Synthesis and Biological Evaluation of a Novel C8-Pyrrolobenzodiazepine (PBD) Adenosine Conjugate. A Study on the Role of the PBD Ring in the Biological Activity of PBD-Conjugates

Here we sought to evaluate the contribution of the PBD unit to the biological activity of PBD-conjugates and, to this end, an adenosine nucleoside was attached to the PBD A-ring C8 position. A convergent approach was successfully adopted for the synthesis of a novel C8-linked pyrrolo(2,1-c)(1,4)benzodiazepine(PBD)-adenosine(ADN) hybrid. The PBD and adenosine (ADN) moieties were synthesized separately and then linked through a pentynyl linker. To our knowledge, this is the first report of a PBD connected to a nucleoside. Surprisingly, the compound showed no cytotoxicity against murine cells and was inactive against Mycobacterium aurum and M. bovis strains and did not bind to guanine-containing DNA sequences, as shown by DNase I footprinting experiments. Molecular dynamics simulations revealed that the PBD–ADN conjugate was poorly accommodated in the DNA minor groove of two DNA sequences containing the AGA-PBD binding motif, with the adenosine moiety of the ligand preventing the covalent binding of the PBD unit to the guanine amino group of the DNA duplex. These interesting findings shed further light on the ability of the substituents attached at the C8 position of PBDs to a ect and modulate the biological and biophysical properties of PBD hybrids

The Prospect of Repurposing Immunomodulatory Drugs for Adjunctive Chemotherapy against Tuberculosis: A Critical Review.

Tuberculosis (TB) remains a global health emergency, with an estimated 2 billion people infected across the world, and 1.4 million people dying to this disease every year. Many aspects of the causative agent, Mycobacterium tuberculosis, make this disease difficult for healthcare and laboratory researchers to fight against, such as unique pathophysiology, latent infection and long and complex treatment regimens, thus causing patient non-compliance with the treatment. Development of new drugs is critical for tackling these problems. Repurposing drugs is a promising strategy for generating an effective drug treatment whilst circumventing many of the challenges of conventional drug development. In this regard, the incorporation of immunomodulatory drugs into the standard regimen to potentiate frontline drugs is found to be highly appealing. Drugs of diverse chemical classes and drug categories are increasingly being evidenced to possess antitubercular activity, both in vitro and in vivo. This article explores and discusses the molecular entities that have shown promise in being repurposed for use in anti-TB adjunctive therapy and aims to provide the most up-to-date picture of their progress

Immunobiology of tubercle bacilli and prospects of immunomodulatory drugs to tackle tuberculosis (TB) and other non-tubercular mycobacterial infections

The COVID-19 pandemic has set back progress made on antimicrobial resistance (AMR). Without urgent re-focus, we risk slowing down drug discovery and providing treatment for drug resistant Mycobacterium tuberculosis. Unique in its immune evasion, dormancy and resuscitation, the causal pathogens of tuberculosis (TB) have demonstrated resistance to antibiotics with efflux pumps and the ability to form biofilms. Repurposing drugs is a prospective avenue for finding new anti-TB drugs. There are many advantages to discovering novel targets of an existing drug, as the pharmacokinetic and pharmacodynamic properties have already been established, they are cost-efficient and can be commercially accelerated for the new development. One such group of drugs are non-steroidal anti-inflammatory drugs (NSAIDs) that are originally known for their ability to supress the host proinflammatory responses. In addition to their anti-inflammatory properties, some NSAIDs have been discovered to have antimicrobial modes of action. Of particular interest is Carprofen, identified to inhibit the efflux mechanism and disrupt biofilm formation in mycobacteria. Due to the complexities of host-pathogens interactions in the lung microbiome, inflammatory responses must carefully be controlled alongside the in vivo actions of the prospective anti-infectives. This critical review explores the potential dual role of a selection of NSAIDs, as an anti-inflammatory and anti-tubercular adjunct to reverse the tide of antimicrobial resistance in existing treatments

Mutation studies of the Gene Encoding YuiC, a Stationary Phase Survival Protein in Bacillus subtilis

