Potent Carbonic Anhydrase Inhibitors Research Articles

Novel benzenesulfonamide-aroylhydrazone conjugates as carbonic anhydrase inhibitors that induce MAPK/ERK-mediated cell cycle arrest and mitochondrial-associated apoptosis in MCF-7 breast cancer cells

A series of novel 4-alkylthio-2-chloro-5-[(2-arylmethylidene)hydrazinecarbonyl]benzenesulfonamide derivatives 3–22 were synthesized and evaluated for their inhibitory activity against human carbonic anhydrase isozymes hCA I, hCA II, hCA IX, and hCA XII. These compounds showed varying degrees of activity against the studied isoenzymes. However, the importance of substituent choice in designing potent carbonic anhydrase inhibitors is highlighted by the strong inhibition profiles of compounds 3 and 10 against hCA IX and the low average KI values for compounds 9 and 10 (134 nM and 77 nM, respectively). All the synthesized compounds were evaluated for their antiproliferative activity toward HeLa, HCT-116, and MCF-7 cell lines. Compounds 9 and 19 exhibited significant activity, particularly against the MCF-7 cell line (IC50 values of 4 μM and 6 μM, respectively). Notably, compound 9 demonstrated a high selectivity index (SI = 8.2) for MCF-7 cells. The antiproliferative effects of compounds 9 and 19 were linked to the induction of cell cycle arrest and apoptosis via the mitochondrial pathway and involved the activation of the MAPK/ERK signaling pathway. Inhibition of MAPK/ERK activity reduced the compounds’ ability to induce cell cycle arrest and apoptosis, indicating the critical role of this pathway. These findings suggest that compounds 9 and 19 are promising candidates for further development as specific and potent anticancer agents targeting the MAPK/ERK pathway.

Synthesis, molecular docking and molecular dynamics simulations, drug-likeness studies, ADMET prediction and biological evaluation of novel pyrazole-carboxamides bearing sulfonamide moiety as potent carbonic anhydrase inhibitors.

Pyrazoles are unique bioactive molecules with a versatile biological profile and they have gained an important place on pharmaceutical chemistry. Pyrazole compounds containing sulfonamide nuclei also attract attention as carbonic anhydrase (CA) inhibitors. In this study, a library of pyrazole-carboxamides were synthesized and the structures of the synthesized molecules were characterized using FT-IR, 1H-NMR, 13C-NMR and HRMS. Then the inhibition effects of newly synthesized molecules on human erythrocyte hCA I and hCA II isoenzymes were investigated. Ki values of the compounds were in the range of 0.063-3.368µM for hCA I and 0.007-4.235µM for hCA II. Molecular docking studies were performed between the most active compounds 6a, 6b and the reference inhibitor, acetazolamide (AAZ) and the hCA I and hCA II receptors to investigate the binding mechanisms between the compounds and the receptors. These compounds showed better interactions than the AAZ. ADMET analyzes were performed for the compounds and it was seen that the compounds did not show AMES toxicity. The stability of the molecular docking results over time was analysed by 50ns molecular dynamics simulations. Molecular dynamics simulations revealed that 6a and 6b exhibited good stability after docking to the binding sites of hCA I and hCA II receptors, with minor conformational changes and fluctuations.

The DFT and in-silico analysis of 2,2′-((1e,1′e)-((3,3′-dimethyl-[1,1′-biphenyl]−4,4′ diyl)bis(azanylylidene))bis(methanylylidene))diphenol molecule

In this study, crystallographic, molecular geometry, surfaces (MEP, Hirshfeld and Frontiers Orbitals-HOMO and LUMO), molecular docking with carbonic anhydrase II-5AML protein, drug-likeness, ADME and vibrational assignment analyses of the molecule were studied in detail and the results were carefully analyzed. The theoretical studies were carried out in DFT/B3LYP/6–311++G(d,p) quantum chemical method and basis set with the Gaussian 09 W package program. Potential energy distributions (PED) were used in conjunction with the VEDA 4 tool to get the vibrational assignments of the title chemical with %10 precision. In solid phase, the FT-IR experimental spectrum was collected at 4000–400 cm−1. Due to the affinity and binding energy of the title molecule to protein active sites, the results showed that our molecule is a potent potential inhibitor of carbonic anhydrase II.

