Rational design, synthesis and testing of novel tricyclic topoisomerase inhibitors for the treatment of bacterial infections part 1.

The alarming reduction in drug effectiveness against bacterial infections has created an urgent need for the development of new antibacterial agents that circumvent bacterial resistance mechanisms. We report here a series of DNA gyrase and topoisomerase IV inhibitors that demonstrate potent activity against a range of Gram-positive and selected Gram-negative organisms, including clinically-relevant and drug-resistant strains. In part 1, we present a detailed structure activity relationship (SAR) analysis that led to the discovery of our previously disclosed compound, REDX05931, which has a minimum inhibitory concentration (MIC) of 0.06 µg mL-1 against fluoroquinolone-resistant Staphylococcus aureus. Although in vitro hERG and CYP inhibition precluded further development, it validates a rational design approach to address this urgent unmet medical need and provides a scaffold for further optimisation, which is presented in part 2.

Rational design, synthesis and testing of novel tricyclic topoisomerase inhibitors for the treatment of bacterial infections part 2.

Building on our previously-reported novel tricyclic topoisomerase inhibitors (NTTIs), we disclose the discovery of REDX07965, which has an MIC90 of 0.5 µg mL-1 against Staphylococcus aureus, favourable in vitro pharmacokinetic properties, selectivity versus human topoisomerase II and an acceptable toxicity profile. The results herein validate a rational design approach to address the urgent unmet medical need for novel antibiotics.

Attenuated stress response to acute restraint and forced swimming stress in arginine vasopressin 1b receptor subtype (Avpr1b) receptor knockout mice and wild-type mice treated with a novel Avpr1b receptor antagonist.

Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin-releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic-pituitary-adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild-type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress-induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild-type mice that had been pre-treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint-stressed knockout male mice, and in Avpr1b-antagonist-treated male wild-type mice. By contrast, plasma CORT levels were not reduced in acutely restraint-stressed female knockout animals, or in female wild-type animals pre-treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic-pituitary-adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors.

Differential effects of the new glucocorticoid receptor antagonist ORG 34517 and RU486 (mifepristone) on glucocorticoid receptor nuclear translocation in the AtT20 cell line.

Glucocorticoid agonists bind to cytoplasmic glucocorticoid receptors (GRs) and subsequently translocate as an agonist-GR complex into the nucleus. In the nucleus the complex regulates the transcription of target genes. A number of GR antagonists (RU486, progesterone, RU40555) have also been shown to induce receptor translocation. These compounds should be regarded as partial agonists. For the nonselective progesterone receptor antagonists, RTI3021-012 and RTI3021-022, it was shown that GR antagonism is possible without the induction of GR translocation. In the present studies, the new GR antagonist, ORG 34517, was investigated for its potential to induce GR translocation and to antagonize corticosterone-induced GR translocation in the AtT20 (mouse pituitary) cell line. ORG 34517 was compared to RU486. In contrast to RU486, ORG 34517 (at doses up to 3 x 10(-7) M) did not induce GR translocation, but was able to block corticosterone (3 x 10(-8) M) induced GR translocation. ORG 34517 can be regarded as a true competitive GR antagonist without partial agonistic activities.

Effect of the glucocorticoid receptor antagonist Org 34850 on basal and stress-induced corticosterone secretion.

The activity of the hypothalamic-pituitary-adrenal (HPA) axis is characterised both by an ultradian pulsatile pattern of glucocorticoid secretion and an endogenous diurnal rhythm. Glucocorticoid feedback plays a major role in regulating HPA axis activity and this mechanism occurs via two different receptors: mineralocorticoid (MR) and glucocorticoid receptors (GR). In the present study, the effects of both acute and subchronic treatment with the GR antagonist Org 34850 on basal and stress-induced HPA axis activity in male rats were evaluated. To investigate the effect of Org 34850 on basal diurnal corticosterone rhythm over the 24-h cycle, an automated blood sampling system collected samples every 10 min. Acute injection of Org 34850 (10 mg/kg, s.c.) did not affect basal or stress-induced corticosterone secretion, but was able to antagonise the inhibitory effect of the glucocorticoid agonist methylprednisolone on stress-induced corticosterone secretion. However, 5 days of treatment with Org 34850 (10 mg/kg, s.c., two times a day), compared to rats treated with vehicle (5% mulgofen in 0.9% saline, 1 ml/kg, s.c.), increased corticosterone secretion over the 24-h cycle and resulted in changes in the pulsatile pattern of hormone release, but had no significant effect on adrenocorticotrophic hormone secretion or on stress-induced corticosterone secretion. Subchronic treatment with Org 34850 did not alter GR mRNA expression in the hippocampus, paraventricular nucleus of the hypothalamus or anterior-pituitary, or MR mRNA expression in the hippocampus. Our data suggest that a prolonged blockade of GRs is required to increase basal HPA axis activity. The changes observed here with ORG 34850 are consistent with inhibition of GR-mediated negative feedback of the HPA axis. In light of the evidence showing an involvement of dysfunctional HPA axis in the pathophysiology of depression, Org 34850 could be a potential treatment for mood disorders.

