Discovery of RC-752, a Novel Sigma-1 Receptor Antagonist with Antinociceptive Activity: A Promising Tool for Fighting Neuropathic Pain.

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.

Discovery of potent indazole-based human glutaminyl cyclase (QC) inhibitors as Anti-Alzheimer's disease agents.

The toxic pyroglutamate form of amyloid-ß (pE-Aß) is important for the pathogenesis of early Alzheimer's disease (AD); therefore, reducing pE-Aß 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 IC50 values of 3.2 nM and 2.3 nM, respectively, both of which were approximately 10-fold more potent than varoglutamstat. In addition, the three inhibitors significantly reduced pE-Aß3-40 levels in an acute animal model after intracerebroventricular (icv) injection and were selective for hQC. Further in vitro pharmacokinetic and toxicity studies, including those investigating cytotoxicity, hERG inhibition, blood-brain barrier (BBB) permeability and metabolic stability, indicated that N-(3-methylindazole-6-yl)-N'-(cyclohexyl)urea derivative exhibited the most promising efficacy, selectivity and drug-like profile; thus, it was evaluated for its in vivo efficacy in an AD model.

Discovery of highly potent human glutaminyl cyclase (QC) inhibitors as anti-Alzheimer's agents by the combination of pharmacophore-based and structure-based design.

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 ß-amyloid (AßΝ3pE) 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 Aß and total Aß 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 AßΝ3pE, small molecule-based QC inhibitors may also provide potential therapeutic options for early-stage AD treatment.

O-GlcNAcylation ameliorates the pathological manifestations of Alzheimer's disease by inhibiting necroptosis.

O-GlcNAcylation (O-linked ß-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, ß-amyloid (Aß) 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 ß-N-acetylglucosaminase. O-GlcNAcylation of RIPK3 suppresses phosphorylation of RIPK3 and its interaction with RIPK1. Moreover, increased O-GlcNAcylation ameliorated AD pathology, including Aß 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 Aß accumulation and neurodegeneration, suggesting O-GlcNAcylation-based treatments as potential interventions for AD.

Discovery of Benzopyridone-Based Transient Receptor Potential Vanilloid 1 Agonists and Antagonists and the Structural Elucidation of Their Activity Shift.

Among a series of benzopyridone-based scaffolds investigated as human transient receptor potential vanilloid 1 (TRPV1) ligands, two isomeric benzopyridone scaffolds demonstrated a consistent and distinctive functional profile in which 2-oxo-1,2-dihydroquinolin-5-yl analogues (e.g., 2) displayed high affinity and potent antagonism, whereas 1-oxo-1,2-dihydroisoquinolin-5-yl analogues (e.g., 3) showed full agonism with high potency. Our computational models provide insight into the agonist-antagonist boundary of the analogues suggesting that the Arg557 residue in the S4-S5 linker might be important for sensing the agonist binding and transmitting signals. These results provide structural insights into the TRPV1 and the protein-ligand interactions at a molecular level.

Discovery of Nonpungent Transient Receptor Potential Vanilloid 1 (TRPV1) Agonist as Strong Topical Analgesic.

Paradoxically, some TRPV1 agonists are, at the organismal level, both nonpungent and clinically useful as topical analgesics. Here, we describe the scaled-up synthesis and characterization in mouse models of a novel, nonpungent vanilloid. Potent analgesic activity was observed in models of neuropathic pain, and the compound blocked capsaicin induced allodynia, showing dermal accumulation with little transdermal absorption. Finally, it displayed much weaker systemic toxicity compared to capsaicin and was negative in assays of genotoxicity.

Discovery of Conformationally Restricted Human Glutaminyl Cyclase Inhibitors as Potent Anti-Alzheimer's Agents by Structure-Based Design.

Alzheimer's disease (AD) is an incurable, progressive neurodegenerative disease whose pathogenesis cannot be defined by one single element but consists of various factors; thus, there is a call for alternative approaches to tackle the multifaceted aspects of AD. Among the potential alternative targets, we aim to focus on glutaminyl cyclase (QC), which reduces the toxic pyroform of ß-amyloid in the brains of AD patients. On the basis of a putative active conformation of the prototype inhibitor 1, a series of N-substituted thiourea, urea, and α-substituted amide derivatives were developed. The structure-activity relationship analyses indicated that conformationally restrained inhibitors demonstrated much improved QC inhibition in vitro compared to nonrestricted analogues, and several selected compounds demonstrated desirable therapeutic activity in an AD mouse model. The conformational analysis of a representative inhibitor indicated that the inhibitor appeared to maintain the Z-E conformation at the active site, as it is critical for its potent activity.

