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This article describes the discovery of novel potent muscarinic receptor antagonists identified during a search for more active histamine H3 receptor (H3R) ligands. The idea was to replace the flexible seven methylene linker with a semirigid 1,4-cyclohexylene or p-phenylene substituted group of the previously described histamine H3R antagonists ADS1017 and ADS1020. These simple structural modifications of the histamine H3R antagonist led to the emergence of additional pharmacological effects, some of which unexpectedly showed strong antagonist potency at muscarinic receptors. This paper reports the routes of synthesis and pharmacological characterization of guanidine derivatives, a novel chemotype of muscarinic receptor antagonists binding to the human muscarinic M2 and M4 receptors (hM2R and hM4R, respectively) in nanomolar concentration ranges. The affinities of the newly synthesized ADS10227 (1-{4-{4-{[4-(phenoxymethyl)cyclohexyl]methyl}piperazin-1-yl}but-1-yl}-1-(benzyl)guanidine) at hM2R and hM4R were 2.8 nM and 5.1 nM, respectively.

Neurodegenerative diseases, e.g., Alzheimer’s disease (AD), are a key health problem in the aging population. The lack of effective therapy and diagnostics does not help to improve this situation. It is thought that ligands influencing multiple but interconnected targets can contribute to a desired pharmacological effect in these complex illnesses. Histamine H3 receptors (H3Rs) play an important role in the brain, influencing the release of important neurotransmitters, such as acetylcholine. Compounds blocking their activity can increase the level of these neurotransmitters. Cholinesterases (acetyl- and butyrylcholinesterase) are responsible for the hydrolysis of acetylcholine and inactivation of the neurotransmitter. Increased activity of these enzymes, especially butyrylcholinesterase (BuChE), is observed in neurodegenerative diseases. Currently, cholinesterase inhibitors: donepezil, rivastigmine and galantamine are used in the symptomatic treatment of AD. Thus, compounds simultaneously blocking H3R and inhibiting cholinesterases could be a promising treatment for AD. Herein, we describe the BuChE inhibitory activity of H3R ligands. Most of these compounds show high affinity for human H3R (Ki &lt, 150 nM) and submicromolar inhibition of BuChE (IC50 &lt, 1 µM). Among all the tested compounds, 19 (E153, 1-(5-([1,1′-biphenyl]-4-yloxy)pentyl)azepane) exhibited the most promising in vitro affinity for human H3R, with a Ki value of 33.9 nM, and for equine&nbsp, serum BuChE, with an IC50 of 590 nM. Moreover, 19 (E153) showed inhibitory activity towards human MAO B with an IC50 of 243 nM. Furthermore, in vivo studies using the Passive Avoidance Task showed that compound 19 (E153) effectively alleviated memory deficits caused by scopolamine. Taken together, these findings suggest that compound 19 can be a lead structure for developing new anti-AD agents.

Dual target ligands are a promising concept for the treatment of Parkinson&rsquo, s disease (PD). A combination of monoamine oxidase B (MAO B) inhibition with histamine H3 receptor (H3R) antagonism could have positive effects on dopamine regulation. Thus, a series of twenty-seven 4-tert-butylphenoxyalkoxyamines were designed as potential dual-target ligands for PD based on the structure of 1-(3-(4-tert-butylphenoxy)propyl)piperidine (DL76). Probed modifications included the introduction of different cyclic amines and elongation of the alkyl chain. Synthesized compounds were investigated for human H3R (hH3R) affinity and human MAO B (hMAO B) inhibitory activity. Most compounds showed good hH3R affinities with Ki values below 400 nM, and some of them showed potent inhibitory activity for hMAO B with IC50 values below 50 nM. However, the most balanced activity against both biological targets showed DL76 (hH3R: Ki = 38 nM and hMAO B: IC50 = 48 nM). Thus, DL76 was chosen for further studies, revealing the nontoxic nature of DL76 in HEK293 and neuroblastoma SH-SY5Ycells. However, no neuroprotective effect was observed for DL76 in hydrogen peroxide-treated neuroblastoma SH-SY5Y cells. Furthermore, in vivo studies showed antiparkinsonian activity of DL76 in haloperidol-induced catalepsy (Cross Leg Position Test) at a dose of 50 mg/kg body weight.

This work describes the microwave assisted synthesis of twelve novel histamine H3 receptor ligands. They display pyrrolo[2,3-d]pyrimidine derivatives with rigidized aliphatic amines as warheads. The compounds were screened for H3R and H4R binding affinities in radioligand displacement assays and the most potent compounds were evaluated for H3R binding properties in vitro and in docking studies. The combination of a rigidized H3R warhead and the pyrrolo[2,3-d]pyrimidine scaffold resulted in selective activity at the H3 receptor with a pKi value of 6.90 for the most potent compound. A bipiperidine warhead displayed higher affinity than a piperazine or morpholine motif, while a naphthyl moiety in the arbitrary region increased affinity compared to a phenyl derivative. The compounds can be starting points for novel, simply synthesized histamine H3 receptor ligands.

