Effects of Topic Delivery of an Inhibitor of Serine Racemase on Laser-Induced Choroidal Vasculopathy.

Purpose: Intravitreal injection of anti-VEGF antibodies remains the primary therapy for exudative age-related macular degeneration (exAMD), although its efficacy is limited. Previous research has demonstrated that both a loss-of-function mutation of srr and the intravenous injection of a serine racemase inhibitor, L-aspartic acid ß-hydroxamate (L-ABH), significantly inhibit laser-induced choroidal neovascularization (CNV) in mice. Given that L-ABH is a small molecule, this study investigated the effects of L-ABH administered via eye drops on CNV, aiming to develop a noninvasive treatment strategy for exAMD. Methods: CNV models in mice and rhesus macaques were established through laser photocoagulation. Seven monkeys were randomly assigned to receive either saline solution or L-ABH eye drops. Intraperitoneal or intravenous injection of fluorescein characterized CNV in both mice and monkeys. Fluorescein fundus angiography was used to assess leakage, whereas optical coherence tomography measured retinal thickness in the monkeys. Results: L-ABH eye drops significantly reduced fluorescein leakage in laser-injured mice (P < 0.001 compared to saline). In laser-injured rhesus macaques, the average percent changes in leakage areas treated with L-ABH were 2.5% ± 25.8% (P = 0.004) and 1.5% ± 75.7% (P = 0.023 compared to saline solution) on day 14 and day 28, respectively. However, L-ABH eye drops did not significantly affect the number of grade IV laser spots or retinal thickness, whereas bevacizumab did. Conclusions: This study demonstrates the potential efficacy of an SRR inhibitor in two animal models of laser-induced CNV. Translational Relevance: This represents the first investigation into the effects of topical delivery of an SRR inhibitor on CNV.

Anti-tumour activity of Panobinostat in oesophageal adenocarcinoma and squamous cell carcinoma cell lines.

BACKGROUND: Oesophageal cancer remains a challenging disease with high mortality rates and few therapeutic options. In view of these difficulties, epigenetic drugs have emerged as potential alternatives for patient care. The goal of this study was to evaluate the effect and biological consequences of Panobinostat treatment, an HDAC (histone deacetylase) inhibitor already approved for treatment of patients with multiple myeloma, in oesophageal cell lines of normal and malignant origin, with the latter being representative of the two main histological subtypes: adenocarcinoma and squamous cell carcinoma. RESULTS: Panobinostat treatment inhibited growth and hindered proliferation, colony formation and invasion of oesophageal cancer cells. Considering HDAC tissue expression, HDAC1 was significantly upregulated in normal oesophageal epithelium in comparison with tumour tissue, whereas HDAC3 was overexpressed in oesophageal cancer compared to non-malignant mucosa. No differences between normal and tumour tissue were observed for HDAC2 and HDAC8 expression. CONCLUSIONS: Panobinostat exposure effectively impaired malignant features of oesophageal cancer cells. Because HDAC3 was shown to be overexpressed in oesophageal tumour samples, this epigenetic drug may represent an alternative therapeutic option for oesophageal cancer patients.

Inhibition of TFEB deacetylation in proximal tubular epithelial cells (TECs) promotes TFEB activation and alleviates TEC damage in diabetic kidney disease.

The inhibition of the autophagolysosomal pathway mediated by transcription factor EB (TFEB) inactivation in proximal tubular epithelial cells (TECs) is a key mechanism of TEC injury in diabetic kidney disease (DKD). Acetylation is a novel mechanism that regulates TFEB activity. However, there are currently no studies on whether the adjustment of the acetylation level of TFEB can reduce the damage of diabetic TECs. In this study, we investigated the effect of Trichostatin A (TSA), a typical deacetylase inhibitor, on TFEB activity and damage to TECs in both in vivo and in vitro models of DKD. Here, we show that TSA treatment can alleviate the pathological damage of glomeruli and renal tubules and delay the DKD progression in db/db mice, which is associated with the increased expression of TFEB and its downstream genes. In vitro studies further confirmed that TSA treatment can upregulate the acetylation level of TFEB, promote its nuclear translocation, and activate the expression of its downstream genes, thereby reducing the apoptosis level of TECs. TFEB deletion or HDAC6 knockdown in TECs can counteract the activation effect of TSA on autophagolysosomal pathway. We also found that TFEB enhances the transcription of Tfeb through binding to its promoter and promotes its own expression. Our results, thus, provide a novel therapeutic mechanism for DKD that the alleviation of TEC damage by activating the autophagic lysosomal pathway through upregulating TFEB acetylation can, thus, delay DKD progression.

