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Neuropathic pain (NP) is a chronic condition resulting from damaged pain-signaling pathways. It is a debilitating disorder that affects up to 10% of the world’s population. Although opioid analgesics are effective in reducing pain, they present severe risks; so, there is a pressing need for non-opioid pain-relieving drugs. One potential alternative is represented by sigma-1 receptor (S1R) antagonists due to their promising analgesic effects. Here, we report the synthesis and biological evaluation of a series of S1R antagonists based on a 2-aryl-4-aminobutanol scaffold. After assessing affinity toward the S1R and selectivity over the sigma-2 receptor (S2R), we evaluated the agonist/antagonist profile of the compounds by investigating their effects on nerve growth factor-induced neurite outgrowth and aquaporin-mediated water permeability in the presence and absence of oxidative stress. (R/S)-RC-752 emerged as the most interesting compound for S1R affinity (Ki S1R = 6.2 ± 0.9) and functional antagonist activity. Furthermore, it showed no cytotoxic effect in two normal human cell lines or in an in vivo zebrafish model and was stable after incubation in mouse plasma. (R/S)-RC-752 was then evaluated in two animal models of NP: the formalin test and the spinal nerve ligation model. The results clearly demonstrated that compound (R/S)-RC-752 effectively alleviated pain in both animal models, thus providing the proof of concept of its efficacy as an antinociceptive agent.

The toxic pyroglutamate form of amyloid-β (pE-Aβ) is important for the pathogenesis of early Alzheimer's disease (AD); therefore, reducing pE-Aβ by inhibiting glutaminyl cyclase (QC) provides a promising strategy for developing disease-modifying AD drugs. In this study, potent and selective QC inhibitors with desirable drug-like properties were discovered by replacing the 3,4-dimethoxyphenyl group in a QC inhibitor with a bioisosteric indazole surrogate. Among them, 3-methylindazole-6-yl and 3-methylindazole-5-yl derivatives with an N-cyclohexylurea were identified as highly potent inhibitors with IC

The inhibition of glutaminyl cyclase (QC) may provide a promising strategy for the treatment of early Alzheimer's disease (AD) by reducing the amount of the toxic pyroform of β-amyloid (AβΝ3pE) in the brains of AD patients. In this work, we identified potent QC inhibitors with subnanomolar IC50 values that were up to 290-fold higher than that of PQ912, which is currently being tested in Phase II clinical trials. Among the tested compounds, the cyclopentylmethyl derivative (214) exhibited the most potent in vitro activity (IC50 = 0.1 nM), while benzimidazole (227) showed the most promising in vivo efficacy, selectivity and druggable profile. 227 significantly reduced the concentration of pyroform Aβ and total Aβ in the brain of an AD animal model and improved the alternation behavior of mice during Y-maze tests. The crystal structure of human QC (hQC) in complex with 214 indicated tight binding at the active site, supporting that the specific inhibition of QC results in potent in vitro and in vivo activity. Considering the recent clinical success of donanemab, which targets AβΝ3pE, small molecule-based QC inhibitors may also provide potential therapeutic options for early-stage AD treatment.

Increased O-GlcNAcylation can confer neuroprotective effects against necroptosis in the context of Alzheimer’s disease., O-GlcNAcylation (O-linked β-N-acetylglucosaminylation) is notably decreased in Alzheimer’s disease (AD) brain. Necroptosis is activated in AD brain and is positively correlated with neuroinflammation and tau pathology. However, the links among altered O-GlcNAcylation, β-amyloid (Aβ) accumulation, and necroptosis are unclear. Here, we found that O-GlcNAcylation plays a protective role in AD by inhibiting necroptosis. Necroptosis was increased in AD patients and AD mouse model compared with controls; however, decreased necroptosis due to O-GlcNAcylation of RIPK3 (receptor-interacting serine/threonine protein kinase 3) was observed in 5xFAD mice with insufficient O-linked β-N-acetylglucosaminase. O-GlcNAcylation of RIPK3 suppresses phosphorylation of RIPK3 and its interaction with RIPK1. Moreover, increased O-GlcNAcylation ameliorated AD pathology, including Aβ burden, neuronal loss, neuroinflammation, and damaged mitochondria and recovered the M2 phenotype and phagocytic activity of microglia. Thus, our data establish the influence of O-GlcNAcylation on Aβ accumulation and neurodegeneration, suggesting O-GlcNAcylation–based treatments as potential interventions for AD.

