Effect of the pseudomonas metabolites HQNO on the Toxoplasma gondii RH strain in vitro and in vivo.

Toxoplasmosis is a widespread disease in humans and animals. Currently, toxoplasmosis chemotherapy options are limited due to severe side effects. There is an urgent need to develop new drugs with better efficacy and few side effects. HQNO, a cytochrome bc1 and type II NADH inhibitor in eukaryotes and bacteria, possesses extensive bioactivity. In this study, the cytotoxicity of HQNO was evaluated in Vero cells. The in vitro effects of HQNO were determined by plaque assay and qPCR assay. To determine the in vivo effect of HQNO, pharmacokinetic experiments and in vivo infection assays were performed in mice. The changes in tachyzoites after HQNO exposure were examined by transmission electron microscopy (TEM), MitoTracker Red CMXRos staining, ROS detection and ATP detection. HQNO inhibited T. gondii invasion and proliferation with an EC50 of 0.995 µM. Pharmacokinetic experiments showed that the Cmax of HQNO (20 mg/kg·bw) was 3560 ± 1601 ng/mL (13.73 µM) in healthy BALB/c mouse plasma with no toxicity in vivo. Moreover, HQNO induced a significant decrease in the parasite burden load of T. gondii in mouse peritoneum. TEM revealed alterations in the mitochondria of T. gondii. Further assays verified that HQNO also decreased the mitochondrial membrane potential (ΔΨm) and ATP levels and enhanced the level of reactive oxygen species (ROS) in T. gondii. Hence, HQNO exerted anti-T. gondii activity, which may be related to the damage to the mitochondrial electron transport chain (ETC).

Discovery of Hexahydrofuro[3,2-b]furans as New Kinase-Selective and Orally Bioavailable JAK3 Inhibitors for the Treatment of Leukemia Harboring a JAK3 Activating Mutant.

Janus kinase 3 (JAK3) is a potential target for the treatment of hematological malignancies. Herein, we report the discovery of a series of new orally bioavailable irreversible JAK3 kinase inhibitors. The representative compound 12n potently inhibited JAK3 kinase activity with an IC50 value of 1.2 nM and was more than 900-fold selective over JAK1, JAK2, and Tyk2. Cell-based assays revealed that 12n significantly suppressed phosphorylation of JAK3 and the downstream effectors STAT3/5 and also robustly restrained proliferation of BaF3 cells transfected with JAK3M511I activating mutation and human leukemia U937 cells harboring JAK3M511I with IC50 values of 22.9 and 20.2 nM, respectively. More importantly, 12n showed reasonable pharmacokinetic (PK) properties, and oral administration of 12n at a dose of 50 mg/kg twice daily led to tumor regression in a U937 cell inoculated xenograft mouse model. Thus, 12n represents a promising lead compound for further optimization to discover new therapeutic agents for hematological malignancies.

Discovery of 8-(6-Methoxypyridin-3-yl)-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1,5-dihydro-4H-[1,2,3]triazolo[4,5-c]quinolin-4-one (CQ211) as a Highly Potent and Selective RIOK2 Inhibitor.

RIOK2 is an atypical kinase implicated in multiple human cancers. Although recent studies establish the role of RIOK2 in ribosome maturation and cell cycle progression, its biological functions remain poorly elucidated, hindering the potential to explore RIOK2 as a therapeutic target. Here, we report the discovery of CQ211, the most potent and selective RIOK2 inhibitor reported so far. CQ211 displays a high binding affinity (Kd = 6.1 nM) and shows excellent selectivity to RIOK2 in both enzymatic and cellular studies. It also exhibits potent proliferation inhibition activity against multiple cancer cell lines and demonstrates promising in vivo efficacy in mouse xenograft models. The crystal structure of RIOK2-CQ211 sheds light on the molecular mechanism of inhibition and informs the subsequent optimization. The study provides a cell-active chemical probe for verifying RIOK2 functions, which may also serve as a leading molecule in the development of therapeutic RIOK2 inhibitors.

Determination and pharmacokinetics study of UK-5099 in mouse plasma by LC-MS/MS.

