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.

Synthesis and biological evaluation of indane-based fluorescent probes for detection of amyloid-ß aggregates in Alzheimer's disease.

In this article, the development of fluorescent imaging probes for the detection of Alzheimer's disease (AD)-associated protein aggregates is described. Indane derivatives with a donor-π-acceptor (D-π-A) structure were designed and synthesized. The probes were evaluated for their ability to bind to ß-amyloid (Aß) protein aggregates, which are a key pathological hallmark of AD. The results showed that several probes exhibited significant changes in fluorescence intensity at wavelengths greater than 600 nm when they were bound to Aß aggregates compared to the Aß monomeric form. Among the tested probes, four D-π-A type indane derivatives showed promising binding selectivity to Aß aggregates over non-specific proteins such as bovine serum albumin (BSA). The molecular docking study showed that our compounds were appropriately located along the Aß fibril axis through the hydrophobic tunnel structure. Further analysis revealed that the most active compound having dimethylaminopyridyl group as an election donor and dicyano group as an electron acceptor could effectively stain Aß plaques in brain tissue samples from AD transgenic mice. These findings suggest that our indane-based compounds have the potential to serve as fluorescent probes for the detection and monitoring of Aß aggregation in AD.

Discovery of novel amidobenzimidazole derivatives as orally available small molecule modulators of stimulator of interferon genes for cancer immunotherapy.

Stimulator of interferon genes (STING) agonists show promise as immunomodulatory agents for cancer therapy. In this study, we report the discovery of a novel orally available STING agonist, SAP-04, that exhibits potent immunomodulatory effects for cancer therapy. By optimizing the amidobenzimidazole core with various pyridine-based heterocyclic substituents, we identified a monomeric variant that displayed more efficient STING agonistic activity than the corresponding dimer. SAP-04 efficiently induced cytokine secretion related to innate immunity by directly binding of the compound to the STING protein, followed by sequential signal transduction for the STING signaling pathway and type I interferon (IFN) responses. Further pharmacological validation in vitro and in vivo demonstrated the potential utility of SAP-04 as an immunomodulatory agent for cancer therapy in vivo. The in vivo anticancer effect was observed in a 4T1 breast tumor syngeneic mouse model through oral administration of the compound. Our findings suggest a possible strategy for developing synthetically accessible monomeric variants as orally available STING agonists.

Structural and functional characterization of USP47 reveals a hot spot for inhibitor design.

USP47 is widely involved in tumor development, metastasis, and other processes while performing a more regulatory role in inflammatory responses, myocardial infarction, and neuronal development. In this study, we investigate the functional and biochemical properties of USP47, whereby depleting USP47 inhibited cancer cell growth in a p53-dependent manner-a phenomenon that enhances during the simultaneous knockdown of USP7. Full-length USP47 shows higher deubiquitinase activity than the catalytic domain. The crystal structures of the catalytic domain, in its free and ubiquitin-bound states, reveal that the misaligned catalytic triads, ultimately, become aligned upon ubiquitin-binding, similar to USP7, thereby becoming ready for catalysis. Yet, the composition and lengths of BL1, BL2, and BL3 of USP47 differ from those for USP7, and they contribute to the observed selectivity. Our study provides molecular details of USP47 regulation, substrate recognition, and the hotspots for drug discovery by targeting USP47.

Identification of (-)-Epigallocateshin gallate derivatives promoting innate immune activation via 2',3'-cyclic GMP-AMP-stimulator of interferon genes pathway.

(-)-Epigallocatehin-3-gallate (EGCG) is a catechin derived from green tea, which has been widely studied for its anti-oxidant and anti-tumor properties. Although EGCG plays important roles in various biological processes, the its effect on the immune system is not fully understood. In this study, we investigated the potential of EGCG as an activator of the stimulator of interferon genes (STING) pathway in the immune system. The cyclic GMP-AMP synthase (cGAS)-2-3-cyclic GMP-AMP (cGAMP)-STING pathway is crucial in the innate immune response to microbial infections, autoimmunity, and anticancer immunity. We confirmed that EGCG enhanced the immune response of cGAMP and identified E2 from 13 synthetic derivatives of EGCG. E2 specifically activated the interferon (IFN) signaling pathway specifically through STING- and cGAMP-dependent mechanisms. These results demonstrate the potential of EGCG and its derivatives as new STING activators that can stimulate the type I interferon response by boosting cGAMP-mediated STING activity.

