Characterization of antimalarial activity of artemisinin-based hybrid drugs.

In response to the spread of artemisinin (ART) resistance, ART-based hybrid drugs were developed, and their activity profile was characterized against drug-sensitive and drug-resistant Plasmodium falciparum parasites. Two hybrids were found to display parasite growth reduction, stage-specificity, speed of activity, additivity of activity in drug combinations, and stability in hepatic microsomes of similar levels to those displayed by dihydroartemisinin (DHA). Conversely, the rate of chemical homolysis of the peroxide bonds is slower in hybrids than in DHA. From a mechanistic perspective, heme plays a central role in the chemical homolysis of peroxide, inhibiting heme detoxification and disrupting parasite heme redox homeostasis. The hybrid exhibiting slow homolysis of peroxide bonds was more potent in reducing the viability of ART-resistant parasites in a ring-stage survival assay than the hybrid exhibiting fast homolysis. However, both hybrids showed limited activity against ART-induced quiescent parasites in the quiescent-stage survival assay. Our findings are consistent with previous results showing that slow homolysis of peroxide-containing drugs may retain activity against proliferating ART-resistant parasites. However, our data suggest that this property does not overcome the limited activity of peroxides in killing non-proliferating parasites in a quiescent state.

Structural and mechanistic insights into the transport of aristolochic acids and their active metabolites by human serum albumin.

Aristolochic acids I and II (AA-I/II) are carcinogenic principles of Aristolochia plants, which have been employed in traditional medicinal practices and discovered as food contaminants. While the deleterious effects of AAs are broadly acknowledged, there is a dearth of information to define the mechanisms underlying their carcinogenicity. Following bioactivation in the liver, N-hydroxyaristolactam and N-sulfonyloxyaristolactam metabolites are transported via circulation and elicit carcinogenic effects by reacting with cellular DNA. In this study, we apply DNA adduct analysis, X-ray crystallography, isothermal titration calorimetry, and fluorescence quenching to investigate the role of human serum albumin (HSA) in modulating AA carcinogenicity. We find that HSA extends the half-life and reactivity of N-sulfonyloxyaristolactam-I with DNA, thereby protecting activated AAs from heterolysis. Applying novel pooled plasma HSA crystallization methods, we report high-resolution structures of myristic acid-enriched HSA (HSAMYR) and its AA complexes (HSAMYR/AA-I and HSAMYR/AA-II) at 1.9 Å resolution. While AA-I is located within HSA subdomain IB, AA-II occupies subdomains IIA and IB. ITC binding profiles reveal two distinct AA sites in both complexes with association constants of 1.5 and 0.5 · 106 M-1 for HSA/AA-I versus 8.4 and 9.0 · 105 M-1 for HSA/AA-II. Fluorescence quenching of the HSA Trp214 suggests variable impacts of fatty acids on ligand binding affinities. Collectively, our structural and thermodynamic characterizations yield significant insights into AA binding, transport, toxicity, and potential allostery, critical determinants for elucidating the mechanistic roles of HSA in modulating AA carcinogenicity.

Vacuum Plasma Treatment Device for Enhancing Fibroblast Activity on Machined and Rough Titanium Surfaces.

This study was conducted to compare the effects of an innovative plasma surface treatment device that does not need a gas supply for titanium disks with two different surface topographies: the prototypical machined surface (MAC) and one of the most diffused roughened ones (SL) obtained through grit blasting and acid etching. A total of 200-MAC and 200-SL titanium disks were used. Each group of disks was divided into four sub-groups of 40 samples each that were subjected to five different tests. Among these, 150-MAC and 150-SL were considered the test group, and they were treated with plasma for 15, 30, and 60 s after being removed from the sterile packaging. On the other hand, 50-MAC and 50-SL were considered the control group, and they were only removed from sterile plastic vials. The samples were analyzed to evaluate the capability of the plasma treatment in influencing protein adsorption, cell adhesion, proliferation, and microbial growth on the test group disks when compared to the untreated disks. Protein adsorption was significantly enhanced after 20 min of plasma treatment for 15 and 30 s on the MAC and SL disks. Plasma treatment for 15 and 30 s significantly increased the level of adhesion in both treated samples after 30 min. Furthermore, the MAC samples showed a significant increase in cell adhesion 4 h after plasma treatment for 15 s. The SEM analysis highlighted that, on the treated samples (especially on the MAC disks), the cells with a polygonal and flat shape prevailed, while the fusiform- and globular-shaped cells were rare. The encouraging results obtained further confirm the effectiveness of plasma treatments on cell adhesion and fibroblast activity.