Computational bioprospecting of phytoconstituents as potential inhibitors for peptide deformylase from Streptococcus oralis: An opportunistic pathogen.

Streptococcus oralis an opportunistic bacterium has been reported to be involved in various blood borne infections like subacute bacterial endocarditis, septicemia, bacterial meningitis and in some cases dental caries too. Among various targets the peptide deformylase, of S.oralis appears to be most potent druggable target as it is involved in protein synthesis is opted for the current study. Due to unavailability of PDB structure of peptide deformylase from S. oralis the study initiates with homology modelling of the protein and 6OW2 of S pneumoniae is considered as the template. Thereafter, Molecular docking, Molecular dynamic simulation, ADME analysis, and MMPBSA analysis was carried out to explore the inhibitory potential of phyto-constituents as potential inhibitors for Peptide deformylase from S.oralis. Actinonin was considered as reference drug. Among 2370 phyto compounds the best observations were recorded for A1-Barrigenol (IMPHY010984) with binding affinity of -8.5 kcal/mol. Calculated RMSD, RMSF, Binding Free Energy for IMPHY010984 averaged at about 0.10 ± 0.03 nm, 0.08 ± 0.05 nm, 131 ± 21 kJ/mol respectively whereas the RMSD, RMSF, Binding Free Energy recorded for reference drug averaged at about 0.19 ± 0.04 nm, 0.11 ± 0.08 nm, -94 ± 18 kJ/mol respectively. Based on in silico observations IMPHY010984 is proved out as superior candidate over reference drug. The study reflects the potential of IMPHY010984 as prophylactic therapeutics for S.oralis.

Synthesis and Biological Evaluation of Novel 2-Aroyl Benzofuran-Based Hydroxamic Acids as Antimicrotubule Agents.

Because of synergism between tubulin and HDAC inhibitors, we used the pharmacophore fusion strategy to generate potential tubulin-HDAC dual inhibitors. Drug design was based on the introduction of a N-hydroxyacrylamide or a N-hydroxypropiolamide at the 5-position of the 2-aroylbenzo[b]furan skeleton, to produce compounds 6a-i and 11a-h, respectively. Among the synthesized compounds, derivatives 6a, 6c, 6e, 6g, 11a, and 11c showed excellent antiproliferative activity, with IC50 values at single- or double-digit nanomolar levels, against the A549, HT-29, and MCF-7 cells resistant towards the control compound combretastatin A-4 (CA-4). Compounds 11a and 6g were also 10-fold more active than CA-4 against the Hela cell line. When comparing the inhibition of tubulin polymerization versus the HDAC6 inhibitory activity, we found that 6a-g, 6i, 11a, 11c, and 11e, although very potent as inhibitors of tubulin assembly, did not have significant inhibitory activity against HDAC6.

Exploring the anti-EBV potential of suberoylanilide hydroxamic acid: Induction of apoptosis in infected cells through suppressing BART gene expression and inducing lytic infection.

Epstein-Barr virus (EBV) is linked to lymphoma and epithelioma but lacks drugs specifically targeting EBV-positive tumors. BamHI A Rightward Transcript (BART) miRNAs are expressed in all EBV-positive tumors, suppressing both lytic infection and host cell apoptosis. We identified suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylase enzymes, as an agent that suppresses BART promoter activity and transcription of BART miRNAs. SAHA treatment demonstrated a more pronounced inhibition of cell proliferation in EBV-positive cells compared to EBV-negative cells, affecting both p53 wild-type and mutant gastric epithelial cells. SAHA treatment enhanced lytic infection in wild-type EBV-infected cells, while also enhancing cell death in BZLF1-deficient EBV-infected cells. It reduced BART gene expression by 85% and increased the expression of proapoptotic factors targeted by BART miRNAs. These findings suggest that SAHA not only induces lytic infection but also leads to cell death by suppressing BART miRNA transcription and promoting the apoptotic program.

