Soluble epoxide hydrolase-targeting PROTAC activates AMPK and inhibits endoplasmic reticulum stress.

Soluble epoxide hydrolase (sEH) is a drug target with the potential for therapeutic utility in the areas of inflammation, neurodegenerative disease, chronic pain, and diabetes, among others. Proteolysis-targeting chimeras (PROTACs) molecules offer new opportunities for targeting sEH, due to its capacity to induce its degradation. Here, we describe that the new ALT-PG2, a PROTAC that degrades sEH protein in the human hepatic Huh-7 cell line, in isolated mouse primary hepatocytes, and in the liver of mice. Remarkably, sEH degradation caused by ALT-PG2 was accompanied by an increase in the phosphorylated levels of AMP-activated protein kinase (AMPK), while phosphorylated extracellular-signal-regulated kinase 1/2 (ERK1/2) was reduced. Consistent with the key role of these kinases on endoplasmic reticulum (ER) stress, ALT-PG2 attenuated the levels of ER stress and inflammatory markers. Overall, the findings of this study indicate that targeting sEH with degraders is a promising pharmacological strategy to promote AMPK activation and to reduce ER stress and inflammation.

A Study of the Activity of Adamantyl Amines against Mutant Influenza A M2 Channels Identified a Polycyclic Cage Amine Triple Blocker, Explored by Molecular Dynamics Simulations and Solid-State NMR.

We compared the anti-influenza potencies of 57 adamantyl amines and analogs against influenza A virus with serine-31 M2 proton channel, usually termed as WT M2 channel, which is amantadine sensitive. We also tested a subset of these compounds against viruses with the amantadine-resistant L26F, V27A, A30T, G34E M2 mutant channels. Four compounds inhibited WT M2 virus in vitro with mid-nanomolar potency, with 27 compounds showing sub-micromolar to low micromolar potency. Several compounds inhibited L26F M2 virus in vitro with sub-micromolar to low micromolar potency, but only three compounds blocked L26F M2-mediated proton current as determined by electrophysiology (EP). One compound was found to be a triple blocker of WT, L26F, V27A M2 channels by EP assays, but did not inhibit V27A M2 virus in vitro, and one compound inhibited WT, L26F, V27A M2 in vitro without blocking V27A M2 channel. One compound blocked only L26F M2 channel by EP, but did not inhibit virus replication. The triple blocker compound is as long as rimantadine, but could bind and block V27A M2 channel due to its larger girth as revealed by molecular dynamics simulations, while MAS NMR informed on the interaction of the compound with M2(18-60) WT or L26F or V27A.

Synthesis and Testing of Analogs of the Tuberculosis Drug Candidate SQ109 against Bacteria and Protozoa: Identification of Lead Compounds against Mycobacterium abscessus and Malaria Parasites.

SQ109 is a tuberculosis drug candidate that has high potency against Mycobacterium tuberculosis and is thought to function at least in part by blocking cell wall biosynthesis by inhibiting the MmpL3 transporter. It also has activity against bacteria and protozoan parasites that lack MmpL3, where it can act as an uncoupler, targeting lipid membranes and Ca2+ homeostasis. Here, we synthesized 18 analogs of SQ109 and tested them against M. smegmatis, M. tuberculosis, M. abscessus, Bacillus subtilis, and Escherichia coli, as well as against the protozoan parasites Trypanosoma brucei, T. cruzi, Leishmania donovani, L. mexicana, and Plasmodium falciparum. Activity against the mycobacteria was generally less than with SQ109 and was reduced by increasing the size of the alkyl adduct, but two analogs were ∼4-8-fold more active than SQ109 against M. abscessus, including a highly drug-resistant strain harboring an A309P mutation in MmpL3. There was also better activity than found with SQ109 with other bacteria and protozoa. Of particular interest, we found that the adamantyl C-2 ethyl, butyl, phenyl, and benzyl analogs had 4-10× increased activity against P. falciparum asexual blood stages, together with low toxicity to a human HepG2 cell line, making them of interest as new antimalarial drug leads. We also used surface plasmon resonance to investigate the binding of inhibitors to MmpL3 and differential scanning calorimetry to investigate binding to lipid membranes. There was no correlation between MmpL3 binding and M. tuberculosis or M. smegmatis cell activity, suggesting that MmpL3 is not a major target in mycobacteria. However, some of the more active species decreased lipid phase transition temperatures, indicating increased accumulation in membranes, which is expected to lead to enhanced uncoupler activity.

Glial cell reactivity and oxidative stress prevention in Alzheimer's disease mice model by an optimized NMDA receptor antagonist.

