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.

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.

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.

Quercetin-glutamic acid conjugate with a non-hydrolysable linker; a novel scaffold for multidrug resistance reversal agents through inhibition of P-glycoprotein.

Previously, we have reported remarkable effect of a quercetin-glutamic acid conjugate to reverse multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer agents through inhibition of P-glycoprotein (Pgp)-mediated drug efflux. Due to the hydrolysable nature, MDR-reversal activity of the quercetin conjugate was attributed to its hydrolysis product, quercetin. However, several lines of evidence demonstrated that the intact quercetin-glutamic acid conjugate has stronger MDR-reversal activity than quercetin. In order to evaluate this hypothesis and to identify a novel scaffold for MDR-reversal agents, we prepared quercetin conjugates with a glutamic acid attached at the 7-O position via a non-hydrolysable linker. Pgp inhibition assay, Pgp ATPase assay, and MDR-reversal activity assay were performed, and the non-hydrolysable quercetin conjugates showed significantly higher activities compared with those of quercetin. Unfortunately, the quercetin conjugates were not as effective as verapamil in Pgp-inhibition and thereby reversing MDR, but it is worth to note that the structurally modified quercetin conjugates with a non-cleavable linker showed significantly improved MDR-reversal activity compared with quercetin. Taken together, the quercetin conjugates with appropriate structural modifications were shown to have a potential to serve as a scaffold for the design of novel MDR-reversal agents.

A Smart Near-Infrared Fluorescence Probe for Selective Detection of Tau Fibrils in Alzheimer's Disease.

Development of a novel, tau-selective smart near-infrared fluorescence (NIRF) probe was attempted by combining the previously identified core scaffold 3,5-dimethoxy-N,N-dimethylaniline-4-yl moiety, with the characteristic donor-π-acceptor architecture of the smart NIRF Aß probes DANIR-2c and MCAAD-3. A series of compounds (2 and 3) were prepared, which were identified as "turn-on" NIRF probes for the visual detection of tau aggregates and Aß fibrils (λem = 650 nm, Stokes shifts = 70-110 nm). In particular, combination of the 3,5-dimethoxy-N,N-dimethylanilin-4-yl moiety and the donor part of MCAAD-3 endowed the resulting probes, 3g and 3h, with significant selectivity toward tau aggregates (selectivity for tau over Aß = 5.7 and 3.8); they showed much higher fluorescence intensities upon binding to tau aggregates (FItau = 49 and 108) than when bound to Aß fibrils (FIAß = 9 and 28). Quantitative analysis of binding affinities and fluorescence properties of 3g and 3h revealed that microenvironment-sensitive molecular rotor-like behavior, rather than binding affinity to the target, is responsible for their selective turn-on fluorescence detection of tau fibrils. Selective fluorescent labeling of tau fibrils by 3g and 3h was further demonstrated by immunofluorescence staining of human Alzheimer's disease brain sections, which showed colocalization of the probes (3g and 3h) and phosphorylated tau antibody.

Dicyanovinyl-substituted J147 analogue inhibits oligomerization and fibrillation of ß-amyloid peptides and protects neuronal cells from ß-amyloid-induced cytotoxicity.

A series of novel J147 derivatives were synthesized, and their inhibitory activities against ß-amyloid (Aß) aggregation and toxicity were evaluated by using the oligomer-specific antibody assay, the thioflavin-T fluorescence assay, and a cell viability assay in the transformed SH-SY5Y cell culture. Among the synthesized J147 derivatives, 3j with a 2,2-dicyanovinyl substituent showed the most potent inhibitory activity against Aß42 oligomerization (IC50 = 17.3 µM) and Aß42 fibrillization (IC50 = 10.5 µM), and disassembled the preformed Aß42 fibrils with an EC50 of 10.2 µM. Finally, we confirmed that 3j is also effective at preventing neurotoxicity induced by Aß42-oligomers as well as Aß42-fibrils.

Quercetin-POM (pivaloxymethyl) conjugates: Modulatory activity for P-glycoprotein-based multidrug resistance.