Discovery of a novel hybrid coumarin-hydroxamate conjugate targeting the HDAC1-Sp1-FOSL2 signaling axis for breast cancer therapy.

BACKGROUND: Breast cancer is one of the most lethal cancers in women. Despite significant advances in the diagnosis and treatment of breast cancer, many patients still succumb to this disease, and thus, novel effective treatments are urgently needed. Natural product coumarin has been broadly investigated since it reveals various biological properties in the medicinal field. Accumulating evidence indicates that histone deacetylase inhibitors (HDACIs) are promising novel anti-breast cancer agents. However, most current HDACIs exhibit only moderate effects against solid tumors and are associated with severe side effects. Thus, to develop more effective HDACIs for breast cancer therapy, hydroxamate of HDACIs was linked to coumarin core, and coumarin-hydroxamate hybrids were designed and synthesized. METHODS: A substituted coumarin moiety was incorporated into the classic hydroxamate HDACIs by the pharmacophore fusion strategy. ZN444B was identified by using the HDACI screening kit and cell viability assay. Molecular docking was performed to explore the binding mode of ZN444B with HDAC1. Western blot, immunofluorescent staining, cell viability, colony formation and cell migration and flow cytometry assays were used to analyze the anti-breast cancer effects of ZN444B in vitro. Orthotopic studies in mouse models were applied for preclinical evaluation of efficacy and toxicity in vivo. Proteomic analysis, dual-luciferase reporter assay, chromatin immunoprecipitation, co-immunoprecipitation, immunofluorescent staining assays along with immunohistochemical (IHC) analysis were used to elucidate the molecular basis of the actions of ZN444B. RESULTS: We synthesized and identified a novel coumarin-hydroxamate conjugate, ZN444B which possesses promising anti-breast cancer activity both in vitro and in vivo. A molecular docking model showed that ZN444B binds to HDAC1 with high affinity. Further mechanistic studies revealed that ZN444B specifically decreases FOS-like antigen 2 (FOSL2) mRNA levels by inhibiting the deacetylase activity of HDAC1 on Sp1 at K703 and abrogates the binding ability of Sp1 to the FOSL2 promoter. Furthermore, FOSL2 expression positively correlates with breast cancer progression and metastasis. Silencing FOSL2 expression decreases the sensitivity of breast cancer cells to ZN444B treatment. In addition, ZN444B shows no systemic toxicity in mice. CONCLUSIONS: Our findings highlight the potential of FOSL2 as a new biomarker and therapeutic target for breast cancer and that targeting the HDAC1-Sp1-FOSL2 signaling axis with ZN444B may be a promising therapeutic strategy for breast cancer.

Synthesis and Evaluation of the First 68Ga-Labeled C-Terminal Hydroxamate-Derived Gastrin-Releasing Peptide Receptor-Targeted Tracers for Cancer Imaging with Positron Emission Tomography.

Gastrin-releasing peptide receptor (GRPR), overexpressed in many solid tumors, is a promising imaging marker and therapeutic target. Most reported GRPR-targeted radioligands contain a C-terminal amide. Based on the reported potent antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH, we synthesized C-terminal hydroxamate-derived [68Ga]Ga-LW02075 ([68Ga]Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH) and [68Ga]Ga-LW02050 ([68Ga]Ga-DOTA-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH), and compared them with the closely related and clinically validated [68Ga]Ga-SB3 ([68Ga]Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt). Binding affinities (Ki) of Ga-SB3, Ga-LW02075, and Ga-LW02050 were 1.20 ± 0.31, 1.39 ± 0.54, and 8.53 ± 1.52 nM, respectively. Both Ga-LW02075 and Ga-LW02050 were confirmed to be GRPR antagonists by calcium release assay. Imaging studies showed that PC-3 prostate cancer tumor xenografts were clearly visualized at 1 h post injection by [68Ga]Ga-SB3 and [68Ga]Ga-LW02050 in PET images, but not by [68Ga]Ga-LW02075. Ex vivo biodistribution studies conducted at 1 h post injection showed that the tumor uptake of [68Ga]Ga-LW02050 was comparable to that of [68Ga]Ga-SB3 (5.38 ± 1.00 vs. 6.98 ± 1.36 %ID/g), followed by [68Ga]Ga-LW02075 (3.97 ± 1.71 %ID/g). [68Ga]Ga-SB3 had the highest pancreas uptake (37.3 ± 6.90 %ID/g) followed by [68Ga]Ga-LW02075 (17.8 ± 5.24 %ID/g), while the pancreas uptake of [68Ga]Ga-LW02050 was only 0.53 ± 0.11 %ID/g. Our data suggest that [68Ga]Ga-LW02050 is a promising PET tracer for detecting GRPR-expressing cancer lesions.