In Alzheimer's disease pathology, several neuronal processes are dysregulated by excitotoxicity including neuroinflammation and oxidative stress (OS). New therapeutic agents capable of modulating such processes are needed to foster neuroprotection. Here, the effect of an optimised NMDA receptor antagonist, UB-ALT-EV and memantine, as a gold standard, have been evaluated in 5XFAD mice. Following treatment with UB-ALT-EV, nor memantine, changes in the calcineurin (CaN)/NFAT pathway were detected. UB-ALT-EV increased neurotropic factors (Bdnf, Vgf and Ngf) gene expression. Treatments reduced astrocytic and microglial reactivity as revealed by glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1) quantification. Interestingly, only UB-ALT-EV was able to reduce gene expression of Trem2, a marker of microglial activation and NF-κB. Pro-inflammatory cytokines Il-1ß, Ifn-γ, Ccl2 and Ccl3 were down-regulated in UB-ALT-EV-treated mice but not in memantine-treated mice. Interestingly, the anti-inflammatory markers of the M2-migroglial phenotype, chitinase-like 3 (Ym1) and Arginase-1 (Arg1), were up-regulated after treatment with UB-ALT-EV. Since iNOS gene expression decreased after UB-ALT-EV treatment, a qPCR array containing 84 OS-related genes was performed. We found changes in Il-19, Il-22, Gpx6, Ncf1, Aox1 and Vim gene expression after UB-ALT-EV. Hence, our results reveal a robust effect on neuroinflammation and OS processes after UB-ALT-EV treatment, surpassing the memantine effect in 5XFAD.

Optimized N-methyl-D-aspartate receptor antagonist exhibits hippocampal proneurogenic effects in aged senescence-accelerated mouse prone 8 mice.

N-methyl-D-aspartate (NMDA) receptor antagonists mediate adult neurogenic effects. Here, the neurogenic effect of a new NMDA receptor antagonist endowed with neuroprotective effects in Alzheimer's disease mice model. Nine-month-old senescence-accelerated mouse prone 8 (SAMP8) with UB-ALT-EV were orally treated. 5-Bromo-2-deoxyuridine (BrdU) (50 mg/kg) was 3× injected I.P. every 2 h. After 28 days of treatment, SAMP8-treated group improved working memory. Moreover, the number of BrdU+ cells and DCX+ cells in the SAMP8 dentate gyrus (DG) was significantly increased. GFAP+ cells were not affected by treatment. Together, these results provided evidence that UB-ALT-EV promotes the survival and proliferation of neural progenitor cells in the aged SAMP8 hippocampus.

Inhibition of NMDA receptors through a membrane-to-channel path.

N-methyl-D-aspartate receptors (NMDARs) are transmembrane proteins that are activated by the neurotransmitter glutamate and are found at most excitatory vertebrate synapses. NMDAR channel blockers, an antagonist class of broad pharmacological and clinical significance, inhibit by occluding the NMDAR ion channel. A vast literature demonstrates that NMDAR channel blockers, including MK-801, phencyclidine, ketamine, and the Alzheimer's disease drug memantine, can bind and unbind only when the NMDAR channel is open. Here we use electrophysiological recordings from transfected tsA201 cells and cultured neurons, NMDAR structural modeling, and custom-synthesized compounds to show that NMDAR channel blockers can enter the channel through two routes: the well-known hydrophilic path from extracellular solution to channel through the open channel gate, and also a hydrophobic path from plasma membrane to channel through a gated fenestration ("membrane-to-channel inhibition" (MCI)). Our demonstration that ligand-gated channels are subject to MCI, as are voltage-gated channels, highlights the broad expression of this inhibitory mechanism.

NMDA receptor antagonists reduce amyloid-ß deposition by modulating calpain-1 signaling and autophagy, rescuing cognitive impairment in 5XFAD mice.

