Structure-based discovery of pyrazole-benzothiadiazole hybrid as human HPPD inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) has attracted increasing attention as a target for treating type I tyrosinemia and other diseases with defects in tyrosine catabolism. Only one commercial drug, 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC), clinically treat type I tyrosinemia, but show some severe side effects in clinical application. Here, we determined the structure of human HPPD-NTBC complex, and developed new pyrazole-benzothiadiazole 2,2-dioxide hybrids from the binding of NTBC. These compounds showed improved inhibition against human HPPD, among which compound a10 was the most active candidate. The Absorption Distribution Metabolism Excretion Toxicity (ADMET) predicted properties suggested that a10 had good druggability, and was with lower toxicity than NTBC. The structure comparison between inhibitor-bound and ligand-free form human HPPD showed a large conformational change of the C-terminal helix. Furthermore, the loop 1 and α7 helix were found adopting different conformations to assist the gating of the cavity, which explains the gating mechanism of human HPPD.

Divergent Construction of Thiochromanes and N-Arylbutanamides via Arylthiodifluoromethyl Radical-Triggered Cascade of Alkenes.

A strategy utilizing silver-catalyzed oxidative decarboxylation radical cascade cyclization of arylthiodifluoroacetic acids with alkenes for the simple and efficient preparation of difluoromethylated thiochromanes and 2,2-disubstituted-N-arylbutanamides derivatives has been developed. This approach includes good functional group tolerance, easily accessible starting materials, and operational simplicity.

New Insights into the Interactions between Herbicides: Trends from Recent Studies.

The use of herbicide combinations is a common practice in modern agriculture. However, unexpected results may be observed due to herbicide and weed diversity, therefore, highlighting the need for a predictive strategy. To this end, a data set was made based on recent studies. This data set included herbicide attributes, such as active ingredient, chemical family, and mode of action, and weed attributes, namely, species, clade, type of leaves, family, and lifespan. Globally, additive interactions (46.30%) were more frequent than antagonistic (29.09%) and synergistic (24.61%) ones. The occurrence of these herbicide interactions with regard to herbicide and weed features is also discussed. Moreover, mesotrione and glyphosate have been, respectively, identified as the most promising or inadequate herbicides in predicting beneficial mixtures. The resulting global trend could guide farmers in their choice of beneficial herbicide companions.

Rational Design of Esterase-Insensitive Fluorogenic Probes for In Vivo Imaging.

Small-molecule fluorogenic probes are indispensable tools for performing research in biomedical fields and chemical biology. Although numerous cleavable fluorogenic probes have been developed to investigate various bioanalytes, few of them meet the baseline requirements for in vivo biosensing for disease diagnosis due to their insufficient specificity resulted from the remarkable esterase interferences. To address this critical issue, we developed a general approach called fragment-based fluorogenic probe discovery (FBFPD) to design esterase-insensitive probes for in vitro and in vivo applications. With the designed esterase-insensitive fluorogenic probe, we successfully achieved light-up in vivo imaging and quantitative analysis of cysteine. This strategy was further extended to design highly specific fluorogenic probes for other representative targets, sulfites, and chymotrypsin. The present study expands the bioanalytical toolboxes available and offers a promising platform to develop esterase-insensitive cleavable fluorogenic probes for in vivo biosensing and bioimaging for the early diagnosis of diseases.

4D label-free proteomic analysis of vitreous from patients with rhegmatogenous retinal detachment.

AIM: To identify metabolites, proteins, and related pathways involved in the etiology of rhegmatogenous retinal detachment (RRD) for use as biomarkers in diagnosing and treating RRD. METHODS: Vitreous specimens were collected and liquid chromatography-tandem mass spectrometry analysis was performed using the four-dimensional label-free technique. Statistically significant differentially expressed proteins, gene ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway representations, and protein interactions were analyzed. RESULTS: Nine specimens were subjected to proteomic analysis. In total, 161 proteins were identified as differentially expressed proteins (DEPs), including 53 upregulated proteins and 108 downregulated proteins. GO functional analysis revealed that some DEPs were enriched in neuron-related terms and membrane protein terms. Moreover, KEGG analysis indicated that the cell adhesion molecule metabolic pathway was associated with the greatest number of DEPs. Finally, the evaluation of protein-protein interaction network revealed that DEPs were clustered in neuronal adhesion, apoptosis, inflammation and immune responses, correct protein folding, and glycolysis. CONCLUSION: Proteomic profiling is useful for the exploration of molecular mechanisms that underlie RRD. This study reveals increased expression levels of proteins related to heat shock protein content, glycolysis, and inflammatory responses in RRD. Knowledge regarding biomarkers of RRD pathogenesis may help to prevent the occurrence of RRD in the future.