Identification of novel pyrrolopyrimidine and pyrrolopyridine derivatives as potent ENPP1 inhibitors.

In an effort to discover novel scaffolds of non-nucleotide-derived Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitors to stimulate the Stimulator of Interferon Genes (STING) pathway, we designed and synthesised pyrrolopyrimidine and pyrrolopyridine derivatives and performed structure-activity relationship (SAR) study. We found 18p possessed high potency (IC50 = 25.0 nM) against ENPP1, and activated STING pathway in a concentration dependent manner. Also, in response to STING pathway activation, cytokines such as IFN-ß and IP-10 were induced by 18p in a concentration dependent manner. Finally, we discovered that 18p causes inhibition of tumour growth in 4T1 syngeneic mouse model. This study provides new insight into the designing of novel ENPP1 inhibitors and warrants further development of small molecule immune modulators for cancer immunotherapy.

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.

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.

Modulation of SETDB1 activity by APQ ameliorates heterochromatin condensation, motor function, and neuropathology in a Huntington's disease mouse model.

The present study describes evaluation of epigenetic regulation by a small molecule as the therapeutic potential for treatment of Huntington's disease (HD). We identified 5-allyloxy-2-(pyrrolidin-1-yl)quinoline (APQ) as a novel SETDB1/ESET inhibitor using a combined in silico and in vitro cell based screening system. APQ reduced SETDB1 activity and H3K9me3 levels in a HD cell line model. In particular, not only APQ reduced H3K9me3 levels in the striatum but it also improved motor function and neuropathological symptoms such as neuronal size and activity in HD transgenic (YAC128) mice with minimal toxicity. Using H3K9me3-ChIP and genome-wide sequencing, we also confirmed that APQ modulates H3K9me3-landscaped epigenomes in YAC128 mice. These data provide that APQ, a novel small molecule SETDB1 inhibitor, coordinates H3K9me-dependent heterochromatin remodelling and can be an epigenetic drug for treating HD, leading with hope in clinical trials of HD.

Identification of highly potent and selective inhibitor, TIPTP, of the p22phox-Rubicon axis as a therapeutic agent for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease linked to oxidative stress, which is associated with significant morbidity. The NADPH oxidase complex (NOX) produces reactive oxygen species (ROS) that are among the key markers for determining RA's pathophysiology. Therefore, understanding ROS-regulated molecular pathways and their interaction is necessary for developing novel therapeutic approaches for RA. Here, by combining mouse genetics and biochemistry with clinical tissue analysis, we reveal that in vivo Rubicon interacts with the p22phox subunit of NOX, which is necessary for increased ROS-mediated RA pathogenesis. Furthermore, we developed a series of new aryl propanamide derivatives consisting of tetrahydroindazole and thiadiazole as p22phox inhibitors and selected 2-(tetrahydroindazolyl)phenoxy-N-(thiadiazolyl)propanamide 2 (TIPTP, M.W. 437.44), which showed considerably improved potency, reaching an IC50 value up to 100-fold lower than an inhibitor that we previously synthesized reported N8 peptide-mimetic small molecule (blocking p22phox-Rubicon interaction). Notably, TIPTP treatment showed significant therapeutic effects a mouse model for RA. Furthermore, TIPTP had anti-inflammatory effects ex vivo in monocytes from healthy individuals and synovial fluid cells from RA patients. These findings may have clinical applications for the development of TIPTP as a small molecule inhibitor of the p22phox-Rubicon axis for the treatment of ROS-driven diseases such as RA.

Development of carbapenem-based fluorogenic probes for the clinical screening of carbapenemase-producing bacteria.

This report describes the synthesis of a library of fluorogenic carbapenemase substrates consisting of carbapenem derivatives, fluorescence dyes, and active cleavable linkers and their evaluation for specifically detecting carbapenemase-producing organisms (CPOs). We synthesized a series of compounds having three different types of linkers such as benzyl ether, carbamate, and amine using hydroxymethyl carbapenem 7a and hydroxyallyl carbapenem 7b as key intermediates. Probe 1b exhibited high stability and a prompt turn-on fluorescence signal upon hydrolysis by carbapenemases. In particular, the screening of clinical samples indicated that the probe 1b exhibited excellent selectivity to the CPOs over other ß-lactamases or non-carbapenemase producing bacteria, which may be of clinical use for the rapid and accurate detection of CPOs for timely diagnosis and treatment.

Synthesis and Biological Evaluation of Disubstituted Pyrimidines as Selective 5-HT2C Agonists.