BACKGROUND: UK-5099 is a potent mitochondrial acetone carrier inhibitor, that exhibits anticancer activity. Recently, the anti-Toxoplasma gondii activity of UK-5099 was proposed, and in vivo studies of its pharmacokinetics in BALB/c mice are necessary to further evaluate the clinical effect of UK-5099. METHODS AND RESULTS: A simple and fast high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis method was established and verified in terms of its linearity, matrix effect, accuracy, precision, recovery and stability. The analytes were separated by an Agilent ZORBAX XDB-C18 column (2.1 × 50 mm, 3.5 µm) at 30 °C. A gradient mobile phase consisting of water with 0.1% formic acid (FA) (phase A) and acetonitrile (ACN) (phase B) was delivered at a flow rate of 0.40 mL·min-1 with an injection volume of 5 µL. A good linear response was obtained in a concentration range of 5-5000 ng·mL-1 (r2 = 0.9947). The lower limit of quantification (LLOQ) was 5 ng·mL-1. The extraction recovery of UK-5099 was greater than 95%. The inter- and intra-day accuracy and precision of the method showed relative standard deviations (RSDs) of less than 15%. This method has been successfully applied to the pharmacokinetic evaluation of UK-5099 in mouse plasma. In health mice, the main pharmacokinetic parameters of UK-5099 after intraperitoneal administration were measured using a noncompartmental model, in which the AUC0-t was 42,103 ± 12,072 ng·h·mL-1 and the MRT0-t was 0.857 ± 0.143 h. The peak concentration (Cmax) was 82,500 ± 20,745 ng·h·mL-1, which occurred at a peak time (Tmax) = 0.250 ± 0.000 h. CONCLUSIONS: A fast and sensitive HPLC-MS/MS method was developed, validated and successfully used for the determination of UK-5099 levels in mice after intraperitoneal administration. This study was the first report of the pharmacokinetic parameters of UK-5099 in mice, which will help to further study the administration of UK-5099 in animals and humans.

Novel Electrophilic Warhead Targeting a Triple-Negative Breast Cancer Driver in Live Cells Revealed by "Inverse Drug Discovery".

The "inverse drug discovery" strategy is a potent means of exploring the cellular targets of latent electrophiles not typically used in medicinal chemistry. Cyclopropenone, a powerful electrophile, is generally used in bio-orthogonal reactions mediated by triarylphosphine or in photo-triggered cycloaddition reactions. Here, we have studied, for the first time, the proteome reactivity of cyclopropenones in live cells and discovered that the cyclopropenone warhead can specifically and efficiently modify a triple-negative breast cancer driver, glutathione S-transferase pi-1 (GSTP1), by covalently binding at the catalytic active site. Further structure optimization and signaling pathway validation have led to the discovery of potent inhibitors of GSTP1.

Myrislignan Induces Redox Imbalance and Activates Autophagy in Toxoplasma gondii.

Toxoplasma gondii (T. gondii) is an important health problem in human and animals, and the highlighting side effects of launched therapeutic chemicals cannot be ignored. Thus, it is urgent to develop new drugs to against the infection. Myrislignan originated from nutmeg exhibited excellent anti-T. gondii activity in vitro and in vivo, and was able to destroy mitochondrial function. However, the exact mechanism of action is still unknown. In this study, combining RNAs deep-sequencing analysis and surface plasmon resonance (SPR) analysis, the differentially expressed genes (DEGs) and high affinity proteins suggested that myrislignan may affect the oxidation-reduction process of T. gondii. Furthermore, the upregulating ROS activity after myrislignan incubation verified that myrislignan destroyed the oxidant-antioxidant homeostasis of tachyzoites. Transmission electron microscopy (TEM) indicated that myrislignan induced the formation of autophagosome-like double-membrane structure. Moreover, monodansyl cadaverine (MDC) staining and western blot further illustrated autophagosome formation. Myrislignan treatment induced a significant reduction in T. gondii by flow cytometry analysis. Together, these findings demonstrated that myrislignan can induce the oxidation-reduction in T. gondii, lead to the autophagy, and cause the death of T. gondii.

Determination of myrislignan levels in BALB/c mouse plasma by LC-MS/MS and a comparison of its pharmacokinetics after oral and intraperitoneal administration.

