CYP3A4 and CYP2C19 genetic polymorphisms and myricetin interaction on tofacitinib metabolism.

Tofacitinib can effectively improve the clinical symptoms of rheumatoid arthritis (RA) patients. In this current study, a recombinant human CYP2C19 and CYP3A4 system was operated to study the effects of recombinant variants on tofacitinib metabolism. Moreover, the interaction between tofacitinib and myricetin was analyzed in vitro. The levels of M9 (the main metabolite of tofacitinib) was detected by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The findings revealed that 11 variants showed significant changes in the levels of M9 compared to CYP3A4.1, while the other variants didn't reveal any remarkable significances. Compared with CYP2C19.1, 11 variants showed increases in the levels of M9, and 10 variants showed decreases. Additionally, it was demonstrated in vitro that the inhibition of tofacitinib by myricetin was a non-competitive type in rat liver microsomes (RLM) and human liver microsomes (HLM). However, the inhibitory mechanism was a competitive type in CYP3A4.18, and mixed type in CYP3A4.1 and .28, respectively. The data demonstrated that gene polymorphisms and myricetin had significant effects on the metabolism of tofacitinib, contributing to important clinical data for the precise use.

Computational Predictive and Electrochemical Detection of Metabolites (CP-EDM) of Piperine.

In this article, we introduce a proof-of-concept strategy, Computational Predictive and Electrochemical Detection of Metabolites (CP-EDM), to expedite the discovery of drug metabolites. The use of a bioactive natural product, piperine, that has a well-curated metabolite profile but an unpredictable computational metabolism (Biotransformer v3.0) was selected. We developed an electrochemical reaction to oxidize piperine into a range of metabolites, which were detected by LC-MS. A series of chemically plausible metabolites were predicted based on ion fragmentation patterns. These metabolites were docked into the active site of CYP3A4 using Autodock4.2. From the clustered low-energy profile of piperine in the active site, it can be inferred that the most likely metabolic position of piperine (based on intermolecular distances to the Fe-oxo active site) is the benzo[d][1,3]dioxole motif. The metabolic profile was confirmed by comparison with the literature, and the electrochemical reaction delivered plausible metabolites, vide infra, thus, demonstrating the power of the hyphenated technique of tandem electrochemical detection and computational evaluation of binding poses. Taken together, we outline a novel approach where diverse data sources are combined to predict and confirm a metabolic outcome for a bioactive structure.

Functional and structural dissection of glycosyltransferases underlying the glycodiversity of wolfberry-derived bioactive ingredients lycibarbarspermidines.

Lycibarbarspermidines are unusual phenolamide glycosides characterized by a dicaffeoylspermidine core with multiple glycosyl substitutions, and serve as a major class of bioactive ingredients in the wolfberry. So far, little is known about the enzymatic basis of the glycosylation of phenolamides including dicaffeoylspermidine. Here, we identify five lycibarbarspermidine glycosyltransferases, LbUGT1-5, which are the first phenolamide-type glycosyltransferases and catalyze regioselective glycosylation of dicaffeoylspermidines to form structurally diverse lycibarbarspermidines in wolfberry. Notably, LbUGT3 acts as a distinctive enzyme that catalyzes a tandem sugar transfer to the ortho-dihydroxy group on the caffeoyl moiety to form the unusual ortho-diglucosylated product, while LbUGT1 accurately discriminates caffeoyl and dihydrocaffeoyl groups to catalyze a site-selective sugar transfer. Crystal structure analysis of the complexes of LbUGT1 and LbUGT3 with UDP, combined with molecular dynamics simulations, revealed the structural basis of the difference in glycosylation selectivity between LbUGT1 and LbUGT3. Site-directed mutagenesis illuminates a conserved tyrosine residue (Y389 in LbUGT1 and Y390 in LbUGT3) in PSPG box that plays a crucial role in regulating the regioselectivity of LbUGT1 and LbUGT3. Our study thus sheds light on the enzymatic underpinnings of the chemical diversity of lycibarbarspermidines in wolfberry, and expands the repertoire of glycosyltransferases in nature.

Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist.

Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

PET Quantification of [18F]VAT in Human Brain and Its Test-Retest Reproducibility and Age Dependence.

Molecular imaging of brain vesicular acetylcholine transporter provides a biomarker to explore cholinergic systems in humans. We aimed to characterize the distribution of, and optimize methods to quantify, the vesicular acetylcholine transporter-specific tracer (-)-(1-(8-(2-[18F]fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-piperidin-4-yl)(4-fluorophenyl)methanone ([18F]VAT) in the brain using PET. Methods: Fifty-two healthy participants aged 21-97 y had brain PET with [18F]VAT. [3H]VAT autoradiography identified brain areas devoid of specific binding in cortical white matter. PET image-based white matter reference region size, model start time, and duration were optimized for calculations of Logan nondisplaceable binding potential (BPND). Ten participants had 2 scans to determine test-retest variability. Finally, we analyzed age-dependent differences in participants. Results: [18F]VAT was widely distributed in the brain, with high striatal, thalamic, amygdala, hippocampal, cerebellar vermis, and regionally specific uptake in the cerebral cortex. [3H]VAT autoradiography-specific binding and PET [18F]VAT uptake were low in white matter. [18F]VAT SUVs in the white matter reference region correlated with age, requiring stringent erosion parameters. Logan BPND estimates stabilized using at least 40 min of data starting 25 min after injection. Test-retest variability had excellent reproducibility and reliability in repeat BPND calculations for 10 participants (putamen, 6.8%; r > 0.93). We observed age-dependent decreases in the caudate and putamen (multiple comparisons corrected) and in numerous cortical regions. Finally, we provide power tables to indicate potential mean differences that can be detected between 2 groups of participants. Conclusion: These results validate a reference region for BPND calculations and demonstrate the viability, reproducibility, and utility of using the [18F]VAT tracer in humans to quantify cholinergic pathways.

Pharmacological profile, phase I metabolism, and excretion time profile of the new synthetic cathinone 3,4-Pr-PipVP.

1-(2,3-Dihydro-1H-inden-5-yl)-2-(piperidin-1-yl)pentan-1-one (3,4-Pr-PipVP), a novel synthetic cathinone (SCat), was first identified in 2022 in Germany. The product was marketed as 1-(bicyclo[4.2.0]octa-1,3,5-trien-3-yl)-2-(pyrrolidin-1-yl)pentan-1-one (3,4-EtPV), a substance not covered by the German New Psychoactive Substances Act (NpSG). Although originally intended to be an exploratory new synthetic cathinone containing the novel bicyclo[4.2.0]octatrienyl function, the compound was subsequently confirmed to contain an indanyl ring system scheduled under generic legislation like the NpSG. However, it is one of only a few marketed SCats carrying a piperidine ring. Inhibition experiments involving norepinephrine, dopamine, and serotonin transporters showed that 3,4-Pr-PipVP was a low potency blocker at all three monoamine transporters compared to related substances such as MDPV. Additionally, pharmacokinetic data were collected from pooled human liver microsomes incubations and from the analysis of authentic urine samples received after oral administration of 5 mg 3,4-Pr-PipVP hydrochloride. Phase I metabolites were tentatively identified in vitro and in vivo using liquid chromatography-time-of-flight mass spectrometry. Main metabolites were formed by metabolic reduction of the carbonyl function with and without additional hydroxylations at the propylene bridge of the molecule. Keto-reduced H2 -3,4-Pr-PipVP and H2 -piperidine-OH-3,4-Pr-PipVP as well as aryl-OH-3,4-Pr-PipVP, and indanyl-OH-piperidine-OH-3,4-Pr-PipVP are suggested as most suitable biomarkers for the detection of 3,4-Pr-PipVP since they were detected for much longer than the parent compound. 3,4-Pr-PipVP could be detected for up to 21 h whereas its metabolites were detectable for up to about 4 days.

pH-Responsive Trihydroxylated Piperidines Rescue The Glucocerebrosidase Activity in Human Fibroblasts Bearing The Neuronopathic Gaucher-Related L444P/L444P Mutations in GBA1 Gene.

