Identification of an Antagonist Targeting G Protein and ß-Arrestin Signaling Pathways of 5-HT7R.

In consideration of the limited number of FDA-approved drugs for autism spectrum disorder (ASD), significant efforts have been devoted to identifying novel drug candidates. Among these, 5-HT7R modulators have garnered considerable attention due to their potential in alleviating autism-like behaviors in ASD animal models. In this study, we designed and synthesized biphenyl-3-ylmethylpyrrolidines 3 and biphenyl-3-yl-dihydroimidazoles 4 as 5-HT7R modulators. Through extensive biological tests of 3 and 4 in G protein and ß-arrestin signaling pathways of 5-HT7R, it was determined that 2-(2'-methoxy-[1,1'-biphenyl]-3-yl)-4,5-dihydro-1H-imidazole 4h acted as a 5-HT7R antagonist in both signaling pathways. In in vivo study with Shank3-/- transgenic (TG) mice, the self-grooming behavior test was performed with 4h, resulting in a significant reduction in the duration of self-grooming. In addition, an immunohistochemical experiment with 4h restored reduced neurogenesis in Shank3-/- TG mice, which is confirmed by the quantification of doublecortin (DCX) positive neurons, suggesting the promising therapeutic potential of 4h.

S-Adenosylmethionine: more than just a methyl donor.

Covering: from 2000 up to the very early part of 2023S-Adenosyl-L-methionine (SAM) is a naturally occurring trialkyl sulfonium molecule that is typically associated with biological methyltransfer reactions. However, SAM is also known to donate methylene, aminocarboxypropyl, adenosyl and amino moieties during natural product biosynthetic reactions. The reaction scope is further expanded as SAM itself can be modified prior to the group transfer such that a SAM-derived carboxymethyl or aminopropyl moiety can also be transferred. Moreover, the sulfonium cation in SAM has itself been found to be critical for several other enzymatic transformations. Thus, while many SAM-dependent enzymes are characterized by a methyltransferase fold, not all of them are necessarily methyltransferases. Furthermore, other SAM-dependent enzymes do not possess such a structural feature suggesting diversification along different evolutionary lineages. Despite the biological versatility of SAM, it nevertheless parallels the chemistry of sulfonium compounds used in organic synthesis. The question thus becomes how enzymes catalyze distinct transformations via subtle differences in their active sites. This review summarizes recent advances in the discovery of novel SAM utilizing enzymes that rely on Lewis acid/base chemistry as opposed to radical mechanisms of catalysis. The examples are categorized based on the presence of a methyltransferase fold and the role played by SAM within the context of known sulfonium chemistry.

Byproduct formation during the biosynthesis of spinosyn A and evidence for an enzymatic interplay to prevent its formation.

Biosynthesis of spinosyn A in Saccharopolyspora spinosa involves a 1,4-dehydration followed by an intramolecular [4 + 2]-cycloaddition catalyzed by SpnM and SpnF, respectively. The cycloaddition also takes place in the absence of SpnF leading to questions regarding its mechanism of catalysis and biosynthetic role. Substrate analogs were prepared with an unactivated dienophile or an acyclic structure and found to be unreactive consistent with the importance of these features for cyclization. The SpnM-catalyzed dehydration reaction was also found to yield a byproduct corresponding to the C11 = C12 cis isomer of the SpnF substrate. This byproduct is stable both in the presence and absence of SpnF; however, relative production of the SpnM product and byproduct could be shifted in favor of the former by including SpnF or the dehydrogenase SpnJ in the reaction. This result suggests a potential interplay between the enzymes of spinosyn A biosynthesis that may help to improve the efficiency of the pathway.

Serotonin Receptors as Therapeutic Targets for Autism Spectrum Disorder Treatment.

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by repetitive and stereotyped behaviors as well as difficulties with social interaction and communication. According to reports for prevalence rates of ASD, approximately 1~2% of children worldwide have been diagnosed with ASD. Although there are a couple of FDA (Food and Drug Administration)-approved drugs for ASD treatment such as aripiprazole and risperidone, they are efficient for alleviating aggression, hyperactivity, and self-injury but not the core symptoms. Serotonin (5-hydroxytryptamine, 5-HT) as a neurotransmitter plays a crucial role in the early neurodevelopmental stage. In particular, 5-HT has been known to regulate a variety of neurobiological processes including neurite outgrowth, dendritic spine morphology, shaping neuronal circuits, synaptic transmission, and synaptic plasticity. Given the roles of serotonergic systems, the 5-HT receptors (5-HTRs) become emerging as potential therapeutic targets in the ASD. In this review, we will focus on the recent development of small molecule modulators of 5-HTRs as therapeutic targets for the ASD treatment.

Evidence for an Enzyme-Catalyzed Rauhut-Currier Reaction during the Biosynthesis of Spinosyn A.