Novel 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide based thiosemicarbazides as potent and selective inhibitors of tumor-associated human carbonic anhydrase IX and XII: Synthesis, cytotoxicity, and molecular modelling studies

In the pursuit of discovering new selective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, a small collection of novel thiosemicarbazides (5a-5t) were designed and synthesized starting from 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide which was evaluated as a potent inhibitor of different CA isoforms in a previous study. The newly synthesized compounds were examined against four human carbonic anhydrases (hCA), namely transmembrane tumor-related hCA IX/XII and cytosolic widespread off-targets hCA I/II. In enzyme inhibition assays, all nineteen compounds display up to ∼340-fold selectivity for hCA IX/XII over off-target isoforms hCA I/II. Four compounds have enzyme inhibition values (Ki) lower than 10 nM against tumor-associated isoforms hCA IX/XII including two compounds in the subnanomolar range (5r and 5s; hCA XII; Ki: 0.69 and 0.87 nM). The potential binding interactions of the most potent compounds against hCA IX and XII, compounds 5s and 5r, respectively, were investigated using ensemble docking and molecular dynamics studies. Cell viability assays using human colorectal adenocarcinoma cell line HT-29 and healthy skin fibroblasts CCD-86Sk show that compound 5e selectively inhibits HT-29 cancer cell proliferation (IC50: 53.32 ± 7.74 µM for HT-29; IC50: 74.64 ± 14.15 µM for CCD-986Sk). Finally, Western blot assays show that compounds 5e and 5r significantly reduce the expression of hCA XII in HT-29 cells. Moreover, 5e shows better cytotoxic activity in hypoxia compared to normoxic conditions. Altogether, the newly designed compounds show stronger inhibition of the tumor-associated hCA IX and XII isoforms and several tested compounds show selective cytotoxicity as well as downregulation of hCA XII expression.

Para-toluenesulfonamide, a novel potent carbonic anhydrase inhibitor, improves hypoxia-induced metastatic breast cancer cell viability and prevents resistance to αPD-1 therapy in triple-negative breast cancer

Overexpression of the hypoxia-induced transmembrane enzyme carbonic anhydrase IX (CA9) has been associated with poor prognosis and chemoresistance in aggressive breast cancer. This study aimed to investigate the involvement of CA9 in the anti-tumor activity of para-toluenesulfonamide (PTS) and elucidate its mechanism of action against breast cancer both in vitro and in vivo. MCF-7 and MDA-MB-231 breast cancer cells were treated with PTS or subjected to hypoxic conditions using cobalt chloride (CoCl2), with acetazolamide serving as a positive control. Additionally, 4T1 breast cancer cell allograft mice were co-treated with PTS and α-programmed cell death 1 (αPD-1) monoclonal antibody for one month. The results demonstrated that PTS effectively reduced cell viability and reversed migration ability in MCF-7 and MDA-MB-231 cells under CoCl2-induced hypoxia. Furthermore, PTS upregulated the expression of apoptosis-related proteins and downregulated CA9, hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial growth factor (VEGF) proteins, possibly through modulation of p38 MAPK and ERK1/2 phosphorylated proteins. In the animal model, PTS100 inhibited tumor growth and lung metastasis in mammary tumor allograft mice, exhibiting synergistic effects when combined with αPD-1 therapy. Collectively, our findings suggest that PTS inhibits breast cancer growth and metastasis through the p38 MAPK/ERK1/2 pathway. Moreover, PTS may have the potential to prevent the development of resistance to αPD-1 therapy in breast cancer.

Exploring novel anticancer pyrazole benzenesulfonamides featuring tail approach strategy as carbonic anhydrase inhibitors