BACKGROUND: We previously demonstrated that the bioactivity of quercetin could be improved through conjugation with a hydrolysable pivaloxymethyl (POM) group. PURPOSE: Present study aimed to evaluate MDR (multidrug resistance)-modulatory activity of the quercetin-POM conjugates. STUDY DESIGN/METHODS: MDR-modulatory activity was determined by measuring cytotoxicity of various anticancer agents to MDR MES-SA/Dx5 cell lines upon combination with the quercetin-POM conjugates. RESULTS: The quercetin-7-O-POM conjugate (7-O-POM-Q) was significantly more potent than quercetin in reversing MDR, which recovered the cytotoxicity of various anticancer agents with EC50 values of 1.1-1.3 µM. A series of mechanistic studies revealed that 7-O-POM-Q competes with verapamil in binding to the same drug-binding site of the major MDR target, Pgp (P-glycoprotein), and inhibits Pgp-mediated drug efflux with a similar potency as verapamil. The physicochemical properties of 7-O-POM-Q were then evaluated, which confirmed that 7-O-POM-Q has remarkably enhanced cellular uptake and intracellular localization compared with quercetin. Additionally, it is noteworthy that 7-O-POM-Q undergoes slow hydrolysis to quercetin over a prolonged period of time. CONCLUSION: The quercetin-POM conjugate showed significantly improved MDR-reversing activity compared with quercetin, which could be attributed to its capacity to maintain high intracellular concentrations.

Water-soluble and cleavable quercetin-amino acid conjugates as safe modulators for P-glycoprotein-based multidrug resistance.

Quercetin-amino acid conjugates with alanine or glutamic acid moiety attached at 7-O and/or 3-O position of quercetin were prepared, and their multidrug resistance (MDR)-modulatory effects were evaluated. A quercetin-glutamic acid conjugate, 7-O-Glu-Q (3a), was as potent as verapamil in reversing MDR and sensitized MDR MES-SA/Dx5 cells to various anticancer drugs with EC50 values of 0.8-0.9 µM. Analysis on Rh-123 accumulation confirmed that 3a inhibits drug efflux by Pgp, and Pgp ATPase assay showed that 3a interacts with the drug-binding site of Pgp to stimulate its ATPase activity. Physicochemical analysis of 3a revealed that solubility, stability, and cellular uptake of quercetin were significantly improved by the glutamic acid promoiety, which eventually dissociates from 3a to produce quercetin and quercetin metabolites in intracellular milieu. Taken together, potent MDR-modulating activity along with intracellular conversion into the natural flavonoid quercetin warrants development of the quercetin-amino acid conjugates as safe MDR modulators.

Novel thienopyrimidinones as mGluR1 antagonists.

There has been much attention to discover mGluR1 antagonists for treating various central nervous system diseases such as seizures and neuropathic pain. Thienopyrimidinone derivatives were designed, synthesized, and biologically evaluated against mGluR1. Among the synthesized compounds, 3-(4-methoxyphenyl)-7-(o-tolyl)thienopyrimidin-4-one 30 exhibited the most potent inhibitory activity with an IC50 value of 45 nM and good selectivity over mGluR5. Also, the selective mGluR1 antagonist 30 showed marginal hERG channel activity (IC50 = 9.87 µM), good profiles to CYP isozymes, and a good pharmacokinetic profile. Overall, the compound 30 was identified as a selective mGluR1 antagonist with a good pharmacokinetic profile, which is probably devoid of cardiac side effect and drug-drug interactions. Therefore, the compound 30 can be expected to be broadly used as mGluR1 antagonistic chemical probe in in vitro and in vivo study for investigating CNS diseases.

Synthesis and biological evaluation of aryloxazole derivatives as antimitotic and vascular-disrupting agents for cancer therapy.

A series of aryloxazole, thiazole, and isoxazole derivatives was synthesized as vascular-targeting anticancer agents. Antiproliferative activity and tumor vascular-disrupting activity of all of the synthesized compounds were tested in vitro using various human cancer cell lines and HUVECs (human umbilical vein endothelial cells). Several compounds with an arylpiperazinyl oxazole core showed excellent cytotoxicity and metabolic stability in vitro. Among this series, two representative compounds (6-48 and 6-51) were selected and tested for the evaluation of anticancer effects in vivo using tumor-bearing mice. Compound 6-48 effectively reduced tumor growth (42.3% reduction in size) at the dose of 100 mg/kg. We believe that compound 6-48 will serve as a good lead compound for antimitotic and vascular-disrupting agents; further investigation to improve the in vivo efficacy of this series is underway.

