Design, synthesis and evaluation of novel LpxC inhibitors containing a hydrazone moiety as Gram-negative antibacterial agents.

LpxC inhibitors are new-type antibacterial agents developed in the last twenty years, mainly against Gram-negative bacteria infections. To enable the development of novel LpxC inhibitors with potent antibacterial activities, several series of compounds were designed and synthesized and their antibacterial activities were evaluated against E. coli ATCC25922, P. aeruginosa ATCC27853, P. aeruginosa clinical isolate PAE 22-1, K. pneumoniae ATCC700603, K. pneumoniae clinical isolate KPN+22-1 in vitro. Compound 6i exhibited significant antibacterial activities against above five Gram-negative bacteria except P. aeruginosa ATCC27853. Moreover, compound 6i exhibited moderate liver microsomal stability and a promising pharmacokinetic profile (AUC0-t = 1050 ng h mL-1, oral bioavailability of 13.3 %) in Sprague-Dawley rats, acceptable PPB, low risk of drug-drug interactions and non-cytotoxic activity against hepatic cell. Collectively, compound 6i could be a promising Gram-negative antibacterial agent for further investigation.

Stability studies of ß-Amino- and ß-Hydroxy difluoromethyl ketones in rat serum and rat liver microsomes.

Although difluoromethyl ketones are used as tools in chemical biology and leads in drug discovery, the metabolic stability of these compounds is generally uncharacterized and must be inferred from in vivo pharmacological assays. In order to address this gap which impedes their wider use, we have synthesized and performed metabolic stability studies for thirty-nine ß-amino and ß-hydroxy difluoromethyl ketones. These investigations provide structure-stability relationships of the difluoromethyl ketones following incubation with rodent serum and liver microsomes.

Extending the shelf life of HLM chips through freeze-drying of human liver microsomes immobilized onto thiol-ene micropillar arrays.

Microfluidic flow reactors functionalized with immobilized human liver microsomes (HLM chips) represent a powerful tool for drug discovery and development by enabling mechanism-based enzyme inhibition studies under flow-through conditions. Additionally, HLM chips may be exploited in streamlined production of human drug metabolites for subsequent microfluidic in vitro organ models or as metabolite standards for drug safety assessment. However, the limited shelf life of the biofunctionalized microreactors generally poses a major barrier to their commercial adaptation in terms of both storage and shipping. The shelf life of the HLM chips in the wetted state is ca. 2-3 weeks only and requires cold storage at 4 °C. In this study, we developed a freeze-drying method for lyophilization of HLMs that are readily immobilized inside microfluidic pillar arrays made from off-stoichiometric thiol-ene polymer. The success of lyophilization was evaluated by monitoring the cytochrome P450 and UDP-glucuronosyltransferase enzyme activities of rehydrated HLMs for several months post-freeze-drying. By adapting the freeze-drying protocol, the HLM chips could be stored at room temperature (protected from light and moisture) for at least 9 months (n = 2 independent batches) and up to 16 months at best, with recovered enzyme activities within 60-120% of the non-freeze-dried control chips. This is a major improvement over the cold-storage requirement and the limited shelf life of the non-freeze-dried HLM chips, which can significantly ease the design of experiments, decrease energy consumption during storage, and reduce the shipping costs with a view to commercial adaptation.

Novel pleuromutilin derivatives conjugated with phenyl-sulfide and boron-containing moieties as potent antibacterial agents against antibiotic-resistant bacteria.

In response to the escalating threat of microbial resistance, a series of novel pleuromutilin derivatives, conjugated with phenyl-sulfide and boron-containing moieties, were designed and synthesized. Most derivatives, especially 14b and 16b, demonstrated significant efficacy against Gram-positive bacteria, including multidrug-resistant strains, as well as pleuromutilin-resistant strains. Compound 16b showed high stability in the liver microsomes of rats and humans, along with acceptable tolerance in vitro and in vivo. Additionally, compound 16b exhibited promising efficacy in MRSA-infected mouse models. Our data highlight the potential of conjugated pleuromutilin derivatives as valuable agents against drug-resistant bacteria.

