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Background: The COVID-19 pandemic has posed significant challenges to global healthcare systems, particularly impacting individuals with pre-existing conditions like hypertension. This study sought to assess the impact of the antihypertensive medications, losartan and enalapril maleate on SARS-CoV-2 infected cells. Vero E6 cells were infected and treated in vitro, evaluating cell viability via the MTT colorimetric assay. Additionally, the study measured relative levels of viral RNA and selected gene messenger RNAs using reverse transcriptase followed by quantitative real-time polymerase chain reaction., Results: The findings revealed that losartan substantially reduced nucleocapsid RNA levels of SARS-CoV-2 to nearly undetectable levels, while enalapril maleate did not demonstrate a significant effect. In response to viral infection, the expression of il-18, p53, p21, and p62 increased compared to uninfected-untreated cells. Notably, il-6 expression was upregulated by both infection and treatments. A comparison between infected cells treated with losartan or enalapril maleate highlighted the presence of distinct profiles in the expression of il-6, p53, p21, and p62., Conclusions: The data from our study suggest that these medications could interfere with certain effects triggered by SARS-CoV-2 infection in Vero E6 cells. However, their influence appears to vary both quantitatively and qualitatively in the modulation of metabolic and signal transduction pathways., (© 2024. The Author(s).)

The increasing prevalence of drug-resistant Mycobacterium tuberculosis strains stimulates the discovery of new drug candidates. Among them are 8-hydroxyquinoline (8HQ) derivatives that exhibited antimicrobial properties. Unfortunately, there is a lack of data assessing possible targets for this class mainly against Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (MtInhA), a validated target in this field. Thus, the main purpose of this study was to identify 8HQ derivatives that are active against M. tuberculosis and MtInhA. Initially, the screening against the microorganism of a small antimicrobial library and its new derivatives that possess some structural similarity with MtInhA inhibitors identified four 7-substituted-8HQ (series 5 - 5a, 5c, 5d and 5i) and four 5-substituted-8HQ active derivatives (series 7 - 7a, 7c, 7d and 7j). In general, the 7-substituted 8-HQs were more potent and, in the enzymatic assay, were able to inhibit MtInhA at low micromolar range. However, the 5-substituted-8-HQs that presented antimycobacterial activity were not able to inhibit MtInhA. These findings indicate the non-promiscuous nature of 8-HQ derivatives and emphasize the significance of selecting appropriate substituents to achieve in vitro enzyme inhibition. Finally, 7-substituted-8HQ series are promising new derivatives for structure-based drug design and further development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The crystallographic structure of the FolB enzyme from Mycobacterium tuberculosis ( Mt FolB), complexed with its inhibitor 8-mercaptoguanine (8-MG), was elucidated at a resolution of 1.95 Å. A novel series of S8-functionalized 8-MG derivatives were synthesised and evaluated as in vitro inhibitors of dihydroneopterin aldolase (DHNA, EC 4.1.2.25) activity of Mt FolB. These compounds exhibited IC 50 values in the submicromolar range. Evaluation of the activity for five compounds indicated their inhibition mode and inhibition constants. Molecular docking analyses were performed to determine the enzyme-inhibitor intermolecular interactions and ligand conformations upon complex formation. The inhibitory activities of all compounds against the M. tuberculosis H37Rv strain were evaluated. Compound 3e exhibited a minimum inhibitory concentration in the micromolar range. Finally, Compound 3e showed no apparent toxicity in both HepG2 and Vero cells. The findings presented herein will advance the quest for novel, specific inhibitors targeting Mt FolB, an attractive molecular target for TB drug development.

There has been a dramatic increase in the identification of non-canonical translation and a significant expansion of the protein-coding genome. Among the strategies used to identify unannotated small Open Reading Frames (smORFs) that encode microproteins, Ribosome profiling (Ribo-Seq) is the gold standard for the annotation of novel coding sequences by reporting on smORF translation. In Ribo-Seq, ribosome-protected footprints (RPFs) that map to multiple genomic sites are removed since they cannot be unambiguously assigned to a specific genomic location. Furthermore, RPFs necessarily result in short (25-34 nucleotides) reads, increasing the chance of multi-mapping alignments, such that smORFs residing in these regions cannot be identified by Ribo-Seq. Moreover, it has been challenging to identify protein evidence for Ribo-Seq. To solve this, we developed Rp3, a pipeline that integrates proteogenomics and Ribosome profiling to provide unambiguous evidence for a subset of microproteins missed by current Ribo-Seq pipelines. Here, we show that Rp3 maximizes proteomics detection and confidence of microprotein-encoding smORFs., (© 2024. The Author(s).)

