Synthesis and biological evaluation of nusbiarylin derivatives as bacterial rRNA synthesis inhibitor with potent antimicrobial activity against MRSA and VRSA.

Bacterial transcription is a valid but underutilized target for antimicrobial agent discovery because of its function of bacterial RNA synthesis. Bacterial transcription factors NusB and NusE form a transcription complex with RNA polymerase for bacterial ribosomal RNA synthesis. We previously identified a series of diarylimine and -amine inhibitors capable of inhibiting the interaction between NusB and NusE and exhibiting good antimicrobial activity. To further explore the structural viability of these inhibitors, coined "nusbiarylins", 36 new derivatives containing diverse substituents at the left benzene ring of inhibitors were synthesized based upon isosteric replacement and the structure-activity relationship concluded from earlier studies. Some of the derivatives displayed good to excellent antibacterial efficacy towards a panel of clinically significant pathogens including methicillin-resistance Staphylococcus aureus (MRSA) and vancomycin-resistance S. aureus (VRSA). In particular, compound 22r exhibited the best antimicrobial activity with a minimum inhibitory concentration (MIC) of 0.5 µg/mL. Diverse mechanistic studies validated the capability of 22r inhibiting the function of NusB protein and bacterial rRNA synthesis. In silico study of drug-like properties also provided promising results. Overall, this series of derivatives showed potential antimicrobial activity and drug-likeness and provided guidance for further optimization.

Nusbiarylins Inhibit Transcription and Target Virulence Factors in Bacterial Pathogen Staphylococcus aureus.

The emergence of multidrug resistance in the clinically significant pathogen Staphylococcus aureus is a global health burden, compounded by a diminishing drug development pipeline, and a lack of approved novel antimicrobials. Our previously reported first-in-class bacterial transcription inhibitors "nusbiarylins" presented a promising prospect towards the discovery of novel antimicrobial agents with a novel mechanism. Here we investigated and characterised the lead nusbiarylin compound, MC4, and several of its chemical derivatives in both methicillin-resistant S. aureus (MRSA) and the S. aureus type strains, demonstrating their capacity for the arrest of growth and cellular respiration, impairment of RNA and intracellular protein levels at subinhibitory concentrations. In some instances, derivatives of MC4 were also shown to attenuate the production of staphylococcal virulence factors in vitro, such as the exoproteins α-toxin and Panton-Valentine Leukocidin (PVL). Trends observed from quantitative PCR assays suggested that nusbiarylins elicited these effects possibly by acting via but not limited to the modulation of global regulatory pathways, such as the agr regulon, which coordinates the expression of S. aureus genes associated with virulence. Our findings encourage the continued development of more potent compounds within this novel family of bacterial transcription inhibitors.

Nusbiarylins, a new class of antimicrobial agents: Rational design of bacterial transcription inhibitors targeting the interaction between the NusB and NusE proteins.

Discovery of antibiotics of a novel mode of action is highly required in the fierce battlefield with multi-drug resistant bacterial infections. Previously we have validated the protein-protein interaction between bacterial NusB and NusE proteins as an unprecedented antimicrobial target and reported the identification of a first-in-class inhibitor of bacterial ribosomal RNA synthesis with antimicrobial activities. In this paper, derivatives of the hit compound were rationally designed based on the pharmacophore model for chemical synthesis, followed by biological evaluations. Some of the derivatives demonstrated the improved antimicrobial activity with the minimum inhibitory concentration (MIC) at 1-2 µg/mL against clinically significant bacterial pathogens. Time-kill kinetics, confocal microscope, ATP production, cytotoxicity, hemolytic property and cell permeability using Caco-2 cells of a representative compound were also measured. This series of compounds were named "nusbiarylins" based on their target protein NusB and the biaryl structure and were expected to be further developed towards novel antimicrobial drug candidates in the near future.

Novel anticancer oridonin derivatives possessing a diazen-1-ium-1,2-diolate nitric oxide donor moiety: Design, synthesis, biological evaluation and nitric oxide release studies.

