An allosteric mechanism for potent inhibition of human ATP-citrate lyase.

ATP-citrate lyase (ACLY) is a central metabolic enzyme and catalyses the ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate and acetyl-CoA1-5. The acetyl-CoA product is crucial for the metabolism of fatty acids6,7, the biosynthesis of cholesterol8, and the acetylation and prenylation of proteins9,10. There has been considerable interest in ACLY as a target for anti-cancer drugs, because many cancer cells depend on its activity for proliferation2,5,11. ACLY is also a target against dyslipidaemia and hepatic steatosis, with a compound currently in phase 3 clinical trials4,5. Many inhibitors of ACLY have been reported, but most of them have weak activity5. Here we report the development of a series of low nanomolar, small-molecule inhibitors of human ACLY. We have also determined the structure of the full-length human ACLY homo-tetramer in complex with one of these inhibitors (NDI-091143) by cryo-electron microscopy, which reveals an unexpected mechanism of inhibition. The compound is located in an allosteric, mostly hydrophobic cavity next to the citrate-binding site, and requires extensive conformational changes in the enzyme that indirectly disrupt citrate binding. The observed binding mode is supported by and explains the structure-activity relationships of these compounds. This allosteric site greatly enhances the 'druggability' of ACLY and represents an attractive target for the development of new ACLY inhibitors.

Potent, selective and reversible hMAO-B inhibition by benzalphthalides: Synthesis, enzymatic and cellular evaluations and virtual docking and predictive studies.

Monoaminooxidases (MAOs) are important targets for drugs used in the treatment of neurological and psychiatric disorders and particularly on Parkinson's Disease (PD). Compounds containing a trans-stilbenoid skeleton have demonstrated good selective and reversible MAO-B inhibition. Here, twenty-two (Z)-3-benzylidenephthalides (benzalphthalides, BPHs) displaying a trans-stilbenoid skeleton have been synthesised and evaluated as inhibitors of the MAO-A and MAO-B isoforms. Some BPHs have selectively inhibited MAO-B, with IC50 values ranging from sub-nM to µM. The most potent compound with IC50 = 0.6 nM was the 3',4'-dichloro-BPH 16, which showed highly selective and reversible MAO-B inhibitory activity. Furthermore, the most selective BPHs displayed a significant protection against the apoptosis, and mitochondrial toxic effects induced by 6-hydroxydopamine (6OHDA) on SH-SY5Y cells, used as a cellular model of PD. The results of virtual binding studies on the most potent compounds docked in MAO-B and MAO-A were in agreement with the potencies and selectivity indexes found experimentally. Additionally, related to toxicity risks, drug-likeness and ADME properties, the predictions found for the most relevant BPHs in this research were within those ranges established for drug candidates.

De novo design of a fluorescence-activating ß-barrel.

The regular arrangements of ß-strands around a central axis in ß-barrels and of α-helices in coiled coils contrast with the irregular tertiary structures of most globular proteins, and have fascinated structural biologists since they were first discovered. Simple parametric models have been used to design a wide range of α-helical coiled-coil structures, but to date there has been no success with ß-barrels. Here we show that accurate de novo design of ß-barrels requires considerable symmetry-breaking to achieve continuous hydrogen-bond connectivity and eliminate backbone strain. We then build ensembles of ß-barrel backbone models with cavity shapes that match the fluorogenic compound DFHBI, and use a hierarchical grid-based search method to simultaneously optimize the rigid-body placement of DFHBI in these cavities and the identities of the surrounding amino acids to achieve high shape and chemical complementarity. The designs have high structural accuracy and bind and fluorescently activate DFHBI in vitro and in Escherichia coli, yeast and mammalian cells. This de novo design of small-molecule binding activity, using backbones custom-built to bind the ligand, should enable the design of increasingly sophisticated ligand-binding proteins, sensors and catalysts that are not limited by the backbone geometries available in known protein structures.

