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.

Molecular modeling studies of [4-(3H-benzoimidazol-5-yl)-pyrimidin-2-yl]-amine-based CDK4 inhibitors.

Aim: CDK4 is a promising target for breast cancer therapy. This study aimed to explore the structure-activity relationship of CDK4 inhibitor abemaciclib analogs and design potent CDK4 inhibitors for breast cancer treatment. Methods & results: A faithful 3D quantitative structure-activity relationship model was established by molecular docking, comparative molecular field analysis and comparative molecular similarity index analysis based on 56 abemaciclib analogs. Molecular dynamics simulation studies revealed the key residues of the interaction between CDK4 and inhibitors. Four novel inhibitors with satisfactory predicted binding affinity to CDK4 were designed. Conclusion: The 3D quantitative structure-activity relationship and molecular dynamics simulation studies provide valuable insight into the development of novel CDK4 inhibitors.

Molecular Dynamics Simulations Based on 1-Phenyl-4-Benzoyl-1-Hydro-Triazole ERRα Inverse Agonists.

Estrogen-related receptor α (ERRα), which is overexpressed in a variety of cancers has been considered as an effective target for anticancer therapy. ERRα inverse agonists have been proven to effectively inhibit the migration and invasion of cancer cells. As few crystalline complexes have been reported, molecular dynamics (MD) simulations were carried out in this study to deepen the understanding of the interaction mechanism between inverse agonists and ERRα. The binding free energy was analyzed by the MM-GBSA method. The results show that the total binding free energy was positively correlated with the biological activity of an inverse agonist. The interaction of the inverse agonist with the hydrophobic interlayer composed of Phe328 and Phe495 had an important impact on the biological activity of inverse agonists, which was confirmed by the decomposition of energy on residues. As Glu331 flipped and formed a hydrogen bond with Arg372 in the MD simulation process, the formation of hydrogen bond interaction with Glu331 was not a necessary condition for the compound to act as an inverse agonist. These rules provide guidance for the design of new inverse agonists.

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.

Binding Mechanism of Thrombin-Ligand Systems Investigated by a Polarized Protein-Specific Charge Force Field and Interaction Entropy Method.

In this study, 2 groups of 10 modified ligand systems with modified P3 and P2 side chains are used to study the binding mechanism with thrombin. Experimental results show that the binding affinity is enhanced by complex ligand side chains. The binding free energy obtained from the polarized protein-specific charge (PPC) force field combined with the newly developed interaction entropy (IE) method is consistent with the experimental values with a high correlation coefficient. On the contrary, poor correlation is obtained using the traditional normal mode (Nmode) method for calculating the entropy change. Furthermore, the binding free energy and hot-spot residue energy are decomposed, and the common hot-spot residues in the two groups of systems are Trp50, Leu96, Ile179, Asp199, Cyx201, Ser226, Trp227, Gly228, and Gly230. The electrostatic and van der Waals interaction energies were found to be the main contributors in the binding energy difference. CH-π and CH-CH interactions of Leu96 ligands are significantly related to the energy change due to the modified side chain, and the hydrogen bond between Asp199 and the ligand provides a strong electrostatic interaction, contributing to the binding free energy. Investigating the B-factor, principal component, and binding pocket also explains the change in the binding affinity caused by the modified side chains in ligands from the viewpoint of conformational change. This study demonstrates that the new IE method is superior to the Nmode method in the predicting binding free energy and emphasizes the importance of electronic polarization in molecular dynamics simulation. Moreover, from the viewpoint of energy and structure analysis, this study reveals the origin of the change in binding free energy in modified ligands with different binding sites.

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.

Theoretical insights into the structural and fluorescence properties of DNA containing fluorescent nucleobases.

Fluorescent base analogues are of great importance as sensitive probes to detect the dynamic structures of DNA. In this research, the structural and photophysical properties of 13-mer oligonucleotides containing 4-aminophthalimide:2,4-diaminopyrimidine (4AP:DAP) (4AP0, 4AP') were characterized using both molecular dynamics simulations and quantum mechanics methods. The results indicate that the 4AP:DAP pair is well adapted to the overall B-DNA structure with higher stability and π-stacking abilities. The structural overlap of 4AP' and 4AP0 with the neighboring adenines only lies in the 5'-direction which results in the structure distortion from native B-DNA. Furthermore, the photophysical properties of the fluorescent base monomers and the B-DNA duplex were explored in detail. A very important result is that the hydrogen bond interaction does not have more effect on the fluorescence band apart from the slight red-shifts. In particular, the identity of the neighboring bases stacked with 4AP has an important effect on the fluorescence band. How the local environment can alter the photophysical features of the nucleobases when they are incorporated into the DNA duplex is elucidated.

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.

