Developing a fast and catalyst-free protocol to form C=N double bond with high functional group tolerance.

The carbon-nitrogen double bond (C=N) is a fundamentally important functional group in organic chemistry. This is largely due to the fact that C=N acts as electrophilic synthon to give nitrogen-containing compounds. Here, we report the condensation of primary amine or hydrazine with very electron-deficient aldehyde to form C=N bond in the absence of any catalysts (metals and acids). The protocol performs at room temperature and applies water as co-solvent. Two hundred examples are presented here. With its intrinsic advantages of wide substrate scopes, excellent efficiency (high yields and short reaction time), operational simplicity, mild condition (room temperature as reaction temperature, no catalysts, no additions, water as co-solvent and opening to air) and available starting materials, the protocol can be compatible with various drugs, prodrugs, dyes and pharmacophores containing primary amino group. In addition, we also successfully apply this protocol to rapidly synthesize the core scaffolds of bioactive molecules.

Lysosomal K+ channel TMEM175 promotes apoptosis and aggravates symptoms of Parkinson's disease.

Lysosomes are degradative organelles and play vital roles in a variety of cellular processes. Ion channels on the lysosomal membrane are key regulators of lysosomal function. TMEM175 has been identified as a lysosomal potassium channel, but its modulation and physiological functions remain unclear. Here, we show that the apoptotic regulator Bcl-2 binds to and inhibits TMEM175 activity. Accordingly, Bcl-2 inhibitors activate the channel in a caspase-independent way. Increased TMEM175 function inhibits mitophagy, disrupts mitochondrial homeostasis, and increases production of reactive oxygen species (ROS). ROS further activates TMEM175 and thus forms a positive feedback loop to augment apoptosis. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD), knockout (KO) of TMEM175 mitigated motor impairment and dopaminergic (DA) neuron loss, suggesting that TMEM175-mediated apoptosis plays an important role in Parkinson's disease (PD). Overall, our study reveals that TMEM175 is an important regulatory site in the apoptotic signaling pathway and a potential therapeutic target for Parkinson's disease (PD).

Feedback control of PLK1 by Apolo1 ensures accurate chromosome segregation.

Stable transmission of genetic material during cell division requires accurate chromosome segregation. PLK1 dynamics at kinetochores control establishment of correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the regulatory mechanism responsible for PLK1 activity in prometaphase has not yet been affirmatively identified. Here we identify Apolo1, which tunes PLK1 activity for accurate kinetochore-microtubule attachments. Apolo1 localizes to kinetochores during early mitosis, and suppression of Apolo1 results in misaligned chromosomes. Using the fluorescence resonance energy transfer (FRET)-based PLK1 activity reporter, we found that Apolo1 sustains PLK1 kinase activity at kinetochores for accurate attachment during prometaphase. Apolo1 is a cognate substrate of PLK1, and the phosphorylation enables PP1γ to inactivate PLK1 by dephosphorylation. Mechanistically, Apolo1 constitutes a bridge between kinase and phosphatase, which governs PLK1 activity in prometaphase. These findings define a previously uncharacterized feedback loop by which Apolo1 provides fine-tuning for PLK1 to guide chromosome segregation in mitosis.

Genetically encoded FRET fluorescent sensor designed for detecting MOF histone acetyltransferase activity in vitro and in living cells.