Here, we describe the synthesis of disubstituted pyrimidine derivatives and their biological evaluation as selective 5-HT2C agonists. To improve selectivity for 5-HT2C over other subtypes, we synthesized two series of disubstituted pyrimidines with fluorophenylalkoxy groups at either the 5-position or 4-position and varying cyclic amines at the 2-position. The in vitro cell-based assay and binding assay identified compounds 10a and 10f as potent 5-HT2C agonists. Further studies on selectivity to 5-HT subtypes and drug-like properties indicated that 2,4-disubstituted pyrimidine 10a showed a highly agonistic effect on the 5-HT2C receptor, with excellent selectivity, as well as exceptional drug-like properties, including high plasma and microsomal stability, along with low CYP inhibition. Thus, pyrimidine 10a could be considered a viable lead compound as a 5-HT2C selective agonist.

Conjugation with Phenylalanine Enhances Autophagy-Inducing Activity of (-)-Epigallocatechin Gallate in Hepatic Cells.

Given the importance of (-)-epigallocatechin gallate (EGCG) as an autophagy-enhancing and thereby lipid-lowering agent, optimization of its activity warrants its therapeutic potential in the treatment of hepatic diseases as well as metabolic disorders. On the basis of our previous observations that structural modifications provided substantial improvements in the bioactivity of EGCG, we investigated the autophagy-enhancing activity of EGCG derivatives. Among 14 EGCG derivatives, E10 with a phenylalanine attached to the D ring of EGCG exhibited the most promising effects in stimulating autophagy in Huh7 cells, which was supported by several lines of evidence: (1) stimulation of autophagy revealed by an increased amount of LC3B-II (4.1 ± 0.8-fold compared to the control) as well as the 2.0 ± 0.1-fold activation of adenosine monophosphate-activated protein kinase in the presence of E10 and (2) E10-stimulated autophagic flux demonstrated by a 1.6 ± 0.4-fold increase in LC3B-II upon co-treatment with chloroquine, 38.1 ± 5.6% reduction of p62/SQSTM1, and an increase in the formation of autophagic compartments visualized by both CYTO-ID staining (3.0 ± 0.1-fold) and tandem RFP-GFP-LC3 fluorescence (2.7 ± 0.4- and 3.2 ± 0.3-fold for green and red fluorescence, respectively). Finally, the autophagy-inducing activity of E10 culminated in a 5.3-fold reduction of hepatic lipid accumulation caused by fatty acids. In all of the assay settings, E10 was consistently 1.3-3.5-fold more potent than EGCG. Taken together, we demonstrated a significant increase in autophagy-stimulating activity of EGCG through structural modifications.

Discovery of ß-Arrestin Biased Ligands of 5-HT7R.

Though many studies have been published about therapeutic potentials of selective 5-HT7R ligands, there have been few biased ligands of 5-HT7R. The development of potent and selective biased ligands of 5-HT7R would be of great help in understanding the relationship between pharmacological effects and G protein/ß-arrestin signaling pathways of 5-HT7R. In order to identify 5-HT7R ligands with biased agonism, we designed and synthesized a series of tetrahydroazepine derivatives 1 and 2 with arylpyrazolo moiety or arylisoxazolo moiety. Through several biological evaluations such as binding affinity, selectivity profile, and functions in G protein and ß-arrestin signaling pathways, 3-(4-chlorophenyl)-1,4,5,6,7,8-hexahydropyrazolo[3,4- d]azepine 1g was discovered as the ß-arrestin biased ligand of 5-HT7R. In an electroencephalogram (EEG) test, 1g increased total non-rapid eye movement (NREM) sleep time and decreased total rapid eye movement (REM) sleep time.

Structural Modification of (-)-Epigallocatechin Gallate (EGCG) Shows Significant Enhancement in Mitochondrial Biogenesis.

