Discovery of Novel Ortho-Aminophenol Derivatives Targeting Lipid Peroxidation with Potent Antiferroptotic Activities.

The concept of ferroptosis inhibition has gained growing recognition as a promising therapeutic strategy for addressing a wide range of diseases. Here, we present the discovery of four series of ortho-aminophenol derivatives as potential ferroptosis inhibitors beginning with the endogenous substance 3-hydroxyanthranilic acid (3-HA) by employing quantum chemistry techniques, in vitro and in vivo assays. Our findings reveal that these ortho-aminophenol derivatives exhibit unique intra-H bond interactions, compelling ortho-amines to achieve enhanced alignment with the aromatic π-system, thereby expanding their activity. Notably, compounds from all four series display remarkable activity against RSL3-induced ferroptosis, showcasing an activity 100 times more than that of 3-HA. Furthermore, these compounds also demonstrate robust in vivo efficacy in protecting mice from kidney ischemia-reperfusion injury and acetaminophen-induced hepatotoxicity. In summary, we provide four distinct series of active scaffolds that significantly expand the chemical space of ferroptosis inhibitors, serving as valuable insights for future structural modifications.

Design, synthesis, and biological evaluation of 2-amino-6-methyl-phenol derivatives targeting lipid peroxidation with potent anti-ferroptotic activities.

The suppression of ferroptosis is emerging as a promising therapeutic strategy for effectively treating a wide range of diseases, including neurodegenerative disorders, organ ischemia-reperfusion injury, and inflammatory conditions. However, the clinical utility of ferroptosis inhibitors is significantly impeded by the limited availability of rational drug designs. In our previous study, we successfully unraveled the efficacy of ferrostatin-1 (Fer-1) attributed to the synergistic effect of its ortho-diamine (-NH) moiety. In this study, we present the discovery of the ortho-hydroxyl-amino moiety as a novel scaffold for ferroptosis inhibitors, employing quantum chemistry as well as in vitro and in vivo assays. 2-amino-6-methylphenol derivatives demonstrated remarkable inhibition of RSL3-induced ferroptosis, exhibiting EC50 values ranging from 25 nM to 207 nM. These compounds do not appear to modulate iron homeostasis or lipid reactive oxygen species (ROS) generation pathways. Nevertheless, they effectively prevent the accumulation of lipid peroxides in living cells. Furthermore, compound 13 exhibits good in vivo activities as it effectively protect mice from kidney ischemia-reperfusion injury. In summary, compound 13 has been identified as a potent ferroptosis inhibitor, warranting further investigation as a promising lead compound.

Endogenous chemicals guard health through inhibiting ferroptotic cell death.

Ferroptosis is a new form of regulated cell death caused by iron-dependent accumulation of lethal polyunsaturated phospholipids peroxidation. It has received considerable attention owing to its putative involvement in a wide range of pathophysiological processes such as organ injury, cardiac ischemia/reperfusion, degenerative disease and its prevalence in plants, invertebrates, yeasts, bacteria, and archaea. To counter ferroptosis, living organisms have evolved a myriad of intrinsic efficient defense systems, such as cyst(e)ine-glutathione-glutathione peroxidase 4 system (cyst(e)ine-GPX4 system), guanosine triphosphate cyclohydrolase 1/tetrahydrobiopterin (BH4) system (GCH1/BH4 system), ferroptosis suppressor protein 1/coenzyme Q10 system (FSP1/CoQ10 system), and so forth. Among these, GPX4 serves as the only enzymatic protection system through the reduction of lipid hydroperoxides, while other defense systems ultimately rely on small compounds to scavenge lipid radicals and prevent ferroptotic cell death. In this article, we systematically summarize the chemical biology of lipid radical trapping process by endogenous chemicals, such as coenzyme Q10 (CoQ10), BH4, hydropersulfides, vitamin K, vitamin E, 7-dehydrocholesterol, with the aim of guiding the discovery of novel ferroptosis inhibitors.

