Transferase Inhibitor Research Articles

Isoprenylation Inhibition Suppresses IL-33-mediated Mast Cell Activation of Allergic Asthma

Abstract Allergic asthma is an inflammatory disease of the airway associated with sensitization to allergens and symptoms such as sneezing, wheezing, and airway hyperresponsiveness. The severity of this disease varies between sex, age, and race; therefore, understanding signaling mechanisms is integral to developing targeted therapeutics. Mast cells (MC), an important effector cell in allergic disease, can be activated by interleukin (IL)-33, an alarmin released after contact with cellular stressors including common allergens. In response to IL-33, MC release inflammatory cytokines and lipid mediators. We previously found that statins suppress IgE-mediated MC activation by inhibiting protein isoprenylation. Therefore, we hypothesized that directly targeting the isoprenylation enzymes farnesyl transferase (FT) and geranylgeranyl transferase (GGT) would suppress IL-33-mediated MC function. We show that the dual transferase inhibitor FGTI-2734 decreased IL-33-mediated inflammatory cytokine secretion, effects that were mimicked by simvastatin. In contrast to the dual inhibitor, suppressing only FT or GGT had no effect on cytokine secretion. These data suggest that inhibiting farnesyl and geranylgeranyl transferases is a potential treatment for IL-33-mediated mast cell activation and allergic disease. Supported by grants from NIH (RO1 AI138495 and RO1 AI164710)

Abstract B024: Combined inhibition of farnesyltransferase and MEK is effective in fusion-negative HRAS-mutant rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, and RAS pathway mutations are the known driver mutations in the majority of fusion-negative (FN) RMS. Studies have demonstrated that HRAS mutations are enriched in infants with FN-RMS and can be associated with inferior outcomes. Using HRAS-mutant RMS models, we have demonstrated that tipifarnib (farnesyl transferase inhibitor, FTI) decreases ERK signaling and decreases in vitro and in vivo tumor growth. The effects of tipifarnib can be incomplete, and limitations may be due to adaptive or acquired resistance, or the presence of concurrent mutations that limit the response to single agents. These features suggest that HRAS-mutated FN-RMS may be sensitive to inhibition with combination therapy that prevents or delays the emergence of resistance. We examined the transcriptional effects of tipifarnib in FN-RMS cell lines using bulk RNA-sequencing to identify therapeutic vulnerabilities that may be exploited by RAS-directed therapies. Analysis of RNA sequencing data revealed downregulation of ERK transcriptional output genes upon treatment with tipifarnib, confirming the role of the MEK-ERK pathway in mediating the response to FTI. Trametinib (MEKi) inhibits tumor growth in xenograft models of FN-RMS but has only modest activity as a single agent. The efficacy of inhibition with FTI and MEKi has not been previously explored in RAS-driven FN-RMS. We therefore tested tipifarnib in combination with trametinib in HRAS-mutant FN-RMS cell lines and utilized in vitro assays to evaluate the effects of the combination on cell growth, differentiation and signaling via RAS effector pathways. In HRAS-mutant cells, we observed additive dose-dependent 2D and 3D growth inhibition in response to the combination. Further, tipifarnib and trametinib more potently reduced ERK phosphorylation than either drug individually, indicating effective RAS pathway inhibition. Additionally, we found that the combination of tipifarnib and trametinib induced myosin heavy chain expression, suggesting both inhibition of proliferation and promotion of myogenic differentiation. In an in vivo model, the combination of tipifarnib and trametinib led to tumor regression which was not seen with either drug alone. Our data suggest that the combination of tipifarnib and trametinib is active in models of HRAS-driven FN-RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with FN-RMS. Citation Format: Patience Odeniyide, Alla Lisok, Elana J. Fertig, Alyza Skaist, Christine A. Pratilas. Combined inhibition of farnesyltransferase and MEK is effective in fusion-negative HRAS-mutant rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B024.

