Search Results (163)

Background/Objectives: The clinically validated multiplex Oncuria bladder cancer (BC) assay quickly and noninvasively identifies disease risk and tracks treatment success by simultaneously profiling 10 protein biomarkers in voided urine samples. Oncuria uses paramagnetic bead-based fluorescence multiplex technology (xMAP^®; Luminex, Austin, TX, USA) to simultaneously measure 10 protein analytes in urine [angiogenin, apolipoprotein E, carbonic anhydrase IX (CA9), interleukin-8, matrix metalloproteinase-9 and -10, alpha-1 anti-trypsin, plasminogen activator inhibitor-1, syndecan-1, and vascular endothelial growth factor]. Methods: In a pilot study (N = 36 subjects; 18 with BC), Oncuria performed essentially identically across three different common analyzers (the laser/flow-based FlexMap 3D and 200 systems, and the LED/image-based MagPix system; Luminex). The current study compared Oncuria performance across instrumentation platforms using a larger study population (N = 181 subjects; 51 with BC). Results: All three analyzers assessed all 10 analytes in identical samples with excellent concordance. The percent coefficient of variation (%CV) in protein concentrations across systems was ≤2.3% for 9/10 analytes, with only CA9 having %CVs > 2.3%. In pairwise correlation plot comparisons between instruments for all 10 biomarkers, R² values were 0.999 for 15/30 comparisons and R² ≥ 0.995 for 27/30 comparisons; CA9 showed the greatest variability (R² = 0.948–0.970). Standard curve slopes were statistically indistinguishable for all 10 biomarkers across analyzers. Conclusions: The Oncuria BC assay generates comprehensive urinary protein signatures useful for assisting BC diagnosis, predicting treatment response, and tracking disease progression and recurrence. The equivalent performance of the multiplex BC assay using three popular analyzers rationalizes test adoption by CLIA (Clinical Laboratory Improvement Amendments) clinical and research laboratories. Full article

Background/Objectives: Human carbonic anhydrases (hCAs) are metalloenzymes involved in essential physiological processes, and their selective inhibition holds therapeutic potential across a wide range of disorders. However, the high degree of structural similarity among isoforms poses a significant challenge for the design of selective inhibitors. In this work, we present a machine learning (ML)-based platform for the isoform-specific prediction and profiling of small molecules targeting hCA I, II, IX, and XII. Methods: By integrating four molecular representations with four ML algorithms, we built 64 classification models, each extensively optimized and validated. The best-performing models for each isoform were applied in a virtual screening campaign for ~2 million compounds. Results: Following a multi-step refinement process, 12 candidates were identified, purchased, and experimentally tested. Several compounds showed potent inhibitory activity in the nanomolar to submicromolar range, with selectivity profiles across the isoforms. To gain mechanistic insights, SHAP-based feature importance analysis and molecular docking supported by molecular dynamics simulations were employed, highlighting the structural determinants of the predicted activity. Conclusions: This study demonstrates the effectiveness of integrating ML, cheminformatics, and experimental validation to accelerate the discovery of selective carbonic anhydrase inhibitors and provides a generalizable framework for activity profiling across enzyme isoforms. Full article

A series of novel derivatives of 3-amino-4-hydroxybenzenesulfonamide was synthesized. As the analyzed compounds possess a sulfonamide group, the affinity of these compounds for human carbonic anhydrases (CAs) was measured by fluorescent thermal shift assay, and compound selectivity for different isoenzymes was identified. The crystal structures of the complexes of compound 25 with CAI and CAII were determined. Additionally, the activity of compounds on the viability of three cancer cell lines—human glioblastoma U-87, triple-negative breast cancer MDA-MB-231, and prostate adenocarcinoma PPC-1—was established using the MTT assay and compared to CAIX-selective and non-selective comparative compounds U-104 and acetazolamide. The half-maximal concentration (EC₅₀) was determined for the identified most active compounds, and their selectivity over fibroblasts was established. Compound 9 (inhibitor of multi-CAs) and compound 21 (not binding to CAs), considered the most promising candidates, were tested in cancer cell 3D cultures (cancer spheroids) by assessing their effect on spheroid growth and viability. Both compounds reduced the viability of spheroids from all cancer cell lines. U-87 and PPC-1 spheroids became looser in the presence of compound 9, while the growth of MDA-MB-231 spheroids was slower compared to the control. Compound 21 reduced the growth of U-87 and MDA-MB-231 3D cultures, with no significant effect on PPC-1 spheroids. Full article

