Design, synthesis and biological evaluation of 3,5-dimethylisoxazole and pyridone derivatives as BRD4 inhibitors.

Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) family, has been recognized as an attractive candidate target for the treatment targeting gene transcription in several types of cancers. In this study, two types of novel compounds were designed, synthesized and evaluated as BRD4 inhibitors. Therein, pyridone derivatives were more effective against BRD4 protein and human leukemia cell lines MV4-11. Among them, compounds 11d, 11e and 11f were the most potential ones with IC50 values of 0.55 µM, 0.86 µM and 0.80 µM against BRD4, and exhibited remarkable antiproliferative activities against MV4-11 cells with IC50 values of 0.19 µM, 0.32 µM and 0.12 µM, respectively. Moreover, in western blot assay, compound 11e induced down-regulation of C-Myc, which is a significant downstream gene of BRD4. Cell cycle analysis assay also showed that compound 11e could block MV4-11 cells at G0/G1 phase. Taken together, our results suggested that compound 11e and its derivatives were a class of novel structural potential BRD4 inhibitors and could serve as lead compounds for further exploration.

Discovery and evaluation of novel nitrodihydroimidazooxazoles as promising anti-tuberculosis agents.

New analogues of antitubercular drug Delamanid were prepared, seeking drug candidates with enhanced aqueous solubility and high efficacy. The strategy involved replacement of phenoxy linker proximal to the 2-nitroimidazooxazole of Delamanid by piperidine fused 5 or 6-membered ring heterocycles (ring A). The new compounds were all more hydrophilic than Delamanid, and several class of analogues showed remarkable activities against M. bovis. And among these series, the tetrahydro-naphthyridine-linked nitroimidazoles displayed excellent antimycobacterial activity against both replicating (MABA) and nonreplicating (LORA) M. tb H37Rv and low cytotoxicity. Compared to Delamanid, these new compounds (6, 7, 45) demonstrated dramatically improved physicochemical properties and are suitable for further in vitro and in vivo evaluation.

Metabolism of SKLB-TB1001, a Potent Antituberculosis Agent, in Animals.

Tuberculosis is a major global health problem, and the emergence of multidrug-resistant and extensively drug-resistant strains has increased the difficulty of treating this disease. Among the novel antituberculosis drugs in the pipeline, decaprenylphosphoryl-beta-d-ribose-2-epimerase (DprE1) inhibitors such as BTZ043 and pBTZ169 exhibited extraordinary antituberculosis potency. Here, the metabolites of the new DprE1 inhibitor SKLB-TB1001 in vivo and its inhibition of cytochrome P450 isoforms and plasma protein binding (PPB) in vitro were studied. The results showed that rapid transformation and high PPB resulted in inadequate exposure in vivo and thus led to the moderate potency of SKLB-TB1001 in vivo This study provided explanations for the discrepant potency of this scaffold in vivo and in vitro Meanwhile, it also provides a rationale for lead optimization of this very promising scaffold of antituberculosis agents to prevent them from being metabolized, thus improving their exposure in vivo.

Effect of C-type lectin 16 on dengue virus infection in Aedes aegypti salivary glands.

C-type lectins (CTLs) are a family of carbohydrate-binding proteins and an important component of mosquito saliva. Although CTLs play key roles in immune activation and viral pathogenesis, little is known about their role in regulating dengue virus (DENV) infection and transmission. In this study, we established a homozygous CTL16 knockout Aedes aegypti mutant line using CRISPR/Cas9 to study the interaction between CTL16 and viruses in mosquito vectors. Furthermore, mouse experiments were conducted to confirm the transmission of DENV by CTL16-/- A. aegypti mutants. We found that CTL16 was mainly expressed in the medial lobe of the salivary glands (SGs) in female A. aegypti. CTL16 knockout increased DENV replication and accumulation in the SGs of female A. aegypti, suggesting that CTL16 plays an important role in DENV transmission. We also found a reduced expression of immunodeficiency and Janus kinase/signal transducer and activator of transcription pathway components correlated with increased DENV viral titer, infection rate, and transmission efficiency in the CTL16 mutant strain. The findings of this study provide insights not only for guiding future investigations on the influence of CTLs on immune responses in mosquitoes but also for developing novel mutants that can be used as vector control tools.

Network pharmacology-based approach uncovers the pharmacodynamic components and mechanism of Fructus Tribuli for improving endothelial dysfunction in hypertension.