The catalog of enzymes known to catalyze the nucleophile-assisted formation of C-C bonds is extremely small, and there is presently no definitive example of a biological Rauhut-Currier reaction. Biosynthesis of the polyketide insecticide spinosyn A in Saccharopolyspora spinosa involves a [4 + 2]-cycloaddition and a subsequent intramolecular C-C bond formation catalyzed by SpnF and SpnL, respectively. Isotope tracer experiments and kinetic isotope effects, however, imply that the SpnL-catalyzed reaction proceeds without initial deprotonation of the substrate. The crystal structure of SpnL exhibits high similarity to SAM-dependent methyltransferases as well as SpnF. The residue Cys60 is also shown to reside in the SpnL active site, and the Cys60Ala SpnL mutant is found to be devoid of activity. Moreover, SpnL is covalently modified at Cys60 and irreversibly inactivated when it is coincubated with a fluorinated substrate analogue designed as a suicide inactivator of nucleophile-assisted C-C bond formation. These results suggest that SpnL catalyzes a biological Rauhut-Currier reaction.

Discovery of G Protein-Biased Antagonists against 5-HT7R.

5-HT7R belongs to a family of G protein-coupled receptors and is associated with a variety of physiological processes in the central nervous system via the activation of the neurotransmitter serotonin (5-HT). To develop selective and biased 5-HT7R ligands, we designed and synthesized a series of pyrazolyl-diazepanes 2 and pyrazolyl-piperazines 3, which were evaluated for binding affinities to 5-HTR subtypes and functional selectivity for G protein and ß-arrestin signaling pathways of 5-HT7R. Among them, 1-(3-(3-chlorophenyl)-1H-pyrazol-4-yl)-1,4-diazepane 2c showed the best binding affinity for 5-HT7R and selectivity over other 5-HTR subtypes. It was also revealed as a G protein-biased antagonist. The self-grooming behavior test was performed with 2c in vivo with Shank3-/- transgenic (TG) mice, wherein 2c significantly reduced self-grooming duration time to the level of wild-type mice. The results suggest that 5-HT7R could be a potential therapeutic target for treating autism spectrum disorder stereotypy.

Modulation of Serotonin Receptors in Neurodevelopmental Disorders: Focus on 5-HT7 Receptor.

Since neurodevelopmental disorders (NDDs) influence more than 3% of children worldwide, there has been intense investigation to understand the etiology of disorders and develop treatments. Although there are drugs such as aripiprazole, risperidone, and lurasidone, these medications are not cures for the disorders and can only help people feel better or alleviate their symptoms. Thus, it is required to discover therapeutic targets in order to find the ultimate treatments of neurodevelopmental disorders. It is suggested that abnormal neuronal morphology in the neurodevelopment process is a main cause of NDDs, in which the serotonergic system is emerging as playing a crucial role. From this point of view, we noticed the correlation between serotonin receptor subtype 7 (5-HT7R) and NDDs including autism spectrum disorder (ASD), fragile X syndrome (FXS), and Rett syndrome (RTT). 5-HT7R modulators improved altered behaviors in animal models and also affected neuronal morphology via the 5-HT7R/G12 signaling pathway. Through the investigation of recent studies, it is suggested that 5-HT7R could be a potential therapeutic target for the treatment of NDDs.

Discovery of G Protein-Biased Ligands against 5-HT7R.

There has been significant attention concerning the biased agonism of G protein-coupled receptors (GPCRs), and it has resulted in various pharmacological benefits. 5-HT7R belongs to a GPCR, and it is a promising pharmaceutical target for the treatment of neurodevelopmental and neuropsychiatric disorders. Based on our previous research, we synthesized a series of 6-chloro-2'-methoxy biphenyl derivatives 1, 2, and 3 with a variety of amine scaffolds. These compounds were evaluated for their binding affinities to 5-HTR subtypes and their functional selectivity toward the Gs protein and the ß-arrestin signaling pathways of 5-HT7R. Among them, 2-(6-chloro-2'-methoxy-[1,1'-biphenyl]-3-yl)-N-ethylethan-1-amine, 2b, was found to be a G-protein-biased ligand of 5-HT7R. In an in vivo study with Shank3 transgenic mice, the self-grooming behavior test was performed with 2b, which increased the duration of self-grooming. The experiments further suggested that 5-HT7R is associated with autism spectrum disorders (ASDs) and could be a therapeutic target for the treatment of stereotypy in ASDs.

Influence of water and enzyme SpnF on the dynamics and energetics of the ambimodal [6+4]/[4+2] cycloaddition.

