Probe Substrates Research Articles

Detection of Bacterial Neutral Ceramidase in Diabetic Foot Ulcers with an Optimized Substrate and Chemoenzymatic Probes

AbstractCeramidases (CDases) are important in controlling skin barrier integrity by regulating ceramide composition and affording downstream signal molecules. While the functions of epidermal CDases are known, roles of neutral CDases secreted by skin‐residing microbes are undefined. Here, we developed a one‐step fluorogenic substrate, S‐B, for specific detection of bacterial CDase activity and inhibitor screening. We identified a non‐hydrolyzable substrate mimic, C6, as the best hit. Based on C6, we designed a photoaffinity probe, JX‐1, which efficiently detects bacterial CDases. Using JX‐1, we identified endogenous low‐abundance PaCDase in a P. aeruginosa monoculture and in a mixed skin bacteria culture. Harnessing both S‐B and JX‐1, we found that CDase activity positively correlates with the relative abundance of P. aeruginosa and is negatively associated with wound area reduction in clinical diabetic foot ulcer patient samples. Overall, our study demonstrates that bacterial CDases are important regulators of skin ceramides and potentially play a role in wound healing.

Novel approaches to characterize individual drug metabolism and advance precision medicine.

Interindividual variability in drug metabolism can significantly affect drug concentrations in the body and subsequent drug response. Understanding an individual's drug metabolism capacity is important for predicting drug exposure and developing precision medicine strategies. The goal of precision medicine is to individualize drug treatment for patients to maximize efficacy and minimize drug toxicity. While advances in pharmacogenomics have improved our understanding of how genetic variations in drug-metabolizing enzymes (DMEs) affect drug response, non-genetic factors are also known to influence drug metabolism phenotypes. This minireview will discuss approaches beyond pharmacogenetic testing to phenotype DMEs- particularly the cytochrome P450 enzymes- in clinical settings. Several phenotyping approaches have been proposed: traditional approaches include phenotyping with exogenous probe substrates and the use of endogenous biomarkers; newer approaches include evaluating circulating non-coding RNAs (ncRNAs) and liquid biopsy-derived markers relevant to DME expression and function. The goals of this minireview are to: 1) provide a high-level overview of traditional and novel approaches to phenotype individual drug metabolism capacity; 2) describe how these approaches are being applied or can be applied to pharmacokinetic studies; and 3) discuss perspectives on future opportunities to advance precision medicine in diverse populations. Significance Statement This minireview provides an overview of recent advances in approaches to characterize individual drug metabolism phenotypes in clinical settings. Herein, we highlight the integration of existing pharmacokinetic biomarkers with novel approaches; also discussed are current challenges and existing knowledge gaps. The article concludes with perspectives on the future deployment of a liquid biopsy-informed physiologically based pharmacokinetic (PBPK) strategy for patient characterization and precision dosing.

A protease activity-based machine-learning approach as a complementary tool for conventional diagnosis of diarrhea-predominant irritable bowel syndrome.

Irritable bowel syndrome (IBS) has no clinically accepted biomarkers even though it affects a large number of individuals worldwide. To address this lack of understanding, we evaluated peptidase activity in fecal samples from 35 patients with diarrheal IBS without symptom exacerbation (IBS-n) and 35 healthy subjects using a library of 384 fluorescent enzymatic substrate probes. IBS-n patients had high trypsin-like peptidase activity for cleavage of C-terminal lysine and arginine residues and low elastase-like activity for cleavage of C-terminal serine and glycine residues. These fluorescent probe library data, together with diagnostic machine-learning techniques, were able to accurately predict IBS-n. This approach can be used to diagnose diseases where no clinically accepted biomarkers exist, in which fecal enzyme activity is altered and also suggests that the development of new therapies targeting enzyme activities is possible.

The Role of Coproporphyrins as Endogenous Biomarkers for Organic Anion Transporting Polypeptide 1B Inhibition - progress from 2016 to 2023.

