Establishment and Validation of an Automated System for the Antifactor IIa Assay: A Case Study of Potency Assessment of a Pharmaceutical Gel Formulation.

Antithrombotic agents and anticoagulant drugs, such as those from the heparin family, are employed in clinical settings for the prevention and treatment of clotting, thromboembolism, and wound healing. The potency assessment of antithrombotic agents is typically conducted using antifactor IIa assay with manual systems which are time-consuming and often lack repeatability. Here, we present a novel automated system that significantly enhances assay repeatability, attaining an outstandingly low relative standard deviation (RSD) % of only 0.6% for repeatability. This system has been applied to a pharmaceutical gel formulation for wound healing developed by Abdi Ibrahim Pharmaceuticals R&D Center as a case study for validation. The automated system demonstrated substantial improvements over manual systems in linearity (R2 = 0.9927), precision, accuracy, specificity, and robustness. The system aligns with the European Pharmacopoeia specifications, promising to enhance quality control across pharmaceutical formulations and conduct absorbance-based end-point assays within the pharmaceutical industry while offering increased throughput and cost-effectiveness.

Differential Expression and Function of SVIP in Breast Cancer Cell Lines and In Silico Analysis of Its Expression and Prognostic Potential in Human Breast Cancer.

The heterogeneity of cancer strongly suggests the need to explore additional pathways to target. As cancer cells have increased proteotoxic stress, targeting proteotoxic stress-related pathways such as endoplasmic reticulum stress is attracting attention as a new anticancer treatment. One of the downstream responses to endoplasmic reticulum stress is endoplasmic reticulum-associated degradation (ERAD), a major degradation pathway that facilitates proteasome-dependent degradation of unfolded or misfolded proteins. Recently, SVIP (small VCP/97-interacting protein), an endogenous ERAD inhibitor, has been implicated in cancer progression, especially in glioma, prostate, and head and neck cancers. Here, the data of several RNA-sequencing (RNA-seq) and gene array studies were combined to evaluate the SVIP gene expression analysis on a variety of cancers, with a particular focus on breast cancer. The mRNA level of SVIP was found to be significantly higher in primary breast tumors and correlated well with its promoter methylation status and genetic alterations. Strikingly, the SVIP protein level was found to be low despite increased mRNA levels in breast tumors compared to normal tissues. On the other hand, the immunoblotting analysis showed that the expression of SVIP protein was significantly higher in breast cancer cell lines compared to non-tumorigenic epithelial cell lines, while most of the key proteins of gp78-mediated ERAD did not exhibit such an expression pattern, except for Hrd1. Silencing of SVIP enhanced the proliferation of p53 wt MCF-7 and ZR-75-1 cells but not p53 mutant T47D and SK-BR-3 cells; however, it increased the migration ability of both types of cell lines. Importantly, our data suggest that SVIP may increase p53 protein levels in MCF7 cells by inhibiting Hrd1-mediated p53 degradation. Overall, our data reveal the differential expression and function of SVIP on breast cancer cell lines together with in silico data analysis.

Potent telomerase activators from a novel sapogenin via biotransformation utilizing Camarosporium laburnicola, an endophytic fungus.

BACKGROUND: Cycloartane-type triterpenoids possess important biological activities, including immunostimulant, wound healing, and telomerase activation. Biotransformation is one of the derivatization strategies of natural products to improve their bioactivities. Endophytic fungi have attracted attention in biotransformation studies because of their ability to perform modifications in complex structures with a high degree of stereospecificity. RESULTS: This study focuses on biotransformation studies on cyclocephagenol (1), a novel cycloartane-type sapogenin from Astragalus species, and its 12-hydroxy derivatives (2 and 3) to obtain new telomerase activators. Since the hTERT protein levels of cyclocephagenol (1) and its 12-hydroxy derivatives (2 and 3) on HEKn cells were found to be notable, biotransformation studies were carried out on cyclocephagenol and its 12-hydroxy derivatives using Camarosporium laburnicola, an endophytic fungus isolated from Astragalus angustifolius. Later, immunoblotting and PCR-based ELISA assay were used to screen starting compounds and biotransformation products for their effects on hTERT protein levels and telomerase activation. All compounds showed improved telomerase activation compared to the control group. CONCLUSIONS: As a result of biotransformation studies, seven new metabolites were obtained and characterized, verifying the potential of C. laburnicola as a biocatalyst. Additionally, the bioactivity results showed that this endophytic biocatalyst is unique in transforming the metabolites of its host to afford potent telomerase activators.

