Discovery of Chemokine CXCL12 Inhibitors by Tandem Application of Virtual Screening and NMR Spectrometry.

The CXC chemokine ligand CXCL12 and its receptor CXCR4 play critical roles in stem-cell homing, infectious diseases, and cancer, which led the CXCL12/CXCR4 signaling axis to attract much attention in drug discovery. CXCR4 is regarded as the primary target while CXCL12 is considered too small to be a druggable target. In this paper, we employed virtual screening approaches and ligand-based NMR screening methods from a SPECS library and in-house natural products to discover new CXCR12 inhibitors. Four natural triterpene saponins were confirmed, and the triterpene sapogenin was identified as the main binding epitope by saturation transfer difference-nuclear magnetic resonance and molecular docking studies. The pentacyclic triterpene scaffold and its elucidated structure-activity relationships provide a new and valuable research direction for the development of novel CXCL12 inhibitors.

Discovery of 1,2,4-triazole derivatives as novel neuroprotectants against cerebral ischemic injury by activating antioxidant response element.

Acute ischemic stroke is an important cause of death and long-term disability worldwide. In this work, we have synthesized a series of derivatives with 3,5­diaryl substituent triazole scaffolds. The derivatives showed favorable protective effective in SNP-induced oxidative stress model, of which compound 5 was the most active. In vivo experiments showed that compound 5 could ameliorate neurological deficits, attenuate infarction sizes, reduce malonaldehyde (MDA) level and increase superoxide dismutase (SOD) level in middle cerebral artery occlusion (MCAO) rats. Preliminary safety evaluation showed that compound 5 exhibited low acute toxicity in BALB/c mice (LD50 greater than 1000 mg/kg). Further investigation indicated that compound 5 was able to scavenge ROS, restore mitochondrial membrane potential and protect PC12 cells from SNP-induced apoptosis. Moreover, compound 5 could initiate transcription of antioxidant response element (ARE) and induced expressions of antioxidative enzymes. Collectively, compound 5 might have the potency of treating acute ischemic stroke.

Design and Discovery of Natural Cyclopeptide Skeleton Based Programmed Death Ligand 1 Inhibitor as Immune Modulator for Cancer Therapy.

Blockade of immune checkpoint PD-1/PD-L1 facilitates the rescue of immune escapes of tumor cells. Though various monoclonal antibodies have been approved for clinical therapy, the development of small molecular inhibitors lags behind antibodies partially owing to the challenges of protein-protein interaction (PPI) blocker design. In this work, we adopted the skeleton of natural cyclopeptidic antibiotics gramicidin S as the start point for PD-1/PD-L1 inhibitor exploring and discovered a series of novel cyclopeptides that could interfere with the PPI of PD-1/PD-L1 based on several rounds of structural design and optimization. The representative active cyclopeptide 66 can bind two PD-L1 and efficiently block the PD-1/PD-L1 interaction, recruit the immune cells to the tumor cells, enhance their killing against tumor cells by promoting the release of granzyme B and perforin, and display significant CD8+ T cell-dependent tumor suppression activity in vivo.

Co-delivery of terbinafine hydrochloride and urea with an in situ film-forming system for nail targeting treatment.

Onychomycosis is a chronic nail disorder consisting of a fungal infection that causes physical and psychosocial discomfort to patients. However, its treatment remains challenging owing to the barrier of the highly keratinized nail plate and the short time that conventional formulations reside on nails. In this work, we developed an in situ film-forming system(IFFS) based on Eudragit® RLPO to co-deliver terbinafine hydrochloride (TBH) and urea, i.e., TBH-urea-RLPO IFFS, with the aim of overcoming the nail barrier, prolonging the residence time, and efficiently treating onychomycosis. The IFFS formulation formed a thin film with good appearance and adhesion upon application in situ. The physical states of TBH and urea in the film were evaluated with polarization microscopy and powder X-ray diffraction. TBH and urea were both amorphousmiscible components within the RLPO film. TBH release from TBH-urea-RLPO IFFS fitted to the Korsmeyer-Pappas model, and the cumulative release at 72 h was significantly higher than that from commercial preparations (Lamisil Pedisan® once). In vitro permeation of TBH from TBH-urea-RLPO IFFS through bovine hoof membranes was evaluated in comparison with the film containing TBH alone (TBH-RLPO) and commercial preparations. The retention and cumulative permeated amount of TBH were significantly enhanced for the TBH-urea-RLPO IFFS (170.80 ± 44.63 µg/cm2vs 75.49 ± 21.50 µg/cm2vs 60.25 ± 27.38 µg/cm2; 61.81 ± 16.09 µg/cm2vs 21.80 ± 11.56 µg/cm2vs 7.91 ± 1.03 µg/cm2, respectively), and the membranes treated with different formulations were observed with SEM and FTIR to identify the denaturing effect of urea on bovine hoof keratin. In vitro antifungal tests against Trichophyton rubrum,Microsporum canis, Fusarium, and Aspergillus fumigatus were cultured on Muller-Hinton agar; the findings indicated that TBH-urea-RLPO IFFS enhanced TBH antifungal activity. Overall, the results support that TBH-urea-RLPO IFFS is an efficient and promising approach for onychomycosis targeting treatment.

