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.

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.

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.

Synthesis and Herbicidal Activity of Triketone-Aminopyridines as Potent p-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

Exploring novel p-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) inhibitors has become one of the most promising research directions in herbicide innovation. On the basis of our tremendous interest in exploiting more powerful HPPD inhibitors, we designed a family of benzyl-containing triketone-aminopyridines via a structure-based drug design (SBDD) strategy and then synthesized them. Among these prepared derivatives, the best active 3-hydroxy-2-(3,5,6-trichloro-4-((4-isopropylbenzyl)amino)picolinoyl)cyclohex-2-en-1-one (23, IC50 = 0.047 µM) exhibited a 5.8-fold enhancement in inhibiting Arabidopsis thaliana (At) HPPD activity over that of commercial mesotrione (IC50 = 0.273 µM). The predicted docking models and calculated energy contributions of the key residues for small molecules suggested that an additional π-π stacking interaction with Phe-392 and hydrophobic contacts with Met-335 and Pro-384 were detected in AtHPPD upon the binding of the best active compound 23 compared with that of the reference mesotrione. Such a molecular mechanism and the resulting binding affinities coincide with the proposed design scheme and experimental values. It is noteworthy that inhibitors 16 (3-hydroxy-2-(3,5,6-trichloro-4-((4-chlorobenzyl)amino)picolinoyl)cyclohex-2-en-1-one), 22 (3-hydroxy-2-(3,5,6-trichloro-4-((4-methylbenzyl)amino)picolinoyl)cyclohex-2-en-1-one), and 23 displayed excellent greenhouse herbicidal effects at 150 g of active ingredient (ai)/ha after postemergence treatment. Furthermore, compound 16 showed superior weed-controlling efficacy against Setaria viridis (S. viridis) versus that of the positive control mesotrione at multiple test dosages (120, 60, and 30 g ai/ha). These findings imply that compound 16, as a novel lead of HPPD inhibitors, possesses great potential for application in specifically combating the malignant weed S. viridis.

Review on the recent progress in the development of fluorescent probes targeting enzymes.

Enzymes are very important for biological processes in a living being, performing similar or multiple tasks in and out of cells, tissues and other organisms at a particular location. The abnormal activity of particular enzyme usually caused serious diseases such as Alzheimer's disease, Parkinson's disease, cancers, diabetes, cardiovascular diseases, arthritis etc. Hence, nondestructive and real-time visualization for certain enzyme is very important for understanding the biological issues, as well as the drug administration and drug metabolism. Fluorescent cellular probe-based enzyme detectionin vitroandin vivohas become broad interest for human disease diagnostics and therapeutics. This review highlights the recent findings and designs of highly sensitive and selective fluorescent cellular probes targeting enzymes for quantitative analysis and bioimaging.

An Activity-Based Fluorogenic Probe Enables Cellular and in Vivo Profiling of Carboxylesterase Isozymes.

Carboxylesterases (CEs) exist as multiple types of isomers in humans, and two major types are CE1 and CE2. They are widely distributed in human tissues and well-known for their important roles in drug metabolism and pathology of various diseases. Thus, the detection of CEs in living systems could provide efficient proof in disease diagnostics, as well as important information regarding chemotherapeutic effects of antitumor drugs and prognosis. To develop a specific probe to discriminate CEs from other hydrolases, especially cholinesterases, is quite challenging due to their structural similarities and substrate specificity. To date, almost all of the fluorescent probes developed for CEs have been constructed with an acetyl group as the recognition unit. Herein we proposed a new design strategy of probe-cavity matching, which led to the identification of a new fluorogenic substrate (termed as HBT-CE) with high specificity toward both CE isomers and improved sensitivity, considering the higher binding affinity and catalysis efficiency. The promising capability of HBT-CE was further demonstrated for endogenous CEs imaging in living cells, zebrafish, and nude mice. In addition, HBT-CE was successfully applied in kinetically monitoring drug-induced CE regulation in cancer cells. All of these findings suggest that HBT-CE is a valuable tool for tracking and imaging endogenous CEs in complex biological systems.

Human Neutrophil Elastase Activated Fluorescent Probe for Pulmonary Diseases Based on Fluorescence Resonance Energy Transfer Using CdSe/ZnS Quantum Dots.