Quercetin-POC conjugates: Differential stability and bioactivity profiles between breast cancer (MCF-7) and colorectal carcinoma (HCT116) cell lines.

In the course of our ongoing efforts to develop novel quercetin conjugates with enhanced stability profiles, we introduced an isopropyloxycarbonylmethoxy (POC) group to 7-OH and/or 3-OH of quercetin and prepared three novel quercetin conjugates. The quercetin-POC conjugates were stable up to 96 h in PBS but slowly hydrolyzed with half-lives of 1-54 h in cell-free culture medium, which is reminiscent of the stability profiles of the previously reported quercetin-POM (pivaloxymethyl) conjugates. However, the quercetin-POC conjugates were more susceptible to passive transport, intracellular hydrolysis, and metabolism in breast cancer (MCF-7) cell line compared with their POM congeners to result in low concentration of quercetin in this cell line and thereby low antiproliferative effect. In contrast, upon incubation with colorectal carcinoma HCT116 cells, the quercetin-POC conjugates were shown to undergo slow hydrolysis and metabolism to maintain concentrations of the active quercetin species high enough to exert enhanced cytotoxicity. Taken together, the quercetin-POC conjugates synthesized in this study exhibited cell type-specific stability as well as bioactivity profiles, which warrants further investigation into the underlying mechanisms and therapeutic potential.

Separation of quercetin's biological activity from its oxidative property through bioisosteric replacement of the catecholic hydroxyl groups with fluorine atoms.

An oxidative property has endowed quercetin with numerous biological benefits, and some of quercetin's biological activities may be related, at least partly, to its antioxidant activity. On the other hand, the oxidative property and associated susceptibility to oxidative decomposition has hampered in-depth investigation of the biological targets as well as underlying mechanisms for quercetin's biological activity. This study was undertaken to separate quercetin's biological activities from its antioxidant properties through bioisosteric replacement of the phenolic hydroxyl groups. The novel quercetin derivative 3',4'-difluoroquercetin (2), thus prepared, showed nonoxidizable property with no attenuation of biological activity. Rather, 2 showed a subtle but significant increase in biological activity compared with quercetin, which might be attributed to its lack of oxidative property. The nonoxidizable nature along with the potent biological activity of the quercetin mimic 2 suggests possible oxidation-independent mechanisms for the biological activities of the quercetin that do not require oxidative formation of the highly electrophilic metabolites.

2,6-Bis-arylmethyloxy-5-hydroxychromones with antiviral activity against both hepatitis C virus (HCV) and SARS-associated coronavirus (SCV).

In this study, as a bioisosteric alternative scaffold of the antiviral aryl diketoacids (ADKs), we used 5-hydroxychromone on which two arylmethyloxy substituents were installed. The 5-hydroxychromones (5b-5g) thus prepared showed anti-HCV activity and, depending on the aromatic substituents on the 2-arylmethyloxy moiety, some of the derivatives (5b-5f) were also active against SCV. In addition, unlike the ADKs which showed selective inhibition against the helicase activity of the SCV NTPase/helicase, the 5-hydroxychromones (5b-5f) were active against both NTPase and helicase activities of the target enzyme. Among those, 3-iodobenzyloxy-substituted derivative 5e showed the most potent activity against HCV (EC(50) = 4 µM) as well as SCV (IC(50) = 4 µM for ATPase activity, 11 µM for helicase activity) and this might be used as a platform structure for future development of the multi-target or broad-spectrum antivirals.

Discovery of piperidinyl aminopyrimidine derivatives as IKK-2 inhibitors.

A serine-threonine kinase IKK-2 plays an important role in activation of NF-κB through phosphorylation of the inhibitor of NF-κB (IκB). As NF-κB is a major transcription factor that regulates genes responsible for cell proliferation and inflammation, development of selective IKK-2 inhibitors has been an important area of anti-inflammatory and anti-cancer research. In this study, to obtain active and selective IKK-2 inhibitors, various substituents were introduced to a piperidinyl aminopyrimidine core structure. The structure-activity relationship study indicated that hydrogen, methanesulfonyl, and aminosulfonyl groups substituted at the piperidinylamino functionality provide high inhibitory activity against IKK-2. Also, morpholinosulfonyl and piperazinosulfonyl group substituted at the aromatic ring attached to the aminopyrimidine core significantly increased the inhibitory activity of the resulting derivatives. In particular, compound 17 with the aromatic piperazinosulfonyl substituent showed the most potent (IC(50)=1.30 µM) and selective (over other kinases such as p38α, p38ß, JNK1, JNK2, JNK3, and IKK-1) inhibitory activity against IKK-2.