We developed an orally active and blood-brain-barrier-permeable benzofuran analogue (8, MDR-1339) with potent antiaggregation activity. Compound 8 restored cellular viability from Aβ-induced cytotoxicity but also improved the learning and memory function of AD model mice by reducing the Aβ aggregates in the brains. Given the high bioavailability and brain permeability demonstrated in our pharmacokinetic studies, 8 will provide a novel scaffold for an Aβ-aggregation inhibitor that may offer an alternative treatment for AD.

A series consisting of 117 2-(halogenated phenyl) acetamide and propanamide analogs were investigated as TRPV1 antagonists. The structure–activity analysis targeting their three pharmacophoric regions indicated that halogenated phenyl A-region analogs exhibited a broad functional profile ranging from agonism to antagonism. Among the compounds, antagonists 28 and 92 exhibited potent antagonism toward capsaicin for hTRPV1 with Ki[CAP] = 2.6 and 6.9 nM, respectively. Further, antagonist 92 displayed promising analgesic activity in vivo in both phases of the formalin mouse pain model. A molecular modeling study of 92 indicated that the two fluoro groups in the A-region made hydrophobic interactions with the receptor.

A series of 1-indazol-3-(1-phenylpyrazol-5-yl)methyl ureas were investigated as hTRPV1 antagonists. The structure–activity relationship study was conducted systematically for both the indazole A-region and the 3-trifluoromethyl/t-butyl pyrazole C-region to optimize the antagonism toward the activation by capsaicin. Among them, the antagonists 26, 50 and 51 displayed highly potent antagonism with Ki(CAP) = 0.4–0.5 nM. Further, in vivo studies in mice indicated that these derivatives both antagonized capsaicin induced hypothermia, consistent with their in vitro activity, and themselves did not induce hyperthermia. In the formalin model, 51 showed anti-nociceptive activity in a dose-dependent manner.

A series of indane-type acetamide and propanamide analogues were investigated as TRPV1 antagonists. The analysis of structure–activity relationship indicated that indane A-region analogues exhibited better antagonism than did the corresponding 2,3-dihydrobenzofuran and 1,3-benzodioxole surrogates. Among them, antagonist 36 exhibited potent and selective antagonism toward capsaicin for hTRPV1 and mTRPV1. Further, in vivo studies indicated that antagonist 36 showed excellent analgesic activity in both phases of the formalin mouse pain model and inhibited the pain behavior completely at a dose of 1 mg/kg in the 2nd phase.

Pyroglutamate-modified amyloid β peptides (pGlu-Aβ) are highly neurotoxic and promote the formation of amyloid plaques. The pGlu-Aβ peptides are generated by glutaminyl cyclase (QC), and recent clinical studies indicate that QC represents an alternative therapeutic target to treat Alzheimer’s disease (AD). We have previously developed a series of QC inhibitors with an extended pharmacophoric scaffold, termed the Arg-mimetic D-region. In the present study, we focused on the structure activity relationship (SAR) of analogues with modifications in the D-region and evaluated their biological activity. Most compounds in this series exhibited potent activity in vitro, and our SAR analysis and the molecular docking studies identified compound 202 as a potential candidate because it forms an additional hydrophobic interaction in the h QC active site. Overall, our study provides valuable insights into the Arg-mimetic pharmacophore that will guide the design of novel QC inhibitors as potential treatments for AD.

Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. We generated an AD Tg pig by somatic cell nuclear transfer (SCNT) using a multi-cistronic vector that harbored three AD-related genes with a total of six well-characterized mutations: hAPP (K670N/M671L, I716V, and V717I), hTau (P301L), and hPS1 (M146V and L286P). Four AD Tg cell lines were established from Jeju black pig ear fibroblasts (JB-PEFs); the resultant JB-PEFAD cells harbored transgene integration, expressed transgene mRNAs, and had normal karyotypes. Tg line #2-1, which expressed high levels of the transgenes, was used for SCNT; cleavage and blastocyst rates of embryos derived from this line were lower than those of Non-Tg. These embryos yielded three piglets (Jeju National University AD-Tg pigs, JNUPIGs) revealed by microsatellite testing to be genetically identical to JB-PEFAD. Transgenes were expressed in multiple tissues, and at especially high levels in brain, and Aβ-40/42, total Tau, and GFAP levels were high in brains of the Tg animals. Five or more copies of transgenes were inserted into chromosome X. This is the first report of an AD Tg pig derived from a multi-cistronic vector.