Long-term successful use of belinostat in a patient with relapsed-refractory angioimmunoblastic lymphoma who has previously been heavily treated.

ABSTRACT: Angioimmunoblastic T-cell lymphoma (AITL) is one of the sub-types of peripheral T-cell lymphomas (PTCLs) that are remarkably refractory and has the potential to have a poor prognosis. The treatment process includes a wide range of treatment modalities, from anthracycline-based regimens that have been used for years to novel agents, such as histone deacetylase inhibitor romidepsin and belinostat. Increased treatment response rates and prolonged survival have been reported in studies with belinostat. Similarly, in this case report, we wanted to share a patient of an advanced age and with a high IPI score, whom we had treated in many treatment lines and maintained a long-term treatment response by administering belinostat.

G protein-coupled estrogen receptor agonist G-1 decreases ADAM10 levels and NLRP3-inflammasome component activation in response to Staphylococcus aureus alpha-hemolysin.

The G protein-coupled estrogen receptor, also known as GPER1 or originally GPR30, is found in various tissues, indicating its diverse functions. It is typically present in immune cells, suggesting its role in regulating immune responses to infectious diseases. Our previous studies have shown that G-1, a selective GPER agonist, can limit the pathogenesis mediated by Staphylococcus aureus alpha-hemolysin (Hla). It aids in clearing bacteria in a mouse skin infection model and restricts the surface display of the Hla receptor, ADAM10 (a disintegrin and metalloprotease 10) in HaCaT keratinocytes. In this report, we delve into the modulation of GPER in human immune cells in relation to the NLRP3 inflammasome. We used macrophage-like differentiated THP-1 cells for our study. We found that treating these cells with G-1 reduces ATP release, decreases the activity of the caspase-1 enzyme, and lessens cell death following Hla intoxication. This is likely due to the reduced levels of ADAM10 and NLRP3 proteins, as well as the decreased display of the ADAM10 receptor in the G-1-treated THP-1 cells. Our studies, along with our previous work, suggest the potential therapeutic use of G-1 in reducing Hla susceptibility in humans. This highlights the importance of GPER in immune regulation and its potential as a therapeutic target.

Trichostatin A Promotes Cytotoxicity of Cisplatin, as Evidenced by Enhanced Apoptosis/Cell Death Markers.

Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, promotes the cytotoxicity of the genotoxic anticancer drug cisplatin, yet the underlying mechanism remains poorly understood. Herein, we revealed that TSA at a low concentration (1 µM) promoted the cisplatin-induced activation of caspase-3/6, which, in turn, increased the level of cleaved PARP1 and degraded lamin A&C, leading to more cisplatin-induced apoptosis and G2/M phase arrest of A549 cancer cells. Both ICP-MS and ToF-SIMS measurements demonstrated a significant increase in DNA-bound platinum in A549 cells in the presence of TSA, which was attributable to TSA-induced increase in the accessibility of genomic DNA to cisplatin attacking. The global quantitative proteomics results further showed that in the presence of TSA, cisplatin activated INF signaling to upregulate STAT1 and SAMHD1 to increase cisplatin sensitivity and downregulated ICAM1 and CD44 to reduce cell migration, synergistically promoting cisplatin cytotoxicity. Furthermore, in the presence of TSA, cisplatin downregulated TFAM and SLC3A2 to enhance cisplatin-induced ferroptosis, also contributing to the promotion of cisplatin cytotoxicity. Importantly, our posttranslational modification data indicated that acetylation at H4K8 played a dominant role in promoting cisplatin cytotoxicity. These findings provide novel insights into better understanding the principle of combining chemotherapy of genotoxic drugs and HDAC inhibitors for the treatment of cancers.