An ultra-fast green ultra-high-performance liquid chromatography-tandem mass spectrometry method for estimating the in vitro metabolic stability of zotizalkib in human liver microsomes.

Zotizalkib (ZTK, TPX-0131) is a fourth-generation highly effective inhibitor of wild-type anaplastic lymphoma kinase (ALK) and ALK-resistant mutations that can penetrate the central nervous system. It exhibited greater potency compared to all five officially approved ALK inhibitors. The aim of this study was to develop a rapid, accurate, eco-friendly, and highly sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for measuring the concentration of ZTK in human liver microsomes (HLMs). The validation aspects of the current UHPLC-MS/MS methodology in the HLMs were conducted in accordance with the bioanalytical method validation standards specified by the US Food and Drug Administration. ZTK and encorafenib were separated using an Agilent C8 column (Eclipse Plus) and an isocratic mobile phase. The calibration curve for the developed ZTK exhibited a linear relationship within the concentration range of 1-3000 ng/mL. The results from the Analytical Green-ness Metric Approach program (0.76) suggested that the created method demonstrated a significant degree of environmental sustainability. The in vitro half-life (t1/2) and intrinsic clearance (Clint) of ZTK were determined to be 15.79 min and 51.35 mL/min/kg, respectively that suggests the ZTK exhibits characteristics similar to those of a medication with a high extraction ratio. These approaches are crucial for the progress of novel pharmaceutical development, especially in improving metabolic stability.

An ultra-fast ultra-high-performance liquid chromatography-tandem mass spectrometry method for estimating the in vitro metabolic stability of palbociclib in human liver microsomes: In silico study for metabolic lability, absorption, distribution, metabolism, and excretion features, and DEREK alerts screening.

Palbociclib (Ibrance; Pfizer) was approved for the management of metastatic breast cancer characterized by hormone receptor-positive/human epidermal growth factor receptor 2 negative status. The objective of this study was to create a fast, precise, environmentally friendly, and highly sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry approach for quantifying palbociclib (PAB) in human liver microsomes with the application for assessing metabolic stability. The validation features were performed in agreement with the bioanalytical method validation standards outlined by the US Food and Drug Administration. The StarDrop software (WhichP450 and DEREK modules) was used in screening the metabolic lability and structural alerts of PAB. The separation of PAB and encorafenib (as an internal standard) was achieved on a C8 column, employing an isocratic mobile phase. The inter-day and intra-day accuracy and precision ranged from -6.00% to 4.64% and from -2.33% to 3.13%, respectively. The constructed calibration curve displayed a linearity in the range of 1-3000 ng/mL. The sensitivity of the established approach was proven by the lower limit of quantification of 0.73 ng/mL. The Analytical GREEness calculator results revealed the high level of greenness of the developed method. The PAB's metabolic stability (t1/2 of 18.5 min and a moderate clearance (Clint) of 44.8 mL/min/kg) suggests a high extraction ratio medication that matched the WhichP450 software results.

Advancing structural elucidation of conjugation drug metabolites in metabolite profiling with novel electron-activated dissociation.

RATIONALE: This study focuses on the advantage of using the novel electron-activated dissociation (EAD) technology on the QTOF system for structural elucidation of conjugation metabolites. In drug metabolite identification, conceptual "boxes" are generally used to represent potential sites of modifications, which are proposed based on MS/MS data. Electron-activated dissociation (EAD) provides unique fragmentation patterns, potentially allowing for more precise localization of the metabolic modification sites compared to CID, particularly for conjugations. METHOD: Known compounds were incubated with rat liver microsomes in the presence of nicotinamide adenine dinucleotide phosphate (NADPH), uridine dihosphate-glucuronic acid (UDPGA), and glutathione. Conjugation metabolites were analyzed using the QTOF system. High-resolution MS/MS spectra were collected using EAD and CID fragmentations along with TOF MS full scan for tested drugs and metabolites. Fragmentation patterns were compared to evaluate their efficiency in structural elucidation. RESULTS: Metabolite profiling identified conjugation metabolites (glucuronides and GSH adducts), using characteristic mass shifts. A comparison of EAD and CID fragmentation revealed EAD-specific fragments for most conjugates. EAD was able to break the relatively stable bonds on parent drug motifs while keeping relatively weak conjugation bonds intact, despite the generally low intensity of EAD. EAD effectively narrowed the conceptual "box" representing modification sites, providing more definitive information on conjugation sites and facilitating the structural elucidation of conjugated metabolites. CONCLUSION: EAD is a powerful tool for metabolite profiling in drug development, particularly for identifying conjugation sites. EAD-enabled MS/MS spectra offer a greater variety of signature fragments compared to CID, resulting in more comprehensive and unique structural information for metabolic modification analysis. Overall, EAD, complementary to CID, has the potential to narrow down potential modification sites, significantly enhancing the precision of conjugation metabolite structure elucidation.

