Characterization of Prejunctional Muscarinic Receptors: Effects on the Release of VIP and Functional Responses and Receptor Expression in the Ovine Submandibular Gland

In the in vivo experiments on anaesthetized sheep, it was presently examined whether muscarinic receptor antagonists with diverse selectivity affect the release of VIP in response to electrical stimulation of the parasympathetic chorda tympanic nerve differently, and if the changes in the release could be associated to altered secretory and vasodilator responses. The location of the muscarinic receptor subtypes was examined also. In the experiments, blood was collected out of the submandibular venous drainage before and during electrical stimulation of chorda tympani nerve in the absence and presence either of pirenzepine or methoctramine. While metchoctramine increased the output of protein, pirenzepine inhibited flow of saliva and increased protein output, vasodilatation, and VIP output. In morphological examinations, the inhibitory muscarinic M4 receptor occurred interacinarily in the gland. It is concluded that prejunctional muscarinic receptors, most likely of the M4 subtype, exert inhibitory modulation of the parasympathetic release of VIP in the ovine submandibular gland

Amyloid Beta Hypothesis: Attention to β- and γ-Secretase Modulators

The amyloid cascade hypothesis poses one possible explanation for the onset and progression of Alzheimer’s disease (AD). With this respect, neurotoxic effect is attributed to soluble and diffusive amyloid-β (Aβ) oligomers. Aβ peptides are produced by proteolytic cleavage of the hydrophobic transmembrane portion of the amyloid precursor protein (APP) by successive action of β- and γ-secretases. Aβ peptides are generated in several isoforms, out of which the most pronounced are Aβ40 and Aβ42 being the major constituents of amyloid plaques found in AD patients’ brains. Since the indisputable evidence pointed out to Aβ oligomers as toxic agents, several pathways to modulate or control the aggregation have been inspected. Given all these aspects, inhibitors of the β- and γ-secretases have gained the most attention. This chapter presents amyloid cascade hypothesis with current progress in the development of β- and γ-secretase modulators to counteract the Aβ burden

6-benzothiazolyl ureas, thioureas and guanidines are potent inhibitors of ABAD/17β-HSD10 and potential drugs for Alzheimer's disease treatment : design, synthesis and in vitro evaluation

Background : The mitochondrial enzyme amyloid beta-binding alcohol dehydrogenase (ABAD) also known as 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) has been connected with the pathogenesis of Alzheimer’s disease (AD). ABAD/ 17β-HSD10 is a binding site for the amyloid-beta peptide (Aβ) inside the mitochondrial matrix where it exacerbates Aβ toxicity. Interaction between these two proteins triggers a series of events leading to mitochondrial dysfunction as seen in AD. Methods : As ABAD’s enzymatic activity is required for mediating Aβ toxicity, its inhibition presents a promising strategy for AD treatment. In this study, a series of new benzothiazolylurea analogues have been prepared and evaluated in vitro for their potency to inhibit ABAD/ 17β-HSD10 enzymatic activity. The most potent compounds have also been tested for their cytotoxic properties and their ability to permeate through blood-brain barrier has been predicted. To explain the structure-activity relationship QSAR and pharmacophore studies have been performed. Results and Conclusions : Compound 12 was identified being the most promising hit compound with good inhibitory activity (IC50 = 3.06 ± 0.40µM) and acceptable cytotoxicity profile comparable to the parent compound of frentizole. The satisfactory physical-chemical properties suggesting its capability to permeate through BBB make compound 12 a novel lead structure for further development and biological assessment.PostprintPeer reviewe

Design, synthesis and in vitro evaluation of benzothiazole-based ureas as potential ABAD/17β-HSD10 modulators for Alzheimer’s disease treatment

