Structure-Based Identification of Kelch-like ECH-Associated Protein 1 as a Pharmacological Target of Electrophile-Containing Catechol-O-Methyltransferase Inhibitors.

Entacapone and nitecapone are electrophile-containing catechol-O-methyltransferase (COMT) inhibitors that are used to treat Parkinson's disease in combination with L-DOPA. It is desirable to investigate whether they can covalently bind to cellular protein targets using their reactive electrophilic warheads. We identified Kelch-like ECH-associated protein 1 (KEAP1), a sensor for oxidative and electrophilic stress, as a potential pharmacological target of both drugs by performing covalent-based reverse docking. We confirmed that both drugs activate nuclear factor erythroid 2-related factor 2 (NRF2) by reversibly modifying C151 on KEAP1. Both drugs can enhance the expression of growth differentiation factor 15 (GDF15) and NRF2 downstream antioxidant response element (ARE) genes, both in vitro and in vivo. Furthermore, both drugs exhibit anti-inflammatory effects in an NRF2-dependent acute gout model. Our findings suggest that these two drugs could be repurposed for the treatment of NRF2-modulated inflammatory diseases, and the 3-methylene-acetylacetone group of nitecapone could serve as a new reversible covalent warhead.

SAR study of 1,2-benzisothiazole dioxide compounds that agonize HIF-2 stabilization and EPO production.

Benzisothiazole dioxide compound was reported to agonize HIF-2 stabilization and improve EPO production, thus conceiving a potential strategy to treat disease with chronic hypoxia exemplified by renal anemia. Herein, on the bases of multiple molecular and cellular assays, a series of benzisothiazole derivatives have been synthesized and their structure-activity relationship was evaluated. The SAR and molecular docking studies have revealed the structural insights on the rational design of HIF-2 agonist and discovered a more potential 5-bromine substituted analogue, which showed 2-4 times improvement of HIF-2 downstream gene transcriptions, including EPO production. The present results suggest the therapeutic potential of the compounds for diseases related to EPO insufficiency.

Structural basis for the allosteric inhibition of hypoxia-inducible factor (HIF)-2 by belzutifan.

Hypoxia-inducible factor (HIF)-2α and its obligate heterodimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT), are both members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor family. Previous studies have identified HIF-2α as a key oncogenic driver in clear cell renal cell carcinoma (ccRCC), rendering it a promising drug target for this type of kidney cancer. Belzutifan is the first HIF-2α inhibitor approved for treating ccRCC and other cancers associated with the von Hippel-Lindau (VHL) disease. However, the detailed inhibitory mechanism of belzutifan at molecular level is still unclear. Here we obtained the crystal structure of HIF-2α-ARNT heterodimer in complex with belzutifan at 2.75 Å resolution. The complex structure shows that belzutifan binds into the PAS-B pocket of HIF-2α, and it destabilizes the dimerization of HIF-2α and ARNT through allosteric effects mainly mediated by the key residue M252 of HIF-2α near the dimer interface. We further explored the inhibitory effects of belzutifan using biochemical and functional assays. The time-resolved fluorescence energy transfer (TR-FRET)-based binding assay showed that belzutifan disrupts the dimerization of HIF-2α and ARNT with a Ki value of 20 nM. The luciferase reporter assay indicated that belzutifan can efficiently inhibit the transcriptional activity of HIF-2α with an IC50 value of 17 nM. Besides, the real-time PCR assay illustrated that belzutifan can reduce the expression of HIF-2α downstream genes in 786-O kidney cancer cells in a dose-dependent manner. Our work reveals the molecular mechanism by which belzutifan allosterically inhibits HIF-2α and provides valuable information for the subsequent drug development targeting HIF-2α. Significance Statement The bHLH-PAS family of transcription factors are an emerging group of small-molecule drug targets. Belzutifan, originally developed by Peloton Therapeutics, is the first FDA-approved drug directly binding to a bHLH-PAS protein, the hypoxia-inducible factor (HIF)-2α. Based on the protein-drug complex structure, biochemical binding assays, and functional profiling of downstream gene expression, this study reveals the regulatory mechanism of how belzutifan allosterically destabilizes HIF-2α's heterodimerization with its obligate partner protein, thus reducing their transcriptional activity that links to tumor progression.