Oxidised adenosine 5'-triphosphate, a P2X(7) antagonist, is toxic to rat cerebellar granule neurones in vitro.

Adenosine 5'-triphosphate (ATP) acts as a neurotransmitter in the central nervous system. Extracellular ATP is also toxic to a number of cell types e.g. via its interaction with P2X membrane receptors, specifically the P2X(7) family member. These results have led to the hypothesis that elevated ATP levels may exacerbate damage during acute neurodegeneration [4]. The aim of this study was to examine the effects of ATP agonists and antagonists on cultured rat cerebellar granule neurones. Neither ATP, nor the P2X agonist benzoylbenzoyl-ATP (BzATP), were toxic when added to primary neurones. However, the P2X(7) antagonist, oxidised ATP (oATP) was highly neurotoxic. This toxicity was inhibited by co-incubation with BzATP. These results demonstrate that oATP is a potent neurotoxin.

Influence of corticotrophin releasing factor on neuronal cell death in vitro and in vivo.

Several studies have demonstrated that antagonists of the corticotrophin releasing factor (CRF) receptor markedly inhibit experimentally induced excitotoxic, ischaemic and traumatic brain injury in the rat, and that CRF expression is elevated in response to experimentally induced stroke or traumatic brain injury. CRF is also induced by the pro-inflammatory cytokine interleukin 1 (IL-1), which participates in various forms of neurodegeneration. The aim of this study was to test the hypothesis that CRF is toxic directly in vivo or in vitro. In primary cultures of rat cortical neurons, exposure to CRF (10 pM-100 nM) for 24 h failed to cause cell death directly, or to modify the neurotoxic effects of N-methyl-D-aspartate (NMDA). Similarly, infusion of CRF (0.3-5 microg) into specific brain regions of the rat did not induce cell death and did not significantly alter the neuronal damage produced by infusion of excitatory amino acids. These data demonstrate that CRF is not directly neurotoxic, and suggest that either CRF mediates neuronal damage by indirect actions (e.g. on the vasculature) and/or that CRF is not the endogenous ligand which contributes to neurodegeneration through activation of CRF receptors.

Caspases mediate C2-ceramide-induced apoptosis of the human oligodendroglial cell line, MO3.13.

The signalling molecule ceramide participates in the sphingomyelin pathway and accumulates intracellularly in response to inflammatory mediators. Here we show that membrane permeable C2-ceramide is apoptogenic in the immortalised human oligodendroglial cell line MO3.13. Apoptosis (defined by cell shrinkage and chromatin condensation) is accompanied by caspase enzyme activation. Immunoblotting analysis of extracts from differentiated MO3.13 cells revealed the presence of caspase-3 proenzyme, activation by cleavage of pro-caspase-3 in cells treated with C2-ceramide and cleavage of the caspase substrates fodrin and rabaptin. Lysates also showed cleavage of a fluorogenic peptide substrate. Addition of the general caspase inhibitor BAF markedly attenuated apoptosis of MO3.13 oligodendroglia. A role for caspase-3-like enzymes in ceramide-induced apoptosis of oligodendroglia may have important implications for approaches to treatment of demyelinating diseases.

Human oligodendroglial cell line, MO3.13, can be protected from apoptosis using the general caspase inhibitor zVAD-FMK.

Recent evidence suggests that the oligodendrocyte cell loss observed in multiple sclerosis sufferers is in part mediated by apoptosis. Here we use a human cell line, MO3.13, as a model system to investigate the biochemical processes involved in oligodendroglial cell death. Treatment with staurosporine kills both naive and differentiated cells in a dose-dependent manner; however, much higher concentrations of staurosporine are required to kill differentiated cells compared to their naive progenitors. Dying cells displayed the typical morphological characteristics of apoptosis, including cell shrinkage and chromatin condensation. Biochemical analysis showed that caspases, a group of enzymes intimately involved in the execution of apoptosis, are activated in both naive and differentiated cells. Western blotting analysis revealed that similar subsets of caspase enzymes were operating and that the substrate cleavage patterns were identical in both naive and differentiated cells. Treatment of MO3.13 cells with the general caspase inhibitor zVAD-FMK protected them from toxin-induced cell death. These results indicate that when an oligodendroglial human cell line is exposed to toxin it dies in an apoptotic manner. In addition, we show that cells can be protected from toxin-induced death using an appropriate inhibitor.