Since the initial clinical study investigating CP-I and CP-III as endogenous biomarkers for OATP inhibition DDIs published in 2016, significant progress has been made in confirming the usefulness of the CPs, particularly the CP-I, as biomarkers in assessing OATP inhibition DDIs. CP-I exhibits selectivity towards OATP1B activity in human subjects with genetic variants of OATP1B1. Its sensitivity to a broad spectrum of clinical OATP1B inhibitors has been established, from weak to vigorous. Dose-dependent CP-I changes in healthy human subjects show agreement with DDI magnitudes of probe substrates by rifampin treatment. PBPK models have been established for concentration changes of plasma CP-I with OATP inhibitors, demonstrating the usefulness of supporting the quantitative translation of the effect of CP-I levels into the DDI risk assessment of potential OATP inhibitors. As plasma CP-I's sensitivity, specificity, and selectivity have been validated in humans, monitoring CP-I levels in single and multiple clinical phase I dose escalation studies is recommended for early assessment of DDI risks and understanding the full dose-response of an investigational drug to OATP inhibitions. A decision tree is proposed to preclude the need to conduct a dedicated DDI study by administering a probe substrate drug to human subjects. Significance Statement The mini-review summarized the validation paths of CP-I and CP-III as biomarkers of OATP1B inhibition in humans for their selectivity, specificity, and sensitivity. The utility of monitoring CP-I to assess drug-drug interactions of OATP1B inhibition in early drug development is proposed. Changes in plasma CP-I in phase I dose range studies can be used to frame plans for late-stage development and facilitate the mechanistic understanding of complex drug-drug interactions.

Phase I study to assess the effect of adavosertib (AZD1775) on the pharmacokinetics of substrates of CYP1A2, CYP2C19, and CYP3A in patients with advanced solid tumors

PurposeAdavosertib may alter exposure to substrates of the cytochrome P450 (CYP) family of enzymes. This study assessed its effect on the pharmacokinetics of a cocktail of probe substrates for CYP3A (midazolam), CYP2C19 (omeprazole), and CYP1A2 (caffeine).MethodsPeriod 1: patients with locally advanced or metastatic solid tumors received ‘cocktail’: caffeine 200 mg, omeprazole 20 mg, and midazolam 2 mg (single dose); period 2: after 7- to 14-day washout, patients received adavosertib 225 mg twice daily on days 1–3 (five doses), with cocktail on day 3. After cocktail alone or in combination with adavosertib administration, 24-h pharmacokinetic sampling occurred for probe substrates and their respective metabolites paraxanthine, 5-hydroxyomeprazole (5-HO), and 1′-hydroxymidazolam (1′-HM). Safety was assessed throughout.ResultsOf 33 patients (median age 60.0 years, range 41–83) receiving cocktail, 30 received adavosertib. Adavosertib co-administration increased caffeine, omeprazole, and midazolam exposure by 49%, 80%, and 55% (AUC0–12), respectively; AUC0–t increased by 61%, 98%, and 55%. Maximum plasma drug concentration (Cmax) increased by 4%, 46%, and 39%. Adavosertib co-administration increased 5-HO and 1′-HM exposure by 43% and 54% (AUC0–12) and 49% and 58% (AUC0–t), respectively; paraxanthine exposure was unchanged. Adavosertib co-administration decreased Cmax for paraxanthine and 5–HO by 19% and 7%; Cmax increased by 33% for 1′-HM. After receiving adavosertib, 19 (63%) patients had treatment-related adverse events (six [20%] grade ≥ 3).ConclusionAdavosertib (225 mg bid) is a weak inhibitor of CYP1A2, CYP2C19, and CYP3A.ClinicalTrials.govNCT03333824

Reducing Hepatotoxicity Mechanism of Radix Wikstroemia Indica by Processing with “Sweat Soaking Method” Using UPLC-MS/MS and a Cocktail Probe Substrate

