Interfering with tumor metabolism is an emerging strategy for treating cancers that are resistant to standard therapies. Featuring a rapid proliferation rate and exacerbated glycolysis, hepatocellular carcinoma (HCC) creates a highly hypoxic microenvironment with excessive production of lactic and carbonic acids. These metabolic conditions promote disease aggressiveness and cancer-related immunosuppression. The pH regulatory molecules work as a bridge between tumor cells and their surrounding milieu. Herein, we show that the pH regulatory molecules CAIX, CAXII and V-ATPase are overexpressed in the HCC microenvironment and that interfering with their pathways exerts antitumor activity. Importantly, the V-ATPase complex was expressed by M2-like tumor-associated macrophages. Blocking ex vivo V-ATPase activity established a less immune-suppressive tumor microenvironment and reversed the mesenchymal features of HCC. Thus, targeting the unique cross-talk between tumor cells and the tumor microenvironment played by pH regulatory molecules holds promise as a strategy to control HCC progression and to reduce the immunosuppressive pressure mediated by the hypoxic/acidic metabolism, particularly considering the potential combination of this strategy with emerging immune checkpoint-based immunotherapies.

Background. Adipose-derived stem cells (ADSCs) are a promising source for the regeneration of bone tissue injuries. At the same time, three-dimensional cultures provide spatial organization of stem cells for optimal intercellular signaling, contact interaction and increase the efficiency of directed osteogenic differentiation prior to further transplantation. The aim of the study was to establish the regenerative potential of mouse adipose-derived stem cells in micromass grafts differentiated into the osteogenic direction to restore the bone injury in mice. Methods. Three-dimensional micromass cultures of murine ADSCs with further differentiation into osteogenic direction were obtained. The migration potential of cells from micromass in vitro and the effectiveness of differentiation by staining for alkaline phosphatase were evaluated. Mice with the model of femoral bone injury were transplanted with ADSCs micromass grafts and 21 days later the lesion site was examined by histological and morphometric methods. Results. The protocols for the cultivation and directed osteogenic differentiation of ADSCs in the three-dimensional micromass culture have been developed. Alkaline phosphatase production was demonstrated in cells that migrated from micromass, confirming the effectiveness of differentiation. In macroscopic examination 21 days after graft transplantation, the defect sites in femur were filled with dense tissue, while in control bones without the use of transplants, the size of the defect by 80 ± 6 % corresponded to the initial diameter and depth of injury. Histological examination of femoral bone lesions in the area of transplantation of micromass grafts revealed the formation of granulation tissue followed by the replacement of defects with newly formed bone tissue with thickening of periosteum and compact bone substance, similar to callus in fracture regeneration. In animals that underwent transplantation of micromass without prior osteogenic differentiation, the diameter of the zone of active regeneration of the diaphysis at the site of injury was 1.3 ± 0.2 mm while in the group with transplantation of directed differentiated graft it was significantly lower (0.37 ± 0.12 mm, p ≤ 0.05). Conclusions. Three-dimensional grafts of adipose-derived multipotent mesenchymal stromal cells cultured in micromass are able to improve bone tissue regeneration in a model of bone injury in mice. In this case, the grafts differentiated into osteogenic direction, provide better morphological indicators of bone recovery, compared with the micromass without prior differentiation.

This study presents the generation of synthetically tractable N-acyl sulfonamide chemical space using a two-step parallel synthetic methodology, providing structurally diverse compounds with high efficiency. The key reaction included the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) combined with primary sulfonamide N-acylation, which proved to be highly effective in generating structurally diverse N-acyl sulfonamides with typically good synthesis success rates (up to 85%) and average yields (up to 61%). The approach demonstrated reliability across different substrates, supporting the efficient construction of a 256-member compound library (out of 73,331 possible combinations) with minor synthetic limitations. Biological evaluations of the synthesized library identified promising inhibitors of human carbonic anhydrase isoforms hCA-IX and hCA-II. Differential scanning fluorimetry and enzymatic assay revealed potent and selective compounds, with some derivatives exhibiting low micromolar IC50 values and noticeable selectivity toward hCA-II. Docking and molecular dynamics simulations provided further insights into binding interactions, confirming the stability of ligand-protein complexes. Thus, this work highlights the synthetic efficiency and pharmacological relevance of 1,2,3-triazole-containing N-acyl sulfonamides, expanding the accessible chemical space for drug discovery.

A synthetically accessible library of N-acyl sulfonamides was constructed using a combination of copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) and N-acylation of primary sulfonamides. The proposed two-step reaction sequence had a high experimentally confirmed synthetic success rate (up to 85%) and gave reasonable product yields (up to 61%). As a result of the validation process, a 262-member compound library (out of >70K accessible combinations) was prepared. Biological profiling of the synthesized library by differential scanning fluorimetry and enzymatic assays identified several low micromolar inhibitors of human carbonic anhydrase. The interaction of the discovered hits with the biological target was studied by docking and molecular dynamics.

N-Acyl sulfonamides are widely recognized carboxylic acid isosteres that can fill the gap related to acidic compounds in chemical catalogs and bioactivity studies. This study presents the generation of synthetically tractable N-acyl sulfonamide chemical space based on the two-step parallel synthetic methodologies yielding structurally diverse compounds with high efficiency. Amide formation and SNAr combined with primary sulfonamide acylation proved to be highly effective in generating structurally diverse N-acyl sulfonamides with typically good synthesis success rates (up to 83%) and average yields (up to 57%). The approach demonstrated reliability across different substrates, supporting the efficient construction of a 526-member compound library (out of 3,258,356 possible combinations) with minor synthetic limitations. Biological evaluations of the synthesized library identified promising inhibitors of human carbonic anhydrase isoforms hCA-IX and hCA-II. Differential scanning fluorimetry screening and enzymatic assays revealed potent and selective compounds, with some derivatives exhibiting low micromolar IC50 values and selectivity towards either hCA-IX or hCA-II. Molecular docking and molecular dynamics simulations provided further insights into binding interactions, confirming the stability of ligand-protein complexes. Thus, this work highlights the synthetic efficiency and pharmacological relevance of N-acyl sulfonamides, expanding the accessible chemical space for drug discovery.