Phylogenetic Analysis of Pyruvate-Ferredoxin Oxidoreductase, a Redox Enzyme Involved in the Pharmacological Activation of Nitro-Based Prodrugs in Bacteria and Protozoa.

The present frontrunners in the chemotherapy of infections caused by protozoa are nitro-based prodrugs that are selectively activated by PFOR-mediated redox reactions. This study seeks to analyze the distribution of PFOR in selected protozoa and bacteria by applying comparative genomics to test the hypothesis that PFOR in eukaryotes was acquired through horizontal gene transfer (HGT) from bacteria. Furthermore, to identify other putatively acquired genes, proteome-wide and gene enrichment analyses were used. A plausible explanation for the patchy occurrence of PFOR in protozoa is based on the hypothesis that bacteria are potential sources of genes that enhance the adaptation of protozoa in hostile environments. Comparative genomics of Entamoeba histolytica and the putative gene donor, Desulfovibrio vulgaris, identified eleven candidate genes for HGT involved in intermediary metabolism. If these results can be reproduced in other PFOR-possessing protozoa, it would provide more validated evidence to support the horizontal transfer of pfor from bacteria.

Prediction of cytochrome P450-mediated bioactivation using machine learning models and in vitro validation.

Cytochrome P450 (P450)-mediated bioactivation, which can lead to the hepatotoxicity through the formation of reactive metabolites (RMs), has been regarded as the major problem of drug failures. Herein, we purposed to establish machine learning models to predict the bioactivation of P450. On the basis of the literature-derived bioactivation dataset, models for Benzene ring, Nitrogen heterocycle and Sulfur heterocycle were developed with machine learning methods, i.e., Random Forest, Random Subspace, SVM and Naïve Bayes. The models were assessed by metrics like "Precision", "Recall", "F-Measure", "AUC" (Area Under the Curve), etc. Random Forest algorithms illustrated the best predictability, with nice AUC values of 0.949, 0.973 and 0.958 for the test sets of Benzene ring, Nitrogen heterocycle and Sulfur heterocycle models, respectively. 2D descriptors like topological indices, 2D autocorrelations and Burden eigenvalues, etc. contributed most to the models. Furthermore, the models were applied to predict the occurrence of bioactivation of an external verification set. Drugs like selpercatinib, glafenine, encorafenib, etc. were predicted to undergo bioactivation into toxic RMs. In vitro, IC50 shift experiment was performed to assess the potential of bioactivation to validate the prediction. Encorafenib and tirbanibulin were observed of bioactivation potential with shifts of 3-6 folds or so. Overall, this study provided a reliable and robust strategy to predict the P450-mediated bioactivation, which will be helpful to the assessment of adverse drug reactions (ADRs) in clinic and the design of new candidates with lower toxicities.

Use of Lentivirus-Based Method for Establishing TK6 Human Cell Lines Expressing Cytochrome P450 and its Applications in Genotoxicity Testing.