BACKGROUND: Myrislignan is a natural product from Myristica sp. with diverse pharmacological activities. Recently, the anti-Toxoplasma gondii (T. gondii) activity of myrislignan has been proposed, and in vivo studies of its pharmacokinetics in BALB/c mice are necessary to further evaluate the clinical effects of myrislignan. RESULTS: In this study, a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify myrislignan levels in mouse plasma using dehydrodiisoeugenol as an internal standard (IS) in positive ion mode. Chromatographic separation of the analytes was achieved using an ACE Ultracore Super C18 analytical column (2.5 µm, 2.1 × 50 mm) at 30 °C. A gradient mobile phase consisting of water (0.1 % formic acid) and acetonitrile (0.1 % formic acid) was delivered at a flow rate of 0.4 mL/min. Myrislignan and the IS eluted at 1.42 and 1.71 min, respectively. A good excellent linear response across the concentration range of 1-1000 ng/mL was achieved (r2 = 0.9973). The lower limit of quantification (LLOQ) was 1 ng/mL, and the inter- and intra-day accuracy and precision of the method showed relative standard deviations (RSDs) less than 10 %. The method was applied to examine the pharmacokinetics of myrislignan in mouse plasma following a single oral administration of 200 mg/kg or intraperitoneal administration of 50 mg/kg myrislignan, and the bioavailability (F) of orally administered myrislignan was only 1.97 % of the bioavailability of intraperitoneally administered myrislignan. CONCLUSIONS: A rapid and sensitive LC-MS/MS method has been was developed, validated and successfully used to determine myrislignan levels in mice after oral or intraperitoneal administration. This study is the first to report the pharmacokinetic parameters of myrislignan in mice and to compare its pharmacokinetics after oral and intraperitoneal administration, which will be useful for further research on the administration of myrislignan in animals and humans.

Licarin-B Exhibits Activity Against the Toxoplasma gondii RH Strain by Damaging Mitochondria and Activating Autophagy.

Toxoplasma gondii is an obligate intracellular pathogen that infects warm-blooded animals and humans. However, side effects limit toxoplasmosis treatment, and new drugs with high efficiency and low toxicity need to be developed. Natural products found in plants have become a useful source of drugs for toxoplasmosis. In this study, twenty natural compounds were screened for anti-T. gondii activity by Giemsa staining or real-time fluorescence quantitative polymerase chain reaction (qPCR) in vitro. Among these, licarin-B from nutmeg exhibited excellent anti-T. gondii activity, inhibiting T. gondii invasion and proliferation in a dose-dependent manner, with an EC50 of 14.05 ± 3.96 µg/mL. In the in vivo, licarin-B treatment significantly reduced the parasite burden in tissues compared to no treatment, protected the 90% infected mice from to death at 50 mg/kg.bw. Flow cytometry analysis suggested a significant reduction in T. gondii survival after licarin-B treatment. Ultrastructural changes in T. gondii were observed by transmission electron microscopy (TEM), as licarin-B induced mitochondrial swelling and formation of cytoplasmic vacuoles, an autophagosome-like double-membrane structure and extensive clefts around the T. gondii nucleus. Furthermore, MitoTracker Red CMXRos, MDC, and DAPI staining showed that licarin-B promoted mitochondrial damage, autophagosome formation, and nuclear disintegration, which were consistent with the TEM observations. Together, these findings indicate that licarin-B is a promising anti-T. gondii agent that potentially functions by damaging mitochondria and activating autophagy, leading to T. gondii death.

Identification of U937JAK3-M511I Acute Myeloid Leukemia Cells as a Sensitive Model to JAK3 Inhibitor.

Mutated JAK3 has been considered a promising target for cancer therapy. Activating mutations of JAK3 are observed in 3.9%-10% of acute myeloid leukemia (AML) patients, but it is unclear whether AML cells are sensitive to JAK3 inhibitors, and no disease-related human AML cell model has been reported. We have identified U937 as the first human AML cell line expressing the JAK3M511I activated mutation and confirmed that JAK3 inhibitors sensitively suppress the proliferation of U937 AML cells.

Identification and Development of 1,4-Diaryl-1,2,3-triazolo-Based Ureas as Novel FLT3 Inhibitors.