Insights into 4-hydroxyphenylpyruvate dioxygenase-inhibitor interactions from comparative structural biology.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) plays a key role in tyrosine metabolism and has been identified as a promising target for herbicide and drug discovery. The structures of HPPD complexed with different types of inhibitors have been determined previously. We summarize the structures of HPPD complexed with structurally diverse molecules, including inhibitors, natural products, substrates, and catalytic intermediates; from these structures, the detailed inhibitory mechanisms of different inhibitors were analyzed and compared, and the key structural factors determining the slow-binding behavior of inhibitors were identified. Further, we propose four subpockets that accommodate different inhibitor substructures. We believe that these analyses will facilitate in-depth understanding of the enzymatic reaction mechanism and enable the design of new inhibitors with higher potency and selectivity.

Enhancement of Biomass Conservation and Bioethanol Production of Sweet Sorghum Silage by Constructing Synergistic Microbial Consortia.

The efficient storage of materials before bioethanol production could be key to improving pretreatment protocol and facilitating biodegradation, in turn improving the cost-effectiveness of biomass utilization. Biological inoculants were investigated for their effects on ensiling performance, biodegradability of silage materials, and final bioethanol yield from sweet sorghum. Two cellulolytic microbial consortia (CF and PY) were used to inoculate silages of sweet sorghum, with and without combined lactic acid bacteria (Xa), for up to 60 days of ensiling. We found that the consortia notably decreased pH and ammonia nitrogen content while increasing lactic acid/acetic acid ratios. The microbes also functioned in synergy with Xa, significantly reducing lignocellulose content and improving biomass preservation. First-order exponential decay models captured the kinetics of nonstructural carbohydrates and suggested high water-soluble carbohydrate (grams per kilogram dry matter [DM]) preservation potential in PY-Xa (33.48), followed by CF-Xa (30.51). Combined addition efficiently improved enzymatic hydrolysis and enhanced bioethanol yield, and sweet sorghum treated with PY-Xa had the highest ethanol yield (28.42 g L-1). Thus, combined bioaugmentation of synergistic microbes provides an effective method of improving biomass preservation and bioethanol production from sweet sorghum silages. IMPORTANCE Ensiling is an effective storage approach to ensure stable year-round supply for downstream biofuel production; it offers combined facilities of storage and pretreatment. There are challenges in ensiling sweet sorghum due to its coarse structure and high fiber content. This study provides a meaningful evaluation of the effects of adding microbial consortia, with and without lactic acid bacteria, on changes in key properties of sweet sorghum. This study highlighted the bioaugmented ensiling using cellulolytic synergistic microbes to outline a cost-effective strategy to store and pretreat sweet sorghum for bioethanol production.

Discovery of Subnanomolar Inhibitors of 4-Hydroxyphenylpyruvate Dioxygenase via Structure-Based Rational Design.

High-potency 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors are usually featured by time-dependent inhibition. However, the molecular mechanism underlying time-dependent inhibition by HPPD inhibitors has not been fully elucidated. Here, based on the determination of the HPPD binding mode of natural products, the π-π sandwich stacking interaction was found to be a critical element determining time-dependent inhibition. This result implied that, for the time-dependent inhibitors, strengthening the π-π sandwich stacking interaction might improve their inhibitory efficacy. Consequently, modification with one methyl group on the bicyclic ring of quinazolindione inhibitors was achieved, thereby strengthening the stacking interaction and significantly improving the inhibitory efficacy. Further introduction of bulkier hydrophobic substituents with higher flexibility resulted in a series of HPPD inhibitors with outstanding subnanomolar potency. Exploration of the time-dependent inhibition mechanism and molecular design based on the exploration results are very successful cases of structure-based rational design and provide a guiding reference for future development of HPPD inhibitors.

Diagnosis of Bacterial Plant Diseases via a Nitroreductase-Activated Fluorescent Sensor.