The aims of this work were to optimize the production of Erwinia carotovora l-asparaginase II enzyme in Escherichia coli by different fed-batch cultivation strategies using a benchtop bioreactor and to evaluate the therapeutic potential of the recombinant enzyme against different acute lymphoblastic leukemia cell lines. The highest enzyme activities (∼98,000 U/L) were obtained in cultures using the DO-stat feeding strategy with induction in 18 h of culture. Under these experimental conditions, the maximum values for recombinant l-asparaginase II (rASNase) yield per substrate, rASNase yield per biomass, and productivity were approximately 1204 U/g glucose , 3660 U/g cells , and 3260 U/(L·h), respectively. This condition was efficient for achieving high yields of the recombinant enzyme, which was purified and used in in vitro antileukemic potential tests. Of all the leukemic cell lines tested, RS4;11 showed the highest sensitivity to rASNase, with an IC 50 value of approximately 0.0006 U/mL and more than 70% apoptotic cells. The study demonstrated that the cultivation strategies used were efficient for obtaining high yield and productivity of rASNase with therapeutic potential inasmuch as cytotoxic activity and induction of apoptosis were demonstrated for this protein., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Astrocytes are glial cells that play key roles in neuroinflammation, which is a common feature in diabetic encephalopathy and aging process. Metformin is an antidiabetic compound that shows neuroprotective properties, including in inflammatory models, but astroglial signaling pathways involved are still poorly known. Interferons α/β are cytokines that participate in antiviral responses and the lack of their signaling increases susceptible to viral infections. Here, we investigated the effects of metformin on astrocytes from hypothalamus, a crucial brain region related to inflammatory processes. Astrocyte cultures were derived from interferon α/β receptor knockout (IFNα/βR -/- ) and wild-type (WT) mice. Metformin did not change the expression of glial fibrillary acidic protein but caused an anti-inflammatory effect by decreasing pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1β), as well as increasing gene expression of anti-inflammatory proteins interleukin-10 and Nrf2 (nuclear factor erythroid derived 2 like 2). However, nuclear factor κB p65 and cyclooxygenase 2 were downregulated in WT astrocytes and upregulated in IFNα/βR -/- astrocytes. AMP-activated protein kinase (AMPK), a molecular target of metformin, was upregulated only in WT astrocytes, while sirtuin 1 increased in both mice models. The expression of inducible nitric oxide synthase was decreased in WT astrocytes and heme oxygenase 1 was increased in IFNα/βR -/- astrocytes. Although loss of IFNα/βR-mediated signaling affects some effects of metformin, our results support beneficial roles of this drug in hypothalamic astrocytes. Moreover, paradoxical response of metformin may involve AMPK. Thus, metformin can mediate glioprotection due its effects on age-related disorders in non-diabetic and diabetic encephalopathy individuals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Tuberculosis is a disease caused by a single pathogen that leads to a death toll estimated to be more than a million per year. Mycobacterium tuberculosis (Mtb), which affects mainly the lungs, spreads by airborne transmission when infectious respiratory particles from an infected human enter the respiratory tract of another person. Despite diagnosis and treatment being well established, the rise of cases of patients infected with Mtb strains with multidrug resistance to the antibiotics used in the regimen against the disease is alarming. Indole used as a core molecule has been described as a promising structure to treat several diseases. 5-Fluoroindole (5-FI) compound, evaluated in the free base and in the hydrochloride (5-FI.HCl) forms, inhibited the growth of pan-sensitive Mtb H37Rv strain in the same range (4.7-29.1 μM) of clinical isolates that have resistance to at least two first-line drugs. Although 5-FI showed no cytotoxicity in Vero and HepG2 cells, high permeability (2.4.10 -6 cm/s) in the PAMPA assay, and high metabolic stability (Cl int 9.0 mL/min/kg) in rat liver microsomes, limited solubility at plasmatic and intestinal pH values prompted formation and employment of its salt form (5-FI.HCl). Although the 5-FI.HCl compound showed increased solubility at pH values of 7.4 and 9.1 and increased stability in aqueous solutions, data for intrinsic clearance (Cl int = 48 mL/min/kg) and a half-life ( t 1/2 = 12 min) showed decreased metabolic stability. As 5-FI.HCl showed both good absorption and ability to reach the systemic circulation of animals without the need to use vehicles containing cosolvents or surfactants, it was chosen to evaluate its effectiveness in the model of tuberculosis in mice. The in vivo results showed the concentration of the compound in plasma increasing within 30 min in the systemic circulation and the capacity of reducing the Mtb burden in the lungs at the concentration of 200 μmol/kg after 21 days of infection, with no toxicity in mice., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Staphylococcus aureus is the etiologic agent of many nosocomial infections, and its biofilm is frequently isolated from medical devices. Moreover, the dissemination of multidrug-resistant (MDR) strains from this pathogen, such as methicillin-resistant S. aureus (MRSA) strains, is a worldwide public health issue. The inhibition of biofilm formation can be used as a strategy to weaken bacterial resistance. Taking that into account, we analysed the ability of marine sponge-associated bacteria to produce antibiofilm molecules, and we found that marine Priestia sp., isolated from marine sponge Scopalina sp. collected on the Brazilian coast, secretes proteins that impair biofilm development from S. aureus. Partially purified proteins (PPP) secreted after 24 hours of bacterial growth promoted a 92% biofilm mass reduction and 4.0 µg/dL was the minimum concentration to significantly inhibit biofilm formation. This reduction was visually confirmed by light microscopy and Scanning Electron Microscopy (SEM). Furthermore, biochemical assays showed that the antibiofilm activity of PPP was reduced by ethylenediaminetetraacetic acid (EDTA) and 1,10 phenanthroline (PHEN), while it was stimulated by zinc ions, suggesting an active metallopeptidase in PPP. This result agrees with mass spectrometry (MS) identification, which indicated the presence of a metallopeptidase from the M28 family. Additionally, whole-genome sequencing analysis of Priestia sp. shows that gene ywad, a metallopeptidase-encoding gene, was present. Therefore, the results presented herein indicate that PPP secreted by the marine Priestia sp. can be explored as a potential antibiofilm agent and help to treat chronic infections., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Utilizing a scaffold-hopping strategy from the drug candidate telacebec, a novel series of 2-(quinolin-4-yloxy)acetamides was synthesized and evaluated as inhibitors of Mycobacterium tuberculosis (Mtb) growth. These compounds demonstrated potent activity against drug-sensitive and multidrug-resistant strains (MIC ≤ 0.02 μM). Leading compounds were evaluated against a known qcrB resistant strain (T313A), and their loss in activity suggested that the cytochrome bc 1 complex is the likely target. Additionally, these structures showed high selectivity regarding mammalian cells (selectivity index > 500) and stability across different aqueous media. Furthermore, some of the synthesized quinolines demonstrated aqueous solubility values that exceeded those of telacebec, while maintaining low rates of metabolism. Finally, a selected compound prevented Mtb growth by more than 1.7 log 10 colony forming units in a macrophage model of tuberculosis (TB) infection. These findings validate the proposed design and introduce new 2-(quinolin-4-yloxy)acetamides with potential for development in TB drug discovery campaigns., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