The human lymphoblastoid cell line TK6 stands out as the most widely employed human cell line in genotoxicity testing, as recommended by various testing guidelines for in vitro assessments. Nevertheless, like many testing cell lines, TK6 lacks functional phase I drug-metabolizing enzymes crucial for chemical genotoxicity evaluations. This protocol introduces a lentivirus-based methodology for establishing a panel of TK6-derived cell lines, each expressing one of 14 cytochrome P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7). The utilization of a lentiviral expression system ensures stable transduction, offering notable advantages such as sustained transgene expression, high transduction efficiency, positive selection feasibility, and user-friendly application. Additionally, we present a detailed procedure for validating the enhanced expression of each CYP in the established cell lines through real-time PCR, western blotting, and mass spectrometry analysis. Lastly, we exemplify the application of these CYP-expressing TK6 cell lines in genotoxicity testing, employing a flow-cytometry-based in vitro micronucleus test. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Lentivirus production and transduction for TK6 cells Support Protocol: Selecting a single clone of CYP-expressing TK6 cells Basic Protocol 2: Validation of CYP expression in TK6 cell lines Basic Protocol 3: Application of transduced cell lines in flow-cytometry-based micronucleus assay.

Renal Glutathione: Dual roles as antioxidant protector and bioactivation promoter.

The tripeptide glutathione (GSH) possesses two key structural features, namely the nucleophilic sulfur and the γ-glutamyl isopeptide bond. The former allows GSH to serve as a critical antioxidant and anti-electrophile. The latter allows GSH to translocate throughout the systemic circulation without being degraded. The kidneys exhibit several unique processes for handling GSH. This includes the extraction of 80% of plasma GSH, in part by glomerular filtration but mostly by transport across the basolateral plasma membrane. Studies on the protective effect of exogenous GSH are summarized, showing the different inherent susceptibility of proximal tubular and distal tubular cells and the impact on pathological or disease states, including hypoxia, diabetic nephropathy, and compensatory renal growth associated with uninephrectomy. Studies on mitochondrial GSH transport show the coordination between the citric acid cycle and oxidative phosphorylation in generating driving forces for both plasma membrane and mitochondrial carriers. The strong protective effects of increasing expression and activity of these carriers against oxidants and mitochondrial toxicants are summarized. Although GSH plays a cytoprotective role in most situations, two distinct exceptions to this are presented. In contrast to expectations, overexpression of the mitochondrial 2-oxoglutarate carrier markedly increased cell death from exposure to the nephrotoxic chemotherapeutic drug cisplatin (CDDP). Another key example of GSH serving a bioactivation role in the kidneys, rather than a detoxification role, is the metabolism of halogenated alkenes such as trichloroethylene (TCE). Although considerable research has gone into this topic, unanswered questions and emerging topics remain and are discussed.

PBTK model-based analysis of CYP3A4 induction and the toxicokinetics of the pyrrolizidine alkaloid retrorsine in man.

Cytochrome P450 (CYP)3A4 induction by drugs and pesticides plays a critical role in the enhancement of pyrrolizidine alkaloid (PA) toxicity as it leads to increased formation of hepatotoxic dehydro-PA metabolites. Addressing the need for a quantitative analysis of this interaction, we developed a physiologically-based toxicokinetic (PBTK) model. Specifically, the model describes the impact of the well-characterized CYP3A4 inducer rifampicin on the kinetics of retrorsine, which is a prototypic PA and contaminant in herbal teas. Based on consumption data, the kinetics after daily intake of retrorsine were simulated with concomitant rifampicin treatment. Strongest impact on retrorsine kinetics (plasma AUC 24 and C max reduced to 67% and 74% compared to the rifampicin-free reference) was predicted directly after withdrawal of rifampicin. At this time point, the competitive inhibitory effect of rifampicin stopped, while CYP3A4 induction was still near its maximum. Due to the impacted metabolism kinetics, the cumulative formation of intestinal retrorsine CYP3A4 metabolites increased to 254% (from 10 to 25 nmol), while the cumulative formation of hepatic CYP3A4 metabolites was not affected (57 nmol). Return to baseline PA toxicokinetics was predicted 14 days after stop of a 14-day rifampicin treatment. In conclusion, the PBTK model showed to be a promising tool to assess the dynamic interplay of enzyme induction and toxification pathways.