Design, synthesis, and biological evaluation of some novel naphthoquinone-glycine/ß-alanine anilide derivatives as noncovalent proteasome inhibitors.

A series of novel noncovalent glycine/ß-alanine anilide derivatives possessing 2-chloronaphthoquinone structure as a pharmacophoric unit were designed, synthesized, and evaluated for their antiproliferative and antiproteasomal activities against MCF-7 cell line, in vitro. According to biological activity results, all the target compounds showed antiproliferative activity in the range of IC50  = 7.10 ± 0.10-41.08 ± 0.14 µM and most of them exhibited inhibitory efficacy with varying ratios against the three catalytic subunits (ß1, ß2, and ß5) presenting caspase-like (C-L), trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities of proteasome. The antiproteasomal activity evaluations revealed that compounds preferentially inhibited the ß5 subunit compared with ß1 and ß2 subunits of the proteasome. Among the compounds, compounds 7 and 9 showed the highest antiproliferative activity with an IC50 value of 7.10 ± 0.10 and 7.43 ± 0.25 µM, respectively. Additionally, compound 7 displayed comparable potency to PI-083 lead compound in terms of ß5 antiproteasomal activity with an inhibition percentage of 34.67 at 10 µM. This compound showed an IC50 value of 32.30 ± 0.45 µM against ß5 subunit. Furthermore, molecular modeling studies of the most active compound 7 revealed key interactions with ß5 subunit. The results suggest that this class of compounds may be beneficial for the development of new potent proteasome inhibitors.

Neuroprotective metabolites via fungal biotransformation of a novel sapogenin, cyclocephagenol.

Cyclocephagenol (1), a novel cycloartane-type sapogenin with tetrahydropyran unit, is only encountered in Astragalus species. This rare sapogenin has never been a topic of biological activity or modification studies. The objectives of this study were; (i) to perform microbial transformation studies on cyclocephagenol (1) using Astragalus endophyte, Alternaria eureka 1E1BL1, followed by isolation and structural characterization of the metabolites; (ii) to investigate neuroprotective activities of the metabolites; (iii) to understand structure-activity relationships towards neuroprotection. The microbial transformation of cyclocephagenol (1) using Alternaria eureka resulted in the production of twenty-one (2-22) previously undescribed metabolites. Oxidation, monooxygenation, dehydration, methyl migration, epoxidation, and ring expansion reactions were observed on the triterpenoid skeleton. Structures of the compounds were established by 1D-, 2D-NMR, and HR-MS analyses. The neuroprotective activities of metabolites and parent compound (1) were evaluated against H2O2-induced cell injury. The structure-activity relationship (SAR) was established, and the results revealed that 1 and several other metabolites had potent neuroprotective activity. Further studies revealed that selected compounds reduced the amount of ROS and preserved the integrity of the mitochondrial membrane. This is the first report of microbial transformation of cyclocephagenol (1).

The role of cycloastragenol at the intersection of NRF2/ARE, telomerase, and proteasome activity.