SpnF is the first monofunctional Diels-Alder/[6+4]-ase that catalyzes a reaction leading to both Diels-Alder and [6+4] adducts through a single transition state. The environment-perturbed transition-state sampling method has been developed to calculate free energies, kinetic isotope effects, and quasi-classical reaction trajectories of enzyme-catalyzed reactions and the uncatalyzed reaction in water. Energetics calculated in this way reproduce the experiment and show that the normal Diels-Alder transition state is stabilized by H bonds with water molecules, while the ambimodal transition state is favored in the enzyme SpnF by both intramolecular hydrogen bonding and hydrophobic binding. Molecular dynamics simulations show that trajectories passing through the ambimodal transition state bifurcate to the [6+4] adduct and the Diels-Alder adduct with a ratio of 1:1 in the gas phase, 1:1.6 in water, and 1:11 in the enzyme. This example shows how an enzyme acts on a vibrational time scale to steer post-transition state trajectories toward the Diels-Alder adduct.

Investigation of the mechanism of the SpnF-catalyzed [4+2]-cycloaddition reaction in the biosynthesis of spinosyn A.

The Diels-Alder reaction is one of the most common methods to chemically synthesize a six-membered carbocycle. While it has long been speculated that the cyclohexene moiety found in many secondary metabolites is also introduced via similar chemistry, the enzyme SpnF involved in the biosynthesis of the insecticide spinosyn A in Saccharopolyspora spinosa is the first enzyme for which catalysis of an intramolecular [Formula: see text]-cycloaddition has been experimentally verified as its only known function. Since its discovery, a number of additional standalone [Formula: see text]-cyclases have been reported as potential Diels-Alderases; however, whether their catalytic cycles involve a concerted or stepwise cyclization mechanism has not been addressed experimentally. Here, we report direct experimental interrogation of the reaction coordinate for the [Formula: see text]-carbocyclase SpnF via the measurement of [Formula: see text]-secondary deuterium kinetic isotope effects (KIEs) at all sites of [Formula: see text] rehybridization for both the nonenzymatic and enzyme-catalyzed cyclization of the SpnF substrate. The measured KIEs for the nonenzymatic reaction are consistent with previous computational results implicating an intermediary state between formation of the first and second carbon-carbon bonds. The KIEs measured for the enzymatic reaction suggest a similar mechanism of cyclization within the enzyme active site; however, there is evidence that conformational restriction of the substrate may play a role in catalysis.

Natural [4 + 2]-Cyclases.

[4 + 2]-Cycloadditions are increasingly being recognized in the biosynthetic pathways of many structurally complex natural products. A relatively small collection of enzymes from these pathways have been demonstrated to increase rates of cyclization and impose stereochemical constraints on the reactions. While mechanistic investigation of these enzymes is just beginning, recent studies have provided new insights with implications for understanding their biosynthetic roles, mechanisms of catalysis, and evolutionary origin.

Chemoenzymatic synthesis of spinosyn A.

Following the biosynthesis of polyketide backbones by polyketide synthases (PKSs), post-PKS modifications result in a significantly elevated level of structural complexity that renders the chemical synthesis of these natural products challenging. We report herein a total synthesis of the widely used polyketide insecticide spinosyn A by exploiting the prowess of both chemical and enzymatic methods. As more polyketide biosynthetic pathways are characterized, this chemoenzymatic approach is expected to become readily adaptable to streamlining the synthesis of other complex polyketides with more elaborate post-PKS modifications.

Structural studies of the spinosyn forosaminyltransferase, SpnP.

Spinosyns A and D (spinosad) are complex polyketide natural products biosynthesized through the cooperation of a modular polyketide synthase and several tailoring enzymes. SpnP catalyzes the final tailoring step, transferring forosamine from a TDP-D-forosamine donor substrate to a spinosyn pseudoaglycone acceptor substrate. Sequence analysis indicated that SpnP belongs to a small group of glycosyltransferases (GTs) that require an auxiliary protein for activation. However, unlike other GTs in this subgroup, no putative auxiliary protein gene could be located in the biosynthetic gene cluster. To learn more about SpnP, the structures of SpnP and its complex with TDP were determined to 2.50 and 3.15 Å resolution, respectively. Binding of TDP causes the reordering of several residues in the donor substrate pocket. SpnP possesses a structural feature that has only been previously observed in the related glycosyltransferase EryCIII, in which it mediates association with the auxiliary protein EryCII. This motif, H-X-R-X5-D-X5-R-X12-20-D-P-X3-W-L-X12-18-E-X4-G, may be predictive of glycosyltransferases that interact with an auxiliary protein. A reverse glycosyl transfer assay demonstrated that SpnP possesses measurable activity in the absence of an auxiliary protein. Our data suggest that SpnP can bind its donor substrate by itself but that the glycosyl transfer reaction is facilitated by an auxiliary protein that aids in the correct folding of a flexible loop surrounding the pseudoaglycone acceptor substrate-binding pocket.