Theoretical insights into the mechanism of ferroptosis suppression via inactivation of a lipid peroxide radical by liproxstatin-1.

Ferroptosis is a recently discovered iron-dependent form of non-apoptotic cell death caused by the accumulation of membrane lipid peroxidation products, which is involved in various pathological conditions of the brain, kidney, liver and heart. A potent spiroquinoxalinamine derivative named liproxstatin-1 is discovered by high-throughput screening, which is able to suppress ferroptosis via lipid peroxide scavenging in vivo. Thus, molecular simulations, density functional theory (DFT) and variational transition-state theory with a small-curvature tunneling (SCT) coefficient are utilized to elucidate the detailed mechanisms of inactivation of a lipid peroxide radical by liproxstatin-1. H-atom abstracted from liproxstatin-1 by a CH3OO˙ radical occurs preferentially at the aromatic amine site (1'-NH) under thermodynamic and frontier molecular orbital analysis. The value of a calculated rate constant at 300 K is up to 6.38 × 103 M-1 S-1, indicating that the quantum tunneling effect is responsible for making a free radical trapping reaction more efficient by liproxstatin-1. The production of a liproxstatin-1 radical is easily regenerated to the active reduced form by ubiquinol in the body to avoid secondary damage by free radicals. A benzene ring and the higher HOMO energy are beneficial to enhance the lipid radical scavenging activity based on the structure-activity relationship study. Overall, the present results provide theoretical insights into the exploration of novel 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.

Mechanistic insight into the regioselectivity of Pd(ii)-catalyzed C-H functionalization of N-methoxy cinnamamide.

Computational studies have been applied to gain insight into the mechanism of Pd(ii) catalyzed α-C-H functionalization of N-methoxy cinnamamide. The results show that the whole catalytic cycle proceeds via sequential six steps, including (i) catalyst Pd(t-BuNC)2 oxidation with O2, (ii) O-H deprotonation, (iii) t-BuNC migratory insertion to the Pd-C bond, (iv) acyl migration, (v) C-H activation and (vi) reductive elimination. The regioselectivity for different C-H activation sites depends on the coordination structures of α-C or ß-C to the palladium(ii) center. The coordination of α-C to the palladium(ii) center shows a regular planar quadrilateral structure, which is stable. However, the ß-C coordinating to the palladium(ii) center mainly exhibits a distorted quadrilateral structure, which is relatively unstable. Thus, the barrier of α-C-H activation is much lower than that of ß-C-H activation. The present results provide a deep understanding of the site-selectivity of C-H activation.

A theoretical insight into the formation mechanisms of C/N-ribonucleosides with pyrimidine and ribose.

The detailed formation mechanisms of C-ribonucleoside and N-ribonucleoside via the reaction of 2,4,6-triaminopyrimidine (TAP) with (d)-ribose in aqueous solution were explored using density functional theory (DFT). The calculations indicate that five isomers (α,ß-furanose, α,ß-pyranose and open-chain aldehyde) of (d)-ribose can exist in equilibrium in aqueous solution. In contrast to cyclic isomers, an open-chain aldehyde is most feasible to react with TAP. In general, the formation pathways of C-nucleoside and N-nucleoside proceed in three steps including nucleophilic addition, dehydration and cyclization. The calculated apparent activation energies are 28.8 kcal mol-1 and 29.2 kcal mol-1, respectively. It suggests that both C- and N-nucleoside can be formed in aqueous solution, which is in good agreement with the experimental results. The water molecule plays an important "H-bridge" role by the hydrogen atom relay. Finally, a model structure of nucleobase, which will be beneficial for the C-C glycosidic bond formation, is proposed.

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.

Theoretical characterization of the conformational features of unnatural oligonucleotides containing a six nucleotide genetic alphabet.

The addition of the unnatural P:Z base pair to the four naturally occurring DNA bases expands the genetic alphabet and yields an artificially expanded genetic information system (AEGIS). Herein, the structural feature of oligonucleotides containing a novel unnatural P:Z base pair is characterized using both molecular dynamics and quantum chemistry. The results show that the incorporation of the novel artificial base pair (P:Z) preserves the global conformational feature of duplex DNA except for some local structures. The Z-nitro group imparts new properties to the groove width, which widens the major groove. The unnatural oligonucleotides containing mismatched base pairs exhibit low stability. This ensures efficient and high-fidelity replication. In general, the incorporation of the P:Z pair strengthens the stability of the corresponding DNA duplex. The calculated results also show that the thermostability originates from both hydrogen interaction and stacking interaction. The Z-nitro group plays an important role in enhancing the stability of the H-bonds and stacking strength of the P:Z pair. Overall, the present results provide theoretical insights in the exploration of artificially expanded genetic information systems.

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.