There is an increasing demand for effective noninvasive diagnosis against common pulmonary diseases, which are rising sharply due to the serious air pollution. Human neutrophil elastase (HNE), a typical protease highly involved in pulmonary inflammatory diseases and lung cancer, is a potential predictor for disease progression. Currently, few of the HNE-targeting probes are applicable in vivo due to the limitation in sensitivity and biocompatibility. Herein, we reported the achievement of in vitro detection and in vivo imaging of HNE by incorporating the HNE-specific peptide substrate, quantum dots (QDs), and organic dyes into the fluorescence resonance energy transfer (FRET) system. The refined nanoprobe, termed QDP, could specifically measure the HNE with excellent sensitivity of 7.15 pM in aqueous solution and successfully image the endogenous and exogenous HNE in living cells. In addition, this nanoprobe enabled HNE imaging in mouse models of lung cancer and acute lung injury, and the HNE activity at high temporal and spatial resolution was continuously monitored. Most importantly, QDP successfully discriminated the serums of patients with lung diseases from those of the healthy controls based on the HNE activity determination. Overall, this study demonstrates the advantages of a FRET-system-based nanoprobe in imaging performance and provides an applicable tool for in vivo HNE detection and pulmonary disease diagnosis.

[Effect of moxibustion on clinical symptoms, peripheral inflammatory indexes and T lymphocyte subsets in COVID-19 patients].

OBJECTIVE: To explore the therapeutic effect and the mechanism of the adjuvant treatment with moxibustion on coronavirus disease 2019 (COVID-19). METHODS: A total of 95 patients with COVID-19 were randomly divided into a moxibustion group (45 cases) and a basic treatment group (50 cases). The routine treatment of western medicine was applied in the patients of both groups. In the moxibustion group, on the base of the treatment of western medicine, moxibustion was applied to Dazhui (GV 14), Feishu (BL 13), Qihai (CV 6) and Zusanli (ST 36), once daily and consecutively for 14 days. At the end of treatment courses, clinical symptom scores for cough, asthmatic breathing, chest oppression and short breath, as well as their remission rates were compared between the two groups before and after treatment. Before and after treatment, the white blood cell (WBC) count, the levels of c-reactive protein (CRP) and interleukin-6 (IL-6) and the absolute number of T lymphocyte subsets, i.e. , and of the peripheral blood were compared in the patients between the two groups. The principal component analysis was adopted to analyze the common data extracted from the above 10 clinical indexes variables and comprehensively evaluate the differences in the therapeutic effect of two regimens. RESULTS: The clinical symptom scores were all decreased after treatment in both of the moxibustion group and the basic treatment group as compared with those before treatment (P<0.05). After treatment, the clinical symptom scores of cough, chest oppression and asthmatic breathing in the moxibustion group were lower significantly than those in the basic treatment group (P<0.05) and the remission rates of cough, chest oppression and asthmatic breathing were higher than the basic treatment group (P<0.05). After treatment, WBC count was increased as compared with that before treatment in either group (P<0.05) and the levels of CRP and IL-6 in the moxibustion group were reduced as compared with those before treatment (P<0.05). The reducing range of IL-6 level in the moxibustion group was larger than the basic treatment group (P<0.05). After treatment, the absolute number of , and T lymphocytes was increased as compared with that before treatment in the moxibustion group (P<0.05), and its increase range was larger than the basic treatment group (P<0.05). The difference value was 33.38 for the score of comprehensive evaluation before and after treatment in the moxbustion group, higher obviously than 8.91 in the basic treatment group. CONCLUSION: On the base of the routine treatment with western medicine, moxibustion therapy supplemented relieves the clinical symptoms, reduces the levels of inflammatory indexes, i.e. IL-6 and CRP as well as improves the absolute number of peripheral T lymphocyte subsets. The clinical therapeutic effect of such regimen with moxibustion supplemented is significantly better than the simple routine treatment of western medicine.

Genetic, epigenetic and biochemical regulation of succinate dehydrogenase function.

Succinate dehydrogenase (SDH), complex II or succinate:quinone oxidoreductase (SQR) is a crucial enzyme involved in both the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS), the two primary metabolic pathways for generating ATP. Impaired function of SDH results in deleterious disorders from cancer to neurodegeneration. SDH function is tailored to meet the energy demands in different cell types. Thus, understanding how SDH function is regulated and how it operates in distinct cell types can support the development of therapeutic approaches against the diseases. In this article we discuss the molecular pathways which regulate SDH function and describe extra roles played by SDH in specific cell types.

Molecular pathogenesis of tumorigenesis caused by succinate dehydrogenase defect.

