Design, synthesis and biological evaluation of deuterated Vismodegib for improving pharmacokinetic properties.

Vismodegib is an oral and high selective hedgehog (Hh) inhibitor used for the treatment of basal cell carcinoma (BCC). In this work, analogs of Vismodegib with deuterium-for-hydrogen replacement at certain metabolically active sites were prepared and found to have a better pharmacokinetic properties in mice. In particular, deuterated compound SKLB-C2211 obviously altered the blood circulation behavior compared to its prototype, which was demonstrated by significantly prolonged blood circulation half-life time (t1/2) and increased AUC0→∞. These results suggested SKLB-C2211 had the potential to be a long-acting inhibitor against Hh signaling pathway, and laid the foundation for the further research of its druggability.

Synthesis and biological activity evaluation of novel amino acid derivatives as potential elicitors against Tomato yellow leaf curl virus.

Disease caused by Tomato yellow leaf curl virus (TYLCV) brings serious production losses of cultivated tomato worldwide. In our previous study, two novel amino acid derivatives exerted satisfactory antiviral activities against TYLCV. In this study, the variation of TYLCV, the transcriptional expression level of Ty-1 and the enzyme activities of POD and PPO in tomato were monitored after treatment with two amino acid derivatives to illustrate the antiviral mechanism. The results showed the symptom severity caused by TYLCV was reduced significantly by two compounds and was associated with the inhibition of viral DNA level at the early stage. Among three levels of concentration, the highest inhibition rate of CNBF-His was 40.66% at 1000 mg/L, for CNBF-Asn, the highest inhibition rate was 36.26% at 2000 mg/L 30 days post-inoculation. Two compounds could also enhance the activities of PPO and POD and the transcriptional expression level of Ty-1 which correlates with plant resistance in tomato. In the field test, two compounds increased the yields of tomato and the maximum increase of yield was 37.66%. This is the first report of novel amino acid derivatives inducing resistance in tomato plant against TYLCV. It is suggested that amino acid derivatives have the potential to be an effective approach against TYLCV in tomato plant.

Development and validation of an HPLC method for determination of Amikacin in water samples by solid phase extraction and pre-column derivatization.

This work presents a rapid and sensitive high performance liquid chromatography method for the determination of amikacin in water samples with solid phase extraction and pre-column derivatization. Amikacin residue was extracted from water samples with solid phase extraction cartridge. Then the extraction solution was derivatized with 4-chloro-3,5-dinitrobenzotrifluoride in the presence of triethylamine at 70°C in 20 min. The amikacin derivative was separated on a C18 column and detected by application of UV detection at 238 nm. The limit of detection is 0.2 µg/L with a signal-to-noise ratio of 3 and linearity is established over the concentration range from 0 to 500.0 µg/L. Recoveries of the amikacin in three types of water samples are from 87.5 % to 99.6 % and RSDs are 2.1 %-4.5 %. This method can be used for the quantification of amikacin residues in water samples.

Endocytic proteins mediating GPR15 receptor internalization provide insight into the underlying mechanisms.

GPR15 is a G protein-coupled receptor involved in immune disorders such as human immunodeficiency virus-induced enteropathy, multiple sclerosis, and colitis. Yet, the important endocytosis mechanism of GPR15 remained unclear. This study determined the participation of endocytic machinery proteins, including Gα proteins, G protein-coupled receptor kinases (GRKs), protein kinase C, arrestins, clathrin, caveolin, and dynamin in GPR15 internalization. The results demonstrate that GPR15 internalization is moderately dependent on GRKs and clathrin, and highly dependent on caveolin and dynamin. Moreover, a bystander arrestin recruitment assay showed that GPR15 recruits arrestin-3 to the cell membrane upon agonist stimulation, although GPR15 internalizes in an arrestin-independent manner. Overall, our study provides novel insights into ß-arrestin recruitment and receptor internalization mechanisms for the recently deorphanized GPR15.

Structure-activity relationship of GPR15L peptide analogues and investigation of their interaction with the GPR15 receptor.