The different response of Brassica napus genotypes to microspore embryogenesis induced by heat shock and trichostatin A is not determined by changes in cell wall structure and composition but by different stress tolerance.

During microspore embryogenesis, microspores are induced to develop into haploid embryos. In Brassica napus, microspore embryogenesis is induced by a heat shock (HS), which initially produces embryogenic structures with different cell wall architectures and compositions, and with different potentials to develop into embryos. The B. napus DH4079 and DH12075 genotypes have high and very low embryo yields, respectively. In DH12075, embryo yield is greatly increased by combining HS and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). However, we show that HS + TSA inhibits embryogenesis in the highly embryogenic DH4079 line. To ascertain why TSA has such different effects in these lines, we treated DH4079 and DH12075 microspore cultures with TSA and compared the cell wall structure and composition of the different embryogenic structures in both lines, specifically the in situ levels and distribution of callose, cellulose, arabinogalactan proteins and high and low methyl-esterified pectin. For both lines, HS + TSA led to the formation of cell walls unfavorable for embryogenesis progression, with reduced levels of arabinogalactan proteins, reduced cell adhesion of inner walls and altered pectin composition. Thus, TSA effects on cell walls cannot explain their different embryogenic response to TSA. We also applied TSA to DH4079 cultures at different times and concentrations before HS application, with no negative effects on embryogenic induction. These results indicate that DH4079 microspores are hypersensitive to combined TSA and HS treatments, and open up new hypotheses about the causes of such hypersensitivity.

Identification and Analysis of Biomarkers Associated with Lipophagy and Therapeutic Agents for COVID-19.

BACKGROUND: Lipids, as a fundamental cell component, play an regulating role in controlling the different cellular biological processes involved in viral infections. A notable feature of coronavirus disease 2019 (COVID-19) is impaired lipid metabolism. The function of lipophagy-related genes in COVID-19 is unknown. The present study aimed to investigate biomarkers and drug targets associated with lipophagy and lipophagy-based therapeutic agents for COVID-19 through bioinformatics analysis. METHODS: Lipophagy-related biomarkers for COVID-19 were identified using machine learning algorithms such as random forest, Support Vector Machine-Recursive Feature Elimination, Generalized Linear Model, and Extreme Gradient Boosting in three COVID-19-associated GEO datasets: scRNA-seq (GSE145926) and bulk RNA-seq (GSE183533 and GSE190496). The cMAP database was searched for potential COVID-19 medications. RESULTS: The lipophagy pathway was downregulated, and the lipid droplet formation pathway was upregulated, resulting in impaired lipid metabolism. Seven lipophagy-related genes, including ACADVL, HYOU1, DAP, AUP1, PRXAB2, LSS, and PLIN2, were used as biomarkers and drug targets for COVID-19. Moreover, lipophagy may play a role in COVID-19 pathogenesis. As prospective drugs for treating COVID-19, seven potential downregulators (phenoxybenzamine, helveticoside, lanatoside C, geldanamycin, loperamide, pioglitazone, and trichostatin A) were discovered. These medication candidates showed remarkable binding energies against the seven biomarkers. CONCLUSIONS: The lipophagy-related genes ACADVL, HYOU1, DAP, AUP1, PRXAB2, LSS, and PLIN2 can be used as biomarkers and drug targets for COVID-19. Seven potential downregulators of these seven biomarkers may have therapeutic effects for treating COVID-19.

Endosymbiosis in trypanosomatids: The bacterium division depends on microtubule dynamism.

Microtubules are components of the cytoskeleton that perform essential functions in eukaryotes, such as those related to shape change, motility and cell division. In this context some characteristics of these filaments are essential, such as polarity and dynamic instability. In trypanosomatids, microtubules are integral to ultrastructure organization, intracellular transport and mitotic processes. Some species of trypanosomatids co-evolve with a symbiotic bacterium in a mutualistic association that is marked by extensive metabolic exchanges and a coordinated division of the symbiont with other cellular structures, such as the nucleus and the kinetoplast. It is already established that the bacterium division is microtubule-dependent, so in this work, it was investigated whether the dynamism and remodeling of these filaments is capable of affecting the prokaryote division. To this purpose, Angomonas deanei was treated with Trichostatin A (TSA), a deacetylase inhibitor, and mutant cells for histone deacetylase 6 (HDAC6) were obtained by CRISPR-Cas9. A decrease in proliferation, an enhancement in tubulin acetylation, as well as morphological and ultrastructural changes, were observed in TSA-treated protozoa and mutant cells. In both cases, symbiont filamentation occurred, indicating that prokaryote cell division is dependent on microtubule dynamism.

