Azobioisosteres of Curcumin with Pronounced Activity against Amyloid Aggregation, Intracellular Oxidative Stress, and Neuroinflammation.

Many (poly-)phenolic natural products, for example, curcumin and taxifolin, have been studied for their activity against specific hallmarks of neurodegeneration, such as amyloid-ß 42 (Aß42) aggregation and neuroinflammation. Due to their drawbacks, arising from poor pharmacokinetics, rapid metabolism, and even instability in aqueous medium, the biological activity of azobenzene compounds carrying a pharmacophoric catechol group, which have been designed as bioisoteres of curcumin has been examined. Molecular simulations reveal the ability of these compounds to form a hydrophobic cluster with Aß42, which adopts different folds, affecting the propensity to populate fibril-like conformations. Furthermore, the curcumin bioisosteres exceeded the parent compound in activity against Aß42 aggregation inhibition, glutamate-induced intracellular oxidative stress in HT22 cells, and neuroinflammation in microglial BV-2 cells. The most active compound prevented apoptosis of HT22 cells at a concentration of 2.5 µm (83 % cell survival), whereas curcumin only showed very low protection at 10 µm (21 % cell survival).

Investigations into neuroprotectivity, stability, and water solubility of 7-O-cinnamoylsilibinin, its hemisuccinate and dehydro derivatives.

Derivatives of the recently described potent neuroprotective 7-O-cinnamoylsilibinin ester were prepared: its hemisuccinate to improve water solubility and the dehydrosilibinin ester that was shown to form in assay media to investigate its role in overall neuroprotective effects. 7-O-Cinnamoyl-2,3-dehydrosilibinin is less neuroprotective than 7-O-cinnamoylsilibinin in a murine hippocampal cell line (HT-22) and we conclude that the dehydrosilibinin derivatives are not the actual carriers of neuroprotective properties, at least in the assay applied. Solubility of the test compounds was determined in shake-flask experiments and the ester's solubility was greatly improved by introduction of a hemisuccinate at the 23-position of silibinin. Time-stability curves in assay media were recorded. The hemisuccinate ester did not act as a prodrug to release 7-O-cinnamoylsilibinin but is the second ester bond to be cleaved. Nevertheless, it still exhibits significant neuroprotection. Therefore, its greatly increased solubility might effectively counterbalance lower in vitro neuroprotection.

Flavonoid-Phenolic Acid Hybrids Are Potent Inhibitors of Ferroptosis via Attenuation of Mitochondrial Impairment.

Cinnamic acid, ferulic acid, and the flavonoids quercetin and taxifolin (dihydroquercetin) are naturally occurring compounds found in plants. They are often referred to as polyphenols and are known, among others, for their pharmacological effects supporting health through the inhibition of aging processes and oxidative stress. To improve their bioavailability, pharmacological activities, and safety, the creation of novel flavonoid-phenolic acid hybrids is an area of active research. Previous work showed that such hybridization products of phenolic acids and flavonoids enhanced the resilience of neuronal cells against oxidative stress in vitro, and attenuated cognitive impairment in a mouse model of Alzheimer's disease (AD) in vivo. Notably, the therapeutic effects of the hybrid compounds we obtained were more pronounced than the protective activities of the respective individual components. The underlying mechanisms mediated by the flavonoid-phenolic acid hybrids, however, remained unclear and may differ from the signaling pathways activated by the originating structures of the respective individual phenolic acids or flavonoids. In this study, we characterized the effects of four previously described potent flavonoid-phenolic acid hybrids in models of oxidative cell death through ferroptosis. Ferroptosis is a type of iron-dependent regulated cell death characterized by lipid peroxidation and mitochondrial ROS generation and has been linked to neurodegenerative conditions. In models of ferroptosis induced by erastin or RSL3, we analyzed mitochondrial (lipid) peroxidation, mitochondrial membrane integrity, and Ca2+ regulation. Our results demonstrate the strong protective effects of the hybrid compounds against ROS formation in the cytosol and mitochondria. Importantly, these protective effects against ferroptosis were not mediated by radical scavenging activities of the phenolic hybrid compounds but through inhibition of mitochondrial complex I activity and reduced mitochondrial respiration. Our data highlight the effects of flavonoid-phenolic acid hybrids on mitochondrial metabolism and further important mitochondrial parameters that collectively determine the health and functionality of mitochondria with a high impact on the integrity and survival of the neuronal cells.