Switch of phosphorylation to O-GlcNAcylation of AhR contributes to vascular oxidative stress induced by benzo[a]pyrene

Benzo[a]pyrene (B[a]P) is a food contaminant toxic for cardiovascular diseases. The nuclear translocation of Arylhydrocarbon receptor (AhR) plays an important role in B[a]P-induced oxidative stress and vascular diseases. We confirmed that B[a]P promoted ROS production in vascular smooth muscle cells (VSMCs) in vitro and in vivo, associated with the nuclear translocation of AhR. It is known that phosphorylation inhibits while dephosphorylation of AhR promotes nuclear translocation of AhR. However, from the posttranslational modification level, the mechanism by which B[a]P activates and regulates the nuclear translocation of AhR is unclear. Co-immunoprecipitation results showed that cytoplasmic AhR was phosphorylated before B[a]P stimulation, and switched to O-GlcNAcylation upon B[a]P 1-h stimulation in VSMCs, suggesting there may be a competitively inhibitory relationship between O-GlcNAcylation and phosphorylation of AhR. Next, siRNAs of O-linked N-acetylglucosamine transferase (OGT), O-GlcNAcase (OGA) and OGA inhibitor PUGNAc were used. SiOGT blocks but siOGA and PUGNAc promote B[a]P -dependent AhR nuclear translocation and oxidative stress. Ser11 may be the competitive binding site for phosphorylation and O-GlcNAcylation of AhR. Phosphorylation-mimic variant inhibits but O-GlcNAcylation of AhR promotes AhR nuclear translocation and oxidative stress. Our findings highlight a new perspective for AhR nuclear translocation regulated by the competitive modification between phosphorylation and O-GlcNAcylation.

Abstract 4897: PRT543, a methyl transferase inhibitor, has potent anti-tumor activity against adenoid cystic carcinoma of salivary glands

Abstract Adenoid Cystic Carcinoma (ACC) is a rare but aggressive malignancy of salivary gland, associated with protracted clinical course and fatal outcome. Treatment modalities are restricted to surgery and/or adjuvant radiotherapy and patients develop recurrence and distant metastasis over time. The absence of effective systematic therapy makes it incurable in advanced stage. ACC display an overall low mutation frequency, therefore very few actionable genetic alterations critical to the ACC development have been recognized. MYB fusion/overexpression is the most frequently found genetic alteration in ACC and is present in ~70% patients. This is followed by activating NOTCH mutations, reported in ~20% patients. Despite the presence of few targetable genetic alterations, recent studies revealed substantial transcriptomic heterogeneity amongst ACC tumors suggesting the role of epigenetic alterations in ACC oncogenesis. Symmetric dimethylation is an important epigenetic mechanism that regulates mRNA splicing, transcription, translation, cell cycle and oncogenic signaling pathways. Protein Arginine Methyl Transferase 5 (PRMT5) is a predominant enzyme for symmetric dimethylation among a family of 9 methyl transferases. In was suggested that PRMT5 plays a role in regulating tumor progression via modulation of MYC signaling, cancer stemness, and a wide array of additional cellular and transcriptional pro-oncogenic processes. While inhibition of PRMTs showed anti-oncogenic response in several preclinical tumor models and a subset of patients with advanced solid malignancies (including ACC), preclinical studies investigating the effect of PRMT5 blockade in ACC remain inadequate. In part, due to the scarcity of ACC specimens and limited availability of the experimental models. PRT543 is a selective and potent small molecule PRMT5 inhibitor that specifically targets PRMT5 among 37 methyl transferases. In the present study, we have investigated the effect this novel agent using a unique collection of ACC cell lines, organoids, and patient derived xenograft mouse models. To our knowledge, this is the first study investigating the therapeutic effect of PRT543 in several preclinical models of ACC. Specifically, we found that PRT543 has potent antitumor activity in in-vitro and in-vivo models with MYB expression and activating NOTCH mutations. Further, based on these observations, we have sequenced a collection of 50 ACC tumor samples to identify the subset of patients who may potentially benefit from PRT543 treatment based on their underlying genetic signatures. This study provides evidence underscoring the role of PRMT5 signaling in ACC and supports the clinical development of PRMT5 inhibitors for this indication. Citation Format: Vasudha Mishra, Alka Singh, Xiangying Chen, Michael Korzinkin, Claudia Wing, Viktoria Sarkisova, Alexandra Ozerova, Oksana Glushchenko, Venkat Thodima, Koichi Ito, Peggy Scherle, Mark Lingen, Rifat Hasina, Alexander Pearson, Ari Rosenberg, Alex Zhavoronkov, Bruce Ruggeri, Nishant Agrawal, Evgeny Izumchenko. PRT543, a methyl transferase inhibitor, has potent anti-tumor activity against adenoid cystic carcinoma of salivary glands. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4897.

Zinc(II)-Sterol Hydrazone Complex as a Potent Anti-Leishmania Agent: Synthesis, Characterization, and Insight into Its Mechanism of Antiparasitic Action.