Background: Radix Wikstroemia indica is a traditional Chinese medicine (TCM) used as anti-inflammatory and anti-tumor drug. However, it has serious hepatotoxicity, "Sweat soaking method" processed could effectively decrease its hepatotoxicity. Objective: The objective of this study is to study the effects of Radix Wikstroemia indica on six kinds of cytochrome P450(CYP450) isozymes of rat liver microsomes before and after processing, and to study the mechanism of Radix Wikstroemia indica processed by the "Sweat soaking method" to reduce liver toxicity in rats. Methods: In this study, the effects of Radix Wikstroemia indica and processed Radix Wikstroemia indica on the six main CYP450 isoforms (2E1, 1A2, 2C6, 2D1, 2C11, and 3A1) were investigated in vitro. Using a cocktail probe of CYP450 isoform-specific substrates and their metabolites, we carried out in vitro enzymatic studies in liver microsomal incubation systems via UPLC-MS/MS. Results: The results showed that the established UPLC-MS/MS method was precise and reliable. Compared with the blank group, the activities of six enzymes in the RWI and PRWI groups were higher than those in the blank group. At the same dose, the enzyme activities of CYP2E1, CYP1A2, CYP2C6, CYP2C11, and CYP3A1 increased with the increase in dose, and the enzyme activities of the RWI group were higher than those of the PRWI group. The enzyme activities of CYP2E1 and CYP1A2 in the Radix Wikstroemia indica group were significantly increased compared with the blank group, CYP3A1 in the RWI high-dose group was higher than that in the blank group and PRWI group with statistical differences (p<0.05 or p<0.01). Conclusion: The processed Radix Wikstroemia indica could reduce liver injury, and its detoxication mechanism might be related to the decrease in enzyme activity of CYP1A2, CYP2E1 and CYP3A1.

Antireflection Coatings Based on PEDOT:PSS Conductive Polymer Using d-Sorbitol Additives for Terahertz Spectroscopy and Imaging.

Optical antireflection has been employed for a variety of applications in terahertz spectroscopy and detectors. However, current methods encounter challenges in terms of cost, bandwidth, structural complexity, and performance. In this study, a low-cost, broadband, and easily processed THz antireflection coating scheme based on the model of impedance-matching effect is proposed, using a 6 wt % d-sorbitol-doped poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (s-PEDOT:PSS) film. By adjusting the thickness of the s-PEDOT:PSS film, these biocompatible conductive polymers enable a significant reduction of Fresnel reflection and operate over a broad bandwidth between 0.2 and 2.2 THz. Applying the antireflective coating to the surface of the sample substrate and electro-optic probe crystal in THz spectroscopy and near-field imaging shows that the spectral resolution is significantly improved, and the devices exhibit more excellent intended performance. The findings of this study could aid in improving the measurement capability of various THz time-domain spectroscopy and imaging system.

A clinical pharmacokinetic drug-drug interaction study between dextromethorphan and emvododstat, a potent anti-SARS-CoV-2 dihydroorotate dehydrogenase inhibitor

PurposeA therapeutic agent that targets both viral replication and the hyper-reactive immune response would offer a highly desirable treatment for severe acute respiratory syndrome corona virus 2 (SARS-CoV-2, coronavirus disease 2019, COVID-19) management. Emvododstat (PTC299; 4-chlorophenyl 6-chloro-1-[4-methoxyphenyl]-1,3, 4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxylate) was found to be a potent inhibitor of immunomodulatory and inflammation-related processes by inhibition of dihydroorotate dehydrogenase to reduce the severity of SARS-CoV-2 infections This drug interaction study was performed to determine if emvododstat was an inhibitor of CYP2D6.MethodsPotential drug-drug interactions between emvododstat and a CYP2D6 probe substrate (dextromethorphan) were investigated by measuring plasma dextromethorphan and metabolite (dextrorphan) concentrations before and after emvododstat administration. On day 1, 18 healthy subjects received an oral dose of 30 mg dextromethorphan followed by a 4-day washout period. On day 5, subjects received an oral dose of 250 mg emvododstat with food. Two hours later, 30 mg dextromethorphan was administered.ResultsWhen given with emvododstat, plasma dextromethorphan concentrations increased substantially, while metabolite levels (dextrorphan) remained essentially the same. Maximum plasma dextromethorphan concentration (Cmax) increased from 2006 to 5847 pg/mL. Dextromethorphan exposure (AUC) increased from 18,829 to 157,400 h·pg/mL for AUC0-last and from 21,585 to 362,107 h·pg/mL for AUC0-inf following administration of emvododstat. When dextromethorphan parameters were compared before and after emvododstat, least squares mean ratios (90% confidence interval) were found to be 2.9 (2.2, 3.8), 8.4 (6.1, 11.5), and 14.9 (10.0, 22.1) for Cmax, AUC0-last, and AUC0-inf, respectively.ConclusionEmvododstat appears to be a strong CYP2D6 inhibitor. No drug-related treatment emergent adverse effects (TEAEs) were considered to be severe or serious.Trial registrationEudraCT 2021-004626-29, 11 May 2021.