In order to discover a novel type of analgesic, we investigated dual activity ligands with TRPV1 antagonism and mu-opioid receptor affinity with the goal of eliciting synergistic analgesia while avoiding the side effects associated with single targeting. Based on a combination approach, a series of 4-benzyl-4-(dimethylamino)piperidinyl analogues were designed, synthesized and evaluated for their receptor activities. Among them, compound 49 exhibited the most promising dual-acting activity toward TRPV1 and the mu-opioid receptor in vitro. In vivo, 49 displayed potent, dose-dependent antinociceptive activity in both the 1st and 2nd phases in the formalin assay. Consistent with its postulated mechanism, we confirmed that in vivo, as in vitro, compound 49 both antagonized TRPV1 and functioned as a mu-opioid agonist. This result indicates that dual-acting TRPV1 antagonist/mu-opioid ligands can be made and represent a new and promising class of analgesic.

Amyloid-β (Aβ) peptide is central to the development of brain pathology in Alzheimer disease (AD) patients. Association with receptors for advanced glycation end-products (RAGE) enables the transport of Aβ peptide from circulating blood to human brain, and also causes the activation of the NF-κB signaling pathway. Here we show that two β-strands of RAGE participate in the interaction with Aβ peptide. Serial deletion analysis of the RAGE V domain indicates that the third and eighth β-strands are required for interaction with Aβ peptide. Site-directed mutagenesis of amino acids located in the third and eighth β-strands abolish the interaction of RAGE with Aβ peptide. Wild-type RAGE activates the NF-κB signaling pathway in response to Aβ peptide treatment, while a RAGE mutant defective in Aβ binding does not. Furthermore, use of peptide for the third β-strand or a RAGE monoclonal antibody that targets the RAGE-Aβ interaction interface inhibited transport of the Aβ peptide across the blood brain barrier in a mice model. These results provide information crucial to the development of RAGE-derived therapeutic reagents for Alzheimer disease.

Current evidence suggests a central role for autophagy in Alzheimer disease (AD), and dysfunction in the autophagic system may lead to amyloid-β (Aβ) accumulation. Using in vitro and in vivo AD models, the present study investigated whether mesenchymal stem cells (MSCs) could enhance autophagy and thus exert a neuroprotective effect through modulation of Aβ clearance In Aβ-treated neuronal cells, MSCs increased cellular viability and enhanced LC3-II expression compared with cells treated with Aβ only. Immunofluorescence revealed that MSC coculture in Aβ-treated neuronal cells increased the number of LC3-II-positive autophagosomes that were colocalized with a lysosomal marker. Ultrastructural analysis revealed that most autophagic vacuoles (AVs) in Aβ-treated cells were not fused with lysosomes, whereas a large portion of autophagosomes were conjoined with lysosomes in MSCs cocultured with Aβ-treated neuronal cells. Furthermore, MSC coculture markedly increased Aβ immunoreactivity colocalized within lysosomes and decreased intracellular Aβ levels compared with Aβ-treated cells. In Aβ-treated animals, MSC administration significantly increased autophagosome induction, final maturation of late AVs, and fusion with lysosomes. Moreover, MSC administration significantly reduced the level of Aβ in the hippocampus, which was elevated in Aβ-treated mice, concomitant with increased survival of hippocampal neurons. Finally, MSC coculture upregulated BECN1/Beclin 1 expression in AD models. These results suggest that MSCs significantly enhance autolysosome formation and clearance of Aβ in AD models, which may lead to increased neuronal survival against Aβ toxicity. Modulation of the autophagy pathway to repair the damaged AD brain using MSCs would have a significant impact on future strategies for AD treatment.

