Investigating the Selectivity of Metalloenzyme Inhibitors in the Presence of Competing Metalloproteins

Metalloprotein inhibitors (MPi) are an important class of therapeutics for the treatment of a variety of diseases, including hypertension, cancer, and HIV/AIDS. However, despite their clinical success, there is an apprehension that MPi may be less selective than other small‐molecule therapeutics and more prone to inhibit off‐target metalloenzymes. We examined the issue of MPi specificity by investigating the selectivity of a variety of MPi against a representative panel of metalloenzymes in the presence of competing metalloproteins (metallothionein, myoglobin, carbonic anhydrase, and transferrin). Our findings reveal that a wide variety of MPi do not exhibit a decrease in inhibitory activity in the presence of large excesses of competing metalloproteins, suggesting that the competing proteins do not titrate the MPi away from its intended target. This study represents a rudimentary but important means to mimic the biological milieu, which contains other metalloproteins that could compete the MPi away from its target. The strategy used in this study may be a useful approach to examine the selectivity of other MPi in development.     

Inhibition of Carbonic Anhydrase IX Suppresses Breast Cancer Cell Motility at the Single-Cell Level

Protein Carbonic Anhydrase IX (CA IX), which is expressed in various hypoxic solid tumors in order to maintain proper pH, is also related to cancer cell adhesion, invasion, and metastasis processes. Here, we investigated whether CA IX inhibition by a highly CA IX selective agent benzenesulfonamide VD11-4-2 triggers changes in individual cell motility. We seeded breast cancer cells on an extracellular matrix-coated glass-bottomed dish and in a microfluidic device with a gradient flow of epidermal growth factor (EGF), tracked individual cell movement, calculated their migration speeds, and/or followed movement direction. Our results showed that the inhibitor VD11-4-2 decreased the speed of CA IX positive breast cancer cells by 20–26% while not affecting non-cancerous cell migration. The inhibitor suppressed the cell migration velocity increment and hindered cells from reaching their maximum speed. VD11-4-2 also reduced CA IX, expressing cell movement towards the growth factor as a chemoattractant. Such a single cell-based migration assay enabled the comprehensive investigation of the cell motility and revealed that VD11-4-2 shows the ability to suppress breast cancer cell migration at a lower concentration than previously tested CA IX inhibitors.     

Carbonic Anhydrase I Is Recognized by an SOD1 Antibody upon Biotinylation of Human Spinal Cord Extracts

We recently reported the presence of a novel 32 kDa protein immunoreactive to a copper, zinc superoxide dismutase (SOD1) antibody within the spinal cord of patients with amyotrophic lateral sclerosis (ALS). This unique protein species was generated by biotinylation of spinal cord tissue extracts to detect conformational changes of SOD1 specific to ALS patients. To further characterize this protein, we enriched the protein by column chromatography and determined its protein identity by mass spectrometry. The protein that gave rise to the 32 kDa species upon biotinylation was identified as carbonic anhydrase I (CA I). Biotinylation of CA I from ALS spinal cord resulted in the generation of a novel epitope recognized by the SOD1 antibody. This epitope could also be generated by biotinylation of extracts from cultured cells expressing human CA I. Peptide competition assays identified the amino acid sequence in carbonic anhydrase I responsible for binding the SOD1 antibody. We conclude that chemical modifications used to identify pathogenic protein conformations can lead to the identification of unanticipated proteins that may participate in disease pathogenesis.     

Role of Carbonic Anhydrase in Cerebral Ischemia and Carbonic Anhydrase Inhibitors as Putative Protective Agents

Ischemic stroke is a leading cause of death and disability worldwide. The only pharmacological treatment available to date for cerebral ischemia is tissue plasminogen activator (t-PA) and the search for successful therapeutic strategies still remains a major challenge. The loss of cerebral blood flow leads to reduced oxygen and glucose supply and a subsequent switch to the glycolytic pathway, which leads to tissue acidification. Carbonic anhydrase (CA, EC 4.2.1.1) is the enzyme responsible for converting carbon dioxide into a protons and bicarbonate, thus contributing to pH regulation and metabolism, with many CA isoforms present in the brain. Recently, numerous studies have shed light on several classes of carbonic anhydrase inhibitor (CAI) as possible new pharmacological agents for the management of brain ischemia. In the present review we summarized pharmacological, preclinical and clinical findings regarding the role of CAIs in strokes and we discuss their potential protective mechanisms.     

