55	8.01E-02	Soluble
ZINC000020509816	1	4	4	2.86	64.36	-3.55	8.01E-02	Soluble
ZINC000032920495	1	4	4	3.14	64.36	-4.00	3.19E-02	Moderately soluble
ZINC000020509949	1	4	4	3.50	64.36	-4.30	1.67E-02	Moderately soluble
ZINC000035477309	1	4	4	2.53	64.36	-3.25	1.52E-01	Soluble
ZINC000035477310	1	4	4	2.54	64.36	-3.25	1.52E-01	Soluble
ZINC000015673863	0	4	6	3.03	55.57	-3.67	6.47E-02	Soluble
ZINC000035537827	0	5	2	1.90	64.80	-2.80	4.8E-01	Soluble
ZINC000020484607	1	4	5	2.55	64.36	-3.14	1.96E-01	Soluble
ZINC000020484604	1	5	5	1.60	73.59	-2.27	1.46E+00	Soluble
ZINC000020484847	1	4	4	2.82	64.36	-3.71	5.97E-02	Soluble
ZINC000020509776	1	4	3	3.26	64.36	-4.05	2.79E-02	Moderately soluble
ZINC000003908545	2	4	5	2.81	75.36	-3.97	3.29E-02	Soluble
ZINC000003908546	2	4	5	2.83	75.36	-3.97	3.29E-02	Soluble
ZINC000020901917	0	5	2	1.51	64.80	-2.49	9.17E-01	Soluble
ZINC000020484748	1	4	4	3.35	64.36	-4.30	1.72E-02	Moderately soluble
ZINC000020510020	1	6	6	2.32	90.66	-3.24	1.89E-01	Soluble
ZINC000020510023	1	6	6	2.36	90.66	-3.24	1.89E-01	Soluble
ZINC000003908532	2	5	5	2.96	75.36	-3.81	4.86E-02	Soluble
ZINC000003908564	1	4	5	2.13	66.57	-2.94	3E-01	Soluble
ZINC000003908535	2	4	6	2.81	75.36	-3.94	3.51E-02	Soluble
ZINC000033028831	1	6	6	1.96	90.66	-2.94	3.66E-01	Soluble
ZINC000033028832	1	6	6	2.03	90.66	-2.94	3.66E-01	Soluble
ZINC000033028795	1	6	5	1.39	76.83	-2.43	1.23E+00	Soluble
ZINC000003908558	2	4	5	2.31	75.36	-3.20	1.63E-01	Soluble
ZINC000013119868	2	4	5	2.34	75.36	-3.20	1.66E-01	Soluble
ZINC000013119870	2	4	5	2.33	75.36	-3.20	1.66E-01	Soluble
ZINC000032920422	1	5	4	2.40	77.25	-3.33	1.49E-01	Soluble
ZINC000003908529	1	4	5	2.72	66.57	-3.83	4.52E-02	Soluble
ZINC000003908536	2	4	5	2.63	75.36	-3.66	6.47E-02	Soluble
ZINC000003908554	2	4	6	2.27	75.36	-3.09	2.13E-01	Soluble
ZINC000003908038	1	4	6	2.38	64.36	-3.07	2.23E-01	Soluble
ZINC000047528273	1	7	6	2.69	64.36	-3.41	1.16E-01	Soluble
ZINC000003908547	2	5	6	2.63	84.59	-3.71	6.3E-02	Soluble
Luminespib				2.63		-3.71	3.02e-05

Table. 3 Drug-likeness of selected ZINC compounds based on Lipinski, Veber, Ghose, and Egan rules.