Searching for new alternatives for treating leishmaniasis, we present the synthesis, characterization, and biological evaluation against Leishmania amazonensis of the new ZnCl2(H3)2 complex. H3 is 22-hydrazone-imidazoline-2-yl-chol-5-ene-3β-ol, a well-known bioactive molecule functioning as a sterol Δ24-sterol methyl transferase (24-SMT) inhibitor. The ZnCl2(H3)2 complex was characterized by infrared, UV-vis, molar conductance measurements, elemental analysis, mass spectrometry, and NMR experiments. The biological results showed that the free ligand H3 and ZnCl2(H3)2 significantly inhibited the growth of promastigotes and intracellular amastigotes. The IC50 values found for H3 and ZnCl2(H3)2 were 5.2 µM and 2.5 µM for promastigotes, and 543 nM and 32 nM for intracellular amastigotes, respectively. Thus, the ZnCl2(H3)2 complex proved to be seventeen times more potent than the free ligand H3 against the intracellular amastigote, the clinically relevant stage. Furthermore, cytotoxicity assays and determination of selectivity index (SI) revealed that ZnCl2(H3)2 (CC50 = 5 μΜ, SI = 156) is more selective than H3 (CC50 = 10 μΜ, SI = 20). Furthermore, as H3 is a specific inhibitor of the 24-SMT, free sterol analysis was performed. The results showed that H3 was not only able to induce depletion of endogenous parasite sterols (episterol and 5-dehydroepisterol) and their replacement by 24-desalkyl sterols (cholesta-5,7,24-trien-3β-ol and cholesta-7,24-dien-3β-ol) but also its zinc derivative resulting in a loss of cell viability. Using electron microscopy, studies on the fine ultrastructure of the parasites showed significant differences between the control cells and parasites treated with H3 and ZnCl2(H3)2. The inhibitors induced membrane wrinkle, mitochondrial injury, and abnormal chromatin condensation changes that are more intense in the cells treated with ZnCl2(H3)2.

Ketone Body β-Hydroxy-Butyrate Sustains Progressive Motility in Capacitated Human Spermatozoa: A Possible Role in Natural Fertility.

Background Calorie restriction is recognized as a useful nutritional approach to improve the endocrine derangements and low fertility profile associated with increased body weight. This is particularly the case for dietary regimens involving ketosis, resulting in increased serum levels of ketone bodies such as β-hydroxy-butyrate (β-HB). In addition to serum, β-HB is detected in several biofluids and β-HB levels in the follicular fluid are strictly correlated with the reproductive outcome in infertile females. However, a possible direct role of ketone bodies on sperm function has not been addressed so far. Methods Semen samples were obtained from 10 normozoospermic healthy donors attending the University Andrology Unit as participants in an infertility survey programme. The effect of β-HB on cell motility in vitro was evaluated on isolated spermatozoa according to their migratory activity in a swim-up selection procedure. The effect of β-HB on spermatozoa undergone to capacitation was also assessed. Results Two hours of exposure to β-HB, 1 mM or 4 mM, proved to be ineffective in modifying the motility of freshly ejaculated spermatozoa isolated according to the migratory activity in a swim-up procedure (all p values > 0.05). Differently, sperm maintenance in 4 mM β-HB after capacitation was associated with a significantly higher percentage of sperm cells with progressive motility compared to β-HB-lacking control (respectively, 67.6 ± 3.5% vs. 55.3 ± 6.5%, p = 0.0158). Succinyl-CoA transferase inhibitor abolished the effect on motility exerted by β-HB, underpinning a major role for this enzyme. Conclusion Our results suggest a possible physiological role for β-HB that could represent an energy metabolite in support of cell motility on capacitated spermatozoa right before encountering the oocyte.

Design of OSMI-4 Analogs Using Scaffold Hopping: Investigating the Importance of the Uridine Mimic in the Binding of OGT Inhibitors.

β-N-Acetylglucosamine transferase (OGT) inhibition is considered an important topic in medicinal chemistry. The involvement of O-GlcNAcylation in several important biological pathways is pointing to OGT as a potential therapeutic target. The field of OGT inhibitors drastically changed after the discovery of the 7-quinolone-4-carboxamide scaffold and its optimization to the first nanomolar OGT inhibitor: OSMI-4. While OSMI-4 is still the most potent inhibitor reported to date, its physicochemical properties are limiting its use as a potential drug candidate as well as a biological tool. In this study, we have introduced a simple modification (elongation) of the peptide part of OSMI-4 that limits the unwanted cyclisation during OSMI-4 synthesis while retaining OGT inhibitory potency. Secondly, we have kept this modified peptide unchanged while incorporating new sulfonamide UDP mimics to try to improve binding of newly designed OGT inhibitors in the UDP-binding site. With the use of computational methods, a small library of OSMI-4 derivatives was designed, prepared and evaluated that provided information about the OGT binding pocket and its specificity toward quinolone-4-carboxamides.