Raloxifene alleviates amyloid-ß-induced cytotoxicity in HT22 neuronal cells via inhibiting oligomeric and fibrillar species formation.

Raloxifene, a selective estrogen receptor modulator, displays benefits for Alzheimer's disease (AD) prevention in postmenopausal women as hormonal changes during menopause have the potential to influence AD pathogenesis, but the underlying mechanism of its neuroprotection is not entirely clear. In this study, the effects of raloxifene on amyloid-ß (Aß) amyloidogenesis were evaluated. The results demonstrated that raloxifene inhibits Aß42 aggregation and destabilizes preformed Aß42 fibrils through directly interacting with the N-terminus and middle domains of Aß42 peptides. Consequently, raloxifene not only reduces direct toxicity of Aß42 in HT22 neuronal cells, but also suppresses expressions of tumor necrosis factor-α and transforming growth factor-ß induced by Aß42 peptides, and then alleviates microglia-mediated indirect toxicity of Aß42 to HT22 neuronal cells. Our results suggested an alternative possible explanation for the neuroprotective activity of raloxifene in AD prevention.

Mitochondria-Targeted Approach: Remarkably Enhanced Cellular Bioactivities of TPP2a as Selective Inhibitor and Probe toward TrxR.

A mitochondria-targeted approach was developed to increase the cellular bioactivities of thioredoxin reductase (TrxR) inhibitors. By being conjugated with a triphenylphosphine (TPP) motif to a previously found TrxR inhibitor 2a, the resulted compound TPP2a can target subcellular mitochondria and efficiently inhibit cellular TrxR, leading to remarkably increased cellular ROS level and mitochondrial apoptosis of HeLa cancer cells. The cellular bioactivities of TPP2a, including its cytotoxicity against a panel of cancer cell lines, dramatically elevated compared with its parental compound 2a. The selectively and covalently interaction of TPP2a with subcellular mitochondrial TrxR was validated by fluorescent microscopy. Moreover, a nonspecific signal quenching coupled strategy was proposed based on the environmentally sensitive fluorescence of TPP2a, which makes it possible to label TrxR by removing the nonspecific backgrounds caused by TPP2a under complex biosettings such as cellular lysates and living cells, implicating a potential of TPP2a for TrxR-specific labeling.

Synergetic skin targeting effect of hydroxypropyl-ß-cyclodextrin combined with microemulsion for ketoconazole.

The objective was to develop a ternary skin targeting system for ketoconazole (KET) using a combined strategy of microemulsion (ME) and cyclodextrin (HP-ß-CD), i.e., KET-CD-ME, which exploits both virtues of cyclodextrin complex and ME to obtain the synergetic effect. KET-CD-ME was formulated using Labrafil M 1944 CS as oil phase, Solutol HS 15 as surfactant, Transcutol P as cosurfactant, and HP-ß-CD solution as aqueous phase. The formulation of KET-CD-ME was optimized and the optimal formulation was characterized in terms of particle size, size distribution, pH value, and viscosity. Long term stability experiment showed that HP-ß-CD could increase the physical stability of ternary system and KET chemical stability. Percutaneous permeation of KET from KET-CD-ME in vitro through rat skin was investigated in comparison with KET microemulsion (KET-ME), KET HP-ß-CD inclusion solution (KET-CD), KET aqueous suspension, and commercial KET cream; the results showed that the combination of ME with HP-ß-CD exhibited significantly synergistic effect on KET deposition within the skin (29.38 ± 1.79 µg/cm(2)) and a slightly synergistic effect on KET penetration through the skin (11.3 µg/cm(2)/h). The enhancement of the combination on skin deposition was further visualized by confocal laser scanning microscope (CLSM). In vitro sensitivity against Candida parapsilosis test indicated that KET-CD-ME enhanced KET antifungal activity mainly owing to the solubilization of HP-ß-CD on KET in the ternary system. Moreover, the interactions between HP-ß-CD and KET in the ternary system were elucidated through microScale thermophoresis (MST) and 2D (1)H NMR spectroscopy. The profiles from MST confirmed the host-guest interactions of HP-ß-CD with KET in the ternary system and a deep insight into the interactions between KET and HP-ß-CD were obtained by means of 2D (1)H NMR spectroscopy. The results indicate that the ternary system of ME combination with HP-ß-CD may be a promising approach for skin targeting delivery of KET.