Acetylation of lysine in the histone H4 N-terminal is one of the most significant epigenetic modifications in cells. Aberrant changes involving lysine acetylation modification are commonly reported in multiple types of cancers. Currently, whether it is for in vivo or in vitro, there are limited approaches for the detection of H4 lysine acetylation levels. In particular, the main problems are the high cost and the cumbersome detection process, such as for radioactive 14C isotope detection. Therefore, there is an important need to develop a simple, fast, and low-cost means of detection. In this study, we reported the development of a gene-coding protein sensor. This protein sensor was designed based on the mechanism of fluorescence resonance energy transfer (FRET). The four kinds of sensors, varying from substrate and linker length, were evaluated, with ~20% increases in response efficiency. Next, sensors with different lysine mutation sites in the substrate sequence or mutation of key amino acids in the binding domain were also analyzed to determine site specificity. We found single-site lysine mutant could not cause a significant decrease in response efficiency. Furthermore, addition of MG149, a histone acetyltransferase inhibitor, resulted in a decrease in the ratio change value. Moreover, histone deacetylase1 HDAC1 was also found to reduce the ratio change values when added to reaction system. Finally, the optimized sensor was applied to living cells and established to provide a sensitive response with overexpression and knockdown of MOF (males absent on the first). These results indicated that the sensor can be used for screening chemical drugs regulating H4 N-terminal lysine acetylation level in vitro, as well as monitoring dynamic changes of lysine acetylation levels in living cells.

Highly selective fluorescence probe with peptide backbone for imaging mercury ions in living cells based on aggregation-induced emission effect.

Mercury is an anthropogenic toxic heavy metal found in the environment. It is highly desirable to develop a fluorescence probe that can selectively and sensitively detect mercury ions using a turn-on response. This paper reports the successful development of a peptide fluorescence probe, TP-2 (TPE-Trp-Pro-Gln-His-Glu-NH2), which uses aggregation-induced emission effects and high selectivity to detect Hg2+. After fluorescence was activated, Hg2+ was efficiently detected using the change in fluorescence intensity. The detection limit for Hg2+ in the buffer solution was 41 nM (R2 = 0.9952). Owing to its high sensitivity, high cell permeability, and low biotoxicity, the probe could perform live cell imaging under biological conditions. This study demonstrated that TP-2 can detect Hg2+ in complex biological environments.

Novel gene-encoded intermolecular FRET sensor for tracking glycosylation of CD147 in living cells.

CD147 is involved in various physiological processes and plays important roles for tumor metastasis. Glycosylation of the protein determines numerous functions of CD147. Up to now, hardly any sensor has been developed for detecting glycosylation of CD147 in live cells. There is a pressing requirement of development of a selective and continuous biosensor for cell imaging. The emergence of gene-encoded fluorescence resonance energy transfer (FRET) sensor provides a new way to develop the sensors to analysts. We designed and constructed novel gene-encoded FRET proteins sensing glycosylation of CD147 by measuring FRET ratio of two intermolecular motifs. With the decrease of CD147 glycosylation level in cells, the FRET ratio increased significantly. The specificity of the sensor targeting to CD147 was also determined by siRNA interference experiment. Finally, continuous living cell image of deglycosylation process of CD147 using the newly developed sensor has been performed successfully. The work not only provides useful tools for analyzing glycosylation of CD147 in living cells, but also implicates alternative strategy for detecting other glycosylated proteins.

Specific epitopes form extensive hydrogen-bonding networks to ensure efficient antibody binding of SARS-CoV-2: Implications for advanced antibody design.

Neutralizing antibody targeting to the SARS-CoV-2 could provide powerful therapies. A neutralizing antibody CC12.1 which was found in SARS-CoV-2 patient samples provides potential protection from disease. The aim of molecular dynamics simulations is to identify key epitopes that are crucial to the antibody binding of SARS-CoV-2 spike glycoprotein receptor binding domain (RBD) to promote the development of superior antibodies. Binding modes of the antibody were investigated and compared with RBD bound receptor ACE2. Key epitopes were revealed and a distal motif of RBD (residue numbers 473-488) was demonstrated by analyzing dynamic trajectories. Compared to the receptor ACE2, conformation of RBD could be better stabilized through additional interaction of antibody with the distal motif of RBD, which was further found driven by electrostatic complementarity. By further analysis of the extensive hydrogen-bonding networks, residues D405, K417, Y421, Y453, L455, R457, Y473, A475, N487, G502, Y505 of RBD, which mainly interacted with CDR H3/L3 and two conserved motifs SNY, SGGS, were identified as key epitopes. Higher binding free energy calculated after point mutations on key residues confirms the crucial role for the specific binding. Subsequently, mutations of VH V98E and VL G68D in CC12.1, which could significantly enhance the binding affinity of the antibody, were also proposed. The results indicate the key epitopes for antibody binding and give explanations for failure of neutralization antibody caused by specific residues mutations on structural basis. Simulations of two point mutations on antibody provide feasible information for advanced antibody design.

