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Aim: To identify potential biomarkers and explore the mechanisms underlying diabetic nephropathy (DN) by integrating machine learning, Mendelian randomization (MR) and experimental validation. Methods: Microarray and RNA‐sequencing datasets (GSE47184, GSE96804, GSE104948, GSE104954, GSE142025 and GSE175759) were obtained from the Gene Expression Omnibus database. Differential expression analysis identified the differentially expressed genes (DEGs) between patients with DN and controls. Diverse machine learning algorithms, including least absolute shrinkage and selection operator, support vector machine‐recursive feature elimination, and random forest, were used to enhance gene selection accuracy and predictive power. We integrated summary‐level data from genome‐wide association studies on DN with expression quantitative trait loci data to identify genes with potential causal relationships to DN. The predictive performance of the biomarker gene was validated using receiver operating characteristic (ROC) curves. Gene set enrichment and correlation analyses were conducted to investigate potential mechanisms. Finally, the biomarker gene was validated using quantitative real‐time polymerase chain reaction in clinical samples from patients with DN and controls. Results: Based on identified 314 DEGs, seven characteristic genes with high predictive performance were identified using three integrated machine learning algorithms. MR analysis revealed 219 genes with significant causal effects on DN, ultimately identifying one co‐expressed gene, carbonic anhydrase II (CA2), as a key biomarker for DN. The ROC curves demonstrated the excellent predictive performance of CA2, with area under the curve values consistently above 0.878 across all datasets. Additionally, our analysis indicated a significant association between CA2 and infiltrating immune cells in DN, providing potential mechanistic insights. This biomarker was validated using clinical samples, confirming the reliability of our findings in clinical practice. Conclusion: By integrating machine learning, MR and experimental validation, we successfully identified and validated CA2 as a promising biomarker for DN with excellent predictive performance. The biomarker may play a role in the pathogenesis and progression of DN via immune‐related pathways. These findings provide important insights into the molecular mechanisms underlying DN and may inform the development of personalized treatment strategies for this disease. [ABSTRACT FROM AUTHOR]

A small library of 79 substituted phenylsulfonamidoalkyl sulfamates, 1b–79b, was synthesized starting from arylsulfonyl chlorides and amino alcohols with different numbers of methylene groups between the hydroxyl and amino moieties yielding intermediates 1a–79a, followed by the reaction of the latter with sulfamoyl chloride. All compounds were screened for their inhibitory activity on bovine carbonic anhydrase II. Compounds 1a–79a showed no inhibition of the enzyme, in contrast to sulfamates 1b–79b. Thus, the inhibitory potential of compounds 1b–79b towards this enzyme depends on the substituent and the substitution pattern of the phenyl group as well as the length of the spacer. Bulkier substituents in the para position proved to be better for inhibiting CAII than compounds with the same substituent in the meta or ortho position. For many substitution patterns, compounds with shorter spacer lengths were superior to those with long chain spacers. Compounds with shorter spacer lengths performed better than those with longer chain spacers for a variety of substitution patterns. The most active compound held inhibition constant as low as Ki= 0.67 µM (for 49b) and a tert-butyl substituent in para position and acted as a competitive inhibitor of the enzyme. [ABSTRACT FROM AUTHOR]

A small library of arylsulfonamido-alkyl sulfamates was prepared by a two-step synthesis from readily available starting materials. The compounds were tested for their ability to inhibit bovine carbonic anhydrase II. Several of them were found as good competitive inhibitors holding Ki values as low as Ki = 0.9 μM (compound 47b). The activity was influenced by the substitution pattern of the arylsulfonamide moiety as well as the length of the spacer to the distal sulfamate group. Molecular docking studies were used to substantiate these findings. For the aryl-substituted analogues, the increase in inhibitory activity for compounds with a shorter spacer can be explained by stabilization via aromatic π-interactions. For the cyclopropyl or methylsulfonyl substituted analogues, their inhibitory activity can be attributed to their reduced steric hindrance. These results provide a basis for designing effective CA II inhibitors.

Human carbonic anhydrase II (hCAII) is a metalloenzyme essential to critical physiological processes in the body. hCA inhibitors are used clinically for the treatment of indications ranging from glaucoma to epilepsy. Targeted protein degraders have emerged as a promising means of inducing the degradation of disease-implicated proteins by using the endogenous quality control mechanisms of a cell. Here, a series of heterobifunctional degrader candidates targeting hCAII were developed from a simple aryl sulfonamide fragment. Degrader candidates were functionalized to produce either cereblon E3 ubiquitin ligase (CRBN) recruiting proteolysis targeting chimeras (PROTACs) or adamantyl-based hydrophobic tags (HyTs). Screens in HEK293 cells identified two PROTAC small-molecule degraders of hCA. Optimization of linker length and composition yielded a degrader with sub-nanomolar potency and sustained depletion of hCAII over prolonged treatments. Mechanistic studies suggest that this optimized degrader depletes hCAII through the same mechanism as previously reported CRBN-recruiting heterobifunctional degraders.

The combination of X‐ray free‐electron lasers (XFELs) with serial femtosecond crystallography represents cutting‐edge technology in structural biology, allowing the study of enzyme reactions and dynamics in real time through the generation of 'molecular movies'. This technology combines short and precise high‐energy X‐ray exposure to a stream of protein microcrystals. Here, the XFEL structure of carbonic anhydrase II, a ubiquitous enzyme responsible for the interconversion of CO2 and bicarbonate, is reported, and is compared with previously reported NMR and synchrotron X‐ray and neutron single‐crystal structures. [ABSTRACT FROM AUTHOR]

Aminoalcohols were converted into the corresponding enantiomeric phenylsulfonamide sulfamates. These compounds proved to be inhibitors of carbonic anhydrase II. Interestingly, a sulfamate derived from (S)-tryptophan was no inhibitor at all while its (R) configurated enantiomer was an excellent inhibitor of carbonic anhydrase II (CA II). A rationale can be deduced from molecular modeling studies. The sulfamates derived from (R) or (S) proline held very low inhibition constants for this enzyme as Ki = 0.77 μM and 0.70 μM, respectively.

