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17 pages, 2038 KiB

Open AccessArticle

Transdermal Delivery of 2-PAM as a Tool to Increase the Effectiveness of Traditional Treatment of Organophosphate Poisoning

by Leysan Vasileva, Gulnara Gaynanova, Irina Zueva, Anna Lyubina, Syumbelya Amerhanova, Daina Buzyurova, Vasily Babaev, Alexandra Voloshina, Konstantin Petrov and Lucia Zakharova

Int. J. Mol. Sci. 2022, 23(23), 14992; https://doi.org/10.3390/ijms232314992 - 30 Nov 2022

Cited by 4 | Viewed by 2334

Abstract

For the first time, the efficacy of post-exposure treatment of organophosphate (OP) poisoning was increased by transdermal delivery of acetylcholinesterase (AChE) reactivator pyridine-2-aldoxime methochloride (2-PAM) as a preventive countermeasure. By selecting the optimal ratio of components, classical transfersomes (based on soybean phosphatidylcholine and [...] Read more.

For the first time, the efficacy of post-exposure treatment of organophosphate (OP) poisoning was increased by transdermal delivery of acetylcholinesterase (AChE) reactivator pyridine-2-aldoxime methochloride (2-PAM) as a preventive countermeasure. By selecting the optimal ratio of components, classical transfersomes (based on soybean phosphatidylcholine and Tween 20) and modified transfersomes (based on soybean phosphatidylcholine, Tween 20 and pyrrolidinium cationic surfactants with different hydrocarbon tail lengths) were obtained for 2-PAM encapsulation. Transfersomes modified with tetradecylpyrrolidinium bromide showed the best results in encapsulation efficiency and sustained release of 2-PAM from vesicles. Using Franz cells, it was found that the incorporation of surfactants into PC liposomes results in a more prolonged release of 2-PAM through the rat skin. Transfersomes containing 2-PAM, after exhaustive physical and chemical characterization, were embedded in a gel based on Carbopol^® 940. A significantly high degree of erythrocyte AChE reactivation (23 ± 7%) was shown for 2-PAM in unmodified transfersomes in vivo. Preliminary transdermal administration of 2-PAM 24 h before emergency post-exposure treatment of OP poisoning leads to an increase in the survival rate of rats from 55% to 90%. Full article

(This article belongs to the Special Issue Enzymes as Targets for Drug Development)

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17 pages, 7802 KiB

Open AccessArticle

Nifedipine Exacerbates Lipogenesis in the Kidney via KIM-1, CD36, and SREBP Upregulation: Implications from an Animal Model for Human Study

by Yen-Chung Lin, Jhih-Cheng Wang, Mai-Szu Wu, Yuh-Feng Lin, Chang-Rong Chen, Chang-Yu Chen, Kuan-Chou Chen and Chiung-Chi Peng

Int. J. Mol. Sci. 2020, 21(12), 4359; https://doi.org/10.3390/ijms21124359 - 19 Jun 2020

Cited by 10 | Viewed by 2777

Abstract

Dysregulation of fatty acid oxidation and accumulation of fatty acids can cause kidney injury. Nifedipine modulates lipogenesis-related transcriptional factor SREBP-1/2 in proximal tubular cells by inhibiting the Adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) pathway in vitro. However, the mechanisms by which nifedipine (NF) [...] Read more.

