Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase.

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.

Dual-Pharmacophore Pyrithione-Containing Cephalosporins Kill Both Replicating and Nonreplicating Mycobacterium tuberculosis.

The historical view of ß-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis (Mtb) in the presence of a ß-lactamase inhibitor. However, most antimycobacterial ß-lactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 ß-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3' that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a ß-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A ß-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent ß-lactam. Thus, the antimycobacterial activity of pyrithione-containing cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states.

Identification of a Mycothiol-Dependent Nitroreductase from Mycobacterium tuberculosis.

The success of Mycobacterium tuberculosis (Mtb) as a pathogen depends on the redundant and complex mechanisms it has evolved for resisting nitrosative and oxidative stresses inflicted by host immunity. Improving our understanding of these defense pathways can reveal vulnerable points in Mtb pathogenesis. In this study, we combined genetic, structural, computational, biochemical, and biophysical approaches to identify a novel enzyme class represented by Rv2466c. We show that Rv2466c is a mycothiol-dependent nitroreductase of Mtb and can reduce the nitro group of a novel mycobactericidal compound using mycothiol as a cofactor. In addition to its function as a nitroreductase, Rv2466c confers partial protection to menadione stress.

N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis.

The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-ß-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.

Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis.

We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a ß-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells.

Benzimidazole-based compounds kill Mycobacterium tuberculosis.

Tuberculosis remains one of the deadliest infectious diseases, killing 1.4 million people annually and showing a rapid increase in cases resistant to multiple drugs. New antibiotics against tuberculosis are urgently needed. Here we describe the design, synthesis and structure-activity relationships of a series of benzimidazole-based compounds with activity against Mycobacterium tuberculosis (Mtb) in a replicating state, a physiologically-induced non-replicating state, or both. Compounds 49, 67, 68, 69, 70, and 72, which shared a 5-nitrofuranyl moiety, exhibited high potency and acceptable selectivity indices (SI). As illustrated by compound 70 (MIC90 < 0.049 µg/mL, SI > 512), the 5-nitrofuranyl group was compatible with minimal cytotoxicity and good intra-macrophage killing, although it lacked non-replicating activity when assessed by CFU assays. Compound 70 had low mutagenic potential by SOS Chromotest assay, making this class of compounds good candidates for further evaluation and target identification.

A time to weep and a time to laugh: humour in the nurse-patient relationship in an adult cancer setting.

PURPOSE: The literature highlights the value of humour in health-care settings. Humour impacts on the physiological, psychosocial and cognitive well-being of a person. The diagnosis of cancer is extremely stressful, and treatments are difficult. Patients and nurses may use humour as a coping mechanism to contend with the stresses caused directly or indirectly by cancer. This study investigated the use of humour during interactions between patients and nurses in an adult cancer ward. METHODS: This study used a modified ethnographic methodology that included fieldwork participant observation and informal interviews (n=30 h) and formal interviews (n=10). In total, 9 nurses and 12 patients participated in participant observation. From these, five nurses and five patients were interviewed. Data were analysed using thematic analysis. RESULTS: Findings highlighted the importance of humour in the nurse-patient relationship. Patients consciously use humour during nurse-patient interactions in an attempt to help nurses cope with the stress they encounter in clinical practice. Patients perceive having a sense of humour as a positive nurse attribute. Nurses expressed concerns regarding compromising professionalism when using humour and felt the need for guidance from senior staff. Constant assessment and reflection help ensure humour is used appropriately in the adult health-care setting. CONCLUSION: The benefits of humour are recognised by both adult cancer patients and nurses. A deeper understanding of patient and nurse perceptions of the use of humour can inform strategies for its therapeutic use in the clinical setting.

Humour in adult cancer care: a concept analysis.