IKKß inhibitor identification: a multi-filter driven novel scaffold.

BACKGROUND: Nuclear factor kappa B (NF-κB) is a chief nuclear transcription factor that controls the transcription of various genes; and its activation is tightly controlled by Inhibitor kappa B kinase (IKK). The irregular transcription of NF-κB has been linked to auto-immune disorders, cancer and other diseases. The IKK complex is composed of three units, IKKα, IKKß, and the regulatory domain NEMO, of which IKKß is well understood in the canonical pathway. Therefore, the inhibition of IKKß by drugs forms the molecular basis for anti-inflammatory drug research. RESULTS: The ligand- and structure-based virtual screening (VS) technique has been applied to identify IKKß inhibitors from the ChemDiv database with 0.7 million compounds. Initially, a 3D-QSAR pharmacophore model has been deployed to greatly reduce the database size. Subsequently, recursive partitioning (RP) and docking filters were used to screen the pharmacophore hits. Finally, 29 compounds were selected for IKKß enzyme inhibition assay to identify a novel small molecule inhibitor of IKKß protein. CONCLUSIONS: In the present investigation, we have applied various computational models sequentially to virtually screen the ChemDiv database, and identified a small molecule that has an IC50 value of 20.3µM. This compound is novel among the known IKKß inhibitors. Further optimization of the hit compound can reveal a more potent anti-inflammatory agent.

Enhanced stability and intracellular accumulation of quercetin by protection of the chemically or metabolically susceptible hydroxyl groups with a pivaloxymethyl (POM) promoiety.

In order to increase stability of quercetin, its metabolically and chemically susceptible hydroxyl groups 7-OH and 3-OH respectively were transiently blocked with a pivaloxymethyl (POM) promoiety to provide two novel quercetin conjugates [7-O-POM-Q, 3-O-POM-Q]. In the absence of stabilizer (ascorbic acid), the synthesized conjugates showed significantly increased stability in cell culture media [t(½) = 4 h, 52 h] compared with quercetin (t(½) < 30 min) and quercetin prodrug 1 (t(½) = 0.8 h). In addition, the quercetin conjugate 2 underwent efficient cellular uptake and intracellular levels of its hydrolysis product, quercetin, were maintained up to 12 h. Stability and intracellular accumulation of were demonstrated by its stabilizer-independent cytostatic effect and induction of apoptotic cell death. Even though was more stable than, it failed to penetrate cell membranes. However, the remarkable stability of warrants further investigation of quercetin conjugates with various promoieties at the 3-OH position.

5-HT3 antagonists under development.

IMPORTANCE OF THE FIELD: The main therapeutic area of 5-HT(3) receptor antagonists is the treatment of chemotherapy-induced nausea and vomiting (CINV), which is the most common and distressing side effects of anticancer treatment. The second major clinical application of 5-HT(3) receptor antagonists is in the treatment of irritable bowel syndrome (IBS). 5-HT(3) antagonists have been widely used and found to decrease gut transit, increase fluid absorption and reduce pain. The uses of 5-HT(3) antagonists are expending to treatment of CNS diseases such as anxiety and sleep disorders as antipsychotics and so on. AREAS COVERED IN THIS REVIEW: The structures, in vitro activities, in vivo effects and some clinical data on 5-HT(3) receptor antagonists under development. WHAT THE READER WILL GAIN: Future research directions in 5-HT(3) antagonists based on the clinical trial data of the pipeline molecules. TAKE HOME MESSAGE: Most drug candidates in clinical trials were discovered in the early 1990s and their patent expiry is imminent. Acquiring intellectual properties of novel 5-HT(3) receptor antagonists with improved efficacies would provide a bright future. Particularly, as the current 5-HT(3) receptor antagonists are classified into only three representative structural families (one third are zacopride-like benzamides and the others are ondansteron-like tricyclic compounds and dolansetron-like bicyclic compounds), structurally diverse compound libraries need to be extensively investigated for identification of novel 5-HT(3) receptor antagonists.