The 57-mer full-length GPR15L(25-81) peptide has been identified as the principal endogenous agonist of the G protein-coupled receptor GPR15. Its main activity resides in the C-terminal 11-mer GPR15L(71-81), which has full efficacy but ~40-fold lower potency than the full-length peptide. Here, we systematically investigated the structure-activity relationship of GPR15L(71-81) by truncations/extensions, alanine-scanning, and N- and C-terminal capping. The synthesized peptide analogues were tested at GPR15 stably expressed in HEK293A cells using a homogenous time-resolved Förster resonance energy transfer-based Gi cAMP functional assay. We show that the C-terminal α carboxyl group and the residues Leu78 , Pro75 , Val74 , and Trp72 are critical for receptor interaction and contribute significantly to the peptide potency. Furthermore, we tested the ability of GPR15L(71-81), C-terminally amidated GPR15L(71-81), and GPR15L(25-81) to activate the three GPR15 receptor mutants in a bioluminescence resonance energy transfer-based G protein activation assay. The results demonstrate that the Lys192 and Glu272 residues in GPR15 are important for the potency of the GPR15L peptide. Overall, our study identifies critical residues in the peptide and receptor sequences for future drug design.

Delineation of the GPR15 receptor-mediated Gα protein signalling profile in recombinant mammalian cells.

The GPR15 receptor is a G protein-coupled receptor (GPCR), which is activated by an endogenous peptide GPR15L(25-81) and a C-terminal peptide fragment GPR15L(71-81). GPR15 signals through the Gi/o pathway to decrease intracellular cyclic adenosine 3',5'-monophosphate (cAMP). However, the activation profiles of the GPR15 receptor within Gi/o subtypes have not been examined. Moreover, whether the receptor can also couple to Gs , Gq/11 and G12/13 is unclear. Here, GPR15L(25-81) and GPR15L(71-81) are used as pharmacological tool compounds to delineate the GPR15 receptor-mediated Gα protein signalling using a G protein activation assay and second messenger assay conducted on living cells. The results show that the GPR15 receptor preferentially couples to Gi/o rather than other pathways in both assays. Within the Gi/o family, the GPR15 receptor activates all the subtypes (Gi1 , Gi2 , Gi3 , GoA , GoB and Gz ). The Emax and activation rates of Gi1, Gi2 , Gi3, GoA and GoB are similar, whilst the Emax of Gz is smaller and the activation rate is significantly slower. The potencies of both peptides toward each Gi/o subtype have been determined. Furthermore, the GPR15 receptor signals through Gi/o to inhibit cAMP accumulation, which could be blocked by the application of the Gi/o inhibitor pertussis toxin.

Metabolically stable diphenylamine derivatives suppress androgen receptor and BET protein in prostate cancer.

Androgen receptor (AR) is a crucial driver of prostate cancer (PC). AR-relevant resistance remains a major challenge in castration-resistant prostate cancer (CRPC). Bromodomain and extra-terminal domain (BET) family are critical AR coregulators. Here, we developed several diphenylamine derivatives and identified compound 7d that disrupted the functions of AR and BET family in prostate cancer and exhibited favorable metabolic stability in vitro and high drug exposure in vivo. We showed 7d not only bound to AR, suppressed transactivation of wild-type AR (wt-AR) and the mutant that mediates Enzalutamide resistance, but also reduced c-Myc protein expression through BET inhibition. In addition, 7d inhibited the proliferation of AR-positive PC cells with favorable selectivity and suppressed AR-V7-expressing VCaP and 22Rv1 xenografts growth in vivo. Collectively, these results indicate the potential of lead compound 7d as an orally available AR and BET inhibitor to treat CRPC and overcome antiandrogen resistance.

Discovery and biological evaluation of darolutamide derivatives as inhibitors and down-regulators of wild-type AR and the mutants.