Engineering bioactive iminosugars with pH-responsive groups is an emerging approach to develop pharmacological chaperones (PCs) able to improve lysosomal trafficking and enzymatic activity rescue of mutated enzymes. The use of inexpensive l-malic acid allowed introduction of orthoester units into the lipophilic chain of an enantiomerically pure iminosugar affording only two diastereoisomers contrary to previous related studies. The iminosugar was prepared stereoselectively from the chiral pool (d-mannose) and chosen as the lead bioactive compound, to develop novel candidates for restoring the lysosomal enzyme glucocerebrosidase (GCase) activity. The stability of orthoester-appended iminosugars was studied by 1 H NMR spectroscopy both in neutral and acidic environments, and the loss of inhibitory activity with time in acid medium was demonstrated on cell lysates. Moreover, the ability to rescue GCase activity in the lysosomes as the result of a chaperoning effect was explored. A remarkable pharmacological chaperone activity was measured in fibroblasts hosting the homozygous L444P/L444P mutation, a cell line resistant to most PCs, besides the more commonly responding N370S mutation.

Mitigating a Bioactivation Liability with an Azetidine-Based Inhibitor of Diacylglycerol Acyltransferase 2 (DGAT2) En Route to the Discovery of the Clinical Candidate Ervogastat.

We recently disclosed SAR studies on systemically acting, amide-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) that addressed metabolic liabilities with the liver-targeted DGAT2 inhibitor PF-06427878. Despite strategic placement of a nitrogen atom in the dialkoxyaromatic ring in PF-06427878 to evade oxidative O-dearylation, metabolic intrinsic clearance remained high due to extensive piperidine ring oxidation as exemplified with compound 1. Piperidine ring modifications through alternate N-linked heterocyclic ring/spacer combination led to azetidine 2 that demonstrated lower intrinsic clearance. However, 2 underwent a facile cytochrome P450 (CYP)-mediated α-carbon oxidation followed by azetidine ring scission, resulting in the formation of ketone (M2) and aldehyde (M6) as stable metabolites in NADPH-supplemented human liver microsomes. Inclusion of GSH or semicarbazide in microsomal incubations led to the formation of Cys-Gly-thiazolidine (M3), Cys-thiazolidine (M5), and semicarbazone (M7) conjugates, which were derived from reaction of the nucleophilic trapping agents with aldehyde M6. Metabolites M2 and M5 were biosynthesized from NADPH- and l-cysteine-fortified human liver microsomal incubations with 2, and proposed metabolite structures were verified using one- and two-dimensional NMR spectroscopy. Replacement of the azetidine substituent with a pyridine ring furnished 8, which mitigated the formation of the electrophilic aldehyde metabolite, and was a more potent DGAT2 inhibitor than 2. Further structural refinements in 8, specifically introducing amide bond substituents with greater metabolic stability, led to the discovery of PF-06865571 (ervogastat) that is currently in phase 2 clinical trials for the treatment of nonalcoholic steatohepatitis.

Transcriptome Analysis Reveals an Essential Role of Exogenous Brassinolide on the Alkaloid Biosynthesis Pathway in Pinellia Ternata.

Pinellia ternata (Thunb.) Druce is a traditional medicinal plant containing a variety of alkaloids, which are important active ingredients. Brassinolide (BR) is a plant hormone that regulates plant response to environmental stress and promotes the accumulation of secondary metabolites in plants. However, the regulatory mechanism of BR-induced alkaloid accumulation in P. ternata is not clear. In this study, we investigated the effects of BR and BR biosynthesis inhibitor (propiconazole, Pcz) treatments on alkaloid biosynthesis in the bulbil of P. ternata. The results showed that total alkaloid content and bulbil yield was enhanced by 90.87% and 29.67% under BR treatment, respectively, compared to the control. We identified 818 (476 up-regulated and 342 down-regulated) and 697 (389 up-regulated and 308 down-regulated) DEGs in the BR-treated and Pcz-treated groups, respectively. Through this annotated data and the Kyoto encyclopedia of genes and genomes (KEGG), the expression patterns of unigenes involved in the ephedrine alkaloid, tropane, piperidine, pyridine alkaloid, indole alkaloid, and isoquinoline alkaloid biosynthesis were observed under BR and Pcz treatments. We identified 11, 8, 2, and 13 unigenes in the ephedrine alkaloid, tropane, piperidine, and pyridine alkaloid, indole alkaloid, and isoquinoline alkaloid biosynthesis, respectively. The expression levels of these unigenes were increased by BR treatment and were decreased by Pcz treatment, compared to the control. The results provided molecular insight into the study of the molecular mechanism of BR-promoted alkaloid biosynthesis.

