Amelioration of Astrocyte-Mediated Neuroinflammation by EI-16004 Confers Neuroprotection in an MPTP-induced Parkinson's Disease Model.

Parkinson's disease (PD) is a neurodegenerative disorder that results in motor impairment due to dopaminergic neuronal loss. The pathology of PD is closely associated with neuroinflammation, which can be characterized by astrocyte activation. Thus, targeting the inflammatory response in astrocytes might provide a novel therapeutic approach. We conducted a luciferase assay on an in-house chemical library to identify compounds with anti-inflammatory effects capable of reducing MPP+-induced NF-κB activity in astrocytes. Among the compounds identified, EI-16004, a novel 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides, exhibited a significant anti-inflammatory effect by significantly reducing MPP+-induced astrocyte activation. Biochemical analysis and docking simulation indicated that EI-16004 inhibited the MPP+-induced phosphorylation of p65 by attenuating ERK phosphorylation, and EI-16004 reduced pro-inflammatory cytokine and chemokine levels in astrocytes. In vivo studies on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model in male C57BL/6 mice showed that EI-16004 ameliorated motor impairment and protected against dopaminergic neuronal loss, and EI-16004 effectively mitigated the MPTP-induced astrocyte activation in striatum (STR) and substantia nigra (SN). These results indicate EI-16004 is a potential neuroprotective agent for the prevention and treatment of astrocyte-mediated neuroinflammatory conditions in PD.

Physicochemical characterization and phase II metabolic profiling of echinochrome A, a bioactive constituent from sea urchin, and its physiologically based pharmacokinetic modeling in rats and humans.

Echinochrome A, a natural naphthoquinone pigment found in sea urchins, is increasingly being investigated for its nutritional and therapeutic value associated with antioxidant, anticancer, antiviral, antidiabetic, and cardioprotective activities. Although several studies have demonstrated the biological effects and therapeutic potential of echinochrome A, little is known regarding its biopharmaceutical behaviors. Here, we aimed to investigate the physicochemical properties and metabolic profiles of echinochrome A and establish a physiologically-based pharmacokinetic (PBPK) model as a useful tool to support its clinical applications. We found that the lipophilicity, color variability, ultraviolet/visible spectrometry, and stability of echinochrome A were markedly affected by pH conditions. Moreover, metabolic and pharmacokinetic profiling studies demonstrated that echinochrome A is eliminated primarily by hepatic metabolism and that four possible metabolites, i.e., two glucuronidated and two methylated conjugates, are formed in rat and human liver preparations. A whole-body PBPK model incorporating the newly identified hepatic phase II metabolic process was constructed and optimized with respect to chemical-specific parameters. Furthermore, model simulations suggested that echinochrome A could exhibit linear disposition profiles without systemic and local tissue accumulation in clinical settings. Our proposed PBPK model of echinochrome A could be a valuable tool for predicting drug interactions in previously unexplored scenarios and for optimizing dosage regimens and drug formulations.

A bioisosteric approach to the discovery of novel N-aryl-N'-[4-(aryloxy)cyclohexyl]squaramide-based activators of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation.

Inhibition of translation initiation has emerging implications for the development of mechanism-based anticancer therapeutics. Phosphorylation of eIF2α is recognized as a key target that regulates the translation initiation cascade. Based on the bioisosteric replacement of urea-derived eIF2α phosphorylation activator 1, a novel series of N-aryl-N'-[4-(aryloxy)cyclohexyl]squaramide derivatives was designed and synthesized; their effects on the activation of eIF2α phosphorylation was assessed systematically. A brief structure-activity relationship analysis was established by stepwise structural optimization of the squaramide series. Subsequently, the antiproliferative activities of the selected analogues were determined in human leukemia K562 cells. We then identified 10 potent eIF2α phosphorylation activators with considerable anticancer activity. The most promising analogues 19 and 40 possessed higher cancer cell selectivity (SI = 6.16 and 4.83, respectively) than parent 1 (SI = 2.20). Finally, protein expression analysis revealed that compounds 19 and 40 induced eIF2α phosphorylation and its downstream effectors ATF4 and CHOP.

Discovery and optimization of novel 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides as bifunctional antidiabetic agents stimulating both insulin secretion and glucose uptake.

A novel series of 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamides was designed, synthesized and evaluated for their biological activities on glucose-stimulated insulin secretion (GSIS). The cytotoxicity of all 41 novel compounds was screened to assess their pharmacological safety in pancreatic ß-cells. A two-step optimization process was carried out to establish the structure-activity relationship for this class and subsequently we identified the most active analogue 26. Further modification study of 26 evidenced the necessity of N-hydrogens in the core architecture. Protein expression analysis suggested that 26 increases insulin secretion via the activation of the upstream effector of pancreatic and duodenal homeobox 1 (PDX-1), which is an important factor promoting GSIS. Moreover, the administration of 26 effectively augmented glucose uptake in C2C12 myotube cells via the suppression of Mitsugumin 53 (MG53), an insulin receptor substrate 1 (IRS-1) ubiquitination E3 ligase.