Aroylated phenylenediamine HO53 modulates innate immunity, histone acetylation and metabolism.

In the current context of antibiotic resistance, the need to find alternative treatment strategies is urgent. Our research aimed to use synthetized aroylated phenylenediamines (APDs) to induce the expression of cathelicidin antimicrobial peptide gene (CAMP) to minimize the necessity of antibiotic use during infection. One of these compounds, HO53, showed promising results in inducing CAMP expression in bronchial epithelium cells (BCi-NS1.1 hereafter BCi). Thus, to decipher the cellular effects of HO53 on BCi cells, we performed RNA sequencing (RNAseq) analysis after 4, 8 and 24h treatment of HO53. The number of differentially expressed transcripts pointed out an epigenetic modulation. Yet, the chemical structure and in silico modeling indicated HO53 as a histone deacetylase (HDAC) inhibitor. When exposed to a histone acetyl transferase (HAT) inhibitor, BCi cells showed a decreased expression of CAMP. Inversely, when treated with a specific HDAC3 inhibitor (RGFP996), BCi cells showed an increased expression of CAMP, indicating acetylation status in cells as determinant for the induction of the expression of the gene CAMP expression. Interestingly, a combination treatment with both HO53 and HDAC3 inhibitor RGFP966 leads to a further increase of CAMP expression. Moreover, HDAC3 inhibition by RGFP966 leads to increased expression of STAT3 and HIF1A, both previously demonstrated to be involved in pathways regulating CAMP expression. Importantly, HIF1α is considered as a master regulator in metabolism. A significant number of genes of metabolic enzymes were detected in our RNAseq data with enhanced expression conveying a shift toward enhanced glycolysis. Overall, we are demonstrating that HO53 might have a translational value against infections in the future through a mechanism leading to innate immunity strengthening involving HDAC inhibition and shifting the cells towards an immunometabolism, which further favors innate immunity activation.

MIP-based electrochemical sensor for highly selective and sensitive determination of entacapone from the triple mixture in tablet dosage form

The aim of this study is to develop a molecularly imprinted polymer (MIP) sensor via photopolymerization for selective and sensitive analysis of entacapone (ENT), a catecholamine-o-methyl transferase (COMT) inhibitor. ENT is used for Parkinson’s disease treatment with levodopa (LEV) and carbidopa (CAR), and it is available in a triple combination tablet dosage form. For the MIP-based electrochemical sensor (4-AP@MIP/GCE) design, 4-aminophenol (4-AP) was selected as the functional monomer. Surface characterization of the 4-AP@MIP/GCE sensor was performed by scanning electron microscopy, electrochemical characterization by CV, and electrochemical impedance spectroscopy (EIS). The linear concentration range was between 1.0 pM and 10.0 pM under optimum conditions for ENT determination. The limit of detection (LOD) of 0.24 pM and the limit of quantification (LOQ) of 0.80 pM were calculated for ENT analysis using 4-AP@MIP/GCE. The accuracy was proven by performing a recovery study on the tablet sample that contains ENT, LEV, and CAR, and the recovery was found to be 99.32%. The selectivity of the 4-AP@MIP/GCE sensor for ENT has been proven by interference studies on LEV, CAR, and their binary mixtures. Looking at the results of the study, it has been proven that the 4-AP@MIP/GCE sensor is a highly selective, sensitive, and specific quantitative option for the analysis of ENT.

Pyrazolones Potentiate Colistin Activity against MCR-1-Producing Resistant Bacteria: Computational and Microbiological Study.