Design and synthesis of aryl-piperidine derivatives as potent and selective PET tracers for cholesterol 24-hydroxylase (CH24H).

Cholesterol 24-hydroxylase (CH24H, CYP46A1) is a cytochrome P450 family enzyme that maintains the homeostasis of brain cholesterol. Soticlestat, a potent and selective CH24H inhibitor, is in development as a therapeutic agent for Dravet syndrome and Lennox-Gastaut syndrome. Herein, we report the discovery of aryl-piperidine derivatives as potent and selective CH24H positron emission tomography (PET) tracers which can be used for dose guidance of a clinical CH24H inhibitor and as a diagnostic tool for CH24H-related pathology. Starting from compound 1 (IC50 = 16 nM, logD = 1.7), which was reported as a CH24H inhibitor with lower lipophilicity, a18F-labeling site (3-fluoroazetidine) was incorporated by structure-based drug design (SBDD) utilizing the co-crystal structure of a compound 1 analog. Subsequent optimization to adjust key parameters for PET tracers, such as potency, lipophilicity, brain penetration, and unbound plasma protein binding, enabled compounds 3f (IC50 = 8.8 nM) and 3g (IC50 = 8.7 nM) as PET imaging candidates. Selectivity of these compounds for CH24H was validated by a brain distribution study using CH24H-WT and KO mice. In non-human primate PET imaging, [18F]3f and [18F]3g showed similar regional uptake in the brain, indicating that these tracers were specific to the CH24H-expressed regions and validated the expression of CH24H in the living brain by different tracers.

Prosopis juliflora piperidine alkaloid extract levels in diet for sheep change energy and nitrogen metabolism and affect enteric methane yield.

BACKGROUND: Ionophore antibiotics improve the efficiency of energy metabolism, which has driven their use as a feed additive in ruminants for decades. Currently, they have not been approved in many countries, generating a challenge for the immediate search for plant extracts with a similar mode of action on rumen metabolism. This study evaluated the effects of enriched Prosopis juliflora (mesquite) piperidine alkaloid extract (MPA) levels as an alternative phytoadditive to sodium monensin (MON) in sheep. RESULTS: The MPA diet did not differ from MON with regard to nutrient intake. A quadratic effect (P < 0.05) was observed for organic matter and neutral detergent fibre digestibility, with respective maximum point at 25.40 and minimum point at 0.95 mg kg-1 MPA. The MPA levels linearly decreased (P < 0.05) faecal nitrogen loss. MPA did not differ from MON with regard to nutrient digestibility, and MPA levels increased (P < 0.05) the proportion of digestible energy and metabolizability from dietary gross energy. The MPA levels linearly decreased (P < 0.05) enteric CH4 production, the yield showing lower (P < 0.05) energy loss as CH4 than MON. CONCLUSION: The results show that MPA levels of 17.3 and 27.8 mg kg-1 are enteric CH4 inhibitors and enhance energy and protein utilization, indicating a promising alternative to MON for ruminants. © 2022 Society of Chemical Industry.

Synthesis and evaluation of tofacitinib analogs designed to mitigate metabolic activation.