The present study aims to perform a comprehensive chemical characterization of the methanol and water extracts of Cephalaria tchihatchewii Boiss. (MECT, WECT) using UPLC-MS/MS, besides evaluating the potential antioxidant activity of these extracts and their effect on some metabolic enzymes. The antioxidant activity was assessed using various assays including DPPH, ABTS, DMPD, FRAP, CUPRAC, and Fe3+ reducing assays. The inhibition effects of the extracts against a-glycosidase, acetylcholinesterase and carbonic anhydrase II enzymes were evaluated. The IC50 values of MECT and WECT were for a-glycosidase were 28.98 and 34.19 µg/mL, for acetylcholinesterase were 21.82 and 26.52 µg/mL, and for carbonic anhydrase, 27.75 and 13.72 µg/mL, respectively. The amounts of phenolic compounds present in MECT and WECT were determined to be 47.00 and 46.00 µg GAE /mg extract, respectively. The amounts of flavonoits present in MECT and WECT were determined to be 60.98 and 49.34 µg KE/mg extract, respectively. The MECT and WECT exhibited distinct activities on DPPH, ABTS, and DMPD. The IC50 values were for DPPH 34.66 and 57.76 µg/mL for ABTS 20.39 and 17.33 µg/mL and for DMPD 53.32 and 57.76 g/mL, respectively. Reducing abilities of extracts were respectively Fe+3 reducing (700:0.377 and 0.680), FRAP (593:0.690 and 0.369), and CUPRAC (450:0.458 and 0.333). UPLCMS/ MS revealed that the major components are MECT (quinic acid 39.844 µg/mL, chlorogenic acid 38.412 µg/mL, fumaric acid 0.301 µg/mL), and WECT (chlorogenic acid 13.639 µg/mL, qunic acid 11.004 µg/mL, cyanidin-3-O-glucoside 0.778 µg/mL). The extracts were determined to have various biological activities, including antioxidant activity and a-glycosidase, acetylcholinesterase, carbonic anhydrase II enzyme inhibition effect. [ABSTRACT FROM AUTHOR]

Earlier studies had shown the potential of modified pentacyclic triterpenes as possible inhibitors of carbonic anhydrase II (CA II). In an extension of our earlier studies, betulin, betulinic acid and, for comparison purposes, glycyrrhetinic acid, ursolic acid and oleanolic acid were therefore converted into the respective acetates and linked to either taurinamide or de-acetylated acetazolamide via a variable linker. In particular, the derivatives 8 and 18 derived from betulinic acid or betulin and provided with a long spacer were found to be strong competitive inhibitors of CA II, thereby holding Ki = 1.27 and 0.20 μM, respectively.

A small library of 79 substituted phenylsulfonamidoalkyl sulfamates, 1b–79b, was synthesized starting from arylsulfonyl chlorides and amino alcohols with different numbers of methylene groups between the hydroxyl and amino moieties yielding intermediates 1a–79a, followed by the reaction of the latter with sulfamoyl chloride. All compounds were screened for their inhibitory activity on bovine carbonic anhydrase II. Compounds 1a–79a showed no inhibition of the enzyme, in contrast to sulfamates 1b–79b. Thus, the inhibitory potential of compounds 1b–79b towards this enzyme depends on the substituent and the substitution pattern of the phenyl group as well as the length of the spacer. Bulkier substituents in the para position proved to be better for inhibiting CAII than compounds with the same substituent in the meta or ortho position. For many substitution patterns, compounds with shorter spacer lengths were superior to those with long chain spacers. Compounds with shorter spacer lengths performed better than those with longer chain spacers for a variety of substitution patterns. The most active compound held inhibition constant as low as Ki = 0.67 μM (for 49b) and a tert-butyl substituent in para position and acted as a competitive inhibitor of the enzyme.

Xianqin Song,1 Yu Song,1 Quanping Ma,2 Kehua Fang,3 Xiaotian Chang1 1Medical Research Center of the Affiliated Hospital of Qingdao University, Qingdao, Shandong, People’s Republic of China; 2Clinical Laboratory, The fourth People’s Hospital of Jinan, Jinan, Shandong, People’s Republic of China; 3Clinical Laboratory of the Affiliated Hospital of Qingdao University, Qingdao, Shandong, People’s Republic of ChinaCorrespondence: Xiaotian Chang, Medical Research Center of the Affiliated Hospital of Qingdao University, Qingdao, Shandong, 266000, People’s Republic of China, Tel/Fax +86 0532-82917331, Email changxt@126.comPurpose: Vascular calcification is a hallmark of atherosclerosis (AS). We and others confirmed that carbonic anhydrase I (CA1) and CA2 increased expression and catalyzed calcium deposition in atherosclerotic aortas. Macrophages have been demonstrated to be strongly related to AS. This study aimed to clarify how and which macrophage subtypes regulate CA1 and CA2 expression to stimulate aortic calcification.Methods and Results: THP-1 cells were induced to form M0, M1 and M2 macrophage subtypes. These cells and their culture supernatants were separately incubated with human vascular smooth muscle cells (VSMCs). Calcification was strongly increased in VSMCs treated with β-GP, a chemical inducer of cellular calcification, following incubation with M1 macrophages or their culture supernatants, and was much higher than that in VSMCs treated with β-GP alone. Meanwhile, the expression of CA1 and CA2, as well as calcification marker genes, including Runx2, BMP-2 and ALP, was increased in VSMCs during this process. TNF-α levels were also increased in the culture medium of M1 macrophages. M0 and M2 macrophages or their supernatants did not significantly stimulate calcification in VSMCs. Following transfection with anti-CA1 or CA2 siRNAs, β-GP-induced VSMCs showed decreased calcification, but the calcification level was partially increased when those VSMCs were incubated with the supernatants of M1 macrophages, while CA1 and CA2 expression as well as TNF-α levels were also elevated. When VSMCs were treated with TNF-α without β-GP induction, calcification and the expression of CA1 and CA2 were also significantly increased.Conclusion: The results of this study suggest that M1 macrophages can increase CA1 and CA2 expression to promote atherosclerotic calcification in VSMCs by secreting TNF-α.Keywords: atherosclerosis, calcification, carbonic anhydrase I, carbonic anhydrase II, macrophages, TNF-&#x03B1

The diterpene isosteviol can easily be synthesized via hydrolysis from stevioside, a renewable resource adding particular utility to its potential for drug synthesis. Of late, there has been an increase in scientific studies focusing on inhibitors of the enzyme carbonic anhydrase II, CA II, since this enzyme is not used just in glaucoma treatment but also in mitigating the side effects of Alzheimer’s disease antibody therapy. The presence of a sulfamate or a sulfonamide group is a key structural feature in many CA II inhibitors. Thus, isosteviol was transformed into sulfamates, either spacered or unspacered, to scrutinize their ability to perform as CA II inhibitors. Three particular derivatives were discovered to be effective inhibitors. As a competitive inhibitor with a Ki = 2.59 μM, a 16-O-acetyl-isosteviol compound holding a 5-amino-1,3,4-thiadiazol-2-yl-amino substituent attached to a succinoyl ester nearly entirely inhibited CA II.