Structure-activity relationship investigation of Phe-Arg mimetic region of human glutaminyl cyclase inhibitors.

Glutamyl cyclase (QC) is a promising therapeutic target because of its involvement in the pathogenesis of Alzheimer's disease. In this study, we developed novel QC inhibitors that contain 3-aminoalkyloxy-4-methoxyphenyl and 4-aminoalkyloxyphenyl groups to replace the previously developed pharmacophore. Several potent inhibitors were identified, showing IC50 values in a low nanomolar range, and were further studied for in vitro toxicity and in vivo activity. Among these, inhibitors 51 and 53 displayed the most potent AßN3pE-40-lowering effects in in vivo acute model with reasonable BBB penetration, without showing cytotoxicity and hERG inhibition. The molecular modeling analysis of 53 indicated that the salt bridge interaction and the hydrogen bonding in the active site provided a high potency. Given the potent activity and favorable BBB penetration with low cytotoxicity, we believe that compound 53 may serve as a potential candidate for anti-Alzheimer's agents.

Potent human glutaminyl cyclase inhibitors as potential anti-Alzheimer's agents: Structure-activity relationship study of Arg-mimetic region.

Pyroglutamate-modified amyloid ß peptides (pGlu-Aß) are highly neurotoxic and promote the formation of amyloid plaques. The pGlu-Aß 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 hQC 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.

Discovery of an Orally Bioavailable Benzofuran Analogue That Serves as a ß-Amyloid Aggregation Inhibitor for the Potential Treatment of Alzheimer's Disease.

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 Aß-induced cytotoxicity but also improved the learning and memory function of AD model mice by reducing the Aß aggregates in the brains. Given the high bioavailability and brain permeability demonstrated in our pharmacokinetic studies, 8 will provide a novel scaffold for an Aß-aggregation inhibitor that may offer an alternative treatment for AD.

Production of transgenic pig as an Alzheimer's disease model using a multi-cistronic vector system.

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 Aß-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.

Discovery of Potent Human Glutaminyl Cyclase Inhibitors as Anti-Alzheimer's Agents Based on Rational Design.

Glutaminyl cyclase (QC) has been implicated in the formation of toxic amyloid plaques by generating the N-terminal pyroglutamate of ß-amyloid peptides (pGlu-Aß) and thus may participate in the pathogenesis of Alzheimer's disease (AD). We designed a library of glutamyl cyclase (QC) inhibitors based on the proposed binding mode of the preferred substrate, Aß3E-42. An in vitro structure-activity relationship study identified several excellent QC inhibitors demonstrating 5- to 40-fold increases in potency compared to a known QC inhibitor. When tested in mouse models of AD, compound 212 significantly reduced the brain concentrations of pyroform Aß and total Aß and restored cognitive functions. This potent Aß-lowering effect was achieved by incorporating an additional binding region into our previously established pharmacophoric model, resulting in strong interactions with the carboxylate group of Glu327 in the QC binding site. Our study offers useful insights in designing novel QC inhibitors as a potential treatment option for AD.

6-Phenoxy-2-phenylbenzoxazoles, novel inhibitors of receptor for advanced glycation end products (RAGE).

Receptor for advanced glycation end products (RAGE) is known to be involved in the transportation of amyloid ß (Aß) peptides and causes the accumulation of Aß in the brain. Moreover, recent studies suggest that the interactions between RAGE and Aß peptides may be the culprit behind Alzheimer's disease (AD). Inhibitors of the RAGE-Aß interactions would not only prevent the accumulation of toxic Aß 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-Aß interactions without causing significant cellular toxicity. Further testing showed that compound 48 suppressed Aß induced toxicity in mouse hippocampal neuronal cells and reduced Aß levels in the brains of a transgenic mouse model of AD after oral administration.

Pyrazole-5-carboxamides, novel inhibitors of receptor for advanced glycation end products (RAGE).

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 Aß-lowering effect of 40 is also described.

Mesenchymal stem cells enhance autophagy and increase ß-amyloid clearance in Alzheimer disease models.

Current evidence suggests a central role for autophagy in Alzheimer disease (AD), and dysfunction in the autophagic system may lead to amyloid-ß (Aß) 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 Aß clearance In Aß-treated neuronal cells, MSCs increased cellular viability and enhanced LC3-II expression compared with cells treated with Aß only. Immunofluorescence revealed that MSC coculture in Aß-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 Aß-treated cells were not fused with lysosomes, whereas a large portion of autophagosomes were conjoined with lysosomes in MSCs cocultured with Aß-treated neuronal cells. Furthermore, MSC coculture markedly increased Aß immunoreactivity colocalized within lysosomes and decreased intracellular Aß levels compared with Aß-treated cells. In Aß-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 Aß in the hippocampus, which was elevated in Aß-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 Aß in AD models, which may lead to increased neuronal survival against Aß 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.