Plant diseases caused by bacteria have become one of the serious problems that threaten human food security, which led to the remarkable reduction of agricultural yields and economic loss. Nitroreductase (NTR), as an important biomarker, is highly expressed in bacteria, and the level of NTR is closely related to the progression of pathogen infection. Therefore, the design of small-molecule fluorescent sensors targeting NTR is of great significance for the detection and diagnosis of plant pathogenic bacteria. In this study, a new fluorescent sensor targeting NTR was discovered and then successfully applied to the imaging of zebrafish and pathogenic bacteria. Most importantly, the developed sensor achieved the real-time diagnosis of Brassica napus L. infected with bacteria, which provides a promising tool for examining the temporal and spatial infection of plant pathogens in precision agriculture.

[Effects of different types of exercise on contractile properties and expressions of MuRF1, PGC-1α /FNDC5 in soleus muscle of unloaded rats].

Objective: To investigate the effects of aerobic versus resistance exercise on soleus muscle contractile properties and the expressions of MuRF1, PGC-1α and FNDC5 in amyotrophic rats after unloading, and the possible molecular biological mechanisms. Methods: Male Wistar rats were randomly divided into recovery group (CT), aerobic exercise group (A), resistance exercise group (R) and control group (C), with 6 rats in each group. The control group did not receive any treatment. The other three groups underwent tail suspension for 2 weeks, and then the recovery group recovered quietly. The aerobic group and the resistance group underwent a 2-week exercise intervention. Exercise plan: the aerobic group rats were treated with treadmill speed corresponding to 65% maximum oxygen uptake (VO2max), 60 min/d, 5 d/w; the rats in the resistance group were allowed to climb the ladder with 65% of the maximum voluntary weight-bearing (MVCC) for 3 times, with a total of 5 sets. Each time had a rest of 1 min, with an interval of 2 min among sets, and 5 d/w. Fasting for 24 hours after the last exercise, the soleus muscle samples were collected to observe the histological changes, test the contractility, and detect MuRF1 and PGC-1α and FNDC5 expressions. Results: compared with the control group, the body weight, soleus muscle wet weight, average cross-sectional area of muscle fibers and muscle contractility of the recovery group were decreased significantly(P<0.01), and the expression of MuRF1 was increased significantly(P<0.01). Compared with the recovery group, the body weight, wet weight of soleus muscle, the average cross-sectional area of muscle fiber and muscle contractility of rats in aerobic group and resistance group were increased (P<0.01), the expression of PGC-1α/FNDC5 was increased (P<0.01) and the expression of MuRF1 was decreased significantly (P<0.01). Compared with the aerobic group, the expression of PGC-1α in soleus muscle of rats in the resistance group was increased (P<0.05), while the expression of MuRF1 was decreased (P<0.05). Conclusion: Aerobic and resistance exercise can significantly improve muscle contractility, upregulate the expression of PGC-1α/FNDC5, and inhibit the expression of MuRF1, indicating that the molecular mechanisms of aerobic and resistance exercise to improve unloaded muscular atrophy may be related to PGC-1α and MuRF1.

Effect of intravitreal ranibizumab on fibrovascular membranes in patients with proliferative diabetic retinopathy.

AIM: To assess the effects of intravitreal ranibizumab (IVR) on angiogenesis and glial activity of the fibrovascular membrane (FVM) in patients with proliferative diabetic retinopathy (PDR). METHODS: Forty-two eyes from 42 patients with PDR requiring vitrectomy were included and divided into two groups: control group (n=16) did not receive IVR, while IVR group (n=26) underwent IVR 5d before vitrectomy. FVM specimens were collected by the same surgeon during the interventions. Histopathological morphology was examined by hematoxylin-eosin (H-E) staining and cell densities in the FVM was assessed. Microvessels were outlined by immunohistochemical staining of CD31 and microvessel density (MVD) assessed as an index of FVM angiogenesis. Dual-color immunofluorescence staining, and confocal microscopy was used to detect co-localization and relative expression levels of glial fibrillary acidic protein (GFAP) and α-smooth muscle actin (α-SMA) as markers of glial-mesenchymal transition (GMT). The GMT index (GI; ratio of relative GFAP/α-SMA expression) was used to semi-quantify the degree of GMT or glial activity of FVMs. RESULTS: H-E staining showed similar vascularization in both groups, with microvessels and scattered stromal cells in the matrix. Infiltrated cell densities did not differ significantly between the two groups (P>0.05). The MVD of the IVR group (130.62±15.46/mm2) was significantly lower than that of the controls (142.25±19.16/mm2, P<0.05). In both groups, all sections were strongly immunostained for GFAP and α-SMA. The Pearson's correlation coefficients (PCC) of intensity of automated pixel count of two markers indicated GFAP and α-SMA co-stained well and GMT participated in the remolding of FVMs in PDR. The mean relative GFAP expression in the IVR group was significantly lower, whereas that of α-SMA was significantly higher than in controls (P<0.05). GI in the IVR group (1.10±0.10) was significantly lower than in the controls (1.21±0.12, P<0.05). CONCLUSION: IVR can reduce angiogenesis, glial activity of FVM and promote glial-fibrotic transformation by reducing MVD and promoting GMT but does not decrease the cell density in patients with PDR.