A novel carbonic anhydrase II (CA II) from erythrocytes of camel (Camelus dromedarius) was purified to homogeneity using affinity chromatography and biochemically characterized. Specific activity of 140.88 U/mg was obtained with 745.17-fold purification and 25.37% yield. The enzyme was a monomer with a lower molecular weight (25 kDa) and lower Zn content (0.50 mol of Zn per mol of protein). The enzyme showed higher optimum temperature (70 °C) and pH (pH 9.0), moreover, it was stable at higher temperatures and strongly alkaline pH as judged by thermodynamic parameters (E a , k d , E d , t 1/2 , D-value, Z-value, ΔH , ΔG and ΔS). The enzyme was inhibited by cations (Al3+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Ni2+, Mg2+ and Zn2+) as well as by anions (Br‾, CH 3 COO‾, ClO 4 ‾, CN‾, F‾, HCO 3 ‾, I‾, N 3 ‾, NO 3 ‾ and SCN‾), some anions (C 6 H 5 O 7 3−, CO 3 2−, SeO 3 ‾ and SO 4 2−) does not affect enzyme activity. Effect of various chemicals on enzyme activity was also investigated. K m , V max , k cat and k cat / K m values for 4-NPA were found to be 1.74 mM, 0.0093 U/mL, 0,0039 s−1 and 0,0023 s−1 mM−1, respectively. With these interesting biochemical properties, camel CA II represents promising candidate for harsh industrial applications, in particular, for a successful biomimetic CO 2 sequestration process. • Purification of a novel CA II from camel erythrocytes was carried out. • Camel erythrocytes CA II was biochemically characterized. • CA II showed higher optimum temperature (70 °C) and pH (pH 9.0). • CA II was stable at higher temperatures and strongly alkaline pH. • CA II represents promising candidate for harsh industrial applications. [ABSTRACT FROM AUTHOR]

This study demonstrates the screening of a collection of twelve 19F-tagged metal-binding pharmacophores (MBPs) against the Zn(ii)-dependent metalloenzyme human carbonic anhydrase II (hCAII) by 19F NMR. The isomorphous replacement of Zn(ii) by Co(ii) in hCAII produces enhanced sensitivity and reveals the potential of 19F NMR-based techniques for metalloenzyme ligand discovery.

Enzymatic reactions through mononuclear metal hydrides are unknown in nature, despite the prevalence of such intermediates in the reactions of synthetic transition-metal catalysts. If metalloenzymes could react through abiotic intermediates like these, then the scope of enzyme-catalysed reactions would expand. Here we show that zinc-containing carbonic anhydrase enzymes catalyse hydride transfers from silanes to ketones with high enantioselectivity. We report mechanistic data providing strong evidence that the process involves a mononuclear zinc hydride. This work shows that abiotic silanes can act as reducing equivalents in an enzyme-catalysed process and that monomeric hydrides of electropositive metals, which are typically unstable in protic environments, can be catalytic intermediates in enzymatic processes. Overall, this work bridges a gap between the types of transformation in molecular catalysis and biocatalysis.

Background: The role of carbonic anhydrase II (CAII) in intrahepatic cholangiocarcinoma (ICC) was investigated and a novel prognostic system combining CAII and preoperative carbohydrate antigen 19‐9 (CA19‐9) was established to predict the survival of patients with ICC after curative resection. Methods: A total of 110 patients who underwent curative‐intent resection for ICC between 2012 and 2020 were retrospectively analyzed. CAII in tumor and peritumor regions was examined by immunohistochemistry, and the relationships between clinicopathological factors and the prognostic value of CAII and CA19‐9 were analyzed. Results: CAII was frequently downregulated in ICC tissues (p <.001). Multivariate analyses indicated that showed that both low CAII expression level and preoperative CA19‐9 ≥236 U/ml were independent risk factors for overall survival (OS) and recurrence‐free survival (RFS) in patients with ICC after radical resection. Survival analysis revealed that patients with high CAII and low CA19‐9 were significantly associated with a better OS and RFS (p <.001). The time‐dependent receiver operating characteristic curves showed that CAII + CA19‐9 had better prognostic predictive ability than CAII or CA19‐9 alone. The nomogram constructed on independent factors including T stage, lymph node metastasis, CA19‐9 (continuous variable), and CAII achieved C‐indexes of 0.754 (95% CI, 0.701–0.807) and 0.730 (0.674–0.785) for OS and RFS, respectively. The calibration curve revealed acceptable agreement between actual and predicted OS and RFS. Conclusions: The combination of CAII and preoperative CA19‐9 is a novel and useful prognostic tool for predicting the survival of patients with ICC after curative resection and guiding clinical decisions. Plain Language Summary: Carbonic anhydrase II (CAII) was frequently downregulated in intrahepatic cholangiocarcinoma (ICC) tissues.Survival analysis revealed that CAII is a novel independent factor for prognosis in patients with ICC after curative resection. CAII could be a useful prognostic marker for patients with ICC after surgery.The combination of CAII and preoperative carbohydrate antigen 19‐9 is a novel and useful prognostic tool for predicting the survival of patients with ICC after curative resection and guiding clinical decisions. Carbonic anhydrase II (CAII) is a novel independent factor for prognosis in patients with intrahepatic cholangiocarcinoma (ICC) after curative resection. Combining CAII and preoperative carbohydrate antigen 19‐9 can more effectively predict the survival of patients with ICC after curative resection, which could influence decision‐making regarding the possible benefit of surgery or other treatment strategies. [ABSTRACT FROM AUTHOR]

The ethyl acetate extract of the marine bacterial strain Streptomyces rochei sp. 81 was purified by silica gel column chromatography to obtain three diketopiperazines (1–3) and one straight chain fatty acid (4). The compounds were identified as cyclo(L-leu-N-ethenethiol-L-pro) (1), cyclo(L-Leu-L-Pro) (2), cyclo(L-Pro-D-Tyr) (3), and 2,4-dimethyldocasonoic acid (4) by HR-ESI-MS and NMR techniques. One new (1) and three known compounds (2–4) were screened for carbonic anhydrase-II (CA-II) inhibition and as a result 1 and 3 were found to be the most active compounds with IC50 of 9.8 ± 0.56 and 10.9 ± 0.88 μM, respectively. The results indicated that these two compounds (1 and 3) can be promising candidates for anticancer drugs. [ABSTRACT FROM AUTHOR]

