The manuscript entitled "X-ray crystallographic and kinetic studies of biguanide containing aryl sulfonamides as Carbonic anhydrase inhibitors" by Baroni C. et al. presents a comprehensive investigation into the design and evaluation of novel inhibitors targeting carbonic anhydrases (CAs), which are crucial enzymes involved in various physiological processes, including acid-base balance, respiration, and fluid secretion. The study is particularly focused on the development of biguanide-containing aryl sulfonamides, which have shown promise as effective CA inhibitors (CAIs). Carbonic anhydrases are metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and protons. They play significant roles in numerous biological processes, making them attractive targets for drug development, particularly in the treatment of conditions such as glaucoma, epilepsy, and cancer. The authors highlight the need for new inhibitors that can selectively target different isoforms of carbonic anhydrases, as existing inhibitors often lack specificity and can lead to side effects and synthesize a series of biguanide-containing aryl sulfonamides, leveraging the structural features of known CA inhibitors to enhance binding affinity and selectivity. The synthesis of these compounds was achieved through established organic chemistry techniques, ensuring high purity and yield.
A stopped-flow kinetic study was performed to evaluate the inhibitory potency of the synthesized compounds against different isoforms of CAs (I, II, VA, IX and XII). The results demonstrated that several of the synthesized biguanide-containing aryl sulfonamides exhibited potent inhibitory activity, with some compounds showing selectivity for hCA XII over hCA II. This selectivity is particularly important for minimizing potential side effects associated with non-specific inhibition. The manuscript also discusses the structure-activity relationship of the synthesized compounds, highlighting how modifications to the chemical structure influenced their inhibitory potency and selectivity. The authors identified specific structural features that contributed to enhanced binding affinity, such as the presence of electron-withdrawing groups and optimal spatial arrangements of functional groups. This information is invaluable for guiding future design efforts aimed at developing even more effective CA inhibitors. To elucidate the binding interactions between the synthesized inhibitor 5b and CA II and XII-mimic, the authors employed X-ray crystallography. The analysis revealed a clear electron density for the benzenesulfonamide group of the inhibitors, confirming their effective binding to the active site of the enzymes. The structural data indicated that the inhibitors formed key interactions with specific amino acid residues, which are critical for their inhibitory activity.
The study concludes that the synthesized biguanide-containing aryl sulfonamides represent a promising class of CAIs with potential therapeutic applications. The combination of X-ray crystallography and kinetic studies provided a robust framework for understanding the mechanisms of inhibition and the factors influencing selectivity. The authors emphasize the importance of continued research in this area, as the development of selective CA inhibitors could lead to significant advancements in the treatment of various diseases.
The manuscript is well-written, well-organized and of great interest. The reviewer suggests it for the publication.

The manuscript entitled "X-ray crystallographic and kinetic studies of biguanide containing aryl sulfonamides as Carbonic anhydrase inhibitors" by Baroni C. et al. presents a comprehensive investigation into the design and evaluation of novel inhibitors targeting carbonic anhydrases (CAs), which are crucial enzymes involved in various physiological processes, including acid-base balance, respiration, and fluid secretion. The study is particularly focused on the development of biguanide-containing aryl sulfonamides, which have shown promise as effective CA inhibitors (CAIs). Carbonic anhydrases are metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and protons. They play significant roles in numerous biological processes, making them attractive targets for drug development, particularly in the treatment of conditions such as glaucoma, epilepsy, and cancer. The authors highlight the need for new inhibitors that can selectively target different isoforms of carbonic anhydrases, as existing inhibitors often lack specificity and can lead to side effects and synthesize a series of biguanide-containing aryl sulfonamides, leveraging the structural features of known CA inhibitors to enhance binding affinity and selectivity. The synthesis of these compounds was achieved through established organic chemistry techniques, ensuring high purity and yield.
A stopped-flow kinetic study was performed to evaluate the inhibitory potency of the synthesized compounds against different isoforms of CAs (I, II, VA, IX and XII). The results demonstrated that several of the synthesized biguanide-containing aryl sulfonamides exhibited potent inhibitory activity, with some compounds showing selectivity for hCA XII over hCA II. This selectivity is particularly important for minimizing potential side effects associated with non-specific inhibition. The manuscript also discusses the structure-activity relationship of the synthesized compounds, highlighting how modifications to the chemical structure influenced their inhibitory potency and selectivity. The authors identified specific structural features that contributed to enhanced binding affinity, such as the presence of electron-withdrawing groups and optimal spatial arrangements of functional groups. This information is invaluable for guiding future design efforts aimed at developing even more effective CA inhibitors. To elucidate the binding interactions between the synthesized inhibitor 5b and CA II and XII-mimic, the authors employed X-ray crystallography. The analysis revealed a clear electron density for the benzenesulfonamide group of the inhibitors, confirming their effective binding to the active site of the enzymes. The structural data indicated that the inhibitors formed key interactions with specific amino acid residues, which are critical for their inhibitory activity.
The study concludes that the synthesized biguanide-containing aryl sulfonamides represent a promising class of CAIs with potential therapeutic applications. The combination of X-ray crystallography and kinetic studies provided a robust framework for understanding the mechanisms of inhibition and the factors influencing selectivity. The authors emphasize the importance of continued research in this area, as the development of selective CA inhibitors could lead to significant advancements in the treatment of various diseases.
The manuscript is well-written, well-organized and of great interest. The reviewer suggests it for the publication.