Melatonin- and Ferulic Acid-Based HDAC6 Selective Inhibitors Exhibit Pronounced Immunomodulatory Effects In Vitro and Neuroprotective Effects in a Pharmacological Alzheimer's Disease Mouse Model.

The structures of melatonin and ferulic acid were merged into tertiary amide-based histone deacetylase 6 (HDAC6) inhibitors to develop multi-target-directed inhibitors for neurodegenerative diseases to incorporate antioxidant effects without losing affinity and selectivity at HDAC6. Structure-activity relationships led to compound 10b as a hybrid molecule showing pronounced and selective inhibition of HDAC6 (IC50 = 30.7 nM, > 25-fold selectivity over other subtypes). This compound shows comparable DPPH radical scavenging ability to ferulic acid, comparable ORAC value to melatonin and comparable Cu2+ chelating ability to EDTA. It also lacks neurotoxicity on HT-22 cells, exhibits a pronounced immunomodulatory effect, and is active in vivo showing significantly higher efficacy in an AD mouse model to prevent both Aß25-35-induced spatial working and long-term memory dysfunction at lower dose (0.3 mg/kg) compared to positive control HDAC6 inhibitor ACY1215 and an equimolar mixture of the three entities ACY1215, melatonin and ferulic acid, suggesting potentially disease-modifying properties.

Development and Application of a Chemical Probe Based on a Neuroprotective Flavonoid Hybrid for Target Identification Using Activity-Based Protein Profiling.

Alzheimer's disease (AD) is the most common form of dementia, and up to now, there are no disease-modifying drugs available. Natural product hybrids based on the flavonoid taxifolin and phenolic acids have shown a promising pleiotropic neuroprotective profile in cell culture assays and even disease-modifying effects in vivo. However, the detailed mechanisms of action remain unclear. To elucidate the distinct intracellular targets of 7-O-esters of taxifolin, we present in this work the development and application of a chemical probe, 7-O-cinnamoyltaxifolin-alkyne, for target identification using activity-based protein profiling. 7-O-Cinnamoyltaxifolin-alkyne remained neuroprotective in all cell culture assays. Western blot analysis showed a comparable influence on the same intracellular pathways as that of the lead compound 7-O-cinnamoyltaxifolin, thereby confirming its suitability as a probe for target identification experiments. Affinity pulldown and MS analysis revealed adenine nucleotide translocase 1 (ANT-1) and sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) as intracellular interaction partners of 7-O-cinnamoyltaxifolin-alkyne and thus of 7-O-esters of taxifolin.

7-O-Esters of taxifolin with pronounced and overadditive effects in neuroprotection, anti-neuroinflammation, and amelioration of short-term memory impairment in vivo.

Alzheimer's disease (AD) is a multifactorial disease and the most common form of dementia. There are no treatments to cure, prevent or slow down the progression of the disease. Natural products hold considerable interest for the development of preventive neuroprotectants to treat neurodegenerative disorders like AD, due to their low toxicity and general beneficial effects on human health with their anti-inflammatory and antioxidant features. In this work we describe regioselective synthesis of 7-O-ester hybrids of the flavonoid taxifolin with the phenolic acids cinnamic and ferulic acid, namely 7-O-cinnamoyltaxifolin and 7-O-feruloyltaxifolin. The compounds show pronounced overadditive neuroprotective effects against oxytosis, ferroptosis and ATP depletion in the murine hippocampal neuron HT22 cell model. Furthermore, 7-O-cinnamoyltaxifolin and 7-O-feruloyltaxifolin reduced LPS-induced neuroinflammation in BV-2 microglia cells as assessed by effects on the levels of NO, IL6 and TNFα. In all in vitro assays the 7-O-esters of taxifolin and ferulic or cinnamic acid showed strong overadditive activity, significantly exceeding the effects of the individual components and the equimolar mixtures thereof, which were almost inactive in all of the assays at the tested concentrations. In vivo studies confirmed this overadditive effect. Treatment of an AD mouse model based on the injection of oligomerized Aß25-35 peptide into the brain to cause neurotoxicity and subsequently memory deficits with 7-O-cinnamoyltaxifolin or 7-O-feruloyltaxifolin resulted in improved performance in an assay for short-term memory as compared to vehicle and mice treated with the respective equimolar mixtures. These results highlight the benefits of natural product hybrids as a novel compound class with potential use for drug discovery in neurodegenerative diseases due to their pharmacological profile that is distinct from the individual natural components.

