血必净联合利奈唑胺注射液对老年重症肺炎患者血清肺表面活性蛋白、基质金属蛋白酶及其组织抑制剂水平的影响

目的:探讨血必净联合利奈唑胺注射液治疗老年重症肺炎患者的临床疗效及对患者血清肺表面活性蛋白(Pulmonary surfactant protein,SP)、基质金属蛋白酶(Matrix metalloproteinases,MMPs)及其组织抑制剂(Matrix metalloproteinases tissue inhibitor,TIMPs)水平的影响。方法:选择我院2015年6月～2017年12月收治的101例老年重症肺炎患者,按随机数字表法分为对照组(n=48)和研究组(n=53)。对照组采用利奈唑胺注射液治疗,研究组在对照组基础上采用血必净治疗。比较两组临床疗效,细菌清除情况,症状缓解时间,治疗前后血清SP、MMPs、TIMPs水平的变化,动脉血气,肺功能,不良反应的发生情况和28天内病死率。结果:治疗后,研究组有效率、细菌清除率均显著高于对照组(均P0.05),发热消失、血常规恢复、痰液颜色改变及胸部影像明显吸收时间均明显短于对照组(P0.05);两组血清SP-A、SP-B、SP-C、SP-D、MMP-2、MMP-9及TIMP-1及TIMP-2、血氧饱和度(blood oxygen saturation,SaO_2)、动脉血二氧化碳分压(arterial blood,PaCO_2)、动脉血二氧化碳分压(arterial blood CO_2 partial pressure of CO_2 partial pressure,PaCO_2)、峰流速(peak velocity of flow,PEF)水平均较治疗前显著下降,而血氧饱和度(blood oxygen saturation,SaO_2)、氧分压(oxygen partial pressure,PaO2)、最大呼气中段流量(maximum tidal midexpiratory flow,MMF)、用力肺活量(forced vital capacity,FVC)均较治疗前明显上升,且研究组以上指标变化较对照组更明显(均P0.05)。两组不良反应的发生情况比较差异无统计学意义(P0.05),而研究组在28天内病死率显著低于对照组(P0.05)。结论:血必净联合利奈唑胺注射液对老年重症肺炎患者的疗效优于单用利奈唑胺注射液治疗,可能与其显著降低患者血清SP、MMPs及TIMPs水平,改善肺功能,降低病死率有关。     

Quantitative assessment of specific carbonic anhydrase inhibitors effect on hypoxic cells using electrical impedance assays

Carbonic anhydrase IX (CA IX) is an important orchestrator of hypoxic tumour environment, associated with tumour progression, high incidence of metastasis and poor response to therapy. Due to its tumour specificity and involvement in associated pathological processes: tumourigenesis, angiogenesis, inhibiting CA IX enzymatic activity has become a valid therapeutic option. Dynamic cell-based biosensing platforms can complement cell-free and end-point analyses and supports the process of design and selection of potent and selective inhibitors. In this context, we assess the effectiveness of recently emerged CA IX inhibitors (sulphonamides and sulphocoumarins) and their antitumour potential using an electrical impedance spectroscopy biosensing platform. The analysis allows discriminating between the inhibitory capacities of the compounds and their inhibition mechanisms. Microscopy and biochemical assays complemented the analysis and validated impedance findings establishing a powerful biosensing tool for the evaluation of carbonic anhydrase inhibitors potency, effective for the screening and design of anticancer pharmacological agents.     

Virtual screening-driven identification of human carbonic anhydrase inhibitors incorporating an original, new pharmacophore

Combinated ligand- and pharmacophore-based virtual screening approaches were used to discover novel potential pharmacophores acting as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs). A free database of commercially available compounds was screened through drug-like filters using a four-point pharmacophore, and followed by docking calculation within the active site of an X-ray structure of isoform CA II. One compound, bearing a trifluoro-dihydroxy-propanone moiety, showed an interesting, selective inhibitory activity in low micromolar range against this isoform versus CA I. The chemical originality of this new pharmacophore can represent an important bioisosteric alternative to the sulfonamido-based functionalities, thus leading to the development of a new class of CAIs.     