Generation of New Artificial Metalloproteins by Cofactor Modification of Native Hemoproteins

Heme can be removed from a number of native hemoproteins, thus forming corresponding apoproteins, each of which provides a site for binding of a metal complex. In one example, myoglobin, an O₂ storage protein, can be reconstituted with iron porphycene to dramatically enhance the O₂ affinity. Although it is known that myoglobin has poor enzymatic activity, the insertion of iron corrole or iron porphycene into apomyoglobin increases its H₂O₂-dependent peroxidase/peroxygenase activities. Furthermore, reconstitution with manganese porphycene promotes hydroxylation of an inert C H bond. It is also of interest to insert a non-porphyrinoid complex into an apoprotein. A cavity of apocytochrome c has been found to bind a diiron carbonyl complex, serving as a functional model of diiron hydrogenase. Aponitrobindin has a rigid β-barrel structure that provides an excellent cavity for covalently anchoring a metal complex. A rhodium complex embedded in the cavity of genetically modified nitrobindin has been found to promote stereoselective polymerization of phenylacetylene.     

Molecular Characterization of Carbonic Anhydrase Genes in Lotus japonicus and Their Potential Roles in Symbiotic Nitrogen Fixation

Carbonic anhydrase (CA) plays a vital role in photosynthetic tissues of higher plants, whereas its non-photosynthetic role in the symbiotic root nodule was rarely characterized. In this study, 13 CA genes were identified in the model legume Lotus japonicus by comparison with Arabidopsis CA genes. Using qPCR and promoter-reporter fusion methods, three previously identified nodule-enhanced CA genes (LjαCA2, LjαCA6, and LjβCA1) have been further characterized, which exhibit different spatiotemporal expression patterns during nodule development. LjαCA2 was expressed in the central infection zone of the mature nodule, including both infected and uninfected cells. LjαCA6 was restricted to the vascular bundle of the root and nodule. As for LjβCA1, it was expressed in most cell types of nodule primordia but only in peripheral cortical cells and uninfected cells of the mature nodule. Using CRISPR/Cas9 technology, the knockout of LjβCA1 or both LjαCA2 and its homolog, LjαCA1, did not result in abnormal symbiotic phenotype compared with the wild-type plants, suggesting that LjβCA1 or LjαCA1/2 are not essential for the nitrogen fixation under normal symbiotic conditions. Nevertheless, the nodule-enhanced expression patterns and the diverse distributions in different types of cells imply their potential functions during root nodule symbiosis, such as CO₂ fixation, N assimilation, and pH regulation, which await further investigations.     

Suppression of Tumor Growth and Cell Migration by Indole-Based Benzenesulfonamides and Their Synergistic Effects in Combination with Doxorubicin

Pharmacological inhibition of the enzyme activity targeting carbonic anhydrases (CAs) demonstrated antiglaucoma and anticancer effects through pH control. Recently, we reported a series of indole-based benzenesulfonamides as potent CA inhibitors. The present study aimed to evaluate the antitumor effects of these compounds against various cancer cell lines, including breast cancer (MDA-MB-231, MCF-7, and SK-BR-3), lung cancer (A549), and pancreatic cancer (Panc1) cells. Overall, more potent cytotoxicity was observed on MCF-7 and SK-BR-3 cells than on lung or pancreatic cancer cells. Among the 15 compounds tested, A6 and A15 exhibited potent cytotoxic and antimigratory activities against MCF-7 and SK-BR-3 cells in the CoCl₂-induced hypoxic condition. While A6 and A15 markedly reduced the viability of control siRNA-treated cells, these compounds could not significantly reduce the viability of CA IX-knockdown cells, suggesting the role of CA IX in their anticancer activities. To assess whether these compounds exerted synergism with a conventional anticancer drug doxorubicin (DOX), the cytotoxic effects of A6 or A15 combined with DOX were analyzed using Chou−Talalay and Bliss independence methods. Our data revealed that both A6 and A15 significantly enhanced the anticancer activity of DOX. Among the tested pairs, the combination of DOX with A15 showed the strongest synergism on SK-BR-3 cells. Moreover, this combination further attenuated cell migration compared to the respective drug. Collectively, our results demonstrated that A6 and A15 suppressed tumor growth and cell migration of MCF-7 and SK-BR-3 cells through inhibition of CA IX, and the combination of these compounds with DOX exhibited synergistic cytotoxic effects on these breast cancer cells. Therefore, A6 and A15 may serve as potential anticancer agents alone or in combination with DOX against breast cancer.     