This study aimed to design potent carbonic anhydrase inhibitors (CAIs) based on pyrazole benzenesulfonamide core. Nine series of substituted pyrazole benzenesulfonamide compounds were synthesized with variable groups like sulphamoyl group as in compounds 4a-e, its bioisosteric carboxylic acid as in compounds 5a-e and 8e, ethyl carboxylate ester as in compounds 6a-e and 9a-e, which were designed as potential prodrugs, isothiazole ring as in compound 7, hydrazide derivative 10e, hydroxamic acid derivatives 11a-e and semicarbazide derivatives 12a-c,e. All the synthesized compounds were investigated for their carbonic anhydrase (CA) inhibitory activity against two human CA isoforms hCA IX and hCA XII and compared to acetazolamide (AAZ). Also, the compounds were assessed for their anticancer activity against 60 cancer cell lines according to the US NCI protocol. Compounds 4b, 5b, 5d, 5e, 6b, 9b, 9e and 11b revealed significant inhibitory activity against both isoforms hCA IX and hCA XII, while 6e, 9d, 11d and 11e showed significant inhibitory activity against hCA XII only compared to acetazolamide as a reference. This would highlight these compounds as promising anticancer drugs. Moreover, compound 6e revealed a remarkable cytostatic activity against CNS cancer cell line (SF-539; TGI = 5.58 μM), renal cancer cell line (786–0; TGI = 4.32 μM) and breast cancer cell line (HS 578 T; TGI = 5.43 μM). Accordingly, compound 6e was subjected to cell cycle analysis and apoptotic assay on the abovementioned cell lines at the specified GI50 (0.45, 0.89 and 1.18 μM, respectively). Also, it revealed the increment of total apoptotic cells percentage in 786–0 (53.19%), SF-539 (46.11%) and HS 578 T (43.55%) relative to the control cells (2.07, 2.64 and 2.52%, respectively). In silico prediction of BBB permeability showed that most of the calculations for compound 6e resulted as BBB (+), which is required for a compound targeting CNS. Further, the interaction of the most active compounds with the key amino acids in the active sites of hCA IX and hCA XII was highlighted by molecular docking analysis.

Discovery of a novel series of potent carbonic anhydrase inhibitors with selective affinity for μ Opioid receptor for Safer and long-lasting analgesia

In this study, we investigated the development of dual-targeted ligands that bind to both μ-opioid receptor (MOR) and carbonic anhydrase (CA) enzymes, using fentanyl structure as a template. We synthesized and evaluated 21 novel compounds with dual-targeted affinity identifying the lead candidate compound 8, showing selective affinity for MOR and potent inhibition of several cytosolic CA isoforms. By means of repeated treatment of 3 daily administrations for 17 days, fentanyl (0.1 mg/kg, subcutaneously) led to tolerance development, pain threshold alterations and withdrawal symptoms in CD-1 mice, as well as astrocyte and microglia activation in the dorsal horn of the lumbar spinal cord. In contrast, compound 8 (0.32 mg/kg s.c.) maintained stable during days its analgesic effect at the higher dose tested with fewer withdrawal symptoms, allodynia development and glial cells activation. Our results suggest that targeting both MOR and CA enzymes can lead to the development of new class of potent analgesic agents with fewer side effects and reduced tolerance development. Further studies are needed to explore the potential mechanisms underlying these effects and to further optimize the therapeutic potential of these compounds.

6-Aminocoumarin oxime-ether/sulfonamides as selective hCA IX and XII inhibitors: Synthesis, evaluation, and molecular dynamics studies.

Carbonic anhydrase isoforms IX and XII are overexpressed in hypoxic tumor cells regulating various physiological processes such as cell proliferation, invasion, and metastasis, resulting in the onset and spread of cancer. Selective inhibition of these enzymes is a promising strategy for anticancer therapy. Coumarin derivatives were identified as selective and potent inhibitors of these isoforms. This study reports 6-aminocoumarin sulfonamide and oxime ether derivatives linked through a chloroacetyl moiety tethered to piperazine and piperidone, respectively, showing selective inhibition against human carbonic anhydrase (hCA) IX and XII with Ki ranging from 0.51 to 1.18 µM and 0.89-4.43 µM. While the sulfonamide derivative 8a exhibited submicromolar inhibition against hCA IX and XII with Ki 0.89 and 0.51 µM, the oxime ether derivatives showed lower activity than the sulfonamides, with the compound 5n inhibiting hCA IX and hCA XII with a Ki of 1.055 and 0.70 µM, respectively. The above results demonstrate the potential of these derivatives as selective, potent inhibitors of carbonic anhydrase IX and XII and provide a foundation for further optimization and development as effective anticancer agents. Further, the binding mode of the synthesized derivatives in the active site were examined using molecular docking and dynamic simulation studies.

Identification of specific carbonic anhydrase inhibitors via in situ click chemistry, phage-display and synthetic peptide libraries: comparison of the methods and structural study.

