A challenging yet critical reaction in chemical synthesis is the selective activation of unactivated carbon-hydrogen bonds in complex molecules. Here we demonstrate the biocatalytic regio- and stereoselective hydroxylation of steroids, including progesterone, testosterone, adrenosterone, androstenedione, and dehydroepiandrosterone (DHEA), using a variant of an engineered bacterial cytochrome P450 enzyme, CYP154C35 from Nocardia otitidiscaviarum. We replaced two amino acid residues within the oxygen-binding groove of the I-helix, and this modification of the acid-alcohol pair to a glutamine and a glutamate set of residues had the effect of converting the enzyme into an efficient peroxygenase (CYP154C35QE). This enzyme performed the biocatalytic oxidation of the steroids stereoselectively to form the 16α-hydroxylated metabolites. In the presence of a substrate, CYP154C35QE was stable to relatively high concentrations of H2O2 but could function catalytically using lower concentrations. The enzyme tolerated 20% DMSO and was not denatured upon being preheated for 1 h at 40 °C. The enzyme was capable of high turnover numbers (∼6500), resulting in complete conversion of the substrate, enabling the formation, purification, and characterization of the enantiopure 16α-hydroxylated steroids at up to 120 mg scale.

Metabolomic analysis provides critical insights into predator-prey interactions, yet the specific metabolites mediating predation strategies and defensive responses across distinct interaction stages remain poorly characterized. Using LC-MS/MS metabolomics, we investigated dynamic metabolic changes in a model intertidal predator-prey system-dogwhelks (Reishia clavigera) and mussels (Mytilus galloprovincialis)-across four predation stages: isolated in separate water (IS), same water without contact (SW), first touch (FT), and touch sustained over 3 h (T3h). Metabolomics results indicated that stage-specific metabolic reprogramming was observed. During the SW vs. IS comparison, dogwhelks upregulated steroid hormone biosynthesis and cysteine/methionine metabolism, producing differentially expressed metabolites (DEMs) such as Endomorphin-1. Conversely, mussels activated arachidonic acid metabolism and purine metabolism, triggering inflammatory responses and impaired stress regulation. At SW proximity without contact, dogwhelks mobilized energy and synthesized predatory toxins, while mussels exhibited metabolic signatures of inflammation and dysregulated self-control. Following physical contact (FT stage), dogwhelks elevated 5-Hydroxy-L-tryptophan (a serotonin precursor), ADP, and Trichloroacetic acid (a shell-dissolving agent), whereas mussels upregulated tryptophan metabolism, alongside stress-alleviating metabolites like Oxybuprocaine and Metamizole. After sustained predation (T3h), dogwhelks prioritized purine metabolism to meet their energy demands, while mussels activated tryptophan metabolism to produce adrenosterone (enhancing muscle mass) and metamizole (an analgesic), suggesting adaptive strategies to mitigate predation trauma. Besides, we conducted experiments on immersed angiotensin and found that it could influence the predation of dogwhelks. These results reveal divergent physiological strategies between predators and preys: dogwhelks optimize energy metabolism and toxin production to sustain predation, while mussels deploy stress-related metabolites, muscle-enhancing compounds, and pain-suppressing agents to resist capture. This study advances our understanding of predator-prey dynamics by linking metabolomic shifts to behavioral and physiological adaptations across interaction stages.

This study characterized cytochrome P450 enzyme CYP154C7 from Streptomyces sp. PAMC26508, emphasizing its capability to hydroxylate steroids, especially at the 16α-position. The enzymatic assay of CYP154C7 demonstrated effective conversion across a pH range of 7.2-7.6, with optimal activity at 30 °C in the Pdx/PdR plus NADH system. Kinetic analysis on most converted steroids (androstenedione and adrenosterone) was performed which shows a greater affinity for androstenedione (K m , 11.06 ± 1.903 μM; V max, 0.0062 ± 0.0002 sec-1) compared to adrenosterone (K m , 34.50 ± 6.2 μM; V max, 0.0119 ± 0.0007 sec-1). A whole-cell system in Escherichia coli, overexpressing recombinant CYP154C7, achieved substantial conversion for steroids, indicating that CYP154C7 can also be used as a potential whole-cell biocatalyst. To gain structural insights, homology models of CYP154C7 and its homologs were constructed using CYP154C5 (PDB ID: 6TO2), refined, validated, and used for docking studies. Comparative docking analysis suggests that lysine (K236) in the active site and tyrosine (Y197) in the substrate access channel of CYP154C7 are crucial for substrate selectivity and catalytic efficiency. This study suggests that CYP154C7 could be a promising candidate for developing modified steroids, providing valuable insights for protein engineering to design commercially useful CYP steroid hydroxylases with diverse substrate specificities.

