ORCID Profile
0000-0002-3071-6531
Current Organisation
The Chinese University of Hong Kong Faculty of Medicine
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Publisher: Elsevier BV
Date: 11-2019
DOI: 10.1016/J.BIOORG.2019.103203
Abstract: 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.
Publisher: Wiley
Date: 31-07-2014
DOI: 10.1111/MMI.12723
Abstract: Acinetobacter species are widely distributed bacteria in the environment, and have recently gained notoriety as opportunistic nosocomial pathogens. Here we characterize a novel RNA polymerase-interacting protein named acidic transcription factor A, AtfA. It is small and highly acidic, and is widely distributed throughout the γ proteobacteria, including other significant pathogens in the genera Moraxella, Pseudomonas, Legionella and Vibrio. In the model species A. baylyi ADP1, deletion of atfA significantly affects expression of over 500 genes, resulting in a large cell phenotype, reduced cell fitness, impaired biofilm formation and twitching motility, and increased sensitivity to antibiotics. Deletion of atfA also causes dramatically enhanced sensitivity to ethanol, which is an important growth promoter and virulence factor in Acinetobacter spp. The results suggest that auxiliary factors of RNA polymerase with important biological roles remain to be discovered.
Publisher: Elsevier BV
Date: 03-2016
DOI: 10.1016/J.BMC.2016.01.040
Abstract: The search for small molecules capable of inhibiting transcription initiation in bacteria has resulted in the synthesis of N,N'-disubstituted hydrazines and imine-carbohydrazides comprised of indole, pyridine, pyrrole, furan and thiophene using the respective trichloroacetyl derivatives, carbohydrazides and aldehydes. Replacement of the indole moiety by smaller heterocycles linked by CONHNC linkers afforded a broad variety of compounds efficiently targeting the RNA polymerase-σ(70)/σ(A) interaction as determined by ELISA and exhibiting increased inhibition of the growth of Escherichia coli compared to Bacillus subtilis in culture. The structural features of the synthesized transcription initiation inhibitors needed for antibacterial activity were identified employing molecular modelling and structure-activity relationship (SAR) studies.
Publisher: Elsevier BV
Date: 09-2017
DOI: 10.1016/J.BMCL.2017.08.036
Abstract: Knoevenagel condensation was employed to generate a set of molecules potentially capable of inhibiting the RNA polymerase-σ
Publisher: MDPI AG
Date: 07-04-2017
Publisher: MyJove Corporation
Date: 20-09-2016
DOI: 10.3791/54256
Publisher: Informa UK Limited
Date: 16-01-2020
Publisher: Elsevier BV
Date: 11-2019
DOI: 10.1016/J.EJMECH.2019.111635
Abstract: The discovery of novel potent neuraminidase (NA) inhibitors remains an attractive approach for treating infectious diseases caused by influenza. In this study, we describe the design and synthesis of novel N-substituted oseltamivir derivatives for probing the 150-cavity which is nascent to the activity site of NA. NA inhibitory studies showed that new derivatives demonstrated the inhibitory activity with IC
Publisher: American Chemical Society (ACS)
Date: 05-07-2013
DOI: 10.1021/CB400231P
Abstract: Antibiotic resistance is a growing global problem, with very few new compounds in development. Bacterial transcription is an underutilized target for antibiotics, which has been attributed to the similarity of the active site of RNA polymerases (RNAPs) across all domains of life and the ease with which resistance can arise through point mutation at multiple sites within this conserved region. In this study we have taken a rational approach to design a novel set of compounds that specifically target the formation of transcription initiation complexes by preventing the unique bacterial σ initiation factor from binding to RNAP. We have identified the region of RNAP to which these compounds bind and demonstrate that one compound, GKL003, has an inhibition constant in the low nanomolar range. This compound has activity against both Gram-positive and -negative organisms, including a community acquired methicillin-resistant strain of the major pathogen Staphylococcus aureus.