Two ß-strands of RAGE participate in the recognition and transport of amyloid-ß peptide across the blood brain barrier.

Amyloid-ß (Aß) 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 Aß peptide from circulating blood to human brain, and also causes the activation of the NF-κB signaling pathway. Here we show that two ß-strands of RAGE participate in the interaction with Aß peptide. Serial deletion analysis of the RAGE V domain indicates that the third and eighth ß-strands are required for interaction with Aß peptide. Site-directed mutagenesis of amino acids located in the third and eighth ß-strands abolish the interaction of RAGE with Aß peptide. Wild-type RAGE activates the NF-κB signaling pathway in response to Aß peptide treatment, while a RAGE mutant defective in Aß binding does not. Furthermore, use of peptide for the third ß-strand or a RAGE monoclonal antibody that targets the RAGE-Aß interaction interface inhibited transport of the Aß 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.

Ligand-based design, synthesis, and biological evaluation of 2-aminopyrimidines, a novel series of receptor for advanced glycation end products (RAGE) inhibitors.

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 Aß 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-Aß 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.

Human serum transthyretin levels correlate inversely with Alzheimer's disease.

Alzheimer's disease (AD) is the fastest growing neurodegenerative disease in the elderly population, and the search for therapeutic targets and diagnostic AD biomarkers is an exigent issue. Because amyloid-ß (Aß) aggregation constitutes the epicenter of AD pathology, Aß-binding proteins that regulate Aß aggregation, such as transthyretin (TTR), have attracted much attention. TTR binds to Aß, prevents its aggregation, and consequently inhibits Aß-induced cellular toxicity. Decreased TTR levels in cerebrospinal fluid (CSF) from AD patients suggest that TTR is a biomarker of AD. But, studies on TTR as a biomarker have focused on CSF; no study has evaluated peripheral levels of TTR in AD. Here, we examined the relationship between serum TTR levels and AD. We measured TTR levels in serum samples from 90 nondemented controls and 111 AD patients and observed significantly lower serum TTR levels in AD (p < 0.001). Notably, females in the control group had lower serum TTR levels compared with male in the control (p = 0.006), while no difference in gender was noted in the AD group. There were no age-related changes in serum TTR levels. Thus, this study demonstrates a clear negative correlation between serum TTR levels and AD, suggesting that TTR is not only involved in AD pathological process but also suggested as possible peripheral biomarker for AD diagnosis in serum level.

Identification of autoantibody against beta-amyloid peptide in the serum of elderly.

Alzheimer's disease (AD) is characterized by two major neurological features: amyloid deposits and neurofibrillary tangles in the brain. According to the amyloid cascade hypothesis, accumulation of amyloid-beta peptide (A-beta) plays a central role in the pathogenesis of AD. Several lines of evidence suggest that antibodies against A-beta play a protective role in the neuropathology of AD. In this study, we describe the purification of an autoantibody against A-beta from human serum using affinity purification method. The purified autoantibody recognized A-beta deposits in the brain of aged Tg2676 mice, an animal model of AD. The serum levels of anti-A-beta autoantibody correlated inversely with age in both AD patients and control non-demented elderly subjects. Furthermore, the levels were significantly lower in AD patients compared with the age-matched control subjects. It is the first time to show the identification of endogenous anti-A-beta autoantibody in human serum and suggesting that serum levels of anti-A-beta autoantibody might be a good biomarker for AD patients.

Reduced serum level of antibodies against amyloid beta peptide is associated with aging in Tg2576 mice.

Both active and passive immunization to eliminate amyloid plaques from the brain of patients with Alzheimer's disease (AD) have confirmed that amyloid beta (Abeta) vaccination does not only result in clearance of Abeta plaques but improves behavioral-cognitive deficits in animal models of AD. In the present study, the levels of naturally occurring serum antibodies against Abeta were measured in Tg2576 mice at various ages using ELISA to determine the relationship between aging and the level of anti-Abeta autoantibody. The level of anti-Abeta antibody fell significantly at the age of 9 months, at the age when amyloid plaques started to appear in the brain of Tg2576 mice, and was persistently low thereafter. However, serum immunoglobulin (Ig) level was elevated in older transgenic mice compared with younger transgenic mice suggesting that the reduced level of anti-Abeta autoantibody was not merely due to deterioration of the immune response in aged Tg2576 mice.