ZINC Code	Lipinski	Ghose	Veber	Egan	ZINC Code	Lipinski	Ghose	Veber	Egan
ZINC000020510095	Yes	Yes	Yes	Yes	ZINC000020510023	Yes	Yes	Yes	Yes
ZINC000035477380	Yes	Yes	Yes	Yes	ZINC000003908532	Yes	Yes	Yes	Yes
ZINC000020509814	Yes	Yes	Yes	Yes	ZINC000003908564	Yes	Yes	Yes	Yes
ZINC000020509816	Yes	Yes	Yes	Yes	ZINC000003908535	Yes	Yes	Yes	Yes
ZINC000032920495	Yes	Yes	Yes	Yes	ZINC000033028831	Yes	Yes	Yes	Yes
ZINC000020509949	Yes	Yes	Yes	Yes	ZINC000033028832	Yes	Yes	Yes	Yes
ZINC000035477309	Yes	Yes	Yes	Yes	ZINC000033028795	Yes	Yes	Yes	Yes
ZINC000035477310	Yes	Yes	Yes	Yes	ZINC000003908558	Yes	Yes	Yes	Yes
ZINC000015673863	Yes	Yes	Yes	Yes	ZINC000013119868	Yes	Yes	Yes	Yes
ZINC000035537827	Yes	Yes	Yes	Yes	ZINC000013119870	Yes	Yes	Yes	Yes
ZINC000020484607	Yes	Yes	Yes	Yes	ZINC000032920422	Yes	Yes	Yes	Yes
ZINC000020484604	Yes	Yes	Yes	Yes	ZINC000003908529	Yes	Yes	Yes	Yes
ZINC000020484847	Yes	Yes	Yes	Yes	ZINC000003908536	Yes	Yes	Yes	Yes
ZINC000020509776	Yes	Yes	Yes	Yes	ZINC000003908554	Yes	Yes	Yes	Yes
ZINC000003908545	Yes	Yes	Yes	Yes	ZINC000003908038	Yes	Yes	Yes	Yes
ZINC000003908546	Yes	Yes	Yes	Yes	ZINC000047528273	Yes	Yes	Yes	Yes
ZINC000020901917	Yes	Yes	Yes	Yes	ZINC000003908547	Yes	Yes	Yes	Yes
ZINC000020484748	Yes	Yes	Yes	Yes	Luminespib	Yes	Yes	Yes	Yes
ZINC000020510020	Yes	Yes	Yes	Yes

Prediction of small molecules binding to Hsp90

Computational-aided methods have been established as a valuable and reliable tool for predicting macromolecule targets of bioactive small molecules⁴⁸. In this regard, online prediction servers have revolutionized drug discovery by providing handy, efficient, and fast tools to screen millions of chemical compounds for their potential protein targets³⁰. As a result, the biological activities of hundreds of small molecules have been identified against several proteins, including kinases and phosphatases⁴⁸. In this work, online servers SwissTargetPredction²⁹, SuperPred 3.0³⁰, PASS³¹, and SEA³² were utilized to conduct prediction studies for binding plausibility of the identified ZINC-compounds to Hsp90. Applying these online servers has shown that, without exception, all selected isoxazoles have the potential to serve as potential inhibitors for Hsp90 activity, Table 4. Consequently, the data from in silico prediction provided the first insights into the prospective functions of these molecules as modulators for Hsp90 ATPase activity.

Table 4. Small molecules in-silico potential binding prediction to Hsp90 obtained by SuperPred, PASS, SwissTargetPredction, and SEA online tools.