We analyzed the ability of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) itself and SARS-CoV-2-IgG immune complexes to trigger human monocyte necroptosis. SARS-CoV-2 was able to induce monocyte necroptosis dependently of MLKL activation. Necroptosis-associated proteins (RIPK1, RIPK3 and MLKL) were involved in SARS-CoV-2N1 gene expression in monocytes. SARS-CoV-2 immune complexes promoted monocyte necroptosis in a RIPK3- and MLKL-dependent manner, and Syk tyrosine kinase was necessary for SARS-CoV-2 immune complex-induced monocyte necroptosis, indicating the involvement of Fcγ receptors on necroptosis. Finally, we provide evidence that elevated LDH levels as a marker of lytic cell death are associated with COVID-19 pathogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Tuberculosis (TB) is one of the main causes of death from a single pathological agent, Mycobacterium tuberculosis (Mtb). In addition, the emergence of drug-resistant TB strains has exacerbated even further the treatment outcome of TB patients. It is thus needed the search for new therapeutic strategies to improve the current treatment and to circumvent the resistance mechanisms of Mtb. The shikimate kinase (SK) is the fifth enzyme of the shikimate pathway, which is essential for the survival of Mtb. The shikimate pathway is absent in humans, thereby indicating SK as an attractive target for the development of anti-TB drugs. In this work, a combination of in silico and in vitro techniques was used to identify potential inhibitors for SK from Mtb (MtSK). All compounds of our in-house database (Centro de Pesquisas em Biologia Molecular e Funcional, CPBMF) were submitted to in silico toxicity analysis to evaluate the risk of hepatotoxicity. Docking experiments were performed to identify the potential inhibitors of MtSK according to the predicted binding energy. In vitro inhibitory activity of MtSK-catalyzed chemical reaction at a single compound concentration was assessed. Minimum inhibitory concentration values for in vitro growth of pan-sensitive Mtb H37Rv strain were also determined. The mixed approach implemented in this work was able to identify five compounds that inhibit both MtSK and the in vitro growth of Mtb., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Herein a series of 4-aminoquinolines were synthesized in an attempt to optimize and study the structural features related to LABIO-17 biological activity, a Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein reductase (MtInhA) inhibitor previously identified by a virtual-ligand-screening approach. Structure-activity relationships led to novel submicromolar inhibitors of MtInhA and potent antitubercular agents. The lead compound is 87-fold more potent as enzymatic inhibitors and 32-fold more potent against M. tuberculosis H37Rv strain in comparison with LABIO-17. These molecules were also active against multidrug-resistant strains, devoid of apparent toxicity to mammalian cells and showed favorable in vitro ADME profiles. Additionally, these compounds were active in an intracellular model of tuberculosis (TB) infection, showed no genotoxicity signals, satisfactory absorption parameters and absence of in vivo acute toxicity. Finally, treatment with selected 4-aminoquinoline for two weeks produced bacteriostatic effect in a murine model of TB. Taken together, these findings indicate that this chemical class may furnish candidates for the future development of drug-sensitive and drug-resistant tuberculosis treatments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Different approaches are in use to improve our knowledge about the causative agent of coronavirus disease (COVID-19). Cell culture-based methods are the better way to perform viral isolation, evaluate viral infectivity, and amplify the virus. Furthermore, next-generation sequencing (NGS) have been essential to analyze a complete genome and to describe new viral species and lineages that have arisen over time. Four naso-oropharyngeal swab samples, collected from April to July of 2020, were isolated and sequenced aiming to produce viral stocks and analyze the mutational profile of the found lineage. B.1.1.33 was the lineage detected in all sequences. Although the samples belong to the same lineage, it was possible to evaluate different mutations found including some that were first described in these sequences, like the S:H655Y and T63N. The results described here can help to elicit how the pandemic started to spread and how it has been evolving in south Brazil., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Tuberculosis (TB) remains one of the leading causes of death due to a single pathogen. The emergence and proliferation of multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) represent compelling reasons to invest in the pursuit of new anti-TB agents. The shikimate pathway, responsible for chorismate biosynthesis, which is a precursor of important aromatic compounds, is required for Mycobacterium tuberculosis growth. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase ( Mtb DAHPS) catalyzes the first step in the shikimate pathway and it is an attractive target for anti-tubercular agents. Here, we used a CRISPRi system to evaluate the DAHPS as a vulnerable target in M. tuberculosis. The silencing of aroG significantly reduces the M. tuberculosis growth in both rich medium and, especially, in infected murine macrophages. The supplementation with amino acids was only able to partially rescue the growth of bacilli, whereas the Aro supplement (aromix) was enough to sustain the bacterial growth at lower rates. This study shows that Mtb DAHPS protein is vulnerable and, therefore, an attractive target to develop new anti-TB agents. In addition, the study contributes to a better understanding of the biosynthesis of aromatic compounds and the bacillus physiology. IMPORTANCE Determining the vulnerability of a potential target allows us to assess whether its partial inhibition will impact bacterial growth. Here, we evaluated the vulnerability of the enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) from M. tuberculosis by silencing the DAHPS-coding aroG gene in different contexts. These results could lead to the development of novel and potent anti-tubercular agents in the near future.

Using cycloalkyl and electron-donating groups to decrease the carbonyl electrophilicity, a novel series of 2-(quinoline-4-yloxy)acetamides was synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (Mtb) growth. Structure-activity relationship studies led to selective and potent antitubercular agents with minimum inhibitory concentrations in the submicromolar range against drug-sensitive and drug-resistant Mtb strains. An evaluation of the activity of the lead compounds against a spontaneous qcrB mutant strain indicated that the structures targeted the cytochrome bc 1 complex. In addition, selected molecules inhibited Mtb growth in a macrophage model of tuberculosis infection. Furthermore, the leading compound was chemically stable depending on the context and showed good kinetic solubility, high permeability, and a low rate of in vitro metabolism. Finally, the pharmacokinetic profile of the compound was assessed after oral administration to mice. To the best of our knowledge, for the first time, a 2-(quinoline-4-yloxy)acetamide was obtained with a sufficient exposure, which may enable in vivo effectiveness and its further development as an antituberculosis drug candidate., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

Enoyl-ACP reductases (ENRs) are enzymes that catalyze the last step of the elongation cycle during fatty acid synthesis. In recent years, new bacterial ENR types were discovered, some of them with structures and mechanisms that differ from the canonical bacterial FabI enzymes. Here, we briefly review the diversity of structural and catalytic properties of the canonical FabI and the new FabK, FabV, FabL, and novel ENRs identified in a soil metagenome study. We also highlight recent efforts to use the newly discovered Fabs as targets for drug development and consider the complex evolutionary history of this diverse set of bacterial ENRs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hopf, Roth, de Souza, Galina, Czeczot, Machado, Basso and Bizarro.)

Flexibility and function are related properties in the study of protein dynamics. Flexibility reflects in the conformational potential of proteins and thus in their functionalities. The presence of interactions between protein-ligands and protein-protein complexes, substrates, and environmental changes can alter protein plasticity, acting from the rearrangement of the side chains of amino acids to the folding/unfolding of large structural motifs. To evaluate the effects of the flexibility in protein systems, we defined the enzyme 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis, or MtInhA, as our target system. MtInhA is biologically active as a tetramer in solution; however, computational studies commonly use the monomer justifying the independence of its active sites due to their distances. However, differences in flexibility between tertiary and quaternary structures could present impact on the size of the active site, influencing the drug discovery process. In this study, we investigated the influence of flexibility restrictions in A- and B-loops of the MtInhA in order to suggest a monomeric structure that describes the conformational behavior of the tetrameric system. Overall, we observed that simulations where restrictions were applied to the A- and B-loops present a more similar behavior to the native structure when compared to unrestricted simulations. Therefore, our work presents a monomeric model of MtInhA, which has conformational characteristics of the biologically active structure. Thus, the data obtained in this work can be applied to the MtInhA system for the generation of more reliable flexible models for molecular docking experiments, and also for the performance of longer simulations by molecular dynamics and with a lower computational cost., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Summary: Genome annotation pipelines traditionally exclude open reading frames (ORFs) shorter than 100 codons to avoid false identifications. However, studies have been showing that these may encode functional microproteins with meaningful biological roles. We developed µProteInS, a proteogenomics pipeline that combines genomics, transcriptomics and proteomics to identify novel microproteins in bacteria. Our pipeline employs a model to filter out low confidence spectra, to avoid the need for manually inspecting Mass Spectrometry data. It also overcomes the shortcomings of traditional approaches that usually exclude overlapping genes, leaderless transcripts and non-conserved sequences, characteristics that are common among small ORFs (smORFs) and hamper their identification., Availability and Implementation: µProteInS is implemented in Python 3.8 within an Ubuntu 20.04 environment. It is an open-source software distributed under the GNU General Public License v3, available as a command-line tool. It can be downloaded at https://github.com/Eduardo-vsouza/uproteins and either installed from source or executed as a Docker image., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tuberculosis remains a global health problem that affects millions of people around the world. Despite recent efforts in drug development, new alternatives are required. Herein, a series of 27 N -(4-(benzyloxy)benzyl)-4-aminoquinolines were synthesized and evaluated for their ability to inhibit the M. tuberculosis H37Rv strain. Two of these compounds exhibited minimal inhibitory concentrations (MICs) similar to the first-line drug isoniazid. In addition, these hit compounds were selective for the bacillus with no significant change in viability of Vero and HepG2 cells. Finally, chemical stability, permeability and metabolic stability were also evaluated. The obtained data show that the molecular hits can be optimized aiming at the development of drug candidates for tuberculosis treatment.

