The Potential Use of Mechanism-Based Enzyme Inactivators in Medicine

Abstract

A new three dimensional representation of enzyme inhibition, applied to Lineweaver-Burk, Hanes and Eadie-Hofstee plots is presented. This type of representation has advantages for enzyme inhibition diagnosis, showing graphic characteristics that pass unnoticed in linear plots.     

Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: Implications for angiotensin II-induced vascular dysfunction

Glutathionylation of the Na⁺-K⁺ pump’s β₁-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na⁺-K⁺ pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na⁺-K⁺ pump activity is an important determinant of vascular tone through effects on [Ca²⁺]_i, we have examined the role of oxidative regulation of the Na⁺-K⁺ pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β₁-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na⁺-K⁺ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K⁺-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na⁺-K⁺ ATPase and decrease in K⁺-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β₁-subunit. Knock-out of FXYD1 dramatically decreased K⁺-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β₁-subunit glutathionylation, and enhanced K⁺-induced vasorelaxation. Ang II inhibits the Na⁺-K⁺ pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump’s β₁-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na⁺-K⁺ pump and may have an important protective role in the vasculature under conditions of oxidative stress.     

A Possible Role of Slips in Cytochrome C Oxidase in the Antioxygen Defense System of the Cell

Evidence is available showing that the coupling efficiency of the proton pump in cytochrome c oxidase of mitochondria can under certain conditions decrease significantly below the maximum attainable value. The view is developed that slips in the proton pump of cytochrome c oxidase represent an intrinsic switch mechanism which regulates the relative contribution of energy transfer and respiratory protection against oxygen toxicity by the oxidase.     

近30年来黄河三角洲植被净初级生产力时空特征及主要影响因素

我国拥有丰富的海岸带蓝色碳汇,准确把握海岸带蓝碳生态系统净初级生产力(NPP)状况,辨识不同人为干扰下蓝碳生态系统NPP的时空分布特征具有重要意义。以黄河三角洲为研究区,以近30a(1987年、1995年、2005年、2016—2017年)为时间尺度,通过遥感手段和现场调查,对黄河三角洲NPP时空变化特征及其主要影响因素进行研究。结果显示:(1)近30年来研究区NPP均值和总量呈现先下降又略微增长的特征,2016—2017年度NPP平均值为294.38g C m~(-2)a~(-1),总量为710.05Gg C/a,表现出显著的季节差异。(2)研究区NPP在各行政区、保护区和地表覆盖类型中均表现出了明显的空间分异性;2016—2017年度NPP分区结果显示,不同分区面积由大到小依次为中生产力区(49.5%)、低生产力区(38.3%)和高生产力区(12.1%)。(3)研究区NPP的时空分异性是地表覆盖类型和植被生长状况共同影响的结果,海陆交互作用、开发利用活动和近年来的生态建设是NPP时空变化的主要影响因素。(4)湿地植被和农田是研究区碳汇的主要贡献者,20世纪90年代以来二者NPP均值逐渐上升,在2016—2016年度分别达570.28g C m~(-2)a~(-1)和335.92g C m~(-2)a~(-1);近30年来,湿地植被NPP总量逐渐减少,农田NPP总量则逐渐增加。湿地植被是海岸带蓝碳的典型载体,农田作为位于滨海地区、由湿地植被转化而来、本身具有较高固碳能力和潜力的碳汇类型,可作为海岸带蓝碳的重要补充。     

Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich’s ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.     

Development of benzoxazole deoxybenzoin oxime and acyloxylamine derivatives targeting innate immune sensors and xanthine oxidase for treatment of gout

Both the inhibition of inflammatory flares and the treatment of hyperuricemia itself are included in the management of gout. Extending our efforts to development of gout therapy, two series of benzoxazole deoxybenzoin oxime derivatives as inhibitors of innate immune sensors and xanthine oxidase (XOD) were discovered in improving hyperuricemia and acute gouty arthritis. In vitro studies revealed that most compounds not only suppressed XOD activity, but blocked activations of NOD-like receptor (NLRP3) inflammasome and Toll-like receptor 4 (TLR4) signaling pathway. More importantly, (E)-1-(6-methoxybenzo[d]oxazol-2-yl)-2-(4-methoxyphenyl)ethanone oxime (5d) exhibited anti-hyperuricemic and anti-acute gouty arthritis activities through regulating XOD, NLRP3 and TLR4. Compound 5d may serve as a tool compound for further design of anti-gout drugs targeting both innate immune sensors and XOD.     

