Development of pyrazolo[1,5-a]pyrimidine-grafted coumarins as selective carbonic anhydrase inhibitors and tubulin polymerization inhibitors with potent anticancer activity

Article

作者: Nocentini, Alessio ; Eldehna, Wagdy M ; Abdel-Aziz, Hatem A ; Abdelrahman, Mohamed A ; Elkotamy, Mahmoud S ; Alkabbani, Mahmoud Abdelrahman ; Giovannuzzi, Simone ; Supuran, Claudiu T ; Abou-Seri, Sahar M

This study presents the design, synthesis, and evaluation of a novel series of coumarin-based compounds (9a-t) as potential anticancer agents. The compounds were strategically designed to inhibit cancer-related carbonic anhydrase (CA) isoforms IX and XII and tubulin polymerization. Two approaches were employed for CA inhibition: utilizing the coumarin motif to occlude the CA active site entrance and incorporating zinc-binding groups (sulfonamide, carboxylic acid, and thiol) to interact with the catalytic zinc ion. The target compounds were also designed to inhibit tubulin polymerization by combining the privileged coumarin and pyrazolo[1,5-a]pyrimidine scaffolds. Biological evaluation of the target compounds (9a-t) revealed that sulfonamide-containing derivatives 9h and 9r exhibited potent inhibitory activity in the low nanomolar range against CA IX (K_i = 23 and 14 nM, respectively) and CA XII (K_i = 6 and 17 nM, respectively). In NCI-60 human tumor cell line screening, compounds 9k, 9m, and 9q demonstrated broad-spectrum anti-proliferative activity in the five-dose assay with MG-MID values of 7.31 μM, 10.68 μM, and 5.92 μM, respectively. Compound 9m showed significant tubulin polymerization inhibition with an IC₅₀ = 5.28 μM, surpassing the efficacy of colchicine. Cell cycle analysis in MDA-MB-231 breast cancer cells revealed G2/M phase arrest for 9m, which induced significant apoptosis and modulated apoptotic markers. Molecular docking studies provided insights into the binding modes of the compounds with CA IX, CA XII, and tubulin. ADMET and toxicity predictions were performed to assess the drug-like properties of the compounds. These findings pave the way for further optimization of the coumarin scaffold to develop dual inhibitors of carbonic anhydrase IX/XII and tubulin polymerization.

2025-03-19·Bioconjugate Chemistry

Molecular Determinants of Affinity and Isoform Selectivity in Protein─Small Molecule Hybrid Inhibitors of Carbonic Anhydrase

Article

作者: Collins, Jon ; Hackel, Benjamin J. ; Schmitz, Zachary ; Blanchard, Paul L. ; Lan, Crystal ; Torres, Sarah W. ; Harthorn, Abbigael

Multiple studies have demonstrated the benefit of engineering hybrid ligands that combine the unique benefits of small molecules and proteins or peptides. However, the molecular complexity of hybrid ligands generates a parameter space so large it cannot be exhaustively explored. We systematically evaluated the impact of one molecular design element, conjugation site, on the discovery of functional protein-small molecule hybrids (PriSMs). We utilized a library of yeast-displayed fibronectin domain variants with amino acid and loop length diversity in the paratope and a single cysteine at one of 18 possible conjugation sites. The protein variants were coupled with maleimide-functionalized acetazolamide and sorted via competitive flow cytometry to discover potent and selective inhibitors of three isoforms of carbonic anhydrase. Deep sequencing of the resultant populations of functional PriSMs revealed an isoform-dependent distribution of conjugation site preferences. The top PriSMs showed potency and selectivity gains up to 23- and 100-fold (in this case, for CA-II vs CA-XII, with a 43-fold selectivity gain for CA-II vs CA-IX) relative to PEG₂-acetazolamide alone. The presented study expands our fundamental understanding of the role of conjugation site in PriSM function and informs future PriSM engineering efforts by highlighting the benefit of conjugation site diversity in PriSM libraries.

2025-03-01·Biochemical and Biophysical Research Communications

Molecular dynamics simulation reveals structural insights into isozyme selectivity of carbonic anhydrase XII inhibitors in hypoxic tumor microenvironment

Article

作者: Gao, Quan-Ze ; Saravanan, Venkatesan ; Kumaradoss, Kathiravan Muthu ; Palani, Sathiya Priya ; Valsaladevi, Anjana Gopi ; Chagaleti, Bharath Kumar

Human carbonic anhydrase (CA) isoenzymes IX and XII are overexpressed in cancer cells, contributing to tumor microenvironment acidification and representing important targets for cancer therapy. In this study, we identified compound V35 (ZINC09419065) as a selective inhibitor of CA IX and CA XII with enhanced binding stability and selectivity compared to standard inhibitors. We analyzed conserved regions in CA I, CA II, CA IX, and CA XII to investigate their isozyme selectivity, revealing critical selectivity determinants at positions 95, 141, and 203. Molecular docking results indicated that V35 interacts robustly with CA XII, forming a metal ion coordination complex with Zn via HIS94, HIS96, HIS119, and THR199, similar to the interaction pattern of standard inhibitor SLC-0111. Molecular dynamics (MD) simulations conducted over 500 ns under hypoxic conditions showed that V35 has high binding stability, with root mean square deviation (RMSD) and fluctuation (RMSF) values comparable to SLC-0111, demonstrating its conformational stability in CA XII. Binding free energy calculations using the MMGBSA method showed that V35 achieves binding free energy of -44.17 kcal/mol with CA XII, closely matching SLC-0111 (-49.41 kcal/mol). Density functional theory (DFT) calculations further highlighted V35's electrostatic potential distribution, supporting its isozyme selectivity. Post-dynamics analysis indicated that the ester functional groups and the inward movement of HIS64 stabilize V35's interactions in CA XII, a feature absent in CA I.

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