Dysregulation of fatty acid oxidation and accumulation of fatty acids can cause kidney injury. Nifedipine modulates lipogenesis-related transcriptional factor SREBP-1/2 in proximal tubular cells by inhibiting the Adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) pathway in vitro. However, the mechanisms by which nifedipine (NF) modulates lipotoxicity in vivo are unclear. Here, we examined the effect of NF in a doxorubicin (DR)-induced kidney injury rat model. Twenty-four Sprague–Dawley rats were divided into control, DR, DR+NF, and high-fat diet (HFD) groups. The DR, DR+NF, and HFD groups showed hypertension and proteinuria. Western blotting and immunohistochemical analysis showed that NF significantly induced TNF-α, CD36, SREBP-1/2, and acetyl-CoA carboxylase expression and renal fibrosis, and reduced fatty acid synthase and AMPK compared to other groups (p < 0.05). Additionally, 18 patients with chronic kidney disease (CKD) who received renal transplants were enrolled to examine their graft fibrosis and lipid contents via transient elastography. Low-density lipoprotein levels in patients with CKD strongly correlated with lipid contents and fibrosis in grafted kidneys (p < 0.05). Thus, NF may initiate lipogenesis through the SREBP-1/2/AMPK pathway and lipid uptake by CD36 upregulation and aggravate renal fibrosis in vivo. Higher low-density lipoprotein levels may correlate with renal fibrosis and lipid accumulation in grafted kidneys of patients with CKD. Full article

(This article belongs to the Special Issue Enzymes as Targets for Drug Development)

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19 pages, 6482 KiB

Open AccessArticle

Nifedipine Upregulates ATF6-α, Caspases -12, -3, and -7 Implicating Lipotoxicity-Associated Renal ER Stress

by Chiung-Chi Peng, Chang-Rong Chen, Chang-Yu Chen, Yen-Chung Lin, Kuan-Chou Chen and Robert Y. Peng

Int. J. Mol. Sci. 2020, 21(9), 3147; https://doi.org/10.3390/ijms21093147 - 29 Apr 2020

Cited by 5 | Viewed by 3173

Abstract

Nifedipine (NF) is reported to have many beneficial effects in antihypertensive therapy. Recently, we found that NF induced lipid accumulation in renal tubular cells. Palmitic acid-induced renal lipotoxicity was found to be partially mediated by endoplasmic reticular (ER) stress, while it can also [...] Read more.

Nifedipine (NF) is reported to have many beneficial effects in antihypertensive therapy. Recently, we found that NF induced lipid accumulation in renal tubular cells. Palmitic acid-induced renal lipotoxicity was found to be partially mediated by endoplasmic reticular (ER) stress, while it can also be elicited by NF in kidney cells; we examined the induction of suspected pathways in both in vitro and in vivo models. NRK52E cells cultured in high-glucose medium were treated with NF (30 µM) for 24–48 h. ER stress-induced lipotoxicity was explored by staining with thioflavin T and Nile red, transmission electron microscopy, terminal uridine nick-end labeling, and Western blotting. ER stress was also investigated in rats with induced chronic kidney disease (CKD) fed NF for four weeks. NF induced the production of unfolded protein aggregates, resulting in ER stress, as evidenced by the upregulation of glucose-regulated protein, 78 kDa (GRP78), activating transcription factor 6α (ATF6α), C/EBP-homologous protein (CHOP), and caspases-12, -3, and -7. In vitro early apoptosis was more predominant than late apoptosis. Most importantly, ATF6α was confirmed to play a unique role in NF-induced ER stress in both models. CKD patients with hypertension should not undergo NF therapy. In cases where it is required, alleviation of ER stress should be considered to avoid further damaging the kidneys. Full article

(This article belongs to the Special Issue Enzymes as Targets for Drug Development)

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16 pages, 4025 KiB

Open AccessArticle

Osteoclast-Released Wnt-10b Underlies Cinacalcet Related Bone Improvement in Chronic Kidney Disease

by Cai-Mei Zheng, Yung-Ho Hsu, Chia-Chao Wu, Chien-Lin Lu, Wen-Chih Liu, Jing-Quan Zheng, Yuh-Feng Lin, Hui-Wen Chiu, Tian-Jong Chang, Jia-Fwu Shyu and Kuo-Cheng Lu

Int. J. Mol. Sci. 2019, 20(11), 2800; https://doi.org/10.3390/ijms20112800 - 8 Jun 2019

Cited by 7 | Viewed by 3647

Abstract

Secondary hyperparathyroidism (SHPT) relates to high turnover bone loss and is responsible for most bone fractures among chronic kidney disease (CKD) patients. Changes in the Wingless/beta-catenin signaling (Wnt/β-catenin) pathway and Wnt inhibitors have been found to play a critical role in CKD related [...] Read more.