Aroma formation and transformation during sealed yellowing process of Pingyang yellow tea.

Yellow tea, a unique type of tea in China which is characterized with yellow color, has gained increasing popularity due to its pleasant taste. However, transformation of aroma compounds during sealed yellowing has been poorly understood. Results of sensory evaluation exhibited that yellowing time was the key factor for flavor and fragrance formation. A total of 52 volatile components during sealed yellowing process of Pingyang yellow soup were further collected and analyzed. The results demonstrated that the sealed yellowing process significantly increased the ratio of alcohol and aldehyde compounds in the aroma volatiles of yellow tea, which were primarily composed of geraniol, linalool, phenylacetaldehyde, linalool oxide and cis-3-hexenol, and their proportion increased with the prolongation of sealed yellowing. Mechanistic speculation revealed that the sealed yellowing process promoted release of alcoholic aroma compounds from their glycoside precursors and enhanced Strecker and oxidative degradation. This study revealed the transformation mechanism of aroma profile during the sealed yellowing process, which would facilitate processing of yellow tea.

Theoretical Insights into the Cotransport Mechanism of GSH with Anticancer Drugs by MRP1.

The multidrug resistance protein MRP1 is an ATP binding cassette (ABC) transporter that confers resistance to many anticancer drugs and regulates redox homeostasis, inflammation, and hormone secretion. MRP1 actively transports compounds across cell membranes, and the presence of glutathione (GSH) is required in many cases. However, the process of MRP1-mediated substrate transportation has been poorly understood. With extensive molecular dynamics simulations, we have found a sandwich-like structure which is generated by GSH, a transmembrane α-helices 11 (TM11)-TM17 axis, and anticancer drugs. This structure is crucial in MRP1 transportation. It triggers the motion of TM11 and TM17, followed by the movement of nucleotide-binding domains 1 (NBD1) and 2 (NBD2), and finally an occluded structure is formed. Trp1246, Lys332, and Phe594 were identified as the main contributors in the formation of the sandwich-like structure. Our findings clearly explain the synergy of GSH with an anticancer drug in MRP1 transportation and have significant meanings for the rational design of novel inhibitors against MRP1.

Theoretical insight into the multiple interactions of quinazoline inhibitors with breast cancer resistance protein (BCRP/ABCG2).

Communicated by Ramaswamy H. Sarma.

Fragment-centric topographic mapping method guides the understanding of ABCG2-inhibitor interactions.

Understanding protein-ligand interactions is crucial to drug discovery and design. However, it would be extremely difficult for the proteins which only have one available apo structure but multiple binding sites. To address this constraint, a fragment-centric topographic mapping method (AlphaSpace software) was employed to map out concave interaction pockets at the assigned protein region. These pockets are used as complementary spaces to screen the known inhibitors for this specific binding site and to guide the molecular docking pose selection as well as protein-ligand interaction analysis. By mapping the shape of central cavity surface, we have tested the strategy against a multi-drug resistant transmembrane protein-ABCG2 to assist in generating a pharmacophore model for its inhibitors that is based on the structure of apo. Classical molecular simulation and accelerated molecular simulation are used to verify the accuracy of inhibitor screening and binding pose selection. Our study not only has gained insight for the development of novel specific ABCG2 inhibitors, but also has provided a general strategy in describing protein-ligand interactions.

Reactions of the Lipid Hydroperoxides With Aminic Antioxidants: The Influence of Stereoelectronic and Resonance Effects on Hydrogen Atom Transfer.

Aminic radical-trapping antioxidants (RTAs), as one of the most important antioxidants, have not received sufficient attention yet. But, an increasing number of aminic RTAs have been identified as ferroptosis inhibitors in recent years, which can potentially mediate many pathological states including inflammation, cancer, neurodegenerative disease, as well as ocular and kidney degeneration. This highlights the importance of aminic RTAs in the field of medicine. Herein, we systematically explored the radical scavenging mechanism of aminic RTAs with a quantum chemical method, particularly emphasizing the role of stereoelectronic factors and resonance factors on the transfer of H-atom and the stability to one-electron oxidation. These theoretical results elucidate the diversity of free radical scavenging mechanisms for aminic RTAs, and has significant implications for the rational design of new aminic RTAs.

