Preferential HDAC6 inhibitors derived from HPOB exhibit synergistic antileukemia activity in combination with decitabine.

Histone deacetylase 6 (HDAC6) is an emerging drug target to treat oncological and non-oncological conditions. Since highly selective HDAC6 inhibitors display limited anticancer activity when used as single agent, they usually require combination therapies with other chemotherapeutics. In this work, we synthesized a mini library of analogues of the preferential HDAC6 inhibitor HPOB in only two steps via an Ugi four-component reaction as the key step. Biochemical HDAC inhibition and cell viability assays led to the identification of 1g (highest antileukemic activity) and 2b (highest HDAC6 inhibition) as hit compounds. In subsequent combination screens, both 1g and especially 2b showed synergy with DNA methyltransferase inhibitor decitabine in acute myeloid leukemia (AML). Our findings highlight the potential of combining HDAC6 inhibitors with DNA methyltransferase inhibitors as a strategy to improve AML treatment outcomes.

Nanoplatform to Investigate Tumor-Initiating Cancer Stem Cells: Breaking the Diagnostic Barrier.

Drug-resistant capacity in a small population of tumor-initiating cancer stem cells (tiCSCs) can be due to aberrant epigenetic changes. However, currently available conventional detection methods are inappropriate and cannot be applied to investigate the scarce population (tiCSCs). In addition, selective inhibitor drugs are shown to reverse epigenetic changes; however, each cancer type is discrete. Hence, it is essential to probe the resultant changes in tiCSCs even after therapy. Therefore, we have developed a multimode nanoplatform to investigate tiCSCs, detect epigenetic changes, and subsequently explore their transformation signals following drug therapy. We performed this by developing a surface-enhanced Raman scattering (SERS)-active nanoplatform integrated with n-dopant using an ultrafast laser ionization technique. The dopant functionalization enhances Raman scattering ability and permits label-free analysis of biomarkers in tiCSCs with the resolution down to the cellular level. Here, we investigated epigenetic biomarkers of tiCSCs in pancreatic and lung cancers. An extended study using inhibitor drugs demonstrates an unexpected increase of tiCSCs from lung cancer; this difference can be attributed to transformation changes in lung tiCSC. Thus, our work brings new insight into the differentiation abilities of CSCs upon epigenetic reversal, emphasizing unique perceptions in cancer treatment.

Genomics-guided targeting of stress granule proteins G3BP1/2 to inhibit SARS-CoV-2 propagation.

SARS-CoV-2 nucleocapsid (N) protein undergoes RNA-induced phase separation (LLPS) and sequesters the host key stress granule (SG) proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and 2 (G3BP1 and G3BP2) to inhibit SG formation. This will allow viral packaging and propagation in host cells. Based on a genomic-guided meta-analysis, here we identify upstream regulatory elements modulating the expression of G3BP1 and G3BP2 (collectively called G3BP1/2). Using this strategy, we have identified FOXA1, YY1, SYK, E2F-1, and TGFBR2 as activators and SIN3A, SRF, and AKT-1 as repressors of G3BP1/2 genes. Panels of the activators and repressors were then used to identify drugs that change their gene expression signatures. Two drugs, imatinib, and decitabine have been identified as putative modulators of G3BP1/2 genes and their regulators, suggesting their role as COVID-19 mitigation agents. Molecular docking analysis suggests that both drugs bind to G3BP1/2 with a much higher affinity than the SARS-CoV-2 N protein. This study reports imatinib and decitabine as candidate drugs against N protein and G3BP1/2 protein.

Enzyme Models-From Catalysis to Prodrugs.