Highly Selective Butyrylcholinesterase Inhibitors with Tunable Duration of Action by Chemical Modification of Transferable Carbamate Units Exhibit Pronounced Neuroprotective Effect in an Alzheimer's Disease Mouse Model.

In this study, the carbamate structure of pseudo-irreversible butyrylcholinesterase (BChE) inhibitors was optimized with regard to a longer binding to the enzyme. A set of compounds bearing different heterocycles (e.g., morpholine, tetrahydroisoquinoline, benzimidazole, piperidine) and alkylene spacers (2 to 10 methylene groups between carbamate and heterocycle) in the carbamate residue was synthesized and characterized in vitro for their binding affinity, binding kinetics, and carbamate hydrolysis. These novel BChE inhibitors are highly selective for hBChE over human acetycholinesterase (hAChE), yielding short-, medium-, and long-acting nanomolar hBChE inhibitors (with a half-life of the carbamoylated enzyme ranging from 1 to 28 h). The inhibitors show neuroprotective properties in a murine hippocampal cell line and a pharmacological mouse model of Alzheimer's disease (AD), suggesting a significant benefit of BChE inhibition for a disease-modifying treatment of AD.

Dual-Acting Cholinesterase-Human Cannabinoid Receptor 2 Ligands Show Pronounced Neuroprotection in Vitro and Overadditive and Disease-Modifying Neuroprotective Effects in Vivo.

We have designed and synthesized a series of 14 hybrid molecules out of the cholinesterase (ChE) inhibitor tacrine and a benzimidazole-based human cannabinoid receptor subtype 2 (hCB2R) agonist and investigated them in vitro and in vivo. The compounds are potent ChE inhibitors, and for the most promising hybrids, the mechanism of human acetylcholinesterase (hAChE) inhibition as well as their ability to interfere with AChE-induced aggregation of ß-amyloid (Aß), and Aß self-aggregation was assessed. All hybrids were evaluated for affinity and selectivity for hCB1R and hCB2R. To ensure that the hybrids retained their agonist character, the expression of cAMP-regulated genes was quantified, and potency and efficacy were determined. Additionally, the effects of the hybrids on microglia activation and neuroprotection on HT-22 cells were investigated. The most promising in vitro hybrids showed pronounced neuroprotection in an Alzheimer's mouse model at low dosage (0.1 mg/kg, i.p.), lacking hepatotoxicity even at high dose (3 mg/kg, i.p.).

One Enzyme To Build Them All: Ring-Size Engineered Siderophores Inhibit the Swarming Motility of Vibrio.

Bacteria compete for ferric iron by producing siderophores, and some microbes engage in piracy by scavenging siderophores of their competitors. The macrocyclic hydroxamate siderophore avaroferrin of Shewanella algae inhibits swarming of Vibrio alginolyticus by evading this piracy. Avaroferrin, as well as related putrebactin and bisucaberin, are produced by the IucC-like synthetases AvbD, PubC, and BibCC. Here, we have established that they are capable of synthesizing not only their native product but also other siderophores. Exploiting this relaxed substrate specificity by synthetic precursors generated 15 different ring-size engineered macrocycles ranging from 18- to 28-membered rings, indicating unprecedented biosynthetic flexibility of the enzymes. Two of the novel siderophores could be obtained in larger quantities by precursor-directed biosynthesis in S. algae. Both inhibited swarming motility of Vibrio and, similar to avaroferrin, the most active one exhibited a heterodimeric architecture. Our results demonstrate the impact of minor structural changes on biological activity, which may trigger the evolution of siderophore diversity.