The development of highly active and selective enzyme inhibitors is one of the priorities of medicinal chemistry. Typically, various high-throughput screening methods are used to find lead compounds from a large pool of synthetic compounds, and these are further elaborated and structurally refined to achieve the desired properties. In an effort to streamline this complex and laborious process, new selection strategies based on different principles have recently emerged as an alternative. Herein, we compare three such selection strategies with the aim of identifying potent and selective inhibitors of human carbonic anhydrase II. All three approaches, in situ click chemistry, phage-display libraries and synthetic peptide libraries, led to the identification of more potent inhibitors when compared to the parent compounds. In addition, one of the inhibitor-peptide conjugates identified from the phage libraries showed greater than 100-fold selectivity for the enzyme isoform used for the compound selection. In an effort to rationalize the binding properties of the conjugates, we performed detailed crystallographic and NMR structural analysis, which revealed the structural basis of the compound affinity towards the enzyme and led to the identification of a novel exosite that could be utilized in the development of isoform specific inhibitors.

Indoline-5-Sulfonamides: A Role of the Core in Inhibition of Cancer-Related Carbonic Anhydrases, Antiproliferative Activity and Circumventing of Multidrug Resistance.

The overexpression and activity of carbonic anhydrase (CA, EC 4.2.1.1) isoforms CA IX and CA XII promote the accumulation of exceeding protons and acidosis in the extracellular tumor environment. Sulfonamides are effective inhibitors of most families of CAs. In this study, using scaffold-hopping, indoline-5-sulfonamide analogs 4a-u of the CA IX-selective inhibitor 3 were designed and synthesized to evaluate their biological properties. 1-Acylated indoline-5-sulfonamides demonstrated inhibitory activity against tumor-associated CA IX and XII with KI values up to 132.8 nM and 41.3 nM. Compound 4f, as one of the most potent inhibitors of CA IX and XII, exhibits hypoxic selectivity, suppressing the growth of MCF7 cells at 12.9 µM, and causes partial inhibition of hypoxia-induced CA IX expression in A431 skin cancer cells. 4e and 4f reverse chemoresistance to doxorubicin of K562/4 with overexpression of P-gp.

Design and synthesis of some new benzoylthioureido phenyl derivatives targeting carbonic anhydrase enzymes

The present study aimed to develop potent carbonic anhydrase inhibitors (CAIs). The design of the target compounds was based on modifying the structure of the ureido-based carbonic anhydrase inhibitor SLC-0111. Six series of a substituted benzoylthioureido core were prepared featuring different zinc-binding groups; the conventional sulphamoyl group 4a–d and 12a–c, its bioisosteric carboxylic acid group 5a–d and 13a–c or the ethyl carboxylate group 6a–d and 14a–c as potential prodrugs. All compounds were assessed for their carbonic anhydrase (CA) inhibitory activity against a panel of four physiologically relevant human CA isoforms hCA I and hCA II, and hCA IX, and hCA XII. Compounds 4a, 4b, 4c, 4d, 5d, 12a, and 12c revealed significant inhibitory activity against hCA I that would highlight these compounds as promising drug candidates for the treatment of glaucoma.

Design and synthesis of benzothiazole-based SLC-0111 analogues as new inhibitors for the cancer-associated carbonic anhydrase isoforms IX and XII

In this work, different series of benzothiazole-based sulphonamides 8a-c, 10, 12, 16a-b and carboxylic acids 14a-c were developed as novel SLC-0111 analogues with the goal of generating potent carbonic anhydrase (CA) inhibitors. The adopted strategy involved replacing the 4-fluorophenyl tail in SLC-0111 with a benzothiazole motif that attached to the ureido linker to produce compounds 8c and its regioisomers 8a-b. In addition, the ureido spacer was elongated by methylene or ethylene groups to afford the counterparts 10 and 12. In turn, the primary sulfamoyl zinc binding group (ZBG) was either substituted or replaced by carboxylic acid functionality in order to provide the secondary sulphonamide-based SLC-0111 analogues 16a-b, and the carboxylic acid derivatives 14a-c, respectively. All compounds (8a-c, 10, 12, 14a-c and 16a-b) were tested for their ability to inhibit CA isoforms CA I, II, IX and XII. Additionally, the in vitro anticancer properties of the developed CAIs were evaluated.