Zinc	Super-Pred	PASS	Swiss	SEA	Zinc	Super-Pred	PASS	Swiss	SEA
ZINC000020510095	Yes	Yes	Yes	Yes	ZINC000020510023	Yes	Yes	Yes	Yes
ZINC000035477380	Yes	Yes	Yes	Yes	ZINC000003908532	Yes	Yes	Yes	Yes
ZINC000020509814	Yes	Yes	Yes	Yes	ZINC000003908564	Yes	Yes	Yes	Yes
ZINC000020509816	Yes	Yes	Yes	Yes	ZINC000003908535	Yes	Yes	Yes	Yes
ZINC000032920495	Yes	Yes	Yes	Yes	ZINC000033028831	Yes	Yes	Yes	Yes
ZINC000020509949	Yes	Yes	Yes	Yes	ZINC000033028832	Yes	Yes	Yes	Yes
ZINC000035477309	Yes	Yes	Yes	Yes	ZINC000033028795	Yes	Yes	Yes	Yes
ZINC000035477310	Yes	Yes	Yes	Yes	ZINC000003908558	Yes	Yes	Yes	Yes
ZINC000015673863	Yes	Yes	Yes	Yes	ZINC000013119868	Yes	Yes	Yes	Yes
ZINC000035537827	Yes	Yes	Yes	Yes	ZINC000013119870	Yes	Yes	Yes	Yes
ZINC000020484607	Yes	Yes	Yes	Yes	ZINC000032920422	Yes	Yes	Yes	Yes
ZINC000020484604	Yes	Yes	Yes	Yes	ZINC000003908529	Yes	Yes	Yes	Yes
ZINC000020484847	Yes	Yes	Yes	Yes	ZINC000003908536	Yes	Yes	Yes	Yes
ZINC000020509776	Yes	Yes	Yes	Yes	ZINC000003908554	Yes	Yes	Yes	Yes
ZINC000003908545	Yes	Yes	Yes	Yes	ZINC000003908038	Yes	Yes	Yes	Yes
ZINC000003908546	Yes	Yes	Yes	Yes	ZINC000047528273	Yes	Yes	Yes	Yes
ZINC000020901917	Yes	Yes	Yes	Yes	ZINC000003908547	Yes	Yes	Yes	Yes
ZINC000020484748	Yes	Yes	Yes	Yes	Luminespib	Yes	Yes	Yes	Yes
ZINC000020510020	Yes	Yes	Yes	Yes

Evaluation of Ligands Potential Inhibition Mechanism by Molecular Docking

To elucidate the potential inhibition mechanism of the selected ZINC compounds against Hsp90, molecular docking simulation studies were launched using the EXPAY SwissDock server. The docking data revealed that the chosen isoxazole compounds target the ATP-binding pocket of Hsp90, which agrees with the previously reported binding mode for Luminespib, Figure 2. This outcome supports the possibility of developing these compounds as potent Hsp90 inhibitors. As presented in Table 5, these molecules' Ligand Binding Energies (LBEs) range from -6.06 to -8.42 Kcal/mol. Furthermore, eleven of these compounds exhibited significant LBE values, -8.00 to -8.42 Kcal/mol, compared to the reference molecule, -7.95 Kcal/mol. This result suggests a higher binding affinity to Hsp90 than the control molecule and further supports the capability of these compounds to be developed into potent Hsp90 inhibitors. A deep analysis of the docking outcomes for the top-ranked compounds and the reference molecule revealed a diverse mode of interactions to Hsp90, Table 6 and Figure 3 and Figure 4. Most selected ZINC compounds form hydrogen bonds with Gly97, reflecting this residue's crucial role in ligand recognition, Figure 3. Hydrophobic interactions with Asn51, Lys58, and Gly97 were frequently observed, which indicates their role in stabilizing the binding to the ligands, Figure 4. Luminespib displayed myriad interactions, including hydrogen bonds with Gly97 and Asp93 and hydrophobic contacts with Met98, Ser52, Leu107, Asp93, and Gly135. On the other side, the top-ranked molecule, ZINC000003908547 of LBE of -8.42 Kcal/mol, forms a hydrogen bond with Asp93, and hydrophobic interaction with Gly97, Asp93, Lys58, Asn51, and Asp54 of Hsp90. ZINC000033028795, the second-top ranked molecule, with LBE of -8.40 Kcal/mol, is engaged to Hsp90 through hydrogen bonds driven by Asn51 and Lys58 residues and hydrophobic interactions through Gly97, Asp93, Lys58, Asn51, and Asp54. Interestingly, Asp93 and Gly97 are involved in hydrogen bonds and hydrophobic interactions with Luminespib and the top-ranked ZINC molecules, ZINC000003908547 and ZINC000003908536, which emphasize their potential as targets for further optimization.

Table 5. The lowest binding energy in Kcal/mol of selected ZINC compounds.