Epilepsy is a chronic neurological disorder characterized by irregular, excessive neuronal excitability, and recurrent seizures that affect millions of patients worldwide. Currently, accessible antiepileptic drugs (AEDs) do not adequately support all epilepsy patients, with around 30% patients not responding to the existing therapies. As lifelong epilepsy treatment is essential, the search for new and more effective AEDs with an enhanced safety profile is a significant therapeutic goal. Seizures are a combination of electrical and behavioral events that can induce biochemical, molecular, and anatomic changes. Therefore, appropriate animal models are required to evaluate novel potential AEDs. Among the large number of available animal models of seizures, the acute pentylenetetrazole (PTZ)-induced myoclonic seizure model is the most widely used model assessing the anticonvulsant effect of prospective AEDs, whereas chronic PTZ-kindled seizure models represent chronic models in which the repeated administration of PTZ at subconvulsive doses leads to the intensification of seizure activity or enhanced seizure susceptibility similar to that in human epilepsy. In this review, we summarized the memory deficits accompanying acute or chronic PTZ seizure models and how these deficits were evaluated applying several behavioral animal models. Furthermore, major advantages and limitations of the PTZ seizure models in the discovery of new AEDs were highlighted. With a focus on PTZ seizures, the major biochemicals, as well as morphological alterations and the modulated brain neurotransmitter levels associated with memory deficits have been illustrated. Moreover, numerous medicinal compounds with concurrent anticonvulsant, procognitive, antioxidant effects, modulating effects on several brain neurotransmitters in rodents, and several newly developed classes of compounds applying computer-aided drug design (CADD) have been under development as potential AEDs. The article details the in-silico approach following CADD, which can be utilized for generating libraries of novel compounds for AED discovery. Additionally, in vivo studies could be useful in demonstrating efficacy, safety, and novel mode of action of AEDs for further clinical development.

The aim was to evaluate the activity of seven medicinal, anti-inflammatory plants at the hH4R with focus on defined chemical compounds from Curcuma longa. Activities were analyzed with membrane preparations from Sf9 cells, transiently expressing the hH4R, Gαi2 and Gβ1γ2 subunits. From the methanolic extract of C. longa curcumin (1), demethoxycurcumin (2) and bis(4-hydroxy-cinnamoyl)methane (3) were isolated, purified with HPLC (elution-time 10.20, 9.66, 9.20 min, respectively) and together with six additional extracts, were characterized via radioligand binding studies at the hH4R. Compounds from C. longa were the most potent ligands at the hH4R. They exhibited estimated K i values of 4.26–6.26 µM (1.57–2.31 µg/mL) (1); 6.66––8.97 µM (2.26–3.04 µg/mL) (2) and 10.24–14.57 µM (3.16–4.49 µg/mL) (3) (95% CI). The estimated K i value of the crude extract of curcuma was 0.50–0.81 µg/mL. Fractionated curcumin and the crude extract surpassed the effect of pure curcumin with a K i value of 5.54 µM or 2.04 µg/mL [95% CI (4.47–6.86 µM), (1.65–2.53 µg/mL)]. Within this study, defined compounds of C. longa were recognized as potential ligands and reasonable lead structures at the hH4R. The mode of anti-inflammatory action of curcumin was further elucidated and the role of extracts in traditional phytomedicine was strengthened.

Isoprenoids constitute one of the most diverse classes of natural products. As a compound class, they are essential to basic metabolic processes including cell-wall biosynthesis, post-translational protein modifications, and signaling. In addition, isoprenoid secondary metabolites are highly valuable natural products with a wide range of biotechnological applications. The biosynthesis of their two universal building blocks, isopentenyl diphosphate and dimethylallyl diphosphate, was thought to proceed exclusively by way of mevalonate as a key intermediate until a novel pathway involving methylerithritol phosphate (MEP) was discovered in the early 1990s. In this review, we describe the seven enzymes of the MEP pathway, along with their discoveries, three-dimensional structures, and mechanisms. The latter include examples of remarkable enzyme catalysis including an unusual cytidilation reaction and the use of iron-sulfur cluster cofactors in reductive ring opening and hydroxy-group elimination. Furthermore, isoprenoid biosynthesis shows a characteristic species distribution. A brief overview highlights the MEP pathway's potential as a selective drug target, which is absent in humans but essential to the survival of many important bacterial and apicomplexan pathogens.