[Microorganism Immobilization Device Using Artificial Siderophores].

Inspired by the mechanism by which microorganisms utilize siderophores to ingest iron, four different FeIII complexes of typical artificial siderophore ligands containing catecholate and/or hydroxamate groups, K3[FeIII-LC3], K2[FeIII-LC2H1], K[FeIII-LC1H2], and [FeIII-LH3], were prepared. They were modified on an Au substrate surface (Fe-L/Au) and applied as microorganism immobilization devices for fast, sensitive, selective detection of microorganisms, where H6LC3, H5LC2H1, H4LC1H2, and H3LH3 denote the tri-catecholate, biscatecholate-monohydroxamate, monocatecholate-bishydroxamate, and tri-hydroxamate type of artificial siderophores, respectively. Their adsorption properties for the several microorganisms were investigated using scanning electron microscopy (SEM), quartz crystal microbalance (QCM), and electric impedance spectroscopy (EIS) methods. The artificial siderophore-iron complexes modified on the Au substrates Fe-LC3/Au, Fe-LC2H1/Au, Fe-LC1H2/Au, and Fe-LH3/Au showed specific microorganism immobilization behavior with selectivity based on the structure of the artificial siderophores. Their specificities corresponded well with the structural characteristics of natural siderophores that microorganisms release from the cell and/or use to take up an iron. These findings suggest that release and uptake are achieved through specific interactions between the artificial siderophore-FeIII complexes and receptors on the cell surfaces of microorganisms. This study revealed that Fe-L/Au systems have specific potential to serve as effective immobilization probes of microorganisms for rapid, selective detection and identification of a variety of microorganisms.

[New Drug Discovery Targeting Iron in Bacterial Infectious Diseases].

Iron is necessary for all living organisms, and bacteria that cause infections in human hosts also need ferrous ions for their growth and proliferation. In the human body, most ferric ions (Fe3+) are tightly bound to iron-binding proteins such as hemoglobin, transferrin, lactoferrin, and ferritin. Pathogenic bacteria express highly specific iron uptake systems, including siderophores and specific receptors. Most bacteria secrete siderophores, which are low-molecular weight metal-chelating agents, to capture Fe3+ outside cell. Siderophores are mainly classified as either catecholate or hydroxamate. Vibrio vulnificus, a Gram-negative pathogenic bacterium, is responsible for serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In our study, we generated deletion mutants of the genes encoding proteins involved in the vulnibactin mediated iron-utilization system, such as ferric-vulnibactin receptor protein (VuuA), periplasmic ferric-vulnibactin binding protein (FatB), ferric-vulnibactin reductase (VuuB), and isochorismate synthase (ICS). ICS and VuuA are required under low-iron conditions for ferric-utilization in M2799, but the alternative proteins FatB and VuuB can function as a periplasmic binding protein and a ferric-chelate reductase, respectively. VatD, which functions as ferric-hydroxamate siderophores periplasmic binding protein, was shown to participate in the ferric-vulnibactin uptake system in the absence of FatB. Furthermore, the ferric-hydroxamate siderophore reductase IutB was observed to participate in ferric-vulnibactin reduction in the absence of VuuB. We propose that ferric-siderophore periplasmic binding proteins and ferric-chelate reductases represent potential targets for drug discovery in the context of infectious diseases.

Reduction of H3K9 methylation by G9a inhibitors improves the development of mouse SCNT embryos.

Removal of somatic histone H3 lysine 9 trimethylation (H3K9me3) from the embryonic genome can improve the efficiency of mammalian cloning using somatic cell nuclear transfer (SCNT). However, this strategy involves the injection of histone demethylase mRNA into embryos, which is limiting because of its invasive and labor-consuming nature. Here, we report that treatment with an inhibitor of G9a (G9ai), the major histone methyltransferase that introduces H3K9me1/2 in mammals, greatly improved the development of mouse SCNT embryos. Intriguingly, G9ai caused an immediate reduction of H3K9me1/2, a secondary loss of H3K9me3 in SCNT embryos, and increased the birth rate of cloned pups about 5-fold (up to 3.9%). G9ai combined with the histone deacetylase inhibitor trichostatin A further improved this rate to 14.5%. Mechanistically, G9ai and TSA synergistically enhanced H3K9me3 demethylation and boosted zygotic genome activation. Thus, we established an easy, highly effective SCNT protocol that would enhance future cloning research and applications.