Evidence for cytochrome P450 3A4-mediated metabolic activation of SCO-267.

SCO-267 is a potent G-protein-coupled receptor 40 agonist that is undergoing clinical development for the treatment of type 2 diabetes mellitus. The current work was undertaken to investigate the bioactivation potential of SCO-267 in vitro and in vivo. Three SCO-267-derived glutathione (GSH) conjugates (M1-M3) were found both in rat and human liver microsomal incubations supplemented with GSH and nicotinamide adenine dinucleotide phosphate. Two GSH conjugates (M1-M2) together with two N-acetyl-cysteine conjugates (M4-M5) were detected in the bile of rats receiving SCO-267 at 10 mg/kg. The identified conjugates suggested the generation of quinone-imine and ortho-quinone intermediates. CYP3A4 was demonstrated to primarily catalyze the bioactivation of SCO-267. In addition, SCO-267 concentration-, time-, and NADPH-dependently inactivated CYP3A in human liver microsomes using testosterone as a probe substrate, along with KI and kinact values of 4.91 µM and 0.036 min-1 , respectively. Ketoconazole (a competitive inhibitor of CYP3A) displayed no significant protective effect on SCO-267-induced CYP3A inactivation. However, inclusion of GSH showed significant protection. These findings revealed that SCO-267 undergoes a facile CYP3A4-catalyzed bioactivation with the generation of quinone-imine and ortho-quinone intermediates, which were assumed to be involved in SCO-267 induced CYP3A inactivation. These findings provide further insight into the bioactivation pathways involved in the generation of reactive, potentially toxic metabolites of SCO-267. Further studies are needed to evaluate the influence of SCO-267 metabolism on the safety of this drug in vivo.

Species-specific Bioactivation of Morpholines as a Causative of Drug Induced Liver Injury Observed in Monkeys

BACKGROUND: Everolimus, an allosteric mechanistic target of rapamycin (mTOR) inhibitor, recently demonstrated the therapeutic value of mTOR inhibitors for Central Nervous System (CNS) indications driven by hyperactivation of mTOR. A newer, potent brain-penetrant analog of everolimus, referred to as (1) in this manuscript [(S)-3-methyl-4-(7-((R)-3-methylmorpholino)-2-(thiazol-4-yl)-3H-imidazo[4,5-b]pyridin-5-yl)morpholine,(1)] catalytically inhibits mTOR function in the brain and increases the lifespan of mice with neuronal mTOR hyperactivation. INTRODUCTION: Early evaluation of the safety of 1 was conducted in cynomolgus monkeys in which oral doses were administered to three animals in a rising-dose fashion (from 2 to 30 mg/kg/day). 1 produced severe toxicity including the evidence of hepatic toxicity, along with non-dose proportional increases in drug exposure. Investigations of cross-species hepatic bioactivation of 1 were conducted to assess whether the formation of reactive drug metabolites was associated with the mechanism of liver toxicity. METHOD: 1 contained two morpholine rings known as structural alerts and can potentially form reactive intermediates through oxidative metabolism. Bioactivation of 1 was investigated in rat, human and monkey liver microsomes fortified with trapping agents such as methoxylamine or potassium cyanide. RESULTS: Our results suggest that bioactivation of the morpholine moieties to reactive intermediates may have been involved in the mechanism of liver toxicity observed with 1. Aldehyde intermediates trappable by methoxylamine were identified in rat and monkey liver microsomal studies. In addition, a total of four cyano conjugates arising from the formation of iminium ion intermediates were observed and identified. These findings may potentially explain the observed monkey toxicity. Interestingly, methoxylamine or cyano adducts of 1 were not observed in human liver microsomes. CONCLUSION: The bioactivation of 1 appears to be species-specific. Circumstantial evidence for the toxicity derived from 1 point to the formation of iminium ion intermediates trappable by cyanide in monkey liver microsomes. The cyano conjugates were only observed in monkey liver microsomes, potentially pointing to cause at least the hepatotoxicity observed in monkeys. In contrast, methoxylamine conjugates were detected in both rat and monkey liver microsomes, with only a trace amount in human liver microsomes. Cyano conjugates were not observed in human liver microsomes, challenging the team on the drugability and progressivity of 1 through drug development. The mechanisms for drug-induced liver toxicity are multifactorial. These results are highly suggestive that the iminium ion may be an important component in the mechanism of liver toxicity 1 observed in the monkey.