ZINC Compound	Lowest Binding Energy (Kcal/mol)	ZINC Compound	Lowest Binding Energy (Kcal/mol)
ZINC000020510095	-7.59	ZINC000020510023	6.06
ZINC000035477380	-7.56	ZINC000003908532	-7.36
ZINC000020509814	-8.33	ZINC000003908564	-6.57
ZINC000020509816	-7.39	ZINC000003908535	-7.18
ZINC000032920495	-7.09	ZINC000033028831	-8.16
ZINC000020509949	-8.24	ZINC000033028832	-8.14
ZINC000035477309	8.00	ZINC000033028795	-8.06
ZINC000035477310	-6.97	ZINC000003908558	-7.85
ZINC000015673863	-7.57	ZINC000013119868	-5.95
ZINC000035537827	-7.03	ZINC000013119870	-6.73
ZINC000020484607	-7.09	ZINC000032920422	-8.19
ZINC000020484604	-7.02	ZINC000003908529	-7.49
ZINC000020484847	-7.70	ZINC000003908536	-8.37
ZINC000020509776	-8.28	ZINC000003908554	-6.3
ZINC000003908545	-7.07	ZINC000003908038	-6.88
ZINC000003908546	-7.68	ZINC000047528273	-7.85
ZINC000020901917	-7.08	ZINC000003908547	-8.42
ZINC000020484748	-7.54	ZINC000020510020	-8.09
Luminespib (reference)	-7.95

Table 6. The lowest binding energy and the interacting residues of the top-ranked ZINC compounds.

ZINC code	Lowest Binding Energy (Kcal/mol)	Interacting Residues
ZINC code	Lowest Binding Energy (Kcal/mol)	Hydrogen Bond	Hydrophobic
ZINC000003908547	-8.42	Asp93	Gly97, Asp93, Lys58, Asn51, Asp54
ZINC000033028795	-8.40	Asn51, Lys58	Asn51, Leu107, Lys58, Gly108
ZINC000003908536	-8.37	Asp93, Gly97	Asn51, Asp93, Lys58, Glu47
ZINC000020509814	-8.33	Gly97	Lys58, Gly97, Asn51
ZINC000020509776	-8.28	Gly97	Asn51, Lys58, Gly97, Leu76
ZINC000020509949	-8.24	Gly97	Met98, Thr184, Leu103, Val150, Leu107
ZINC000032920422	-8.19	Gly97, Met98	Lys58, Gly97, Gly108, Thr109, Asp54
ZINC000033028832	-8.14	Gly97	Lys58, Asn51, Gly97, Gly108
ZINC000033028831	-8.16	Met98	Lys58, Asn51, Gly137, Gly97
ZINC000020510020	-8.09	Gly135	Gly135, Asn51, Lys58, Gly108
ZINC000035477309	-8.00	Gly97	Lys58, Gly135, Gly97, Asn51
Luminespib	-7.92	Gly97, Asp93	Met98, Ser52, Leu107, Asp93, Gly135

The breakthroughs in deep-learning technologies have established in silico approaches as a versatile and reliable tool in all stages of drug discovery, from small molecule screening to examining their potential biological activities^30,32,46,48. This study employed the chemical structure of the clinically verified Hsp90 inhibitor, Luminespib, and computer-aided techniques to identify isoxazole-based molecules as potential inhibitors for Hsp90 oncogenic activity. Of 400 top-ranked ZINC compounds, 36 were selected for further investigation by target prediction online servers and molecular docking simulation. Collectively, all shortlisted molecules have shown potential inhibition roles against Hsp90 activity. Moreover, eleven of these molecules exhibited more binding affinities to Hsp90 than the control molecule, Luminespib. Molecular docking simulations revealed that a combination of hydrogen bonds and hydrophobic interaction to Hsp90 critical residues such as Asp93, Gly97, and Lys58 drives the potential activity of these ZINC compounds. Further investigation of the role of these residues in modulating Hsp90 activity and the optimization of these compounds' structural and pharmacokinetic properties could lead to novel, effective anticancer agents targeting this chaperone machinery.

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The authors declare potential competing interests as follows: NA

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Virtual screening and molecular docking characterization of Isoxazole-based molecules as potential Hsp90 inhibitors: In silico insight

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