Secondary hyperparathyroidism (SHPT) relates to high turnover bone loss and is responsible for most bone fractures among chronic kidney disease (CKD) patients. Changes in the Wingless/beta-catenin signaling (Wnt/β-catenin) pathway and Wnt inhibitors have been found to play a critical role in CKD related bone loss. A calcimimetic agent, cinacalcet, is widely used for SHPT and found to be similarly effective for parathyroidectomy clinically. A significant decrease in hip fracture rates is noted among US hemodialysis Medicare patients since 2004, which is probably related to the cinacalcet era. In our previous clinical study, it was proven that cinacalcet improved the bone mineral density (BMD) even among severe SHPT patients. In this study, the influence of cinacalcet use on bone mass among CKD mice was determined. Cinacalcet significantly reduced the cortical porosity in femoral bones of treated CKD mice. It also improved the whole-bone structural properties through increased stiffness and maximum load. Cinacalcet increased femoral bone wingless 10b (Wnt10b) expression in CKD mice. In vitro studies revealed that cinacalcet decreased osteoclast bone resorption and increased Wnt 10b release from osteoclasts. Cinacalcet increased bone mineralization when culturing the osteoblasts with cinacalcet treated osteoclast supernatant. In conclusion, cinacalcet increased bone quantity and quality in CKD mice, probably through increased bone mineralization related with osteoclast Wnt 10b secretion. Full article

(This article belongs to the Special Issue Enzymes as Targets for Drug Development)

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11 pages, 2086 KiB

Open AccessArticle

Novel Peptide-Based Inhibitors for Microtubule Polymerization in Phytophthora capsici

by Sang-Choon Lee, Sang-Heon Kim, Rachel A. Hoffmeister, Moon-Young Yoon and Sung-Kun Kim

Int. J. Mol. Sci. 2019, 20(11), 2641; https://doi.org/10.3390/ijms20112641 - 29 May 2019

Cited by 8 | Viewed by 2797

Abstract

The plant disease Phytophthora blight, caused by the oomycete pathogen Phytophthora capsici, is responsible for major economic losses in pepper production. Microtubules have been an attractive target for many antifungal agents as they are involved in key cellular events such as cell [...] Read more.

The plant disease Phytophthora blight, caused by the oomycete pathogen Phytophthora capsici, is responsible for major economic losses in pepper production. Microtubules have been an attractive target for many antifungal agents as they are involved in key cellular events such as cell proliferation, signaling, and migration in eukaryotic cells. In order to design a novel biocompatible inhibitor, we screened and identified inhibitory peptides against alpha- and beta-tubulin of P. capsici using a phage display method. The identified peptides displayed a higher binding affinity (nanomolar range) and improved specificity toward P. capsici alpha- and beta-tubulin in comparison to Homo sapiens tubulin as evaluated by fluorometric analysis. One peptide demonstrated the high inhibitory effect on microtubule formation with a nanomolar range of IC₅₀ values, which were much lower than a well-known chemical inhibitor—benomyl (IC₅₀ = 500 µM). Based on these results, this peptide can be employed to further develop promising candidates for novel antifungal agents against Phytophthora blight. Full article

(This article belongs to the Special Issue Enzymes as Targets for Drug Development)

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15 pages, 4242 KiB

Open AccessArticle

Nifedipine Modulates Renal Lipogenesis via the AMPK-SREBP Transcriptional Pathway

by Yen-Chung Lin, Mai-Szu Wu, Yuh-Feng Lin, Chang-Rong Chen, Chang-Yu Chen, Chang-Jui Chen, Che-Chou Shen, Kuan-Chou Chen and Chiung-Chi Peng

Int. J. Mol. Sci. 2019, 20(7), 1570; https://doi.org/10.3390/ijms20071570 - 29 Mar 2019

Cited by 26 | Viewed by 4723

Abstract

Lipid accumulation in renal cells has been implicated in the pathogenesis of obesity-related kidney disease, and lipotoxicity in the kidney can be a surrogate marker for renal failure or renal fibrosis. Fatty acid oxidation provides energy to renal tubular cells. Ca²⁺ is [...] Read more.