Enzymes are highly specific biological catalysts that accelerate the rate of chemical reactions within the cell. Our knowledge of how enzymes work remains incomplete. Computational methodologies such as molecular mechanics (MM) and quantum mechanical (QM) methods play an important role in elucidating the detailed mechanisms of enzymatic reactions where experimental research measurements are not possible. Theories invoked by a variety of scientists indicate that enzymes work as structural scaffolds that serve to bring together and orient the reactants so that the reaction can proceed with minimum energy. Enzyme models can be utilized for mimicking enzyme catalysis and the development of novel prodrugs. Prodrugs are used to enhance the pharmacokinetics of drugs; classical prodrug approaches focus on alternating the physicochemical properties, while chemical modern approaches are based on the knowledge gained from the chemistry of enzyme models and correlations between experimental and calculated rate values of intramolecular processes (enzyme models). A large number of prodrugs have been designed and developed to improve the effectiveness and pharmacokinetics of commonly used drugs, such as anti-Parkinson (dopamine), antiviral (acyclovir), antimalarial (atovaquone), anticancer (azanucleosides), antifibrinolytic (tranexamic acid), antihyperlipidemia (statins), vasoconstrictors (phenylephrine), antihypertension (atenolol), antibacterial agents (amoxicillin, cephalexin, and cefuroxime axetil), paracetamol, and guaifenesin. This article describes the works done on enzyme models and the computational methods used to understand enzyme catalysis and to help in the development of efficient prodrugs.

Silylation of Deoxynucleotide Analog Yields an Orally Available Drug with Antileukemia Effects.

DNA methyltransferase inhibitors have improved the prognosis of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, because these agents are easily degraded by cytidine deaminase (CDA), they must be administered intravenously or subcutaneously. Recently, two orally bioavailable DNA methyltransferase inhibitors, CC-486 and ASTX727, were approved. In previous work, we developed 5-O-trialkylsilylated decitabines that resist degradation by CDA. However, the effects of silylation of a deoxynucleotide analog and enzymatic cleavage of silylation have not been fully elucidated. Enteric administration of OR21 in a cynomolgus monkey model led to high plasma concentrations and hypomethylation, and in a mouse model, oral administration of enteric-coated OR21 led to high plasma concentrations. The drug became biologically active after release of decitabine (DAC) from OR21 following removal of the 5'-O-trisilylate substituent. Toxicities were tolerable and lower than those of DAC. Transcriptome and methylome analysis of MDS and AML cell lines revealed that OR21 increased expression of genes associated with tumor suppression, cell differentiation, and immune system processes by altering regional promoter methylation, indicating that these pathways play pivotal roles in the action of hypomethylating agents. OR21 induced cell differentiation via upregulation of the late cell differentiation drivers CEBPE and GATA-1 Thus, silylation of a deoxynucleotide analog can confer oral bioavailability without new toxicities. Both in vivo and in vitro, OR21 exerted antileukemia effects, and had a better safety profile than DAC. Together, our findings indicate that OR21 is a promising candidate drug for phase I study as an alternative to azacitidine or decitabine.

Epigenetic modulators combination with chemotherapy in breast cancer cells.

Despite the concerning adverse effects on tumour development, epigenetic drugs are very promising in cancer treatment. The aim of this study was to compare the differential effects of standard chemotherapy regimens (FEC: 5-fluorouracil plus epirubicine plus cyclophosphamide) in combination with epigenetic modulators (decitabine, valproic acid): (a) on gene methylation levels of selected tumour biomarkers (LINE-1, uPA, PAI-1, DAPK); (b) their expression status (uPA and PAI-1); (c) differentiation status (5meC and H3K27me3). Furthermore, cell survival as well as changes concerning the invasion capacity were monitored in cell culture models of breast cancer (MCF-7, MDA-MB-231). A significant overall decrease of cell survival was observed in the FEC-containing combination therapies for both cell lines. Methylation results showed a general tendency towards increased demethylation of the uPA and PAI-1 gene promoters for the MCF-7 cells, as well as the proapoptotic DAPK gene in the treatment regimens for both cell lines. The uPA and PAI-1 antigen levels were mainly increased in the supernatant of FEC-only treated MDA-MB-231 cells. DAC-only treatment induced an increase of secreted uPA protein in MCF-7 cell culture, while most of the VPA-containing regimens also induced uPA and PAI-1 expression in MCF-7 cell fractions. Epigenetically active substances can also induce a re-differentiation in tumour cells, as shown by 5meC, H3K27me3 applying ICC. SIGNIFICANCE OF THE STUDY: Epigenetic modulators especially in the highly undifferentiated and highly malignant MDA-MB-231 tumour cells significantly reduced tumour malignancy thus; further clinical studies applying specific combination therapies with epigenetic modulators may be warranted.