Receptor for advanced glycation end products (RAGE) is known to be involved in the transportation of amyloid β (Aβ) peptides and causes the accumulation of Aβ in the brain. Moreover, recent studies suggest that the interactions between RAGE and Aβ peptides may be the culprit behind Alzheimer's disease (AD). Inhibitors of the RAGE-Aβ interactions would not only prevent the accumulation of toxic Aβ in the brain, and but also block the progress of AD, therefore, have the potential to provide a 'disease-modifying therapy'. In this study, we have developed a series of 6-phenoxy-2-phenylbenzoxazole analogs as novel inhibitors of RAGE. Among these derivatives, we found several effective inhibitors that block the RAGE-Aβ interactions without causing significant cellular toxicity. Further testing showed that compound 48 suppressed Aβ induced toxicity in mouse hippocampal neuronal cells and reduced Aβ levels in the brains of a transgenic mouse model of AD after oral administration.

The structure-activity relationships for the 'B-region' of N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues have been investigated as TRPV1 receptor antagonists. A docking model of potent antagonist 2 with the sensor region of TRPV1 is proposed.

Isosteric replacement of the phenolic hydroxyl group in potent vanilloid receptor (VR1) agonists with the alkylsulfonamido group provides a series of compounds which are effective antagonists to the action of the capsaicin on rat VR1 heterologously expressed in Chinese hamster ovary (CHO) cells. In particular, compound 61, N-[2-(3,4-dimethylbenzyl)-3-pivaloyloxypropyl]-N'-[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea was a full antagonist against capsaicin, displayed a K(i) value of 7.8 nM (compared to 520 nM for capsazepine and 4 nM for 5-iodoRTX), and showed excellent analgesic activity in mice. Structure-activity analysis of the influence of modifications in the A- and C-regions of 4-methylsulfonamide ligands on VR1 agonism/antagonism indicated that 3-fluoro substitution in the A-region and a 4-tert-butylbenzyl moiety in the C-region favored antagonism, whereas a 3-methoxy group in the A-region and 3-acyloxy-2-benzylpropyl moieties in the C-region favored agonism.

In an effort to develop novel inhibitors of receptor for advanced glycation end products (RAGE) for the treatment of Alzheimer's disease, a series of pyrazole-5-carboxamides were designed, synthesized and biologically evaluated. Analyses of the extensive structure–activity relationship (SAR) led us to identify a 4-fluorophenoxy analog (40) that exhibited improved in vitro RAGE inhibitory activity and more favorable aqueous solubility than the parent 2-aminopyrimidine, 1. Surface plasmon resonance (SPR) and molecular docking study strongly supported the RAGE inhibitory activity of pyrazole-5-carboxamides. The brain Aβ-lowering effect of 40 is also described.

Using the approach of ligand-based drug design, we discovered a novel series of 4,6-disubstituted 2-aminopyrimidines as RAGE inhibitors. In transgenic mouse models of AD, one of the 4,6-bis(4-chlorophenyl)pyrimidine analogs, 59, significantly lowered the concentration of toxic soluble Aβ in the brain and improved cognitive function. SPR analysis confirmed the direct binding of 59 with RAGE, which should contribute to its biological activities via inhibition of the RAGE–Aβ interaction. We also predicted the binding mode of the 4,6-bis(4-chlorophenyl)pyrimidine analogs to the RAGE V-domain through flexible docking study.

To investigate how neuronal activity in the prefrontal cortex changes in an animal model of schizophrenia, we recorded single unit activity in the medial prefrontal cortex of urethane-anesthetized and awake rats following methamphetamine (MA) administration. Systemic MA injection (4 mg/kg, IP) induced inconsistent changes, that is, both enhancement and reduction, in unit discharge rate, with a subset of neurons transiently (<30 min) elevating their activities. The direction of firing rate change was poorly predicted by the mean firing rate or the degree of burst firing during the baseline period. Also, simultaneously recorded units showed opposite directions of firing rate change, indicating that recording location is a poor predictor of the direction of firing rate change. These results raise the possibility that systemic MA injection induces random bidirectional changes in prefrontal cortical unit activity, which may underlie some of MA-induced psychotic symptoms.

The structure-activity relationships for the 'A-region' of N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues have been investigated as TRPV1 receptor antagonists. The 2-halogen analogues showed enhanced antagonism compared to the prototype antagonist.