In vitro characterization of protonitazene metabolites, using human liver microsomes, and first application to two urines collected from death cases.

Protonitazene, or N,N-diethyl-5-nitro-2-[(4-propoxyphenyl)methyl]-1H-benzimidazole-1-ethanamine, is a novel synthetic opioid, which belongs to the nitazene family. Over the last four years, nitazenes have re-emerged on the new psychoactive substances market and have been reported in several fatal intoxication cases. The metabolism of several nitazene analogues have already been studied, but to date, no data exists regarding protonitazene. The aim of the study was the detection of protonitazene and its metabolites in authentic human urine collected in two fatal intoxication cases, comparing the data after in vitro incubation with human liver microsomes, and subsequent analysis by ultra-performance liquid chromatography-tandem mass spectrometry and ultra-performance liquid chromatography-high-resolution mass spectrometry. Protonitazene metabolites, including N-desethyl-protonitazene, 5-amino-protonitazene and 4-hydroxy-nitazene, were characterized in vitro and were identified in the urine of both cases. The ratios between metabolites and parent protonitazene, higher than 1, were calculated to estimate the proportionality of metabolites. The results suggest that testing protonitazene metabolites should increase the window detection of exposure to protonitazene.

Bioisosteric replacement strategy leads to novel DNA gyrase B inhibitors with improved potencies and properties.

The identification of novel 4-hydroxy-2-quinolone-3-carboxamide antibacterials with improved properties is of great value for the control of antibiotic resistance. In this study, a series of N-heteroaryl-substituted 4-hydroxy-2-quinolone-3-carboxamides were developed using the bioisosteric replacement strategy. As a result of our research, we discovered the two most potent GyrB inhibitors (WBX7 and WBX18), with IC50 values of 0.816 µM and 0.137 µM, respectively. Additional antibacterial activity screening indicated that WBX18 possesses the best antibacterial activity against MRSA, VISA, and VRE strains, with MIC values rangingbetween0.5and 2 µg/mL, which was 2 to over 32 times more potent than that of vancomycin. In vitro safety and metabolic stability, as well as in vivo pharmacokinetics assessments revealed that WBX18 is non-toxic to HUVEC and HepG2, metabolically stable in plasma and liver microsomes (mouse), and displays favorable in vivo pharmacokinetic properties. Finally, docking studies combined with molecular dynamic simulation showed that WBX18 could stably fit in the active site cavity of GyrB.

Discovery of a Novel Lidocaine Metabolite by Human Liver Microsome and Identification of Microbial Species Which Produces the Same Metabolite.

Preparation of drug metabolites at the milligram scale is essential for determining the structure and toxicity of drug metabolites. However, their preparation using recombinant proteins and human liver microsomes (HLM) is often difficult because of technical and ethical issues. Reproducing human drug metabolism in food-derived microorganisms may be useful for overcoming these challenges. In this study, we identified an unknown metabolite of the anaesthetic drug lidocaine, which is metabolised by HLM. By screening for lidocaine metabolic activity in five types of foods (blue cheese, shiitake mushroom, natto, yoghurt, and dry yeast), we found that bacteria isolated from natto reproduced the lidocaine metabolic reaction that occurs in HLM. A fraction containing the unknown lidocaine metabolite was prepared through mass cultivation of a Bacillus subtilis standard strain, ethyl acetate extraction, open column chromatography, and HPLC purification. We identified the unknown metabolite as 3-(2,6-dimethylphenyl)-1-ethyl-2-methyl-4-imidazolidinone using NMR. Our results showed that food-derived microorganisms can produce large amounts of human drug metabolites via large-scale cultivation. Additionally, food microorganisms that can reproduce drug metabolism in humans can be used to examine drug metabolites at a low cost and without ethical issues.