O-Phenylenediamine: a privileged pharmacophore of ferrostatins for radical-trapping reactivity in blocking ferroptosis.

Ferroptosis is a non-apoptotic, iron dependent form of regulated cell death that is characterized by the accumulation of lipid hydroperoxides. It has drawn considerable attention owing to its putative involvement in diverse neurodegenerative diseases. Ferrostatins are the first identified inhibitors of ferroptosis and they inhibit ferroptosis by efficiently scavenging free radicals in lipid bilayers. However, their further medicinal application has been limited due to the deficient knowledge of the lipid peroxyl radical-trapping mechanism. In this study, experimental and theoretical methods were performed to illustrate the possible lipid hydroperoxide inhibition mechanism of ferrostatins. The results show that an ortho-amine (-NH) moiety from ferrostatins can simultaneously interact with lipid radicals, and then form a planar seven-membered ring in the transition state, and finally present greater reactivity. NBO analysis shows that the formed planar seven-membered ring forces ortho-amines into better alignment with the aromatic π-system. It significantly increases the magnitudes of amine conjugation and improves spin delocalization in the transition state. Additionally, a classical H-bond type interaction was discovered between a radical and an o-NH group as another transition state stabilizing effect. This type of radical-trapping mechanism is novel and has not been found in diphenylamine or traditional polyphenol antioxidants. It can be said that o-phenylenediamine is a privileged pharmacophore for the design and development of ferroptosis inhibitors.

Solvent Mediating a Switch in the Mechanism for Rhodium(III)-Catalyzed Carboamination/Cyclopropanation Reactions between N-Enoxyphthalimides and Alkenes.

Recently, a new synthetic methodology of rhodium-catalyzed carboamination/cyclopropanation from the same starting materials at different reaction conditions has been reported. It provides an efficient strategy for the stereospecific formation of both carbon- and nitrogen-based functionalities across an alkene. Herein we carried out a detailed theoretical mechanistic exploration for the reactions to elucidate the switch between carboamination and cyclopropanation as well as the origin of the chemoselectivity. Instead of the experimentally proposed RhIII-RhI-RhIII catalytic mechanism, our results reveal that the RhIII-RhV-RhIII mechanism is much more favorable in the two reactions. The chemoselectivity is attributed to a combination of electronic and steric effects in the reductive elimination step. The interactions between alkene and the rhodacycle during the alkene migration insertion control the stereoselectivity in the carboamination reactions. The present results disclose a dual role of the methanol solvent in controlling the chemoselectivity.

Effect of allosteric molecules on structure and drug affinity of HIV-1 protease by molecular dynamics simulations.

Recent experiments show that small molecules can bind onto the allosteric sites of HIV-1 protease (PR), which provides a starting point for developing allosteric inhibitors. However, the knowledge of the effect of such binding on the structural dynamics and binding free energy of the active site inhibitor and PR is still lacking. Here, we report 200ns long molecular dynamics simulation results to gain insight into the influences of two allosteric molecules (1H-indole-6-carboxylic acid, 1F1 and 2-methylcyclohexano, 4D9). The simulations demonstrate that both allosteric molecules change the PR conformation and stabilize the structures of PR and the inhibitor; the residues of the flaps are sensitive to the allosteric molecules and the flexibility of the residues is pronouncedly suppressed; the additions of the small molecules to the allosteric sites strengthen the binding affinities of 3TL-PR by about 12-15kal/mol in the binding free energy, which mainly arises from electrostatic term. Interestingly, it is found that the action mechanisms of 1F1 and 4D9 are different, the former behaviors like a doorman that keeps the inhibitor from escape and makes the flaps (door) partially open; the latter is like a wedge that expands the allosteric space and meanwhile closes the flaps. Our data provide a theoretical support for designing the allosteric inhibitor.