Background/Objectives: The need for dual-targeted enzyme inhibitors is critical in addressing complex diseases like Alzheimer’s and glaucoma. Imidazothiadiazole and chalcone moieties are known for diverse bioactivities. This study aimed to develop novel imidazothiadiazole–chalcone hybrids as potential inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and human carbonic anhydrase isoforms (hCAs), specifically hCA I and hCA II. Methods: Four hybrid molecules (8a–8d) were synthesized and structurally confirmed via ¹H NMR, ¹³C NMR, FT-IR, MS, and elemental analysis techniques. Their enzyme inhibitory activities were assessed using Ellman’s and Verpoorte’s methods. Molecular docking and 100 ns molecular dynamics (MD) simulations were conducted to examine binding interactions. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties were predicted using the pkCSM platform. Results: All compounds showed strong enzyme inhibition: AChE (K_i: 3.86–11.35 nM), BChE (K_i: 1.01–1.78 nM), hCA I (K_i: 45.13–81.24 nM), and hCA II (K_i: 36.08–52.45 nM). Docking analyses confirmed favorable binding, particularly with active-site residues. MD simulations demonstrated stable interactions throughout 100 ns. Compound 8a exhibited the highest cholinesterase inhibition, while compounds 8d and 8c were the most potent against hCA I and hCA II, respectively. The ADMET results showed high absorption and acceptable safety, with mild mutagenicity or cardiotoxicity concerns in select compounds. Conclusions: These findings suggest that imidazothiadiazole–chalcone hybrids are promising multi-target enzyme inhibitors. Their potent activity, structural stability, and pharmacokinetic potential support their further development for therapeutic use in neurodegenerative and ocular diseases. Full article

Saliva was used as non-invasive alternative to blood for diagnosing pathophysiological conditions. This study aimed to assess changes in protein profile in people with obesity after bariatric surgery and to assess the impact of exercise on these changes. The saliva proteome was determined from two-dimensional gels of twenty adults (ten people with normal weight and ten people with obesity). The effects of bariatric surgery and exercise were assessed. A decrease in body weight, body mass index, and waist-to-height ratio was observed after bariatric surgery. Low levels of carbonic anhydrase VI (CA-VI), short palate, lung, and nasal epithelium clone 2 (SPLUNC2), and haptoglobin were observed. One month after bariatric surgery, spots of haptoglobin and SPLUNC2 increased, although one CA-VI spot decreased. Zn-alpha-2 glycoprotein, immunoglobulin chains, and actin-related protein-3, which are high in people with obesity, decreased 1 month after bariatric surgery. Five months after bariatric surgery, the most significant change was the amylase decrease. The exercise-induced changes in salivary proteins increased SPLUNC, CA-VI, type S cystatins, actin cytoplasmic 1, and zinc alpha-2 glycoprotein levels and decrease Ig kappa chain C region and Rab GDP dissociation inhibitor beta. It can be concluded that the salivary proteins change between people with normal weight vs. patients with obesity, as well as after bariatric surgery and exercise programmes. Salivary proteins may be useful biomarkers in non-invasive samples for monitoring and assessing the impact of interventions on people with obesity. Full article

Acetazolamide (AZA) is a validated carbonic anhydrase inhibitor (CAI) that has the potential for use in various therapeutic applications. Herein, we report a novel AZA-loaded biodegradable nanodelivery system that was proven to enhance the antibacterial efficacy of the drug against Gram-negative bacteria, such as Escherichia coli. Carbonic anhydrases (CA, EC 4.2.1.1) in E. coli play a crucial role in bacterial metabolism and CO₂/HCO₃⁻ balance; therefore, they represent a suitable target for antimicrobial strategies. The nanoparticles were obtained using a green synthetic protocol that allowed conjugation of a natural fatty acid to hyaluronic acid (HA) under solvent-free conditions. Full characterization of the micellar aggregates was performed (diameter of the micelles, zeta potential, and drug release study). In vitro studies demonstrated that AZA loaded in HA-based nanoparticles significantly inhibited E. coli growth at concentrations as low as 0.5 µg/mL, whereas higher concentrations of free AZA were required, as previously reported. Additionally, encapsulated AZA disrupted glucose consumption in E. coli, indicating its profound impact on bacterial metabolism. These findings suggest that the HA–palmitate nanoparticle not only enhances the delivery and efficacy of AZA but also offers a strategy to affect bacterial metabolism. Full article