Androgen receptor (AR) has been a target of prostate cancer (PC) for nearly six decades. Recently, downregulating or degrading AR and the mutants especially the splice variant 7 (AR-V7) lacking ligand binding domain (LBD) emerged as an advantageous therapeutic approach to overcome drug resistance. Here, the structural modification of darolutamide resulted in the discovery of dual-action AR inhibitors and down-regulators. Unlike other traditional AR antagonists targeting the AR-LBD, compounds 4k and 4b not only inhibit the activities of wt-AR and AR-F876L mutant but also downregulate the protein expression of full-length (AR-full) and AR variant 7 (AR-V7) at mRNA level. In cell proliferation assays, compounds 4k and 4b exhibited better antiproliferative activities than darolutamide and enzalutamide against AR-V7-positive 22Rv1 cells and VCaP cells. In addition, 4k demonstrated better antitumor activity than clinically used enzalutamide in castration-resistant VCaP xenograft model. Collectively, combining the activities of AR inhibition and downregulation, compound 4k is proposed as an advantageous lead compound to disrupt AR signaling and overcome resistance.

Effects of anoxia on survival and gene expression in Bactrocera dorsalis.

The oriental fruit fly (Bactrocera dorsalis) larvae may commonly experience a hypoxia microenvironment and have evolved the ability to survive in the low oxygen condition with some physiological and biochemical mechanisms. However, little is known about the response of B. dorsalis to hypoxia or anoxia. In this study, the effect of anoxia on the survival of B. dorsalis was investigated. The results showed that the B. dorsalis larvae were quite tolerant to anoxia conditions and can tolerate up to 24 h of anoxia exposure without a significant reduction in survival, 100% mortality was reached after 84 h of anoxia exposure. The cDNA of hypoxia inducible factor (HIF) 1α and HIF-1ß is 2912 and 3618 bp in length, encoding 766 and 648 amino acid residues, respectively. Both HIF-1α and HIF-1ß contain conserved basic helix-loop-helix (bHLH) domain and Per-Arnt-Sim (PAS) domain. HIF-1α can be induced by hypoxia, whereas HIF-1ß expression was not significantly changed with the oxygen concentration. Three major heat shock proteins (Hsps) expression increased significantly during anoxia and recovery and Hsp70 was the most responsive to anoxia. Four superoxide dismutase (SOD) genes expression were also up-regulated during anoxia exposure. These data suggest that B. dorsalis has a strategy to induce HIF-1α and HIF-1-responsive genes to survive in the low oxygen condition.

Discovery of novel inhibitors of RNA silencing suppressor P19 based on virtual screening.

To control plant viruses, viral RNA silencing suppressors are important drug targets due to their key roles in interfering antiviral RNA silencing. Here we have presented a strategy, combining virtual and experimental screening, to discover the inhibitors of viral suppressor. By docking 157 026 compounds from a natural product library into P19 model, eighteen candidate compounds were selected. Candidates VS2, VS12, VS14 and VS15 displayed strong binding ability to P19 in the surface plasmon resonance imaging assay with K D values of 136.2, 111.6, 81.2 and 124.5 nM, respectively. Then the inhibition activities of these inhibitors on the association between P19 and siRNA were also affirmed by electrophoretic mobility shift assay. Moreover, the antiviral effects on plants showed that compounds VS14 and VS15 both exhibited antiviral activities against Tomato bushy stunt virus (TBSV) in vivo with inhibition rates of 32.35% and 16.61% in 11 dpi, respectively. This strategy would be a powerful tool for the discovery of novel antiviral agents and provide new insights into the control of plant viruses.

Synthesis and evaluation of novel thiazole-based derivatives as selective inhibitors of DNA-binding domain of the androgen receptor.

A series of thiazole-based inhibitors selectively targeting DNA-binding domain of the androgen receptor (AR) were synthesized and evaluated, and the SAR data were summarized. We identified a novel compound SKLB-C2807 that effectively inhibited the human prostate cancer cell line LNCaP/AR with the IC50 value of 0.38 µm without significant antiproliferative effects on other cell lines PC-3 (AR-negative), SW620, MCF-7 (ER-positive), and L-O2 (non-cancerous). This compound also considerably decreased the expression of prostate-specific antigen. Its binding mode to the AR-DBD was studied. These efforts lay the foundation for developing the next generation of anti-androgens.

Design, Synthesis, and Biological Evaluation of Dimorpholine Substituted Thienopyrimidines as Potential Class I PI3K/mTOR Dual Inhibitors.