Aging is well-characterized by the gradual decline of cellular functionality. As redox balance, proteostasis, and telomerase systems have been found to be associated with aging and age-related diseases, targeting these systems with small compounds has been considered a promising therapeutic approach. Cycloastragenol (CA), a small molecule telomerase activator obtained from Astragalus species, has been reported to positively affect several age-related pathophysiologies, but the mechanisms underlying CA activity have yet to be reported. Here, we presented that CA increased NRF2 nuclear localization and activity leading to upregulation of cytoprotective enzymes and attenuation of oxidative stress-induced ROS levels. Furthermore, CA-mediated induction of telomerase activity was found to be regulated by NRF2. CA not only increased the expression of hTERT but also its nuclear localization via upregulating the Hsp90-chaperon complex. In addition to modulating nuclear hTERT levels at unstressed conditions, CA alleviated oxidative stress-induced mitochondrial hTERT levels while increasing nuclear hTERT levels. Concomitantly, H2O2-induced mitochondrial ROS level was found to be significantly decreased by CA administration. Our data also revealed that CA strongly enhanced proteasome activity and assembly. More importantly, the proteasome activator effect of CA is dependent on the induction of telomerase activity, which is mediated by NRF2 system. In conclusion, our results not only revealed the cross-talk among NRF2, telomerase, and proteasome systems but also that CA functions at the intersection of these three major aging-related cellular pathways.

Novel regulation mechanism of adrenal cortisol and DHEA biosynthesis via the endogen ERAD inhibitor small VCP-interacting protein.

Endoplasmic reticulum-associated degradation (ERAD) is a well-characterized mechanism of protein quality control by removal of misfolded or unfolded proteins. The tight regulation of ERAD is critical for protein homeostasis as well as lipid metabolism. Although the mechanism is complex, all ERAD branches converge on p97/VCP, a key protein in the retrotranslocation step. The multifunctionality of p97/VCP relies on its multiple binding partners, one of which is the endogenous ERAD inhibitor, SVIP (small VCP-interacting protein). As SVIP is a promising target for the regulation of ERAD, we aimed to assess its novel physiological roles. We revealed that SVIP is highly expressed in the rat adrenal gland, especially in the cortex region, at a consistently high level during postnatal development, unlike the gradual increase in expression seen in developing nerves. Steroidogenic stimulators caused a decrease in SVIP mRNA expression and increase in SVIP protein degradation in human adrenocortical H295R cells. Interestingly, silencing of SVIP diminished cortisol secretion along with downregulation of steroidogenic enzymes and proteins involved in cholesterol uptake and cholesterol biosynthesis. A certain degree of SVIP overexpression mainly increased the biosynthesis of cortisol as well as DHEA by enhancing the expression of key steroidogenic proteins, whereas exaggerated overexpression led to apoptosis, phosphorylation of eIF2α, and diminished adrenal steroid hormone biosynthesis. In conclusion, SVIP is a novel regulator of adrenal cortisol and DHEA biosynthesis, suggesting that alterations in SVIP expression levels may be involved in the deregulation of steroidogenic stimulator signaling and abnormal adrenal hormone secretion.

Induction of Divergent Cell Death Pathways by Urea and Carbohydrazide Derivatives.

BACKGROUND: The complexity of cancer biology and the development of chemotherapy resistance are two main obstacles to cancer treatment and necessitate novel anticancer molecules that target different cell death pathways. Modulation of Endoplasmic Reticulum (ER) stress and subsequent activation of the Unfolded Protein Response (UPR) has been proposed as a potential chemotherapeutic target, as prolonged ER stress can lead to cell death via apoptosis or necrosis. OBJECTIVE: The present study aims to evaluate the molecular mechanism underlying the cytotoxic activity of selected urea and carbohydrazide derivatives. METHODS: Cell proliferation assays were performed on HeLa, Capan-1, MCF-7, HCC-1937, and MRC-5 cell lines by WST-1 assay. The expression levels of selected ER stress, autophagy, and apoptosis marker proteins were compared by immunoblotting to characterize the underlying mechanism of cytotoxicity. Flow cytometry was used to detect apoptosis. RESULTS: Of the tested cytotoxic compounds, 3a, 4a, 5a, 6a, and 1b dramatically and 5b moderately increased ER stress-related CHOP protein levels. Interestingly, 5b but not 3a, 4a, 5a, 6a, or 1b increased the expression of proapoptotic proteins such as cleaved PARP-1 and cleaved caspase-3 and -7. The flow-cytometry analysis further confirmed that the cytotoxic activity of 5b but not the other compounds is mediated by apoptosis, demonstrated by a significant increase in the percentage of late apoptotic cells (7-AAD/annexin V double-positive cells). CONCLUSION: Our results suggest that changing a substituent from trifluoromethyl to nitro in urea and carbohydrazide core structure alters the cell death mechanism from apoptosis to an apoptosis-independent cell death pathway. This study shows an example of how such simple modifications of a core chemical structure could cause the induction of divergent cell death pathways.