The polymyxin colistin is a last line antibiotic for extensively resistant Gram-negative bacteria. Colistin binding to lipid A disrupts the Gram-negative outer membrane, but mobile colistin resistance (mcr) gene family members confer resistance by catalyzing phosphoethanolamine (PEA) transfer onto lipid A, neutralizing its negative charge to reduce colistin interactions. Multiple mcr isoforms have been identified in clinical and environmental isolates, with mcr-1 being the most widespread and mcr-3 being common in South and East Asia. Preliminary screening revealed that treatment with pyrazolones significantly reduced mcr-1, but not mcr-3, mediated colistin resistance. Molecular dynamics (MD) simulations of the catalytic domains of MCR-1 and a homology model of MCR-3, in different protonation states of active site residues H395/H380 and H478/H463, indicate that the MCR-1 active site has greater water accessibility than MCR-3, but that this is less influenced by changes in protonation. MD-optimized structures of MCR-1 and MCR-3 were used in virtual screening of 20 pyrazolone derivatives. Docking of these into the MCR-1/MCR-3 active sites identifies common residues likely to be involved in protein-ligand interactions, specifically the catalytic threonine (MCR-1 T285, MCR-3 T277) site of PEA addition, as well as differential interactions with adjacent amino acids. Minimal inhibitory concentration assays showed that the pyrazolone with the lowest predicted binding energy (ST3f) restores colistin susceptibility of mcr-1, but not mcr-3, expressing Escherichia coli. Thus, simulations indicate differences in the active site structure between MCR-1 and MCR-3 that may give rise to differences in pyrazolone binding and so relate to differential effects upon producer E. coli. This work identifies pyrazolones as able to restore colistin susceptibility of mcr-1-producing bacteria, laying the foundation for further investigations of their activity as phosphoethanolamine transferase inhibitors as well as of their differential activity toward mcr isoforms.

HBP/O-GlcNAcylation Metabolic Axis Regulates Bone Resorption Outcome

Osteoclasts play a key role in the regulation of bone mass and are highly active metabolically. Here we show that a metabolic reprogramming toward the hexosamine biosynthetic pathway (HBP) is required not only for osteoclast differentiation but also to determine the bone resorption mode during physiological and pathological bone remodeling. We found that pharmacological inhibition of O-GlcNAc transferase (OGT) significantly reduced protein O-GlcNAcylation and osteoclast differentiation. Accordingly, genetic deletion of OGT also inhibited osteoclast formation and downregulated critical markers related to osteoclasts differentiation and function (NFATc1, αvintegrin, cathepsin K). Indeed, cells treated with OSMI-1, an OGT inhibitor, also reduced nuclear translocation of NFATc1. Furthermore, the addition of exogenous N-acetylglucosamine (GlcNAc) strongly increased osteoclast formation and demineralization ability. Strikingly, our data show for the first time that O-GlcNAcylation facilitates an aggressive trench resorption mode in human cells. The incubation of osteoclasts with exogenous GlcNAc increases the percentage of erosion by trench while having no effect on pit resorption mode. Through time-lapse recording, we documented that osteoclasts making trenches moving across the bone surface are sensitive to GlcNAcylation. Finally, osteoclast-specific Ogt-deficient mice show increased bone density and reduced inflammation-induced bone loss during apical periodontitis model. We show that osteoclast-specific Ogt-deficient mice are less susceptible to develop bacterial-induced periapical lesion. Consistent with this, Ogt-deleted mice showed a decreased number of tartrate-resistant acid phosphatase-positive cells lining the apical periodontitis site. In summary, here we describe a hitherto undiscovered role of the HBP/O-GlcNAcylation axis tuning resorption mode and dictating bone resorption outcome.

A Review of Genetic and Gene Therapy for Parkinson's Disease.

Parkinson's disease (PD) is a syndrome with deterioration of neurons, with its onset starting in the '20s, known as the young beginning of Parkinson's to the late inception of the ailment in the 60s. The majority of the environmental risk associated with PD is age. The pathophysiology of PD is related to the accretion of synuclein alpha (SNCA) protein leading to toxicity. This toxicity further leads to a depletion in dopamine levels, creating both motor and non-motor symptoms. PD is the combination of genetic and environmental risk factors. Linkage and association studies provided data on autosomal dominant and recessive genes linked to PD. Current treatment regimes involve using levodopa, catechol-O-methyl transferase inhibitors, anticholinergics, and monoamine oxidase B (MAO-B) inhibitors. Genetic treatment is done by identifying possible targets. Gene therapy includes silencing, replacing, or correcting the flawed gene with a good gene. This therapy has the advantage of eliminating significant PD symptoms with fewer to no adverse effects than conventional treatment. These targets are organized into disease-modifying or non-disease modifying. The distinction between these two is that disease-modifying treatment stops the degeneration of neurons, while non-disease modifying treatment involves dopaminergic enzyme expression. In non-modifying targets, aromatic L-amino acid decarboxylase (AADC) therapy is used but not as a standalone, so the presentation of AADC, tyrosine hydroxylase (TH), and GTP cyclohydrolase 1 (GCH) is done together as a tricistronic system. With these developments, a drug named prosavin is under clinical phase 1 trial. Disease-modifying targets involve glial cell-derived neurotrophic factor (GDNF). Direct GDNF delivery reduces PD symptoms. This GDNF infusion technique works with a tetracycline-controlled transactivator. Gene therapy introduction into the treatment of PD would be beneficial as there would be lesser adverse effects seen as linked with conventional treatment involving levodopa, MAO-B inhibitors, and anticholinergics, among a few. This article discusses the genetic basis and genetic model of therapy for PD.