Vibrational Spectra and Molecular Vibrational Behaviors of Dibenzyl Disulfide, Dibenzyl Sulphide and Bibenzyl.

The vibration spectroscopy (Raman and infrared) of widely concerned molecules in sulfur corrosion phenomenon (Dibenzyl Disulfide, Dibenzyl Sulphide, and Bibenzyl) is detailedly analyzed based on density functional theory and experimental measurement. The dominant conformations of these molecules are determined according to Boltzmann distribution in relative Gibbs free energy. Additionally, noncovalent interaction analysis is conducted to indicate intramolecular interaction. Vibration normal mode is assigned based on potential energy distribution, which comprehensively reveals the molecular vibrational behaviors. Conformations weighted spectra are obtained and compared with experimentally measured spectra. We found that experimental spectra are in good agreement with the theoretical spectra in B3LYP-D3(BJ)/6-311G** level with a frequency correction factor. Furthermore, the divergence among these molecules is discussed. The vibrational behavior of the methylene group in the molecule shows a trend with the presence of the sulfur atom.

Copper-Catalyzed Cross-Coupling of Benzylic C-H Bonds and Azoles with Controlled N-Site Selectivity.

Azoles are important motifs in medicinal chemistry, and elaboration of their structures via direct N-H/C-H coupling could have broad utility in drug discovery. The ambident reactivity of many azoles, however, presents significant selectivity challenges. Here, we report a copper-catalyzed method that achieves site-selective cross-coupling of pyrazoles and other N-H heterocycles with substrates bearing (hetero)benzylic C-H bonds. Excellent N-site selectivity is achieved, with the preferred site controlled by the identity of co-catalytic additives. This cross-coupling strategy features broad scope for both the N-H heterocycle and benzylic C-H coupling partners, enabling application of this method to complex molecule synthesis and medicinal chemistry.

Novel 4-Hydroxybenzyl Adducts in Human Hemoglobin: Structures and Mechanisms of Formation.

Humans are exposed to large numbers of electrophiles from their diet, the environment, and endogenous physiological processes. Adducts formed at the N-terminal valine of hemoglobin are often used as biomarkers of human exposure to electrophilic compounds. We previously reported the formation of hemoglobin N-terminal valine adducts (added mass, 106.042 Da) in the blood of human smokers and nonsmokers and identified their structure as 4-hydroxybenzyl-Val. In the present work, mass spectrometry-based proteomics was utilized to identify additional sites for 4-hydroxybenzyl adduct formation at internal nucleophilic amino acid side chains within hemoglobin. Hemoglobin isolated from human blood was treated with para-quinone methide (para-QM) followed by global nanoLC-MS/MS and targeted nanoLC-MS/MS to identify amino acid residues containing the 4-hydroxybenzyl modification. Our experiments revealed the formation of 4-hydroxybenzyl adducts at the αHis20, αTyr24, αTyr42, αHis45, ßSer72, ßThr84, ßThr87, ßSer89, ßHis92, ßCys93, ßCys112, ßThr123, and ßHis143 residues (in addition to N-terminal valine) through characteristic MS/MS spectra. These amino acid side chains had variable reactivity toward para-QM with αHis45, αTyr42, ßCys93, ßHis92, and ßSer72 forming the largest numbers of adducts upon exposure to para-QM. Two additional mechanisms for formation of 4-hydroxybenzyl adducts in humans were investigated: exposure to 4-hydroxybenzaldehyde (4-HBA) followed by reduction and UV-mediated reactions of hemoglobin with tyrosine. Exposure of hemoglobin to a 5-fold molar excess of 4-HBA followed by reduction with sodium cyanoborohydride produced 4-hydroxybenzyl adducts at several amino acid side chains of which αHis20, αTyr24, αTyr42, αHis45, ßSer44, ßThr84, and ßHis92 were verified in targeted mass spectrometry experiments. Similarly, exposure of human blood to ultraviolet radiation produced 4-hydroxybenzyl adducts at αHis20, αTyr24, αTyr42, αHis45, ßSer44, ßThr84, and ßSer89. Overall, our results reveal that 4-hydroxybenzyl adducts form at multiple nucleophilic sites of hemoglobin and that para-QM is the most likely source of these adducts in humans.