ETHNOPHARMACOLOGICAL RELEVANCE: Fructus Tribuli (FT), a traditional Chinese medicinal herbal, has been used for the clinical treatment of cardiovascular diseases for many years and affects vascular endothelial dysfunction (ED) in patients with hypertension. AIM OF THE STUDY: This study aimed to demonstrate the pharmacodynamic basis and mechanisms of FT for the treatment of ED. MATERIALS AND METHODS: The present study used ultra-high-performance liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) to analyze and identify the chemical components of FT. The active components in blood were determined after the oral administration of FT by comparative analysis to blank plasma. Then, based on the active components in vivo, network pharmacology was performed to predict the potential targets of FT in treating ED. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were also performed, and component-target-pathway networks were constructed. Interactions between the major active components and main targets were verified by molecular docking. Moreover, spontaneously hypertensive rats (SHRs) were divided into the normal, model, valsartan, low-dose FT, medium-dose FT, and high-dose FT experimental groups. In pharmacodynamic verification studies, treatment effects on blood pressure, serum markers (nitric oxide [NO], endothelin-1 [ET-1,], and angiotensin Ⅱ [Ang Ⅱ)]) of ED, and endothelial morphology of the thoracic aorta were evaluated and compared between groups. Finally, the PI3K/AKT/eNOS pathway was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot of the thoracic aorta of rats in each group to detect the mRNA expression of PI3K, AKT, and eNOS and the protein expression of PI3K, AKT, p-AKT, eNOS, and p-eNOS. RESULTS: A total of 51 chemical components were identified in FT, and 49 active components were identified in rat plasma. Thirteen major active components, 22 main targets, and the PI3K/AKT signaling pathway were screened by network pharmacology. The animal experiment results showed that FT reduced systolic blood pressure and ET-1 and Ang Ⅱ levels and increased NO levels in SHRs to varying degrees. The therapeutic effects were positively correlated with the oral dose of FT. Hematoxylin-eosin (HE) staining confirmed that FT could alleviate the pathological damage of the vascular endothelium. qRT-PCR and Western blot analysis confirmed that up-regulated expression of the PI3K/AKT/eNOS signaling pathway could improve ED. CONCLUSIONS: In this study, the material basis of FT was comprehensively identified, and the protective effect on ED was confirmed. FT had a treatment effect on ED through multi-component, multi-target, and multi-pathways. It also played a role by up-regulating the PI3K/AKT/eNOS signaling pathway.

Terrestrosin D, a spirostanol saponin from Tribulus terrestris L. with potential hepatorenal toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Fructus Tribuli (FT) has been commonly used as a traditional medicine for thousands of years. With the diverse uses of FT, more attention has been paid to its hepatorenal toxicity. However, the compounds causing the hepatorenal toxicity of FT remain undetermined. Terrestrosin D (TED), a major spirostanol saponin isolated from FT, may exert hepatorenal toxicity. AIM OF THE STUDY: This study aimed to evaluate the potential hepatorenal toxicity of TED, and preliminarily explore the possible mechanism of TED-induced hepatorenal toxicity. MATERIALS AND METHODS: Cytotoxicity assays, a repeated-dose 28-day in-vivo study, a toxicokinetic study, and a tissue distribution study were used to evaluate the potential hepatorenal toxicity of TED. Furthermore, network pharmacology was applied to preliminarily explore the possible mechanism of TED-induced hepatorenal toxicity. RESULTS: Both the in vitro and in vivo studies showed that the spirostanol saponin TED had potential hepatorenal toxicity. Nonetheless, hepatorenal toxicity induced by oral treatment with TED at a dosage range of 5 - 15 mg/kg daily for 28 consecutive days to Sprague-Dawley (SD) rats was reversible after 14 days of TED withdrawal. The toxicokinetic study demonstrated that the systematic exposure of SD rats to TED had an accumulation phenomenon and a dose-dependent trend after a 28-day repeated-dose oral administration. The tissue distribution study revealed that TED had a targeted distribution in the liver and kidneys accompanied by a phenomenon of accumulation in SD rats. Network pharmacology combined with molecular docking methods was used to screen for the key targets (HSP90AA1, CNR1, and DRD2) and the key pathways of TED-induced hepatorenal toxicity. CONCLUSIONS: The spirostanol saponin TED, a major spirostanol saponin isolated from FT, had potential hepatorenal toxicity.