Label-free detection and identification of single bacteria via terahertz near-field imaging.

In recent years, terahertz (THz) imaging has attracted much attention because of its ability to obtain physical and chemical information in a label-free, noninvasive and nonionizing manner. However, the low spatial resolution of traditional THz imaging systems and the weak dielectric response of biological samples hinder the application of this technology in the biomedical field. In this paper, we report a new THz near-field imaging method for a single bacteria, through the coupling effect of nanoscale radius of probe and platinum gold substrate, which greatly enhances THz near-field signal of biological samples. A THz super-resolution image of bacteria has been successfully obtained by strictly controlling the relevant test parameters such as tip parameters and driving amplitude. By analyzing and processing the THz spectral image, the morphology and inner structure of bacteria have been observed. The method has been used to detect and identify Escherichia coli represented by Gram-negative bacteria and Staphylococcus aureus represented by Gram-positive bacteria. This application provides a new label-free, noninvasive and nonionizing testing protocol for the detection of single bacteria.

Evaluation of Drug-Drug Interaction Potential of Enarodustat (JTZ-951) Using a Cytochrome P450 Probe Cocktail.

The drug interaction potential of enarodustat (doses: 25, 50mg) on the activity of cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4 was evaluated after once-daily administration for 15 days in a phase 1 multiple-ascending-dose study in healthy subjects. Probe substrates specific for the enzymes, i.e.,caffeine (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4), were administered orally as a cocktail with (day15) and without (day -3) enarodustat. Drug interaction was based on geometric mean maximum plasma concentration (Cmax ) and area under the plasma concentration-time curve from the time of dosing to infinity (AUCinf ) ratios (day 15/day -3) for CYP1A2, 2C9, 2C19, 2D6, 3A4, and urinary excretion of dextromethorphan metabolite dextrorphan for CYP2D6. At the 2 enarodustat doses, for caffeine, the geometric mean ratios (range) for Cmax and AUCinf were 0.99-1.06 and 1.61-1.63, respectively. The ratios for peak concentrations and total exposures were 0.98-1.07 and 0.71-1.78 for tolbutamide and omeprazole, respectively. For dextrorphan the Cmax and AUCinf ratios were 0.83-0.90 and 1.02-1.04, respectively. The mean dextrorphan cumulative amount excreted into the urine from the time of dosing to 24 hours values on day -3 and day 15 were 8.25 mg and 8.20 mg at the lower dose, and 9.40 mg and 9.51 mg at the higher dose. The ratios for midazolam Cmax and AUCinf were 1.42-1.63. Overall, there was a lack of enarodustat dose dependency regarding the geometric mean ratios and 90% confidence intervals and urinary excretion of dextrorphan. There were some cases where the 90% confidence intervals at the 2 enarodustat doses were outside the 0.80-1.25 range, but changes in the geometric mean ratios were all <2-fold.

The Potentially Significant Role of CYP3A-Mediated Oxidative Metabolism of Dabigatran Etexilate and its Intermediate Metabolites in Drug-Drug Interaction Assessments Using Microdose Dabigatran Etexilate.