Acetylated triterpenoids betulin, oleanolic acid, ursolic acid, and glycyrrhetinic acid were converted into their succinyl-spacered acetazolamide conjugates. These conjugates were screened for their inhibitory activity onto carbonic anhydrase II and their cytotoxicity employing several human tumor cell lines and non-malignant fibroblasts. As a result, the best inhibitors were derived from betulin and glycyrrhetinic acid while those derived from ursolic or oleanolic acid were significantly weaker inhibitors but also of diminished cytotoxicity. A betulin-derived conjugate held a Ki = 0.129 μM and an EC50 = 8.5 μM for human A375 melanoma cells. [ABSTRACT FROM AUTHOR]

A series of hydrazine-1-carbothioamides derivatives (3a–3j) were synthesized and analyzed for inhibitory potential towards bovine carbonic anhydrase II (b-CA II) and 15-lipoxygenase (15-LOX). Interestingly, four derivatives, 3b, 3d, 3g, and 3j, were found to be selective inhibitors of CA II, while other derivatives exhibited CA II and 15-LOX inhibition. In silico studies of the most potent inhibitors of both b-CA II and 15-LOX were carried out to find the possible binding mode of compounds in their active site. Furthermore, MD simulation results confirmed that these ligands are stably bound to the two targets, while the binding energy further confirmed the inhibitory effects of the 3h compound. As these compounds may have a role in particular diseases, the reported compounds are of great relevance for future applications in the field of medicinal chemistry. [ABSTRACT FROM AUTHOR]

The principle of isosteres or bioisosteres in medicinal chemistry is a central and essential concept in modern drug discovery. For example, carboxylic acids are often replaced by bioisosteres to mitigate issues related to lipophilicity or acidity while retaining acidic characteristics in addition to hydrogen bond donor/acceptor abilities. Separately, the development of metal-binding pharmacophores (MBPs) for binding to the active site metal ion in metalloenzymes of therapeutic interest is an emerging area in the realm of fragment-based drug discovery (FBDD). The direct application of the bioisostere concept to MBPs has not been well-described or systematically investigated. Herein, the picolinic acid MBP is used as a case study for the development of MBP isosteres (so-called MBIs). Many of these isosteres are novel compounds, and data on their physicochemical properties, metal binding capacity, and metalloenzyme inhibition characteristics are presented. The results show that MBIs of picolinic acid generally retain metal coordinating properties and exhibit predictable metalloenzyme inhibitory activity while possessing a broad range of physicochemical properties (e.g., p Ka, log P). These findings demonstrate the use of bioisosteres results in an untapped source of metal binding functional groups suitable for metalloenzyme FBDD. These MBIs provide a previously unexplored route for modulating the physicochemical properties of metalloenzyme inhibitors and improving their drug-likeness.

What is known and objective: Red man syndrome (RMS) is a non‐IgE‐mediated anaphylactoid adverse event frequently witnessed after a rapid infusion of vancomycin. This study aims to unravel drugs and associated off‐label targets that induce RMS by exploiting FDA Adverse Event Reporting System (FAERS) and Pharmacovigilance/Pharmacogenomics Insilico Pipeline (PHARMIP). Methods: The case/non‐case retrospective observational study was conducted in the FAERS database. Reporting odds ratio (ROR) and proportional reporting ratio (PRR) data mining algorithms were used to evaluate the strength of the signal. The off‐label targets of the drugs with potential signals were obtained using online servers by applying a similarity ensemble approach and a reverse pharmacophore database, which was further validated by molecular docking studies. Results and discussion: Oritavancin exhibited a strong positive signal (PRR:1185.20 and ROR:1256), which suggests a higher risk for causing RMS. The literature search revealed the involvement of the MRGPRX2 gene in the development of RMS. PHARMIP study unearthed Carbonic anhydrase II (CA2) as the common off‐label target among the drugs causing RMS. The results obtained from molecular docking studies reinforced the findings as mentioned earlier, wherein the highest docking score was disinterred for oritavancin (−9.4 for MRGPRX2 and − 8.7 for CA2). What is new and conclusion: Many antibiotics and other classes of medications have been discovered in the quest for drugs that may induce RMS, although a causal relationship could not be established. The implication of MRGPX2 and CA2 in the initial stages of pathogenesis necessitates the development of inhibitors that could be used as potential therapeutic agents against RMS. [ABSTRACT FROM AUTHOR]

Myocardial infarction (MI) is a significant threat to human health and life. Xue-Fu-Zhu-Yu Decoction (XFZYD), a renowned traditional Chinese medicine prescription for treating myocardial infarction, is known to play a significant role in the management of MI. However, its mechanism of action remains unclear. Through network pharmacology analysis of compound-target interactions, we have identified Carbonic Anhydrase II (CA2) as a critical target for XFZYD in the treatment of MI. Subsequently, we will embark on a target-based drug design approach with a focus on CA2 as the key target: Pharmacophore modeling: Two pharmacophore models were developed and validated to screen for small molecules with CA2 inhibitory features. Virtual screening: Based on two pharmacophore models, small molecules with the property of binding to the CA2 target were screened from a virtual screening library. Molecular docking: Molecular docking was employed to identify small molecules with stable binding affinity to CA2. ADMET prediction: ADMET models were utilized to screen for small molecules with favorable pharmacological properties. Molecular dynamics: Molecular dynamics simulations were further conducted to analyze the binding modes of the selected small molecules with CA2, ultimately resulting in the identification of Ligand 3 and Ligand 5 as small molecule inhibitors targeting CA2. Finally, the mechanisms underlying the anti-MI effects were discussed. The primary objective of this article is to uncover the mechanism by which XFZYD acts on MI and utilize it for drug development. These findings provide novel avenues for the development of anti-MI drugs.Communicated by Ramaswamy H. Sarma.