Experimental and thermodynamic modeling decitabine anti cancer drug solubility in supercritical carbon dioxide.

Design and development of efficient processes for continuous manufacturing of solid dosage oral formulations is of crucial importance for pharmaceutical industry in order to implement the Quality-by-Design paradigm. Supercritical solvent-based manufacturing can be utilized in pharmaceutical processing owing to its inherent operational advantages. However, in order to evaluate the possibility of supercritical processing for a particular medicine, solubility measurement needs to be carried out prior to process design. The current work reports a systematic solubility analysis on decitabine as an anti-cancer medicine. The solvent is supercritical carbon dioxide at different conditions (temperatures and pressures), while gravimetric technique is used to obtain the solubility data for decitabine. The results indicated that the solubility of decitabine varies between 2.84 × 10-05 and 1.07 × 10-03 mol fraction depending on the temperature and pressure. In the experiments, temperature and pressure varied between 308-338 K and 12-40 MPa, respectively. The solubility of decitabine was plotted against temperature and pressure, and it turned out that the solubility had direct relation with the pressure due to the effect of pressure on solvating power of solvent. The effect of temperature on solubility was shown to be dependent on the cross-over pressure. Below the cross-over pressure, there is a reverse relation between temperature and solubility, while a direct relation was observed above the cross-over pressure (16 MPa). Theoretical study was carried out to correlate the solubility data using several thermodynamic-based models. The fitting and model calibration indicated that the examined models were of linear nature and capable to predict the measured decitabine solubilities with the highest average absolute relative deviation percent (AARD %) of 8.9%.

Immuno-DNA binding directed template-free DNA extension and enzyme catalysis for sensitive electrochemical DNA methyltransferase activity assay and inhibitor screening.

Sensitive and accurate determination of DNA methyltransferase (DNA Mtase) activity is highly pursued for understanding fundamental biological processes related to DNA methylation, clinical disease diagnosis and drug discovery. Herein, we propose a new electrochemical immuno-DNA sensing platform for DNA Mtase activity assay and inhibitor screening. After homogeneous DNA methylation by CpG methyltransferase (M.SssI Mtase), the methylated DNA can be specifically recruited onto an electrode via its immunological binding with the immobilized anti-5-methylcytosine antibody. The recruited methylated DNA was simultaneously used as a substrate to facilitate successive template-free DNA extension and enzyme catalysis for the dual-step signal amplification of DNA Mtase activity. The developed immuno-DNA sensing strategy effectively integrates solution-phase DNA methylation, surface affinity binding recognition, and successive template-free DNA extension and enzyme catalysis-based signal amplification, rendering a highly specific, sensitive and accurate assay of DNA Mtase activity. A low detection limit of 0.039 U mL-1 could be achieved with a high selectivity. It was also applied for efficient evaluation of various inhibitors. Current affinity recognition of the immobilized antibody with methylated DNA switches the sensing platform into a DNA operation interface, facilitating the opportunity for combining various DNA-based signal amplification strategies to improve the detection performance. It would be used as a general strategy for the analysis of DNA Mtase activity, inhibitors and more analytes, and is anticipated to show potential for applications in disease diagnosis and drug discovery.

Abnormal methylation of PIK3AP1 was involved in regulating the immune inflammatory response of GES-1 cells induced by Helicobacter pylori.