Adrenosterone (Androst-4-ene-3,11,17-trione, 11OXO) is forbidden in sports according to the Prohibited List of the World Anti-Doping Agency. The administration of 11OXO may be detected by monitoring the urinary concentrations of its main human metabolites 11β-hydroxy-androsterone and 11β-hydroxy-etiocholanolone. Preliminary urinary concentration and concentration ratio thresholds have been established for sports drug testing purposes, but adaptations are desirable as the suggested limits would result in numerous suspicious findings due to naturally elevated concentrations and ratios. Recently, the metabolism of 11-oxo-testosterone (KT) was investigated in the context of anti-doping research, resulting in a preliminary urinary concentration threshold and a confirmation procedure based on the determination of carbon isotope ratios (CIRs). Gas chromatography coupled to isotope ratio mass spectrometry was employed to investigate the CIRs of selected steroids. As KT is also a metabolite of 11OXO, the developed protocols for KT have been tested to elucidate their potential to detect the administration of 11OXO after a single oral dose of 100 mg. In order to further improve the analytical approach, the threshold for urinary concentrations of KT was re-investigated by employing a reference population of n = 5232 routine doping control samples. Quantification of urinary steroids was conducted by employing gas chromatography coupled to triple quadrupole mass spectrometry. Derived from these, a subset of n = 106 samples showing elevated concentrations of KT was investigated regarding their CIRs. By means of this, potentially positive samples due to the illicit administration of 11OXO or KT could be excluded, and the calculation of reference population-derived thresholds for the concentrations and CIR of KT was possible. Based on the results, the urinary concentration threshold for KT is suggested to be established at 130 ng/mL.

Hibernating Spermophilus dauricus experiences minor muscle atrophy, which is an attractive anti-disuse muscle atrophy model. Integrated metabolomics and proteomics analysis was performed on the hibernating S. dauricus during the pre-hibernation (PRE) stage, torpor (TOR) stage, interbout arousal (IBA) stage, and post-hibernation (POST) stage. Time course stage transition-based (TOR vs. PRE, IBA vs. TOR, POST vs. IBA) differential expression analysis was performed based on the R limma package. A total of 14 co-differential metabolites were detected. Among these, l-cystathionine, l-proline, ketoleucine, serine, and 1-Hydroxy-3,6,7-Trimethoxy-2, 8-Diprenylxanthone demonstrated the highest levels in the TOR stage; Beta-Nicotinamide adenine dinucleotide, Dihydrozeatin, Pannaric acid, and Propionylcarnitine demonstrated the highest levels in the IBA stage; Adrenosterone, PS (18:0/14,15-EpETE), S-Carboxymethylcysteine, TxB2, and 3-Phenoxybenzylalcohol demonstrated the highest levels in the POST stage. Kyoto Encyclopedia of Genes and Genomes pathways annotation analysis indicated that biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism were co-differential metabolism pathways during the different stages of hibernation. The stage-specific metabolism processes and integrated enzyme-centered metabolism networks in the different stages were also deciphered. Overall, our findings suggest that (1) the periodic change of proline, ketoleucine, and serine contributes to the hindlimb lean tissue preservation; and (2) key metabolites related to the biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism may be associated with muscle atrophy resistance. In conclusion, our co-differential metabolites, co-differential metabolism pathways, stage-specific metabolism pathways, and integrated enzyme-centered metabolism networks are informative for biologists to generate hypotheses for functional analyses to perturb disuse-induced muscle atrophy.

