Hepatocyte Uptake and Loss Assay (HUpLA): Proof-of-Concept of an All-in-One System for Measuring Hepatic Influx, Egress, and Metabolic Clearance Based on the Extended Clearance Concept.

Hepatic clearance (CLH) prediction is a critical parameter to estimate human dose. However, CLH underpredictions are common, especially for slowly metabolized drugs, and may be attributable to drug properties that pose challenges for conventional in vitro ADME assays, resulting in non-valid data, which prevents in-vitro-to-in-vivo extrapolation and CLH predictions. Other processes, including hepatocyte and biliary distribution via transporters, can also play significant roles in CLH Recent advances in understanding the interplay of metabolism and drug transport for clearance processes have aided in developing the Extended Clearance Model (ECM). In this study, we demonstrate proof-of-concept of a novel two-step assay enabling measurement of multiple kinetic parameters from a single experiment in plated human primary hepatocytes with and without transporter and CYP inhibitors - the Hepatocyte Uptake and Loss Assay (HUpLA). HUpLA accurately predicted the CLH of 8 of the 9 drugs (within 2-fold of the observed CLH). Distribution clearances were within 3-fold of observed literature values in standard uptake and efflux assays. In comparison, the conventional suspension hepatocyte stability assay poorly predicted the CLH CLH of only 2 drugs were predicted within 2-fold of the observed CLH Therefore, HUpLA is advantageous by enabling the measurement of enzymatic and transport processes concurrently within the same system, alleviating the need for applying scaling factors independently. The use of primary human hepatocytes enables physiologically relevant exploration of transporter-enzyme interplay. Most importantly, HUpLA shows promise as a sensitive measure for low-turnover drugs. Further evaluation across different drug characteristics is needed to demonstrate method robustness. Significance Statement HUpLA involves measuring four commonly derived in vitro hepatic clearance endpoints. Since endpoints are generated within a single test system, it blunts experimental error originating from assays otherwise conducted independently. A key advance is the concept of removing drug-containing media following intracellular drug loading, enabling measurement of drug reappearance rate in media, as well as measurement of loss of total drug in the test system unencumbered by background quantities of drug in media otherwise present in a conventional assay.

Assumptions Underlying Hepatic Clearance Models: Recognizing the Influence of Saturable Protein Binding on Driving Force Concentration and Discrimination Between Models of Hepatic Clearance.

One underlying assumption of hepatic clearance models is often underappreciated. Namely, plasma protein binding is assumed to be nonsaturable within a given drug concentration range, dependent only on protein concentration and equilibrium dissociation constant. However, in vitro hepatic clearance experiments often use low albumin concentrations that may be prone to saturation effects, especially for high-clearance compounds, where the drug concentration changes rapidly. Diazepam isolated perfused rat liver literature datasets collected at varying concentrations of albumin were used to evaluate the predictive utility of four hepatic clearance models (the well-stirred, parallel tube, dispersion, and modified well-stirred model) while both ignoring and accounting for potential impact of saturable protein binding on hepatic clearance model discrimination. In agreement with previous literature findings, analyses without accounting for saturable binding showed poor clearance prediction using all four hepatic clearance models. Here we show that accounting for saturable albumin binding improves clearance predictions across the four hepatic clearance models. Additionally, the well-stirred model best reconciles the difference between the predicted and observed clearance data, suggesting that the well-stirred model is an appropriate model to describe diazepam hepatic clearance when considering appropriate binding models. SIGNIFICANCE STATEMENT: Hepatic clearance models are vital for understanding clearance. Caveats in model discrimination and plasma protein binding have sparked an ongoing scientific discussion. This study expands the understanding of the underappreciated potential for saturable plasma protein binding. Fraction unbound must correspond to relevant driving force concentration. These considerations can improve clearance predictions and address hepatic clearance model disconnects. Importantly, even though hepatic clearance models are simple approximations of complex physiological processes, they are valuable tools for clinical clearance predictions.

Precursor-directed biosynthesis of micacocidin derivatives with activity against Mycoplasma pneumoniae.

Micacocidin is a promising natural product for the treatment of Mycoplasma pneumoniae infections. In the biosynthesis of this antibiotic, a fatty acid-AMP ligase (FAAL) activates the starter unit hexanoic acid as acyl-adenylate and forwards it to an iteratively acting polyketide synthase. Biochemical analysis of the FAAL revealed an extended substrate tolerance, thereby opening the door for the modification of a micacocidin residue that is barely accessible via semisynthesis. A total of six new analogues were generated by precursor-directed biosynthesis in this study and profiled against M. pneumoniae.

Ralfuranone Is Produced by an Alternative Aryl-Substituted γ-Lactone Biosynthetic Route in Ralstonia solanacearum.

The aryl-substituted γ-lactones ralfuranones A and B were isolated after feeding L-[1-(13)C]-phenylalanine to a liquid culture of the plant pathogenic bacterium Ralstonia solanacearum. (13)C NMR analysis demonstrated specific enrichment of the label at position 2 of the γ-lactone. This labeling pattern is consistent with a biosynthetic mechanism that includes direct cyclization of two monomeric phenylpyruvate precursors into an α,ß-substituted lactone, but incompatible with a terphenylquinone intermediate. As the latter was shown as an intermediate in allantofuranone biosynthesis, we conclude that aryl-substituted γ-lactones can be assembled via divergent biosynthetic routes.