Dabigatran etexilate (DABE), a double ester prodrug of dabigatran (DAB), is a probe substrate of intestinal P-glycoprotein (P-gp) commonly used in clinical drug-drug interaction (DDI) studies. When compared to its therapeutic dose at 150 mg, microdose DABE (375 µg) showed approximately 2-fold higher in DDI magnitudes with CYP3A/P-gp inhibitors. In this study, we conducted several in vitro metabolism studies to demonstrate that DABE at a theoretical gut concentration after microdosing significantly underwent NADPH-dependent oxidation (~40-50%) in parallel to carboxylesterase-mediated hydrolysis in human intestinal microsomes. Furthermore, NADPH-dependent metabolism of its intermediate monoester BIBR0951 was also observed in both human intestinal and liver microsomes, accounting for 100% and 50% of total metabolism, respectively. Metabolite profiling using LC-MS/MS confirmed the presence of several novel oxidative metabolites of DABE and of BIBR0951 in the NADPH-fortified incubations. CYP3A was identified as the major enzyme catalyzing the oxidation of both compounds. The metabolism of DABE and BIBR0951 was well described by Michaelis-Menten kinetics, with Km ranging 1-3 µM, significantly below the expected concentrations following the therapeutic dose of DABE. Overall, the present results suggested that CYP3A played a significant role in the presystemic metabolism of DABE and BIBR0951 following microdose DABE administration, thus attributing partly to the apparent overestimation in the DDI magnitude observed with the CYP3A/P-gp inhibitors. Therefore, DABE at the microdose, unlike the therapeutic dose, would likely be a less predictive tool and should be considered as a clinical dual substrate for P-gp and CYP3A when assessing potential P-gp-mediated impacts by dual CYP3A/P-gp inhibitors. Significance Statement This is the first study demonstrating a potentially significant role of CYP-mediated metabolism of the prodrug DABE following a microdose, but not a therapeutic dose. This additional pathway coupled with its susceptibility to P-gp may make DABE a clinical dual substrate for both P-gp and CYP3A at a microdose. The study also highlights the need for better characterization of the pharmacokinetics and metabolism of a clinical DDI probe substrate over the intended study dose range for proper result interpretations.

Intestinal organoids as an in vitro platform to characterize disposition, metabolism, and safety profile of small molecules

Intestinal organoids derived from LGR5+ adult stem cells allow for long-term culturing, more closely resemble human physiology than traditional intestinal models, like Caco-2, and have been established for several species.Here we evaluated intestinal organoids for drug disposition, metabolism, and safety applications. Enterocyte-enriched human duodenal organoids were cultured as monolayers to enable bidirectional transport studies. 3D enterocyte-enriched human duodenal and colonic organoids were incubated with probe substrates of major intestinal drug metabolizing enzymes (DMEs). To distinguish human intestinal toxic (high incidence of diarrhea in clinical trials and/or black box warning related to intestinal side effects) from non-intestinal toxic compounds, ATP-based cell viability was used as a readout, and compounds were ranked based on their IC50 values in relation to their 30-times maximal total plasma concentration (Cmax). To assess if rat and dog organoids reproduced the respective in vivo intestinal safety profiles, ATP-based viability was assessed in rat and dog organoids and compared to in vivo intestinal findings when available.Human duodenal monolayers discriminated high and low permeable compounds and demonstrated functional activity for the main efflux transporters Multi drug resistant protein 1 (MDR1, P-glycoprotein P-gp) and Breast cancer resistant protein (BCRP). Human 3D duodenal and colonic organoids also showed metabolic activity for the main intestinal phase I and II DMEs. Organoids derived from specific intestinal segments showed activity differences in line with reported DMEs expression. Undifferentiated human organoids accurately distinguished all but one compound from the test set of non-toxic and toxic drugs. Cytotoxicity in rat and dog organoids correlated with preclinical toxicity findings and observed species sensitivity differences between human, rat, and dog organoids.In conclusion, the data suggest intestinal organoids are suitable in vitro tools for drug disposition, metabolism, and intestinal toxicity endpoints. The possibility to use organoids from different species, and intestinal segment holds great potential for cross-species and regional comparisons.