(-)-Epigallocatechin-3-gallate (EGCG) is known as a mitochondria-targeted molecule that can prevent mitochondrial deterioration and induce mitochondrial biogenesis by modulating key regulators of mitochondrial metabolism. In this study, we tackled whether derivatization of EGCG could result in enhancement of its effects on mitochondrial biogenesis. EGCG, EGCG peracetate (AcEGCG), and its 4″- O-alkyl substituted congeners prepared by previously reported procedures were biologically evaluated. Interestingly, EGCG and AcEGCG were only marginally effective in inducing mitochondrial biogenesis, while AcEGCG congeners with an alkyl group at the 4″- O position showed significantly increased biological activity compared to their parent compound. Among these series, 3f with a methyl-branched carbonate chain at the 4″- O position of the AcEGCG scaffold showed the most enhancement in inducing mitochondrial biogenesis. Hepa1-6 cells treated with 3f exhibited increases in both mitochondrial mass (1.5 times) and relative mtDNA content to nDNA (1.5 times). As a mitochondrial biogenesis enhancer, 3f also increased expression levels of regulators for mitochondrial function, including PGC-1α (4.0 fold), p-AMPK (2.5 fold), SIRT1 (4.2 fold), ERRα (1.8 fold), NRF-1 (1.6 fold), NRF-2 (1.7 fold), and mtTFA (1.6 folds). Investigation of oxidative phosphorylation by mitochondria in the presence of 3f revealed that 3f increased the NAD+/NADH ratio, the amount of cytochrome c, ATP synthesis, and oxygen consumption in Hepa1-6 cells by 2.2, 1.4, 1.5, and 2.1 fold, respectively. Taken together, these results warrant an extensive structure-activity relationship study for EGCG derivatives to develop novel mitochondrial biogenesis enhancers.

Identification of Optically Active Pyrimidine Derivatives as Selective 5-HT2C Modulators.

A series of pyrimidine derivatives 4a-i were synthesized and evaluated for their binding affinities towards 5-HT2C receptors. With regard to designed molecules 4a-i, the influence of the size of alkyl ether and the absolute configuration of a stereogenic center on the 5-HT2C binding affinity and selectivity was studied. The most promising diasteromeric mixtures 4d and 4e were selected in the initial radioligand binding assay and they were further synthesized as optically active forms starting from optically active alcohols 5d and 5e, prepared by an enzymatic kinetic resolution. Pyrimidine analogue (R,R)-4e displayed an excellent 5-HT2C binding affinity with good selectivity values against a broad range of other 5-HT receptor subtypes.

A Difluoroboron ß-Diketonate Probe Shows "Turn-on" Near-Infrared Fluorescence Specific for Tau Fibrils.

Tau aggregation in neuronal cells has recently received significant attention as a robust predictor of the progression of Alzheimer's disease (AD) because of its proven correlation with the degree of cognitive impairment in AD patients. Accordingly, noninvasive imaging of tau aggregates has been highlighted as a promising diagnostic tool for AD. We have previously identified a tau-specific "turn-on" near-infrared fluorescent (NIRF) probe (1), and, in this study, structural modification was performed to optimize its physicochemical as well as fluorescence properties. Thus, a series of fluorescent dyes (2a-2j) composed of a variously substituted difluoroboron ß-diketonate and an N,N-dimethylaniline moiety linked by a length-extendable π-bridge were prepared. Among those, isobutyl-substituted difluoroboron ß-ketonate with a π-conjugated 1,4-butadienyl linker (2e) showed the most promising properties as a tau-specific NIRF probe. Compared with 1, the "turn-on" fluorescence of 2e was more specific to tau fibrils, and it showed 8.8- and 6.2-times higher tau-over-Aß and tau-over-BSA specificity, respectively. Also, the fluorescence intensity of 2e upon binding to tau fibrils was substantially higher (∼2.9 times) than that observed from 1. The mechanism for tau-specificity of 2e was investigated, which suggested that the molecular rotor-like property of 2e enables specific recognition of the microenvironment of tau aggregates to emit strong fluorescence. In transgenic cell lines stably expressing GFP-tagged tau proteins, 2e showed good colocalization with tau-GFP. Moreover, the fluorescence from 2e exhibited almost complete overlap with p-Tau antibody staining in the human AD brain tissue section. Collectively, these observations demonstrate the potential of 2e as a tau-specific fluorescent dye in both in vitro and ex vivo settings.

Neuropathic pain-alleviating effects of pyrazole-conjugated arylsulfonamides as 5-HT6 receptor antagonists.

A novel series of arylsulfonylaminomethyl-3-(1-phenyl-5-isopropyl)pyrazoles was evaluated for serotonin receptor subtype 6 (5-HT6R) antagonistic effects in vitro. We also investigated their neuropathic pain-alleviating effects in vivo using a rat spinal nerve ligation (SNL) model. Bicyclic aromatic sulfonamino groups, such as naphthalene and quinolin-substituted derivatives, showed good 5-HT6 inhibitory activity in vitro. Among them, selected compounds, 12 and 13, having 8-quinoylsulfonamino groups, showed potent neuropathic pain-alleviating effects in the rat model.