Overstimulation of N-methyl-D-aspartate receptors (NMDARs) is the leading cause of brain excitotoxicity and often contributes to neurodegenerative diseases such as Alzheimer's Disease (AD), the most common form of dementia. This study aimed to evaluate a new NMDA receptor antagonist (UB-ALT-EV) and memantine in 6-month-old female 5XFAD mice that were exposed orally to a chronic low-dose treatment. Behavioral and cognitive tests confirmed better cognitive performance in both treated groups. Calcium-dependent protein calpain-1 reduction was found after UB-ALT-EV treatment but not after memantine. Changes in spectrin breakdown products (SBDP) and the p25/p35 ratio confirmed diminished calpain-1 activity. Amyloid ß (Aß) production and deposition was evaluated in 5XFAD mice and demonstrated a robust effect of NMDAR antagonists on reducing Aß deposition and the number and size of Thioflavin-S positive plaques. Furthermore, glycogen synthase kinase 3ß (GSK3ß) active form and phosphorylated tau (AT8) levels were diminished after UB-ALT-EV treatment, revealing tau pathology improvement. Because calpain-1 is involved in autophagy activation, autophagic proteins were studied. Strikingly, results showed changes in the protein levels of unc-51-like kinase (ULK-1), beclin-1, microtubule-associated protein 1A/1B-light chain 3(LC3B-II)/LC3B-I ratio, and lysosomal-associated membrane protein 1 (LAMP-1) after NMDAR antagonist treatments, suggesting an accumulation of autophagolysosomes in 5XFAD mice, reversed by UB-ALT-EV. Likewise, treatment with UB-ALT-EV recovered a WT mice profile in apoptosis markers Bcl-2, Bax, and caspase-3. In conclusion, our results revealed the potential neuroprotective effect of UB-ALT-EV by attenuating NMDA-mediated apoptosis and reducing Aß deposition and deposition jointly with the autophagy rescue to finally reduce cognitive alterations in a mice model of familial AD.

Design, synthesis, and in vitro and in vivo characterization of new memantine analogs for Alzheimer's disease.

Currently, of the few accessible symptomatic therapies for Alzheimer's disease (AD), memantine is the only N-methyl-d-aspartate receptor (NMDAR) blocker approved by the FDA. This work further explores a series of memantine analogs featuring a benzohomoadamantane scaffold. Most of the newly synthesized compounds block NMDARs in the micromolar range, but with lower potency than previously reported hit IIc, results that were supported by molecular dynamics simulations. Subsequently, electrophysiological studies with the more potent compounds allowed classification of IIc, a low micromolar, uncompetitive, voltage-dependent, NMDAR blocker, as a memantine-like compound. The excellent in vitro DMPK properties of IIc made it a promising candidate for in vivo studies in Caenorhabditis elegans (C. elegans) and in the 5XFAD mouse model of AD. Administration of IIc or memantine improved locomotion and rescues chemotaxis behavior in C. elegans. Furthermore, both compounds enhanced working memory in 5XFAD mice and modified NMDAR and CREB signaling, which may prevent synaptic dysfunction and modulate neurodegenerative progression.

A Novel NMDA Receptor Antagonist Protects against Cognitive Decline Presented by Senescent Mice.

Alzheimer's disease (AD) is the leading cause of dementia. Non-competitive N-Methyl-D-aspartate (NMDA) receptor antagonist memantine improved cognition and molecular alterations after preclinical treatment. Nevertheless, clinical results are discouraging. In vivo efficacy of the RL-208, a new NMDA receptor blocker described recently, with favourable pharmacokinetic properties was evaluated in Senescence accelerated mice prone 8 (SAMP8), a mice model of late-onset AD (LOAD). Oral administration of RL-208 improved cognitive performance assessed by using the three chamber test (TCT), novel object recognition test (NORT), and object location test (OLT). Consistent with behavioural results, RL-208 treated-mice groups significantly changed NMDAR2B phosphorylation state levels but not NMDAR2A. Calpain-1 and Caspase-3 activity was reduced, whereas B-cell lymphoma-2 (BCL-2) levels increased, indicating reduced apoptosis in RL-208 treated SAMP8. Superoxide Dismutase 1 (SOD1) and Glutathione Peroxidase 1 (GPX1), as well as a reduction of hydrogen peroxide (H2O2), was also determined in RL-208 mice. RL-208 treatment induced an increase in mature brain-derived neurotrophic factor (mBDNF), prevented Tropomyosin-related kinase B full-length (TrkB-FL) cleavage, increased protein levels of Synaptophysin (SYN) and Postsynaptic density protein 95 (PSD95). In whole, these results point out to an improvement in synaptic plasticity. Remarkably, RL-208 also decreased the protein levels of Cyclin-Dependent Kinase 5 (CDK5), as well as p25/p35 ratio, indicating a reduction in kinase activity of CDK5/p25 complex. Consequently, lower levels of hyperphosphorylated Tau (p-Tau) were found. In sum, these results demonstrate the neuroprotectant role of RL-208 through NMDAR blockade.

DMT1 Inhibitors Kill Cancer Stem Cells by Blocking Lysosomal Iron Translocation.

Cancer stem cells (CSC) constitute a cell subpopulation in solid tumors that is responsible for resistance to conventional chemotherapy, metastasis and cancer relapse. The natural product Salinomycin can selectively target this cell niche by directly interacting with lysosomal iron, taking advantage of upregulated iron homeostasis in CSC. Here, inhibitors of the divalent metal transporter 1 (DMT1) have been identified that selectively target CSC by blocking lysosomal iron translocation. This leads to lysosomal iron accumulation, production of reactive oxygen species and cell death with features of ferroptosis. DMT1 inhibitors selectively target CSC in primary cancer cells and circulating tumor cells, demonstrating the physiological relevance of this strategy. Taken together, this opens up opportunities to tackle unmet needs in anti-cancer therapy.