Succinate dehydrogenase (SDH), also named as complex II or succinate:quinone oxidoreductases (SQR) is a critical enzyme in bioenergetics and metabolism. This is because the enzyme is located at the intersection of oxidative phosphorylation and tricarboxylic acid cycle (TCA); the two major pathways involved in generating energy within cells. SDH is composed of 4 subunits and is assembled through a multi-step process with the aid of assembly factors. Not surprisingly malfunction of this enzyme has marked repercussions in metabolism leading to devastating tumors such as paraganglioma and pheochromocytoma. It is already known that mutations in the genes encoding subunits lead to tumorigenesis, but recent discoveries have indicated that mutations in the genes encoding the assembly factors also contribute to tumorigenesis. The mechanisms of pathogenesis of tumorigenesis have not been fully understood. However, a multitude of signaling pathways including succinate signaling was determined. We, here discuss how defective SDH may lead to tumor development at the molecular level and describe how yeast, as a model system, has contributed to understanding the molecular pathogenesis of tumorigenesis resulting from defective SDH.

The assembly of succinate dehydrogenase: a key enzyme in bioenergetics.

Succinate dehydrogenase (SDH) also known as complex II or succinate:quinone oxidoreductase is an enzyme involved in both oxidative phosphorylation and tricarboxylic acid cycle; the processes that generate energy. SDH is a multi-subunit enzyme which requires a series of proteins for its proper assembly at several steps. This enzyme has medical significance as there is a broad range of human diseases from cancers to neurodegeneration related to SDH malfunction. Some of these disorders have recently been linked to defective assembly factors, reinvigorating further research in this area. Apart from that this enzyme has agricultural importance as many fungicides have been/will be designed targeting specifically this enzyme in plant fungal pathogens. In addition, we speculate it might be possible to design novel fungicides specifically targeting fungal assembly factors. Considering the medical and agricultural implications of SDH, the aim of this review is an overview of the SDH assembly factors and critical analysis of controversial issues around them.

Activity-Based Near-Infrared Fluorogenic Probe for Enabling in Vitro and in Vivo Profiling of Neutrophil Elastase.

Neutrophil elastase (NE), a typical hematopoietic serine protease, has significant roles in inflammatory and immune responses, and thus is highly associated with various diseases such as acute lung injury (ALI) and lung cancer. Rapid and accurate measurement of NE activity in biological systems is particularly important for understanding the role of NE in inflammatory diseases, as well as clinical diagnosis. However, the specific detection and noninvasive imaging of NE in vivo remains a challenge. To address this issue, a small-molecule substrate based near-infrared fluorogenic probe (NEP) for NE was constructed via incorporating pentafluoroethyl as the recognition group with a hemicyanine dye-based fluorophore. This initially quenched probe possesses more than 25-fold red fluorescence enhancement upon the catalysis of human NE, and the detection limit is about 29.6 ng/mL. In addition, the high specificity and the long emission wavelength (λemmax = 700 nm) of NEP allowed the direct monitoring of NE-trafficking, exogenous NE uptake, and endogenous NE upregulation at the cellular level. Moreover, the successful spatiotemporal imaging of NE in ALI model mice also made it a promising new tool in clinical diagnosis for ALI and other lung diseases.

Preoperative neutrophil-to-lymphocyte ratio is a predictor of survival of epithelial ovarian cancer: a systematic review and meta-analysis of observational studies.

Inflammation plays an important role in the development and progression of epithelial ovarian cancer (EOC). However, no meta-analysis has comprehensively and quantitatively investigated the prognostic value of the neutrophil-to-lymphocyte ratio (NLR) in EOC patients. Therefore, we performed a meta-analysis to quantify the prognostic impact of this biomarker. We searched the PubMed and Web of Science databases from their inception through December 31, 2016, and examined observational studies evaluating the association of preoperative NLR with progression-free survival (PFS) and overall survival (OS) of EOC patients. A random-effects model was used to summarize hazard ratios (HRs) with 95% confidence intervals (CIs). Twelve retrospective cohort studies including 3,154 EOC patients were identified. Elevated NLR in EOC patients was associated with worse PFS (summarized HR=1.80; 95% CI = 1.22-2.65; I2 = 79.1%) and OS (summarized HR = 1.72; 95% CI = 1.18-2.51; I2 = 73.5%) compared with low NLR. No evidence of publication bias was detected by funnel plot analysis and formal statistical tests. Although the results were robust in all subgroup analyses, not all results were statistically significant. We determined that adjustments for CA-125 level and performance status might be sources of heterogeneity. These combined results indicate that preoperative NLR is an important predictor of prognosis in EOC patients. Since the high heterogeneity and retrospective study design of included studies, these results require further validation with prospective cohort and trials enrolling larger patient populations and conducting longer follow-up examinations.

Discovery of a butyrylcholinesterase-specific probe via a structure-based design strategy.

