Taking advantage of the interaction between the sulfoxide and heme cofactor to develop indoleamine 2, 3-dioxygenase 1 inhibitors.

Taking advantage of key interactions between sulfoxide and heme cofactor, we used the sulfoxide as the anchor functional group to develop two series of indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitors: 2-benzylsulfinylbenzoxazoles (series 1) and 2-phenylsulfinylbenzoxazoles (series 2). In vitro enzymatic screening shows that both series can inhibit the activity of IDO1 in low micromolar (series 1) or nanomolar (series 2) levels. They also show inhibitory selectivity between IDO1 and tryptophan 2, 3-dioxygenase 2. Interestingly, although series 1 is less potent IDO1 inhibitors of these two series, it exhibited stronger inhibitory activity toward kynurenine production in interferon-γ stimulated BxPC-3 cells. Enzyme kinetics and binding studies demonstrated that 2-sulfinylbenzoxazoles are non-competitive inhibitors of tryptophan, and they interact with the ferrous form of heme. These results demonstrated 2-sulfinylbenzoxazoles as type II IDO1 inhibitors. Furthermore, molecular docking studies supports the sulfoxide being of the key functional group that interacts with the heme cofactor. Compound 22 (series 1) can inhibit NO production in a concentration dependent manner in lipopolysaccharides (LPS) stimulated RAW264.7 cells, and can relieve pulmonary edema and lung injury in LPS induced mouse acute lung injury models.

Insights Into the Detection Selectivity of Redox and Non-redox Based Probes for the Superoxide Anion Using Coumarin and Chromone as the Fluorophores.

In this study, we evaluated the applicability of various superoxide anion sensors which were designed based on either redox or non-redox mechanisms. Firstly, both redox- and non-redox-based superoxide anion probes were designed and synthesized using either coumarin or chromone as the fluorophores, and the photophysical properties of these probes were measured. Subsequently, the sensing preference of both types of probes toward various reactive oxygen species (ROS) was evaluated. We found that non-redox-based O2 •- probes exhibited broad sensing ability toward various ROS. By contrast, redox based O2 •- probes showed a clear reactivity hierarchy which was well correlated to the oxidizing strength of the ROS. Lastly, the detection selectivity of redox-based O2 •- recognizing probes was also observed when balancing various factors, such as reactant ROS concentrations, temperature, and changing reaction transformation rates. Herein, we concluded the selectivity advantage of redox-based O2 •- probes.

Synthesis and evaluation of benzenesulfonic acid derivatives as human neutrophil elastase (hNE) inhibitors.

Herein we report our investigation concerning the development of Human neutrophil elastase (hNE) inhibitors for the treatment of Acute Respiratory Distress Syndrome (ARDS). Various benzenesulfonic acid derived compounds were synthesized and evaluated as competitive inhibitors of hNE. Biological screening revealed that compound 4f shows moderate inhibitory activity (IC50 = 35.2 µM) against hNE. Compound 4f was also superimposed onto the active center of hNE to understand the binding mode.

Determining Essential Requirements for Fluorophore Selection in Various Fluorescence Applications Taking Advantage of Diverse Structure-Fluorescence Information of Chromone Derivatives.

Herein, we report our work exploring the essential requirements for fluorophore selection during the development of various fluorescence applications. We assembled a library of chromone-derived fluorophores with diverse structure-fluorescence properties, which allowed us to choose the fluorophore pairs with similar structures but differing fluorescence properties and compared the performance of the selected fluorophore pairs in three types of commonly used fluorescence applications. We found that the selection standard of a suitable fluorophore is variable depending on the application. (1) In fluorescence imaging, fluorophores with strong and constant fluorescence under various conditions, such as a large pH range, are preferred. Notably, (2) in the detection of bioactive species, fluorophores with relatively lower fluorescence quantum yield favor the detection sensitivity. Furthermore, (3) in enzymatic assays employing fluorescence, the key parameter is the binding affinity between the fluorophore and the enzyme.

Tetramethylpyrazine ameliorates isoflurane-induced cognitive dysfunction by inhibiting neuroinflammation via miR-150 in rats.