Liposome Click Membrane Permeability Assay for Identifying Permeable Peptides.

PURPOSE: To develop a novel, target agnostic liposome click membrane permeability assay (LCMPA) using liposome encapsulating copper free click reagent dibenzo cyclooctyne biotin (DBCO-Biotin) to conjugate azido modified peptides that may effectively translocate from extravesicular space into the liposome lumen. METHOD: DBCO-Biotin liposomes were prepared with egg phosphatidylcholine and cholesterol by lipid film rehydration, freeze/thaw followed by extrusion. Size of DBCO-Biotin liposomes were characterized with dynamic light scattering. RESULTS: The permeable peptides representing energy independent mechanism of permeability showed higher biotinylation in LCMPA. Individual peptide permeability results from LCMPA correlated well with shifts in potency in cellular versus biochemical assays (i.e., cellular/ biochemical ratio) demonstrating quantitative correlation to intracellular barrier in intact cells. CONCLUSION: The study provides a novel membrane permeability assay that has potential to evaluate energy independent transport of diverse peptides.

Design and synthesis of a monocyclic derivative as a selective ACC1 inhibitor by chemical modification of biphenyl ACC1/2 dual inhibitors.

A structure-activity relationship (SAR) study towards novel ACC1-selective inhibitors was carried out by modifying the molecular length of the linker in biaryl derivative 1 g, an ACC1/2 dual inhibitor. Ultimately, this leads us to discover novel phenoxybenzyloxy derivative 1i as a potent ACC1-selective inhibitor. Further chemical modification of this scaffold to improve cellular potency as well as physicochemical and pharmacokinetic (PK) properties produced N-2-(pyridin-2-ylethyl)acetamide derivative 1n, which showed highly potent ACC1-selective inhibition as well as sufficient PK profile for further in vivo evaluations. Oral administration of 1n significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at doses of 100 mg/kg. Accordingly, our novel series of potent ACC1-selective inhibitors represents a set of useful orally-available research tools, as well as potential therapeutic agents for cancer and fatty acid-related diseases.

The targetable nanoparticle BAF312@cRGD-CaP-NP represses tumor growth and angiogenesis by downregulating the S1PR1/P-STAT3/VEGFA axis in triple-negative breast cancer.

BACKGROUND: Overexpressed vascular endothelial growth factor A (VEGFA) and phosphorylated signal transducer and activator of transcription 3 (P-STAT3) cause unrestricted tumor growth and angiogenesis of breast cancer (BRCA), especially triple-negative breast cancer (TNBC). Hence, novel treatment strategy is urgently needed. RESULTS: We found sphingosine 1 phosphate receptor 1 (S1PR1) can regulate P-STAT3/VEGFA. Database showed S1PR1 is highly expressed in BRCA and causes the poor prognosis of patients. Interrupting the expression of S1PR1 could inhibit the growth of human breast cancer cells (MCF-7 and MDA-MB-231) and suppress the angiogenesis of human umbilical vein endothelial cells (HUVECs) via affecting S1PR1/P-STAT3/VEGFA axis. Siponimod (BAF312) is a selective antagonist of S1PR1, which inhibits tumor growth and angiogenesis in vitro by downregulating the S1PR1/P-STAT3/VEGFA axis. We prepared pH-sensitive and tumor-targeted shell-core structure nanoparticles, in which hydrophilic PEG2000 modified with the cyclic Arg-Gly-Asp (cRGD) formed the shell, hydrophobic DSPE formed the core, and CaP (calcium and phosphate ions) was adsorbed onto the shell; the nanoparticles were used to deliver BAF312 (BAF312@cRGD-CaP-NPs). The size and potential of the nanoparticles were 109.9 ± 1.002 nm and - 10.6 ± 0.056 mV. The incorporation efficacy for BAF312 was 81.4%. Results confirmed BAF312@cRGD-CaP-NP could dramatically inhibit tumor growth and angiogenesis in vitro and in MDA-MB-231 tumor-bearing mice via downregulating the S1PR1/P-STAT3/VEGFA axis. CONCLUSIONS: Our data suggest a potent role for BAF312@cRGD-CaP-NPs in treating BRCA, especially TNBC by downregulating the S1PR1/P-STAT3/VEGFA axis.