A new underlying mechanism for the neuroprotective effect of bosutinib: Reverting toxicity-induced PARylation in SIN1-mediated neurotoxicity.

Increased levels of reactive oxygen and nitrogen species play an important role in the development and progression of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. The overproduction of these highly reactive chemical species leads to DNA damage and subsequent activation of the poly(ADP-ribose)polymerase (PARP) enzyme. Several studies have demonstrated the potential use of PARP inhibitors for neuroprotection. We previously reported that the dual Src/Abl kinase inhibitor bosutinib (BOS) decreases PARP activity and acts as a chemosensitizer in cancer cells. In this study, we evaluated the neuroprotective potential of BOS with respect to its inhibitory effect on cellular poly(ADP-ribos)ylation (PARylation) using a 3-morpholinosydnonimine (SIN1)-mediated cellular toxicity model. Our data suggest that pretreatment with BOS, especially at lower doses, significantly decreased the level of SIN1-induced cellular PARylation. This regulation pattern of PARylation was found to be associated with the protective effect of BOS against SIN1 on the viability of retinoic acid-differentiated SH-SY5Y cells. Furthermore, while PARP-1 expression was decreased, phosphorylation of SAPK/JNK was not reverted at the observed neuroprotective doses of BOS. In conclusion, we suggest a novel mechanism for the neuroprotective effect of BOS involving the inhibition of cellular PARylation.

Exploring of tumor-associated carbonic anhydrase isoenzyme IX and XII inhibitory effects and cytotoxicities of the novel N-aryl-1-(4-sulfamoylphenyl)-5-(thiophen-2-yl)-1H-pyrazole-3-carboxamides.

A series of novel N-aryl-1-(4-sulfamoylphenyl)-5-(thiophen-2-yl)-1H-pyrazole-3-carboxamides was synthesized and examined as inhibitors of cytosolic (human) hCA I and hCA II, and cancer-related transmembrane hCA IX and hCA XII isoenzymes. AC2 was the most selective inhibitor towards cancer-related hCA IX while AC8 and AC9 selectively inhibited hCA XII over off-target isoenzymes. Anticancer effects of the compounds were evaluated towards human oral squamous cell carcinoma (OSCC) cell lines, human mesenchymal normal oral cells, breast (MCF7), prostate (PC3), non-small cell lung carcinoma cells (A549), and non-tumoral fetal lung fibroblast cells (MRC5). Compounds moderately showed cytotoxicity towards cancer cell lines. Among others, AC6 showed cell-specific cytotoxic activity and induced apoptosis in a dose-dependent manner without a significant change in the cell cycle distribution of MCF7. These results suggest that pyrazole-3-carboxamides need further molecular modification to increase their anticancer drug candidate potency.

Telomerase activators from 20(27)-octanor-cycloastragenol via biotransformation by the fungal endophytes.

Cycloastragenol [20(R),24(S)-epoxy-3ß,6α,16ß,25-tetrahydroxycycloartane] (CA), the principle sapogenol of many cycloartane-type glycosides found in Astragalus genus, is currently the only natural product in the anti-aging market as telomerase activator. Here, we report biotransformation of 20(27)-octanor-cycloastragenol (1), a thermal degradation product of CA, using Astragalus species originated endophytic fungi, viz. Penicillium roseopurpureum, Alternaria eureka, Neosartorya hiratsukae and Camarosporium laburnicola. Fifteen new biotransformation products (2-16) were isolated, and their structures were established by NMR and HRESIMS. Endophytic fungi were found to be capable of performing hydroxylation, oxidation, ring cleavage-methyl migration, dehydrogenation and Baeyer-Villiger type oxidation reactions on the starting compound (1), which would be difficult to achieve by conventional synthetic methods. In addition, the ability of the metabolites to increase telomerase activation in Hekn cells was evaluated, which showed from 1.08 to 12.4-fold activation compared to the control cells treated with DMSO. Among the compounds tested, 10, 11 and 12 were found to be the most potent in terms of telomerase activation with 12.40-, 7.89- and 5.43-fold increase, respectively (at 0.1, 2 and 10 nM concentrations, respectively).