The inhibition and binding of three metal-binding pharmacophores (MBPs), 2-hydroxycyclohepta-2,4,6-trien-1-one (tropolone), 2-mercaptopyridine-N-oxide (1,2-HOPTO), and 2-hydroxycyclohepta-2,4,6-triene-1-thione (thiotropolone) to human carbonic anhydrase II (hCAII) and a mutant protein hCAII L198G were investigated. These MBPs displayed bidentate coordination to the active site Zn(II) metal ion, but the MBPs respond to the mutation of L198G differently, as characterized by inhibition activity assays and X-ray crystallography. The L198G mutation increases the active site volume thereby decreasing the steric pressure exerted on MBPs upon binding, allowing changes in MBP coordination to be observed. When comparing the binding mode of tropolone to thiotropolone or 1,2-HOPTO (O,O versus O,S donor sets), structural modifications of the hCAII active site were shown to have a stronger effect on MBPs with an O,O versus O,S donor set. These findings were corroborated with density functional theory (DFT) calculations of model coordination complexes. These results suggest that the MBP binding geometry is a malleable interaction, particularly for certain ligands, and that the identity of the donor atoms influences the response of the ligand to changes in the protein active site environment. Understanding underlying interactions between a MBP and a metalloenzyme active site may aid in the design and development of potent metalloenzyme inhibitors.

This study uses mutants of human carbonic anhydrase (HCAII) to examine how changes in the organization of water within a binding pocket can alter the thermodynamics of protein-ligand association. Results from calorimetric, crystallographic, and theoretical analyses suggest that most mutations strengthen networks of water-mediated hydrogen bonds and reduce binding affinity by increasing the enthalpic cost and, to a lesser extent, the entropic benefit of rearranging those networks during binding. The organization of water within a binding pocket can thus determine whether the hydrophobic interactions in which it engages are enthalpy-driven or entropy-driven. Our findings highlight a possible asymmetry in protein-ligand association by suggesting that, within the confines of the binding pocket of HCAII, binding events associated with enthalpically favorable rearrangements of water are stronger than those associated with entropically favorable ones.

In this work, we were going to generate first- and second-order electrochemical and spectroscopic data to investigate inhibition of the carbonic anhydrase II (CA II) by acetazolamide (AZ), dorzolamide (DZ) and methazolamide (MZ) which enabled us to have a deep insight to the mechanism of these inhibitions. MCR-ALS and PARAFAC were chosen for chemometric analyses of the data and after data resolution, spectral and voltammetric profiles and concentration profiles were obtained which helped us to obtain qualitative information about inhibition of the CA II which was completed by molecular docking and hard-modeling of the data. Our results showed that order of the inhibition of the CA II was occurred according to MZ > DZ > AZ. According to the results of inhibition of the CA II, the MZ was used to develop a novel amperometric method for determination of the CA II which was not electroactive. The sensor response was linearly correlated with concentration of the CA II in the range of 0–20 nM with a limit of detection (LOD) of 0.21 nM and a sensitivity of 0.57 μA nM−1. The developed method was stable, selective and sensitive for the determination of the CA II.

(1) Background: The benefit of using inhibitors of carbonic anhydrase (CA), such as acetazolamide, in the treatment of epilepsy has previously been described. (2) Methods: In this paper, the effect on CA of the most well-known antiepileptic drugs was studied in vitro and in vivo. The effects, after chronic treatment, of carbamazepine, phenytoin, valproate, primidone, clonazepam, and ethosuximide were studied in vitro on purified CA, isozyme I (CA I) and CA, and isozyme II (CA II) activity and in vivo on epileptic erythrocyte CA I and CA II activity. (3) Results: In vitro results showed that all antiepileptic drugs reduced purified CA II activity according to dose–response relationships and slightly inhibited CA I activity. In vivo results showed that the chronic administration of antiseizure drugs induced a progressive reduction in erythrocyte CA II activity in all the groups studied. This study shows that CA II inhibition can be induced both in vitro and in vivo by major antiepileptic agents as it might be one of the effective mechanisms of these anticonvulsant drugs. (4) Conclusions: The decrease in CA II activity in epileptic patients after antiseizure treatment suggests the involvement of CA II in the pathogenesis of epilepsy. [ABSTRACT FROM AUTHOR]

A novel series of hydantoins incorporating phthalimides has been synthesised by condensation of activated phthalimides with 1-aminohydantoin and investigated for their inhibitory activity against a panel of human (h) carbonic anhydrase (CA, EC 4.2.1.1): the cytosolic isoforms hCA I, hCA II, and hCA VII, secreted isoform hCA VI, and the transmembrane hCA IX, by a stopped-flow CO 2 hydrase assay. Although all newly developed compounds were totally inactive on hCA I and mainly ineffective towards hCA II, they generally exhibited moderate repressing effects on hCA VI, VII, and IX with K I s values in the submicromolar to micromolar ranges. The salts 3a and 3b , followed by derivative 5 , displayed the best inhibitory activity of all the evaluated compounds and their binding mode was proposed in silico . These compounds can also be considered interesting starting points for the development of novel pharmacophores for this class of enzyme inhibitors.

We perform structural analysis and calculate the vibrational energy diffusivities, represented as heat‐communication map, between a carbonic anhydrase II and its binding inhibitor benzosulfonamide, to identify critical residues involved in the secondary and tertiary adaptations surrounding the active site, such as HID95, HIE118, HID93, THR197, and HIE198. Notably, the water molecules surrounding the active sites play an essential part in the energy flow. The stability of the docked ligand is assessed using its structural deviation and free energy calculation. Additionally, density functional theory calculations are used to evaluate specific binding energies, resulting in prominent residues such as THR197, THR198, and HIE118 with high binding potential to the benzosulfonamide ligand. [ABSTRACT FROM AUTHOR]

Enzymes are catalysts of biological processes. Significant insight into their catalytic mechanisms has been obtained by relating site-directed mutagenesis studies to kinetic activity assays. However, revealing the detailed relationship between structural modifications and functional changes remains challenging owing to the lack of information on reaction intermediates and of a systematic way of connecting them to the measured kinetic parameters. Here, a systematic approach to investigate the effect of an active-site-residue mutation on a model enzyme, human carbonic anhydrase II (CA II), is described. Firstly, structural analysis is performed on the crystallographic intermediate states of native CA II and its V143I variant. The structural comparison shows that the binding affinities and configurations of the substrate (CO2) and product (HCO3−) are altered in the V143I variant and the water network in the water-replenishment pathway is restructured, while the proton-transfer pathway remains mostly unaffected. This structural information is then used to estimate the modifications of the reaction rate constants and the corresponding free-energy profiles of CA II catalysis. Finally, the obtained results are used to reveal the effect of the V143I mutation on the measured kinetic parameters (kcat and kcat/Km) at the atomic level. It is believed that the systematic approach outlined in this study may be used as a template to unravel the structure–function relationships of many other biologically important enzymes.

Testing of an expanded, 800-compound set of analogues of the earlier described Strecker-type α-aminonitriles (selected from publicly available Enamine Ltd. Screening Collection) in thermal shift assay against bovine carbonic anhydrase (bCA) led to further validation of this new class of inhibitors and identification a new, refined chemotype represented by inhibitors with 10-improved potency.