Design, synthesis and cell imaging of a simple peptide-based probe for the selective detection of RNA.

Here we present a novel peptide-based fluorescent "turn-on" molecule P1 for detecting RNA, in a double or single strand, AU-rich or CG-rich. Both computational and experimental studies indicate that the detection efficiency depends on the binding affinity of P1 and conformational changes. P1 could be applied for cell imaging without any additional transfection vectors. Selective detection of RNA in cells was determined by RNase digestion. Successful application of P1 for RNA imaging in cell mitosis reveals that it may have broad applications in research, biotechnology and medical science.

Discovery and Biological Evaluation of CD147 N-Glycan Inhibitors: A New Direction in the Treatment of Tumor Metastasis.

N-glycosylation is instrumental to the regulation of CD147 functions, including the maturation of CD147, secretion of matrix metalloproteinases (MMPs), and promotion of tumor metastasis. Glycosylated CD147 is highly expressed in various cancer types, participates in metastasis, and is associated with the poor prognosis of malignant tumors. However, to date, there has been little development of target-specific inhibitors for CD147 glycosylation. In this work, we report a strategy for discovering CD147 glycosylation inhibitors through computer-aided screening and inhibition assays. Four compounds were screened as potential CD147 glycosylation inhibitors. Of these, compound 72 was finally identified as the best candidate. Further experiments confirmed that compound 72 inhibited the production of MMPs and the metastasis of cancer cells in the Hela cell line. Results further suggest that compound 72 could promote the expression of E-cadherin by targeting CD147, thereby inhibiting tumor migration. Finally, the structures of the other potential CD147 N-glycosylation inhibitors may eventually provide guidance for future optimization.

Repurposing Low-Molecular-Weight Drugs against the Main Protease of Severe Acute Respiratory Syndrome Coronavirus 2.

The coronavirus disease pandemic caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected the global healthcare system. As low-molecular-weight drugs have high potential to completely match interactions with essential SARS-CoV-2 targets, we propose a strategy to identify such drugs using the fragment-based approach. Herein, using ligand- and protein-observed fragment screening approaches, we identified niacin and hit 1 binding to the catalytic pocket of the main protease (Mpro) of SARS-CoV-2, thereby modestly inhibiting the enzymatic activity of Mpro. We further searched for low-molecular-weight drugs containing niacin or hit 1 pharmacophores with enhanced inhibiting activity, e.g., carmofur, bendamustine, triclabendazole, emedastine, and omeprazole, in which omeprazole is the only one binding to the C-terminal domain of SARS-CoV-2 Mpro. Our study demonstrates that the fragment-based approach is a feasible strategy for identifying low-molecular-weight drugs against the SARS-CoV-2 and other potential targets lacking specific drugs.

Cannabinoids Rescue Cocaine-Induced Seizures by Restoring Brain Glycine Receptor Dysfunction.

Cannabinoids are reported to rescue cocaine-induced seizures (CISs), a severe complication in cocaine users. However, the molecular targets for cannabinoid therapy of CISs remain unclear. Here, we report that the systemic administration of cannabinoids alleviates CISs in a CB1/CB2-receptor-independent manner. In HEK293 cells and cortical neurons, cocaine-induced dysfunction of the glycine receptor (GlyR) is restored by cannabinoids. Such restoration is blocked by GlyRα1S296A mutation. Consistently, the therapeutic effects of cannabinoids on CISs are also eliminated in GlyRα1S296A mutant mice. Based on molecular dynamic simulation, the hydrogen-bonding interaction between cocaine and the GlyR is weakened by cannabinoid docking. Without altering cocaine distribution across the brain, cannabinoids significantly suppress cocaine-exaggerated neuronal excitability in the prefrontal cortex (PFC) and hippocampus by rehabilitating extra-synaptic GlyR function. Microinjection of cannabinoids into the PFC and hippocampus restores cocaine-puzzled neural activity and alleviates CISs. These findings suggest that using GlyR-hypersensitive cannabinoids may represent a potential therapeutic strategy for treating CISs.