2a, a novel curcumin analog, sensitizes cisplatin-resistant A549 cells to cisplatin by inhibiting thioredoxin reductase concomitant oxidative stress damage.

(1E,4Z,6E)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(5-methylfuran-2-yl)hepta-1,4,6-trien-3-one (2a), a novel curcumin analog, was previously synthesized in our laboratory as a potential thioredoxin reductase (TrxR) inhibitor with excellent growth inhibitory effects on several TrxR over-expressed cancer cells. In this study, our further studies show that 2a is able to inhibit the growth of cisplatin-resistant A549 (A549/CDDP) cells much more effectively in a dose-dependent manner than that of A549 cells in antiproliferative activity experiments. Moreover, 2a-pretreated A549/CDDP cells are sensitive to cisplatin treatment, which is accompanied by the inhibition of TrxR activity in A549/CDDP cells. As a consequence of targeting TrxR, 2a in turn remarkably up-regulates intracellular reactive oxygen species level, depletes glutathione (GSH), and reduces the GSH/GSSG ratio, suggesting that the intracellular redox balance is shifted to a more oxidative state. Consequently, concomitant with the cell growth inhibition of 2a, apoptosis is induced by 2a probably through increased oxidative stress in A549/CDDP cells. In conclusion, these observations demonstrated that TrxR inhibitors would be promising drugs to achieve a successful combinatory or single cancer chemotherapy.

Novel sorbicillin analogues from the marine fungus Trichoderma sp. associated with the seastar Acanthaster planci.

Two novel sorbicillinoid analogues, (4'Z)-sorbicillin (1) and (2S)-2,3-dihydro-7-hydroxy-6-methyl-2-[(E)-prop-1-enyl]-chroman-4-one (2), together with three known compounds, (2S)-2,3-dihydro-7-hydroxy-6,8-dimethyl-2-[(E)-prop-1-enyl]-chroman-4-one (3), sorbicillin (4), and 2',3'-dihydrosorbicillin (5), were isolated from the culture broth of the fungus Trichoderma sp. associated with the seastar Acanthaster planci. Their structures were determined by analysis of the NMR and MS data. Compound I was the first example with a Z-configuration of the C-4'/C-5' double bond in the sorbyl side chain. Compounds 2 and 3 were uncommon monomeric sorbicillinoids with a cyclic sorbyl chain. 2, 3 and 5 showed moderate cytotoxic activities against various cancer cell lines.

Synthesis, cytotoxicity of new 4-arylidene curcumin analogues and their multi-functions in inhibition of both NF-κB and Akt signalling.

A series of new 4-arylidene curcumin analogues (4-arylidene-1,7-bisarylhepta-1,6-diene-3,5-diones) were synthesized and found to be potent antiproliferative agents against a panel of cancer cell lines at submicromolar to low micromolar concentrations by SRB assay. Their inhibitory abilities against NF-κB was evaluated by High Content Analysis (HCA) based immunofluorescence assay; and the Akt signalling inhibition was determined by fluorescence polarization assay and western blot respectively. The Structure-Activity Relationship was discussed. Our results revealed that 4-arylidene curcumin analogues may work in a multi-targets manner in cancer cell.

Synthesis and cytotoxicity evaluation of biaryl-based chalcones and their potential in TNFα-induced nuclear factor-κB activation inhibition.

A series of biaryl-based chalcones were designed as a combination of the natural chalcone and biphenyl moieties, and synthesized by two step chemistry involving Knoevenagel reaction and microwave assistant Suzuki coupling. Sulforhodamine B (SRB) assay was performed to evaluate the cell viability inhibitory abilities of these compounds against five cancer cell lines (A549, CNE2, SW480, MCF-7, and HepG2) from different tissues. Their Nuclear Factor-κB (NF-κB) nuclear translocation inhibitory activities were further investigated by High Content Analysis (HCA) based assay. Most of the compounds showed moderate to strong anticancer and NF-κB nuclear translocation inhibition activities and potent compounds were found.

Synthesis and identification of new 4-arylidene curcumin analogues as potential anticancer agents targeting nuclear factor-κB signaling pathway.

A series of curcumin analogues including new 4-arylidene curcumin analogues (4-arylidene-1,7-bisarylhepta-1,6-diene-3,5-diones) were synthesized. Cell growth inhibition assays revealed that most 4-arylidene curcumin analogues can effectively decrease the growth of a panel of lung cancer cells at submicromolar and low micromolar concentrations. High content analysis technology coupled with biochemical studies showed that this new class of 4-arylidene curcumin analogues exhibits significantly improved NF-κB inhibition activity over the parent compound curcumin, at least in part by inhibiting IκB phosphorylation and degradation via IKK blockage; selected 4-arylidene curcumin analogues also reduced the tumorigenic potential of cancer cells in a clonogenic assay.