Here, we present an in vivo drug screening protocol using a zebrafish model of metastasis for the identification of anti-metastatic drugs. A tamoxifen-controllable Twist1a-ER[T2] transgenic zebrafish line was established to serve as a platform for the identification. By crossing Twist1a-ER[T2] with xmrk (a homolog of hyperactive form of the epidermal growth factor receptor) transgenic zebrafish, which develop hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish show spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen and tail regions in five days, through induction of epithelial to mesenchymal transition (EMT). This rapid and high-frequency induction of cell dissemination makes it possible to perform an in vivo drug screen for the identification of anti-metastatic drugs targeting metastatic dissemination of cancer cells. The protocol evaluates the suppressor effect of a test drug on metastasis in five days, by comparing the frequencies of the fish showing abdominal and distant dissemination patterns in the test drug-treated group with those in the vehicle-treated group. Our study previously identified that adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1), has a suppressor effect on cell dissemination in the model. Furthermore, we validated that a pharmacologic and genetic inhibition of HSD11β1 suppressed metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model. Taken together, this protocol opens new routes for the identification of anti-metastatic drugs. Graphical overview Timing Day 0: Zebrafish spawning Day 8: Primary tumor induction Day 11: Chemical treatment Day 11.5: Metastatic dissemination induction in the presence of a test chemical Day 16: Data analysis.

The anabolic properties of 11-hydroxyandrostenedione (OHA4) and its physiologically active metabolites 11-ketotestosterone (KT) and 11-ketodihydrotestosterone (KDHT) have been discussed in several recent publications. Especially KT has become readily available via internet-based providers. No doping control methods for the detection of KT or KDHT exist, neither on the initial testing procedure level nor as confirmatory assay. Probing for the misuse of adrenosterone, the prohormone of OHA4, has already been addressed, and the suggested marker for its misuse was mainly the urinary concentration of 11-hydroxyandrosterone (OHA). In addition, for confirmation purposes, the carbon isotope ratios (CIR) were taken into consideration. The urinary concentration of OHA is highly variable, and the endogenous dilution after exogenous administration may therefore be considerable; hence, described approaches resulted in short detection times. In this study, the human metabolism of KT was investigated in order to provide additional means for the detection of KT and its prohormone OHA4. Two volunteers (one female and one male) orally administered 20 mg of KT each, and urine samples were collected for 5 days. Urinary concentrations of KT and its metabolites were investigated, and a reference population encompassing 220 male and female athletes was investigated in order to elucidate preliminary thresholds. As confirmation procedure, an isotope ratio mass spectrometry-based method was developed in order to determine the CIR of KT and relevant metabolites. The developed methods enabled the detection of exogenous KT for more than 20 h after a single oral administration, which is comparable to a single oral testosterone administration.

CYP105D18 supports H2O2 as an oxygen surrogate for catalysis well and shows high H2O2 resistance capacity. We report the hydroxylation of different steroids using H2O2 as a cosubstrate. Testosterone was regiospecifically hydroxylated to 2β-hydroxytestosterone. Based on the experimental data and molecular docking, we predicted that hydroxylation of methyl testosterone and nandrolone would occur at position 2 in the A-ring, while hydroxylation of androstenedione and adrenosterone was predicted to occur in the B-ring. Further, structure-guided rational design of the substrate access channel was performed with the mutagenesis of residues S63, R82, and F184. Among the mutants, S63A showed a marked decrease in product formation, while F184A showed a significant increase in product formation in testosterone, nandrolone, methyl testosterone, androstenedione, and adrenosterone. The catalytic efficiency (kcat/Km) toward testosterone was increased 1.36-fold in the F184A mutant over that in the wild-type enzyme. These findings might facilitate the potential use of CYP105D18 and further engineering to establish the basis of biotechnological applications. IMPORTANCE The structural modification of steroids is a challenging chemical reaction. Modifying the core ring and the side chain improves the biological activity of steroids. In particular, bacterial cytochrome P450s are used as promiscuous enzymes for the activation of nonreactive carbons of steroids. In the present work, we reported the H2O2-mediated hydroxylation of steroids by CYP105D18, which also overcomes the use of expensive cofactors. Further, exploring the substrate access channel and modifying the bulky amino acid F184A increase substrate conversion while modifying the substrate recognizing amino acid S63 markedly decreases product formation. Exploring the substrate access channel and the rational design of CYP105D18 can improve the substrate conversion, which facilitates the engineering of P450s for industrial application.