Oridonin (1) is a complex ent-kaurane diterpenoid with unique antitumor profile, which is isolated from Isodon rubescens. In order to develop novel derivatives of oridonin with improved potency, a series of nitric oxide (NO)-releasing oridonin derivatives were synthesized by coupling diazeniumdiolates with oridonin and its semisynthesized analogues. Their in vitro antiproliferative activity, nitric oxide release ability, and preliminary anticancer mechanism were further evaluated. The results displayed that all the target compounds exhibited potent antiproliferative activities, with IC50 values ranging from 1.84 to 17.01µM. Besides, it was observed that in most cases, the antiproliferative activity correlated well with levels of intracellular NO release. More interestingly, preliminary mechanism studies revealed that the most potent compound 14d induced apoptosis and arrested the cell cycle at the S phase in Bel-7402 cells, which is different from parent compound oridonin. Together, the above promising results warrant the further development of oridonin/NO hybrids as potential antitumor leads.

Discovery of Novel Polycyclic Heterocyclic Derivatives from Evodiamine for the Potential Treatment of Triple-Negative Breast Cancer.

Evodiamine (Evo) is a quinazolinocarboline alkaloid found in Evodia rutaecarpa and exhibits moderate antiproliferative activity. Herein, we report using a scaffold-hopping approach to identify a series of novel polycyclic heterocyclic derivatives based on Evo as the topoisomerase I (Top1) inhibitor for the treatment of triple-negative breast cancer (TNBC), which is an aggressive subtype of breast cancer with limited treatment options. The most potent compound 7f inhibited cell growth in a human breast carcinoma cell line (MDA-MB-231) with an IC50 value of 0.36 µM. Further studies revealed that Top1 was the target of 7f, which directly induced irreversible Top1-DNA covalent complex formation or induced an oxidative DNA lesion through an indirect mechanism mediated by reactive oxygen species. More importantly, in vivo studies showed that 7f exhibited potent antitumor activity in a TNBC-patient-derived tumor xenograft model. These results suggest that compound 7f deserves further investigation as a promising candidate for the treatment of TNBC.

HPLC, quantitative NMR and HRMS spectroscopic data of nusbiarylins as a new class of antimicrobial agents.

Bacterial transcription is a valid but underutilized target for antimicrobial agent discovery [1]. Nusbiarylins are the first-in-class bacterial ribosomal RNA synthesis inhibitors that possess potent activity against various types of multidrug-resistant bacteria with a novel mode of action by targeting the interaction of bacterial transcription factors NusB and NusE [2]. To facilitate the characterization of nusbiarylin derivatives produced by other researchers, high-performance liquid chromatography (HPLC) profiles, quantitative nuclear magnetic resonance (qNMR) and high-resolution mass spectrometry (HRMS) spectroscopic data were presented for the quick determination of purity and characterization of 95 nusbiarylin compounds. The data presented in this article supplement the 1H and 13C NMR data provided previously [3,4], and assist the reproduction of nusbiarylins for chemical, biological and drug discovery research.

Simple Method for Studying in Vitro Protein-Protein Interactions Based on Protein Complementation and Its Application in Drug Screening Targeting Bacterial Transcription.

Protein-protein interactions (PPIs) underpin essential cellular processes of all organisms and are increasingly considered as drug targets. A number of techniques have been established to study PPIs; however, development of a simple and cost-effective method for in vitro high throughput screening of PPI inhibitors is still in demand or desirable. We report herein a simple method based on protein complementation for the in vitro study of PPIs, as well as screening of inhibitors against the PPI of interest. We have validated this system utilizing bacterial transcription factors NusB and NusE. Three derivatives of an inhibitor targeting the NusB-NusE interaction were synthesized and characterized with the system, which showed specific inhibition and antimicrobial activities. We have further confirmed the system with the RNA polymerase-σ interaction and an inhibitor. This system is expected to be suitable for more extensive high throughput screening of large chemical libraries. Additionally, our vector system can be easily adapted to study other PPI pairs, followed by inhibitor screening for hit identification in the application of early stage drug discovery.

Design, synthesis and biological evaluation of antimicrobial diarylimine and -amine compounds targeting the interaction between the bacterial NusB and NusE proteins.