Gastric epithelial cells (GES-1) stimulated by Helicobacter pylori (H. pylori) would affect the expression of related genes and induce the immune response of the cells. Abnormal methylation of DNA was one of the main causes. The aim of this study was to investigate phosphoinositol-3-kinase adaptor protein 1(PIK3AP1), which was screened from the chip data as an immune gene candidate to against the inflammatory response of cells caused by H. pylori infection. PIK3AP1 plays a key role in PI3K/AKT signaling pathway. The gene chip analysis and experimental results confirmed that PIK3AP1 expression was downregulated and PIK3AP1 promoter was hypermethylated after H. pylori stimulation in GES-1 cells. Meanwhile, the expression level of PIK3AP1 was significantly upregulated after 5-aza-dc treatment, and its expression was higher after 5-aza-dc and H. pylori co-treatment than that of H. pylori treatment but lower than that of 5-aza-dc treatment. Therefore, hypermethylation was the main reason for the down-regulation of PIK3AP1 after H. pylori stimulation. In addition, the intervention of PIK3AP1 inhibited the expression of downstream gene AKT, and suppressing the expression of the immunoinflammatory gene IL-6 in GES-1 cells. Furthermore, the intervention of PIK3AP1 would promote cell proliferation. In summary, hypermethylation of the PIK3AP1 promoter was accompanied by reduction of the expression level of PIK3AP1 in GES-1 cells by H. pylori stimulation. The expression of PIK3AP1, AKT, and IL-6 genes was positively correlated, Meanwhile, the PIK3AP1 can affect the proliferation of GES-1 cells. These results would be helpful to understand the innate immune response function of PIK3AP1 to pathogenic bacterial infection in the stomach.

Novel prodrugs of decitabine with greater metabolic stability and less toxicity.

BACKGROUND: DNA demethylation therapy is now used in practice for hematological tumors and is being developed for solid tumors. Nevertheless, it is difficult to achieve stable pharmacokinetics with the current DNA-demethylating agents, azacitidine (AZA) and decitabine (DAC), because of their rapid deamination by cytidine deaminase in vivo and spontaneous hydrolytic cleavage. Here, we aimed to develop metabolically stable prodrugs of AZA and DAC as novel DNA-demethylating agents. RESULTS: Thirty-five 5'-O-trialkylsilylated AZAs/DACs were synthesized with potential resistance to deamination. Out of these, 11 compounds exhibited demethylating activity similar to that of DAC and guadecitabine, and a suitable aqueous solubility. Pharmacokinetic analysis in mice showed that OR-2003 displayed the highest serum concentration and the area under the curve in an intraperitoneal experiment, whereas OR-2100 exhibited high stability to cytidine deaminase. Treatment of cells with OR-2003 and OR-2100 depleted DNA methyltransferase 1 completely and induced both gene-specific and genome-wide demethylation. The treatment suppressed the growth of multiple types of cancer cells and induced re-expression of tumor suppressor genes. The anti-tumor effect and DNA demethylation effect of OR-2003 and OR-2100 were comparable to that of DAC with fewer adverse effects in vivo. CONCLUSIONS: We developed two novel prodrugs of DAC that exhibited greater stability, comparable DNA demethylation activity, and less toxicity. These compounds are expected to overcome the difficulty in achieving stable pharmacokinetics in patients, leading to maximum DNA demethylation activity with minimum adverse effects.

Clinical update on hypomethylating agents.

Hypomethylating agents (HMAs), azacitidine and decitabine, are standards of care in higher-risk myelodysplastic syndromes and in acute myeloid leukemia patients ineligible for intensive therapy. Over the last 10 years, research efforts have sought to better understand their mechanism of action, both at the molecular and cellular level. These efforts have yet to robustly identify biomarkers for these agents. The clinical activity of HMAs in myeloid neoplasms has been firmly established now but still remains of limited magnitude. Besides optimized use at different stages of the disease, most of the expected clinical progress with HMAs will come from the development of second-generation compounds orally available and/or with improved pharmacokinetics, and from the search, so far mostly empirical, of HMA-based synergistic drug combinations.

Di-O-lauroyl-decitabine-lipid nanocapsules: toward extending decitabine activity.