Rolling circle transcription and CRISPR/Cas12a-assisted versatile bicyclic cascade amplification assay for sensitive uracil-DNA glycosylase detection

Uracil-DNA glycosylase (UDG) is pivotal in maintaining genome integrity and aberrant expressed UDG is highly relevant to numerous diseases. Sensitive and accurate detecting UDG is critically significant for early clinical diagnosis. In this research, we demonstrated a sensitive UDG fluorescent assay based on rolling circle transcription (RCT)/CRISPR/Cas12a-assisted bicyclic cascade amplification strategy. Target UDG catalyzed to remove uracil base of DNA dumbbell-shape substrate probe (SubUDG) to produce an apurinic/apyrimidinic (AP) site, at which SubUDG was cleaved by apurinic/apyrimidinic endonuclease (APE1) subsequently. The exposed 5′-PO4 was ligated with the free 3′-OH terminus to form an enclosed DNA dumbbell-shape substrate probe (E-SubUDG). E-SubUDG functioned as a template can actuate T7 RNA polymerase-mediated RCT signal amplification, generating multitudes of crRNA repeats. The resultant Cas12a/crRNA/activator ternary complex activated the activity of Cas12a, causing a significantly enhanced fluorescence output. In this bicyclic cascade strategy, target UDG was amplified via RCT and CRISPR/Cas12a, and the whole reaction was completed without complex procedures. This method enabled sensitive and specific monitor UDG down to 0.0005 U/mL, screen corresponding inhibitors, and analyze endogenous UDG in A549 cells at single-cell level. Importantly, this assay can be extended to analyze other DNA glycosylase (hAAG and Fpg) by altering the recognition site in DNA substrates probe rationally, thereby offering a potent tool for DNA glycosylase-associated clinical diagnosis and biomedical research.

Evaluation potential effects of Picroside II on cytochrome P450 enzymes in vitro and in vivo

Ethnopharmacology relevancePicrorhiza scrophulariiflora Pennell, a well-known Chinese herb, has been traditionally utilized as an antioxidant and anti-inflammatory agent. One of its main bioactive components is Picroside II, a glycoside derivative. However, there is limited information on the effects of Picroside II on the activity of cytochrome P450 (CYP) enzymes nor on potential herb-drug interactions are rarely studied. Aim of the studyThe purpose of the study was to investigate the effects of Picroside II on the activity of cytochrome P450 enzymes in vitro and in vivo and its potential herb-drug interactions. Materials and methodsSpecific probe substrates were employed to assess the effect of Picroside II on the activity of P450 enzymes. The inhibitory effects of Picroside II on CYP enzymes were assayed both in human (i.e., 1A, 2C9, 2C19, 2D6, 2E1, and 3A) and rat (i.e., 1A, 2C6/11, 2D1, 2E1, and 3A) liver microsomes in vitro. The inductive effects were investigated in rats following oral gavage of 2.5 mg/kg and 10 mg/kg Picroside II. A specific Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) method was developed to determine the formation of specific metabolites. ResultsEnzyme inhibition results showed that Picroside II (0.5–200 μM) had no evident inhibitory effects on rat and human liver microsomes in vitro. Interestingly, the administration of multiple doses of 10 mg/kg Picroside II inhibited the activity of CYP2C6/11 by reducing the rate of formation of 4-hydroxydiclofenac and 4-hydroxymephenytoin, while Picroside II at 2.5 mg/kg increased the activity of CYP3A by promoting the formation of 1-hydroxymidazolam and 6-hydroxychlorzoxazone in rats. In addition, there were negligible effects on CYP1A, CYP2D1, and CYP2E1 in rats. ConclusionsThe results indicated that Picroside II modulated the activities of CYP enzymes and was involved in CYP2C and CYP3A medicated herb-drug interactions. Therefore, careful monitoring is necessary when Picroside II is used in combination with related conventional drugs.

Unambiguous Measurement of Local Hole Current in Organic Semiconductors Using Conductive Atomic Force Microscopy

Conductive atomic force microscopy (C-AFM) is a widely used tool for studying the charge transport properties of organic semiconductor films with nanoscale resolution. Local hole current is commonly measured by electrically contacting the film with a high work function C-AFM probe on top and an underlying electrode coated with a hole transport layer. The two voltage polarities, corresponding to the probe injection and substrate injection of holes, are both found in the C-AFM literature; nevertheless, there has been a lack of consideration about the possible influence of voltage polarity on image contrast and charge transport mechanisms. By analyzing local hole current maps and current–voltage curves for three organic semiconductors (a small molecule and two polymers), we find that probe and substrate injection leads to drastically different hole current maps and charge transport mechanisms. Specifically, the substrate injection of holes exhibits ohmic characteristics at low voltages and space-charge-limited current behavior at elevated voltages. Conversely, the probe injection of holes leads to injection-limited current that is sensitive to the state of the probe–sample interface. These measurements provide a blueprint for ensuring that C-AFM measurements are unambiguously probing the bulk properties of organic semiconductor films.