Deacetyl-mycoepoxydiene, isolated from plant endophytic fungi Phomosis sp. demonstrates anti-microtubule activity in MCF-7 cells.

Deacetyl-mycoepoxydiene (DM), a novel secondary metabolite produced by the plant endophytic fungi Phomosis sp., induced the reorganization of cytoskeleton in actively growing MCF-7 cells by promoting polymerization of tubulin. DM could induce cell cycle arrest at G2/M in MCF-7 cells. Additionally, DM-induced apoptosis was characterized with up-regulating caspase-3, Bax, caspase-9, parp, and p21 while down-regulating Bcl-2 activation. DM conferred dose- and time-dependent inhibitory effects upon cell proliferation of MCF-7 cells both in cultured cells and nude mice with human breast carcinoma xenografts. The results obtained from these in vitro and in vivo models provide new data revealing the potential for DM as a novel microtubule inhibitor.

Baicalin attenuates focal cerebral ischemic reperfusion injury through inhibition of nuclear factor κB p65 activation.

Baicalin is a flavonoid compound purified from plant Scutellaria baicalensis Georgi. We aimed to evaluate the neuroprotective effects of baicalin against cerebral ischemic reperfusion injury. Male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 24 h. Baicalin at doses of 50, 100 and 200 mg/kg was intravenously injected after ischemia onset. Twenty-four hours after reperfusion, the neurological deficit was scored and infarct volume was measured. Hematoxylin and eosin (HE) staining was performed to analyze the histopathological changes of cortex and hippocampus neurons. We examined the levels of NF-κB p65 in ischemic cortexes by Western blot analysis and RT-PCR assay. The results showed that the neurological deficit scores were significantly decreased from 2.0 ± 0.7 to 1.2 ± 0.4 and the volume of infarction was reduced by 25% after baicalin injection. Histopathological examination showed that the increase of neurons with pycnotic shape and condensed nuclear in cortex and hippocampus were not observed in baicalin treated animals. Further examination showed that NF-κB p65 in cortex was increased after ischemia reperfusion injury, indicating the molecular mechanism of ischemia reperfusion injury. The level of NF-κB p65 was decreased by 73% after baicalin treatment. These results suggest that baicalin might be useful as a potential neuroprotective agent in stroke therapy. The neuroprotective effects of baicalin may relate to inhibition of NF-κB p65.

Anti-proliferative effects of recombinant iron superoxide dismutase on HepG2 cells via a redox-dependent PI3k/Akt pathway.

The coding sequence for an iron superoxide dismutase (fe-sod) was amplified from the Nostoc commune genome. Recombinant Fe-SOD was overexpressed in Escherichia coli, accounting for approximately 76% of total bacterial protein. Fe-SOD was purified from bacterial lysate by Ni-NTA column chromatography and used to generate an anti-SOD antibody. The purified Fe-SOD was encapsulated in liposomes and delivered to HepG2 liver tumor cells to eliminate cellular superoxide anions. The SOD-loaded cells exhibited lower reactive oxygen species (ROS) levels and higher reduced glutathione (GSH) levels. In Fe-SOD-treated cells, the cell cycle was delayed in the G(1) phase, and HepG2 cell growth slowed in association with dephosphorylation of the serine-threonine kinase Akt. Low-dose H(2)O(2) stimulated Akt phosphorylation, implying that Akt activation in HepG2 cells is redox-sensitive. Akt phosphorylation was abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitors, suggesting that PI3K is an upstream mediator of Akt activation in HepG2 cells. This study provides insight into recombinant Fe-SOD-induced signaling mechanisms in liver tumor cells and suggests the feasibility of using Fe-SOD as an antitumor agent.