Synthesis, Molecular Docking and Preliminary Antileukemic Activity of 4-Methoxybenzyl Derivatives Bearing Imidazo[2,1-b][1,3,4]thiadiazole.

In this study, we synthesized 22 compounds in a series with various substitution on imidazo[2,1-b][1,3,4]thiadiazole. The potential cytotoxic activity of these compounds investigated in leukemia cell lines by Differential Nuclear Staining (DNS). Our results identified two compounds, 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate and 6-(4-chlorophenyl)-2-(4-methoxybenzyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde, exhibited the most cytotoxic effect against murine leukemia cells (L1210), human T-lymphocyte cells (CEM) and human cervix carcinoma cells (HeLa) with IC50 values ranging between 0.79 and 1.6 µM. The results indicate that 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate is inducing phosphatidylserine externalization and caspase-3 activation which are both a hallmark of apoptosis. Docking studies showed that 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate binds within the active sites of transforming growth factor beta (TGF-ß) type I receptor kinase domain by strong hydrogen binding and hydrophobic interactions.

Novel (+)-Neoisopulegol-Based O-Benzyl Derivatives as Antimicrobial Agents.

Discovery of novel antibacterial agents with new structures, which combat pathogens is an urgent task. In this study, a new library of (+)-neoisopulegol-based O-benzyl derivatives of aminodiols and aminotriols was designed and synthesized, and their antimicrobial activity against different bacterial and fungal strains were evaluated. The results showed that this new series of synthetic O-benzyl compounds exhibit potent antimicrobial activity. Di-O-benzyl derivatives showed high activity against Gram-positive bacteria and fungi, but moderate activity against Gram-negative bacteria. Therefore, these compounds may serve a good basis for antibacterial and antifungal drug discovery. Structure-activity relationships were also studied from the aspects of stereochemistry of the O-benzyl group on cyclohexane ring and the substituent effects on the ring system.

Shoot Multiplication and Callus Induction of Labisia pumila var. alata as Influenced by Different Plant Growth Regulators Treatments and Its Polyphenolic Activities Compared with the Wild Plant.

This study aims to investigate whether the in vitro-cultured L. pumila var. alata has higher antioxidant activity than its wild plant. An 8-week-old L. pumila var. alata nodal segment and leaf explants were cultured onto Murashige and Skoog (MS) medium supplemented with various cytokinins (zeatin, kinetin, and 6-benzylaminopurine (BAP)) for shoot multiplication and auxins (2,4-dichlorophenoxyacetic acid (2,4-D) and picloram) for callus induction, respectively. The results showed that 2 mg/L zeatin produced the optimal results for shoot and leaf development, and 0.5 mg/L 2,4-D produced the highest callus induction results (60%). After this, 0.5 mg/L 2,4-D was combined with 0.25 mg/L cytokinins and supplemented to the MS medium. The optimal results for callus induction (100%) with yellowish to greenish and compact texture were obtained using 0.5 mg/L 2,4-D combined with 0.25 mg/L zeatin. Leaves obtained from in vitro plantlets and wild plants as well as callus were extracted and analyzed for their antioxidant activities (DPPH and FRAP methods) and polyphenolic properties (total flavonoid and total phenolic content). When compared with leaf extracts of in vitro plantlets and wild plants of L. pumila var. alata, the callus extract displayed significantly higher antioxidant activities and total phenolic and flavonoid content. Hence, callus culture potentially can be adapted for antioxidant and polyphenolic production to satisfy pharmaceutical and nutraceutical needs while conserving wild L. pumila var. alata.