Ion Bombardment-Induced Nanoarchitectonics on Polyetheretherketone Surfaces for Enhanced Nanoporous Bioactive Implants.

In spite of the biocompatible, nontoxic, and radiolucent properties of polyetheretherketone (PEEK), its biologically inert surface compromises its use in dental, orthopedic, and spine fusion industries. Many efforts have been made to improve the biological performance of PEEK implants, from bioactive coatings to composites using titanium alloys or hydroxyapatite and changing the surface properties by chemical and physical methods. Directed plasma nanosynthesis (DPNS) is an atomic-scale nanomanufacturing technique that changes the surface topography and chemistry of solids via low-energy ion bombardment. In this study, PEEK samples were nanopatterned by using argon ion irradiation by DPNS to yield active nanoporous biomaterial surface. PEEK surfaces modified with two doses of low and high fluence, corresponding to 1.0 × 1017 and 1.0 × 1018 ions/cm2, presented pore sizes of 15-25 and 60-90 nm, respectively, leaving exposed PEEK fibers and an increment of roughness of nearly 8 nm. The pores per unit area were closely related for high fluence PEEK and low fluence PEEK surfaces, with 129.11 and 151.72 pore/µm2, respectively. The contact angle significantly decreases in hydrophobicity-hydrophilicity tests for the irradiated PEEK surface to ∼46° from a control PEEK value of ∼74°. These super hydrophilic substrates had 1.6 times lower contact angle compared to the control sample revealing a rough surface of 20.5 nm only at higher fluences when compared to control and low fluences of 12.16 and 14.03 nm, respectively. These super hydrophilic surfaces in both cases reached higher cell viability with ∼13 and 34% increase, respectively, compared to unmodified PEEK, with an increased expression of alkaline phosphatase at 7 days on higher fluences establishing a higher affinity for preosteblasts with increased cellular activity, thus revealing successful and improved integration with the implant material, which can potentially be used in bone tissue engineering.

Current Trend and New Opportunities for Multifunctional Bio-Scaffold Fabrication via High-Pressure Foaming.

Biocompatible and biodegradable foams prepared using the high-pressure foaming technique have been widely investigated in recent decades as porous scaffolds for in vitro and in vivo tissue growth. In fact, the foaming process can operate at low temperatures to load bioactive molecules and cells within the pores of the scaffold, while the density and pore architecture, and, hence, properties of the scaffold, can be finely modulated by the proper selection of materials and processing conditions. Most importantly, the high-pressure foaming of polymers is an ideal choice to limit and/or avoid the use of cytotoxic and tissue-toxic compounds during scaffold preparation. The aim of this review is to provide the reader with the state of the art and current trend in the high-pressure foaming of biomedical polymers and composites towards the design and fabrication of multifunctional scaffolds for tissue engineering. This manuscript describes the application of the gas foaming process for bio-scaffold design and fabrication and highlights some of the most interesting results on: (1) the engineering of porous scaffolds featuring biomimetic porosity to guide cell behavior and to mimic the hierarchical architecture of complex tissues, such as bone; (2) the bioactivation of the scaffolds through the incorporation of inorganic fillers and drugs.

Development and preclinical evaluation of bioactive nerve conduits for peripheral nerve regeneration: A comparative study.