The role of O-GlcNAcylation in mRNA nuclear export studied by single-molecule super-resolution microscopy.

Characterizing the relationship between the nuclear export of mRNA and the O-GlcNAcylation of nuclear pore proteins is a critical process for understanding the dynamic effects of post-translational modifications on membrane protein structures. Specifically, it was reported that the concentration of O-GlcNAcylated nucleoporins (Nups) in the nuclear pore complexes (NPCs) can be significantly altered by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) inhibitor drugs. Combining two single-molecule super-resolution microscopy methods, STORM and SPEED microscopy, we found that the amount of messenger ribonucleoprotein particles (mRNPs) that successfully export through the NPC is approximately doubled when increasing the O-GlcNAcylation in the NPC. Reversely, five-fold less mRNPs can exit the nucleus to reach the cytoplasm with the decreased O-GlcNAcylation of Nups. Moreover, a virtual 3D imaging by SPEED microscopy revealed that the spatial distributions of these altered O-GlcNAcylated Nups colocalized well with the locations of Nups that are rich in phenylalanine-glycine (FG) repeats, suggesting the passage of mRNPs through the selective permeability barrier in the NPC is regulated by the amount of O-GlcNAcylated FG-Nups.

Drug Repurposing to Inhibit Histamine N-Methyl Transferase.

Lower activity of the histaminergic system is associated with neurological disorders, including Alzheimer's disease (AD). Thus, the enhancement of histaminergic neurotransmission by inhibition of histamine N-methyl transferase (HNMT), which degrades histamine, appears as an important approach. For this purpose, rigid and flexible molecular docking studies of 185 FDA-approved drugs with the HNMT enzyme were carried out to select two compounds to perform molecular dynamics (MD) simulations to evaluate the binding free energies and stability of the enzyme-drug complexes. Finally, an HNMT inhibition assay was performed to corroborate their effect towards HNMT. Molecular docking studies with HNMT allowed the selection of dihydroergotamine and vilazodone since these molecules showed the lowest Gibbs free energy values. Analysis of the binding mode of vilazodone showed interactions with the binding pocket of HNMT with Glu28, Gln143, and Asn283. In contrast, dihydroergotamine binds to the HNMT active site in a different location, apparently because it is overall the more rigid ligand compared to flexible vilazodone. HNMT inhibitory activity for dihydroergotamine and vilazodone was corroborated (IC50 = 72.89 μM and 45.01 μM, respectively) by in vitro assays. Drug repurposing of HNMT was achieved by employing computational studies.

Pathophysiology of Allergic Rhinitis with Future Therapeutic Targets- An Update

Rhinitis is defined by a combination of two or more nasal symptoms (running, blocking, itching, and sneezing). Allergic Rhinitis (AR) occurs when these symptoms are due to Immunoglobulin E (IgE)-mediated inflammation following exposure to the allergen. The current review article has attempted to revisit the pathophysiologic basis of this disease in order to understand the likely therapeutic targets. Controlling histone acetylation with histone deacetylase inhibitors, Deoxyribonucleic acid (DNA) hypermethylation with DNA methyl transferase inhibitors and post-transcriptional gene expression with micro Ribonucleic Acid (miRNA) mimetics which target epigenetic changes, may aid in the treatment of AR. The allergen-induced nasal hypersecretion in allergic patients can also be definitively blocked by vidian neurectomy. Probiotics are non pathogenic microorganisms that are assumed to exert a positive influence on host health and physiology which may aid in the treatment of AR. Reversing or targeting the epigenetic changes in susceptible individuals can help prevent sensitisation of the individual and better treatment outcome in patients with AR, help cure the symptoms and lack of drug dependance.

Glucose Deprivation Promotes Cancer Cell Invasion by Varied Expression of EMT Structural Proteins and Regulatory Molecules in MDA-MB-231 Triple-Negative Breast Cancer Cells.