Nanobioelectrocatalysis Using Human Liver Microsomes and Cytochrome P450 Bactosomes: Pyrenyl-Nanocarbon Electrodes.

Human liver microsomes containing various drug-metabolizing cytochrome P450 (P450) enzymes, along with their NADPH-reductase bound to phospholipid membranes, were absorbed onto 1-pyrene butylamine pi-pi stacked with amine-functionalized multiwalled carbon nanotube-modified graphite electrodes. The interfaced microsomal biofilm demonstrated direct electrochemical communication with the underlying electrode surface and enhanced oxygen reduction electrocatalytic activity typical of heme enzymes such as P450s over the unmodified electrodes and nonenzymatic currents. Similar enhancements in currents were observed when the bioelectrodes were constructed with recombinant P450 2C9 (single isoform) expressed bactosomes. The designed liver microsomal and 2C9 bactosomal bioelectrodes successfully facilitated the electrocatalytic conversion of diclofenac, a drug candidate, into 4'-hydroxydiclofenac. The enzymatic electrocatalytic metabolite yield was several-fold greater on the modified electrodes than on the unmodified bulk graphite electrodes adsorbed with a microsomal or bactosomal film. The nonenzymatic metabolite production was less than the enzymatically catalyzed metabolite yield in the designed microsomal and bactosomal biofilm electrodes. To test the throughput potential of the designed biofilms, eight-electrode array configurations were tested with the microsomal and bactosomal biofilms toward electrochemical 4'-hydroxydiclofenac metabolite production from diclofenac. The stability of the designed microsomal bioelectrode was assessed using nonfaradaic impedance spectroscopy over 40 h, which indicated good stability.

Metabolic pathway-based self-assembled Au@MXene liver microsome electrochemical biosensor for rapid screening of aflatoxin B1.

Cytochrome P450 enzymes (CYPs) catalyze the production of aflatoxin B1 (AFB1) metabolites, which play an important role in carcinogenesis. In this study, we report a simple electrochemical liver-microsome-based biosensor using a composite of gold nanoparticles adsorbed on MXene (Au@MXene) for rapid screening of AFB1. Rat liver microsomes (RLMs) were directly adsorbed on the Au@MXene nanocomposite. The high conductivity, large specific surface area, and good biocompatibility of the Au@MXene nanocomposite enabled the direct electron transfer between the RLMs and the electrode and maintained the biological activity of the enzyme in the RLMs to a large extent. The metabolic behavior of the RLM biosensor that was developed for the electrocatalyst of AFB1 to its hydroxylation metabolite aflatoxin M1 (AFM1) was confirmed. Based on the change in the electrical signal generated by this metabolic behavior, we established the relationship between AFB1 content and amperometric (I-t) current signal. When the AFB1 concentration ranged from 0.01 µM to 50 µM, the AFB1 concentration was linearly related to the electrical signal with a limit of detection of 2.8 nM. The results of the recovery experiments for corn samples showed that the recovery and accuracy of the sensor were consistent with the UPLC-MS/MS method.

Identification of Optimal Urinary Biomarkers of Synthetic Cannabinoids BZO-HEXOXIZID, BZO-POXIZID, 5F-BZO-POXIZID, and BZO-CHMOXIZID for Illicit Abuse Monitoring.