Few models exist that allow for rapid and effective screening of anti-metastasis drugs. Here, we present a drug screening protocol utilizing gastrulation of zebrafish embryos for identification of anti-metastasis drugs. Based on the evidence that metastasis proceeds through utilizing the molecular mechanisms of gastrulation, we hypothesized that chemicals interrupting zebrafish gastrulation might suppress the metastasis of cancer cells. Thus, we developed a phenotype-based chemical screen that uses epiboly, the first morphogenetic movement in gastrulation, as a marker. The screen only needs zebrafish embryos and enables hundreds of chemicals to be tested in five hours by observing the epiboly progression of chemical-treated embryos. In the screen, embryos at the two-cell stage are firstly corrected and then developed to the sphere stage. The embryos are treated with a test chemical and incubated in the presence of the chemical until vehicle-treated embryos develop to the 90% epiboly stage. Finally, positive 'hit' chemicals that interrupt epiboly progression are selected by comparing epiboly progression of the chemical-treated and vehicle-treated embryos under a stereoscopic microscope. A previous study subjected 1,280 FDA-approved drugs to the screen and identified adrenosterone and pizotifen as epiboly-interrupting drugs. These were validated to suppress metastasis of breast cancer cells in mice models of metastasis. Furthermore, 11β-hydroxysteroid dehydrogenase 1 (HSD11β1) and serotonin receptor 2C (HTR2C), the primary targets of adrenosterone and pizotifen, respectively, promoted metastasis through induction of epithelial-mesenchymal transition (EMT). Therefore, this screen could be converted into a chemical genetic screening platform for identification of metastasis-promoting genes. Graphical abstract.

The histological process of gonadal differentiation, together with the endocrine changes of sex steroid hormones and some of their precursors, was studied in hatchery-produced greater amberjack Seriola dumerili from 101 until 408 days post-hatching (dph), with samplings conducted every 50 days. Histological processing showed that sex differentiation began at 101 dph with the formation of the ovarian cavity in females, while the presumptive males did not yet contain any germ cells in their gonad. At 150 dph, we observed the first germ cells in the developing testes. Sex differentiation in almost all sampled individuals was complete at 408 dph. No size dimorphism was observed between the sexes, and the sex ratio was 1:1, suggesting that there was no influence of early rearing in captivity on sex differentiation. Plasma concentrations of adrenosterone (Ad), androstenedione (Δ4), 11-ketotestosterone (11ΚΤ), testosterone (Τ), estradiol (Ε2), progesterone (P4) and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) were measured in males and females with the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) to examine their role in the sex differentiation process. From the seven hormones, the only one that exhibited differences between the sexes was 11-KT and the plasma 11-KT concentration was found to be a useful indication of greater amberjack sex. Variations were observed in the mean values of Ad, Δ4, 11-KT, T, P4 and 17,20βP over time in one or both sexes, indicating their involvement in the sex differentiation process.

Here we reveal a simple generation of deuterium halide (DX) from common and inexpensive reagents readily available in a synthetic chemistry laboratory, i.e. prenyl-, allyl-, and propargyl halides, under mild conditions. We envisaged that in situ generation of an acid, deuterium halide, would be useful for acid-catalyzed reactions and could be employed for organocatalytic deuteration. The present work reports a metal-free method for deuterium labeling covering a broad range of substrate including phenolic compounds (i.e. flavonoids and stilbenes), indoles, pyrroles, carbonyl compounds, and steroids. This method was also applied for commonly used drugs such as loxoprofen, haloperidol, stanolone, progesterone, androstenedione, donepezil, ketorolac, adrenosterone, cortisone, pregnenolone, and dexamethasone. A gram-scale chromatography-free synthesis of some deuterated compounds is demonstrated in this work. This work provides a simple, clean and by-product-free, site-selective deuteration, and the deuterated products are obtained without chromatographic separation. When applying these initiators for other acid-catalyzed reactions, the deuterium isotope effects of DX may provide products which are different from those obtained from reactions using common acids. Although the mechanism of the spontaneous transformation of prenyl halides to acid is unclear, this overlooked chemistry may be useful for many reactions.