Discovery of quinolone derivatives as antimycobacterial agents.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), is an important public health issue. Current first-line drugs administered to TB patients have been in use for over 40 years, whereas second-line drugs display strong side effects and poor compliance. Additionally, designing effective regimens to treat patients infected with multi- and extremely-drug-resistant (MDR and XDR) strains of TB is challenging. In this report, we screened our compound library and identified compound 1 with antituberculosis activity and a minimal inhibitory concentration (MIC) against M. tuberculosis of 20 µg mL-1. Structure optimization and the structure-activity relationship of 1 as the lead compound enabled the design and synthesis of a series of quinolone derivatives, 6a1-6a2, 6b1-6b36, 6c1, 6d1-6d14, 7a1-7a2, 7b1-7b2, 7c1, 8a1-8a5, 9a1-9a4 and 10a1-10a6. These compounds were evaluated in vitro for anti-tubercular activity against the M. tuberculosis H37Rv strain. Among them, compounds 6b6, 6b12 and 6b21 exhibited MIC values in the range of 1.2-3 µg mL-1 and showed excellent activity against the tested MDR-TB strain (MIC: 3, 2.9 and 0.9 µg mL-1, respectively). All three compounds were non-toxic toward A549 and Vero cells (>100 and >50 µg mL-1, respectively). In addition, an antibacterial spectrum test carried out using compound 6b21 showed that this compound specifically inhibits M. tuberculosis. These can serve as a new starting point for the development of anti-TB agents with therapeutic potential.

Generating mutant Aedes aegypti mosquitoes using the CRISPR/Cas9 system.

Implementation of CRISPR/Cas9 methodologies for mosquito gene editing has not yet become widespread. This protocol details the procedure for Aedes aegypti mosquito gene editing using homology-directed repair and fluorescent marker insertion, which facilitates the generation and screening of mutant mosquito lines for gene function testing. We describe optimized methods for single guide RNA plasmid preparation, homologous recombination donor plasmid construction, embryo microinjection, and precise gene knock-in confirmation. We also provide general guidance for establishing mutant mosquito lines. For details on the practical use and execution of this protocol, please refer to Li et al. (2020).

Transgenic Expression of Human C-Type Lectin Protein CLEC18A Reduces Dengue Virus Type 2 Infectivity in Aedes aegypti.

The C-type lectins, one family of lectins featuring carbohydrate binding domains which participate in a variety of bioprocesses in both humans and mosquitoes, including immune response, are known to target DENV. A human C-type lectin protein CLEC18A in particular shows extensive glycan binding abilities and correlates with type-I interferon expression, making CLEC18A a potential player in innate immune responses to DENV infection; this potential may provide additional regulatory point in improving mosquito immunity. Here, we established for the first time a transgenic Aedes aegypti line that expresses human CLEC18A. This expression enhanced the Toll immune pathway responses to DENV infection. Furthermore, viral genome and virus titers were reduced by 70% in the midgut of transgenic mosquitoes. We found significant changes in the composition of the midgut microbiome in CLEC18A expressing mosquitoes, which may result from the Toll pathway enhancement and contribute to DENV inhibition. Transgenic mosquito lines offer a compelling option for studying DENV pathogenesis, and our analyses indicate that modifying the mosquito immune system via expression of a human immune gene can significantly reduce DENV infection.

Identification of a new series of benzothiazinone derivatives with excellent antitubercular activity and improved pharmacokinetic profiles.

Nitrobenzothiazinone (BTZ) is a promising scaffold with potent activity against M. tuberculosis by inhibiting decaprenylphosphoryl-beta-d-ribose 2'-oxidase (DprE1). But unfavorable durability poses a challenge to further development of this class of agents. Herein, a series of BTZs bearing a variety of different substituents at the C-2 position were designed and synthesized. Compounds were screened for their antimycobacterial activity against Mycobacterium tuberculosis H37Ra and were profiled for metabolic stability, plasma protein-binding capacity and pharmacokinetics in vivo. In general, these new BTZs containing N-piperazine, N-piperidine or N-piperidone moiety have excellent antitubercular activity and low cytotoxicity. Several of the compounds showed improved microsomal stability and lower plasma protein-binding, opening a new direction for further lead optimization. And we obtained compound 3o, which maintained good anti-tuberculosis activity (MIC = 8 nM) and presented better in vitro ADME/T and in vivo pharmacokinetic profiles than reported BTZ compound PBTZ169, which may serve as a candidate for the treatment of tuberculosis.