A novel class of multitarget anti-Alzheimer benzohomoadamantane‒chlorotacrine hybrids modulating cholinesterases and glutamate NMDA receptors.

The development of multitarget compounds against multifactorial diseases, such as Alzheimer's disease, is an area of very intensive research, due to the expected superior therapeutic efficacy that should arise from the simultaneous modulation of several key targets of the complex pathological network. Here we describe the synthesis and multitarget biological profiling of a new class of compounds designed by molecular hybridization of an NMDA receptor antagonist fluorobenzohomoadamantanamine with the potent acetylcholinesterase (AChE) inhibitor 6-chlorotacrine, using two different linker lengths and linkage positions, to preserve or not the memantine-like polycyclic unsubstituted primary amine. The best hybrids exhibit greater potencies than parent compounds against AChE (IC50 0.33 nM in the best case, 44-fold increased potency over 6-chlorotacrine), butyrylcholinesterase (IC50 21 nM in the best case, 24-fold increased potency over 6-chlorotacrine), and NMDA receptors (IC50 0.89 µM in the best case, 2-fold increased potency over the parent benzohomoadamantanamine and memantine), which suggests an additive effect of both pharmacophoric moieties in the interaction with the primary targets. Moreover, most of these compounds have been predicted to be brain permeable. This set of biological properties makes them promising leads for further anti-Alzheimer drug development.

Palladium-catalyzed cocyclotrimerization of arynes with a pyramidalized alkene.

The metal-catalyzed [2+2+2] cocycloaddition of arynes with pyramidalized alkenes is presented. The generation of a highly reactive pyramidalized alkene in the presence of a large excess of in situ-produced arynes led to the corresponding cocyclotrimerization (1 : 2)-adducts in good yields, establishing the first example of a palladium-based reaction of a pyramidalized alkene.

Pharmacological and Electrophysiological Characterization of Novel NMDA Receptor Antagonists.

This work reports the synthesis and pharmacological and electrophysiological evaluation of new N-methyl-d-aspartic acid receptor (NMDAR) channel blocking antagonists featuring polycyclic scaffolds. Changes in the chemical structure modulate the potency and voltage dependence of inhibition. Two of the new antagonists display properties comparable to those of memantine, a clinically approved NMDAR antagonist.

Parenchymal and airway diseases caused by asbestos.

PURPOSE OF REVIEW: The extensive industrial use of asbestos for many decades has been linked to development of benign and malignant pleuropulmonary disease. This review summarizes newer evidence and ongoing controversies that exist in the literature regarding asbestos-related parenchymal and airway diseases. RECENT FINDINGS: Asbestosis represents a significant respiratory problem despite the improvement in the workplace hygiene and a decrease in use of asbestos. The management of asbestosis remains challenging as currently there is no specific treatment. The role of asbestos exposure alone as a cause of chronic airway obstruction remains uncertain. The relationship between lung cancer and asbestos exposure alone and in combination with smoking has also been investigated. The benefit of screening for asbestos-related pleuropulmonary disease remains uncertain as does the use of computed tomography scanning for the purpose of screening. SUMMARY: Future studies will help clarify the clinical issues and shape screening strategies for asbestos-exposed individuals.

C-reactive protein and copeptin: prognostic predictors in chronic obstructive pulmonary disease exacerbations.

PURPOSE OF REVIEW: Acute exacerbation of chronic obstructive pulmonary disease (COPD) is a natural event in the course of COPD and remains a major cause of morbidity and mortality associated with this disease. Clinical criteria that define an acute exacerbation are subjective and open for debate. Identifying biomarkers that would be easily measured and followed in patients with acute exacerbation of COPD seems to be highly attractive. The aim of this review is to assess the role of biomarkers, C-reactive protein (CRP) and copeptin, as prognostic predictors in COPD exacerbations. RECENT FINDINGS: Many pulmonary biomarkers have been extensively studied in the recent years. CRP and copeptin have gained particular interest. Recent data suggest that CRP is elevated during an acute exacerbation of COPD but CRP alone is neither sensitive nor specific in predicting clinical severity or outcome. Copeptin increases during acute exacerbation of COPD and may correlate with disease severity. SUMMARY: Further studies are needed to determine the role of CRP and copeptin as biomarkers that aid in diagnosis and clinical outcome in acute exacerbation of COPD.