Discovery of antimicrobial agents with a novel model of action is in urgent need for the clinical management of multidrug-resistant bacterial infections. Recently, we reported the identification of a first-in-class bacterial ribosomal RNA synthesis inhibitor, which interrupted the interaction between the bacterial transcription factor NusB and NusE. In this study, a series of diaryl derivatives were rationally designed and synthesized based on the previously established pharmacophore model. Inhibitory activity against the NusB-NusE binding, circular dichroism of compound treated NusB, antimicrobial activity, cytotoxicity, hemolytic property and cell permeability using Caco-2 cells were measured. Structure-activity relationship and quantitative structure-activity relationship were also concluded and discussed. Some of the derivatives demonstrated improved antimicrobial activity than the hit compound against a panel of clinically important pathogens, lowering the minimum inhibition concentration to 1-2 µg/mL against Staphylococcus aureus, including clinical strains of methicillin-resistant Staphylococcus aureus at a level comparable to some of the marketed antibiotics. Given the improved antimicrobial activity, specific inhibition of target protein-protein interaction and promising pharmacokinetic properties without significant cytotoxicity, this series of diaryl compounds have high potentials and deserve for further studies towards a new class of antimicrobial agents in the future.

Design, synthesis, and biological evaluation of NAD(P)H: Quinone oxidoreductase (NQO1)-targeted oridonin prodrugs possessing indolequinone moiety for hypoxia-selective activation.

The enzyme NQO1 is a potential target for selective cancer therapy due to its overexpression in certain hypoxic tumors. A series of prodrugs possessing a variety of cytotoxic diterpenoids (oridonin and its analogues) as the leaving groups activated by NQO1 were synthesized by functionalization of 3-(hydroxymethyl)indolequinone, which is a good substrate of NQO1. The target compounds (29a-m) exhibited relatively higher antiproliferative activities against NQO1-rich human colon carcinoma cells (HT-29) and human lung carcinoma (A549) cells (IC50 = 0.263-2.904 µM), while NQO1-defficient lung adenosquamous carcinoma cells (H596) were less sensitive to these compounds, among which, compound 29h exhibited the most potent antiproliferative activity against both A549 and HT-29 cells, with IC50 values of 0.386 and 0.263 µM, respectively. Further HPLC and docking studies demonstrated that 29h is a good substrate of NQO1. Moreover, the investigation of anticancer mechanism showed that the representative compound 29h affected cell cycle and induced NQO1 dependent apoptosis through an oxidative stress triggered mitochondria-related pathway in A549 cells. Besides, the antitumor activity of 29h was also verified in a liver cancer xenograft mouse model. Biological evaluation of these compounds concludes that there is a strong correlation between NQO1 enzyme and induction of cancer cell death. Thus, this suggests that some of the target compounds activated by NQO1 are novel prodrug candidates potential for selective anticancer therapy.

A Novel Potent Anticancer Compound Optimized from a Natural Oridonin Scaffold Induces Apoptosis and Cell Cycle Arrest through the Mitochondrial Pathway.

The cytotoxicity of the natural ent-kaurene diterpenoid, oridonin, has been extensively studied. However, the application of oridonin for cancer therapy was hampered primarily by its moderate potency. In this study, a series of oridonin A-ring modified analogues, and their derivatives bearing various substituents on 14-OH position, were designed, synthesized, and evaluated for anticancer efficacy. Some of the derivatives were significantly more potent than oridonin against both drug-sensitive and drug-resistant cancer cells. The most potent compound, 13p, was 200-fold more efficacious than oridonin in MCF-7 cancer cells. Furthermore, 13p induced apoptosis and cell cycle arrest at the G2/M phase. A decrease in mitochondrial membrane potential and an increase in Bax/Bcl-2 ratio, accompanied by activated caspase-3 cleavage, were observed in MCF-7 cells after treatment with 13p, suggesting that the mitochondrial pathway was involved in the 13p-mediated apoptosis. Moreover, 13p significantly inhibited tumor growth in mouse xenograft models and had no observable toxic effect.