A peptide-based fluorescent sensor for selective imaging of glutathione in living cells and zebrafish.

Monitoring and imaging glutathione (GSH) in living systems is an essential tool to determine the key roles of GSH in biological pathways, but most fluorescent sensors can only be used in vitro because of their potential biotoxicity. Here, a peptide-based fluorescent sensor, FP, has been successfully designed and synthesized based on the biocompatibility of the peptide backbone and low toxicity. The design strategy of FP contains a specific spatial structure of the peptide sequence which selectively binds to Cu2+, triggering fluorescence quenching. Interestingly, the fluorescence of FP can be fully restored by GSH, due to the strong binding between Cu2+ and the GSH sulfhydryl groups. Finally, the sensor is highly sensitive and selective for imaging GSH both in vitro and in vivo with low toxicity. Thus, FP with its strong "on-off-on" fluorescence changes is a powerful way to image GSH both in cells and zebrafish larvae to study the GSH pathway.

Human Hyperekplexic Mutations in Glycine Receptors Disinhibit the Brainstem by Hijacking GABAA Receptors.

Hyperekplexia disease is usually caused by naturally occurring point mutations in glycine receptors (GlyRs). However, the γ-aminobutyric acid type A receptor (GABAAR) seems to be also involved regarding the therapeutic basis for hyperekplexia using benzodiazepines, which target GABAARs but not GlyRs. Here, we show that the function of GABAARs was significantly impaired in the hypoglossal nucleus of hyperekplexic transgenic mice. Such impairment appeared to be mediated by interaction between GABAAR and mutant GlyR. The GABAAR dysfunction was caused only by mutant GlyR consisting of homomeric α1 subunits, which locate primarily at pre- and extra-synaptic sites. In addition, the rescue effects of diazepam were attenuated by Xli-093, which specifically blocked diazepam-induced potentiation on α5-containing GABAAR, a major form of pre- and extra-synaptic GABAAR in the brainstem. Thus, our results suggest that the pre- and extra-synaptic GABAARs could be a potential therapeutic target for hyperekplexia disease caused by GlyR mutations.

Identification of the hot spot residues for pyridine derivative inhibitor CCT251455 and ATP substrate binding on monopolar spindle 1 (MPS1) kinase by molecular dynamic simulation.

Protein kinase monopolar spindle 1 plays an important role in spindle assembly checkpoint at the onset of mitosis. Over expression of MPS1 correlated with a wide range of human tumors makes it an attractive target for finding an effective and specific inhibitor. In this work, we performed molecular dynamics simulations of protein MPS1 itself as well as protein bound systems with the inhibitor and natural substrate based on crystal structures. The reported orally bioavailable 1 h-pyrrolo [3,2-c] pyridine inhibitors of MPS1 maintained stable binding in the catalytic site, while natural substrate ATP could not stay. Comparative study of stability and flexibility of three systems reveals position shifting of ß-sheet region within the catalytic site, which indicates inhibition mechanism was through stabilizing the ß-sheet region. Binding free energies calculated with MM-GB/PBSA method shows different binding affinity for inhibitor and ATP. Finally, interactions between protein and inhibitor during molecular dynamic simulations were measured and counted. Residue Gly605 and Leu654 were suggested as important hot spots for stable binding of inhibitor by molecular dynamic simulation. Our results reveal an important position shifting within catalytic site for non-inhibited proteins. Together with hot spots found by molecular dynamic simulation, the results provide important information of inhibition mechanism and will be referenced for designing novel inhibitors.