Cancer is one of the biggest leading causes of death worldwide. With increased accumulation of environmental factors like radiation and mutagens, causative mutation in the gene pool is increasing day by day in the human population. Metastasis and its resistance to a wide variety of treatments are the reasons which make cancer difficult to treat. It has already been reported that cancer cells introduce smart modifications in their metabolism and signaling pathways which sense changes in cancer microenvironment and help them survive under harsh conditions. The pattern of expression of these signaling molecules and their structural counterparts involved in cancer metastasis need to be investigated. We used different approaches to study the effect of several levels of glucose deprivation on cancer metastasis in the highly resistant breast cancer model cell line MDA-MB-231 grown in high- (25-mM) and low- (5-mM) glucose medium. Microscopic observations have shown that these cancer cells attach to the surface faster in the presence of high-compared to low-glucose concentrations. These observations were supported by varied expression of genes involved in this morphological transition. Under low-glucose concentration, the expression of epithelial-to-mesenchymal transition (EMT) structural proteins and regulatory molecules such as SLUG, ZEB, HIFα1A, STAT3, and VIM was downregulated, whereas expression of G9a (a histone-modifying enzyme, histone methyl transferase inhibitor), Snail, FBPase, MMP13, and PKM2 was upregulated. This resulted in the turning on of resistance mechanisms in MDA-MB-231 cancer cells enabling them to cope with the stressed tumor microenvironments leading to increased cancer invasion and migration. The increased invasion as shown by trans-well study and loose spheroids in 3D spheroid study showed how metastasis is triggered in MDA-MB-231 under glucose starvation. Our data suggests the devastating outcome of nutrient deprivation on cancer progression, which was previously thought to be supportive in cancer control.

Anti-cancer effect of entacaponeon esophageal cancer cells via apoptosis induction and cell cycle modulation.

Esophageal cancer (EC) is the sixth leading cause of cancer-related death, despite many advances in treatment, the survival of patients still remains poor. In recent years, the N6-methyladenosine (m6A) has been introduced as one of the most important modifications at the epitranscriptome level, with an important role in the mRNA regulation in various diseases, such as cancers. The m6A is regulated by different factors, including FTO as a demethylase. The m6A modification, especially through FTO overexpression has an oncogenic role in different cancer types such as EC. Recent studies showed that entacapone, a catechol-o-methyl transferase (COMT) inhibitor currently applied for Parkinson's disease, can inhibit FTO enzyme. In this study, we aimed to investigate the effect of entacapone as an FTO inhibitor on the m6A level and also apoptosis and cell cycle response in KYSE-30 and YM-1 of esophageal squamous cancer cell (ESCC) lines. Cell toxicity and IC50 of entacapone were evaluated using The MTT assay in YM-1 and KYSE-30 cells. Cells were treated into two groups: DMSO (control) and entacapone (mean IC50 ). Total RNA was extracted, and m6A levels were measured via the ELISA method. Subsequently, the apoptosis and cell cycle dys-regulation were detected by annexin-V-FITC/PI staining and PI staining via flow cytometry. Entacapone has the cytotoxicity effect on both esophageal cancer cell lines compared to normal PBMC cells. As well, entacapone treatment (140 μM) can induce apoptosis (KYSE-30: 50%. YM-1:22.6%) and has a modulatory effect on cell cycle progression in both YM-1 and KYSE-30 cells (p-value<.05). However, no significant difference in the m6A concentration was observed. Our findings suggested that entacapone has the inhibitory effect on ESCC cell lines through induction of the apoptosis and modulation of the cell cycle without toxicity on the normal PBMC.