Tetramethylpyrazine (TMP) has neuroprotective effects in the pathogenesis of some human diseases, such as Parkinson's disease. The present study aimed to investigate the role of TMP in isoflurane-induced cognitive dysfunction in rats, and further identify the mechanisms involved in the protective effects of TMP. The Morris water maze test was used to evaluate the cognitive function of rats exposed to isoflurane or treated with TMP. ELISA was conducted to evaluate the effects of isoflurane or TMP on neuroinflammation. The expression of microRNA-150 (miR-150) was measured using reverse transcription-quantitative PCR, and the potential target genes of miR-150 were predicted and verified. The impaired cognitive function induced by isoflurane in the rats was significantly ameliorated by treatment with TMP. In addition, TMP treatment in rats attenuated neuroinflammation caused by isoflurane. The expression of miR-150 was inhibited by isoflurane exposure, but was enhanced by TMP treatment in rats. Furthermore, the overexpression of miR-150 alleviated the isoflurane-induced cognitive dysfunction and neuroinflammation, while the neuroprotective effects of TMP were significantly abrogated by the knockdown of miR-150. AKT3 was a direct target of miR-150, and its mRNA expression was significantly decreased by the overexpression of miR-150 in isoflurane- and TMP-treated rats. These results demonstrated the protective effects of TMP against isoflurane-induced cognitive dysfunction, which were achieved by attenuating neuroinflammation via the regulation of the miR-150/AKT3 pathway. In addition, miR-150 may serve as a novel therapeutic target for the alleviation of cognitive dysfunction induced by anesthetics.

Taking advantage of the aromatisation of 7-diethylamino-4-methyl-3,4-dihydrocoumarin in the fluorescence sensing of superoxide anion.

The aromatisation of 7-diethylamino-3,4-dihydrocoumarin provides an alternative fluorescent probing technique to selectively detect the concentration of superoxide anion in solution. In addition, we reported the advantage of evaluating O2˙- sensing probes in anhydrous DMSO instead of in aqueous buffers when using KO2 as the surrogate of O2˙-.

Design and Synthesis of Indoleamine 2,3-Dioxygenase 1 Inhibitors and Evaluation of Their Use as Anti-Tumor Agents.

Indoleamine 2,3-dioxygenase (IDO) 1 is the key enzyme for regulating tryptophan metabolism and is an important target for interrupting tumor immune escape. In this study, we designed four series of compounds as potential IDO1 inhibitors by attaching various fragments or ligands to indole or phenylimidazole scaffolds to improve binding to IDO1. The compounds were synthesized and their inhibitory activities against IDO1 and tryptophan 2,3-dioxygenase were evaluated. The cytotoxicities of the compounds against two tumor cell lines were also determined. Two compounds with a phenylimidazole scaffold (DX-03-12 and DX-03-13) showed potent IDO1 inhibition with IC50 values of 0.3-0.5 µM. These two IDO1 inhibitors showed low cell cytotoxicity, which indicated that they may exert their anti-tumor effect via immune modulation. Compound DX-03-12 was investigated further by determining the in vivo pharmacokinetic profile and anti-tumor efficacy. The pharmacokinetic study revealed that DX-03-12 had satisfactory properties in mice, with rapid absorption, moderate plasma clearance (∼36% of hepatic blood flow), acceptable half-life (∼4.6 h), and high oral bioavailability (∼96%). Daily oral administration of 60 mg/kg of compound DX-03-12 decreased tumor growth by 72.2% after 19 days in a mouse melanoma cell B16-F10 xenograft model compared with the untreated control. Moreover, there was no obvious weight loss in DX-03-12-treated mice. In conclusion, compound DX-03-12 is a potent lead compound for developing IDO1 inhibitors and anti-tumor agents.

A Cinchona Alkaloid Antibiotic That Appears To Target ATP Synthase in Streptococcus pneumoniae.

Optochin, a cinchona alkaloid derivative discovered over 100 years ago, possesses highly selective antibacterial activity toward Streptococcus pneumoniae. Pneumococcal disease remains the leading source of bacterial pneumonia and meningitis worldwide. The structure-activity relationships of optochin were examined through modification to both the quinoline and quinuclidine subunits, which led to the identification of analogue 48 with substantially improved activity. Resistance and molecular modeling studies indicate that 48 likely binds to the c-ring of ATP synthase near the conserved glutamate 52 ion-binding site, while mechanistic studies demonstrated that 48 causes cytoplasmic acidification. Initial pharmacokinetic and drug metabolism analyses of optochin and 48 revealed limitations of these quinine analogues, which were rapidly cleared, resulting in poor in vivo exposure through hydroxylation pendants to the quinuclidine and O-dealkylation of the quinoline. Collectively, the results provide a foundation to advance 48 and highlight ATP synthase as a promising target for antibiotic development.