The differential scanning fluorimetry (DSF) screening of 5.692 fragments in combination with benzenesulfonamide (BSA) against bovine carbonic anhydrase (bCA) delivered >100 hits that either caused, on their own, a significant thermal shift (ΔTm, °C) in the protein melting temperature or significantly influenced the thermal shift observed for BSA alone. Three hits based on 1,2,3-triazole moiety represent the periphery of the recently reported potent inhibitors of hCA II, IX and XII which were efficacious in vivo. Such a re-discovery of suitable BSA periphery essentially validates the new fragment-based approach to the discovery of future CAIs. Structures of other validated fragment hits are reported.

A small library of ten new Nimesulide-iminothiazolines conjugates was synthesized by the reduction of nitro group of Nimesulide followed by conversion into variously substituted acyl thioureas. Heterocyclization of the latter with phenacyl bromide afforded the products (7a-j) in good to excellent yields and high purity. The newly synthezied (7a-j) were screend for inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carbonic anhydrase I (hCA I) and carbonic anhydrase II (hCA II). Most of the synthesized molecules were more effective than standard inhibitors tacrine, and acetazolamide against AchE, BchE and against CA I and CA II respectively. Compounds 7 h, 7f, 7d and 7i were the most potent compounds against hCA I&II. Whilst compounds 7a, 7d and 7f showed highest inhibition against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) when compared with standard inhibitor Tacrine. In silico studies were also performed to find the type of interactions. Molecular docking was accomplished to explore the putative binding mode of interactions of selective inhibitors. Finally, the ADMET analysis of the molecules was also performed.

Acetylated triterpenoids betulin, oleanolic acid, ursolic acid, and glycyrrhetinic acid were converted into their succinyl-spacered acetazolamide conjugates. These conjugates were screened for their inhibitory activity onto carbonic anhydrase II and their cytotoxicity employing several human tumor cell lines and non-malignant fibroblasts. As a result, the best inhibitors were derived from betulin and glycyrrhetinic acid while those derived from ursolic or oleanolic acid were significantly weaker inhibitors but also of diminished cytotoxicity. A betulin-derived conjugate held a Ki = 0.129 μM and an EC50 = 8.5 μM for human A375 melanoma cells.

A series of hydrazine-1-carbothioamides derivatives (3a–3j) were synthesized and analyzed for inhibitory potential towards bovine carbonic anhydrase II (b-CA II) and 15-lipoxygenase (15-LOX). Interestingly, four derivatives, 3b, 3d, 3g, and 3j, were found to be selective inhibitors of CA II, while other derivatives exhibited CA II and 15-LOX inhibition. In silico studies of the most potent inhibitors of both b-CA II and 15-LOX were carried out to find the possible binding mode of compounds in their active site. Furthermore, MD simulation results confirmed that these ligands are stably bound to the two targets, while the binding energy further confirmed the inhibitory effects of the 3h compound. As these compounds may have a role in particular diseases, the reported compounds are of great relevance for future applications in the field of medicinal chemistry.

Purpose: To evaluate the clinical characteristics of patients with acute zonal occult outer retinopathy (AZOOR), according to the presence or absence of anti-retinal antibodies (ARAs) that are frequently detected in autoimmune retinopathy. Methods: Retrospective observational case series. This study included 33 patients with acute-stage AZOOR who had been followed up for more than 6 months after the initial visit. The median follow-up period was 26 months. Immunoblot analyses were used to detect autoantibodies for recoverin, carbonic anhydrase II, and α-enolase in serum from these patients. Main outcome measures comprised clinical factors at the initial and final visits, including best-corrected visual acuity, mean deviation on Humphrey perimetry, and retinal morphology, which were statistically compared between patients with AZOOR who exhibited ARAs and those who did not. Results: At least one serum ARA was detected in 42% of patients with AZOOR. There were no significant differences in clinical factors between the two groups, including follow-up period, best-corrected visual acuity and mean deviation at the initial and final visits, a-wave amplitude on single-flash electroretinography at the initial visit, and frequencies of improvement of the macular ellipsoid zone and AZOOR recurrence. Conclusions: Our findings suggest that the presence of ARAs did not influence visual outcomes or outer retinal morphology in patients with AZOOR. [ABSTRACT FROM AUTHOR]

Carbonic anhydrases (CA), having Zn2+ metal atoms, are responsible for the catalysis of CO2 and water to bicarbonate and protons. Any abnormality in the functioning of these enzymes may lead to morbidities such as glaucoma and different types of cancers including brain, renal and pancreatic carcinomas. To cope with the lack of presence of a promising therapeutic agent against these cancers, searching for an efficient and suitable carbonic anhydrase inhibitor is crucial. In the current study, ten novel 3-ethylaniline hybrid imino-thiazolidinones were synthesized and characterized by FTIR, NMR (1H, 13C), and mass spectrometry. Synthesis was carried out by diethyl but-2-ynedioate cyclization and different acyl thiourea substitutions of 3-ethyl amine. The CA (II) enzyme inhibition profile for all synthesized derivatives was determined. It was observed that compound 6e demonstrated highest inhibition of CA-II with an IC50 value of 1.545 ± 0.016 µM. In order to explore the pharmacophoric properties and develop structure activity relationship, in silico screening was performed. In silico investigations included density functional theory (DFT) studies, pharmacophore-guided model development, molecular docking, molecular dynamic (MD) simulations, and prediction of drug likeness scores. DFT investigations provided insight into the electronic characteristics of compounds, while molecular docking determined the binding orientation of derivatives within the CA-II active site. Compounds 6a, 6e, and 6g had a reactive profile and generated stable protein-ligand interactions with respective docking scores of −6.12, −6.99, and −6.76 kcal/mol. MD simulations were used to evaluate the stability of the top-ranked complex. In addition, pharmacophore-guided modeling demonstrated that compound 6e produced the best pharmacophore model (HHAAARR) compared to standard brinzolamide. In vitro and in silico investigations anticipated that compound 6e would be an inhibitor of carbonic anhydrase II with high efficacy. Compound 6e may serve as a potential lead for future synthesis that can be investigated at the molecular level, and additional in vivo studies are strongly encouraged.