Potential therapeutic effects of curcumin: relationship to microtubule-associated proteins 2 in Aß1-42 insult.

Curcumin can bind senile plaques and promote disaggregation of existing amyloid deposits and prevent aggregation of new amyloid deposits. Curcumin can also reverse distorted and curvy neurites around senile plaques and repair the neuritic abnormalities. We hypothesized whether altered neurite morphologies resulting from Aß production had anything to do with the changes of expression of microtubule-associated protein 2 (MAP2), but curcumin could reverse damaged neurites by upregulation of MAP2 expression. In present study we designed and chemically synthesized curcumin and its six derivatives. After screening the protective effect of curcumin and derivatives, we found that the viability of SK-N-SH cell model induced by Aß1-42 was significantly increased by curcumin and Cur1, and the expression of MAP-2 protein was obviously up-regulated in immunocytochemical staining and Western blot. The cell morphologies, including the number of neurites, neurite growth and neurite extension, were significantly improved. Cur1 showed more significant protective effect on SK-N-SH cells than curcumin. Our study revealed for the first time that the neuroprotective effect of curcumin and curcumin derivatives not only directly depends on their special chemical constitution, but they can resist to Aß damage by up-regulation of MAP-2 expression. In view of the special advantages of curcumin and Cur1, we reasonably believe that curcumin and Cur1 may be considered as an ideal therapeutic agent for the treatment of AD.

New fluorescent trans-dihydrofluoren-3-ones from aldol-Robinson annulation-regioselective addition involved one-pot reaction.

An unexpected discovery of new trans-4-acetyl-1,9-dimethyl-4,4a-dihydro-3H-fluoren-3-ones from one pot reactions of benzaldehydes and acetylacetone is described. The synthetic mechanism and stereochemistry were discussed. These new derivatives exhibit good fluorescent properties in solutions.

5-N-methylated quindoline derivatives as telomeric g-quadruplex stabilizing ligands: effects of 5-N positive charge on quadruplex binding affinity and cell proliferation.

A series of 5-N-methyl quindoline (cryptolepine) derivatives (2a- x) as telomeric quadruplex ligands was synthesized and evaluated. The designed ligands possess a positive charge at the 5- N position of the aromatic quindoline scaffold. The quadruplex binding of these compounds was evaluated by circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assay, polymerase chain reaction (PCR) stop assay, nuclear magnetic resonance (NMR), and molecular modeling studies. Introduction of a positive charge not only significantly improved the binding ability but also induced the selectivity toward antiparallel quadruplex, whereas the nonmethylated derivatives tended to stabilize hybrid-type quadruplexes. NMR and molecular modeling studies revealed that the ligands stacked on the external G-quartets and the positively charged 5- N atom could contribute to the stabilizing ability. Long-term exposure of human cancer cells to 2r showed a remarkable cessation in population growth and cellular senescence phenotype and accompanied by a shortening of the telomere length.

Synthesis and evaluation of curcumin analogues as potential thioredoxin reductase inhibitors.

Series of curcumin derivatives were synthesized; the inhibitory activities on thioredoxin reductase (TrxR) of all analogues were evaluated by DTNB assay in vitro. It is found that most of the analogues can inhibit TrxR in the low micromolar range; Structure-activity relationship analysis reveals that analogues with furan moiety have excellent inhibitory effect on TrxR in an irreversible manner, indicating that the furan moiety may serve as a possible pharmacophore during the interaction of curcumin analogues with TrxR. The effect of selected curcuminoids on growth of different TrxR overexpressed cancer cell lines was also investigated and discussed.

Alpha-glucosidase inhibition of natural curcuminoids and curcumin analogs.

Natural curcumin (1), demethoxycurcumin (2) and bisdemethoxycurcumin (3) isolated from Curcuma longa (turmeric), and synthetic curcumin analogs (A(1-7), B(1-7), C(1-6) and D(1-7)) were evaluated in vitro for the alpha-glucosidase inhibitory activity via UV and circular dichroism (CD) spectroscopy. The results indicated that natural curcuminoid compound 3 showed a remarkable inhibitory effect with IC(50) of 23.0 microM, and the synthetic compounds A(2), B(2), C(2) and D(2) showed potent inhibitory effects with IC(50) of 2.8, 2.6, 1.6 and 8.2 microM, respectively. Kinetic study exhibited that the mechanism of alpha-glucosidase inhibition of both 3 and C(2) was non-competitive. The structure activity relationship revealed that the ortho dihydroxyl groups could form a more tight interaction with alpha-glucosidase to exert more potential inhibitory activities.