Organic arsenicals target thioredoxin reductase followed by oxidative stress and mitochondrial dysfunction resulting in apoptosis.

Considering the vital role of cellular redox state, more and more researches focus on the design of drugs targeting thioredoxin reductase (TrxR), an important enzyme in maintaining the balance of cellular redox. Here two organic arsenicals, 2-(((4-(1,3,2-dithiarsinan-2-yl) phenyl) imino) methyl) phenol (PIM-PAO-PDT) and N-(4-(1,3,2-dithiarsinan-2-yl) phenyl)-2-hydroxybenzamide (PAM-PAO-PDT), bearing the S-As-S chemical scaffold and different linking groups have been synthesized, and both of them show the better inhibitory activity and selectivity towards HL-60 cells. Importantly, it is illustrated that they can target TrxR selectively and inhibit its activity via the disturbance for Cys83 and Cys88 located in conserved active sites. Afterwards, the cells suffer from the burst of ROS, consumption of antioxidants and high sensitivity for oxidants, which further damage the mitochondria leading to dysfunction including the collapse of membrane potential, ATP level decline, mitochondrial membrane swelling, MPTP opening, Ca2+ and cytochrome c release. Then the mitochondria-dependent apoptosis is triggered by PIM-PAO-PDT and PAM-PAO-PDT, which can also be deterred in the presence of NAC, DTT or LA. Although the organic arsenicals can suppress TrxR activity, the following oxidative stress and mitochondrial dysfunction are the main causes for apoptosis.

Identification of the quinolinedione inhibitor binding site in Cdc25 phosphatase B through docking and molecular dynamics simulations.

Cdc25 phosphatase B, a potential target for cancer therapy, is inhibited by a series of quinones. The binding site and mode of quinone inhibitors to Cdc25B remains unclear, whereas this information is important for structure-based drug design. We investigated the potential binding site of NSC663284 [DA3003-1 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5, 8-dione] through docking and molecular dynamics simulations. Of the two main binding sites suggested by docking, the molecular dynamics simulations only support one site for stable binding of the inhibitor. Binding sites in and near the Cdc25B catalytic site that have been suggested previously do not lead to stable binding in 50 ns molecular dynamics (MD) simulations. In contrast, a shallow pocket between the C-terminal helix and the catalytic site provides a favourable binding site that shows high stability. Two similar binding modes featuring protein-inhibitor interactions involving Tyr428, Arg482, Thr547 and Ser549 are identified by clustering analysis of all stable MD trajectories. The relatively flexible C-terminal region of Cdc25B contributes to inhibitor binding. The binding mode of NSC663284, identified through MD simulation, likely prevents the binding of protein substrates to Cdc25B. The present results provide useful information for the design of quinone inhibitors and their mechanism of inhibition.

Discovery of Cdc25A Lead Inhibitors with a Novel Chemotype by Virtual Screening: Application of Pharmacophore Modeling Based on a Training Set with a Limited Number of Unique Components.

Cdc25 phosphatase was studied as an attractive target for cancer therapy. Multiple pharmacophore models with the unique core features of classic quinone inhibitors and those of novel inhibitors were used to discover a novel lead inhibitor. A total of 21 compounds with qualified physical properties were screened from the Maybridge HitFinder database containing 14 400 compounds by pharmacophore models. Four compounds were found to inhibit Cdc25A activity by more than 50 % at a concentration of 100 µm. Among these compounds, KM10389 (N-{2-[(furan-2-ylmethyl)thio]ethyl}-2-[(4-hydroxy-6-propylpyrimidin-2-yl)thio]acetamide) showed high inhibitory activity with an IC50 value of 7.9 µm. Selective cytotoxicity toward HeLa cells was observed with an IC50 value of 66.3 µm, whereas the IC50 value for HEK293 cells was higher than 100 µm. Blocking of the G1/S transition was also observed for HeLa cells in the presence of the compound by increasing the G1 phase by 16.15 %. Together with compounds HTS02435 and HTS01205, a novel lead inhibitor structure was identified and analyzed by a molecular docking study. The implication of virtual screening by using different pharmacophore models representing the different features is fully discussed.