Ni-Catalyzed Arylbenzylation of Alkenylarenes: Kinetic Studies Reveal Autocatalysis by ZnX2 *.

We report a Ni-catalyzed regioselective arylbenzylation of alkenylarenes with benzyl halides and arylzinc reagents. The reaction furnishes differently substituted 1,1,3-triarylpropyl structures that are reminiscent of the cores of oligoresveratrol natural products. The reaction is also compatible for the coupling of internal alkenes, secondary benzyl halides and variously substituted arylzinc reagents. Kinetic studies reveal that the reaction proceeds with a rate-limiting single-electron-transfer process and is autocatalyzed by in-situ-generated ZnX2 . The reaction rate is amplified by a factor of three through autocatalysis upon addition of ZnX2 .

Isolation of new phytometabolites from Alpinia galanga willd rhizomes.

The current research was aimed to isolate newer phyto-metabolites from rhizomes of Alpinia galanga plant. Study involved preparation of Alpinia galanga rhizome methanolic extract, followed by normal phase column chromatography assisted isolation of new phytometabolites (using different combinations of chloroform and methanol), and characterization (by UV, FTIR, 13C-NMR, 1H-NMR, COSY, DEPT and Mass spectrometry). The isolation and characterization experiment offered two phytometabolites: an ester (Ag-1) and tetrahydronapthalene type lactone (Ag-2). Present study concludes and reports the two phytometabolites, benzyl myristate (Ag-1) and 3-Methyl-6α, 8ß-diol-7-carboxylic acid tetralin-11, 9ß-olide (Ag-2) for the first time in Alpinia galanga rhizome. The study recommends that these phytometabolites Ag-1 and Ag-2 can be utilized as effective analytical biomarkers for identification, purity and quality control of this plant in future.

Site-Selective Copper-Catalyzed Azidation of Benzylic C-H Bonds.

Site selectivity represents a key challenge for non-directed C-H functionalization, even when the C-H bond is intrinsically reactive. Here, we report a copper-catalyzed method for benzylic C-H azidation of diverse molecules. Experimental and density functional theory studies suggest the benzyl radical reacts with a CuII-azide species via a radical-polar crossover pathway. Comparison of this method with other C-H azidation methods highlights its unique site selectivity, and conversions of the benzyl azide products into amine, triazole, tetrazole, and pyrrole functional groups highlight the broad utility of this method for target molecule synthesis and medicinal chemistry.

Multifunctional Reactive MALDI Matrix Enabling High-Lateral Resolution Dual Polarity MS Imaging and Lipid C═C Position-Resolved MS2 Imaging.

Local lipid variations in tissues are readily revealed with mass spectrometry imaging (MSI) methods, and the resulting lipid distributions serve as bioanalytical signatures to reveal cell- or tissue-specific lipids. Comprehensive MSI lipid mapping requires measurements in both ion polarities. Additionally, structural lipid characterization is necessary to link the lipid structure to lipid function. Whereas some structural elements of lipids are readily derived from high-resolution mass spectrometry (MS) and tandem-MS (MSn), the localization of C═C double bonds (DBs) requires specialized fragmentation and/or functionalization methods. In this work, we identify a multifunctional matrix-assisted laser desorption/ionization (MALDI) matrix for spatially resolved lipidomics investigations that reacts with lipids in Paternò-Büchi (PB) reactions during laser irradiation facilitating DB-position assignment and allows dual-polarity high-resolution MALDI-MSI and MALDI MS2I studies. By screening 12 compounds for improved ionization efficiency in positive-/negative-ion mode and the functionalization yield compared to the previously introduced reactive MALDI matrix benzophenone, 2-benzoylpyridine (BzPy) is identified as the best candidate. The new matrix enables DB localization of authentic standards belonging to 12 lipid classes and helps to assign 133/58 lipid features in positive-/negative-ion mode from mouse cerebellum tissue. The analytical capabilities of BzPy as a multifunctional MALDI-MSI matrix are demonstrated by imaging endogenous and PB-functionalized lipids in mouse kidney sections with 7 µm lateral resolution in both ion modes. Tracking diagnostic lipid DB-position fragment ions in mouse pancreatic tissue with down to 10 µm pixel size allows us to identify the islets of Langerhans associated with lipid isomer upregulation and depletion.