Refined structure of the acetazolamide complex of human carbonic anhydrase II at 1.9 Å

The binding of acetazolamide to human carbonic anhydrase II (HCA II) has been investigated by X-ray crystallography. The atomic positions of the enzyme inhibitor complex have been refined at 1.9 Å resolution using the least squares refinement program package PROLSQ. The crystallographic R-factor is 17.6%. The bound inhibitor is clearly resolved in the active site of the enzyme. The acetazolamide amine group is bound as a fourth ligand to the zinc ion, the other three are all histidine residues. In addition to van der Waals' interactions and the previously described binding of the sulphonamide group, the inhibitor forms a hydrogen bond from the carbonyl oxygen of the acetylamido group to the amino group of Gln 92.     

Kinetic and in silico studies of hydroxy-based inhibitors of carbonic anhydrase isoforms I and II

A series of hydroxy and phenolic compounds have been assayed for the inhibition of two physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic human isozymes I and II. The investigated molecules showed inhibition constants in the range of 1.07–4003 and 0.09–31.5?μM at the hCA I and hCA II enzymes, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico studies were also applied. Molecular docking scores of the studied compounds are compared using three different scoring algorithms, namely Glide/SP, Glide/XP and Glide/IFD. In addition, different ADME (absorption, distribution, metabolism and excretion) analysis was performed. All the examined compounds were found within the acceptable range of pharmacokinetic profiles.     

A protein is involved in accessibility of the inhibitor acetazolamide to the carbonic anhydrase(s) in the cyanobacterium Synechocystis PCC 6803

A gene, zam (for resistance to acetazolamide), controlling resistance to the carbonic anhydrase inhibitor acetazolamide, is described. It has been cloned from a spontaneous mutant, AZA^r-5b, isolated from the cyanobacterium Synechocystis PCC 6803, for its resistance to this drug (Bédu et al., Plant Physiol 93: 1312–1315, 1990). This mutant, besides its resistance to acetazolamide, displayed an absence of catalysed oxygen exchange activity on whole cells, suggestive of a deficiency in carbonic anhydrase activity. The gene was isolated by screening a genomic library of AZA^r-5b, and selecting for the capacity to transfer the AZA^r phenotype to wild-type cells. A system leading to forced homologous recombination in the host chromosome, using a platform vector, was devised in order to bypass direct selection difficulties. The putative encoded protein, 782 amino acids long, showed some homology with four eukaryotic and prokaryotic proteins involved in different cellular processes, one of them suppressing a phosphatase deficiency. The mutated allele of AZA^r-5b showed an in-frame 12 nucleotide duplication, which should not interfere with translation, and might result from transposition of a mobile element. Integration into a wild-type genome of either the spontaneous mutated allele or one inactivated by insertional mutagenesis conferred the character of resistance, but not the deficiency in oxygen exchange, indicating that the two phenotypic aspects of AZA^r-5b corresponded to two independent mutations. A working hypothesis explaining the phenotypes of the mutants is that the presence of the Zam protein would be necessary for the inhibitor to reach (one of) the two carbonic anhydrases present in this strain. This, however, would be a secondary action, the physiological role of the protein still being cryptic.     

Discovering novel carbonic anhydrase type IX (CA IX) inhibitors from seven million compounds using virtual screening and in vitro analysis

Carbonic anhydrase type IX (CA IX) enzyme is mostly over expressed in different cancer cell lines and tumor tissues. Potent CA IX inhibitors can be effective for adjusting the pH imbalance in tumor cells. In the present work, we represented the successful application of high throughput virtual screening (HTVS) of large dataset from ZINC database included of ～7 million compounds to discover novel inhibitors of CA IX. HTVS and molecular docking were performed using consequence Glide/standard precision (SP), extra precision (XP) and induced fit docking (IFD) molecular docking protocols. For each compound, docking code calculates a set of low-energy poses and then exhaustively scans the binding pocket of the target with small compounds. Novel CA IX inhibitor candidates were suggested based on molecular modeling studies and a few of them were tested using in vitro analysis. These compounds were determined as good inhibitors against human CA IX target with K_i in the range of 0.85–1.58?μM. In order to predict the pharmaceutical properties of the selected compounds, ADME (absorption, distribution, metabolism and excretion) analysis was also carried out.     