Optimization, Characterization, and Comparison of Two Luciferase-Expressing Mouse Glioblastoma Models.

Glioblastoma (GBM) is the most aggressive brain cancer. To model GBM in research, orthotopic brain tumor models, including syngeneic models like GL261 and genetically engineered mouse models like TRP, are used. In longitudinal studies, tumor growth and the treatment response are typically tracked with in vivo imaging, including bioluminescence imaging (BLI), which is quick, cost-effective, and easily quantifiable. However, BLI requires luciferase-tagged cells, and recent studies indicate that the luciferase gene can elicit an immune response, leading to tumor rejection and experimental variation. We sought to optimize the engraftment of two luciferase-expressing GBM models, GL261 Red-FLuc and TRP-mCherry-FLuc, showing differences in tumor take, with GL261 Red-FLuc cells requiring immunocompromised mice for 100% engraftment. Immunohistochemistry and MRI revealed distinct tumor characteristics: GL261 Red-FLuc tumors were well-demarcated with densely packed cells, high mitotic activity, and vascularization. In contrast, TRP-mCherry-FLuc tumors were large, invasive, and necrotic, with perivascular invasion. Quantifying the tumor volume using the HALO® AI analysis platform yielded results comparable to manual measurements, providing a standardized and efficient approach for the reliable, high-throughput analysis of luciferase-expressing tumors. Our study highlights the importance of considering tumor engraftment when using luciferase-expressing GBM models, providing insights for preclinical research design.

Ralfuranone thioether production by the plant pathogen Ralstonia solanacearum.

Ralfuranones are aryl-substituted furanone secondary metabolites of the Gram-negative plant pathogen Ralstonia solanacearum. New sulfur-containing ralfuranone derivatives were identified, including the methyl thioether-containing ralfuranone D. Isotopic labeling in vivo, as well as headspace analyses of volatiles from R. solanacearum liquid cultures, established a mechanism for the transfer of an intact methylthio group from L-methionine or α-keto-γ-methylthiobutyric acid. The methylthio acceptor molecule ralfuranone I, a previously postulated biosynthetic intermediate in ralfuranone biosynthesis, was isolated and characterized by NMR. The highly reactive Michael acceptor system of this intermediate readily reacts with various thiols, including glutathione.

Regulation of autotrophic CO2 fixation in the archaeon Thermoproteus neutrophilus.

Thermoproteus neutrophilus, a hyperthermophilic, chemolithoautotrophic, anaerobic crenarchaeon, uses a novel autotrophic CO(2) fixation pathway, the dicarboxylate/hydroxybutyrate cycle. The regulation of the central carbon metabolism was studied on the level of whole cells, enzyme activity, the proteome, transcription, and gene organization. The organism proved to be a facultative autotroph, which prefers organic acids as carbon sources that can easily feed into the metabolite pools of this cycle. Addition of the preferred carbon sources acetate, pyruvate, succinate, and 4-hydroxybutyrate to cultures resulted in stimulation of the growth rate and a diauxic growth response. The characteristic enzyme activities of the carbon fixation cycle, fumarate hydratase, fumarate reductase, succinyl coenzyme A (CoA) synthetase, and enzymes catalyzing the conversion of succinyl-CoA to crotonyl-CoA, were differentially downregulated in the presence of acetate and, to a lesser extent, in the presence of other organic substrates. This regulation pattern correlated well with the differential expression profile of the proteome as well as with the transcription of the encoding genes. The genes encoding phosphoenolpyruvate (PEP) carboxylase, fumarate reductase, and four enzymes catalyzing the conversion of succinyl-CoA to crotonyl-CoA are clustered. Two putative operons, one comprising succinyl-CoA reductase plus 4-hydroxybutyrate-CoA ligase genes and the other comprising 4-hydroxybutyryl-CoA dehydratase plus fumarate reductase genes, were divergently transcribed into leaderless mRNAs. The promoter regions were characterized and used for isolating DNA binding proteins. Besides an Alba protein, a 18-kDa protein characteristic for autotrophic Thermoproteales that bound specifically to the promoter region was identified. This system may be suitable for molecular analysis of the transcriptional regulation of autotrophy-related genes.

Ralfuranone biosynthesis in Ralstonia solanacearum suggests functional divergence in the quinone synthetase family of enzymes.

Ralstonia solanacearum is a destructive crop plant pathogen and produces ralfuranone, i.e., a monophenyl-substituted furanone. Extensive feeding experiments with (13)C-labeled L-phenylalanine now proved that all carbon atoms of the heterocycle derive, after deamination, from this aromatic amino acid. A genetic locus was identified which encodes the aminotransferase RalD and the furanone synthetase RalA. The latter is a tridomain nonribosomal peptide synthetase (NRPS)-like enzyme which was characterized (1) biochemically by the ATP-pyrophosphate exchange assay, and (2) genetically through gene inactivation and transcriptional analysis in axenic culture and in planta. This is the first study to our knowledge on the biochemical and genetic basis of R. solanacearum secondary metabolism. It implies new chemistry for NRPSs, as RalA-mediated biosynthesis requires C-C-bond and subsequent C-O-bond formation to establish the furanone ring system.