Quantitative Evaluation of in Vivo Target Efficacy of Anti-tumor Agents via an Immunofluorescence and EdU Labeling Strategy.

Current methods used to evaluate in vivo target efficacy of selected compound include western blot to semi-quantitatively analyze protein expression. However, problems arise as it is difficult to compare in vivo target efficacy of anti-tumor agents with the same mode of action. It is therefore desirable to develop a protocol that can quantitatively display in vivo target efficacy while also providing other useful information. In this study EdU labeling was used to mark out the proliferating area. The tumor tissue was accordingly divided into proliferating and non-proliferating areas. Fifteen tumor related proteins were stained by immunofluorescence and were found to express in either the proliferating or non-proliferating areas. This allows the quantitative analysis of protein expressions within the precise area. With simple image analysis, our method gave precise percent changes of protein expression and cell proliferation between the drugs treated group and the control group. Additional information, such as, the status of protein expression can also be obtained. This method exhibits high sensitivity, and provides a quantitative approach for in vivo evaluation of target efficacy.

Exploration of the Fluorescent Properties and the Modulated Activities against Sirtuin Fluorogenic Assays of Chromenone-Derived Natural Products.

Chromenone-derived natural products include chromones (flavone, isoflavone) and coumarins. Chromenone compounds not only exhibit impressive biological activities, but also are an important resource of experimentally used fluorophores, such as, 7-amino-4-methylcoumarin (AMC). Various chromenone compounds have reported to have weak fluorescence, and this has the potential to interfere with the measurements during AMC fluorogenic assays and result in non-robust assay readouts. Several flavones and isoflavones were found as SIRT1 activators, while fluorogenic sirtuin assays utilized AMC labelled peptides as the substrates. In this study we investigated whether the fluorescent properties of chromenone-derived natural products interrupt the measurement of SIRT1/2 modulated activities. We found that the reported SIRT1 activators: flavones were detected with the SIRT1 activation activity, but isoflavones were not detected with SIRT1 activation activity, and instead that they were found to be fluorogenic compounds. Another chromenone compound, osthole, exhibited a moderate SIRT2 inhibitory activity with an IC50 of 10 µM. In conclusion, the fluorescent properties of these chromenone compounds do affect the measurement of the sirtuin activities of both inhibitors and activators. However, if the possible fluorescence properties are mitigated in the assay readout, these fluorogenic assays enable the screening of activity modulators.

Virtual Screening of Small Molecular Inhibitors against DprE1.

Decaprenylphosphoryl-ß-d-ribose oxidase (DprE1) is the flavoprotein subunit of decaprenylphosphoryl-d-ribose epimerase involved in cell wall synthesis in Mycobacterium tuberculosis and catalyzes the conversion of decaprenylphosphoryl ribose to decaprenylphosphoryl arabinose. DprE1 is a potential target against tuberculosis, including multidrug-resistant tuberculosis. We identified potential DprE1 inhibitors from the ChemDiv dataset through virtual screening based on pharmacophore and molecular docking. Thirty selected compounds were subjected to absorption, distribution, metabolism, excretion, and toxicity prediction with the Discovery Studio software package. Two compounds were obtained as hits for inhibiting DprE1 activity in M. tuberculosis and are suitable for further in vitro and in vivo evaluation.

Geniposide Increases Unfolded Protein Response-Mediating HRD1 Expression to Accelerate APP Degradation in Primary Cortical Neurons.