Aims: YuiC is a stationary phase survival (Sps) protein from the Firmicute Bacillus subtilis that possesses muralytic activity to cleave bacterial cell-wall peptidoglycan. It has a small lytic transglycosylase (MltA) fold analogous to the resuscitation promoting factors (Rpfs) of Actinobacteria which have a hybrid of a mini lysozyme and soluble lytic transglycosylase (Slt35/70) fold. The present study aimed at identifying key residues of YuiC/Sps that are catalytically active and studying the effect of B. subtilis cell growth upon sps/yuiC deletion. Methodology & Results: Four forms of mutated yuiC were created through Site-directed, Ligase-Independent Mutagenesis Polymerase Chain Reaction (SLIM PCR) that include the substitutions of D129A, D151A, D162A and K102A. These individual mutated yuiC genes were cloned and expressed in the Escherichia coli BL21 (DE3) expression system and subsequently purified to homogeneity using affinity, cation exchange and size exclusion chromatography. The D129A variant was shown to be insoluble, indicating its role in maintaining the right protein folding of YuiC. The remaining three variants resulted in soluble proteins but were inactive on zymograms indicating that they may be responsible for catalysis. B. subtilis cells harbouring individual sps genes (yuiC, yabE, yocH and yorM) knocked out showed stationary phase defects and altered colony morphologies compared to the wild type. Conclusion: This study has identified the key residues involved in catalysis of YuiC, which are the D151, D162 and K102. These are conserved in Sps domains. The catalytic mechanism of YuiC is similar to the mechanism reported for Neisseria gonorrhoeae MltA. sps/yuiC knock outs have implied that each sps/yuiC has a significant role on B. subtilis late growth stage. Significance and Impact of study: The B. subtilis YuiC/Sps model has given an insight into Sps functions in the final growth stage of the Firmicutes, which members include etiologic agents of anthrax, botulism and listeriosis. Inhibition of Sps protein may inactivate pathogen replication and facilitate entrance into a non-contagious dormant sporulation stage

Antitubercular specific activity of ibuprofen and the other 2-arylpropanoic acids using the HT-SPOTi whole-cell phenotypic assay

Objectives: Lead antituberculosis (anti-TB) molecules with novel mechanisms of action are urgently required to fuel the anti-TB drug discovery pipeline. The aim of this study was to validate the use of the high-throughput spot culture growth inhibition (HT-SPOTi) assay for screening libraries of compounds against Mycobacterium tuberculosis and to study the inhibitory effect of ibuprofen (IBP) and the other 2-arylpropanoic acids on the growth inhibition of M tuberculosis and other mycobacterial species. Methods: The HT-SPOTi method was validated not only with known drugs but also with a library of 47 confirmed anti-TB active compounds published in the ChEMBL database. Three over-the-counter non-steroidal anti-inflammatory drugs were also included in the screening. The 2-arylpropanoic acids, including IBP, were comprehensively evaluated against phenotypically and physiologically different strains of mycobacteria, and their cytotoxicity was determined against murine RAW264.7 macrophages. Furthermore, a comparative bioinformatic analysis was employed to propose a potential mycobacterial target. Results: IBP showed antitubercular properties while carprofen was the most potent among the 2-arylpropanoic class. A 3,5-dinitro-IBP derivative was found to be more potent than IBP but equally selective. Other synthetic derivatives of IBP were less active, and the free carboxylic acid of IBP seems to be essential for its anti-TB activity. IBP, carprofen and the 3,5-dinitro-IBP derivative exhibited activity against multidrug-resistant isolates and stationary phase bacilli. On the basis of the human targets of the 2-arylpropanoic analgesics, the protein initiation factor infB (Rv2839c) of M tuberculosis was proposed as a potential molecular target. Conclusions: The HT-SPOTi method can be employed reliably and reproducibly to screen the antimicrobial potency of different compounds. IBP demonstrated specific antitubercular activity, while carprofen was the most selective agent among the 2-arylpropanoic class. Activity against stationary phase bacilli and multidrug-resistant isolates permits us to speculate a novel mechanism of antimycobacterial action. Further medicinal chemistry and target elucidation studies could potentially lead to new therapies against TB

Overexpression and functional characterization of an ABC transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis

The genes encoding ABC transporters occupy 2.5% of the genome of Mycobacterium tuberculosis . However, none of these putative ABC transporters has been characterized so far. We describe the development of expression systems for simultaneous expression of the ATP binding protein DrrA and the membrane integral protein DrrB which together behave as a functional doxorubicin efflux pump. Doxorubicin uptake in Escherichia coli or Mycobacterium smegmatis expressing DrrAB was inhibited by reserpine, an inhibitor of ABC transporters. The localization of DrrA to the membrane depended on the simultaneous expression of DrrB. ATP binding was positively regulated by doxorubicin and daunorubicin. At the same time, DrrB appeared to be sensitive to proteolysis when expressed alone in the absence of DrrA. Simultaneous expression of the two polypeptides was essential in order to obtain a functional doxorubicin efflux pump. Expression of DrrAB in E. coli conferred 8-fold increased resistance to ethidium bromide, a cationic compound. 2',7'-bis-(2-carboxyethyl)-5(-and 6)-carboxyfluorescein (BCECF), a neutral compound also behaved as a substrate of the reconstituted efflux pump. When expressed in M. smegmatis, DrrAB conferred resistance to a number of clinically relevant, structurally unrelated antibiotics. The resistant phenotype could be reversed by verapamil and reserpine, two potent inhibitors of ABC transporters