酰胺类物质辅助变性碳酸脱水酶复性及动力学

研究了酰胺类物质对促进变性牛碳酸脱水酶（CAB）的复性作用,考察了在较低盐酸胍浓度下添加剂浓度对复性收率的影响,并利用三级反应动力学方程对酰胺类化合物辅助CAB复性的动力学过程进行了深入分析.结果表明,含有酰胺基团的多种添加剂均能有效促进CAB复性;酰胺化合物与盐酸胍的作用相似,即复性速率常数k_N值均大于无添加剂时的自发复性,且随添加剂浓度增加而降低;但聚集体生成速率常数k_A值则在最佳添加剂浓度下出现最小值,从而使k_N/k_A值最大,复性收率最大.     

Roles of Carbonic Anhydrases and Carbonic Anhydrase Related Proteins in Zebrafish

During recent decades, zebrafish (Danio rerio) have become one of the most important model organisms in which to study different physiological and biological phenomena. The research field of carbonic anhydrases (CAs) and carbonic anhydrase related proteins (CARPs) is not an exception to this. The best-known function of CAs is the regulation of acid–base balance. However, studies performed with zebrafish, among others, have revealed important roles for these proteins in many other physiological processes, some of which had not yet been predicted in the light of previous studies and suggestions. Examples include roles in zebrafish pigmentation as well as motor coordination. Disruption of the function of these proteins may generate lethal outcomes. In this review, we summarize the current knowledge of CA-related studies performed in zebrafish from 1993–2021 that was obtained from PubMed search.     

Inhibitory Effects of Sulfonamide Derivatives on the β-Carbonic Anhydrase (MpaCA) from Malassezia pachydermatis,a Commensal,Pathogenic Fungus Present in Domestic Animals

Fungi are exposed to various environmental variables during their life cycle, including changes in CO₂ concentration. CO₂ has the potential to act as an activator of several cell signaling pathways. In fungi, the sensing of CO₂ triggers cell differentiation and the biosynthesis of proteins involved in the metabolism and pathogenicity of these microorganisms. The molecular machineries involved in CO₂ sensing constitute a promising target for the development of antifungals. Carbonic anhydrases (CAs, EC 4.2.1.1) are crucial enzymes in the CO₂ sensing systems of fungi, because they catalyze the reversible hydration of CO₂ to proton and HCO₃^-. Bicarbonate in turn boots a cascade of reactions triggering fungal pathogenicity and metabolism. Accordingly, CAs affect microorganism proliferation and may represent a potential therapeutic target against fungal infection. Here, the inhibition of the unique β-CA (MpaCA) encoded in the genome of Malassezia pachydermatis, a fungus with substantial relevance in veterinary and medical sciences, was investigated using a series of conventional CA inhibitors (CAIs), namely aromatic and heterocyclic sulfonamides. This study aimed to describe novel candidates that can kill this harmful fungus by inhibiting their CA, and thus lead to effective anti-dandruff and anti-seborrheic dermatitis agents. In this context, current antifungal compounds, such as the azoles and their derivatives, have been demonstrated to induce the selection of resistant fungal strains and lose therapeutic efficacy, which might be restored by the concomitant use of alternative compounds, such as the fungal CA inhibitors.     

Immobilization of carbonic anhydrase by embedding and covalent coupling into nanocomposite hydrogel containing hydrotalcite

Poly(acrylic acid-co-acrylamide)/hydrotalcite (PAA-AAm/HT) nanocomposite hydrogels activated by N-hydroxysuccinimide (NHS) in the presence of N, N'-dicyclohexylcarbodiimide (DCC) were used to immobilize carbonic anhydrase (CA) by embedding and covalent coupling. Cryo Scanning Electron Microscope (CryoSEM) proved the presence of free water in the porous network structures of the swollen hydrogels. Fluorescence microscopy indicated the existence of the immobilized enzyme in the hydrogels. Compared with un-activated hydrogels, activated hydrogels could improve the amount of the immobilization of enzyme, and maximum enzyme loading is about 4.6 mg/g of support for the activated hydrogels. The porous embedding and multi-point covalent linkage between enzyme and hydrogels strengthened the secondary structure stability of enzyme and thus enhanced enzyme stability in the presence of organic solvent and at high temperature. The immobilized enzyme in the activated hydrogel with enhanced structural stability offers great potential as a method to stabilize enzyme for various applications.     