Publisher: CSIRO Publishing
Date: 2005
DOI: 10.1071/FP04224
Abstract: Correlative physiological evidence suggests that membrane transport into storage parenchyma cells is a key step in determining hexose levels accumulated in tomato (Lycopersicon esculentum Mill.) fruit (Ruan et al. 1997). Expression of three previously identified hexose transporter genes (LeHT1, 2 and 3) demonstrated that LeHT3, and to a lesser extent LeHT1, are the predominant transporters expressed in young fruit (10 d after anthesis DAA). Expression of both transporters dropped sharply until 24 DAA, after which only LeHT3 expression remained at detectable levels through to fruit ripening. LeHT2 was not expressed substantially until the onset of fruit ripening. For fruit at both 10 and 30 DAA, LeHT3 transcripts were detected in storage parenchyma cells of the outer pericarp tissue, but not in vascular bundles or the first layer of parenchyma cells surrounding these bundles. In contrast to LeHT gene expression, hexose transporter protein levels were maximal between 20 and 30 DAA, which corresponded to the period of highest hexose accumulation. The delayed appearance of transporter protein is consistent with some form of post-transcriptional regulation. Based on these analyses, LeHT3 appears to be responsible for the rapid hexose accumulation in developing tomato fruit.
Publisher: Elsevier BV
Date: 2008
DOI: 10.1016/J.PLASMID.2007.11.001
Abstract: Tandem affinity purification has become a valuable tool for the isolation of protein complexes. Here we describe the construction and use of a series of plasmid vectors for Gram positive bacteria. The vectors utilize the SPA tag as well as variants containing a 3C rather than the TEV protease site as 3C protease has been shown to work efficiently at the low temperatures (4 degrees C) used to isolate protein complexes. In addition, a further vector incorporates a GST moiety in place of the 3xFLAG of the SPA tag which provides an additional tagging option for situations where SPA binding may be inefficient. The vectors are all compatible with previously constructed fluorescent protein fusion vectors enabling construction of a suite of affinity and fluorescently tagged genes using a single PCR product.
Publisher: American Chemical Society (ACS)
Date: 05-03-2019
DOI: 10.1021/ACSINFECDIS.9B00020
Abstract: 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.
Publisher: EMBO
Date: 14-08-2009
Publisher: American Society for Microbiology
Date: 03-2016
Abstract: Transcription, the first step of gene expression, is carried out by the enzyme RNA polymerase (RNAP) and is regulated through interaction with a series of protein transcription factors. RNAP and its associated transcription factors are highly conserved across the bacterial domain and represent excellent targets for broad-spectrum antibacterial agent discovery. Despite the numerous antibiotics on the market, there are only two series currently approved that target transcription. The determination of the three-dimensional structures of RNAP and transcription complexes at high resolution over the last 15 years has led to renewed interest in targeting this essential process for antibiotic development by utilizing rational structure-based approaches. In this review, we describe the inhibition of the bacterial transcription process with respect to structural studies of RNAP, highlight recent progress toward the discovery of novel transcription inhibitors, and suggest additional potential antibacterial targets for rational drug design.
Publisher: American Chemical Society (ACS)
Date: 05-10-2016
DOI: 10.1021/ACSINFECDIS.5B00058
Abstract: Bacterial transcription is a proven target for antibacterial research. However, most of the known inhibitors targeting transcription are from natural extracts or are hits from screens where the binding site remains unidentified. Using an RNA polymerase holoenzyme homology structure from the model Gram-positive organism Bacillus subtilis, we created a pharmacophore model and used it for in silico screening of a publicly available library for compounds able to inhibit holoenzyme formation. The hits demonstrated specific affinity to bacterial RNA polymerase and excellent activity using in vitro assays and showed no binding to the equivalent structure from human RNA polymerase II. The target specificity in live cells and antibacterial activity was demonstrated in microscopy and growth inhibition experiments. This is the first ex le of targeted inhibitor development for a bacterial RNA polymerase, outlining a complete discovery process from virtual screening to biochemical validation. This approach could serve as an appropriate platform for the future identification of inhibitors of bacterial transcription.