This work was supported by the Ministry of Health of the Czech Republic (no. NV15-28967A), Charles University in Prague (no. GAUK B-CH/992214, SVV 260 291) and the Alzheimer’s Society (specifically The Barcopel Foundation). This research is part-funded by the MSD Scottish Life Sciences fund.Amyloid-beta peptide (Aβ) has been recognized to interact with numerous proteins, which may lead to pathological changes in cell metabolism of Alzheimer’s disease (AD) patients. One such known metabolic enzyme is mitochondrial amyloid-binding alcohol dehydrogenase (ABAD), also known as 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10). Altered enzyme function caused by the Aβ-ABAD interaction, was previously shown to cause mitochondrial distress and a consequent cytotoxic effect, therefore providing a feasible target in AD drug development. Based on previous frentizole derivatives studies, we report two novel series of benzothiazolyl ureas along with novel insights into the structure and activity relationships for inhibition of ABAD. Two compounds ( 37 , 39 ) were identified as potent ABAD inhibitors, where compound 39 exhibited comparable cytotoxicity with the frentizole standard; however, one-fold higher cytotoxicity than the parent riluzole standard. The calculated and experimental physical chemical properties of the most potent compounds showed promising features for blood-brain barrier penetration.PostprintPeer reviewe

THE DIFFERENCE IN THE MUSHROOMS YIELD (Agaricus bisporus Imbach) GROWN USING TWO DIFFERENT CACing METHODS

Cilj pokusa bio je ustanoviti postoji li razlika u prinosu šampinjona, ako se u proizvodnji koriste dvije CACing metode. U pokusu su korištene metoda sa „spawnom“ i metoda s proraslim kompostom. Rezultati pokusa pokazali su da nije bilo statistički značajne razlike između navedenih metoda. Stoga se može zaključiti da su obadvije metode dobre. Problem bi se mogao javiti kod umiješanja proraslog komposta u pokrivku zbog moguće zaraze patogenima, dok s druge strane „spawn“ materijal je skuplja opcija. Stoga bi možda ipak bolja bila opcija umiješanja „spawna“ s obzirom na sigurnost po pitanju patogena.The aim of the experiment was to determine the mushrooms yield difference using two CACing methods. In the experiment“spawn” and overgrowing compost methods were used. The test results showed that there were no statistically significant differences between these methods. It can be concluded that both methods are good. The problem could occur in blending overgrowing compost because of possible contamination with pathogens, while on the other hand "spawn" material is a more expensive option. Therefore, it might still be a better option blending "spawn" to protect from pathogens

Synthesis and evaluation of frentizole-based indolyl thiourea analogues as MAO/ABAD inhibitors for Alzheimer’s disease treatment

This work was supported by the Ministry of Health of the Czech Republic (no. NV15-28967A), the Charles University in Prague (SVV 260 291), COST Action CM1103 (STSM 15879 and 17487) and CA15135, University of Hradec Kralove (Faculty of Informatics and Management, project Excellence 2015), University of St Andrews (undergraduate project funding to D.Z.), Biotechnology and Biological Sciences Research Council (BBSRC; no. BB/J01446X/1), the Alzheimer’s Society and the Barcopel Foundation.Alzheimer’s disease (AD) is a neurodegenerative disorder associated with an excessive accumulation of amyloid-beta peptide (Aβ). Based on the multifactorial nature of AD, preparation of multi-target-directed ligands presents a viable option to address more pathological events at one time. A novel class of asymmetrical disubstituted indolyl thioureas have been designed and synthesized to interact with monoamine oxidase (MAO) and/or amyloid-binding alcohol dehydrogenase (ABAD). The design combines the features of known MAO inhibitors scaffolds (e.g. rasagiline or ladostigil) and a frentizole moiety with potential to interact with ABAD. Evaluation against MAO identified several compounds that inhibited in the low to moderate micromolar range. The most promising compound ( 19 ) inhibited human MAO-A and MAOB with IC50 values of 6.34 μM and 0.30 μM, respectively. ABAD activity evaluation did not show any highly potent compound, but the compound series allowed identification of structural features to assist the future development of ABAD inhibitors. Finally, several of the compounds were found to be potent inhibitors of horseradish peroxidase (HRP), preventing the use of the Amplex™ Red assay to detect hydrogen peroxide produced by MAO, highlighting the need for serious precautions when using an enzyme-coupled assay.PostprintPeer reviewe