The serotonin receptor subtype 7 (5-HT7R) is clearly involved in behavioral functions such as learning/memory, mood regulation and circadian rhythm. Recent discoveries proposed modulatory physiological roles for serotonergic systems in reward-guided behavior. However, the interplay between serotonin (5-HT) and dopamine (DA) in reward-related behavioral adaptations needs to be further assessed. TP-22 is a recently developed arylpiperazine-based 5-HT7R agonist, which is also showing high affinity and selectivity towards D1 receptors. Here, we report that TP-22 displays D1 receptor antagonist activity. Moreover, we describe the first in vivo tests with TP-22: first, a pilot experiment (assessing dosage and timing of action) identified the 0.25 mg/kg i.v. dosage for locomotor stimulation of rats. Then, a conditioned place preference (CPP) test with the DA-releasing psychostimulant drug, methylphenidate (MPH), involved three rat groups: prior i.v. administration of TP-22 (0.25 mg/kg), or vehicle (VEH), 90 min before MPH (5 mg/kg), was intended for modulation of conditioning to the white chamber (saline associated to the black chamber); control group (SAL) was conditioned with saline in both chambers. Prior TP-22 further increased the stimulant effect of MPH on locomotor activity. During the place-conditioning test, drug-free activity of TP-22+MPH subjects remained steadily elevated, while VEH+MPH subjects showed a decline. Finally, after a priming injection of TP-22 in MPH-free conditions, rats showed a high preference for the MPH-associated white chamber, which conversely had vanished in VEH-primed MPH-conditioned subjects. Overall, the interaction between MPH and pre-treatment with TP-22 seems to improve both locomotor stimulation and the conditioning of motivational drives to environmental cues. Together with recent studies, a main modulatory role of 5-HT7R for the processing of rewards can be suggested. In the present study, TP-22 proved to be a useful psychoactive tool to better elucidate the role of 5-HT7R and its interplay with DA in reward-related behavior.

Autism spectrum disorder (ASD) is a complex heterogeneous neurodevelopmental disorder characterized by social and communicative impairments, as well as repetitive and restricted behaviors (RRBs). With the limited effectiveness of current pharmacotherapies in treating repetitive behaviors, the present study determined the effects of acute systemic treatment of the novel multi-targeting ligand ST-2223, with incorporated histamine H3 receptor (H3R) and dopamine D2/D3 receptor affinity properties, on ASD-related RRBs in a male Black and Tan BRachyury (BTBR) mouse model of ASD. ST-2223 (2.5, 5, and 10 mg/kg, i.p.) significantly mitigated the increase in marble burying and self-grooming, and improved reduced spontaneous alternation in BTBR mice (all p &lt, 0.05). Similarly, reference drugs memantine (MEM, 5 mg/kg, i.p.) and aripiprazole (ARP, 1 mg/kg, i.p.), reversed abnormally high levels of several RRBs in BTBR (p &lt, 0.05). Moreover, ST-2223 palliated the disturbed anxiety levels observed in an open field test (all p &lt, 0.05), but did not restore the hyperactivity parameters, whereas MEM failed to restore mouse anxiety and hyperactivity. In addition, ST-2223 (5 mg/kg, i.p.) mitigated oxidative stress status by decreasing the elevated levels of malondialdehyde (MDA), and increasing the levels of decreased glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) in different brain parts of treated BTBR mice (all p &lt, 0.05). These preliminary in vivo findings demonstrate the ameliorative effects of ST-2223 on RRBs in a mouse model of ASD, suggesting its pharmacological prospective to rescue core ASD-related behaviors. Further confirmatory investigations on its effects on various brain neurotransmitters, e.g., dopamine and histamine, in different brain regions are still warranted to corroborate and expand these initial data.

A newly developed series of non-imidazole histamine H3 receptor (H3R) antagonists (1&ndash, 16) was evaluated in vivo for anticonvulsant effects in three different seizure models in Wistar rats. Among the novel H3R antagonists examined, H3R antagonist 4 shortened the duration of tonic hind limb extension (THLE) in a dose-dependent fashion in the maximal electroshock (MES)-induced seizure and offered full protection against pentylenetetrazole (PTZ)-induced generalized tonic-clonic seizure (GTCS), following acute systemic administration (2.5, 5, 10, and 15 mg/kg, i.p.). However, only H3R antagonist 13, without appreciable protective effects in MES- and PTZ-induced seizure, fully protected animals in the strychnine (STR)-induced GTCS following acute systemic pretreatment (10 mg/kg, i.p.). Moreover, the protective effect observed with H3R antagonist 4 in MES-induced seizure was completely abolished when animals were co-administered with the H3R agonist (R)-&alpha, methylhistamine (RAMH, 10 mg/kg, i.p.). However, RAMH failed to abolish the full protection provided by the H3R antagonist 4 in PTZ-induced seizure and H3R antagonist 13 in STR-induced seizure. Furthermore, in vitro antiproliferative effects or possible metabolic interactions could not be observed for compound 4. Additionally, the predictive in silico, as well as in vitro, metabolic stability for the most promising H3R antagonist 4 was assessed. The obtained results show prospective effects of non-imidazole H3R antagonists as innovative antiepileptic drugs (AEDs) for potential single use against epilepsy.