A class of 1,4-diaryl-1,2,3-triazolo-based ureas were synthesized and developed as novel FLT3 inhibitors. The representative compound 28 strongly inhibited FLT3-ITD kinase (IC50 = 32.8 nM) and isogenic BaF3-FLT3-ITD cell (GI50 = 0.6 nM). It exhibited potent inhibition against FLT3-ITD positive MV4-11 (GI50 = 3.0 nM) and MOLM-13 (GI50 = 5.9 nM) cell lines and high selectivity over FLT3-WT cell lines. It also displayed good pharmacokinetics properties and demonstrated promising oral in vivo efficacy in a MV4-11 cell xenografted mouse model. It might be a potent lead compound for further development to treat FLT3-ITD driven acute myloid leukemia.

Myrislignan Exhibits Activities Against Toxoplasma gondii RH Strain by Triggering Mitochondrial Dysfunction.

Toxoplasma gondii is a widespread obligatory parasitic protozoon that infects nearly all warm-blooded animals and causes toxoplasmosis. However, the current treatments for toxoplasmosis are limited by severe side effects. Myrislignan is a natural product from Myristica fragrans Houtt with wide pharmacological activities. In the current study, we tested the anti-T. gondii activity of myrislignan both in vitro and in vivo and explored its potential mechanism of action. The cytotoxicity of myrislignan in African green monkey kidney (Vero) cells was assessed using Cell Counting Kit-8 (CCK-8) assays. The in vitro effects of myrislignan on T. gondii were determined by quantitative PCR and Giemsa staining. An in vivo murine model of T. gondii infection was used to determine the efficacy of myrislignan. The changes in tachyzoites after myrislignan exposure were examined by electron microscopy. The impact of myrislignan on mitochondrial function in tachyzoites was assessed by MitoTracker Red CMXRos staining and an ATP detection kit. In vitro, myrislignan inhibited T. gondii tachyzoite proliferation with a 50% effective concentration of 32.41 µg/ml, and reduced the invasion of cells by tachyzoites (14.63 and 1.92% invasion rates for control and 70 µg/ml myrislignan, respectively). Importantly, myrislignan had no significant cytotoxicity against Vero cells at concentrations less than 132 µg/ml. In addition, surface shrinkage and mitochondrial damage were observed in tachyzoites after myrislignan exposure. The reduced ΔΨm and ATP levels in tachyzoites treated with myrislignan further confirmed mitochondrial damage. In the in vivo murine model, myrislignan treatment significantly reduced the parasite burden in tissues compared to no treatment. In conclusion, myrislignan had potent anti-T. gondii activities both in vitro and in vivo, and these activities might involve the interruption of mitochondrial function. These data suggest that myrislignan might be a useful compound for the treatment of toxoplasmosis.

Determination and pharmacokinetics study of oxyclozanide suspension in cattle by LC-MS/MS.

BACKGROUND: Oxyclozanide is an anthelmintic drug that is widely used to treat fasciolosis. However, the pharmacokinetics of oxyclozanide in cattle are not yet clearly understood. The present study was designed to develop a sensitive method to determine oxyclozanide levels in cattle plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and to study its pharmacokinetics for application in cattle. RESULTS: A simple and rapid HPLC-MS/MS analytical method was established and validated to quantify oxyclozanide levels in cattle plasma using niclosamide as the internal standard (IS) in negative ion mode. Chromatographic separation of the analytes was achieved using a C18 analytical column (75 × 4.6 mm, 2.7 µm) at 30 °C. The mobile phase comprised 0.01% v/v acetic acid (HOAc) diluted in water:acetonitrile (MeCN) (90:10% v/v) and 5 mM ammonium formate in methanol (MeOH):MeCN (75:25, v/v) at a 10:90 ratio (v/v) and was delivered at a flow rate of 0.4 mL min- 1. A good linear response across the concentration range of 0.02048-25.600 µg/mL was achieved (r2 = 0.994). The method was validated with respect to linearity, matrix effect, accuracy, precision, recovery and stability. The lower limit of quantification (LLOQ) was 0.020 µg/mL, and the extraction recovery was > 98% for oxyclozanide. The inter- and intra-day accuracy and precision of the method showed the relative standard deviation (RSD) less than 10%. The method was successfully applied to an assessment of the pharmacokinetics of oxyclozanide in cattle plasma. In healthy cattle, a single oral dose of an oxyclozanide suspension followed the one-compartment model, with a half-life (T1/2) of 64.40 ± 30.18 h, a plasma clearance rate (CL/F) of 11.426 ± 2.442 mL/h/kg, and an average area under the curve (AUC) of 965.608 ± 220.097 h*µg/mL. The peak concentration (Cmax) was 15.870 ± 2.855 µg/mL, which occurred at a peak time (Tmax) = 22.032 ± 3.343 h. CONCLUSIONS: A reliable, accurate HPLC-MS/MS analytical method was established in our study and successful applied to study the pharmacokinetics of oxyclozanide in cattle plasma. These results will be useful for further evaluations of the pharmacokinetic properties of oxyclozanide or for monitoring therapeutic drugs in animals.