Teriflunomide Is an Indirect Human Constitutive Androstane Receptor (CAR) Activator Interacting With Epidermal Growth Factor (EGF) Signaling

The constitutive androstane receptor (CAR) is a nuclear receptor involved mainly in xenobiotic and endobiotic metabolism regulation. CAR is activated directly by its ligands via the ligand binding domain (LBD) or indirectly by inhibition of the epidermal growth factor (EGF) signaling. We found that leflunomide (LEF) and its main metabolite teriflunomide (TER), both used for autoimmune diseases treatment, induce the prototype CAR target gene CYP2B6 in primary human hepatocytes. As TER was discovered to be an EGF receptor antagonist, we sought to determine if TER is an indirect activator of CAR. In primary human hepatocytes and in differentiated HepaRG cells, we found that LEF and TER up-regulate CAR target genes CYP2B6 and CYP3A4 mRNAs and enzymatic activities. TER stimulated CAR+A mutant translocation into the nucleus but neither LEF nor TER activated the CAR LBD, CAR3 variant or pregnane X receptor (PXR) in gene reporter assays. Interestingly, TER significantly up-regulated CAR mRNA expression, a result which could be a consequence of both EGF receptor and ELK-1 transcription factor inhibition by TER or by TER-mediated activation of glucocorticoid receptor (GR), an upstream hormonal regulator of CAR. We can conclude that TER is a novel indirect CAR activator which through EGF inhibition and GR activation controls both detoxification and some intermediary metabolism genes

Development of submicromolar 17β-HSD10 inhibitors and their in vitro and in vivo evaluation

Funding: This study was supported by the Ministry of Education, Youth, and Sports of the Czech Republic (project ESF no. CZ.02.1.01/0.0/0.0/18_069/0010054), by the University of Hradec Kralove (Faculty of Science, no. SV2103‐2022), by Charles University, Prague, Czech Republic (project Cooperatio, research area Neurosciences), by the project MH CZ-DRO VFN64165, and by MH CZ - DRO (UHHK, 00179906), by the Ministry of Defence of the Czech Republic (Faculty of Military Health Sciences Hradec Kralove) under the grant entitled the “Long-term organization development plan - Medical Aspects of Weapons of Mass Destruction”, and by the RS MacDonald Charitable Trust and Rosetrees Trust.17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) is a multifunctional mitochondrial enzyme and putative drug target for the treatment of various pathologies including Alzheimer's disease or some types of hormone-dependent cancer. In this study, a series of new benzothiazolylurea-based inhibitors were developed based on the structure-activity relationship (SAR) study of previously published compounds and predictions of their physico-chemical properties. This led to the identification of several submicromolar inhibitors (IC50 ∼0.3 μM), the most potent compounds within the benzothiazolylurea class known to date. The positive interaction with 17β-HSD10 was further confirmed by differential scanning fluorimetry and the best molecules were found to be cell penetrable. In addition, the best compounds weren't found to have additional effects for mitochondrial off-targets and cytotoxic or neurotoxic effects. The two most potent inhibitors 9 and 11 were selected for in vivo pharmacokinetic study after intravenous and peroral administration. Although the pharmacokinetic results were not fully conclusive, it seemed that compound 9 was bioavailable after peroral administration and could penetrate into the brain (brain-plasma ratio 0.56).Peer reviewe

Synthesis and biological evaluation of benzochromenopyrimidinones as cholinesterase inhibitors and potent antioxidant, non-hepatotoxic agents for Alzheimer’s disease

We report herein the straightforward two-step synthesis and biological assessment of novel racemic benzochromenopyrimidinones as non-hepatotoxic, acetylcholinesterase inhibitors with antioxidative properties. Among them, compound 3Bb displayed a mixed-type inhibition of human acetylcholinesterase (IC50 = 1.28 ± 0.03 μM), good antioxidant activity, and also proved to be non-hepatotoxic on human HepG2 cell line.JMC thanks Government of Spain for support (SAF2016-65586-R), JJ and OS thank MH CZ- DRO (UHHK 00179906).We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)