Background/Objectives: α-carbonic anhydrase (α-TcCA) has emerged as a promising drug target in T. cruzi, the causative agent of Chagas disease in the Americas. Sulfonamides, known inhibitors of CAs, bind to the zinc ion on the enzyme’s active site. This study proposes the repositioning of sulfonamide-based drugs to identify new trypanocidal agents. Method: Ligand-based virtual screening and molecular docking analysis were performed on FDA-approved drugs targeting α-TcCA. These compounds were evaluated in vitro and ex vivo against the A1 and NINOA strains, followed by enzymatic assays. Results: Four sulfonylureas were selected: glimepiride (Glim), acetohexamide (Ace), gliclazide (Glic), and tolbutamide (Tol). Ace and Tol had half-maximal inhibitory concentration (IC₅₀) values similar or better than reference drugs against the NINOA strain in the epimastigote and trypomastigote stages, while Glic and Glim had the highest activity against the A1 strain (epimastigotes and amastigotes). Notably, Ace had the highest trypanocidal activity against all stages in NINOA, with IC₅₀ values of 6.5, 46.5, and 46 μM for epimastigotes, trypomastigotes, and amastigotes, respectively. Additionally, Ace inhibited α-TcCA with K_I = 5.6 μM, suggesting that its trypanocidal effect is associated to the enzyme inhibition. Conclusions: This study supports the repositioning of FDA-approved sulfonamide-based hypoglycaemic agents as trypanocidal compounds. Future studies should focus on structural modifications to improve selectivity. Integrating docking, parasitological, and enzymatic data is crucial for optimizing drug candidates for Chagas disease. Full article

In the search for new selective inhibitors of human carbonic anhydrase (hCA), particularly the cancer-associated isoforms hCA IX and hCA XII, a series of 4-substituted pyridine-3-sulfonamides was synthesized using the “click” CuAAC reaction, proven by X-ray crystallography, and evaluated for their inhibitory activity against hCA I, hCA II, hCA IX, and hCA XII. Additional molecular docking studies and cytostatic activity assays on three cancer cell lines were conducted. The compounds exhibited a broad range of inhibitory activity, with K_I reaching 271 nM for hCA II, 137 nM for hCA IX, and 91 nM for hCA XII. Notably, compound 4 demonstrated up to 5.9-fold selectivity toward the cancer-associated hCA IX over the ubiquitous hCA II, while compound 6 exhibited a remarkable 23.3-fold selectivity between transmembrane isoforms hCA IX and hCA XII. Molecular docking studies have shown the possibility of selective interaction with the hydrophilic or lipophilic half of the active site, what results from the adjacent (3,4) position of the “tail” in relation to the sulfonamide group. Full article

Background—Aggressive solid tumors are commonly characterized by both basic intracellular pH and acidic extracellular pH, which increase cell survival and proliferation. As carbonic anhydrases IX/XII are involved in this pH regulation, their inhibition is an appealing approach in cancer therapy, avoiding cancer cell survival and proliferation. Substituted coumarins are selective non-classical CA IX and CA XII inhibitors. Methods—In this study, new 7-hydroxycoumarinamides were synthesized and assayed for CA inhibition and antiproliferative activity. Results—All of the coumarinamides showed human CA IX and CA XII selective inhibition over the off-target CA I and CA II isoforms. Coumarin acts as a suicide inhibitor because its heterocyclic ring can be hydrolyzed by CA esterase activity to give the corresponding 2-hydroxycinnamic acid derivative which blocks the entrance of the active site. The 2-hydroxycinnamic acid derivatives deriving from the most potent and selective coumarinamides were docked into CA IX and XII to better understand the activity and selectivity against the two CA isoforms. The most active coumarinamides also produced a decrease of A549 cell proliferation and were able to arrest cells at the G1/S checkpoint. Conclusions—These results may open new perspectives for developing coumarin-based CA IX/XII inhibitors. Full article