微生物碳酸酐酶在岩溶发育中的研究现状及展望

碳酸酐酶是生物体中普遍存在的一种金属酶,能催化CO<,2>可逆的水合反应,微生物是碳酸酐酶的重要来源之一.综述了国内外微生物碳酸酐酶在岩溶发育中的研究现状,阐述了碳酸酐酶对碳循环的影响及其在石漠化治理中的作用,并对微生物碳酸酐酶在岩溶发育中的研究进行了展望.     

碳酸酐酶微生物沉积碳酸钙修复水泥基材料表面裂缝

采用琼脂作为载体将细菌菌株和营养物质涂刷在水泥石表面,为碳酸酐酶微生物酶化反应过程创造适宜环境以加速矿化,7d后在水泥石表面形成连续密实的沉淀膜层,X射线衍射分析表明该膜层中碳酸钙为方解石晶型。通过扫描电子显微镜观察到碳酸钙颗粒呈花生状,碳酸钙膜与水泥石表面黏结紧密。此外,改变水泥石表面裂缝宽度,对水泥石表面吸水性能进行测试,结果表明:当裂缝宽度小于100μm时,微生物覆膜修复后水泥石表面初始吸水速率和毛细吸水系数大大降低,修复效果明显;当裂缝宽度为100~200μm时,微生物覆膜修复能力有限;当裂缝宽度大于200μm时,微生物覆膜修复无效果。碳酸酐酶微生物诱导矿化修复过程既吸收大气中的温室气体CO2,也不产生任何有害物质,具有优异的环境友好特性。     

碳酸酐酶固定及在二氧化碳捕集应用研究进展

化石燃料的使用是全球气候变暖的主要原因,二氧化碳捕集、利用及封存(CCUS)技术能够有效减少碳排放,缓解气候变化压力。化学吸收法是CO₂捕集的重要方法之一,具有分离效率高、成本低等优点,但存在解吸过程中消耗能量较多、长期使用造成设备腐蚀性等问题。利用碳酸酐酶(CA)强化化学方法吸收CO₂,可以提高CO₂吸收效率,有效解决传统工艺中的热能损失,逐渐成为CO₂捕集与封存研究中的热点。但CA自身热稳定性低、可重复性差,需对其进行固定化以提高稳定性和活性。重点介绍了CA的固定化方法及常用载体材料,总结了CA在强化CO₂捕集中的作用机理,讨论了其在CO₂捕集中的应用,并对该技术的未来发展方向作出了展望。固定化载体与方法会影响固定化酶的性质,故CA在固定化时要选择合适的载体与方法。CA固定化方法包括吸附法、包埋法、共价结合法和交联法4种,各有优劣,应根据酶所应用的领域来选择合适的固定化方法。常用固定化载体有天然高分子、无机载体材料等,选择时应综合考虑载体的理化性质和工业应用能力。CO₂捕集过程中,CA主要通过促进化学溶剂吸收CO₂和诱导CO₂矿化生成碳酸钙两方面强化捕集效率。未来研究方向应集中于开发具有更高活性和稳定性的新型CA、制备廉价高性能载体材料和进一步探究CA的内在因素和外界条件对其工业应用产生的阻碍。     

Impairment of Hypoxia-Induced CA IX by Beta-Blocker Propranolol—Impact on Progression and Metastatic Potential of Colorectal Cancer Cells

The coexistence of cancer and other concomitant diseases is very frequent and has substantial implications for treatment decisions and outcomes. Beta-blockers, agents that block the beta-adrenergic receptors, have been related also to cancers. In the model of multicellular spheroids formed by colorectal cancer cells we described a crosstalk between beta-blockade by propranolol and tumour microenvironment. Non-selective beta-blocker propranolol decreased ability of tumour cells to adapt to hypoxia by reducing levels of HIF1α and carbonic anhydrase IX in 3D spheroids. We indicated a double action of propranolol in the tumour microenvironment by inhibiting the stability of HIF1α, thus mediating decrease of CA IX expression and, at the same time, by its possible effect on CA IX activity by decreasing the activity of protein kinase A (PKA). Moreover, the inhibition of β-adrenoreceptors by propranolol enhanced apoptosis, decreased number of mitochondria and lowered the amount of proteins involved in oxidative phosphorylation (V-ATP5A, IV-COX2, III-UQCRC2, II-SDHB, I-NDUFB8). Propranolol reduced metastatic potential, viability and proliferation of colorectal cancer cells cultivated in multicellular spheroids. To choose the right treatment strategy, it is extremely important to know how the treatment of concomitant diseases affects the superior microenvironment that is directly related to the efficiency of anti-cancer therapy     