Nano-formulation of Ethambutol with multifunctional Graphene Oxide and magnetic nanoparticles retains Its anti-tubercular activity with prospects of improving chemotherapeutic efficacy

Tuberculosis (TB) is a dreadful bacterial disease, infecting millions of human and cattle every year worldwide. More than 50 years after its discovery, ethambutol continues to be an effective part of the World Health Organization’s recommended frontline chemotherapy against TB. However, the lengthy treatment regimens consisting of a cocktail of antibiotics affect patient compliance. There is an urgent need to improve the current therapy so as to reduce treatment duration and dosing frequency. In this study, we have designed a novel anti-TB multifunctional formulation by fabricating graphene oxide with iron oxide magnetite nanoparticles serving as a nano-carrier on to which ethambutol was successfully loaded. The designed nanoformulation was characterised using various analytical techniques. The release of ethambutol from anti-TB multifunctional nanoparticles formulation was found to be sustained over a significantly longer period of time in phosphate buffer saline solution at two physiological pH (7.4 and 4.8). Furthermore, the nano-formulation showed potent anti-tubercular activity while remaining non-toxic to the eukaryotic cells tested. The results of this in vitro evaluation of the newly designed nano-formulation endorse its further development in vivo

Bioactive compounds from the Bornean endemic plant Goniothalamus longistipetes

The present study aimed to screen plants for bioactive compounds with potential antibacterial activities. In our efforts to evaluate plants from Borneo, we isolated and elucidated the structures of four natural products from the bioactive fraction of a chloroform extract of Goniothalamus longistipetes using various chromatographic and spectroscopic techniques. The bioactive compounds were identified as a known styryllactone, (+)-altholactone ((2S,3R,3aS,7aS)-3-hydroxy-2-phenyl-2,3,3a,7a-tetrahydrobenzo-5(4H)-5-one) (1), a new styryllactone, (2S,3R,3aS,7aS)-3-hydroxy-2-phenyl-2,3,3a,7a-tetrahydrobenzo-5(4H)-5-one) (2) as well as a new alkaloid, 2,6-dimethoxyisonicotinaldehyde (3) and a new alkenyl-5-hydroxyl-phenyl benzoic acid (4). 1 and 4 showed broad-spectrum anti-bacterial activities against Gram-positive and Gram-negative bacteria as well as acid-fast model selected for this study. Compound 2 only demonstrated activities against Gram-positive bacteria whilst 3 displayed selective inhibitory activities against Gram-positive bacterial strains. Additionally, their mechanisms of anti-bacterial action were also investigated. Using Mycobacterium smegmatis as a fast-growing model of tubercle bacilli, compounds 1, 2 and 4 demonstrated inhibitory activities against whole-cell drug efflux and biofilm formation; two key intrinsic mechanisms of antibiotic resistance. Interestingly, the amphiphilic compound 4 exhibited inhibitory activity against the conjugation of plasmid pKM101 in Escherichia coli using a plate conjugation assay. Plasmid conjugation is a mechanism by which Gram-positive and Gram-negative-bacteria acquire drug resistance and virulence. These results indicated that bioactive compounds isolated from Goniothalamus longistipetes can be potential candidates as ‘hits’ for further optimisation

Weighted gene co-expression network analysis identifies key modules and hub genes associated with mycobacterial infection of human macrophages

Tuberculosis (TB) is still a leading cause of death worldwide. Treatments remain unsatisfactory due to an incomplete understanding of the underlying host–pathogen interactions during infection. In the present study, weighted gene co-expression network analysis (WGCNA) was conducted to identify key macrophage modules and hub genes associated with mycobacterial infection. WGCNA was performed combining our own transcriptomic results using Mycobacterium aurum-infected human monocytic macrophages (THP1) with publicly accessible datasets obtained from three types of macrophages infected with seven different mycobacterial strains in various one-to-one combinations. A hierarchical clustering tree of 11,533 genes was built from 198 samples, and 47 distinct modules were revealed. We identified a module, consisting of 226 genes, which represented the common response of host macrophages to different mycobacterial infections that showed significant enrichment in innate immune stimulation, bacterial pattern recognition, and leukocyte chemotaxis. Moreover, by network analysis applied to the 74 genes with the best correlation with mycobacteria infection, we identified the top 10 hub-connecting genes: NAMPT, IRAK2, SOCS3, PTGS2, CCL20, IL1B, ZC3H12A, ABTB2, GFPT2, and ELOVL7. Interestingly, apart from the well-known Toll-like receptor and inflammation-associated genes, other genes may serve as novel TB diagnosis markers and potential therapeutic targets

Correction to: Lu et al. weighted gene co-expression network analysis identifies key modules and hub genes associated with mycobacterial infection of human macrophages. antibiotics 2021, 10, 97

The authors wish to make the following corrections to this paper [...