Design, synthesis, and bioactivity evaluation of macrocyclic benzo[b]pyrido[4,3-e][1,4]oxazine derivatives as novel Pim-1 kinase inhibitors.

Pim-1 kinase is a serine/threonine kinase which is vital in many tumors. The Pim-1 inhibitor 10-DEBC and its derivatives discovered in our previous work were modified through macrocyclization strategy. A series of benzo[b]pyridine[4,3-e][1,4]oxazine macrocyclic compounds were designed, synthesized, and evaluated as novel Pim-1 kinase inhibitors. Among these compounds, compound H5 exhibited the highest activity with an IC50 value of 35 nM. In addition, the crystal complex structure of Pim-1 kinase bound with compound H3 was determined, and the structure-activity relationship of these macrocyclic compounds was analyzed, which provides the structural basis of further optimization of novel macrocyclic Pim-1 kinase inhibitors..

Identification of oleoylethanolamide as an endogenous ligand for HIF-3α.

Hypoxia-inducible factors (HIFs) are α/ß heterodimeric transcription factors modulating cellular responses to the low oxygen condition. Among three HIF-α isoforms, HIF-3α is the least studied to date. Here we show that oleoylethanolamide (OEA), a physiological lipid known to regulate food intake and metabolism, binds selectively to HIF-3α. Through crystallographic analysis of HIF-3 α/ß heterodimer in both apo and OEA-bound forms, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics (MD) simulations, and biochemical and cell-based assays, we unveil the molecular mechanism of OEA entry and binding to the PAS-B pocket of HIF-3α, and show that it leads to enhanced heterodimer stability and functional modulation of HIF-3. The identification of HIF-3α as a selective lipid sensor is consistent with recent human genetic findings linking HIF-3α with obesity, and demonstrates that endogenous metabolites can directly interact with HIF-α proteins to modulate their activities, potentially as a regulatory mechanism supplementary to the well-known oxygen-dependent HIF-α hydroxylation.

Engineering and elucidation of the lipoinitiation process in nonribosomal peptide biosynthesis.

Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformational states of starter condensation domains during the reaction cycle of the lipoinitiation process. Our studies provide further insights into the mechanism of lipoinitiation and the engineering of nonribosomal peptide synthetases.

Ultrahigh ß-phase content poly(vinylidene fluoride) with relaxor-like ferroelectricity for high energy density capacitors.

Poly(vinylidene fluoride)-based dielectric materials are prospective candidates for high power density electric storage applications because of their ferroelectric nature, high dielectric breakdown strength and superior processability. However, obtaining a polar phase with relaxor-like behavior in poly(vinylidene fluoride), as required for high energy storage density, is a major challenge. To date, this has been achieved using complex and expensive synthesis of copolymers and terpolymers or via irradiation with high-energy electron-beam or γ-ray radiations. Herein, a facile process of pressing-and-folding is proposed to produce ß-poly(vinylidene fluoride) (ß-phase content: ~98%) with relaxor-like behavior observed in poly(vinylidene fluoride) with high molecular weight > 534 kg mol-1, without the need of any hazardous gases, solvents, electrical or chemical treatments. An ultra-high energy density (35 J cm-3) with a high efficiency (74%) is achieved in a pressed-and-folded poly(vinylidene fluoride) (670-700 kg mol-1), which is higher than that of other reported polymer-based dielectric capacitors to the best of our knowledge.

Acid/base-controllable fluorescent molecular switches based on cryptands and basic N-heteroaromatics.

Two kinds of fluorescent BMP32C10-based cryptands 1 and 2 have been developed. Cryptand 1 contains a binaphthol group, while cryptand 2 bears a coumarin group in their third arms. Based on this design, novel self-assemblies constructed from cryptand 1 or 2 and basic N-heteroaromatic guests 3-6 were successfully obtained. Moreover, the threading/dethreading processes of the host-guest complexes could be well switched by the alternate addition of acid/base, and accompanied by concurrent changes in fluorescence.