Carbonic anhydrase II (CAII) is one of the zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide, leading to the formation of bicarbonate and proton. CAII plays a significant role in health and disease. For example, CAII helps to maintain eye pressure while regulating the pH of the tumor microenvironment, and by extension, contributing to cancer progression. Owing to its remarkable role in cancer, visual health, and other human diseases, CAII can serve as an attractive therapeutic target. We report an original study based on high-throughput virtual screening of natural compounds from the ZINC database in search of potential inhibitors of CAII. We selected the hits based on the physicochemical, absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties, pan-assay interference compound (PAINS) patterns, and interaction analysis. Importantly, two natural compounds were identified, ZINC08918123 and ZINC00952700, bearing considerable affinity and specific interactions to the residues of the CAII-binding pocket with well-organized conformational fitting compatibility. We investigated the conformational dynamics of CAII in complex with the identified compounds through molecular dynamics simulation, which revealed the formation of a stable complex preserved throughout the 100 ns trajectories. The stability of the protein/ligand complexes is maintained by significant numbers of noncovalent interactions throughout the simulations. In conclusion, natural compounds identified in the present study specifically and computer-assisted drug design broadly offer a reliable resource and strategy to discover potential promising therapeutic inhibitors of CAII to cure various cancers and glaucoma after further experimental validation and clinical studies. [ABSTRACT FROM AUTHOR]

The binding of a series of metal-binding pharmacophores (MBPs) related to the ligand 1-hydroxypyridine-2-(1H)-thione (1,2-HOPTO) in the active site of human carbonic anhydrase II (hCAII) has been investigated. The presence and/or position of a single methyl substituent drastically alters inhibitor potency and can result in coordination modes not observed in small-molecule model complexes. It is shown that this unexpected binding mode is the result of a steric clash between the methyl group and a highly ordered water network in the active site that is further stabilized by the formation of a hydrogen bond and favorable hydrophobic contacts. The affinity of MBPs is dependent on a large number of factors including donor atom identity, orientation, electrostatics, and van der Waals interactions. These results suggest that metal coordination by metalloenzyme inhibitors is a malleable interaction and that it is thus more appropriate to consider the metal-binding motif of these inhibitors as a pharmacophore rather than a "chelator". The rational design of inhibitors targeting metalloenzymes will benefit greatly from a deeper understanding of the interplay between the variety of forces governing the binding of MBPs to active site metal ions.

The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins.

Acquired idiopathic generalized anhidrosis (AIGA) is characterized by anhidrosis/hypohidrosis without other autonomic and neurological dysfunctions. It has been believed that AIGA patients usually present no significant morphological alterations in the secretory portion of eccrine glands consisting of clear, dark and myoepithelial cells. However, we have recently revealed morphological damage of eccrine glands in AIGA patients by immunohistochemistry. Moreover, inhibitory side‐effects against carbonic anhydrase II (CA II) by the antiepileptic reagent topiramate have been reported to cause heat intolerance mimicking AIGA. To determine the precise morphological changes and CA II expression in eccrine glands of AIGA patients, electron microscopic observation and immunohistochemistry were applied to skin of both anhidrotic (non‐sweating) and normohidrotic (sweating‐preserved) sites, taken from each patient clinically diagnosed with AIGA. We found consistent clear cell injury in eccrine glands in anhidrotic skin samples of AIGA patients. Electron micrographs demonstrated edematous, swollen and destructive damage in clear cells of eccrine glands from non‐sweating areas of almost all AIGA patients. Immunohistochemically, clear cells showed reduced CA II expression that was heterogeneously distributed in non‐sweating skin. Some areas showed almost complete loss of CA II expression in spite of preserved dark cells, and others showed mild or moderate loss of it. Selective destruction of clear cells resulting in heterogenous atrophy in AIGA patients may be important to elucidate its etiology. [ABSTRACT FROM AUTHOR]

Combating pathological conditions related to hyperactivity of enzymes remains a formidable challenge for health. Small molecules therapy constitutes one of the means to circumvent the medical disorders resulting from enzyme hyperactivity. In this regard, we have synthesized structurally diverse amino acid hybrid Schiff bases (5a–5l and 10a–10k) and evaluated them for carbonic anhydrase II, α-glucosidase, and urease inhibitory potential. These new chemical scaffolds showed variable efficacies against the selected enzymes. The results indicated that compounds 5b (11.8 ± 1.33 µM), 10i (83.3 ± 1.13 µM), and 10f (88.2 ± 2.27 µM) are the most active scaffolds against carbonic anhydrase II, α-glucosidase, and urease, respectively. A structure–activity relationship revealed the most structural features contributing to the overall activities. Molecular docking suggested that these compounds possess excellent binding interactions with the active site residues of the targets by interacting through hydrogen bonding, π–π, and π–cation interactions. [ABSTRACT FROM AUTHOR]

In the current investigation, the chemical composition of the essential oils (EOs) from the stem and leaves of Acridocarpus orientalis A. Juss. were evaluated by gas chromatography (GC) and gas-chromatography-mass spectrometry (GC-MS). The main volatile constituents identified in the essential oil of stem were rich in methyl 8-pimaren-18-oate (43.8 %), octacosane (5.8 %), heptacosane (4.6 %) and hexacosane (4.1 %), methyl dehydroabietate (3.9 %) and methyl pimar-8(14)-en-18-oate (3.6 %), while tetracosane (16.6 %), heptacosane (16.4 %), docosane (13.9 %), hentriacontane (13.5 %), heneicosane (10.9 %) and α-pinene (7.7 %) were abundant in the EO of leaves. Both essential oils, screened against urease, α-glucosidase and carbonic anhydrase II (CA-II) enzyme inhibitory activities, were found active against CA-II and inactive against urease and α-glucosidase enzymes. The leaves of AO were found more active only against CA-II enzyme having IC50 values of 45.6 ± 1.0 μg/mL than stem (156.3 ± 1.5 μg/mL). The results determined that the EO of leaves could be a promising target for anti-cancer treatment. This is the first report, up to the best of the author's knowledge, on the chemical composition and CA-II activity of this essential oil. [ABSTRACT FROM AUTHOR]

Dihydro [2,3D] pyridine substituted enaminosulfonamide compounds have been synthesized and their effects on carbonic anhydrase II (hCAII) have been evaluated. Pyrido [2,3 d] pyrimidines were synthesized from barbituric acid derivatives, malonanitrile, aldehyde derivatives in basic condition and then hydrolyzed with hydrochloric acid. The targeted compounds were synthesized from amino sulfanilamide, dihydro [2,3D] pyridine compounds, and triethylorthoformate. ¹H NMR, 13C NMR, FT-IR and elemental analysis were used for the structural analysis of the compounds. The half maximal inhibitory concentration (IC50) values of the compounds were determined to be between 27.03 and 104.39 μM for hCA II and 19.85-76.64 μM for Ki. [ABSTRACT FROM AUTHOR]