Tofacitinib (TFT), a JAK inhibitor used for the treatment of rheumatoid arthritis and other diseases, is associated with severe liver injury that is believed to be caused by its reactive aldehyde or epoxide metabolites. In this study, we synthesized six tofacitinib analogs designed to avoid the formation of reactive metabolites and evaluated their JAK3 inhibitory activity, metabolic stability, CYP3A time-dependent inhibition, and cytotoxicity. Our data indicated that purine analog 3, which showed little inhibition of CYP3A and cytotoxicity and inhibited JAK3 in the nanomolar range, could be a safer drug candidate than TFT. In addition, the results of the bioactivation study using TFT and its analogs suggest that the epoxide metabolite might contribute to TFT-induced CYP3A4 mechanism-based inhibition and hepatic toxicity.

Review of the development of BTK inhibitors in overcoming the clinical limitations of ibrutinib.

Bruton's tyrosine kinase (BTK) regulates multiple important signaling pathways and plays a key role in the proliferation, survival, and differentiation of B-lineage cells and myeloid cells. BTK is a promising target for the treatment of hematologic malignancies. Ibrutinib, the first-generation BTK inhibitor, was approved to treat several B-cell malignancies. Despite the remarkable potency and efficacy of ibrutinib against various lymphomas and leukemias in the clinics, there are also some clinical limitations, such as off-target toxicities and primary/acquired drug resistance. As strategies to overcome these challenges, second- and third-generation BTK inhibitors, BTK-PROTACs, as well as combination therapies have been explored. In this review, we summarize clinical developments of the first-, second- and third-generation BTK inhibitors, as well as recent advances in BTK-PROTACs and ibrutinib-based combination therapies.

Site-Specific Labeling of Endogenous Proteins Using CoLDR Chemistry.

Chemical modifications of native proteins can affect their stability, activity, interactions, localization, and more. However, there are few nongenetic methods for the installation of chemical modifications at a specific protein site in cells. Here we report a covalent ligand directed release (CoLDR) site-specific labeling strategy, which enables the installation of a variety of functional tags on a target protein while releasing the directing ligand. Using this approach, we were able to label various proteins such as BTK, K-RasG12C, and SARS-CoV-2 PLpro with different tags. For BTK we have shown selective labeling in cells of both alkyne and fluorophores tags. Protein labeling by traditional affinity methods often inhibits protein activity since the directing ligand permanently occupies the target binding pocket. We have shown that using CoLDR chemistry, modification of BTK by these probes in cells preserves its activity. We demonstrated several applications for this approach including determining the half-life of BTK in its native environment with minimal perturbation, as well as quantification of BTK degradation by a noncovalent proteolysis targeting chimera (PROTAC) by in-gel fluorescence. Using an environment-sensitive "turn-on" fluorescent probe, we were able to monitor ligand binding to the active site of BTK. Finally, we have demonstrated efficient CoLDR-based BTK PROTACs (DC50 < 100 nM), which installed a CRBN binder onto BTK. This approach joins very few available labeling strategies that maintain the target protein activity and thus makes an important addition to the toolbox of chemical biology.

Securinine induces mitotic block in cancer cells by binding to tubulin and inhibiting microtubule assembly: A possible mechanistic basis for its anticancer activity.

AIM: Analysis of the anticancer and antimitotic activity of the plant derived alkaloid securinine along with its effect on the organization of cellular microtubules as well as its binding with purified goat brain tubulin in-vitro. MATERIALS AND METHODS: The cytotoxicity of securinine on different cell lines was conducted using SRB assay. The effect of securinine on the cellular microtubules was analyzed using immunofluorescence microscopy. The binding of securinine on purified goat brain tubulin was evaluated using fluorescent spectroscopy. KEY FINDINGS: Securinine effectively prevented the proliferation of cervical, breast and lung cancer cells with an IC50 of 6, 10 and 11 µM respectively and induced minimal toxicity in HEK cell line. Securinine at concentrations higher than IC50 induced significant depolymerization in interphase and mitotic microtubules and it suppressed the reassembly of cold depolymerized spindle microtubules in HeLa cells. In the wound healing assay, securinine effectively suppressed the migration of HeLa cells to close the wound. Securinine bound to tubulin with a Kd of 9.7 µM and inhibited the assembly of tubulin into microtubules. The treatment with securinine induced a mitochondrial dependent ROS response in HeLa cells which enhanced the cytotoxic effect of securinine. The result from gene expression studies indicates that securinine induced apoptosis in MCF-7 cells through p53 dependent pathway. SIGNIFICANCE: Considering the strong anticancer and anti-metastatic property and low toxicity in non-malignant cell lines, we suggest that securinine can be used as a chemotherapeutic drug either alone or in combination with other known anticancer molecules.