Lipid accumulation in renal cells has been implicated in the pathogenesis of obesity-related kidney disease, and lipotoxicity in the kidney can be a surrogate marker for renal failure or renal fibrosis. Fatty acid oxidation provides energy to renal tubular cells. Ca²⁺ is required for mitochondrial ATP production and to decrease reactive oxygen species (ROS). However, how nifedipine (a calcium channel blocker) affects lipogenesis is unknown. We utilized rat NRK52E cells pre-treated with varying concentrations of nifedipine to examine the activity of lipogenesis enzymes and lipotoxicity. A positive control exposed to oleic acid was used for comparison. Nifedipine was found to activate acetyl Coenzyme A (CoA) synthetase, acetyl CoA carboxylase, long chain fatty acyl CoA elongase, ATP-citrate lyase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG CoA) reductase, suggesting elevated production of cholesterol and phospholipids. Nifedipine exposure induced a vast accumulation of cytosolic free fatty acids (FFA) and stimulated the production of reactive oxygen species, upregulated CD36 and KIM-1 (kidney injury molecule-1) expression, inhibited p-AMPK activity, and triggered the expression of SREBP-1/2 and lipin-1, underscoring the potential of nifedipine to induce lipotoxicity with renal damage. To our knowledge, this is the first report demonstrating nifedipine-induced lipid accumulation in the kidney. Full article

(This article belongs to the Special Issue Enzymes as Targets for Drug Development)

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20 pages, 6461 KiB

Open AccessArticle

Production and Characterization of Recombinant Wild Type Uricase from Indonesian Coelacanth (L. menadoensis) and Improvement of Its Thermostability by In Silico Rational Design and Disulphide Bridges Engineering

by Sakda Yainoy, Thanawat Phuadraksa, Sineewanlaya Wichit, Maprang Sompoppokakul, Napat Songtawee, Virapong Prachayasittikul and Chartchalerm Isarankura-Na-Ayudhya

Int. J. Mol. Sci. 2019, 20(6), 1269; https://doi.org/10.3390/ijms20061269 - 13 Mar 2019

Cited by 12 | Viewed by 4852

Abstract

The ideal therapeutic uricase (UOX) is expected to have the following properties; high expression level, high activity, high thermostability, high solubility and low immunogenicity. The latter property is believed to depend largely on sequence identity to the deduced human UOX (dH-UOX). Herein, we [...] Read more.

The ideal therapeutic uricase (UOX) is expected to have the following properties; high expression level, high activity, high thermostability, high solubility and low immunogenicity. The latter property is believed to depend largely on sequence identity to the deduced human UOX (dH-UOX). Herein, we explored L. menadoensis uricase (LM-UOX) and found that it has 65% sequence identity to dH-UOX, 68% to the therapeutic chimeric porcine-baboon UOX (PBC) and 70% to the resurrected ancient mammal UOX. To study its biochemical properties, recombinant LM-UOX was produced in E. coli and purified to more than 95% homogeneity. The enzyme had specific activity up to 10.45 unit/mg, which was about 2-fold higher than that of the PBC. One-litre culture yielded purified protein up to 132 mg. Based on homology modelling, we successfully engineered I27C/N289C mutant, which was proven to contain inter-subunit disulphide bridges. The mutant had similar specific activity and production yield to that of wild type (WT) but its thermostability was dramatically improved. Up on storage at −20 °C and 4 °C, the mutant retained ~100% activity for at least 60 days. By keeping at 37 °C, the mutant retained ~100% activity for 15 days, which was 120-fold longer than that of the wild type. Thus, the I27C/N289C mutant has potential to be developed for treatment of hyperuricemia. Full article

(This article belongs to the Special Issue Enzymes as Targets for Drug Development)