AIMS: To report an analysis of the concept of humour in adult cancer care. BACKGROUND: Humour is a form of communication which is present in the adult cancer setting. Numerous studies show the multi-dimensional value of humour in cancer care. A clear conceptual understanding, however, of what it represents is lacking. DESIGN: Walker and Avant's framework was used to guide this concept analysis. DATA SOURCES: Literature searches included bibliographic databases, internet, and manual searches. REVIEW METHODS: Literature published from 1990 to the present was reviewed. Thematic analysis was carried out to identify critical attributes and antecedents. RESULTS: Based on the analysis, a definition of humour in adult cancer nursing is proposed. Humour is a subjective emotional response, resulting from the recognition and expression of incongruities of a comic, absurd and impulsive situation, remark, character, or action, which enhances feelings of closeness or togetherness when shared in the context of trust between the patient and nurse and may be used as a coping mechanism in a stressful situation such as the adult cancer care setting. CONCLUSION: The analysis provides an understanding of the concept of humour in the adult cancer setting and includes a theoretical illustration of its critical attributes. This concept analysis provides a forum for discussion with reference to the use of humour in adult cancer nursing care. Further exploration is recommended to determine the meaning of humour and its nature across different care settings.

Nonsteroidal anti-inflammatory drug sensitizes Mycobacterium tuberculosis to endogenous and exogenous antimicrobials.

Existing drugs are slow to eradicate Mycobacterium tuberculosis (Mtb) in patients and have failed to control tuberculosis globally. One reason may be that host conditions impair Mtb's replication, reducing its sensitivity to most antiinfectives. We devised a high-throughput screen for compounds that kill Mtb when its replication has been halted by reactive nitrogen intermediates (RNIs), acid, hypoxia, and a fatty acid carbon source. At concentrations routinely achieved in human blood, oxyphenbutazone (OPB), an inexpensive anti-inflammatory drug, was selectively mycobactericidal to nonreplicating (NR) Mtb. Its cidal activity depended on mild acid and was augmented by RNIs and fatty acid. Acid and RNIs fostered OPB's 4-hydroxylation. The resultant 4-butyl-4-hydroxy-1-(4-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione (4-OH-OPB) killed both replicating and NR Mtb, including Mtb resistant to standard drugs. 4-OH-OPB depleted flavins and formed covalent adducts with N-acetyl-cysteine and mycothiol. 4-OH-OPB killed Mtb synergistically with oxidants and several antituberculosis drugs. Thus, conditions that block Mtb's replication modify OPB and enhance its cidal action. Modified OPB kills both replicating and NR Mtb and sensitizes both to host-derived and medicinal antimycobacterial agents.

Genome-wide screen for Mycobacterium tuberculosis genes that regulate host immunity.

In spite of its highly immunogenic properties, Mycobacterium tuberculosis (Mtb) establishes persistent infection in otherwise healthy individuals, making it one of the most widespread and deadly human pathogens. Mtb's prolonged survival may reflect production of microbial factors that prevent even more vigorous immunity (quantitative effect) or that divert the immune response to a non-sterilizing mode (qualitative effect). Disruption of Mtb genes has produced a list of several dozen candidate immunomodulatory factors. Here we used robotic fluorescence microscopy to screen 10,100 loss-of-function transposon mutants of Mtb for their impact on the expression of promoter-reporter constructs for 12 host immune response genes in a mouse macrophage cell line. The screen identified 364 candidate immunoregulatory genes. To illustrate the utility of the candidate list, we confirmed the impact of 35 Mtb mutant strains on expression of endogenous immune response genes in primary macrophages. Detailed analysis focused on a strain of Mtb in which a transposon disrupts Rv0431, a gene encoding a conserved protein of unknown function. This mutant elicited much more macrophage TNFα, IL-12p40 and IL-6 in vitro than wild type Mtb, and was attenuated in the mouse. The mutant list provides a platform for exploring the immunobiology of tuberculosis, for example, by combining immunoregulatory mutations in a candidate vaccine strain.

Acid-susceptible mutants of Mycobacterium tuberculosis share hypersusceptibility to cell wall and oxidative stress and to the host environment.