Tendinopathy is often described as a complex and multifactorial condition which affects tendons. Tendon disorders are marked by a reduction in mechanical function, accompanied by pain and swelling. At the molecular level, tendinopathy leads to oxidative stress-driven inflammation, increased cell death, disruption of extracellular matrix balance, abnormal growth of capillaries and arteries, and degeneration of collagen formation. Here, we report an innovative approach to modulate oxidative stress during tendinopathy based on sulfonamide-based Carbonic Anhydrase Inhibitors—carbon monoxide releasing molecules (CAI–CORMs) hybrids endowed with dual carbon monoxide (CO) releasing activity and carbonic anhydrase (CA) inhibition. The synthesised compounds have been studied in a model of human Achilles tendon-derived cells stimulated by H₂O₂. Among the library, compound 1c and, to a greater extent, compound 1a, showed to be extremely effective in terms of restoration of cell metabolic activity and cell proliferation due to their capacity to release CO and inhibit the CA isoforms involved in inflammatory processes in the nanomolar range. Moreover, 1a can restore collagen type 1 secretion under pro-oxidant conditions. Full article

The present study aims to create spiro-N-(4-sulfamoyl-phenyl)-1,3,4-thiadiazole-2-carboxamide derivatives with anticancer activities. The in vitro anticancer evaluation showed that only the novel spiro-acenaphthylene tethered-[1,3,4]-thiadiazole (compound 1) exhibited significant anticancer efficacy as a selective inhibitor of tumor-associated isoforms of carbonic anhydrase. Compound 1 demonstrated considerable efficacy against the renal RXF393, colon HT29, and melanoma LOX IMVI cancer cell lines, with IC₅₀ values of 7.01 ± 0.39, 24.3 ± 1.29, and 9.55 ± 0.51 µM, respectively. In comparison, doxorubicin exhibited IC₅₀ values of 13.54 ± 0.82, 13.50 ± 0.71, and 6.08 ± 0.32 µM for the corresponding cell lines. Importantly, compound 1 exhibited lower toxicity to the normal WI 38 cell line than doxorubicin, with IC₅₀ values of 46.20 ± 2.59 and 18.13 ± 0.93 µM, respectively, indicating greater selectivity of the target compound compared to the standard anticancer agent doxorubicin. Also, mechanistic experiments demonstrated that compound 1 exhibits inhibitory activity against human carbonic anhydrase hCA IX and XII, with IC₅₀ values of 0.477 ± 0.03 and 1.933 ± 0.11 μM, respectively, indicating enhanced selectivity for cancer-associated isoforms over cytosolic isoforms hCA I and II, with IC₅₀ values of 7.353 ± 0.36 and 12.560 ± 0.74 μM, respectively. Cell cycle studies revealed that compound 1 caused G1 phase arrest in RXF393 cells, and apoptosis experiments verified a substantial induction of apoptosis with significant levels of early and late apoptosis, as well as necrosis (11.69%, 19.78%, and 3.66%, respectively), comparable to those induced by the conventional cytotoxic agent doxorubicin, at 9.91%, 23.37%, and 6.16%, respectively. Molecular docking experiments confirmed the strong binding affinity of compound 1 to the active sites of hCA IX and XII, highlighting significant interactions with zinc-binding groups and hydrophobic residues. These findings underscore the target compound’s potential as a viable anticancer agent via targeting CA. Full article

Toxoplasma gondii, the causative agent of toxoplasmosis, is a protozoan parasite capable of infecting a wide range of hosts, posing significant health risks, particularly to immunocompromised individuals and congenital transmission. Current therapeutic options primarily target the active tachyzoite stage but are limited by issues such as toxicity and incomplete efficacy. As a result, there is an urgent need for alternative therapies that can selectively target parasite-specific mechanisms critical for metabolic processes and host–parasite interactions. In this context, α-carbonic anhydrase (Tg_CA), an enzyme essential for T. gondii survival has emerged as a promising drug target. Tg_CA was successfully expressed and purified to evaluate its susceptibility to sulfonamide-based inhibitors, represented by compounds 1–24 and the AAZ–HCT series. These inhibitors demonstrated a broad spectrum of activity, with KI values ranging from 17.8 to 8450 nM. Several compounds exhibited moderate to high potency against Tg_CA; however, concerns regarding selectivity arose because of the inhibition of human isoforms, particularly CA I and CA II. Thus, although some inhibitors showed strong activity against Tg_CA, optimizing selectivity remains crucial for minimizing off-target effects and improving therapeutic efficacy. Further structural modifications may enhance selectivity and advance the development of effective treatments for toxoplasmosis. Full article