BACKGROUND: Acute myeloid leukemia mainly affects adult patients. Complete remission for patients younger than 60 years, who are candidates for standard induction therapy, is achieved in 60%-80% of cases. However, the prognosis is still poor for older patients, who are unfit for intensive chemotherapy, and only a few therapies are available. Hypomethylating agents, such as decitabine, are approved for such patients. The current dosing regimen consists of one administration per day, for 5 days, each 4 weeks. METHODS: Here, we present the synthesis of a decitabine prodrug, combined with its encapsulation into a lipid-based nanocapsule formulation. Decitabine (C12)2 was synthetized, then loaded into nanocapsules. Its stability in phosphate buffer ans human plasma was checked. Its activity was evaluated by Cell proliferation assays and cell-cycle analysis on human erythroleukemia cells. Then its pharmacokinetics was determined on a rat model. RESULTS: Decitabine (C12)2 was obtained with a yield of 50%. Drug loading into nanocarriers of 27.45±0.05 nm was 5.8±0.5 mg/mL. The stability of decitabine was improved and its activity on leukemia cells was not altered. Finally, pharmacokinetics studies showed a prolonged mean residence time of the drug. CONCLUSION: Decitabine (C12)2 as a prodrug showed high encapsulation efficiency, a good stability in plasma with no impact on its activity on leukemia cells and improved pharmacokinetics.

Development and validation of LC-MS/MS methods for the quantification of the novel anticancer agent guadecitabine and its active metabolite ß­decitabine in human plasma, whole blood and urine.

Guadecitabine (SGI-110), a dinucleotide of ߭decitabine and deoxyguanosine, is currently being evaluated in phase II/III clinical trials for the treatment of hematological malignancies and solid tumors. This article describes the development and validation of bioanalytical assays to quantify guadecitabine and its active metabolite ߭decitabine in human plasma, whole blood and urine using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Since ߭decitabine is rapidly metabolized further by cytidine deaminase, plasma and whole blood samples were kept on ice-water after collection and stabilized with tetrahydrouridine (THU) directly upon sample collection. Sample preparation consisted of protein precipitation for plasma and whole blood and dilution for urine samples and was further optimized for each matrix and analyte separately. Final extracts were injected onto a C6-phenyl column for guadecitabine analysis, or a Nova-Pak Silica column for ߭decitabine analysis. Gradient elution was applied for both analytes using the same eluents for each assay and detection was performed on triple quadrupole mass spectrometers operating in the positive ion mode (Sciex QTRAP 5500 and QTRAP 6500). The assay for guadecitabine was linear over a range of 1.0-200 ng/mL (plasma, whole blood) and 10-2000 ng/mL (urine). For ߭decitabine the assay was linear over a range of 0.5-100 ng/mL (plasma, whole blood) and 5-1000 ng/mL (urine). The presented methods were successfully validated according to the latest FDA and EMA guidelines for bioanalytical method validation and applied in a guadecitabine clinical mass balance trial in patients with advanced cancer.

Improved locus-specific unmethylated controls for MS-HRM analysis derived from 5-aza-2-deoxycytidine-treated DNA.

We report two restriction enzyme-based approaches for generating clean locus-specific unmethylated controls for methylation-sensitive high-resolution melting (MS-HRM) analyses. These unmethylated standards are derived from DNA treated with the demethylating agent 5-aza-2-deoxycytidine (5-Aza-dc). By using them, we overcome a limitation of 5-Aza-dc treatment - incomplete demethylation at various genomic regions. When 5-Aza-dc-treated DNA is used directly as unmethylated MS-HRM standard, partially demethylated DNA can give false methylation results. MS-HRM assay differentiates between methylated and unmethylated bisulfite-treated DNA based on the different melting profiles of PCR products amplified from them. To estimate test sample methylation levels, test sample melting profiles are compared to those of methylation standards. With our pure unmethylated controls, adequate standards of known methylation levels can be prepared for single-locus MS-HRM.

Development, validation, and clinical application of a high-performance liquid chromatography-tandem mass spectrometry assay for the quantification of total intracellular ß-decitabine nucleotides and genomic DNA incorporated ß-decitabine and 5-methyl-2'-deoxycytidine.