In vivo fluorescent screening for HPPD-targeted herbicide discovery.

BACKGROUND: 4-Hydroxyphenylpyruvate dioxygenase (HPPD), playing a critical role in vitamin E and plastoquinone biosynthesis in plants, has been recognized as one of the most important targets for herbicide discovery for over 30 years. Structure-based rational design of HPPD inhibitors has received more and more research interest. However, a critical challenge in the discovery of new HPPD inhibitors is the common inconsistency between molecular-level HPPD-based bioevaluation and the weed control efficiency in fields, due to the unpredictable biological processes of absorption, distribution, metabolism, and excretion. RESULTS: In this study, we developed a fluorescent-sensing platform of efficient in vivo screening for HPPD-targeted herbicide discovery. The refined sensor has good capability of in situ real-time fluorescence imaging of HPPD in living cells and zebrafish. More importantly, it enabled the direct visible monitoring of HPPD inhibition in plants in a real-time manner. CONCLUSION: We developed a highly efficient in vivo fluorescent screening method for HPPD-targeted herbicide discovery. This discovery not only offers a promising tool to advance HPPD-targeted herbicide discovery, but it also demonstrates a general path to develop the highly efficient, target-based, in vivo screening for pesticide discovery. © 2022 Society of Chemical Industry.

Synthesis of polychloromethylated and halogenated spiro[5,5]trienones via dearomative spirocyclization of biaryl ynones.

We disclosed a selective polychloromethylation and halogenation reaction of alkynes via a radical addition/spirocyclization cascade sequence, in which polyhaloalkanes were used as the precursor for polyhalomethyl and halogen radicals. Using this strategy, a series of valuable halogen-, CHCl2- or CCl3-containing spiro[5,5]trienones were synthesized in good yields with good functional group tolerance in one pot under simple and mild conditions. It is noted that an unprecedented halogenation instead of dibromomethylation was achieved when CH2Br2 was used in this work.

Development of Small-Molecule Fluorescent Probes Targeting Enzymes.

As biological catalysts, enzymes are vital in controlling numerous metabolic reactions. The regulation of enzymes in living cells and the amount present are indicators of the metabolic status of cell, whether in normal condition or disease. The small-molecule fluorescent probes are of interest because of their high sensitivity and selectivity, as well as their potential for automated detection. Fluorescent probes have been useful in targeting particular enzymes of interest such as proteases and caspases. However, it is difficult to develop an ideal fluorescent probe for versatile purposes. In the future, the design and synthesis of enzyme-targeting fluorescent probes will focus more on improving the selectivity, sensitivity, penetration ability and to couple the fluorescent probes with other available imaging molecules/technologies.

Catalyst- and Oxidizing Reagent-Free Electrochemical Benzylic C(sp3)-H Oxidation of Phenol Derivatives.

A site-selective electrochemical approach for the benzylic C(sp3)-H oxidation reaction of phenol derivatives along with hydrogen evolution has been developed. The protocol proceeds in an easily available undivided cell at room temperature under catalyst- and oxidizing reagent-free conditions. The corresponding aryl aldehydes and ketones are obtained in satisfactory yields, and the gram-scale synthesis is easy to be carried out.