In severe peripheral nerve injuries, nerve conduits (NCs) are good alternatives to autografts/allografts; however, the results the available devices guarantee for are still not fully satisfactory. Herein, differently bioactivated NCs based on the new polymer oxidized polyvinyl alcohol (OxPVA) are compared in a rat model of sciatic nerve neurotmesis (gap: 5 mm; end point: 6 weeks). Thirty Sprague Dawley rats are randomized to 6 groups: Reverse Autograft (RA); Reaxon®; OxPVA; OxPVA + EAK (self-assembling peptide, mechanical incorporation); OxPVA + EAK-YIGSR (mechanical incorporation); OxPVA + Nerve Growth Factor (NGF) (adsorption). Preliminarily, all OxPVA-based devices are comparable with Reaxon® in Sciatic Functional Index score and gait analysis; moreover, all conduits sustain nerve regeneration (S100, ß-tubulin) without showing substantial inflammation (CD3, F4/80) evidences. Following morphometric analyses, OxPVA confirms its potential in PNI repair (comparable with Reaxon®) whereas OxPVA + EAK-YIGSR stands out for its myelinated axons total number and density, revealing promising in injury recovery and for future application in clinical practice.

Utilization of Local Microorganisms as Bioactivators to Produce Organic Fertilizers and Analysis of Molecular Bacterial Diversity.

<b>Background and Objective:</b> Local micro organism (LMO) is the result of the fermentation of various mixtures of organic matter. One of the organic materials used, based on the local wisdom of West Sumatra, is tapai (fermented Cassava), which is used as a bio activator in the manufacture of organic fertilizer. The research aims to produce organic fertilizers that meet national quality standards in terms of the physical and chemical quality of fertilizers as well as to determine the diversity of bacteria in bio activators through next-generation sequencing analysis. <b>Materials and Methods:</b> The organic ingredients for bio activators, cow feces as basic fertilizer ingredients, materials for analyzing bacterial diversity, LMO gDNA was extracted using ZymoBIOMICS DNA Miniprep Kit DNA and sequenced using Oxford Nanopore Technology. <b>Results:</b> On a scale of 1-3, the physical quality of organic fertilizers had an average value of 2.58 for smell, 2.83 for texture and 2.58 for color. The chemical quality of organic fertilizers is C-organic (23.56%), nitrogen (1.60%), carbon and nitrogen ratio (14.75%), phosphate (0.47%) and potassium (0.64%). The results of the analysis of bacteria on the bioactivator consisted of 7 phyla, 9 families, 45 genres and 297 species. The most common species is <i>Lentilactobacillus hilgardii</i> (62%). <b>Conclusion:</b> The organic fertilizer produced using the mole tapai bio activator complies with Indonesian national standard 19-7030-2004 based on physical and chemical parameters. The type of bacteria that dominates the bioactivator is the lactic acid bacteria group, which reaches 90%.

Investigation on the in vitro metabolism of bicyclol using liver microsomes, hepatocytes and human recombinant cytochrome P450 enzymes.

In vitro metabolism of bicyclol was studied using liver microsomes, hepatocytes and human recombinant cytochrome P450 enzymes. Liquid chromatography-benchtop orbitrap mass spectrometry technique was utilised to identify the metabolites.A total of 19 metabolites, including 5 new metabolites (M2, M3, M4, M5 and M16) were tentatively identified. Among these metabolites, M6&M8 (demethylenation), M9&M10 (demethylation) and M19 (glucuronidation) were the major metabolites.In glutathione (GSH)-supplemented liver microsomes, 5 new GSH conjugates were found and tentatively identified. The formation was assumed to be through demethylenation of methylenedioxyphenyl to form catechol derivatives, which further underwent oxidation to form ortho-quinone intermediates, reacting with GSH to form stable adducts.CYP3A4 and 2C19 were demonstrated to be the major enzymes responsible for the bioactivation of bicyclol.This study provided valuable information on the metabolic fate of bicyclol in liver microsomes and hepatocytes, and the bioactivation pathways were reported for the first time, which would be helpful for us to understand the potential drug-drug interactions and the possible side effect of this drug.