Background: Gut microbiota-derived short-chain fatty-acid (SFCA) acetate protects mice against RSV A2 strain infection by increasing interferon-β production and expression of interferon-stimulated genes (ISGs). However, the role of SFCA in RSV infection using strains isolated from patients is unknown., Methods: We first used RSV clinical strains isolated from infants hospitalized with RSV bronchiolitis to investigate the effects of in vitro SCFA-acetate treatment of human pulmonary epithelial cells. We next examined whether SCFA-acetate treatment is beneficial in a mouse model of RSV infection using clinical isolates. We sought to investigate the relationship of gut microbiota and fecal acetate with disease severity among infants hospitalized with RSV bronchiolitis, and whether treating their respiratory epithelial cells with SCFA-acetate ex-vivo impacts viral load and ISG expression. We further treated epithelial cells from SARS-CoV-2 infected patients with SCFA-acetate., Findings: In vitro pre-treatment of A549 cells with SCFA-acetate reduced RSV infection with clinical isolates and increased the expression of RIG-I and ISG15. Animals treated with SCFA-acetate intranasally recovered significantly faster, with reduction in the RSV clinical isolates viral load, and increased lung expression of IFNB1 and the RIG-I. Experiments in RIG-I knockout A549 cells demonstrated that the protection relies on RIG-I presence. Gut microbial profile was associated with bronchiolitis severity and with acetate in stool. Increased SCFA-acetate levels were associated with increasing oxygen saturation at admission, and shorter duration of fever. Ex-vivo treatment of patients' respiratory cells with SCFA-acetate reduced RSV load and increased expression of ISGs OAS1 and ISG15, and virus recognition receptors MAVS and RIG-I, but not IFNB1. These SCFA-acetate effects were not found on cells from SARS-CoV-2 infected patients., Interpretation: SCFA-acetate reduces the severity of RSV infection and RSV viral load through modulation of RIG-I expression., Funding: FAPERGS (FAPERGS/MS/CNPq/SESRS no. 03/2017 - PPSUS 17/2551-0001380-8 and COVID-19 20/2551-0000258-6); CNPq 312504/2017-9; CAPES) - Finance Code 001., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

This study aimed to develop single-step purification and immobilization processes on cellulosic supports of β-galactosidase from Kluyveromyces sp. combined with the Cellulose-Binding Domain (CBD) tag. After 15 min of immobilization, with an enzymatic load of 150 U/g support , expressed activity values reached 106.88 (microcrystalline cellulose), 115.03 (alkaline nanocellulose), and 108.47 IU/g (acid nanocellulose). The derivatives produced were less sensitive to the presence of galactose in comparison with the soluble purified enzyme. Among the cations assessed (Na + , K + , Mg 2+ , and Ca 2+ ), magnesium provided the highest increase in the enzymatic activity of β-galactosidases immobilized on cellulosic supports. Supports and derivatives showed no cytotoxic effect on the investigated cell cultures (HepG2 and Vero). Derivatives showed high operational stability in the hydrolysis of milk lactose and retained from 53 to 64% of their hydrolysis capacity after 40 reuse cycles. This study obtained biocatalyzers with promising characteristics for application in the food industry. Biocatalyzers were obtained through a low-cost one-step sustainable bioprocess of purification and immobilization of a β-galactosidase on cellulose via CBD., (Copyright © 2022 Elsevier B.V. All rights reserved.)

For the first time, this work reported the one-step purification and targeted immobilization process of a β-galactosidase (Gal) with the Cellulose Binding Domain (CBD) tag, by binding it to different magnetic cellulose supports. The process efficiency after β-galactosidase-CBD immobilization on magnetic cellulose-based supports showed values of approximately 90% for all evaluated enzymatic loads. Compared with free Gal, derivatives showed affinity values between β-galactosidase and the substrate 1.2 × higher in the lactose hydrolysis of milk. β-Galactosidase-CBD's oriented immobilization process on supports increased the thermal stability of the immobilized enzyme by up to 7 × . After 15 cycles of reuse, both enzyme preparations showed a relative hydrolysis percentage of 50% of lactose in milk. The oriented immobilization process developed for purifying recombinant proteins containing the CBD tag enabled the execution of both steps simultaneously and quickly and the obtention of β-galactosidases with promising catalytic characteristics for application in the food and pharmaceutical industries., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The epidemiological importance of mycobacterial species is indisputable, and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important bacterial metabolites, represents a set of targets for inhibitors of Mycobacterium tuberculosis growth. The aroA -encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene disruption, gene knockdown, point mutations (D61W, R134A, E321N), and kinetic analysis to evaluate aroA gene essentiality and vulnerability of its protein product, EPSPS, from Mycolicibacterium ( Mycobacterium ) smegmatis ( Ms EPSPS). We demonstrate that aroA -deficient cells are auxotrophic for aromatic amino acids (AroAAs) and that the growth impairment observed for aroA -knockdown cells grown on defined medium can be rescued by AroAA supplementation. We also evaluated the essentiality of selected Ms EPSPS residues in bacterial cells grown without AroAA supplementation. We found that the catalytic residues R134 and E321 are essential, while D61, presumably important for protein dynamics and suggested to have an indirect role in catalysis, is not essential under the growth conditions evaluated. We have also determined the catalytic efficiencies ( K cat / K m ) of recombinant wild-type (WT) and mutated versions of Ms EPSPS (D61W, R134A, E321N). Our results suggest that drug development efforts toward EPSPS inhibition may be ineffective if bacilli have access to external sources of AroAAs in the context of infection, which should be evaluated further. In the absence of AroAA supplementation, aroA from M. smegmatis is essential, its essentiality is dependent on Ms EPSPS activity, and Ms EPSPS is vulnerable. IMPORTANCE We found that cells from Mycobacterium smegmatis, a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l-tryptophan, l-phenylalanine, and l-tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs. The depleted enzyme, the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes the sixth step of shikimate pathway. Depletion of this enzyme inside cells was performed by disrupting or silencing the EPSPS-encoding aroA gene. Finally, we evaluated the essentiality of specific residues from EPSPS that are important for its catalytic activity, determined with experiments of enzyme kinetics using recombinant EPSPS mutants.