Substituted benzyloxytricyclic compounds as retinoic acid-related orphan receptor gamma t (RORγt) agonists.

Substituted benzyloxy aryl compound 2 was identified as an RORγt agonist. Structure based drug design efforts resulted in a potent and selective tricyclic compound 19 which, when administered orally in an MC38 mouse tumor model, demonstrated a desired pharmacokinetic profile as well as a dose-dependent pharmacodynamic response. However, no perceptible efficacy was observed in this tumor model at the doses investigated.

1,2-cis-Selective glucosylation enabled by halogenated benzyl protecting groups.

We report on our initial results from a systematic effort to implement electron-withdrawing protecting groups and Lewis basic solvents/additives as an approach to 1,2-cis(α)-selective O-glucosylation. 1,2-cis-Selective O-glucosylations are reported with thioglucosides and glucosyl trichloroacetimidates and a range of acceptors. A correlation between electron-withdrawing effects and 1,2-cis selectivity has been established. This phenomenon may prove to be broadly applicable in the area of chemical O-glycosylation.

Design and synthesis of 3-benzylaminocoumarin-7-O-sulfamate derivatives as steroid sulfatase inhibitors.

Steroid sulfatase (STS) is a sulfatase enzyme that catalyzes the conversion of sulfated steroid precursors to free steroid. The inhibition of STS could abate estrogenic steroids that stimulate the proliferation and development of breast cancer, and therefore STS is a potential target for adjuvant endocrine therapy. In this study, a series of 3-benzylaminocoumarin-7-O-sulfamate derivatives targeting STS were designed and synthesized. Structure-relationship activities (SAR) analysis revealed that attachment of a benzylamino group at the 3-position of coumarin improved inhibitory activity. Compound 3j was found to have the highest inhibition activity against human placenta isolated STS (IC50  0.13 µM) and MCF-7 cell lines (IC50 1.35 µM). Kinetic studies found compound 3j to be an irreversible inhibitor of STS, with KI and kinact value of 86.9 nM and 158.7 min-1, respectively.

Discrimination and Identification of Aroma Profiles and Characterized Odorants in Citrus Blend Black Tea with Different Citrus Species.

Citrus blend black teas are popular worldwide, due to its unique flavor and remarkable health benefits. However, the aroma characteristics, aroma profiles and key odorants of it remain to be distinguished and cognized. In this study, the aroma profiles of 12 representative samples with three different cultivars including citrus (Citrus reticulata), bergamot (Citrus bergamia), and lemon (Citrus limon) were determined by a novel approach combined head space-solid phase microextraction (HS-SPME) with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS). A total of 348 volatile compounds, among which comprised esters (60), alkenes (55), aldehydes (45), ketones (45), alcohols (37), aromatic hydrocarbons (20), and some others were ultimately identified. The further partial least squares discrimination analysis (PLS-DA) certified obvious differences existed among the three groups with a screening result of 30 significant differential key volatile compounds. A total of 61 aroma-active compounds that mostly presented green, fresh, fruity, and sweet odors were determined in three groups with gas chromatography-olfactometry/mass spectrometry (GC-O/MS) assisted analysis. Heptanal, limonene, linalool, and trans-ß-ionone were considered the fundamental odorants associated with the flavors of these teas. Comprehensive analysis showed that limonene, ethyl octanoate, copaene, ethyl butyrate (citrus), benzyl acetate, nerol (bergamot) and furfural (lemon) were determined as the characterized odorants for each type.