Mycobacterium tuberculosis can persist in macrophage phagosomes that acidify to a pH of approximately 4.5 after activation of the macrophage with gamma interferon. How the bacterium resists the low pH of the acidified phagosome is incompletely understood. A screen of 10,100 M. tuberculosis transposon mutants for mutants hypersensitive to pH 4.5 led to the discovery of 21 genes whose disruption attenuated survival of M. tuberculosis at a low pH (41). Here, we show that acid-sensitive M. tuberculosis mutants with transposon insertions in Rv2136c, Rv2224c, ponA2, and lysX were hypersensitive to antibiotics, sodium dodecyl sulfate, heat shock, and reactive oxygen and nitrogen intermediates, indicating that acid resistance can be associated with protection against other forms of stress. The Rv2136c mutant was impaired in intrabacterial pH homeostasis and unable to maintain a neutral intrabacterial pH in activated macrophages. The Rv2136c, Rv2224c, and ponA2 mutants were attenuated in mice, with the Rv2136c mutant displaying the most severe level of attenuation. Pathways utilized by M. tuberculosis for acid resistance and intrabacterial pH maintenance are potential targets for chemotherapy.

Identification of a copper-binding metallothionein in pathogenic mycobacteria.

A screen of a genomic library from Mycobacterium tuberculosis (Mtb) identified a small, unannotated open reading frame (MT0196) that encodes a 4.9-kDa, cysteine-rich protein. Despite extensive nucleotide divergence, the amino acid sequence is highly conserved among mycobacteria that are pathogenic in vertebrate hosts. We synthesized the protein and found that it preferentially binds up to six Cu(I) ions in a solvent-shielded core. Copper, cadmium and compounds that generate nitric oxide or superoxide induced the gene's expression in Mtb up to 1,000-fold above normal expression. The native protein bound copper within Mtb and partially protected Mtb from copper toxicity. We propose that the product of the MT0196 gene be named mycobacterial metallothionein (MymT). To our knowledge, MymT is the first metallothionein of a Gram-positive bacterium with a demonstrated function.

Chemical inhibitors of TNF signal transduction in human neutrophils point to distinct steps in cell activation.

Chemical screening identified three small compounds that selectively inhibited activation of the respiratory burst (RB) of human neutrophils in response to tumor necrosis factor (TNF) and formylated peptide but not phorbol ester and spared the ability of neutrophils to kill bacteria. These compounds partially inhibited TNF-triggered cytoskeletal rearrangements without blocking adhesion or transmigation of polymorphonuclear neutrophils through TNF-activated monolayers of endothelial cells. The compounds were nontoxic to neutrophils and endothelial cells. They had no direct inhibitory effect on the tyrosine kinases Src, Syk, or Pyk2. However, their differential effects on cell spreading, bacteria-induced RB, TNF-induced degranulation, TNF-induced protein tyrosine phosphorylation, and TNF-induced Syk activation suggested that each may act on different elements of neutrophil signaling pathways.

Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils.

Through chemical screening, we identified a pyrazolone that reversibly blocked the activation of phagocyte oxidase (phox) in human neutrophils in response to tumor necrosis factor (TNF) or formylated peptide. The pyrazolone spared activation of phox by phorbol ester or bacteria, bacterial killing, TNF-induced granule exocytosis and phox assembly, and endothelial transmigration. We traced the pyrazolone's mechanism of action to inhibition of TNF-induced intracellular Ca2+ elevations, and identified a nontransmembrane ("soluble") adenylyl cyclase (sAC) in neutrophils as a Ca2+-sensing source of cAMP. A sAC inhibitor mimicked the pyrazolone's effect on phox. Both compounds blocked TNF-induced activation of Rap1A, a phox-associated guanosine triphosphatase that is regulated by cAMP. Thus, TNF turns on phox through a Ca2+-triggered, sAC-dependent process that may involve activation of Rap1A. This pathway may offer opportunities to suppress oxidative damage during inflammation without blocking antimicrobial function.

Medicines management in care homes for older people: the nurse's role.

Increasing attention is being paid to the inappropriate use of medicines in UK care homes for older people. While polypharmacy may sometimes be necessary, older people can be particularly sensitive to the effects of medicines due to physiological chances, while the effect of a mixture of medicines is often unknown. The harmful consequences of inappropriate prescribing for older people unnecessarily add to overall health-care expenditure and the workload of staff. Community nurses can play an important role in attenuating some of these problems while enhancing the quality of life of older people in care homes. The growing population of older people and burgeoning health-care expenditure on medicines demands that greater attention is given to medicine management among older people so that the efficacy of therapeutic regimes are maximized.