Altered proteostasis induced by amyloid peptide aggregation and hyperphosphorylation of tau protein, is a prominent feature of Alzheimer's disease, which highlights the occurrence of endoplasmic reticulum stress and triggers the activation of the unfolded protein response (UPR), a signaling pathway that enforces adaptive programs to sustain proteostasis. In this study, we investigated the role of geniposide in the activation of UPR induced by high glucose in primary cortical neurons. We found that high glucose induced a significant activation of UPR, and geniposide enhanced the effect of high glucose on the phosphorylation of IRE1α, the most conserved UPR signaling branch. We observed that geniposide induced the expression of HRD1, an ubiquitin-ligase E3 in a time dependent manner, and amplified the expression of HRD1 induced by high glucose in primary cortical neurons. Suppression of IRE1α activity with STF-083010, an inhibitor of IRE1 phosphorylation, prevented the roles of geniposide on the expression of HRD1 and APP degradation in high glucose-treated cortical neurons. In addition, the results from RNA interfere on HRD1 revealed that HRD1 was involved in geniposide regulating APP degradation in cortical neurons. These data suggest that geniposide might be benefit to re-establish proteostasis by enhancing the UPR to decrease the load of APP in neurons challenged by high glucose.

A Suzuki-Miyaura method for labelling proliferating cells containing incorporated BrdU.

The 5-bromo-2'-deoxyuridine (BrdU) incorporation cell proliferation assay is the most commonly used method for assessing DNA replication. The current detection of BrdU in cells relies on antibody immunostaining, but has various limitations including low antibody specificity and poor tissue penetration. In this study, we utilised a Suzuki-Miyaura reaction to develop a chemical method to label cellular BrdU with fluorescent boronic acid probes. The coupling conditions were optimised for complex cellular environments, and the key observation was the need to use oxygen scavengers and zerovalent palladium to prevent side reactions and increase the rate of coupling. The reliability and specificity of the BrdU Suzuki-Miyaura labelling method were verified under various biological conditions. The applicability of the BrdU Suzuki-Miyaura labelling methodology was also investigated, and we show that labelling cellular BrdU is highly sensitive and reliable, which is comparable to the ideal performance of BrdU immunostaining. Moreover, the Suzuki-Miyaura reaction protocol provides high BrdU recognition specificity. Taken together, the BrdU Suzuki-Miyaura labelling protocol provides an attractive alternative to the more traditional cell proliferation assay.

A Mild Aqueous Sonogashira Reaction as a Fluorescent Labeling Strategy for 5-Bromide-2'-Deoxyuridine.

C5-modified uridines are a valuable class of nucleoside analogues, both as potent chemotherapy agents and through their use as the conjunction site in DNA labeling strategies. As an important C5-modified uridine, BrdU has been used in cell proliferation assays since the 1980s. Currently, the detection of BrdU relies on traditional immunostaining; however, this approach has its limitations. Thus, it is desirable, albeit difficult, to develop chemistry methods to fluorescently label BrdU in a cellular context. In the present study, we report our efforts toward developing a robust chemistry methodology for BrdU fluorescent labeling. The Sonogashira reaction was chosen as the key reaction, and various alkynyl groups (aliphatic or aryl) containing fluorescent dyes were synthesized to cross-couple with BrdU. Various bases and catalyst systems were screened to evaluate the optimum conditions. A mild aqueous Sonogashira reaction (K2PdCl4, S-Phos, n-Bu4N⁺OH-, Sodium d-isoascorbate, EtOH/H2O = 1:1, 37 °C, Ar) was obtained to enable high-yielding BrdU fluorescent labeling.

Synthesis and Evaluation of Novel Benzofuran Derivatives as Selective SIRT2 Inhibitors.

A series of benzofuran derivatives were designed and synthesized, and their inhibitory activites were measured against the SIRT1-3. The enzymatic assay showed that all the compounds showed certain anti-SIRT2 activity and selective over SIRT1 and SIRT3 with IC50 (half maximal inhibitory concentration) values at the micromolar level. The preliminary structure-activity relationships were analyzed and the binding features of compound 7e (IC50 3.81 µM) was predicted using the CDOCKER program. The results of this research could provide informative guidance for further optimizing benzofuran derivatives as potent SIRT2 inhibitors.

A Morphological identification cell cytotoxicity assay using cytoplasm-localized fluorescent probe (CLFP) to distinguish living and dead cells.