Pharmacophore-Oriented Discovery of Novel 1,2,3-Benzotriazine-4-one Derivatives as Potent 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a functional protein existing in almost all aerobic organisms. In the field of agricultural chemicals, HPPD is acknowledged to be one of the crucial targets for herbicides at present due to its unique bio-function in plants. In the Auto Core Fragment in silico Screening (ACFIS) web server, a potential HPPD inhibitor featuring 1,2,3-benzotriazine-4-one was screened out via a pharmacophore-linked fragment virtual screening (PFVS) method. Molecular simulation studies drove the process of "hit-to-lead" optimization, and a family of 1,2,3-benzotriazine-4-one derivatives was synthesized. Consequently, 6-(2-hydroxy-6-oxocyclohex-1-ene-1-carbonyl)-5-methyl-3-(2-methylbenzyl)benzo[d][1,2,3]triazin-4(3H)-one (15bu) was identified to be the best HPPD inhibitor (IC50 = 36 nM) among the 1,2,3-benzotriazine-4-one derivatives, which had over 8-fold improvement of enzyme inhibition compared with the positive control mesotrione (IC50 = 289 nM). Crystallography information for the AtHPPD-15bu complex revealed several important interactions of the ligand bound upon the target protein, i.e., the bidentate chelating interaction of the triketone motif with the metal ion of AtHPPD, a tight π-π stacking interaction consisting of the1,2,3-benzotriazine-4-one moiety and two benzene rings of Phe-424 and Phe-381, and the polydirectional hydrophobic contacts consisting of the ortho-CH3-benzyl group of the core scaffold and some hydrophobic residues. Furthermore, compound 15bu displayed 100% inhibition against the five species of target weeds at the tested dosage, which was comparable to the weed control of mesotrione. Collectively, the fused 1,2,3-benzotriazine-4-one-triketone hybrid is a promising chemical tool for the development of more potent HPPD inhibitors and provides a valuable lead compound 15bu for herbicide innovation.

Rational Redesign of Enzyme via the Combination of Quantum Mechanics/Molecular Mechanics, Molecular Dynamics, and Structural Biology Study.

Increasing demands for efficient and versatile chemical reactions have prompted innovations in enzyme engineering. A major challenge in engineering α-ketoglutarate-dependent oxygenases is to develop a rational strategy which can be widely used for directly evolving the desired mutant to generate new products. Herein, we report a strategy for rational redesign of a model enzyme, 4-hydroxyphenylpyruvate dioxygenase (HPPD), based on quantum mechanics/molecular mechanics (QM/MM) calculation and molecular dynamic simulations. This strategy enriched our understanding of the HPPD catalytic reaction pathway and led to the discovery of a series of HPPD mutants producing hydroxyphenylacetate (HPA) as the alternative product other than the native product homogentisate. The predicted HPPD-Fe(IV)═O-HPA intermediate was further confirmed by the crystal structure of Arabidopsis thaliana HPPD/S267W complexed with HPA. These findings not only provide a good understanding of the structure-function relationship of HPPD but also demonstrate a generally applicable platform for the development of biocatalysts.

Pyroglutamate Aminopeptidase I Promotes Hepatocellular Carcinoma via IL-6/STAT3 Activation as Revealed by a Specific Biosensor.

As a global health challenge, hepatocellular carcinoma (HCC) is strongly associated with chronic inflammation. Targeting inflammation, particularly inflammatory factors, is regarded as an important strategy for HCC diagnosis and treatment. Pyroglutamic aminopeptidase I (PGP-I), a common exopeptidase, was recently identified as a novel inflammatory cytokine in cells. However, whether PGP-I is involved in HCC development and can be regarded as a biomarker remains unclear. To address this issue, endogenous PGP-I was imaged in live cells and in vivo, and the related biochemical and pathological processes were analyzed accordingly with a newly developed fluorogenic PGP-I biosensor. Bioimaging with the specific biosensor demonstrated the aberrant expression of PGP-I in HCC cell lines and tumor-bearing nude mice. Moreover, overexpression of PGP-I in HCC cells promoted tumor progression, whereas knockdown of PGP-I significantly suppressed tumor cell growth and migration. The activity of PGP-I was further identified to be highly related to the phosphorylation of STAT3, which could be impeded by the natural product parthenolide. Collectively, these findings suggest that PGP-I, which can promote hepatocellular tumor progression through the classical inflammation-/tumor-related IL-6/STAT3 pathway, may serve as a potential HCC biomarker and therapeutic target.

Electrochemical Trifluoromethylthiolation and Spirocyclization of Alkynes with AgSCF3: Access to SCF3-Containing Spiro[5,5]trienones.

A novel and efficient strategy for trifluoromethylthiolation and dearomatization of activated alkynes with stable and readily available AgSCF3 has been developed. Reported herein is the unprecedented electrochemical generation of the SCF3 radical in the absence of persulfate for the synthesis of SCF3-containing spiro[5,5]trienones in good yields via a 6-exo-trig radical cyclization.