The dihydroneopterin aldolase (DHNA, EC 4.1.2.25) activity of FolB protein is required for the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde (GA) in the folate pathway. FolB protein from Mycobacterium tuberculosis ( Mt FolB) is essential for bacilli survival and represents an important molecular target for drug development. S8-functionalized 8-mercaptoguanine derivatives were synthesised and evaluated for inhibitory activity against Mt FolB. The compounds showed IC 50 values in the submicromolar range. The inhibition mode and inhibition constants were determined for compounds that exhibited the strongest inhibition. Additionally, molecular docking analyses were performed to suggest enzyme-inhibitor interactions and ligand conformations. To the best of our knowledge, this study describes the first class of Mt FolB inhibitors.

Tuberculosis has been described as a global health crisis since the 1990s, with an estimated 1.4 million deaths in the last year. Herein, a series of 20 1 H -indoles were synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (Mtb) growth. Furthermore, the top hit compounds were active against multidrug-resistant strains, without cross-resistance with first-line drugs. Exposing HepG2 and Vero cells to the molecules for 72 h showed that one of the evaluated structures was devoid of apparent toxicity. In addition, this 3-phenyl-1 H -indole showed no genotoxicity signals. Finally, time-kill and pharmacodynamic model analyses demonstrated that this compound has bactericidal activity at concentrations close to the Minimum Inhibitory Concentration, coupled with a strong time-dependent behavior. To the best of our knowledge, this study describes the activity of 3-phenyl-1 H -indole against Mtb for the first time.

Human thymidine phosphorylase (hTP) is overexpressed in several solid tumors and is commonly associated with aggressiveness and unfavorable prognosis. 6-(((1,3-Dihydroxypropan-2-yl)amino)methyl)-5-iodopyrimidine-2,4(1H,3H)-dione (CPBMF-223) is a noncompetitive hTP inhibitor, which has been described as a tumor angiogenesis inhibitor. The present study investigated the effects of CPBMF-223 in a xenograft tumor induced by human colorectal carcinoma cells (HCT-116). Additionally, CPBMF-223 capacity to reduce cell migration, its toxicological profile, and pharmacokinetic characteristics, were also evaluated. The intraperitoneal treatment with CPBMF-223 markedly prevented the relative tumor growth with an efficacy similar to that observed for 5-fluorouracil. Interestingly, number of vessels were significantly decreased in the treated groups. Moreover, CPBMF-223 significantly reduced the migration of cell line HCT-116. In the Ames assay and in an acute oral toxicity test, the molecule did not alter any evaluated parameter. Using the zebrafish toxicity model, cardiac and locomotor parameters were slightly changed. Regarding the pharmacokinetics profile, CPBMF-223 showed clearance of 9.42 L/h/kg after intravenous administration, oral bioavailability of 13.5%, and a half-life of 0.75 h. Our findings shed new light on the role of hTP in colorectal cancer induced by HCT-116 cell in mice, pointing out CPBMF-223 as, hopefully, a promising drug candidate., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Antimalarial drugs have been suggested as promising scaffolds with anti-tubercular activities. In this work, we demonstrated, for the first time, the effectiveness of tafenoquine against mycobacteria. Firstly, tafenoquine inhibited the growth of Mycobacterium smegmatis and Mycobacterium tuberculosis with lower MICs values as compared to other antimalarial drugs, such as mefloquine, chloroquine, and primaquine. Importantly, tafenoquine was active against three multi-drug resistant strains of M. tuberculosis with MIC values similar to pan-sensitive strains, suggesting that tafenoquine is capable of evading the major mechanisms of resistance found in drug-resistant clinical isolates of M. tuberculosis. Importantly, tafenoquine displayed a synergistic effect when combined with mefloquine. In addition, tafenoquine displayed an improved activity compared to the groups treated with both isoniazid and rifampicin in the six-week nutrient starved M. tuberculosis cultures. This finding suggests that further investigations of tafenoquine against dormant mycobacteria are worth pursuing. Moreover, different concentrations of tafenoquine ranging from 1.25 to 80 μM displayed different effects against M. tuberculosis, from moderate (reduction of a 1.8 log CFU/mL) to potent bactericidal (reduction of a 4.2 log CFU/mL) activities. Tafenoquine may represent a hit for further drug optimization and for future clinical development as a new anti-mycobacterial agent, especially in cases of resistant and/or dormant forms of tuberculosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) has been described as a global health crisis since the second half of the 1990s. Mycobacterium tuberculosis (Mtb), the etiologic agent of TB in humans, is a very successful pathogen, being the main cause of death in the population among infectious agents. In 2019, it was estimated that around 10 million individuals were contaminated by this bacillus and about 1.2 million succumbed to the disease. In recent years, our research group has reported the design and synthesis of quinoline derivatives as drug candidates for the treatment of TB. These compounds have demonstrated potent and selective growth inhibition of drug-susceptible and drug-resistant Mtb strains. Herein, a new synthetic approach was established providing efficient and rapid access (15 min) to a series of 4-alkoxy-6-methoxy-2-methylquinolines using ultrasound energy. The new synthetic protocol provides a simple procedure utilizing an open vessel system that affords the target products at satisfactory yields (45-84%) and elevated purities (≥95%). The methodology allows the evaluation of a larger number of molecules in assays against the bacillus, facilitating the determination of the structure-activity relationship with a reduced environmental cost.

Potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65) is a potent inhibitor of the uridine phosphorylase 1 (UPP1) enzyme. Its non-ionized analog has already demonstrated biological properties by reducing adverse effects caused by the chemotherapeutic 5-fluorouracil (5-FU). In addition, it has been demonstrated that uridine inhibits inflammation and fibrosis in bleomycin lung injury, decreasing collagen production. The purpose of this study was to investigate the in vitro and in vivo effects of CPBMF65 on activated hepatic stellate cells (HSC) and on carbon tetrachloride-induced liver fibrosis in mice. After incubation with CPBMF65, decreased cell proliferation and phenotype reversion were observed in vitro. In addition, CPBMF65 promoted a protective effect on tetrachloride-induced liver fibrosis in mice, demonstrated by its antifibrotic and anti-inflammatory actions. The results of the present study indicate that the UPP1 inhibitor (CPBMF65) may have potential as a novel therapeutic agent for the treatment of liver fibrosis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Tuberculosis (TB) is one of the most fatal diseases and is responsible for the infection of millions of people around the world. Most recently, scientific frontiers have been engaged to develop new drugs that can overcome drug-resistant TB. Following this direction, using a designed scaffold based on the combination of two separate pharmacophoric groups, a series of menadione-derived selenoesters was developed with good yields. All products were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv and attractive results were observed, especially for the compounds 8a, 8c and 8f (MICs 2.1, 8.0 and 8.1 μM, respectively). In addition, 8a, 8c and 8f demonstrated potent in vitro activity against multidrug-resistant clinical isolates (CDCT-16 and CDCT-27) with promising MIC values ranging from 0.8 to 3.1 μM. Importantly, compounds 8a and 8c were found to be non-toxic against the Vero cell line. The SI value of 8a (>23.8) was found to be comparable to that of isoniazid (>22.7), which suggests the possibility of carrying out advanced studies on this derivative. Therefore, these menadione-derived selenoesters obtained as hybrid compounds represent promising new anti-tubercular agents to overcome TB multidrug resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Hepatocellular carcinoma (HCC) is the most prevalent type of tumor among primary liver tumors and is the second highest cause of cancer-related deaths worldwide. Current therapies are controversial, and more research is needed to identify effective treatments. A new synthetic compound, potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65), is a potent inhibitor of the human uridine phosphorylase-1 (hUP1) enzyme, which controls the cell concentration of uridine (Urd). Urd is a natural pyrimidine nucleoside involved in cellular processes, such as RNA synthesis. In addition, it is considered a promising biochemical modulator, as it may reduce the toxicity caused by chemotherapeutics without impairing its anti-tumor activity. Thus, the objective of this study is to evaluate the effects of CPBMF65 on the proliferation of the human hepatocellular carcinoma cell line (HepG2). Cell proliferation, cytotoxicity, apoptosis, senescence, autophagy, intracellular Urd levels, cell cycle arrest, and drug resistance were analyzed. Results demonstrate that, after incubation with CPBMF65, HepG2 cell proliferation decreased, mainly through cell cycle arrest and senescence, increasing the levels of intracellular Urd and maintaining cell proliferation reduced during chronic treatment. In conclusion, results show, for the first time, the ability of a hUP1 inhibitor (CPBMF65) to reduce HepG2 cell proliferation through cell cycle arrest and senescence.