An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding.

We have isolated a major integral membrane protein from Golgi-derived coatomer-coated vesicles. This 24-kDa protein, p24, defines a family of integral membrane proteins with homologs present in yeast and humans. In addition to sequence similarity, all p24 family members contain a motif with the characteristic heptad repeats found in coiled coils. When the yeast p24 isoform, yp24A, is knocked out in a strain defective for vesicle fusion, a dramatic reduction in the accumulation of transport vesicles is observed. Together, these results indicate a role for this protein family in the budding of coatamer-coated and other species of coated vesicles.

Post-translational processing of Schizosaccharomyces pombe YPT5 protein. In vitro and in vivo analysis of processing mutants.

SpYPT5p is a member of the rab/YPT small GTP-binding protein family, which is believed to be involved in the regulation of intracellular trafficking. The protein sequence terminates with a CXC motif, and in our previous report (Newman, C. M. H., Giannakouros, T., Hancock, J. F., Fawell, E. H., Armstrong, J., and Magee, A. I. (1992) J. Biol. Chem. 267, 11329-11336) we have shown that SpYPT5p is prenylated both in vivo and in vitro, where geranylgeranylation was confirmed, and carboxyl-methylated. In order to dissect the role of prenylation of each cysteine, we have generated C-terminal mutants where either one or both cysteine(s) were replaced by serine and expressed them in vitro in reticulocyte lysates and in vivo in transfected COS cells. Our results suggest that both cysteines of the CXC motif are prenylated but that the rate of prenylation of the two cysteines is different. The upstream cysteine was found to be preferentially prenylated in reticulocyte lysates unless cytosol from COS cells was added. A separate activity could therefore be required for prenylation of the second cysteine, or the presence of an additional factor is needed to allow accumulation of doubly prenylated SpYPT5p. However, the modification of the upstream cysteine is not a prerequisite for the prenylation of the other. Furthermore, gene replacement in Schizosaccharomyces pombe revealed that each cysteine of the CXC motif can individually support function. Carboxyl methylation occurred only on protein which had been prenylated on the C-terminal cysteine and was required for efficient membrane binding in vitro.

Function of the ypt2 gene in the exocytic pathway of Schizosaccharomyces pombe.

The ypt2 gene of the fission yeast Schizosaccharomyces pombe encodes a member of the ypt/rab family of small GTP-binding proteins, related in sequence to Sec4p of Saccharomyces cerevisiae but closer to mammalian rab8. We have introduced a mutation into the gene corresponding to a mutation identified in ypt1, in which a conserved valine residue was altered to asparagine. The mutated ypt2 gene was introduced into the S. pombe genome by gene replacement. The resulting strain was temperature-sensitive for growth. Normal growth was restored by introduction of a plasmid-borne wild-type ypt2 cDNA or by cDNA for rab8 but not by various other rab or ypt sequences. At restrictive temperature the mutant cells accumulated the secretory protein acid phosphatase in a form that appeared to be fully glycosylated and acquired a population of vesicles detectable by electron microscopy. Thus the ypt2 protein, and by inference rab8, appear to function in the last stage of the secretory pathway.

Schizosaccharomyces pombe ypt5: a homologue of the rab5 endosome fusion regulator.

The ypt/rab proteins are a family of small GTP-binding proteins thought to be required for different stages of membrane traffic. From the fission yeast Schizosaccharomyces pombe we have isolated and characterized ypt5, a gene encoding a homologue of rab5, a mammalian protein apparently involved in regulating fusion of early endosomes. Recombinant ypt5 protein bound GTP. The ypt5 gene was found to be essential for viability on minimal media, but ypt5-disrupted cells grew slowly on some rich media and accumulated a population of small vesicles not observed in wild-type cells. Canine rab5 cDNA could replace the ypt5 gene in S. pombe and restore normal growth and viability. Ypt5 protein expressed in mammalian cells colocalized with the transferrin receptor to early endosomes. Thus, molecular aspects of the early endocytic pathway may be conserved between mammalian cells and S. pombe and hence may be amenable to genetic analysis.