Functional evaluation of vandetanib metabolism by CYP3A4 variants and potential drug interactions in vitro.

AIM: To investigate the enzymatic properties of cytochrome P450 3A4 (CYP3A4) variants and their ability to metabolize vandetanib (VNT) in vitro, and to study potential drug interactions in combination with VNT. METHOD: Recombinant CYP3A4 cell microsomes were prepared using a Bac-to-Bac baculovirus expression system. Enzymatic reactions were carried out, and the metabolites were determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: The activities of 27 CYP3A4 variants were determined to assess the degree of VNT metabolism that occurred. Analysis indicated that there was enhanced intrinsic clearance (Vmax/Km, CLint) for eight variants (CYP3A4.2, 3, 9, 15, 16, 29, 32, and 33), while there was a significant decrease in CYP3A4.5, 7, 8, 10-14, 17-20, 23, 24, 28, 31, and 34. Compared with CYP3A4.1, no significant differences were found for CYP3A4.6 and 30. Furthermore, the relative clearances were compared between VNT and cabozantinib, which were all metabolized by CYP3A4 with the same indications. When combined with ketoconazole, which is a CYP inhibitor, obvious differences were observed in the potency of VNT between different variants, including CYP3A4.2, 15, and 18. CONCLUSION: This comprehensive assessment of CYP3A4 variants provides significant insights into the allele-specific metabolism of VNT and drug interactions in vitro. We hope that these comprehensive data will provide references and predictions for the clinical application of VNT.

Neuroprotective effects of minocycline and KML29, a potent inhibitor of monoacylglycerol lipase, in an experimental stroke model: a small-animal positron emission tomography study.

Hypoxia caused by ischemia induces acidosis and neuroexcitotoxicity, resulting in neuronal death in the central nervous system (CNS). Monoacylglycerol lipase (MAGL) is a modulator of 2-arachidonoylglycerol (2-AG), which is involved in retrograde inhibition of glutamate release in the endocannabinoid system. In the present study, we used positron emission tomography (PET) to monitor MAGL-positive neurons and neuroinflammation in the brains of ischemic rats. Additionally, we performed PET imaging to evaluate the neuroprotective effects of an MAGL inhibitor in an ischemic injury model. Methods: Ischemic-injury rat models were induced by intraluminal right middle cerebral artery occlusion (MCAO). PET studies of the brains of the ischemic rats were performed at several experimental time points (pre-occlusion, days 2, 4, and 7 after the MCAO surgery) using [11C]SAR127303 for MAGL and [18F]FEBMP for 18 kDa translocator protein (TSPO, a hall-mark of neuroinflammation). Medication using minocycline (a well-known neuroprotective agent) or KML29 (a potent MAGL inhibitor) was given immediately after the MCAO surgery and then daily over the subsequent three days. Results: PET imaging of the ischemic rats using [11C]SAR127303 showed an acute decline of radioactive accumulation in the ipsilateral side at two days after MCAO surgery (ratio of the area under the curve between the ipsilateral and contralateral sides: 0.49 ± 0.04 in the cortex and 0.73 ± 0.02 in the striatum). PET imaging with [18F]FEBMP, however, showed a moderate increase in accumulation of radioactivity in the ipsilateral hemisphere on day 2 (1.36 ± 0.11), and further increases on day 4 (1.72 ± 0.15) and day 7 (1.99 ± 0.06). Treatment with minocycline or KML29 eased the decline in radioactive accumulation of [11C]SAR127303 for MAGL (minocycline-treated group: 0.82 ± 0.06 in the cortex and 0.81 ± 0.05 in the striatum; KML29-treated group: 0.72 ± 0.07 in the cortex and 0.88 ± 0.04 in the striatum) and increased uptake of [18F]FEBMP for TSPO (minocycline-treated group: 1.52 ± 0.21 in the cortex and 1.56 ± 0.11 in the striatum; KML29-treated group: 1.63 ± 0.09 in the cortex and 1.50 ± 0.17 in the striatum). In MCAO rats, minocycline treatment showed a neuroprotective effect in the sensorimotor cortex suffering from severe hypoxic injury, whereas KML29 treatment saved neurons in the striatum, including bundles of myelinated axons. Conclusions: PET imaging allowed visualization of the different neuroprotective effects of minocycline and KML29, and indicated that combination pharmacotherapy using these drugs may be an effective therapy in acute ischemia.