(3,4-Dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and its derivatives as carbonic anhydrase isoenzymes inhibitors

In this study, we have synthesised (3,4-dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and a series of its derivatives (5, 13–16) and tested the ability of these compounds to inhibit two metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, hCA I and hCA II. The synthesised compounds showed inhibitory effect on hCA I and hCA II isozymes. The results showed that synthesised compounds (5, 13–16) demonstrated the best inhibition activity against hCA I (IC₅₀: 3.22–54.28 μM) and hCA II (IC₅₀: 18.52–142.01 μM). The compound 14 showed the highest inhibiton effect against hCA I (IC₅₀: 3.22 μM; K_i: 1.19?±?1.4 μM). On the other hand, the compound 13 showed the highest inhibiton effect against hCA II (IC₅₀: 18.52 μM; K_i: 3.25?±?1.13 μM).     

7-Aryl-triazolyl-substituted sulfocoumarins are potent,selective inhibitors of the tumor-associated carbonic anhydrase IX and XII

Sulfocoumarins behave as interesting inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Here, we report a new series of 7-substituted derivatives which were obtained by the click chemistry approach from 7-propargyloxy-sulfocoumarin and aryl azides incorporating halogens, hydroxy, methoxy and carboxyl moieties in their molecules. The new compounds were screened for the inhibition on four physiologically relevant human CA (hCA) isoforms, the cytosolic hCA I and II and the transmembrane tumor-associated hCA IX and XII. The new compounds did not inhibit the cytosolic isoforms but were low nanomolar inhibitors of the tumor-associated ones hCA IX and XII.     

Isatin: a privileged scaffold for the design of carbonic anhydrase inhibitors

The isatin scaffold is the constitutive fragment of several natural and synthetic bioactive molecules. Albeit several benzene sulphonamide-based carbonic anhydrase inhibitors (CAIs) have been reported, only recently isatin benzene sulphonamides have been studied and proposed as CAIs. In this study we have designed, synthesised, and evaluated the biological activity of a series of differently substituted isatin-based benzene sulphonamides which have been designed for the inhibition of carbonic anhydrase isoforms. The activity of all the synthesised compounds was evaluated towards human carbonic anhydrase I, II, IX, and XII isozymes. Our results indicate that the nature and position of substituents on the isatin ring can modulate both activity and isozyme selectivity.     

The inhibitory effect of boric acid on hypoxia-regulated tumour-associated carbonic anhydrase IX

Carbonic anhydrases (EC 4.2.1.1) catalyse the reversible hydration of CO₂ into bicarbonate and protons. As a hypoxia-sensitive and tumour-associated isoform, isoform CA IX, is significantly overexpressed in various malignancies, being a validated target for new anticancer/antimetastatic drugs. A multitude of studies has shown that CA IX inhibition decreases cancer cell proliferation and metastasis through pHe/pHi modulation and enhancement of ferroptosis among others. Numerous studies demonstrated increased efficacy of cytotoxic drugs combined with CA inhibitors (CAIs) in various cancer types. We tested the inhibitory effect of boric acid (BA), an inorganic Lewis acid, on CA IX as well as other isoforms (CA I, II, and XII). BA acted as a millimolar in vitro CAI, decreased proliferation of two cancer cell lines, although not strong correlations between the in vitro inhibition and in vivo effects were observed. The mechanism of antiproliferative action of BA should be investigated in more detail.