Enhanced response speed and selectivity of fluorescein-based H2S probe via the cleavage of nitrobenzene sulfonyl ester assisted by ortho aldehyde groups.

In this work, we developed three fluorescent probes (F-1, F-2, and F-3) based on fluorescein, mono-formylated fluorescein, and bis-formylated fluorescein for hydrogen sulfide (H2S) detection. The probe F-3, which bears two aldehyde groups, exhibited the fastest response. This fast response is attributed to the ortho effect of the aldehyde group, which enables fast nucleophilic addition of H2S to an aldehyde group and subsequent intramolecular thiolysis of dinitrophenyl ether. In addition, the aldehyde groups on F-3 react with biothiols (e.g., cysteine, homocysteine) to form thiazolidine diastereomers, which suppress the fluorescence of fluorescein. The introduction of two aldehyde groups also resulted in high selectivity of F-3 towards H2S. Furthermore, good linearity was observed between F-3 fluorescence intensity at 510nm and H2S concentration in the range of 0-10µM. F-3 exhibited a detection limit as low as 0.024µM. Confocal laser scanning micrographs of HeLa cells incubated with F-3 confirmed that F-3 is cell-permeable and can successfully detect H2S in living cells.

Synthesis of a highly HOCl-selective fluorescent probe and its use for imaging HOCl in cells and organisms.

During infection, nicotinamide adenine dinucleotide phosphate-oxidase of innate immune cells generates important microbicidal reactive oxygen species (ROS) such as hypochlorous acid (HOCl) to kill the invading pathogens. However, excess amounts of HOCl induce oxidative damage of functional biomolecules such as DNA and proteins, which may cause chronic inflammatory diseases. Herein, we outline protocols for the preparation of a rhodamine-based HOCl probe, as well as applications thereof, with which to detect HOCl in living cells and organisms. The probe (R19S) can be prepared from a commercially available rhodamine, rhodamine 6G, in two steps. When R19S is treated with HOCl, the sulfur atom is replaced by an oxygen atom, resulting in opening of the lactone ring; thus, nonfluorescent R19S is converted to highly fluorescent rhodamine 19 (R19). R19S exhibits high selectivity for HOCl over other ROS and high sensitivity in a weakly acidic environment. In addition, we describe fluorescence imaging assays of HOCl in mouse neutrophils and Drosophila targeted using this probe. The approximate amount of time required to synthesize the probe is 2-3 d, after which it can be used for up to 5 h in the bioimaging of living cells.

Recent progress in the development of fluorescent, luminescent and colorimetric probes for detection of reactive oxygen and nitrogen species.

Reactive oxygen (ROS) and nitrogen (RNS) species cause oxidative and nitrosative stresses, respectively. These stresses are implicated not only in diverse physiological processes but also in various pathological processes, including cancer and neurodegenerative disorders. In addition, some ROS and RNS in the environment are pollutants that threaten human health. As a consequence of these effects, sensitive methods, which can be employed to selectively monitor ROS and RNS in live cells, tissues and organisms as well as in environmental samples, are needed so that their biological roles can be understood and their concentrations in environmental samples can be determined. In this review, fluorescent, luminescent and colorimetric ROS and RNS probes, which have been developed since 2011, are comprehensively discussed.

The synthesis of UDP-selective fluorescent probe and its imaging application in living cells.

A perylene-based probe was developed for uridine diphosphate (UDP) sensing and cell imaging. The probe presented about 4-fold fluorescence enhancement in the presence or absence of 100equiv UDP. The selectivity toward UDP over other phosphor-containing anions was observed. The selective UDP sensing was speculated to be related to the binding affinities of Zn(2+) ions in sensor with the uridine and phosphate moieties of UDP. Furthermore, this probe was also applied to image of UDP in living cells.