Multifunctional 5,6-dimethoxybenzo[d]isothiazol-3(2H)-one-N-alkylbenzylamine derivatives with acetylcholinesterase,monoamine oxidases and β-amyloid aggregation inhibitory activities as potential agents against Alzheimer’s disease

A series of 5,6-dimethoxybenzo[d]isothiazol-3(2H)-one-N-alkylbenzylamine derivatives were designed, synthesized and evaluated as potential multifunctional agents for the treatment of Alzheimer’s disease (AD). The in vitro assays indicated that most of these derivatives were selective AChE inhibitors with good multifunctional properties. Among them, compounds 11b and 11d displayed comprehensive advantages, with good AChE (IC₅₀?=?0.29?±?0.01?μM and 0.46?±?0.02?μM, respectively), MAO-A (IC₅₀?=?8.2?±?0.08?μM and 7.9?±?0.07?μM, respectively) and MAO-B (IC₅₀?=?20.1?±?0.16?μM and 43.8?±?2.0% at 10?μM, respectively) inhibitory activities, moderate self-induced Aβ_1–42 aggregation inhibitory potency (35.4?±?0.42% and 48.0?±?1.53% at 25?μM, respectively) and potential antioxidant activity. In addition, the two representative compounds displayed high BBB permeability in vitro. Taken together, these multifunctional properties make 11b and 11d as a promising candidate for the development of efficient drugs against AD.     

Methoxycamalexins and related compounds: Syntheses,antifungal activity and inhibition of brassinin oxidase

The phytoalexin camalexin is a competitive inhibitor of brassinin oxidase, an enzyme that detoxifies the phytoalexin brassinin and is produced by an economically important plant pathogen. For this reason, the camalexin scaffold has guided the design of inhibitors of brassinin detoxification. To further understand the structure–activity relationships of camalexin related compounds, the syntheses of monomethoxy and dimethoxycamalexins were undertaken. Four monomethoxy camalexins together with 4,6-dimethoxy and 5,7-dimethoxy camalexins were prepared from the corresponding methoxyindoles using the Ayer's method. The dimethoxy derivatives were prepared from the corresponding dimethoxyindole-3-thiocarboxamides using the Hantzsch reaction; however, this method did not work for the syntheses of 4,6-dimethoxy and 5,7-dimethoxycamalexins due to the lower reactivities of the corresponding indole-3-thiocarboxamides. The antifungal activity and brassinin oxidase inhibitory activity of all methoxycamalexins and ten camalexin related compounds were investigated. Among the 20 compounds evaluated, monomethoxycamalexins were stronger antifungals than the dimethoxy derivatives. However, remarkably, 5,6-dimethoxycamalexin, 6,7-dimethoxycamalexin and 5-methoxycamalexin displayed the strongest inhibitory activity against brassinin oxidase, while 4,5-dimethoxycamalexin displayed no inhibitory effect. Altogether the structure–activity relationships of camalexin related compounds suggest that the targets for fungal growth inhibition and brassinin oxidase inhibition are unrelated and emphasize that brassinin oxidase inhibitors do not need to be antifungal.     

Design,synthesis and evaluation of 4′-OH-flurbiprofen-chalcone hybrids as potential multifunctional agents for Alzheimer’s disease treatment

A series of 4′-OH-flurbiprofen-chalcone hybrids were designed, synthesized and evaluated as potential multifunctional agents for the treatment of Alzheimer’s disease. The biological screening results indicated that most of these hybrids exhibited good multifunctional activities. Among them, compounds 7k and 7m demonstrated the best inhibitory effects on self-induced Aβ_1–42 aggregation (60.0% and 78.2%, respectively) and Cu²⁺-induced Aβ_1–42 aggregation (52.4% and 95.0%, respectively). Moreover, these two representative compounds also exhibited good antioxidant activities, MAO inhibitions, biometal chelating abilities and anti-neuroinflammatory activities in vitro. Furthermore, compound 7m displayed appropriate blood-brain barrier permeability. These multifunctional properties highlight compound 7k and 7m as promising candidates for further development of multi-functional drugs against AD.     

Synthesis,structure-activity relationships,and mechanistic studies of 5-arylazo-tropolone derivatives as novel xanthine oxidase (XO) inhibitors

Xanthine oxidase (XO) is an enzyme that contains molybdenum at the active site and catalyzes the oxidation of purine bases to uric acid. Even though XO inhibitors are widely used for the treatment of hyperuricemia and gout, only very few such compounds are clinically used as drugs for the treatment of these diseases. Given the unique physicochemical properties of tropolone, i.e., its chelating effect and the pKa value that is similar to that of carboxylic acid, we have synthesized 22 5-arylazotropolone derivatives as potential XO inhibitors. In vitro enzyme-inhibitory assays for XO revealed that 3-nitro derivative 1j showed the most potent XO inhibitory activity, which is by one order of magnitude more potent than allopurinol. An enzyme-kinetic study revealed that 1j inhibited the production of uric acid by XO both competitively and non-competitively. A docking-simulation study of 1j with XO suggested that the carbonyl and hydroxyl groups of the tropolone ring interact with the hydroxy group that acts as a ligand for molybdenum and the amino acid residues around the active site of XO.