Publisher: Elsevier BV
Date: 10-2019
DOI: 10.1016/J.LFS.2019.116818
Abstract: Considering the potential oral administration sequences and role of microbiota for metformin (MET) and berberine (BBR) during anti-diabetic treatments, the current study aimed to investigate the pharmacokinetic interactions between MET and BBR in rats after oral administration at different sequences and impacts of microbiota on such interactions. Sprague-Dawley rats were ided into five groups as per what was orally administered to them: MET (G1)/BBR (G2) at 200 mg/kg, BBR 2-hour (h) after dosing MET (G3), MET 2-h after dosing BBR (G4) or MET with BBR at the same time (G5) followed by monitoring their pharmacokinetic profiles. Further in vitro incubations mimicking the above five treatments in rat intestinal content (G1R-G5R), human fecalase (G1H-G5H) and selected bacteria (G1B-G5B) were conducted for both MET and BBR (10 μg/ml for G1R/H-G5R/H and 50 μM for G1B-G5B) up to 24-h. Concentrations of MET and BBR were analyzed by LC/MS/MS. Although BBR was barely measurable in vivo, it significantly increased systemic exposure of MET in G3/G4. Consistent with pharmacokinetic findings, sequential in vitro incubations of MET and BBR in both rat intestinal content and human fecalase demonstrated significant increase on MET persisted after 24-h incubation in G3R/H & G4R/H. Moreover, post-dose (G3B) and pre-dose (G4B) of BBR decreased the MET degradation significantly in most selected bacteria. Our finding for the first time demonstrated the significant effect of sequential co-administration of BBR and MET on their pharmacokinetic interactions, which could be related to their microbiota mediated metabolisms in gastrointestinal tract (GI).
Publisher: Elsevier BV
Date: 09-2014
DOI: 10.1016/J.PLASMID.2014.06.002
Abstract: We describe a dual vector-based system for overproduction of recombinant Escherichia coli RNA polymerase (RNAP). A cleavable deca-histidine tag (His10) was incorporated into the C-terminus of the β' subunit to facilitate protein purification. Unique restriction sites were introduced into the genes encoding the β and β' subunits (rpoB and rpoC, respectively), facilitating mutation of functionally significant subunit fragments through insertion of modified PCR fragments into the appropriate vector. RNAP with an R275A substitution in the β' subunit, which is essential for interaction with transcription initiation factor σ, was generated and exhibited reduced activity compared to native recombinant RNAP.
Publisher: Informa UK Limited
Date: 09-2010
Publisher: Elsevier BV
Date: 04-2015
DOI: 10.1016/J.BMC.2015.02.037
Abstract: Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of several classes of mono-indole and mono-benzofuran inhibitors that targeted the essential protein-protein interaction between RNA polymerase core and σ(70)/σ(A) factors in bacteria. In this study, the reaction of indole-2-, indole-3-, indole-7- and benzofuran-2-glyoxyloyl chlorides with amines and hydrazines afforded a variety of glyoxyloylamides and glyoxyloylhydrazides. Similarly, condensation of 2- and 7-trichloroacetylindoles with amines and hydrazines delivered amides and hydrazides. The novel molecules were found to inhibit the RNA polymerase-σ(70)/σ(A) interaction as measured by ELISA, and also inhibited the growth of both Gram-positive and Gram-negative bacteria in culture. Structure-activity relationship (SAR) studies of the mono-indole and mono-benzofuran inhibitors suggested that the hydrophilic-hydrophobic balance is an important determinant of biological activity.
Publisher: Elsevier BV
Date: 12-2020
Publisher: American Society for Microbiology
Date: 04-08-2014
DOI: 10.1128/JB.02020-14
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C4OB00460D
Abstract: The synthesis of novel bis-indole amides and glyoxylamides as bacterial transcription complex formation inhibitors and their structure–activity relationships are discussed.
Publisher: Oxford University Press (OUP)
Date: 17-02-2015
DOI: 10.1093/NAR/GKV108
Publisher: American Chemical Society (ACS)
Date: 07-07-2020
Publisher: American Chemical Society (ACS)
Date: 28-08-2017
DOI: 10.1021/ACS.BIOCHEM.7B00349
Abstract: We report the discovery of the first bacterial ribosomal RNA (rRNA) synthesis inhibitor that has specific antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). A pharmacophore model was constructed on the basis of the protein-protein interaction between essential bacterial rRNA transcription factors NusB and NusE and employed for an in silico screen to identify potential leads. One compound, (E)-2-{[(3-ethynylphenyl)imino]methyl}-4-nitrophenol (MC4), demonstrated antimicrobial activity against a panel of S. aureus strains, including MRSA, without significant toxicity to mammalian cells. MC4 resulted in a decrease in the rRNA level in bacteria, and the target specificity of MC4 was confirmed at the molecular level. Results obtained from this work validated the bacterial rRNA transcription machinery as a novel antimicrobial target. This approach may be extended to other factors in rRNA transcription, and MC4 could be applied as a chemical probe to dissect the relationship among MRSA infection, MRSA growth rate, and rRNA synthesis, in addition to its therapeutic potential.