Synthesis and Biological Evaluation of Coumarin Carboxamides as Selective and Potent Inhibitors of Carbonic Anhydrases IX and XII

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the reversible hydration of carbon dioxide to bicarbonate and proton. Inhibition of isoforms IX and XII could aid in the amelioration of cancer. A series of coumarin carboxamides (6a-j) were synthesized and were assayed against hCA isoforms I, II, IX, and XII. Thin Layer Chromatography (TLC) analysis was done by utilizing Merck silica gel 60 F254 aluminum plates. Stuart Digital Melting Point Apparatus (SMP 30) was used in determining the melting points of the compounds, which are uncorrected. High Resolution Mass Spectra (HRMS) were determined by Agilent QTOF mass spectrometer 6540 series instrument and were performed using ESI techniques at 70eV. All the compounds selectively inhibited isoforms IX and XII as against hCAs I and II. Compounds 6a-e exhibited the best inhibitory profiles against hCA IX (Ki < 25 nM). The isoform hCA XII was effectively inhibited by all compounds showing the Ki values less than 65 nM. The Compounds 6a, 6b, 6g, 6h, and 6j exhibited Ki values less than 10 nM. The binding interactions of the most potent compounds, 6a and 6b, were investigated through docking studies with hCAs IX and XII. These compounds may be utilized as useful starting points for the design and development of selective and potent hCA IX and XII inhibitors.

Metal Chelators Ethylenediaminetetraacetic acid (EDTA) and N,N,N′,N′‐tetrakis(2‐pyridinylmethyl)‐1,2‐ethanediamine (TPEN) Interfere with Carbonic Anhydrase (CA) Activity in Wilbur‐Anderson Assay (WAa)

In our effort to set up a laboratory for undergraduate students with the tentative title ”Studying enzymatic activity of Carbonic Anhydrase”, we successfully determined carbonic anhydrase activity and demonstrated its inhibition by Acetazolamide, a potent CA inhibitor, using the Wilbur‐Anderson assay (WAa) using 6 experimental set‐ups with a class of 24 students.WAa hinges upon determining the time T (in seconds) required for 4 mL of saturated with carbon dioxide (CO2) water to lower pH from 8.3 to 6.3 of the mixture when added to 6 mL of 0.02M Tris buffer at 0°C without (T0) and with enzyme (TE). Activity (A) is defined as A = 2*(T0/TE‐1) assay units (AU). In n = 16 blank trials, T0 = 75.0±10.7s (Mean±Standard deviation), and with 0.001mg of CA in n = 12 trials, TE = 50.2±8.1s, A = 0.99±0.66 AU (p < 0.001). When for a test we used EDTA, a carboxylic high‐affinity metal ion chelator, to inactivate this Zn2+‐containing enzyme, we unexpectedly encountered that in the presence of the sodium salt of EDTA (Na2EDTA): 1) T was significantly slowed in a concentration‐dependent manner: for 5µM, 50µM, and 500µM, T was 84.3±7.9s (n = 10, p < 0.05), 108.5±.6s (n = 7, p < 0.001), and 130.8±22.7s (n = 10, p < 0.001), respectively; and 2) unexpectedly, in the context of the previous finding, Na2EDTA significantly enhanced CA activity (TE = 38.5±4.0s, A = 1.84 AU, n = 10, p < 0.001) in the presence of 5µM Na2EDTA that was abrogated by 10µM Acetazolamide, a potent CA inhibitor (T = 138.3±10.4s, n = 7, p < 0.001). TPEN, a pyridinic non‐carboxylic metal ion chelator that is more potent in metal ion chelation that EDTA, significantly slowed T0 (99.5±15.9s, n = 4) similar to Na2EDTA, and significantly enhance CA activity (TE = 41.1±3.8, n = 4, p < 0.05) with equal potency.This indicates that principally chemically different metal ion chelators EDTA and TPEN interfere with the determination of CA activity in the context of WAa. Studies to explain these phenomena are underway with the hypotheses that the contaminating metal ions that catalyze CO2 hydration/dehydration without CA are responsible for the observed phenomena.