Cell cytotoxicity assays include cell activity assays and morphological identification assays. Currently, all frequently used cytotoxicity assays belong to cell activity assays but suffer from detection limitations. Morphological identification of cell death remains as the gold standard, although the method is difficult to scale up. At present there is no generally accepted morphological identification based cell cytotoxicity assay. In this study, we applied previous developed cell cytoplasm-localized fluorescent probe (CLFP) to display cell morphologies. Under fluorescence microscopy, the fluorescence morphology and intensity of living cells are distinct from dead cells. Based on these characters we extracted the images of living cells from series of samples via computational analysis. Thus, a novel cell morphological identification cytotoxicity assay (CLFP assay) is developed. The performance of the CLFP assay was similar to cell activity assay (MTT assay), but the accuracy of the CLFP assay was superior when measuring the cytotoxicity of active compounds.

Structural Simplification of Bedaquiline: the Discovery of 3-(4-(N,N-Dimethylaminomethyl)phenyl)quinoline-Derived Antitubercular Lead Compounds.

Bedaquiline (BDQ) is a novel and highly potent last-line antituberculosis drug that was approved by the US FDA in 2013. Owing to its stereo-structural complexity, chemical synthesis and compound optimization are rather difficult and expensive. This study describes the structural simplification of bedaquiline while preserving antitubercular activity. The compound's structure was split into fragments and reassembled in various combinations while replacing the two chiral carbon atoms with an achiral linkage instead. Four series of analogues were designed; these candidates retained their potent antitubercular activity at sub-microgram per mL concentrations against both sensitive and multidrug-resistant (MDR) Mycobacterium tuberculosis strains. Six out of the top nine MIC-ranked candidates were found to inhibit mycobacterial ATP synthesis activity with IC50 values between 20 and 40 µm, one had IC50 >66 µm, and two showed no inhibition, despite their antitubercular activity. These results provide a basis for the development of chemically less complex, lower-cost bedaquiline derivatives and describe the identification of two derivatives with antitubercular activity against non-ATP synthase related targets.

A high-resolution method to assess cell multinucleation with cytoplasm-localized fluorescent probes.

Cell multinucleation is closely related to chromosomal instability. We report a simple, convenient method to assess cell multinucleation with cytoplasm-localized fluorescent probes (CLFP) which is superior to conventional nuclear staining methods. The CLFP method provides high-resolution images that enable the accurate calculation of the number of nuclear fragments.

Development of 3-alkyl-6-methoxy-7-hydroxy-chromones (AMHCs) from natural isoflavones, a new class of fluorescent scaffolds for biological imaging.

Starting from 7-hydroxyisoflavones, we developed a new class of fluorescent scaffolds, 3-alkyl-6-methoxy-7-hydroxy-chromones (AMHCs, MW∼ 205.19, λab∼ 350 nm, λem∼ 450 nm) via a trial and error process. AMHCs have the advantages of being a small molecular moiety, having strong fluorescence in basic buffers, reasonable solubility and stability, non-toxicity, and are conveniently linked to pharmacophores. AMHCs were successfully used in fluorescence microscopy imaging of cells and tissues.

Discovery of potent and selective sirtuin 2 (SIRT2) inhibitors using a fragment-based approach.

Sirtuin 2 (SIRT2) is one of the sirtuins, a family of NAD(+)-dependent deacetylases that act on a variety of histone and non-histone substrates. Accumulating biological functions and potential therapeutic applications have drawn interest in the discovery and development of SIRT2 inhibitors. Herein we report our discovery of novel SIRT2 inhibitors using a fragment-based approach. Inspired by the purported close binding proximity of suramin and nicotinamide, we prepared two sets of fragments, namely, the naphthylamide sulfonic acids and the naphthalene-benzamides and -nicotinamides. Biochemical evaluation of these two series provided structure-activity relationship (SAR) information, which led to the design of (5-benzamidonaphthalen-1/2-yloxy)nicotinamide derivatives. Among these inhibitors, one compound exhibited high anti-SIRT2 activity (48 nM) and excellent selectivity for SIRT2 over SIRT1 and SIRT3. In vitro, it also increased the acetylation level of α-tubulin, a well-established SIRT2 substrate, in both concentration- and time-dependent manners. Further kinetic studies revealed that this compound behaves as a competitive inhibitor against the peptide substrate and most likely as a noncompetitive inhibitor against NAD(+). Taken together, these results indicate that we have discovered a potent and selective SIRT2 inhibitor whose novel structure merits further exploration.