Regioselective synthesis of 7-O-esters of the flavonolignan silibinin and SARs lead to compounds with overadditive neuroprotective effects.

A series of neuroprotective hybrid compounds was synthesized by conjugation of the flavonolignan silibinin with natural phenolic acids, such as ferulic, cinnamic and syringic acid. Selective 7-O-esterfication without protection groups was achieved by applying the respective acyl chlorides. Sixteen compounds were obtained and SARs were established by evaluating antioxidative properties in the physicochemical FRAP assay, as well as in a cell-based neuroprotection assay using murine hippocampal HT-22 cells. Despite weak activities in the FRAP assay, esters of the α,ß-unsaturated acids showed pronounced overadditive effects at low concentrations greatly exceeding the effects of equimolar mixtures of silibinin and the respective acids in the neuroprotection assay. Cinnamic and ferulic acid esters (5a and 6a) also showed overadditive effects regarding inhibition of microglial activation, PC12 cell differentiation, in vitro ischemia as well as anti-aggregating abilities against Aß42 peptide and τ protein. Remarkably, the esters of ferulic acid with silybin A and silybin B (11a and 11b) showed a moderate but significant difference in both neuroprotection and in their anti-aggregating capacities. The results demonstrate that non-toxic natural antioxidants can be regioselectively connected as esters with medium-term stability exhibiting very pronounced overadditive effects in a portfolio of biological assays.

Structure-Activity Relationships and Computational Investigations into the Development of Potent and Balanced Dual-Acting Butyrylcholinesterase Inhibitors and Human Cannabinoid Receptor 2 Ligands with Pro-Cognitive in Vivo Profiles.

The enzyme butyrylcholinesterase (BChE) and the human cannabinoid receptor 2 (hCB2R) represent promising targets for pharmacotherapy in the later stages of Alzheimer's disease. We merged pharmacophores for both targets into small benzimidazole-based molecules, investigated SARs, and identified several dual-acting ligands with a balanced affinity/inhibitory activity and an excellent selectivity over both hCB1R and hAChE. A homology model for the hCB2R was developed based on the hCB1R crystal structure and used for molecular dynamics studies to investigate binding modes. In vitro studies proved hCB2R agonism. Unwanted µ-opioid receptor affinity could be designed out. One well-balanced dual-acting and selective hBChE inhibitor/hCB2R agonist showed superior in vivo activity over the lead CB2 agonist with regards to cognition improvement. The data shows the possibility to combine a small molecule with selective and balanced GPCR-activity/enzyme inhibition and in vivo activity for the therapy of AD and may help to rationalize the development of other dual-acting ligands.

One Enzyme, Three Metabolites: Shewanella algae Controls Siderophore Production via the Cellular Substrate Pool.

Shewanella algae B516 produces avaroferrin, an asymmetric hydroxamate siderophore, which has been shown to inhibit swarming motility of Vibrio alginolyticus. We aimed to elucidate the biosynthesis of this siderophore and to investigate how S. algae coordinates the production of avaroferrin and its two symmetric counterparts. We reconstituted the reaction in vitro with the main enzyme AvbD and the putative biosynthetic precursors, and demonstrate that multispecificity of this enzyme results in the production of all three cyclic hydroxamate siderophores that were previously isolated as natural products from S. algae. Surprisingly, purified AvbD exhibited a clear preference for the larger cadaverine-derived substrate. In live cells, however, siderophore ratios are maximized toward avaroferrin production, and we demonstrate that these siderophore ratios are the result of a regulation on substrate pool level, which may allow rapid evolutionary adaptation to environmental changes. Our results thereby give insights into a unique evolutionary strategy toward metabolite diversity.