Nonsteroidal antiinflammatory drugs alter antibiotic susceptibility and expression of virulence-related genes and protein A of Staphylococcus aureus

Background/aim: Nonsteroidal antiinflammatory drugs (NSAIDs) including diclofenac, naproxen, ibuprofen, acetylsalicylic acid, and acetaminophen have been shown to have antimicrobial effects on various microorganisms. The aim of this study was to investigate the antibacterial effects of NSAIDs on Staphylococcus aureus. Materials and methods: Susceptibilities of S. aureus strains to NSAIDs with or without antimicrobials (moxifloxacin, vancomycin, ciprofloxacin, clindamycin, and gentamicin) were determined using the microdilution method and disk diffusion test. Expression levels of genes in the presence of drugs were investigated by real-time quantitative RT-PCR (qRT-PCR), and immunoblotting analysis was performed for staphylococcal protein A (SpA). Results: Our results showed that all NSAIDs were active against S. aureus strains with MIC values ranging from 195 µg/mL to 6250 µg/ mL. NSAIDs increased the antibiotic susceptibility of the strains, and diclofenac was found to be more effective than the other drugs. Drugs showed different effects on expression levels of virulence factor and/or regulatory genes. Immunoblotting analysis of SpA protein was mostly in accordance with qRT-PCR results. Conclusion: The regulatory/virulence factor genes and proteins of S. aureus investigated in this study may be reasonable targets for these drugs, and we suggest that the data may contribute to the field of infection control and antimicrobial resistance.

Divergent Modulation of Proteostasis in Prostate Cancer.

Proteostasis regulates key cellular processes such as cell proliferation, differentiation, transcription, and apoptosis. The mechanisms by which proteostasis is regulated are crucial and the deterioration of cellular proteostasis has been significantly associated with tumorigenesis since it specifically targets key oncoproteins and tumor suppressors. Prostate cancer (PCa) is the second most common cause of cancer death in men worldwide. Androgens mediate one of the most central signaling pathways in all stages of PCa via the androgen receptor (AR). In addition to their regulation by hormones, PCa cells are also known to be highly secretory and are particularly prone to ER stress as proper ER function is essential. Alterations in various complex signaling pathways and cellular processes including cell cycle control, transcription, DNA repair, apoptosis, cell adhesion, epithelial-mesenchymal transition (EMT), and angiogenesis are critical factors influencing PCa development through key molecular changes mainly by posttranslational modifications in PCa-related proteins, including AR, NKX3.1, PTEN, p53, cyclin D1, and p27. Several ubiquitin ligases like MDM2, Siah2, RNF6, CHIP, and substrate-binding adaptor SPOP; deubiquitinases such as USP7, USP10, USP26, and USP12 are just some of the modifiers involved in the regulation of these key proteins via ubiquitin-proteasome system (UPS). Some ubiquitin-like modifiers, especially SUMOs, have been also closely associated with PCa. On the other hand, the proteotoxicity resulting from misfolded proteins and failure of ER adaptive capacity induce unfolded protein response (UPR) that is an indispensable signaling mechanism for PCa development. Lastly, ER-associated degradation (ERAD) also plays a crucial role in prostate tumorigenesis. In this section, the relationship between prostate cancer and proteostasis will be discussed in terms of UPS, UPR, SUMOylation, ERAD, and autophagy.

Synthesis and evaluation of pyridinium-hydrazone derivatives as potential antitumoral agents.