Discovery and SAR studies of 3-amino-4-(phenylsulfonyl)tetrahydrothiophene 1,1-dioxides as non-electrophilic antioxidant response element (ARE) activators.

The transcription factor NRF2 controls resistance to oxidative insult and is thus a key therapeutic target for treating a number of disease states associated with oxidative stress and aging. We previously reported CBR-470-1, a bis-sulfone which activates NRF2 by increasing the levels of methylglyoxal, a metabolite that covalently modifies NRF2 repressor KEAP1. Here, we report the design, synthesis, and structure activity relationship of a series of bis-sulfones derived from this unexplored chemical template. We identify analogs with sub-micromolar potencies, 7f and 7g, as well as establish that efficacious NRF2 activation can be achieved by non-toxic analogs 7c, 7e, and 9, a key limitation with CBR-470-1. Further efforts to identify non-covalent NRF2 activators of this kind will likely provide new insight into revealing the role of central metabolism in cellular signaling.

Pharmacokinetic Evaluation of Metabolic Drug Interactions between Repaglinide and Celecoxib by a Bioanalytical HPLC Method for Their Simultaneous Determination with Fluorescence Detection.

Since diabetes mellitus and osteoarthritis are highly prevalent diseases, combinations of antidiabetic agents like repaglinide (REP) and non-steroidal anti-inflammatory drugs (NSAID) like celecoxib (CEL) could be commonly used in clinical practice. In this study, a simple and sensitive bioanalytical HPLC method combined with fluorescence detector (HPLC-FL) was developed and fully validated for simultaneous quantification of REP and CEL. A simple protein precipitation procedure and reversed C18 column with an isocratic mobile phase (mixture of ACN and pH 6.0 phosphate buffer) were employed for sample preparation and chromatographic separation. The fluorescence detector was set at a single excitation/emission wavelength pair of 240 nm/380 nm. The linearity (10-2000 ng/mL), accuracy, precision, extraction recovery, matrix effect, and stability for this method were validated as per the current FDA guidance. The bioanalytical method was applied to study pharmacokinetic interactions between REP and CEL in vivo, successfully showing that concurrent administration with oral REP significantly altered the pharmacokinetics of oral CEL. Furthermore, an in vitro metabolism and protein binding study using human materials highlighted the possibility of metabolism-based interactions between CEL and REP in clinical settings.

Discovery of 2-aryloxy-4-amino-quinazoline derivatives as novel protease-activated receptor 2 (PAR2) antagonists.

Protease-activated receptor 2 (PAR2) is a member of G protein-coupled receptor and its activation initiates diverse inflammatory responses. Recent studies suggest that antagonists of PAR2 may provide a novel therapeutic strategy for inflammatory diseases. In this study, we have developed a series of 2-aryloxy-4-amino-quinazoline derivatives as PAR2 antagonists and examined their effects against LPS-induced inflammatory responses in RAW 264.7 macrophages. Among these derivatives, compound 2f displayed the greatest antagonistic activity with the IC50 value of 2.8µM. Binding modes of the newly identified PAR2 antagonists were analyzed by molecular docking using IFD/MM-GBSA methods in the putative binding site of PAR2 homology model. Moreover, 2f demonstrated significant inhibitory effects on the LPS-activated pro-inflammatory mediators including nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) through the regulation of various intracellular signaling pathways involving nuclear factor-κB (NF-κB), activator protein 1 (AP-1) and the mitogen-activated protein kinases (MAPK). Furthermore, administration of 2f significantly reduced the mortality of LPS-induced sepsis in mice. These results provide useful insights into the development of novel PAR2 antagonists with anti-inflammatory activity in vitro and in vivo.

Discovery and biological evaluation of tetrahydrothieno[2,3-c]pyridine derivatives as selective metabotropic glutamate receptor 1 antagonists for the potential treatment of neuropathic pain.

Metabotropic glutamate receptor 1 (mGluR1) has been a prime target for drug discovery due to its heavy involvement in various brain disorders. Recent studies suggested that mGluR1 is associated with chronic pain and can serve as a promising target for the treatment of neuropathic pain. In an effort to develop a novel mGluR1 antagonist, we designed and synthesized a library of compounds with tetrahydrothieno[2,3-c]pyridine scaffold. Among these compounds, compound 9b and 10b showed excellent antagonistic activity in vitro and demonstrated pain-suppressing activity in animal models of pain. Both compounds were orally active, and compound 9b exhibited a favorable pharmacokinetic profile in rats. We believe that these compounds can provide a promising lead compound that is suitable for the potential treatment of neuropathic pain.