We report herein the structure-based design and application of a fluorogenic molecular probe (BChE-FP) specific to butyrylcholinesterase (BChE). This probe was rationally designed by mimicking the native substrate and optimized stepwise by manipulating the steric feature and the reactivity of the designed probe targeting the structural difference of the active pockets of BChE and AChE. The refined probe, BChE-FP, exhibits high specificity toward BChE compared to AChE, producing about 275-fold greater fluorescence enhancement upon the catalysis by BChE. Thus, BChE-FP is a specific BChE probe identified by the structure-based design and it can discriminate BChE from AChE. Furthermore, it has been successfully applied for imaging the endogenous BChE in living cells, as well as BChE inhibitor screening and characterization under physiological conditions.

Nonpeptide-Based Small-Molecule Probe for Fluorogenic and Chromogenic Detection of Chymotrypsin.

We report herein a nonpeptide-based small-molecule probe for fluorogenic and chromogenic detection of chymotrypsin, as well as the primary application for this probe. This probe was rationally designed by mimicking the peptide substrate and optimized by adjusting the recognition group. The refined probe 2 exhibits good specificity toward chymotrypsin, producing about 25-fold higher enhancement in both the fluorescence intensity and absorbance upon the catalysis by chymotrypsin. Compared with the most widely used peptide substrate (AMC-FPAA-Suc) of chymotrypsin, probe 2 shows about 5-fold higher binding affinity and comparable catalytical efficiency against chymotrypsin. Furthermore, it was successfully applied for the inhibitor characterization. To the best of our knowledge, probe 2 is the first nonpeptide-based small-molecule probe for chymotrypsin, with the advantages of simple structure and high sensitivity compared to the widely used peptide-based substrates. This small-molecule probe is expected to be a useful molecular tool for drug discovery and chymotrypsin-related disease diagnosis.

Detection of Intracellular Selenol-Containing Molecules Using a Fluorescent Probe with Near-Zero Background Signal.

Selenocysteine (Sec), encoded as the 21st amino acid, is the predominant chemical form of selenium that is closely related to various human diseases. Thus, it is of high importance to identify novel probes for sensitive and selective recognition of Sec and Sec-containing proteins. Although a few probes have been reported to detect artificially introduced selenols in cells or tissues, none of them has been shown to be sensitive enough to detect endogenous selenols. We report the characterization and application of a new fluorogenic molecular probe for the detection of intracellular selenols. This probe exhibits near-zero background fluorescence but produces remarkable fluorescence enhancement upon reacting with selenols in a fast chemical reaction. It is highly specific and sensitive for intracellular selenium-containing molecules such as Sec and selenoproteins. When combined with flow cytometry, this probe is able to detect endogenous selenols in various human cancer cells. It is also able to image endogenous selenol-containing molecules in zebrafish under a fluorescence microscope. These results demonstrate that this molecular probe can function as a useful molecular tool for intracellular selenol sensing, which is valuable in the clinical diagnosis for human diseases associated with Sec-deficiency or overdose.

Efficient synthesis and antiproliferative activity of novel thioether-substituted flavonoids.

As widely occurring natural products, flavonoids are an important source for drug discovery, due to their structural diversity and broad-spectrum biological activity. In this work, a library of novel, thioether-substituted flavonoids with diverse heterocyclic groups was synthesized via a microwave-assisted procedure with the advantages of good yields, short times, mild conditions and ready isolation of the products. Their antiproliferative activities were evaluated against six cancer cell lines, HCCLM-7, Hela, MDA-MB-435S, SW-480, Hep-2, and MCF-7 by the MTT-based assay. Compared with the positive control 5-fluorouracil, three compounds, 6a, 6b and 6j were successfully identified as the most promising candidates, due to their higher potency and broad-spectrum bioactivity with IC50 values in the range of 0.43 µM-6.7 µM.

Rieske iron-sulfur protein of the cytochrome bc(1) complex: a potential target for fungicide discovery.

The cytochrome bc(1) complex (complex III, cyt bc(1)) is an essential component of cellular respiration. Cyt bc(1) has three core subunits that are required for its catalytic activity: cytochrome b, cytochrome c(1), and the Rieske iron-sulfur protein (ISP). Although most fungicides inhibit this enzyme by binding to the cytochrome b subunit, resistance to these fungicides has developed rapidly due to their widespread application. Resistance is mainly associated with mutations in cytochrome b, the only subunit encoded by mitochondrial DNA. Recently, the flexibility and motion of the ISP and its essential role in electron transfer have received intense attention; this leads us to propose a new classification of cyt bc(1) inhibitors (three types of Q(o) inhibitors) that mobilize, restrict, or fix the rotation of the ISP. Importantly, the strengths of the ISP-inhibitor interactions correlate with inhibitor activity and the development of resistance to Q(o) inhibitors, thereby offering clues for designing novel cyt bc(1) inhibitors with high potency and a low risk of resistance.