Evaluation of indole-picolinamide hybrid molecules as carbonic anhydrase-II inhibitors: Biological and computational studies

The present study reports the successful synthesis and evaluation of a series of indole-picolinamide hybrids as potent inhibitors of bovine carbonic anhydrase II, a promising therapeutic target for the treatment of neurological disorders, osteoporosis, glaucoma, cancer, and obesity. Various multistep synthetic approaches were utilized to access the desired structures having exclusive sites for chemical modifications and diverse spots for biological interactions between the ligand and enzyme. Compound 1a was observed as a potent inhibitor of the bovine CA-II with an IC50 value of 0.0440 ± 0.009 µM. The inhibition potency was about 22-fold higher than the standard drug acetazolamide (IC50 = 0.9618 ± 0.180 µM). Several impactful structure-activity relationships were deduced that enlighten the crucial role of substituents as well as the key pharmacophores involved in the inhibition process. Molecular docking tools reinforced the in vitro assay results. The most active compound 1a was also investigated for anticancer potential using sulforhodamine B assay and results showed two-fold efficacy against HeLa cells when compared to standard drug cisplatin (IC50 = 8.045 ± 3.791 µg/mL and 4.128 ± 1.473 µg/mL, respectively). These findings were also confirmed by flow cytometry and comet tests. Moreover, fluorescence microscopy with DAPI staining revealed apoptosis as a mechanism of cancer cell death.

A family of amphiphilic dioxidovanadium(V) hydrazone complexes as potent carbonic anhydrase inhibitors along with anti-diabetic and cytotoxic activities.

A family of dioxidovanadium(V) complexes (1-4) of the type [Na(H2O)x]+[VVO2(HL1-4)]- (x = 4, 4.5 and 7) where HL2- represents the dianionic form of 2-hydroxybenzoylhydrazone of 2-hydroxyacetophenone (H2L1, complex 1), 2-hydroxy-5-methylacetophenone (H2L2, complex 2), 2-hydroxy-5-methoxyacetophenone (H2L3, complex 3) and 2-hydroxy-5-chloroacetophenone (H2L4, complex 4), have been synthesized and characterized by analytical and spectral methods. These complexes exhibited the potential abilities to suppress the erythrocytes carbonic anhydrase enzymatic activity in type 1 and type 2 diabetic patients (in vitro), promising antidiabetic activity against T2 diabetic mice (in vivo). They also exhibited significant cytotoxic activity against cervical cancer (SiHa) cells (in vitro) as the IC50 value of complexes 1, 2 and 4 is substantially lower than the value found for cisplatin while that of 3 is comparable and follow the order: 4 < 1 < 2 < 3 and can kill the cells by apoptosis via the generation of reactive oxygen species (ROS). The complexes are soluble both in water and octanol media and also non-toxic at working concentrations. The antidiabetic activity of these four complexes follows the order: 4 > 2 > 1 > 3 while both the carbonic anhydrase and cytotoxic activity follow the order: 4 > 1 > 2 > 3 suggesting that complex 4, containing electron withdrawing Cl atom is the most reactive while 3 with electron donating OCH3 group is the least reactive species. The molecular docking study on hCA-I and hCA-II demonstrates that complexes interact via hydrogen bonding as well as different types of π-stacking.

Synthesis, carbonic anhydrase inhibition, anticancer activity, and molecular docking studies of 1,3,4-oxadiazole derivatives.

In this work, we have synthesized various organic compounds possessing 1,3,4-oxadiazole as a core structure and the structure of the newly synthesized target compounds has been revealed using different analytical approaches such as FT-IR, LCMS, and NMR (proton and carbon), respectively. The in vitro carbonic anhydrase potentials of these synthesized 17 different analogues were investigated. The result suggests that compound 7g, a 3-pyridine substituted analogue with an IC50 of 0.1µM, was found to have the most potent carbonic inhibitory activity (11-fold more active) than the positive control (acetazolamide) with an IC50 of 1.1 ± 0.1µM. Besides, among the series 7(a-q) approved in the identification of four potent carbonic anhydrase inhibitors with the IC50 standards varies from 0.1 to 1.0 ± 0.1µM. Additionally, the non-competitive behaviour for potent compound 7g was analysed using the Lineweaver-Burk plot from the kinetic study. Furthermore, the anticancer activity of all the synthesized compounds screened against B16F10 melanoma cells using the MTT assay method. Additionally, the molecular docking studies revealed that 7g inhibitor shows good binding energy as well as good binding interaction pattern along with enzyme.