High prevalence of fasciolosis and evaluation of the efficacy of anthelmintics against Fasciola hepatica in buffaloes in Guangxi, China.

Fasciola hepatica is a common parasite of grazing livestock in Guangxi Zhuang Autonomous Region in China, but its prevalence has not been studied. While triclabendazole is commonly used to treat F. hepatica infection in China, oxyclozanide has never been used. This study investigated the prevalence of F. hepatica infections in buffaloes in the Guangxi and evaluated the efficacy of oxyclozanide and triclabendazole as treatments. In the prevalence study, a total of 767 individual faecal samples were obtained from 58 farms in Guangxi to detect the prevalence of F. hepatica, and the total rate of infection was 87.35%. A subset of 277 infected buffaloes from these farms were randomly divided into 3 groups. Group 1 (n = 101) was treated with oxyclozanide at 10 mg/kg.bw; group 2 (n = 94) was treated with triclabendazole (12 mg/kg.bw); and group 3 (n = 82) was untreated. Faecal samples were taken on days 0, 7, 14, 21 and 28. Whole blood and serum were collected on days 0 and 14. Anthelmintic efficacy was assessed using faecal egg count reduction (FECR), buffaloes positive by coprology reduction (BPCR) as well as post-treatment improvement in biochemical and haematological indicators. After 28 days treatment, group 1 and 2 showed FECR% values above 98%, and BPCR% values of 97.03% and 77.66%, respectively. In addition, the biochemical indicators and haematological parameters were improved at 14 days post-treatment compared with those before treatment. These results indicate a high prevalence of F. hepatica in Guangxi, demonstrate that oxyclozanide and triclabendazole are effective against F. hepatica infection in buffaloes, and indicate that oxyclozanide could be used in China as an alternative drug.

Licochalcone A: An effective and low-toxicity compound against Toxoplasma gondii in vitro and in vivo.

Toxoplasma gondii, an obligate intracellular protozoan, is the causative agent of toxoplasmosis, which can cause serious public health problems. The current drugs used to treat toxoplasmosis have many limitations. This study evaluated the anti-T. gondii activity and potential mechanism of Licochalcone A (Lico A) in vitro and in vivo. The safe concentration of Lico A in HFF cells was determined by MTT cell viability assays. The presence of T. gondii was assessed by qPCR and Giemsa staining. Azithromycin and sulfadiazine, commonly used effective treatments, served as drug controls. T. gondii ultrastructural alterations were observed by electron microscopy. The anti-T. gondii activity of Lico A was evaluated using an in vivo mouse infection model. In vitro, Lico A had no negative effect on host cell viability at concentrations below 9 µg/mL; however, it did inhibit T. gondii proliferation in a dose-dependent manner, with a 50% inhibitory concentration (IC50) of 0.848 µg/mL. Electron microscopy analyses indicated substantial structural and ultrastructural changes in tachyzoites after Lico A treatment. Nile Red staining assays demonstrated that Lico A caused lipid accumulation. Lico A treatment significantly increased the survival rate of BALB/c mice infected with T. gondii. Lico A achieved the same therapeutic effect as a commonly used clinical drugs (combination of sulfadiazine, pyrimethamine and folinic acid). In conclusion, Lico A has strong anti-T. gondii activity in vitro and in vivo and might be developed into a new anti-T. gondii drug. Moreover, Lico A may exert these effects by interfering with lipid metabolism in the parasite.