Identification of Binding Modes for Amino Naphthalene 2-Cyanoacrylate (ANCA) Probes to Amyloid Fibrils from Molecular Dynamics Simulations.

The amino naphthalene 2-cyanoacrylate (ANCA) probe is a kind of fluorescent amyloid binding probe that can report different fluorescence emissions when bound to various amyloid deposits in tissue, while their interactions with amyloid fibrils remain unclear due to the insoluble nature of amyloid fibrils. Here, all-atom molecular dynamics simulations were used to investigate the interaction between ANCA probes with three different amyloid fibrils. Two common binding modes of ANCA probes on Aß40 amyloid fibrils were identified by cluster analysis of multiple simulations. The van der Waals and electrostatic interactions were found to be major driving forces for the binding. Atomic contacts analysis and binding free energy decomposition results suggested that the hydrophobic part of ANCA mainly interacts with aromatic side chains on the fibril surface and the hydrophilic part mainly interacts with positive charged residues in the ß-sheet region. By comparing the binding modes with different fibrils, we can find that ANCA adopts different conformations while interacting with residues of different hydrophobicity, aromaticity, and electrochemical properties in the ß-sheet region, which accounts for its selective mechanism toward different amyloid fibrils.

A novel peptide-based fluorescence chemosensor for selective imaging of hydrogen sulfide both in living cells and zebrafish.

Hydrogen sulfide (H2S) plays an important role as a signaling compound (gasotransmitter) in living systems. However, the development of an efficient imaging chemosensor of H2S in live animals is a challenging field for chemists. Herein, a novel peptide-based fluorescence chemosensor L-Cu was designed and synthesized on the basis of the copper chelating with the peptide ligand (FITC-Ahx-Ser-Pro-Gly-His-NH2, L), and its H2S sensing ability has been evaluated both in living cells and zebrafish. The peptide backbone and Cu2+-removal sensing mechanism are used to deliver rapid response time, high sensitivity, and good biocompatibility. After a fast fluorescence quench by Cu2+ coordinated with L, the fluorescence of L is recovered by adding S2- to form insoluble copper sulfide in aqueous solution with a detection limit for hydrogen sulfide measured to be 31nM. Furthermore, the fluorescence chemosensor L-Cu showed excellent cell permeation and low biotoxicity to realize the intracellular biosensing, L-Cu has also been applied to image hydrogen sulfide in live zebrafish larvae. We expect that this peptide-based fluorescence chemosensor L-Cu can be used to study H2S-related chemical biology in physiological and pathological events.

Fluorescence "on-off-on" peptide-based chemosensor for the selective detection of Cu2+ and S2- and its application in living cell bioimaging.

Copper ions are known to be very important for homeostasis, which is critical for the metabolism and development of living organisms. In addition, sulfide ions, as an important endogenously produced gasotransmitter, have been proved to be implicated in a variety of physiological functions such as anti-apoptosis, vasodilation, antioxidation, and anti-inflammation. Herein, we report the development of a novel fluorescence chemosensor (L) based on a tetra-peptide conjugated with dansyl groups as a promising analytical tool for detecting Cu2+ and S2- in 100% aqueous solutions, which exhibits excellent cell biotoxicity and intracellular biosensing ability. The chemosensor L displays an "on-off-on" response type fluorescence change upon the addition of Cu2+ and S2- to aqueous media and living cells. Moreover, L displays high selectivity and sensitivity with the detection limits for Cu2+ and S2- measured to be 88 nM and 75 nM, respectively. This study raises the new possibility of a highly selective and sensitive peptide-based fluorescence chemosensor for multifunctional detection, including cation and anions, using a successive fluorescence response strategy in environmental and biological systems.