Dysfunctional signaling of the PI3K/AKT/mTOR pathway in cancer and its crucial role in cell growth and survival have made it a much desired target for cancer therapeutics. A series of dimorpholine substituted thienopyrimidine derivatives had been prepared and evaluated in vitro and in vivo. Among them, compound 14o was identified as a dual Class I PI3K and mTOR kinase inhibitor, which had an approximately 8-fold improvement in mTOR inhibition relative to the class I PI3K inhibitor 1 (pictilisib, GDC-0941). Western blot analysis confirmed the 14o mechanistic modulation of the cellular PI3K/AKT/mTOR pathway through inhibiting phosphorylation of both AKT and S6 in human cancer cell lines. In addition, 14o demonstrated significant efficacy in SKOV-3 and U87MG tumor xenograft models without causing significant weight loss and toxicity.

The bioaccumulation and biotransformation of synthetic estrogen quinestrol in crucian carp.

The occurrence and fate of endocrine disrupting chemicals (EDCs) in aquatic species have attracted close attention during the last decades. In this study, the bioaccumulation and biotransformation of synthetic estrogen quinestrol, one of the typical EDCs, in the plasma and liver of crucian carp, were investigated by a newly developed and validated reversed-phase high performance liquid chromatography with fluorescent detection method. Crucian carp were exposed to quinestrol in concentration of 2, 10, 50, 100 µg/L (5.49, 27.43, 137.17, 274.34 nmol/L) for 60 days. After 60 days' exposure, the concentrations of quinestrol found in liver and plasma were in the range of 0.25-0.69 mg/kg and 0.19-0.30 mg/L respectively, positively correlated with the exposure concentrations ranged 2-100 µg/L (5.49-274.34 nmol/L). There was a negative correlation between the bio-accumulation ratios and the exposure concentrations of quinestrol. 17α-Ethinylestradiol was also found in liver and plasma, and the concentrations were 0.02-0.19 mg/kg and 0.37-0.96 mg/L, respectively. The results indicated that quinestrol can be accumulated and transformed to 17α-ethinylestradiol in crucian carp. Moreover, exposure to quinestrol caused oxidative damages to crucian carp and the content of malondialdehyde increased in all treatment concentrations.

Preparation and characterization of novel functionalized prochloraz microcapsules using silica-alginate-elements as controlled release carrier materials.

Controlled release formulation of pesticides is an effective approach to achieve the desirable purpose of increasing the utilization of pesticides and reducing the environmental residuals. In this work, a novel functionalized microcapsule using silica cross-linked with alginate, and some beneficial elements to crops, was prepared. The microcapsules were structurally characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy. The results showed that the microcapsules had a high loading efficiency of prochloraz (about 30% w/w) and could effectively protect prochloraz against degradation under UV irradiation and alkaline conditions, showed sustainable release for at least 60 days, and also likely increased disease resistance due to the element on the surface. Given the advantages of the microcapsules, this delivery system may be extended to other photosensitive or pH-sensitive pesticides in the future.

Determination of malondialdehyde in biological fluids by high-performance liquid chromatography using rhodamine B hydrazide as the derivatization reagent.

Malondialdehyde (MDA) is a biomarker for lipid peroxidation, and studies of sensitive and selective analytical methods for it are very important for pathological research. The aim of this work was to develop and validate a novel HPLC method for the quantification of MDA in biological fluids using rhodamine B hydrazide (RBH) as the derivatization reagent. After pretreatment and derivatization in acid medium at 50 °C for 40 min, the RBH-derivatized MDA was separated on a Kromasil C18 column at 25 °C and detected by a fluorescence detector at excitation wavelength of 560 nm and emission wavelength of 580 nm. The results showed linearity in the range of 0.8-1500.0 nM with a detection limit of 0.25 nM (S/N = 3). The recovery of MDA from plasma and urine was 91.50 to 99.20%, with a relative standard deviation range of 1.45 to 3.26%. In comparison to other methods reported for the determination of MDA, the proposed method showed superiority in simplicity, more sensitivity, shorter derivatization time, and less interference. The developed method was applied to quantification of MDA in human biological fluids collected from five volunteers with a concentration range of 24.62-245.00 nM.