Because only 0.01% of prokaryotic genospecies can be cultured and in situ observations are often impracticable, culture-independent methods are required to understand microbial life and harness potential applications of microbes. Here, we report a methodology for the production of proteins with desired functions based on single amplified genomes (SAGs) from unculturable species. We use this method to resurrect an alcohol dehydrogenase (ADH/D1) from an uncharacterized halo-thermophilic archaeon collected from a brine pool at the bottom of the Red Sea. Our crystal structure of 5,6-dihydroxy NADPH-bound ADH/D1 combined with biochemical analyses reveal the molecular features of its halo-thermophily, its unique habitat adaptations, and its possible reaction mechanism for atypical oxygen activation. Our strategy offers a general guide for using SAGs as a source for scientific and industrial investigations of “microbial dark matter.”

Phloretin hydrolase from Eubacterium ramulus (Phy) catalyzes the hydrolysis of the dihydrochalcone phloretin to phloroglucinol and phloretic acid, performing a formal retro- Friedel–Crafts acylation reaction on its substrate. Its closest sequence homolog, of 25 % amino acid sequence identity, is diacetyl phloroglucinol hydrolase (Phlg) from Pseudomonas fluorescens, which catalyses a similar, hydrolytic, de-acylation of its substrate. The structure of Phlg has been determined and a catalytic mechanism proposed (J Biol Chem 285:4603–4611, 2010). In order to compare the catalytic characteristics of Phy with Phlg, the gene encoding Phy was expressed and the enzyme purified and crystallised. An X-ray fluorescence scan identified zinc within the crystals. A homology model of Phy, based on the structure of Phlg (PDB code 3HWP), informed the construction of a point mutant library of the enzyme, targeting residues shared with Phlg that are thought to be involved in zinc binding and the recognition of acyl and phenol functionality on the aromatic ring of the substrates. Mutation of His123, His251, Glu154 and Glu255 (conserved zinc binding residues) resulted in variants that were either poorly expressed, or of much reduced activity; Mutation of Tyr115 and His203, thought to bind the phenol groups in the 1-and 3-positions of the phloroglucinol ring respectively, resulted in variants of 15-fold reduced activity and an inactive variant. These results are suggestive of conservation of some aspects of mechanism and substrate recognition between Phy and Phlg, and of the catalytic characteristics of Zn-dependent C–C hydrolases of this type in general.

Although CC bond hydrolases are distributed widely in Nature, they has as yet have received only limited attention in the area of biocatalysis compared to their counterpart the C-heteroatom hydrolases, such as lipases and proteases. However, the substrate range of CC hydrolases, and their non-dependence on cofactors, suggest that these enzymes may have considerable potential for applications in synthesis. In addition, hydrolases such as the β-diketone hydrolase from Rhodococcus (OCH) are known, that catalyse the formation of interesting chiral intermediates. Further enzymes, such as kynureninase and a meta-cleavage product hydrolase (MhpC), are able to catalyse carbon-carbon bond formation, suggesting wider applications in biocatalysis than previously envisaged. In this review, the distribution, catalytic characteristics and applications of CC hydrolases are described, with a view to assessing their potentialfor use in biocatalytic processes in the future.

Crotonase superfamily enzymes catalyze a wide variety of reactions, including hydrolytic C-C bond cleavage in symmetrical β-diketones by 6-oxo camphor hydrolase (OCH) from Rhodococcus sp. The organic solvent tolerance and temperature stability of OCH and its structurally related ortholog Anabaena β-diketone hydrolase have been investigated. Both enzymes showed excellent tolerance toward organic solvents; for instance, even in the presence of 80% (v/v) THF or dioxane, OCH was still active. In most solvent mixtures, except methanol, the stereospecificity was conserved (>99% e.e. of product), hence neither the type of solvent nor its concentration appeared to have an effect on the stereoselectivity of the enzyme. Attempts to correlate the observed activities with log P, functional solvent group or denaturing capacity (DC) of the solvent were only successful in the case of DC for water miscible solvents. This study represents the first investigation of organic solvent stability for members of the crotonase superfamily.