DNA hypermethylation is an epigenetic event that is commonly found in malignant cells and is used as a therapeutic target for ß-decitabine (ß-DEC) containing hypomethylating agents (eg Dacogen® and guadecitabine). ß-DEC requires cellular uptake and intracellular metabolic activation to ß-DEC triphosphate before it can get incorporated into the DNA. Once incorporated in the DNA, ß-DEC can exert its hypomethylating effect by trapping DNA methyltransferases (DNMTs), resulting in reduced 5-methyl-2'-deoxycytidine (5mdC) DNA content. ß-DEC DNA incorporation and its effect on DNA methylation, however, have not yet been investigated in patients treated with ß-DEC containing therapies. For this reason, we developed and validated a sensitive and selective LC-MS/MS method to determine total intracellular ß-DEC nucleotide (ß-DEC-XP) concentrations, as well as to quantify ß-DEC and 5mdC DNA incorporation relative to 2'-deoxycytidine (2dC) DNA content. The assay was successfully validated according to FDA and EMA guidelines in a linear range from 0.5 to 100 ng/mL (ß-DEC), 50 to 10,000 ng/mL (2dC), and 5 to 1,000 ng/mL (5mdC) in peripheral blood mononuclear cell (PBMC) lysate. An additional calibrator at a concentration of 0.1 ng/mL was added for ß-DEC to serve as a limit of detection (LOD). Clinical applicability of the method was demonstrated in patients treated with guadecitabine. Our data support the use of the validated LC-MS/MS method to further explore the intracellular pharmacokinetics in patients treated with ß-DEC containing hypomethylating agents.

Nanogel-mediated delivery of a cocktail of epigenetic drugs plus doxorubicin overcomes drug resistance in breast cancer cells.

Epigenetic modifications (e.g., DNA methylation or histone deacetylation) are commonly implicated in cancer chemoresistance. We previously showed that pretreating resistant MCF-7/ADR breast cancer cells with a demethylating agent (5-aza-2'-deoxycytidine (DAC)) or with an inhibitor of histone deacetylase (suberoylanilide hydroxamic acid (SAHA)) sensitized resistant cells to doxorubicin (DOX) treatment. However, even with increasing doses of DOX, a fraction of resistant cells remained nonresponsive to this pretreatment (~ 25% pretreated with DAC, ~ 45% with SAHA). We hypothesized that pretreating resistant cells with a combination of epigenetic drugs (DAC + SAHA) could more effectively overcome drug resistance. We postulated that delivery of epigenetic drugs encapsulated in biodegradable nanogels (NGs) would further enhance their efficacy. MCF-7/ADR cells were first treated with a single drug vs. a combination of epigenetic drugs, either as solutions or encapsulated in NGs, then subjected to DOX, either in solution or in NGs. Antiproliferative data showed that pretreatment with epigenetic drugs in NGs, then with DOX in NGs, was most effective in overcoming resistance; this treatment inhibited cell growth by > 90%, even at low doses of DOX. Cell cycle analysis showed that a major fraction of cells treated with a cocktail of epigenetic drugs + DOX, all in NG formulations, remained in the G2/M cell cycle arrest phase for a prolonged period. The mechanism of better efficacy of epigenetic drugs in NGs could be attributed to their sustained effect. A similar strategy could be developed for other cancer cells in which drug resistance is due to epigenetic modifications.

Metabolic Recruitment and Directed Evolution of Nucleoside Triphosphate Uptake in Escherichia coli.

We report the design and elaboration of a selection protocol for importing a canonical substrate of DNA polymerase, thymidine triphosphate (dTTP) in Escherichia coli. Bacterial strains whose growth depend on dTTP uptake, through the action of an algal plastid transporter expressed from a synthetic gene inserted in the chromosome, were constructed and shown to withstand the simultaneous loss of thymidylate synthase and thymidine kinase. Such thyA tdk dual deletant strains provide an experimental model of tight nutritional containment for preventing dissemination of microbial GMOs. Our strains transported the four canonical dNTPs, in the following order of preference: dCTP > dATP ≥ dGTP > dTTP. Prolonged cultivation under limitation of exogenous dTTP led to the enhancement of dNTP transport by adaptive evolution. We investigated the uptake of dCTP analogues with altered sugar or nucleobase moieties, which were found to cause a loss of cell viability and an increase of mutant frequency, respectively. E. coli strains equipped with nucleoside triphosphate transporters should be instrumental for evolving organisms whose DNA genome is morphed chemically by fully substituting its canonical nucleotide components.