A CpG methylation-powered dynamic three-dimensional-DNAzyme walker for single-molecule monitoring of multiple cytosine-C5 methyltransferases

Cytosine-C5 methyltransferases (MTases) maintain methylation status in CpG islands (CpGIs), and their abnormal activities are implicated in various diseases and cancers. Simultaneous monitoring of multiple cytosine-C5 MTases has remained challenging due to diversity of DNA substrates and scarcity of methylation-independent restriction endonucleases (MIREs). Herein, we construct a CpG methylation-powered dynamic three-dimensional-DNAzyme walker for single-molecule monitoring of multiple cytosine-C5 MTases. The presence of M.SssI and M.CviPI catalyzes cytosines-C5 methylation in hairpin probes to form catalytic substrates of GlaI. Then GlaI specifically cleaves 5-mCs to yield two metal-specific DNAzymes. Upon the addition of substrate probes (SP1/SP2)-functionalized AuNPs, two DNAzymes selectively bind with SP1 and SP2, and autonomously complete 3D-DNAzymes walking with assistance of cofactors Mg2+ and Zn2+, inducing cyclic cleavage of substrate probes and dissociation of abundant Cy5 and Cy3 from AuNPs. Cy5 and Cy3 are simply measured through single-molecule imaging, with Cy5 indicating M.SssI and Cy3 indicating M.CviPI. This nanosensor achieves a limit of detection (LOD) of 3.944 × 10-9 U/μL for M.SssI and 5.046 × 10-9 U/μL for M.CviPI. It can discriminate cytosine-C5 MTases members, screen potential inhibitors, and quantify multiple cytosine-C5 MTases in human serum and diverse cell lysates, providing a powerful platform for biomedical research and clinical diagnostics.

Visualization and Comparison of the Level of Apurinic/Apyrimidinic Endonuclease 1 in Live Normal/Cancerous and Neuron Cells with a Fluorescent Nanoprobe.

As a major apurinic/apyrimidinic endonuclease and a redox signaling protein in human cells, APE1 plays a crucial role in cellular function and survival. The relationship between alterations of APE1 expression and subcellular localization and the initiation, development and treatment of various cancers has received extensive attention. However, comparing the in-vivo activity of APE1 in normal and cancerous breast live cells remains challenging due to the low efficiency of commonly used liposome transfection methods in delivering DNA substrate probes into human normal breast epithelial cells (MCF-10A). In this work, we develop a DNA/RNA hybrid-based small magnetic fluorescent nanoprobe (25 ± 3 nm) that can be taken up by various live cells under magnetic transfection. The D0/R-nanoprobe demonstrates an outstanding specificity toward APE1 and strong resistance to the cellular background interference. Using this nanoprobe, we are not only able to visualize the intracellular activity of APE1 in breast ductal carcinoma (MCF-7) live cells, but also demonstrate the APE1 activity in MCF-10A live cells for the first time. The method is then extended to observe the changes in APE1 levels in highly metabolically active neuroendocrine cells under normal conditions and severe attacks by reactive oxygen species in real-time. The fluorescent nanoprobe provides a useful tool for studying the dynamic changes of intracellular APE1 in normal or cancerous live cells. It also displays the potential for visible and controllable release of miRNA drugs within live cells for therapeutic purposes.

