Presentations

Abstract

Introduction:

Mitotic inhibitors have substantial implications in the prevention of cancer cell growth and proliferation by binding to α/ß-tubulin heterodimer to inhibit a multitude of cellular functions resulting in apoptosis. Colchicine, a compound originally extracted from Colchicum autumnale, is a mitotic inhibitor that binds at the interface of the α/ß tubulin heterodimer. Despite colchicine's anticancer properties, it has a narrow therapeutic index due to its nonspecific inhibition of tubulin polymerization in noncancerous cells 1. Combretastatin A-4 (CA-4), a natural product, extracted from Combretum caffrum, was found to be a highly potent antimitotic, antiproliferative, antivascular, antiangiogenic agent, binds at the colchicine binding site of α/ß tubulin, and inhibits multidrug resistant cancer cell growth1. Notably, greater cytotoxic potency is associated with the cis-CA-4 stereoisomer; however, it suffers from poor water solubility that prevents its further development into the clinic. We have discovered a unique boronic acid isostere CA-4 analog with the advantage in improved anticancer cell proliferation and water solubility compared to CA-4 2. Boronic acid containing compounds are normally stable and remain protonated under physiological conditions3. With the evidence of a FDA approved protease inhibitor, boronic acid containing dipeptide Bortezomib (VELCADE), we performed a molecule docking of α/ß tubulin-ligand interaction to guide the design and synthesis a novel series of CA-4 boronic acid derivatives. In our docking model, a boronic acid group is designed as an isostere group to replace the hydroxy group at the C-ring of CA-4. Various position of boronic acid substituted on the phenyl ring of CA-4, as well as replacement the phenyl ring with a pyridine ring were docked in our model. Binding energies were calculated and compared with CA-4. Docking was validated by in vitro evaluation of the synthesized novel boronic acid compounds including the assay of colchicine replacement, inhibition of tubulin polymerization and breast cancer cell MCF-7 proliferation. This study aimed to investigate protein-ligand interactions using molecular docking to establish how the analogues of CA-4 boronic acid containing compound interact with the colchicine binding pocket of the α/ß tubulin heterodimer. These interactions were then compared to the ability to inhibit tubulin polymerization and MCF7 cell growth, serving as a valuable tool in our drug discovery effort.

Methods:

AutoDock Vina 1.2.0 was used to perform molecular docking of compounds into a high-resolution tubulin heterodimer structure (PDB ID: 6XER). Colchicine (CID: 2833), phenstatin (CID: 9948888), and cis-combretastatin A-4 (CID: 5351344) structures were obtained to perform docking. Docking boron-containing compounds remains a challenge due to a lack of reasonable parameters for boron in most docking software; thus, all boron atoms were changed to carbon atoms4. SMILES for the synthesized compounds were converted to mol files with 3D coordinates using OpenBabel. The tubulin heterodimer was edited to remove the second heterodimer, stathmin domain, and other compounds and ions (water, guanosine-5'-triphosphate, guanosine-5'-diphosphate, sulfate ions, and magnesium ions). Docking was performed using a box of x = 22.62383, y = 20.6082, z = 22.8357 size with a center x= -6.64202, y = -9.81822, z = 40.4171, capturing the colchicine binding pocket. The docking position with the lowest dock score and orientated in a similar manner as colchicine was chosen for each compound.

Results:

Colchicine was re-docked to confirm the docking model. Both compounds 1 and 18 exhibited greater cytotoxic potency when compared to compounds CA-4 and phenstatin. CA-4 was found to form a hydrogen bond of 3.154 Å between the second methoxy oxygen of the A ring and cysteine 239 of the beta chain, this interaction resulted in a dock score of -7.0. Docking phenstatin produced 2 hydrogen bonds with ß tubulin-a 2.509 Å hydrogen bond between the second methoxy oxygen and cysteine 239, and the carbonyl oxygen interacted with lysine 252 which formed a hydrogen bond of 3.067 Å. This resulted in a dock score of -7.4. The docking study indicated that compounds 1 and 18 depicted greater stability than the original compounds, CA-4 and phenstatin. Compound 1's C ring methoxy groups formed two hydrogen bonds with cysteine 239 of the beta chain resulting in a dock score of -7.2. The second methoxy oxygen of the A ring of compound 18 formed a hydrogen bond with cysteine 239 of the beta chain (2.640 Å). Unlike phenstatin, hydrogen bonding was observed between asparagine 256 of the beta chain and the carbonyl oxygen of compound 18 (2.650 Å) and alanine 248 of the beta chain and the hydroxyl oxygen of the C ring of compound 18 (2.368 Å). Despite increased stability due a greater number of hydrogen bonds, compound 18 did not have improved tubulin polymerization inhibition when compared to phenstatin. This suggests the significance of the carbonyl oxygen hydrogen bonding with Lys 252 of the ß chain of tubulin. Despite this, no correlation was determined between compound binding energy and inhibition of MCF-7 cell growth or inhibition of tubulin polymerization, likely due to a low sample size. A strong correlation was found between compound inhibition of tubulin polymerization and MCF-7 cell growth.

Conclusion:

Molecular docking indicates boronic acid bioisosteres of CA-4 have significant potential as mitotic inhibitors and tubulin polymerization inhibitors. The original CA-4 boronic acid derivative, compound 1, and compound 18 were found to dock with greater stability when compared to CA-4 and phenstatin. This docking model mirrored in vitro assessment of compound potency to inhibit tubulin polymerization and MCF 7 cell growth. These clinically relevant novel compounds may provide a new class of anticancer drugs and suggest the importance of investigating the use of boronic acids in drug design. Likewise, this docking model further suggests the relevance of molecular docking to characterize the colchicine binding pocket to design compounds with specificity to residues within the α/ß tubulin heterodimer that may be necessary for antimitotic effects.