1,3-di-4-piperidylpropane derivatives as potential acetyl cholinesterase antagonists: Molecular docking, synthesis, and biological evaluation.

Acetylcholine esterase (AChE) is a key biological target responsible for the management of cholinergic transmission, and its inhibitors are used for the therapy of Alzheimer's disease. In the present study, a small library of molecules with 1,3-di-4-piperidylpropane nucleus were docked on AChE. The selected compounds were synthesized and evaluated for their enzyme inhibition. P25 and P17 expressed significantly higher AChE inhibition than standards with IC50 values of 0.591µM and 0.625µM, respectively. Binding mode of derivatives in the active site of AChE revealed dual binding of molecules in peripheral anionic site (PAS) and catalytic anionic site (CAS) of enzyme cavity.

Pharmacological and metabolic characterization of the novel synthetic opioid brorphine and its detection in routine casework.

After their first emergence in 2009, Novel synthetic opioids (NSO) have become an emerging class of New Psychoactive Substances (NPS) on the market for these new drugs. So far, 67 NSO have been reported to the Early Warning system of the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). It is presumed that NSO mainly target the four known opioid receptors, i.e. the µ-opioid (MOR), the δ-opioid (DOR), the κ-opioid (KOR) and nociceptin receptors and that their consumption can result in serious adverse effects such as massive respiratory depression or death. In the present study we investigated the in vivo and in vitro metabolism of brorphine, a NSO that was first identified on the NPS market in August 2019 in the United States, using both a pooled human liver microsome assay and real forensic case samples. For the detection of metabolites LC-HR-MS/MS was used and quantification of brorphine was performed using an LC-MS/MS method. Additionally, we pharmacologically characterized brorphine regarding its activation of the MOR and KOR via G protein recruitment using the [35S]-GTPγS assay. In forensic urine samples, 14 distinct metabolites were identified, whereas in blood only four metabolites could be found. The pooled human liver microsome assay generated six distinct in vitro phase I metabolites. The most prominent in vivo metabolite was formed by N-oxydation, whereas the main in vitro metabolite was formed by hydroxylation. The pharmacological characterization at the MOR and KOR revealed brorphine to be a potent MOR agonist and a weak, partial KOR agonist in the [35S]-GTPγS assay.

Discovery of Soticlestat, a Potent and Selective Inhibitor for Cholesterol 24-Hydroxylase (CH24H).

Cholesterol 24-hydroxylase (CH24H, CYP46A1), a brain-specific cytochrome P450 (CYP) family enzyme, plays a role in the homeostasis of brain cholesterol by converting cholesterol to 24S-hydroxycholesterol (24HC). Despite a wide range of potential of CH24H as a drug target, no potent and selective inhibitors have been identified. Here, we report on the structure-based drug design (SBDD) of novel 4-arylpyridine derivatives based on the X-ray co-crystal structure of hit derivative 1b. Optimization of 4-arylpyridine derivatives led us to identify 3v ((4-benzyl-4-hydroxypiperidin-1-yl)(2,4'-bipyridin-3-yl)methanone, IC50 = 7.4 nM) as a highly potent, selective, and brain-penetrant CH24H inhibitor. Following oral administration to mice, 3v resulted in a dose-dependent reduction of 24HC levels in the brain (1, 3, and 10 mg/kg). Compound 3v (soticlestat, also known as TAK-935) is currently under clinical investigation for the treatment of Dravet syndrome and Lennox-Gastaut syndrome as a novel drug class for epilepsies.