Integrative Analysis of Histone Acetylation Regulated CYP4F12 in Esophageal Cancer Development.

Current therapeutic strategies for esophageal cancer (EC) patients have yielded limited improvements in survival rates. Recent research has highlighted the influence of drug metabolism enzymes on both drug response and EC development. Our study aims to identify specific drug metabolism enzymes regulated by histone acetylation and to elucidate its molecular and clinical features. CYP4F12 exhibited a notable upregulation subsequent to trichostatin A treatment as evidenced by RNA sequencing analysis conducted on the KYSE-150 cell line. The change in gene expression was associated with increased acetylation level of histone 3 K18 and K27 in the promoter. The regulation was dependent on p300. In silicon analysis of both The Cancer Genome Atlas esophageal carcinoma and GSE53624 dataset suggested a critical role of CYP4F12 in EC development, because CYP4F12 was downregulated in tumor tissues and predicted better disease-free survival. Gene ontology analysis has uncovered a robust correlation between CYP4F12 and processes related to cell migration, as well as its involvement in cytosine-mediated immune activities. Further investigation into the relationship between immune cells and CYP4F12 expression has indicated an increased level of B cell infiltration in samples with high CYP4F12 expression. CYP4F12 was also negatively correlated with the expression of inhibitory checkpoints. An accurate predictive nomogram model was established combining with clinical factors and CYP4F12 expression. In conclusion, CYP4F12 was crucial in EC development, and targeting CYP4F12 may improve the therapeutic efficacy of current treatment in EC patients. SIGNIFICANCE STATEMENT: CYP4F12 expression was downregulated in esophageal cancer (EC) patients and could be induced by trichostatin A. During EC development, CYP4F12 was linked to reduced cell migration and increased infiltration of B cells. CYP4F12 also is a biomarker as prognostic predictors and therapeutic guide in EC patients.

Replacement of the hydroxamic acid group in the selective HDAC8 inhibitor PCI-34051.

PCI-34051 is a valuable tool to interrogate the therapeutic effects of selective inhibition of HDAC8. However, it has not advanced to clinical trials, perhaps due to poor PK or off-target effects. We hypothesized that the presence of a hydroxamic acid (HA) group in PCI-34051 contributed to its lack of advancement. Therefore, we replaced the HA in the PCI-34051 scaffold with a series of moieties that have the potential to bind to Zn and evaluated their activity in a HDAC8 assay. Surprisingly, none of the replacements effectively mimicked the HA, and analogs lost significant potency. Evaluation of the analogs' affinity to Zn indicated that none had affinity for Zn within the same range as the HA. These studies point to the difficulty in the application of bioisosteric replacements for Zn binding motifs.

Anticancer efficacy triggered by synergistically modulating the homeostasis of anions and iron: Design, synthesis and biological evaluation of dual-functional squaramide-hydroxamic acid conjugates.

Targeting the homeostasis of anions and iron has emerged as a promising therapeutic approach for the treatment of cancers. However, single-targeted agents often fall short of achieving optimal treatment efficacy. Herein we designed and synthesized a series of novel dual-functional squaramide-hydroxamic acid conjugates that are capable of synergistically modulating the homeostasis of anions and iron. Among them, compound 16 exhibited the most potent antiproliferative activity against a panel of selected cancer cell lines, and strong in vivo anti-tumor efficacy. This compound effectively elevated lysosomal pH through anion transport, and reduced the levels of intracellular iron. Compound 16 could disturb autophagy in A549 cells and trigger robust apoptosis. This compound caused cell cycle arrest at the G1/S phase, altered the mitochondrial function and elevated ROS levels. The present findings clearly demonstrated that synergistic modulation of anion and iron homeostasis has high potentials in the development of promising chemotherapeutic agents with dual action against cancers.