O‐GlcNAcylation modulates GLUT1 levels at the blood‐brain barrier

AbstractBackgroundGlucose transporter 1 (GLUT1) at the blood‐brain barrier (BBB) is essential for glucose uptake into the brain. Previous studies have shown that GLUT1 levels are reduced at the BBB in Alzheimer’s disease (AD) post‐mortem brain, and perhaps explains the decreased glucose utilization observed in AD (Sweeney et al., Nat Rev Neurol, 2018). Individuals with a genetic reduction of GLUT1, known as GLUT1‐deficiency syndrome, were treated with increased blood glucose with no long‐lasting increase in brain glucose. Diabetes is a risk factor for AD and AD is sometimes referred to as Type‐3 diabetes due to elevated blood glucose levels. Taken together, these ideas provoked us to investigate connections between glucose and GLUT1 levels. Here, we tested if the glucose‐dependent post‐translational modification, O‐linked‐N‐acetylglucosaminylation (O‐GlcNAcylation), is the missing link. We hypothesized that increasing glucose concentrations will elevate O‐GlcNAcylation and decrease GLUT1 levels at the BBB. Furthermore, previous studies have shown that decreased GLUT1 levels result in reduced levels of the low‐density lipoprotein receptor‐related protein‐1 (LRP‐1) (Winkler, Nat Neurosci, 2015). Thus, we also are determining if altering glucose levels not only changes GLUT1 levels, but also other key BBB receptors implicated in AD.MethodsMouse brain endothelial (bEnd.3) cells were treated with vehicle (glucose 5.6 mmol), DMSO, the O‐GlcNAcase inhibitor Thiamet‐G (TMG, 1 µM), or the O‐GlcNAc transferase inhibitor OSMI‐1 (20 µM), for 24 and 48 hours. Next, the protein expression of O‐GlcNAc and GLUT1 were measured by western blot.ResultsAs previously reported, TMG increased and OSMI‐1 decreased O‐GlcNAcylation at both 24 and 48 h, compared to vehicle. Notably, TMG treatment significantly reduced and OSMI‐1 increased GLUT1 expression at both time points.ConclusionGLUT1 levels are modulated through O‐GlcNAcylation at BBB. When blood glucose levels are high, O‐GlcNAcylation will be high, thereby reducing GLUT1 at the BBB and brain glucose uptake. This is likely a homeostatic mechanism to prevent spikes in brain glucose levels when blood sugar levels are elevated.

A stable hepatitis D virus-producing cell line for host target and drug discovery

Chronic hepatitis D is the most aggressive form of chronic viral hepatitis. It is caused by super-infection of hepatitis B virus (HBV)-infected hepatocytes with hepatitis D virus (HDV). While the recent conditional approval of bulevirtide for HDV treatment offers a new therapeutic modality in Europe, there is an unmet medical need to further improve therapy. A more detailed characterization of virus-host interactions is needed for the identification of novel therapeutic targets. Addressing this need, we engineered a new stably-transformed cell line, named HuH7-2C8D, producing high titer recombinant HDV and allowing the study of viral particles morphogenesis and infectivity. Using this culture system, where viral propagation by re-infection is limited, we observed an increased accumulation of edited version of the viral genomes within secreted HDV viral particles over time that is accompanied with a decrease in viral particle infectivity. We confirmed the interaction of HDV proteins with a previously described host factor in HuH7-2C8D cells and additionally showed that these cells are suitable for co-culture assays with other cell types such as macrophages. Finally, the use of HuH7-2C8D cells allowed to confirm the dual antiviral activity of farnesyl transferase inhibitors, including the clinical candidate lonafarnib, against HDV. In conclusion, we have established an easy-to-handle cell culture model to investigate HDV replication, morphogenesis, and host interactions. HuH7-2C8D cells are also suitable for high-throughput antiviral screening assays for the development of new therapeutic strategies.

Evaluation of geranylgeranyl diphosphate synthase inhibition as a novel strategy for the treatment of osteosarcoma and Ewing sarcoma.

Rab GTPases are critical regulators of protein trafficking in the cell. To ensure proper cellular localization and function, Rab proteins must undergo a posttranslational modification, termed geranylgeranylation. In the isoprenoid biosynthesis pathway, the enzyme geranylgeranyl diphosphate synthase (GGDPS) generates the 20-carbon isoprenoid donor (geranylgeranyl pyrophosphate [GGPP]), which is utilized in the prenylation of Rab proteins. We have pursued the development of GGDPS inhibitors (GGSI) as a novel means to target Rab activity in cancer cells. Osteosarcoma (OS) and Ewing sarcoma (ES) are aggressive childhood bone cancers with stagnant survival statistics and limited treatment options. Here we show that GGSI treatment induces markers of the unfolded protein response (UPR) and triggers apoptotic cell death in a variety of OS and ES cell lines. Confirmation that these effects were secondary to cellular depletion of GGPP and disruption of Rab geranylgeranylation was confirmed via experiments using exogenous GGPP or specific geranylgeranyl transferase inhibitors. Furthermore, GGSI treatment disrupts cellular migration and invasion in vitro. Metabolomic profiles of OS and ES cell lines identify distinct changes in purine metabolism in GGSI-treated cells. Lastly, we demonstrate that GGSI treatment slows tumor growth in a mouse model of ES. Collectively, these studies support further development of GGSIs as a novel treatment for OS and ES.