The global epidemic of tuberculosis (TB) imposes a sustained epidemiologic vigilance and investments in research by governments. Mycobacterium tuberculosis , the main causative agent of TB in human beings, is a very successful pathogen, being the main cause of death in the population among infectious agents. In 2018, ~10 million individuals were contaminated with this bacillus and became ill with TB, and about 1.2 million succumbed to the disease. Most of the success of the M. tuberculosis to linger in the population comes from its ability to persist in an asymptomatic latent state into the host and, in fact, the majority of the individuals are unaware of being contaminated. Even though TB is a treatable disease and is curable in most cases, the treatment is lengthy and laborious. In addition, the rise of resistance to first-line anti-TB drugs elicits a response from TB research groups to discover new chemical entities, preferably with novel mechanisms of action. The pathway to find a new TB drug, however, is arduous and has many barriers that are difficult to overcome. Fortunately, several approaches are available today to be pursued by scientists interested in anti-TB drug development, which goes from massively testing chemical compounds against mycobacteria, to discovering new molecular targets by genetic manipulation. This review presents some difficulties found along the TB drug development process and illustrates different approaches that might be used to try to identify new molecules or targets that are able to impair M. tuberculosis survival., (Copyright © 2020 Dalberto, de Souza, Abbadi, Neves, Rambo, Ramos, Macchi, Machado, Bizarro and Basso.)

Using a classical molecular simplification approach, a series of 36 quinolines were synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (M. tuberculosis) growth. Structure-activity relationship (SAR) studies leaded to potent antitubercular agents, with minimum inhibitory concentration (MIC) values as low as 0.3 μM against M. tuberculosis H37Rv reference strain. Furthermore, the lead compounds were active against multidrug-resistant strains, without cross-resistance with some first- and second-line drugs. Testing the molecules against a spontaneous mutant strain containing a single mutation in the qcrB gene (T313A) indicated that the synthesized quinolines targeted the cytochrome bc 1 complex. In addition, leading compounds were devoid of apparent toxicity to HepG2 and Vero cells and showed moderate elimination rates in human liver S9 fractions. Finally, the selected structures inhibited M. tuberculosis growth in a macrophage model of tuberculosis infection. Taken together, these data indicate that this class of compounds may furnish candidates for the future development of antituberculosis drugs., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020. Published by Elsevier Masson SAS.)

Roughly a third of the world's population is estimated to have latent Mycobacterium tuberculosis infection, being at risk of developing active tuberculosis (TB) during their lifetime. Given the inefficacy of prophylactic measures and the increase of drug-resistant M. tuberculosis strains, there is a clear and urgent need for the development of new and more efficient chemotherapeutic agents, with selective toxicity, to be implemented on patient treatment. The component enzymes of the shikimate pathway, which is essential in mycobacteria and absent in humans, stand as attractive and potential targets for the development of new drugs to treat TB. This review gives an update on published work on the enzymes of the shikimate pathway and some insight on what can be potentially explored towards selective drug development.

Transglutaminases (TGases) are a class of transferases widely used in the food and biotechnology industries. In this work, we describe the production of recombinant Bacillus amyloliquefaciens TGase in Escherichia coli, obtaining the protein in its soluble and active form. In order to reduce TGase activity inside host cells and consequently its toxicity, we constructed a bicistronic plasmid containing the B. amyloliquefaciens TGase gene fused to the inhibitory Streptomyces caniferus prodomain. To make the enzyme active and avoid the need of prodomain removal in vitro, we also cloned the 3C protease gene into the same plasmid. After a fast single-step purification protocol, we obtained a partially purified recombinant TGase with 37 mU/mg protein activity, that crosslinked bovine serum albumin (BSA). This is the first report on the expression of B. amyloliquefaciens TGase in E. coli in its mature and active form., (Copyright © 2019 Elsevier Inc. All rights reserved.)

New effective compounds to treat tuberculosis are urgently needed. IQG-607 is an orally active anti-tuberculosis drug candidate, with promising preliminary safety profile and anti-mycobacterial activity in both in vitro and in vivo models of tuberculosis infection. Here, we evaluated the mutagenic and genotoxic effects of IQG-607, and its interactions with CYP450 isoforms. Moreover, we describe for the first time a combination study of IQG-607 in Mycobacterium tuberculosis-infected mice. Importantly, IQG-607 had additive effects when combined with the first-line anti-tuberculosis drugs rifampin and pyrazinamide in mice. IQG-607 presented weak to moderate inhibitory potential against CYP450 isoforms 3A4, 1A2, 2C9, 2C19, 2D6, and 2E1. The Salmonella mutagenicity test revealed that IQG-607 induced base pair substitution mutations in the strains TA100 and TA1535. However, in the presence of human metabolic S9 fraction, no mutagenic effect was detected in any strain. Additionally, IQG-607 did not increase micronucleus frequencies in mice, at any dose tested, 25, 100, or 250 mg/kg. The favorable activity in combination with first-line drugs and mild to moderate toxic events described in this study suggest that IQG-607 represents a candidate for clinical development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: The present paper aims to identify and characterize studies that assess the repercussions of parental rearing styles on development of psychopathological symptoms and to examine the possible relations between parental rearing behaviors and development of early maladaptive schemas (EMS)., Methods: A systematic search was conducted on the PsychNet, BVS, Scopus, Web of Science and PubMed databases for empirical studies published up to 2018 in Portuguese, English, or Spanish that investigated and characterized the effects of parental rearing styles., Results: The electronic search identified 321 articles on the various different databases, only 22 of which met the criteria for inclusion and were read in full. Correlations were found between EMS and maternal rejection, parental rearing styles and depression in the studies. EMS were also found to act as mediators in the relationship between parental rearing styles and/or education and dysfunctional symptoms during personality development., Conclusions: In the studies selected, parental rearing styles stood out from other variables that influence personality development and activation of schematic patterns. It is therefore important to highlight the importance of conducting studies in this area to provide information that can promote care and prevention strategies in early childhood.