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16 pages, 2529 KiB

Open AccessArticle

Novel C15 Triene Triazole, D-A Derivatives Anti-HepG2, and as HDAC2 Inhibitors: A Synergy Study

by Zhiwen Qi, Chengzhang Wang, Jianxin Jiang and Caie Wu

Int. J. Mol. Sci. 2018, 19(10), 3184; https://doi.org/10.3390/ijms19103184 - 16 Oct 2018

Cited by 9 | Viewed by 3251

Abstract

A series of novel C15 urushiol derivatives were designed by introducing a pechmann structure and F-, Cl-, and Br-nitro substituents with different electronic properties into its alkyl side chain, as well as a triazolyl functional group in its aromatic oxide. Their chemical structures [...] Read more.

A series of novel C15 urushiol derivatives were designed by introducing a pechmann structure and F-, Cl-, and Br-nitro substituents with different electronic properties into its alkyl side chain, as well as a triazolyl functional group in its aromatic oxide. Their chemical structures were determined based on the analysis of the NMR (nuclear magnetic resonance) spectroscopic and mass spectrometric data. The results showed that compound 4 exhibited a strong inhibition of the HepG2 cell proliferation (half maximal inhibitory concentration (IC50): 2.833 μM to human hepatocellular carcinoma (HepG2), and 80.905 μM to human normal hepatocytes (LO2)). Furthermore, it had an excellent synergistic effect with levopimaric acid. The nitrogen atom of the triazole ring formed a hydrogen-bonding interaction with Gly103, Gly154, and Tyr308, which made compound 4 bind to histone deacetylase (HDAC)2 more tightly. One triazole ring and His33 formed a π–π stacking effect; the other, whose branches were deep into the pocket, further enhanced the interaction with HDAC2. Meanwhile, compound 4 involved a hydrophobic interaction with the residues Phe210 and Leu276. The hydrophobic interaction and π–π stacking provided powerful van der Waals forces for the compounds. Full article

(This article belongs to the Special Issue Enzymes as Targets for Drug Development)

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19 pages, 2971 KiB

Open AccessArticle

Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii

by Elena Maresi, Giacomo Janson, Silvia Fruncillo, Alessandro Paiardini, Rosario Vallone, Paola Dominici and Alessandra Astegno

Int. J. Mol. Sci. 2018, 19(7), 2111; https://doi.org/10.3390/ijms19072111 - 20 Jul 2018

Cited by 9 | Viewed by 3861

Abstract

Sulfur-containing amino acids play essential roles in many organisms. The protozoan parasite Toxoplasma gondii includes the genes for cystathionine β-synthase and cystathionine γ-lyase (TgCGL), as well as for cysteine synthase, which are crucial enzymes of the transsulfuration and de novo pathways for cysteine [...] Read more.

Sulfur-containing amino acids play essential roles in many organisms. The protozoan parasite Toxoplasma gondii includes the genes for cystathionine β-synthase and cystathionine γ-lyase (TgCGL), as well as for cysteine synthase, which are crucial enzymes of the transsulfuration and de novo pathways for cysteine biosynthesis, respectively. These enzymes are specifically expressed in the oocyst stage of T. gondii. However, their functionality has not been investigated. Herein, we expressed and characterized the putative CGL from T. gondii. Recombinant TgCGL almost exclusively catalyses the α,γ-hydrolysis of l-cystathionine to form l-cysteine and displays marginal reactivity toward l-cysteine. Structure-guided homology modelling revealed two striking amino acid differences between the human and parasite CGL active-sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of Asn360 to Ser demonstrated the importance of this residue in modulating the specificity for the catalysis of α,β- versus α,γ-elimination of l-cystathionine. Replacement of Ser77 by Glu completely abolished activity towards l-cystathionine. Our results suggest that CGL is an important functional enzyme in T. gondii, likely implying that the reverse transsulfuration pathway is operative in the parasite; we also probed the roles of active-site architecture and substrate binding conformations as determinants of reaction specificity in transsulfuration enzymes. Full article

(This article belongs to the Special Issue Enzymes as Targets for Drug Development)

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