Two curcumin analogs inhibited the function and protein expression of breast cancer resistance protein: in vitro and in vivo studies

1. Two curcumin analogs, (1E,6E)-1,7-bis(3,5-diethyl-4-hydroxyphenyl)hepta-1,6-diene-3,5- dione (N17) and its prodrug ((1E,6E)-3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2,6-diethyl-4,1- phenylene)bis(3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate) (N17′), were evaluated as breast cancer resistance protein (BCRP) inhibitors. 2. MDCKII-BCRP and MDCKII-WT were used to evaluate the modulation effects of N17 and N17′ on BCRP and to explore the relevant mechanism. Sprague-Dawley rats were orally administered rosuvastatin (ROS), a probe substrate of BCRP, without and with N17′ (100 mg/kg) to investigate the effect of N17′ on ROS pharmacokinetics. 3. In cell studies, N17 and N17′ were substrates of BCRP, and they decreased the activity and protein expression of BCRP. In rat study, N17′ increased the systemic exposure of ROS by 218% (p = 0.058). 4. N17 and N17′ are potential BCRP inhibitors and will be promising candidates for overcoming the BCRP-mediated multidrug resistance.

In vitro validation of an in vivo phenotyping drug cocktail for major drug transporters in humans

PurposeCocktails of transporter probe drugs are used in vivo to assess transporter activity and respective drug-drug interactions. An inhibitory effect of components on transporter activities should be ruled out. Here, for a clinically tested cocktail consisting of adefovir, digoxin, metformin, sitagliptin, and pitavastatin, inhibition of major transporters by individual probe substrates was investigated in vitro. MethodsTransporter transfected HEK293 cells were used in all evaluations. Cell-based assays were applied for uptake by human organic cation transporters 1/2 (hOCT1/2), organic anion transporters 1/3 (hOAT1/3), multidrug and toxin extrusion proteins 1/2K (hMATE1/2K), and organic anion transporter polypeptide 1B1/3 (hOATP1B1/3). For P-glycoprotein (hMDR1) a cell-based efflux assay was used whereas an inside-out vesicle-based assay was used for the bile salt export pump (hBSEP). All assays used standard substrates and established inhibitors (as positive controls). Inhibition experiments using clinically achievable concentrations of potential perpetrators at the relevant transporter expression site were carried out initially. If there was a significant effect, the inhibition potency (Ki) was studied in detail. ResultsIn the inhibition tests, only sitagliptin had an effect and reduced hOCT1- and hOCT2- mediated metformin uptake and hMATE2K mediated MPP+ uptake by more than 70%, 80%, and 30%, respectively. The ratios of unbound Cmax (observed clinically) to Ki of sitagliptin were low with 0.009, 0.03, and 0.001 for hOCT1, hOCT2, and hMATE2K, respectively. ConclusionThe inhibition of hOCT2 in vitro by sitagliptin is in agreement with the borderline inhibition of renal metformin elimination observed clinically, supporting a dose reduction of sitagliptin in the cocktail.

A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

Pregnancy-mediated physiological and biochemical changes contribute to alterations in the pharmacokinetics of certain drugs. There is a paucity of data on the systematic evaluation of the underlying mechanisms. The objective of the current study was to examine the impact of changes in circulating and tissue hormonal concentrationduring the late stage of pregnancy on the activity and expression of hepatic cytochrome P450 (CYP) enzymes using a cocktail probe approach. Freshly isolated primary human hepatocytes were incubated with third trimester physiologic (plasma) and projected liver (ten-fold higher) concentrations of female hormones: progesterone (2µM), estradiol (0.3µM), estriol (0.8µM), estrone (0.2µM), 17α-hydroxyprogesterone (0.1µM), and human growth hormone (0.005µM). The metabolic activity of the hepatocytes was assessed using a cocktail of isozyme-specific P450 probe substrates (CYP1A2 (phenacetin), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4 (testosterone)). A validated LC-MS/MS assay was used to measure the corresponding metabolite concentrations. CYP450 protein and mRNA levels were measured using western blot and qRT-PCR, respectively. Female hormones at projected third-semester hepatic concentrations significantly enhanced mRNA and protein expression and increased the metabolic activity of CYP3A4. The expression and activity of other CYP450 enzymes studied were not affected by mixtures of female hormones at concentrations used. The increased activity of CYP3A4 is consistent with the clinically observed increase in clearance of CYP3A4 substrates during pregnancy. Overall expression and activity of CYP450 isozymes are differentially regulated during pregnancy.