Sulfonamides is an important class of compounds, which can be used as carbonic anhydrase inhibitors. Nine different benzenesulfonamide compounds were synthesized, and their inhibitory effects on carbonic anhydrase II were studied by esterase method and molecular docking. The results showed that compounds (IId)–(IIg) with nitro and acetamide groups on the benzene ring exhibited excellent carbonic anhydrase II inhibitory activities. Molecular docking showed that compared with the control inhibitor acetazolamide, the compounds (IId)–(IIg) docked at the carbonic anhydrase II active site and showed higher binding energy and stronger binding ability. The physical and chemical properties of all compounds were studied by Molinspiration, which showed outstanding drug-like properties and ADME properties. Cytotoxicity assay results showed that compounds (IIe) and (IIf) were almost non-toxic to HepG2 and RAW264.7 cells. In conclusion, the compounds (IIe) and (IIf) have a certain application prospect as new inhibitors of carbonic anhydrase II. [ABSTRACT FROM AUTHOR]

A series of novel pyrazole-dicarboxamides were synthesized from pyrazole-3,4-dicarboxylic acid chloride and various primary and secondary sulfonamides. The structures of the new compounds were confirmed by FT-IR, 1 H-NMR, 13 C-NMR, and HRMS. Then the inhibition effects of newly synthesized molecules on human erythrocyte hCA I and hCA II isoenzymes were investigated. K i values of the compounds were in the range of 0.024-0.496 µM for hCA I and 0.006-5.441 µM for hCA II. Compounds 7a and 7i showed nanomolar level of inhibition of hCA II, and these compounds exhibited high selectivity for this isoenzyme. Molecular docking studies were performed between the most active compounds 7a, 7b, 7i, and the reference inhibitor AAZ and the hCAI and hCAII to investigate the binding mechanisms between the compounds and the isozymes. These compounds showed better interactions than the AAZ. ADMET and drug-likeness analyses for the compounds have shown that the compounds can be used pharmacologically in living organisms., (© 2024 Wiley Periodicals LLC.)

Various carbonic anhydrase (CA) enzyme isoforms are known today. In addition to the use of CA inhibitors as diuretics, antiepileptics and antiglaucoma agents, the inhibition of other specific isoforms of CA was reported to have clinical benefits in cancers. In this study, two groups of 1,3,4-thiadiazole derivatives were designed and synthesized to act as human CA I and II ( h CA I and h CA II) inhibitors. The activities of these compounds were tested in vitro and evaluated in silico studies. The activity of the synthesized compounds was also tested against acetylcholinesterase (AChE) to evaluate the relation of the newly designed structures to the activity against AChE. The synthesized compounds were analyzed by 1 H NMR, 13 C NMR and high-resolution mass spectroscopy (HRMS). The results displayed a better activity of all the synthesized compounds against h CA I than that of the commonly used standard drug, Acetazolamide (AAZ). The compounds also showed better activity against h CA II, except for compounds 5b and 6b . Only compounds 6a and 6c showed superior activity against AChE compared to the standard agent, tacrine (THA). In silico studies, including absorption, distribution, metabolism and excretion (ADME) and drug-likeness evaluation, molecular docking, molecular dynamic simulations (MDSs) and density functional theory (DFT) calculations, were compatible with the in vitro results and presented details regarding the structure-activity relationship.Communicated by Ramaswamy H. Sarma.

Activation of human carbonic anhydrase II (hCA II) holds great promise for treating memory loss symptoms associated with Alzheimer's disease. Despite its importance, the activation mechanism of hCA II has been largely overlooked in favor of the well-studied inhibition mechanism. To address this unexplored realm, we use first-principles calculations to tease out the activation mechanism of hCA II using 2-(2-aminoethyl)-pyridine (2-2AEPy), a promising in vitro activator. We explored both stepwise and concerted mechanisms via both available nitrogen sites of 2-2AEPy: (i) aminoethyl group (Nα) and (ii) pyridine ring (Nβ). Our results show that a concerted mechanism via Nα holds the key to hCA II activation. The activation process of the concerted mechanism exhibits the characteristics of an exergonic reaction, wherein the transition state resembles the reactant with a notably low imaginary frequency of 452.4i cm -1 and barrier height of 5.2 kcal mol -1 . Such meager transition barriers propel the activation of hCA II at in vivo temperatures. These findings initiate future research into hCA II activation mechanisms and the development of efficient activators, which may lead to promising therapeutic interventions for Alzheimer's disease.

Drug discovery, in particular optimization of candidates using medicinal chemistry, is generally guided by structural biology. However, for optimizing binding kinetics, relevant for efficacy and off‐target effects, information on protein motion is important. Herein, we demonstrate for the prototypical textbook example of an allegedly "rigid protein" that substantial active‐site dynamics have generally remained unrecognized, despite thousands of medicinal‐chemistry studies on this model over decades. Comparing cryogenic X‐ray structures, solid‐state NMR on micro‐crystalline protein at room temperature, and solution NMR structure and dynamics, supported by MD simulations, we show that under physiologically relevant conditions the pocket is in fact shaped by pronounced open/close conformational‐exchange dynamics. The study, which is of general significance for pharmacological research, evinces a generic pitfall in drug discovery routines. [ABSTRACT FROM AUTHOR]

The differential scanning fluorimetry (DSF) screening of 5.692 fragments in combination with benzenesulfonamide (BSA) against bovine carbonic anhydrase (bCA) delivered >100 hits that either caused, on their own, a significant thermal shift (ΔTm, °C) in the protein melting temperature or significantly influenced the thermal shift observed for BSA alone. Three hits based on 1,2,3-triazole moiety represent the periphery of the recently reported potent inhibitors of hCA II, IX and XII which were efficacious in vivo. Such a re-discovery of suitable BSA periphery essentially validates the new fragment-based approach to the discovery of future CAIs. Structures of other validated fragment hits are reported. [ABSTRACT FROM AUTHOR]

Testing of an expanded, 800-compound set of analogues of the earlier described Strecker-type α-aminonitriles (selected from publicly available Enamine Ltd. Screening Collection) in thermal shift assay against bovine carbonic anhydrase (bCA) led to further validation of this new class of inhibitors and identification a new, refined chemotype represented by inhibitors with 10-improved potency. [ABSTRACT FROM AUTHOR]