BACKGROUND: The continuous introduction of new synthetic cannabinoid (SC) subtypes and analogues remains a major problem worldwide. Recently, a new "OXIZID" generation of SCs surfaced in seized materials across various countries. Hence, there is an impetus to identify urinary biomarkers of the OXIZIDs to detect their abuse. METHODS: We adapted our previously reported two-pronged approach to investigate the metabolite profiles and disposition kinetics of 4 OXIZID analogues, namely, BZO-HEXOXIZID (MDA-19), BZO-POXIZID (5C-MDA-19), 5F-BZO-POXIZID (5F-MDA-19), and BZO-CHMOXIZID (CHM-MDA-19). First, bottom-up in vitro incubation experiments comprising metabolite identification, metabolic stability, and reaction phenotyping were performed using human liver microsomes and recombinant human cytochrome P450 enzymes. Second, top-down analysis of authentic urine samples from drug abusers was performed to corroborate the in vitro findings and establish a panel of urinary biomarkers. RESULTS: A total of 42 to 51 metabolites were detected for each OXIZID, and their major metabolic pathways included N-alkyl and phenyl hydroxylation, oxidative defluorination (for 5F-BZO-POXIZID), oxidation to ketone and carboxylate, amide hydrolysis, and N-dealkylation. The OXIZIDs were metabolically unstable, mainly metabolized by cytochromes P3A4, P3A5, and P2C9, and demonstrated mechanism-based inactivation of cytochrome P3A4. Integrating with the results of 4 authentic urine samples, the parent drug and both N-alkyl and phenyl mono-hydroxylated metabolites of each OXIZID were determined as suitable urinary biomarkers. CONCLUSIONS: Drug enforcement agencies worldwide may apply these biomarkers in routine monitoring procedures to identify abusers and counter the escalation of OXIZID abuse.

Metabolic and immune-sensitive contacts between lipid droplets and endoplasmic reticulum reconstituted in vitro.

Coordinated cell function requires a variety of subcellular organelles to exchange proteins and lipids across physical contacts that are also referred to as membrane contact sites. Such organelle-to-organelle contacts also evoke interest because they can appear in response to metabolic changes, immune activation, and possibly other stimuli. The microscopic size and complex, crowded geometry of these contacts, however, makes them difficult to visualize, manipulate, and understand inside cells. To address this shortcoming, we deposited endoplasmic reticulum (ER)-enriched microsomes purified from rat liver or from cultured cells on a coverslip in the form of a proteinaceous planar membrane. We visualized real-time lipid and protein exchange across contacts that form between this ER-mimicking membrane and lipid droplets (LDs) purified from the liver of rat. The high-throughput imaging possible in this geometry reveals that in vitro LD-ER contacts increase dramatically when the metabolic state is changed by feeding the animal and also when the immune system is activated. Contact formation in both cases requires Rab18 GTPase and phosphatidic acid, thus revealing common molecular targets operative in two very different biological pathways. An optical trap is used to demonstrate physical tethering of individual LDs to the ER-mimicking membrane and to estimate the strength of this tether. These methodologies can potentially be adapted to understand and target abnormal contact formation between different cellular organelles in the context of neurological and metabolic disorders or pathogen infection.

Comparative chemical and biological hydrolytic stability of homologous esters and isosteres.

Esters are one of the major functional groups present in the structures of prodrugs and bioactive compounds. Their presence is often associated with hydrolytic lability. In this paper, we describe a comparative chemical and biological stability of homologous esters and isosteres in base media as well as in rat plasma and rat liver microsomes. Our results provided evidence for the hydrolytic structure lability relationship and demonstrated that the hydrolytic stability in plasma and liver microsome might depend on carboxylesterase activity. Molecular modelling studies were performed in order to understand the experimental data. Taken together, the data could be useful to design bioactive compounds or prodrugs based on the correct choice of the ester subunit, addressing compounds with higher or lower metabolic lability.

Assessment of the intracellular distribution of copper in liver specimens from cats.