Xanthine Dehydrogenase Is a Modulator of Dopaminergic Neurodegeneration in Response to Bacterial Metabolite Exposure in C. elegans.

Oxidative stress is a contributing factor to Parkinson's disease (PD). Considering the prevalence of sporadic PD, environmental exposures are postulated to increase reactive oxygen species and either incite or exacerbate neurodegeneration. We previously determined that exposure to the common soil bacterium, Streptomyces venezuelae (S. ven), enhanced oxidative stress and mitochondrial dysfunction in Caenorhabditis elegans, leading to dopaminergic (DA) neurodegeneration. Here, S. ven metabolite exposure in C. elegans was followed by RNA-Seq analysis. Half of the differentially identified genes (DEGs) were associated with the transcription factor DAF-16 (FOXO), which is a key node in regulating stress response. Our DEGs were enriched for Phase I (CYP) and Phase II (UGT) detoxification genes and non-CYP Phase I enzymes associated with oxidative metabolism, including the downregulated xanthine dehydrogenase gene, xdh-1. The XDH-1 enzyme exhibits reversible interconversion to xanthine oxidase (XO) in response to calcium. S. ven metabolite exposure enhanced XO activity in C. elegans. The chelation of calcium diminishes the conversion of XDH-1 to XO and results in neuroprotection from S. ven exposure, whereas CaCl2 supplementation enhanced neurodegeneration. These results suggest a defense mechanism that delimits the pool of XDH-1 available for interconversion to XO, and associated ROS production, in response to metabolite exposure.

Enzymatic activation in vitamin D signaling - Past, present and future.

Vitamin D signaling is important in regulating calcium homeostasis essential for bone health but also displays other functions in cells of several tissues. Disturbed vitamin D signaling is linked to a large number of diseases. The multiple cytochrome P450 (CYP) enzymes catalyzing the different hydroxylations in bioactivation of vitamin D3 are crucial for vitamin D signaling and function. This review is focused on the progress achieved in identification of the bioactivating enzymes and their genes in production of 1α,25-dihydroxyvitamin D3 and other active metabolites. Results obtained on species- and tissue-specific expression, catalytic reactions, substrate specificity, enzyme kinetics, and consequences of gene mutations are evaluated. Matters of incomplete understanding regarding the physiological roles of some vitamin D hydroxylases are critically discussed and the authors will give their view of the importance of each enzyme for vitamin D signaling. Roles of different vitamin D receptors and an alternative bioactivation pathway, leading to 20-hydroxylated vitamin D3 metabolites, are also discussed. Considerable progress has been achieved in knowledge of the vitamin D3 bioactivating enzymes. Nevertheless, several intriguing areas deserve further attention to understand the pleiotropic and diverse activities elicited by vitamin D signaling and the mechanisms of enzymatic activation necessary for vitamin D-induced responses.

Preclinical evaluation of chemically reactive metabolites and mitigation of bioactivation in drug discovery.

The formation of reactive metabolites (RMs) is thought to be one of the pathogeneses for some idiosyncratic adverse drug reactions (IADRs) which are considered one of the leading causes of some drug attritions and/or recalls. Minimizing or eliminating the formation of RMs via chemical modification is a useful tactic to reduce the risk of IADRs and time-dependent inhibition (TDI) of cytochrome P450 enzymes (CYPs). The RMs should be carefully handled before making a go-no-go decision. Herein, we highlight the role of RMs in the occurrence of IADRs and CYP TDI, the risk of structural alerts, the approaches of RM assessment at the discovery stage and strategies to minimize or eliminate RM liability. Finally, some considerations for handling a RM-positive drug candidate are suggested.