4-[1-Ethyl-1-methylhexy]-phenol induces apoptosis and interrupts Ca2+ homeostasis via ROS pathway in Sertoli TM4 cells.

Biological effect of an individual nonylphenol (NP) isomer extremely relies upon the side chain structure. This research was designed to evaluate the impact of NP isomer, 4-[1-ethyl-1-methylhexy]-phenol (NP65), on Sertoli cells in vitro. Sertoli TM4 cells were exposed to various concentration (0, 0.1, 1, 10, or 20μM) of NP65 for 24h, and the outcomes indicated that treatment of NP65 induced reactive oxygen species (ROS) generation, oxidative stress, and apoptosis for Sertoli TM4 cells. In addition, it was found that NP65 exposure affected homeostasis of Ca2+ in Sertoli TM4 cells by increasing cytoplasm [Ca2+]i, inhibiting Ca2+-ATPase activity and decreasingcyclic adenosine monophosphate (cAMP) concentration. Pretreatment with ROS scavenger, N-acetylcysteine (NAC), attenuated NP65-induced oxidative stress as well as apoptosis for TM4 cells. Furthermore, NAC blocked NP65-induced disorders of Ca2+ homeostasis by attenuating the growth of intracellular [Ca2+]i and the inhibition of Ca2+-ATPase and cAMP activities. Thus, we have demonstrated that NP65 induced apoptosis as well as acted as a potent inhibitor of Ca2+-ATPase activity and resulted in disorder of Ca2+ homeostasis in Sertoli TM4 cells; ROS participated in the process. Our results supported the view that oxidative stress acted an essential role within the development of apoptosis and Ca2+ overload in TM4 cells as a consequence of NP65 stimulation.

Discovery of Kinase and Carbonic Anhydrase Dual Inhibitors by Machine Learning Classification and Experiments.

A multi-target small molecule modulator is advantageous for treating complicated diseases such as cancers. However, the strategy and application for discovering a multi-target modulator have been less reported. This study presents the dual inhibitors for kinase and carbonic anhydrase (CA) predicted by machine learning (ML) classifiers, and validated by biochemical and biophysical experiments. ML trained by CA I and CA II inhibitor molecular fingerprints predicted candidates from the protein-specific bioactive molecules approved or under clinical trials. For experimental tests, three sulfonamide-containing kinase inhibitors, 5932, 5946, and 6046, were chosen. The enzyme assays with CA I, CA II, CA IX, and CA XII have allowed the quantitative comparison in the molecules’ inhibitory activities. While 6046 inhibited weakly, 5932 and 5946 exhibited potent inhibitions with 100 nM to 1 μM inhibitory constants. The ML screening was extended for finding CAs inhibitors of all known kinase inhibitors. It found XMU-MP-1 as another potent CA inhibitor with an approximate 30 nM inhibitory constant for CA I, CA II, and CA IX. Differential scanning fluorimetry confirmed the direct interaction between CAs and small molecules. Cheminformatics studies, including docking simulation, suggest that each molecule possesses two separate functional moieties: one for interaction with kinases and the other with CAs.

Synthesis, molecular modelling and QSAR study of new N-phenylacetamide-2-oxoindole benzensulfonamide conjugates as carbonic anhydrase inhibitors with antiproliferative activity

In continuation of our previous studies to optimise potent carbonic anhydrase inhibitors, two new series of isatin N-phenylacetamide based sulphonamides were synthesised and screened for their human (h) carbonic anhydrase (EC 4.2.1.1) inhibitory activities against four isoforms hCA I, hCA II, hCA IX and hCA XII. The indole-2,3-dione derivative 2h showed the most effective inhibition profile against hCAI and hCA II (KI = 45.10, 5.87 nM) compared to acetazolamide (AAZ) as standard inhibitor. Moreover, 2h showed appreciable inhibition activity against the tumour-associated hCA XII, similar to AAZ showing KI of 7.91 and 5.70 nM, respectively. The analogs 3c and 3d showed good cytotoxicity effects, and 3c revealed promising selectivity towards lung cell line A549. Molecular docking was carried out for 2h and 3c to predict their binding conformations and affinities towards the hCA I, II, IX and XII isoforms.