The intracellular distribution of copper in the liver has been investigated in dogs and humans. However, this has not been reported in cats. This study aimed to assess the intracellular copper distribution in liver specimens from cats with a range of hepatic copper concentrations. Twenty-nine frozen liver specimens from cats were included. Each liver specimen was divided into two pieces for overall copper quantification and tissue fractionation. The copper concentrations in liver specimens and liver fractions were measured by flame atomic absorption spectroscopy. Five specimens had copper concentrations < 100 µg/g dry weight, eight had copper concentrations between 100 and 180 µg/g, 14 had copper concentrations between 181 and 700 µg/g, and two had copper concentrations >700 µg/g. Only one specimen had positive copper staining. Regardless of the overall concentrations, copper was mostly found in the cytosolic fraction followed by the nuclear, large granule, and microsomal fractions. Our findings indicate that similarly to other species, intracellular copper is predominantly found in the cytosolic and nuclear fractions in cats. The distribution in cats with copper-loaded conditions, such as primary copper hepatopathy, was not assessed but warrants evaluation.

Discovery of novel paeonol-based derivatives against skin inflammation in vitro and in vivo.

T-LAK-cell-originated protein kinase (TOPK), a novel member of the mitogen-activated protein kinase family, is considered an effective therapeutic target for skin inflammation. In this study, a series (A - D) of paeonol derivatives was designed and synthesised using a fragment growing approach, and their anti-inflammatory activities against lipopolysaccharide (LPS)-induced nitric oxide production in RAW264.7 cells were tested. Among them, compound B12 yielded the best results (IC50 = 2.14 µM) with low toxicity (IC50 > 50 µM). Preliminary mechanistic studies indicated that this compound could inhibit the TOPK-p38/JNK signalling pathway and phosphorylate downstream related proteins. A murine psoriasis-like skin inflammation model was used to determine its therapeutic effect.

Structure-based optimization of hydroxylactam as potent, cell-active inhibitors of lactate dehydrogenase.

Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.

4-Oxooctahydroquinoline-1(2H)-carboxamides as hepatitis B virus (HBV) capsid core protein assembly modulators.

Hepatitis B virus (HBV) core protein, the building block of the HBV capsid, plays multiple roles in viral replication, and is an attractive target for development of antiviral agents with a new mechanism of action. In addition to the heteroaryldihydropyrimidines (HAPs), sulfamoylbenzamides (SBAs), dibenzothiazepine derivatives (DBTs), and sulfamoylpyrrolamides (SPAs) that inhibit HBV replication by modulation of viral capsid assembly and are currently under clinical trials for the treatment of chronic hepatitis B (CHB), other chemical structures with activity to modulate HBV capsid assembly have also been explored. Here we describe our continued optimization of a benzamide originating from our high throughput screening. A new bicyclic carboxamide lead featuring an electron deficient non-planar core structure was discovered. Evaluations of its ADMET (absorption, distribution, metabolism, excretion and toxicity) and pharmacokinetic (PK) profiles demonstrate improved metabolic stability and good bioavailability.

Enhancing monoamine oxidase B inhibitory activity via chiral fluorination: Structure-activity relationship, biological evaluation, and molecular docking study.

Parkinson's disease (PD) is a common neurodegenerative disease among the elderly. Currently, monoamine oxidase B (MAO-B) inhibitors are extensively used for PD in clinics. In this work, a series of novel chiral fluorinated pyrrolidine derivatives were designed and synthesized. In vitro biological evaluations revealed that compound D5 was the most potent, selective MAO-B inhibitor (IC50 = 0.019 µM, MAO-A/MAO-B selectivity index = 2440), which was 10-fold than that of miracle drug safinamide (IC50 = 0.163 µM, MAO-A/MAO-B selectivity index = 172). It was verified that the enhanced hydrophobic interaction of D5 improved the activity against MAO-B in molecular docking study. Besides, D5 exhibited excellent metabolic properties and pharmacokinetic profiles in monkeys and rats. Moreover, D5 displayed more efficacious than safinamide in vivo models. In the MPTP-induced PD mouse model, D5 significantly alleviated DA deficits and increased the effect of levodopa on dopamine concentration in the striatum. Meanwhile, D5 produced a prominent reduction in tremulous jaw movements induced by galantamine. Accordingly, we present D5 as a novel, highly potent, and selective MAO-B inhibitor for PD therapy.