The hydrazones of 4-hydrazinylpyridinium bearing alkylphenyl groups on pyridinium nitrogen were synthesized and evaluated for their cytotoxic activity against MCF-7, PC3, U2OS, and HEK293 cell lines by Wst1 cell proliferation assay. Cytotoxic activity results indicated that d derivatives having butylene chain; 4 and 5 series having naphthalene and anthracene ring systems showed high cytotoxic activity (IC50  = 3.27-8.54 µm) on cancer cells. 3d (4-(2-(4-hydroxybenzylidene)hydrazinyl)-1-(4-phenylbutyl)pyridinium bromide) was the most cytotoxic compound with IC50 value of 3.27 µm against MCF-7. The most active derivatives (1d, 2d, 3d, 4, and 5 series) were selected to investigate for the effects on autophagy by analyzing the expression of autophagy marker proteins. The conversion of LC3-I to its lipidated form LC3-II is essential for autophagy and related to autophagosomes. According to our results, all tested compounds except for 3d induced lipidated form LC3-II accumulation. Then, the effects of the compounds on p62 protein level were also analyzed by the immunoblotting as the autophagy inhibition results in accumulation of p62. Further molecular mechanistic studies including morphological analysis and live-death assays indicated that all tested compounds (1d, 2d, 3d, 4, and 5 series) are potent antitumoral molecules and all except for 3d have potential to inhibit autophagic flux.

Design, Synthesis and Evaluation of the Biological Activities of Some New Carbohydrazide and Urea Derivatives.

OBJECTIVES: Urea and carbohydrazide derivatives are important compounds exhibiting cytotoxic activities. In this study, a series of new urea and carbohydrazide derivatives containing an pyridine ring were synthesized and evaluated for cytotoxic activity. MATERIALS AND METHODS: The proposed structures of the synthesized compounds were confirmed using elemental analysis, IR, and 1H-NMR spectroscopic techniques. The cytotoxic potencies of synthesized compounds were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) on BRCA mutant-carrying HCC1937 and Capan-1 cell lines, as well as on MCF7, HeLa, and MRC5 cells. RESULTS: 3a, 3b, 3c and 3d showed cytotoxic activity against all cancer cell lines. CONCLUSION: Our data indicate that compounds 3a-d are more selective to cancer cells compared with nontumoral fibroblasts; however, these compounds are not more potent on HR defective cells with BRCA mutants.

Dual-Prevention for UV-Induced Skin Damage: Incorporation of Melatonin-Loaded Elastic Niosomes into Octyl Methoxycinnamate Pickering Emulsions.

Incorporation of antioxidants into sunscreens is a logical approach, yet co-delivery of them with UV filters is a challenge. Here, we purposed a combination therapy, in which the chemical UV filter, octyl methoxycinnamate, was accumulated on upper skin while the antioxidant, melatonin, can penetrate deeper layers to show its effects. Melatonin-loaded elastic niosomes and octyl methoxycinnamate Pickering emulsion were prepared separately. Lyophilized elastic niosomes were dispersed into the Pickering emulsion to prepare the proposed combination formulation. The characterization studies of the formulations revealed that elastic niosomes can be prepared with tunable nanometer sizes, whereas Pickering emulsions can encapsulate the UV filter in micrometer-sized droplets. Melatonin-loaded elastic niosomes prepared with Tween80/Span80 mixture were 146 nm with a PI of 0.438, and 58.42% entrapment efficiency was achieved. The mean diameter size of the combination formulation was 27.8 µm. Ex vivo permeation studies revealed that 7.40% of octyl methoxycinnamate and 58% of melatonin were permeated through the rat skin while 27.6% octyl methoxycinnamate and 37% of melatonin accumulated in the skin after 24 h. Cell culture studies with real-time cell analyzer showed that the proposed formulation consist of melatonin-loaded elastic niosomes and octyl methoxycinnamate Pickering emulsion had no negative effect on the cell proliferation and viability. According to α,α-diphenyl-ß-picrylhydrazyl free radical scavenging method, the proposed formulation showed as high antioxidant activity as melatonin itself. It is concluded that the proposed formulation would be a promising dual therapy for UV-induced skin damage with co-delivery strategy.