A peptidomimetic containing a thiazolo ring-fused 2-pyridone (C10) has now been reported to inhibit hypoxia-induced tolerance to isoniazid (INH) in Mycobacterium tuberculosis (Flentie et al., Proc. Natl. Acad. Sci. U. S. A., 2019). The C10 compound could also potentiate the bactericidal activity in aerobically grown bacilli, prevented selection of drug-resistant strains, and reversed INH resistance in katG (catalase-peroxidase) mutants., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Overexpressed human thymidine phosphorylase (hTP) has been associated with cancer aggressiveness and poor prognosis by triggering proangiogenic and antiapoptotic signaling. Designed as transition-state analogues by mimicking the oxacarbenium ion, novel pyrimidine-2,4-diones were synthesized and evaluated as inhibitors of hTP activity. The most potent compound (8g) inhibited hTP in the submicromolar range with a noncompetitive inhibition mode with both thymidine and inorganic phosphate substrates. Furthermore, compound 8g was devoid of apparent toxicity to a panel of mammalian cells, showed no genotoxicity signals, and had low probability of drug-drug interactions and moderate in vitro metabolic rates. Finally, treatment with 8g (50 mg/(kg day)) for 2 weeks (5 days/week) significantly reduced tumor growth using an in vivo glioblastoma model. To the best of our knowledge, this active compound is the most potent in vitro hTP inhibitor with a kinetic profile that cannot be reversed by the accumulation of any enzyme substrates.

New effective compounds for tuberculosis treatment are needed. This study evaluated the effects of a series of quinoxaline-derived chalcones against laboratorial strains and clinical isolates of M. tuberculosis. Six molecules, namely N5, N9, N10, N15, N16, and N23 inhibited the growth of the M. tuberculosis H37Rv laboratorial strain. The three compounds (N9, N15 and N23) with the lowest MIC values were further tested against clinical isolates and laboratory strains with mutations in katG or inhA genes. From these data, N9 was selected as the lead compound for further investigation. Importantly, this chalcone displayed a synergistic effect when combined with moxifloxacin. Noteworthy, the anti-tubercular effects of N9 did not rely on inhibition of mycolic acids synthesis, circumventing important mechanisms of resistance. Interactions with cytochrome P450 isoforms and toxic effects were assessed in silico and in vitro. The chalcone N9 was not predicted to elicit any mutagenic, genotoxic, irritant, or reproductive effects, according to in silico analysis. Additionally, N9 did not cause mutagenicity or genotoxicity, as revealed by Salmonella/microsome and alkaline comet assays, respectively. Moreover, N9 did not inhibit the cytochrome P450 isoforms CYP3A4/5, CYP2C9, and CYP2C19. N9 can be considered a potential lead molecule for development of a new anti-tubercular therapeutic agent., Competing Interests: The authors have declared that no competing interests exist.

Using a classical hybridization approach, a series of 1H-benzo[d]imidazoles and 3,4-dihydroquinazolin-4-ones were synthesized (39 examples) and evaluated as inhibitors of Mycobacterium tuberculosis growth. Chemical modification studies yielded potent antitubercular agents with minimum inhibitory concentration (MIC) values as low as 0.24 μM against M. tuberculosis H37Rv strain. Further, the synthesized compounds were active against four drug-resistant strains containing different levels of resistance for the first line drugs. These molecules were devoid of apparent toxicity to HepG2, HaCat, and Vero cells with IC 50s  > 30 μM. Viability in mammalian cell cultures was evaluated using MTT and neutral red assays. In addition, some 3,4-dihydroquinazolin-4-ones showed low risk of cardiac toxicity, no signals of neurotoxicity or morphological alteration in zebrafish (Danio rerio) toxicity models. 3,4-Dihydroquinazolin-4-ones 9q and 9w were considered the lead compounds of these series of molecules with MIC values of 0.24 μM and 0.94 μM against M. tuberculosis H37Rv, respectively. Taken together, these data indicate that this class of compounds may furnish candidates for future development of novel anti-TB drugs., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP), encoded by deoD gene (Rv3307), is an enzyme from the purine salvage pathway, which has been widely studied as a molecular target for the development of inhibitors with potential antimycobacterial activity. However, the role of MtPNP in tuberculosis pathogenesis and dormancy is still unknown. The present work aims to construct a deoD knockout strain from M. tuberculosis, to evaluate the role of MtPNP in the growth of M. tuberculosis under oxygenated condition and in a dormancy model, and to assess whether deoD gene is important for M. tuberculosis invasion and growth in macrophages. The construction of a knockout strain for deoD gene was confirmed at DNA level by PCR and protein level by Western blot and LC-MS/MS. The deoD gene is not required for M. tuberculosis growth and survival under oxygenated and hypoxic conditions. The disruption of deoD gene did not affect mycobacterial ability to invade and grow in RAW 264.7 cells under the experimental conditions employed here., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The emergence of strains of Mycobacterium tuberculosis resistant to isoniazid (INH) has underscored the need for the development of new anti-tuberculosis agents. INH is activated by the mycobacterial katG -encoded catalase-peroxidase, forming an acylpyridine fragment that is covalently attached to the C4 of NADH. This isonicotinyl-NAD adduct inhibits the activity of 2- trans -enoyl-ACP(CoA) reductase (InhA), which plays a role in mycolic acid biosynthesis. A metal-based INH analog, Na 3 [Fe II (CN) 5 (INH)]·4H 2 O, IQG-607, was designed to have an electronic redistribution on INH moiety that would lead to an intramolecular electron transfer to bypass KatG activation. HPLC and EPR studies showed that the INH moiety can be oxidized by superoxide or peroxide yielding similar metabolites and isonicotinoyl radical only when associated to IQG-607, thereby supporting redox-mediated drug activation as a possible mechanism of action. However, IQG-607 was shown to inhibit the in vitro activity of both wild-type and INH-resistant mutant InhA enzymes in the absence of KatG activation. IQG-607 given by the oral route to M. tuberculosis -infected mice reduced lung lesions. Experiments using early and late controls of infection revealed a bactericidal activity for IQG-607. HPLC and voltammetric methods were developed to quantify IQG-607. Pharmacokinetic studies showed short half-life, high clearance, moderate volume of distribution, and low oral bioavailability, which was not altered by feeding. Safety and toxic effects of IQG-607 after acute and 90-day repeated oral administrations in both rats and minipigs showed occurrence of mild to moderate toxic events. Eight multidrug-resistant strains (MDR-TB) were resistant to IQG-607, suggesting an association between katG mutation and increasing MIC values. Whole genome sequencing of three spontaneous IQG-607-resistant strains harbored katG gene mutations. MIC measurements and macrophage infection experiments with a laboratorial strain showed that katG mutation is sufficient to confer resistance to IQG-607 and that the macrophage intracellular environment cannot trigger the self-activation mechanism. Reduced activity of IQG-607 against an M. tuberculosis strain overexpressing S94A InhA mutant protein suggested both the need for KatG activation and InhA as its target. Further efforts are suggested to be pursued toward attempting to translate IQG-607 into a chemotherapeutic agent to treat tuberculosis.