Publisher: Elsevier BV
Date: 09-2015
DOI: 10.1016/J.YMETH.2015.05.005
Abstract: Very few clinically available antibiotics target bacterial RNA polymerase (RNAP) suggesting it is an underutilized target. The advent of detailed structural information of RNAP holoenzyme (HE) has allowed the design and in silico screening of novel transcription inhibitors. Here, we describe our approach for the design and testing of small molecule transcription inhibitors that work by preventing the interaction between the essential transcription initiation factor σ and RNAP. With the appropriate structural information this approach can be easily modified to other essential protein-protein interactions.
Publisher: Elsevier BV
Date: 09-2019
DOI: 10.1016/J.EJMECH.2019.05.090
Abstract: 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.
Publisher: Elsevier BV
Date: 05-2008
DOI: 10.1016/J.PEP.2008.01.006
Abstract: We describe a vector-based system for the production of recombinant Bacillus subtilis RNA polymerase. The recombinant enzyme is C-terminally tagged with nine consecutive histidine residues resulting in about 90% pure enzyme in a single nickel-affinity purification step. The vectors permitted production of recombinant enzyme lacking an omega subunit or containing either the omega(1) (YkzG) or omega(2) (YloH) subunits. In transcription time-course assays all of the recombinant enzymes exhibited identical activity to native RNAP. The modular assembly of the artificial RNA polymerase operon permits ready mutation of any subunit and incorporation into the recombinant enzyme, which will enable new functional/structural studies with this enzyme.
Publisher: MDPI AG
Date: 09-08-2019
DOI: 10.3390/MOLECULES24162902
Abstract: Novel antimicrobial classes are in desperate need for clinical management of infections caused by increasingly prevalent multi-drug resistant pathogens. The protein-protein interaction between bacterial RNA polymerase (RNAP) and the housekeeping sigma initiation factor is essential to transcription and bacterial viability. It also presents a potential target for antimicrobial discovery, for which a hit compound (C3) was previously identified from a pharmacophore model-based in silico screen. In this study, the hit compound was experimentally assessed with some rationally designed derivatives for the antimicrobial activities, in particular against Streptococcus pneumoniae and other pathogens. One compound, C3-005, shows dramatically improved activity against pneumococci compared to C3. C3-005 also attenuates S. pneumoniae toxin production more strongly than existing classes of antibiotics tested. Here we demonstrate a newly validated antimicrobial agent to address an overlooked target in the hit-to-lead process, which may pave the way for further antimicrobial development.
Publisher: Elsevier BV
Date: 08-2015
DOI: 10.1016/J.EJMECH.2015.06.021
Abstract: Nitric oxide (NO) and its auto-oxidation products are known to disrupt normal bacterial function and NO releasing molecules have the potential to be developed as antibacterial leads in drug discovery. We have designed and synthesized a series of novel nitrated compounds by combining NO releasing groups with ocotillol-type triterpenoids, which have previously demonstrated activity only against Gram-positive bacteria. The in vitro NO release capacity and antibacterial activity were sequentially evaluated and the data showed that most of the synthesized compounds could release nitric oxide. Compound 16a, 17a and 17c, with nitrated aliphatic esters at C-3 position, displayed higher NO release than other analogues, correlating to their good antibacterial activity, in which 17c demonstrated broad-spectrum activity against both Gram positive and -negative bacteria, as well as excellent synergism at sub-minimum inhibitory concentration when using with kanamycin and chlor henicol. Furthermore, the epifluorescent microscopic study indicated that the ocotillol-type triterpenoid core may induce NO release on the bacterial membrane. Our results demonstrate that nitrated substitutions at C-3 of ocotillol-type derivatives could provide an approach to expand their antibacterial spectrum, and that ocotillol-type triterpenoids may also be developed as appropriate carriers for NO donors in antibacterial agent discovery with low cytotoxicity.
Publisher: MDPI AG
Date: 11-08-2020
DOI: 10.3390/IJMS21165772
Abstract: 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.
Location: Hong Kong
No related grants have been discovered for Xiao Yang.