Proteomic Profiling of the First Human Dental Pulp Mesenchymal Stem/Stromal Cells from Carbonic Anhydrase II Deficiency Osteopetrosis Patients

Osteopetrosis is a hereditary disorder characterized by sclerotic, thick, weak, and brittle bone. The biological behavior of mesenchymal cells obtained from osteopetrosis patients has not been well-studied. Isolated mesenchymal stem/stromal cells from dental pulp (DP-MSSCs) of recently extracted deciduous teeth from osteopetrosis (OP) patients and healthy controls (HCs) were compared. We evaluated whether the dental pulp of OP patients has a population of MSSCs with similar multilineage differentiation capability to DP-MSSCs of healthy subjects. Stem/progenitor cells were characterized using immunohistochemistry, flow cytometry, and proteomics. Our DP-MSSCs were strongly positive for CD44, CD73, CD105, and CD90. DP-MSSCs obtained from HC subjects and OP patients showed similar patterns of proliferation and differentiation as well as gene expression. Proteomic analysis identified 1499 unique proteins with 94.3% similarity in global protein fingerprints of HCs and OP patients. Interestingly, we observed subtle differences in expressed proteins of osteopetrosis disease-related in pathways, including MAPK, ERK 1/2, PI3K, and integrin, rather than in the stem cell signaling network. Our findings of similar protein expression signatures in DP-MSSCs of HC and OP patients are of paramount interest, and further in vivo validation study is needed. There is the possibility that OP patients could have their exfoliating deciduous teeth banked for future use in regenerative dentistry.     

Quantitative Proteomic Profiling of Small Molecule Treated Mesenchymal Stem Cells Using Chemical Probes

The differentiation of human adipose derived stem cells toward a neural phenotype by small molecules has been a vogue topic in the last decade. The characterization of the produced cells has been explored on a broad scale, examining morphological and specific surface protein markers; however, the lack of insight into the expression of functional proteins and their interactive partners is required to further understand the extent of the process. The phenotypic characterization by proteomic profiling allows for a substantial in-depth analysis of the molecular machinery induced and directing the cellular changes through the process. Herein we describe the temporal analysis and quantitative profiling of neural differentiating human adipose-derived stem cells after sub-proteome enrichment using a bisindolylmaleimide chemical probe. The results show that proteins enriched by the Bis-probe were identified reproducibly with 133, 118, 126 and 89 proteins identified at timepoints 0, 1, 6 and 12, respectively. Each temporal timepoint presented several shared and unique proteins relative to neural differentiation and their interactivity. The major protein classes enriched and quantified were enzymes, structural and ribosomal proteins that are integral to differentiation pathways. There were 42 uniquely identified enzymes identified in the cells, many acting as hubs in the networks with several interactions across the network modulating key biological pathways. From the cohort, it was found by gene ontology analysis that 18 enzymes had direct involvement with neurogenic differentiation.     

Exploration of the binding mode of indanesulfonamides as selective inhibitors of human carbonic anhydrase type VII by targeting Lys 91

Convulsions are common neurological disorders in clinical medicine and are triggered by several mechanisms. The enhancement of neuronal excitability can be related, among other factors, to GABAergic depolarization. Carbonic anhydrase (CA) VII contributes to this electrophysiological behavior by providing bicarbonate anion, which can mediate current through channels coupled to GABA(A) receptors. Among the cytosolic CAs, the mechanism of action and inhibition of CA VII is less understood. We present herein the pharmacological evaluation of both enantiomers of an indanesulfonamide compound substituted by a pentafluorophenyl moiety against CA VII and five other human CA isoforms to evaluate their selectivity. The investigated compounds are powerful inhibitors of hCA VII, with K(i) values in the range of 1.7-3.3 nM, but their selectivity needs to be improved. A molecular modeling study was conducted to rationalize the structure-activity relationships and provide useful insight into the future design of selective hCA VII inhibitors.