The 2-(quinolin-4-yloxy)acetamides (QOAs) have been reported to be promising molecules for tuberculosis treatment. Recent studies demonstrated their potent antimycobacterial activity, biological stability and synergism with rifampicin. The identification of the molecular target is an essential step towards the development of a novel drug candidate. Here, we report the target identification of the QOAs. We found that these compounds are active against Mycobacterium tuberculosis clinical isolates resistant to isoniazid, rifampicin, ethambutol, streptomycin and ethionamide. The initial evidence that DNA gyrase might be the target of QOAs, based on high minimum inhibitory concentration (MIC) values against ofloxacin-resistant clinical isolates and structural similarities with fluoroquinolones, was discarded by experiments performed with M. tuberculosis GyrA point mutant, DNA gyrase supercoiling inhibition assay and overexpression of DNA gyrase. We selected spontaneous mutants for our lead compound 21 and observed that these strains were also resistant to all QOA derivatives. The genomes of the spontaneous mutants were sequenced, and the results revealed a single mutation in qcrB gene (T313A), which indicates that the QOAs target the cytochrome bc 1 complex. The protein-compound interaction was further investigated by molecular docking. These findings reinforce the relevance of these compounds as promising candidates for the treatment of multidrug-resistant tuberculosis., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

The role, if any, played by the kinin system in tuberculosis infection models, either in vivo or in vitro, was investigated. The effects of Mycobacterium tuberculosis infection on C57BL/6 wild type, B 1 R-/-, B 2 R-/- and double B 1 R/B 2 R knockout mice were evaluated. Immunohistochemistry analysis was carried out to assess B 1 R and B 2 R expression in spleens and lungs of M. tuberculosis-infected mice. In addition, in vitro experiments with M. tuberculosis-infected macrophages were performed. The in vivo effects of HOE-140 and SSR240612 on the mice model of infection were also evaluated. Infected B 2 R-/- mice exhibited increased splenomegaly, whereas decreased spleen weight in infected double B 1 R/B 2 R knockout mice was observed. The bacterial load, determined as colony-forming units, did not differ in the spleens and lungs of the studied mouse strains. Importantly, immunohistochemical analysis revealed that B 1 R was upregulated in both spleens and lungs of infected mice. M. tuberculosis-infected macrophages incubated with SSR240612, alone or in combination with des-Arg 9 -BK, for four days, displayed a marked inhibitory effect on CFU counts. However, the pre-incubation of the selective B 1 R (des-Arg 9 -BK and SSR240612) and B 2 R (BK and HOE-140) agonists and antagonists, respectively, did not significantly affect the bacterial loads. A statistically significant reduction in the CFU of M. tuberculosis in lungs and spleens of animals treated with SSR240612, but not with HOE-140, was observed. Further efforts should be pursued to clarify whether or not SSR240612 might be considered an option for the treatment of tuberculosis., (Copyright © 2018. Published by Elsevier Ltd.)

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB) represents a major threat to global health. Isoniazid (INH) is a prodrug used in the first-line treatment of tuberculosis. It undergoes oxidation by a catalase-peroxidase KatG, leading to generation of an isonicotinoyl radical that reacts with NAD(H) forming the INH-NADH adduct as the active metabolite. A redox-mediated activation of isoniazid using an iron metal complex was previously proposed as a strategy to overcome isoniazid resistance due to KatG mutations. Here, we have prepared a series of iron metal complexes with isoniazid and analogues, containing alkyl substituents at the hydrazide moiety, and also with pyrazinamide derivatives. These complexes were activated by H 2 O 2 and studied by ESR and LC-MS. For the first time, the formation of the oxidized INH-NAD adduct from the pentacyano(isoniazid)ferrate(II) complex was detected by LC-MS, supporting a redox-mediated activation, for which a mechanistic proposition is reported. ESR data showed all alkylated hydrazides, in contrast to non-substituted hydrazides, only generated alkyl-based radicals. The structural modifications did not improve minimal inhibitory concentration (MIC) against MTB in comparison to isoniazid iron complex, providing support to isonicotinoyl radical formation as a requirement for activity. Nonetheless, the pyrazinoic acid hydrazide iron complex showed redox-mediated activation using H 2 O 2 with generation of a pyrazinoyl radical intermediate and production of pyrazinoic acid, which is in fact the active metabolite of pyrazinamide prodrug. Thereby, this strategy can also unveil new opportunities for activation of this type of drug., (Copyright © 2017 Elsevier Inc. All rights reserved.)

IQG-607 is a metal complex previously reported as a promising anti-tuberculosis (TB) drug against isoniazid (INH)-resistant strains of Mycobacterium tuberculosis Unexpectedly, we found that INH-resistant clinical isolates were resistant to IQG-607. Spontaneous mutants resistant to IQG-607 were subjected to whole-genome sequencing, and all sequenced colonies carried alterations in the katG gene. The katG (S315T) mutation was sufficient to confer resistance to IQG-607 in both MIC assays and inside macrophages. Moreover, overexpression of the InhA(S94A) protein caused IQG-607's resistance., (Copyright © 2018 American Society for Microbiology.)

IQG-607 is an analog of isoniazid with anti-tuberculosis activity. This work describes the development and validation of an HPLC method to quantify pentacyano(isoniazid)ferrate(II) compound (IQG-607) and the pharmacokinetic studies of this compound in mice. The method showed linearity in the 0.5-50μg/mL concentration range (r=0.9992). Intra- and inter-day precision was <5%, and the recovery ranged from 92.07 to 107.68%. IQG-607 was stable in plasma for at least 30days at -80°C and, after plasma processing, for 4h in the auto-sampler maintained on ice (recovery >85%). The applicability of the method for pharmacokinetic studies was determined after intravenous (i.v.) and oral (fasted and fed conditions) administration to mice. IQG-607 levels in plasma were quantified at time points for up to 2.5h. A short half-life (t 1/2 ) (1.14h), a high clearance (CL) (3.89L/h/kg), a moderate volume of distribution at steady state (Vdss) of 1.22L/kg, were observed after i.v. (50mg/kg) administration. Similar results were obtained for oral administration (250mg/kg) under fasted and fed conditions. The oral bioavailability (F), approximately 4%, was not altered by feeding. Plasma protein binding was 88.87±0.9%. The results described here provide novel insights into a pivotal criterion to warrant further efforts to be pursued towards attempts to translate this chemical compound into a chemotherapeutic agent to treat TB., (Copyright © 2017 Elsevier B.V. All rights reserved.)