Acinetobacter baumannii is a Gram-negative opportunistic pathogen responsible for severe hospital-associated infections. Owing to its ability to develop resistance to a wide range of antibiotics, novel therapeutic strategies are urgently needed. One promising approach is to target bacterial carbonic anhydrases (CAs; EC 4.2.1.1), which are enzymes critical for various metabolic processes. The genome of A. baumannii encodes a β-CA (βAbauCA), which is essential for producing bicarbonate ions required in the early stages of uridine triphosphate (UTP) synthesis, a precursor for the synthesis of peptidoglycans, which are vital components of the bacterial cell wall. This study aimed to inhibit βAbauCA in vitro, with the potential to impair the vitality of the pathogen in vivo. We conducted sequence and structural analyses of βAbauCA to explore its differences from those of human CAs. Additionally, kinetic and inhibition studies were performed to investigate the catalytic efficiency of βAbauCAβ and its interactions with sulfonamides and their bioisosteres, classical CA inhibitors. Our results showed that βAbauCA has a turnover rate higher than that of hCA I but lower than that of hCA II and displays distinct inhibition profiles compared to human α-CAs. Based on the obtained data, there are notable differences between the inhibition profiles of the human isoforms CA I and CA II and bacterial βAbauCA. This could open the door to designing inhibitors that selectively target bacterial β-CAs without affecting human α-CAs, as well as offer a novel strategy to weaken A. baumannii and other multidrug-resistant pathogens. Full article

Carbonic anhydrases (CAs) are a group of zinc-containing enzymes involved in many physiological processes through their role in the maintenance of the equilibrium between bicarbonate and CO₂ levels. Human carbonic anhydrases (hCAs) are recognized as important drug targets due to their major implication in the development of diseases including cancer. Sulfanilamide derivatives have been widely studied and have shown remarkable efficiency in inhibiting carbonic anhydrases, with the presence of SO₂NH₂ in their structure. Therefore, the sulfonamide moiety is considered as the leading scaffold in the search for new hCA inhibitors. Moreover, the introduction of an enaminone to sulfonamide-based CA inhibitors showed an enhancement of inhibitory activity. In this context, we were interested in the in silico investigation of benzenesulfonamide derivatives containing β-enaminone that were synthesized from dicarbonyl compounds and sulfanilamide under microwave irradiation. The in silico assessment includes a molecular docking simulation against hCA II (PDB: 2AW1). The docked ligands showed good docking score values (−8.099 and −7.053 kcal.mol⁻¹), which indicates a good stability of the studied compounds within the active site. Further, significant interactions with the residues of the active site were observed, including metal coordination with Zn 262, an H-bond with Thr 199, and pi–pi stacking with the side chain of His94, which are considered as the key interactions for CA inhibition. A complementary in silico study that involved ADMET prediction was performed to learn more about the pharmacokinetic properties and the toxicity of the products in order to comprehend their ability to become drug-candidates. Full article

Propolis is very significant in terms of its phytochemical content and biological activity among bee products. In this study, the antioxidant activities (total phenolic and flavonoid, Fe³⁺, Cu²⁺ (CUPRAC), Fe³⁺-TPTZ (FRAP) reducing, and DPPH^•, ABTS^•+ scavenging assays) of propolis collected from the Bitlis province of Türkiye were determined. In addition, the carbonic anhydrase I and II isoenzymes (hCA I and hCA II), α-glycosidase, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) inhibition activity and phytochemical profile of propolis and mineral content were determined by LC-MS/MS and ICP-MS, respectively. In propolis, 31 phytochemicals were found, and the highest concentration of acacetin (23.604 mg/g) was detected. It is seen that the phytochemicals in propolis provide antioxidant properties. The mineral content was screened for 18 elements and determined to be rich in the elements that make up the salt content. Total phenolic content was 215.14 mg GAE/g, and total flavonoid content was 79.11 mg QE/g. The Fe³⁺ reduction result was 0.940 (µg/mL), CUPRAC 1.183 (µg/mL), FRAP 0.963 (µg/mL), DPPH^• scavenging IC₅₀: 16.7 (µg/mL), and ABTS IC₅₀: 8.01 (µg/mL). hCA I enzyme inhibition results in IC₅₀: 7.19 (µg/mL), hCA II 8.15, AChE 5.17, BChE 7.50, and α-Glycosidase 5.72. As a result of this study, it was determined that Bitlis propolis has high antioxidant properties and a rich phytochemical content. It was also observed that it is an effective enzyme inhibitor against epilepsy, glaucoma, Alzheimer’s, and diabetes, which are important diseases, and it can be evaluated in the treatment of these diseases and drug production. Full article