In Vitro Activity of Novel Topoisomerase Inhibitors against Francisella tularensis and Burkholderia pseudomallei.

Antimicrobial resistance is a global issue, and the investigation of alternative therapies that are not traditional antibiotics are warranted. Novel bacterial type II topoisomerase inhibitors (NBTIs) have recently emerged as a novel class of antibiotics with reduced potential for cross-resistance to fluoroquinolones due to their novel mechanism of action. This study investigated the in vitro activity of a series of cyclohexyl-oxazolidinone bacterial topoisomerase inhibitors against type strains of Francisella tularensis and Burkholderia pseudomallei. Broth microdilution, time-kill, and cell infection assays were performed to determine activity against these biothreat pathogens. Two candidates were identified that demonstrated in vitro activity in multiple assays that in some instances was equivalent to ciprofloxacin and doxycycline. These data warrant the further evaluation of these novel NBTIs and future iterations in vitro and in vivo.

Discovery and structure-activity relationships of a novel oxazolidinone class of bacterial type II topoisomerase inhibitors.

There is an increasingly urgent and unmet medical need for novel antibiotic drugs that tackle infections caused by multidrug-resistant (MDR) pathogens. Novel bacterial type II topoisomerase inhibitors (NBTIs) are of high interest due to limited cross-resistance with fluoroquinolones, however analogues with Gram-negative activity often suffer from hERG channel inhibition. A novel series of bicyclic-oxazolidinone inhibitors of bacterial type II topoisomerase were identified which display potent broad-spectrum anti-bacterial activity, including against MDR strains, along with an encouraging in vitro safety profile. In vivo proof of concept was achieved in a A. baumannii mouse thigh infection model.

Teixobactin and Its Analogues: A New Hope in Antibiotic Discovery.

Increasing bacterial resistance against current antibiotics and lack of new molecules to combat bacterial resistance are key challenges to global health. There is, therefore, a continuing need to develop new antibiotics. Teixobactin, a cyclic undecapeptide, displays excellent antibacterial activities against a range of pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis. Interestingly, it operates by multiple modes of actions and is bactericidal toward S. aureus without detectable resistance. This unique combination of wide Gram-positive activity coupled with its inability to elicit resistance make teixobactin a very attractive molecule for antimicrobial therapeutic development. This Viewpoint discusses teixobactin, its analogues, and the challenges and opportunities associated with their future development.

Novel Bacterial Topoisomerase Inhibitors with Potent Broad-Spectrum Activity against Drug-Resistant Bacteria.

The novel bacterial topoisomerase inhibitor class is an investigational type of antibacterial inhibitor of DNA gyrase and topoisomerase IV that does not have cross-resistance with the quinolones. Here, we report the evaluation of the in vitro properties of a new series of this type of small molecule. Exemplar compounds selectively and potently inhibited the catalytic activities of Escherichia coli DNA gyrase and topoisomerase IV but did not block the DNA breakage-reunion step. Compounds showed broad-spectrum inhibitory activity against a wide range of Gram-positive and Gram-negative pathogens, including biodefence microorganisms and Mycobacterium tuberculosis No cross-resistance with fluoroquinolone-resistant Staphylococcus aureus and E. coli isolates was observed. Measured MIC90 values were 4 and 8 µg/ml against a panel of contemporary multidrug-resistant isolates of Acinetobacter baumannii and E. coli, respectively. In addition, representative compounds exhibited greater antibacterial potency than the quinolones against obligate anaerobic species. Spontaneous mutation rates were low, with frequencies of resistance typically <10-8 against E. coli and A. baumannii at concentrations equivalent to 4-fold the MIC. Compound-resistant E. coli mutants that were isolated following serial passage were characterized by whole-genome sequencing and carried a single Arg38Leu amino acid substitution in the GyrA subunit of DNA gyrase. Preliminary in vitro safety data indicate that the series shows a promising therapeutic index and potential for low human ether-a-go-go-related gene (hERG) inhibition (50% inhibitory concentration [IC50], >100 µM). In summary, the compounds' distinct mechanism of action relative to the fluoroquinolones, whole-cell potency, low potential for resistance development, and favorable in vitro safety profile warrant their continued investigation as potential broad-spectrum antibacterial agents.

Discovery and structure-activity relationships of a novel isothiazolone class of bacterial type II topoisomerase inhibitors.

There is an urgent and unmet medical need for new antibacterial drugs that tackle infections caused by multidrug-resistant (MDR) pathogens. During the course of our wider efforts to discover and exploit novel mechanism of action antibacterials, we have identified a novel series of isothiazolone based inhibitors of bacterial type II topoisomerase. Compounds from the class displayed excellent activity against both Gram-positive and Gram-negative bacteria with encouraging activity against a panel of MDR clinical Escherichia coli isolates when compared to ciprofloxacin. Representative compounds also displayed a promising in vitro safety profile.

Efficacy of a Novel Tricyclic Topoisomerase Inhibitor in a Murine Model of Neisseria gonorrhoeae Infection.

There is an urgent need for new antibiotics to treat multidrug-resistant Neisseria gonorrhoeae In this report, the microbiology, in vivo pharmacokinetics, and efficacy of REDX05931, a representative novel tricyclic topoisomerase inhibitor, were evaluated. REDX05931 demonstrated high oral bioavailability in mice and reduced N. gonorrhoeae infection after a single dose in a mouse model of gonorrhea. These data support the potential of this series of small molecules as a new treatment for drug-resistant gonorrheal infections.

Optimisation of pharmacokinetic properties to afford an orally bioavailable and selective V1A receptor antagonist.

The previously described lead compound 5 is a potent and selective V(1A) antagonist with affinity at both the rat and human receptor, but displays poor oral bioavailability and moderate clearance. We report herein the successful optimisation of the pharmacokinetic (PK) properties to afford the potent, selective, orally bioavailable and CNS penetrant compound 15f. A custom optimisation approach was required which demonstrated the value of using early, rapid in vivo PK studies to show improvements in oral exposure. Such assays may be of particular value where low oral bioavailability is anticipated to be multifactorial (e.g., permeability, gut wall metabolism and/or transport) where satisfactory modelling of in vitro data is likely to be difficult within a drug discovery context.

Synthesis and SAR studies of novel 2-(6-aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists.

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) antagonists are described. 2-(6-Aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and OT. Optimised compound 16 shows a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.

Identification and optimisation of novel sulfonamide, selective vasopressin V1B receptor antagonists.

The synthesis and preliminary structure-activity relationships (SAR) of a novel class of vasopressin V(1B) receptor antagonists are described. Hit compound 5, identified via high throughput screening of the corporate collection, showed good activity in a V(1B) binding assay (K(i) 63 nM) but did not possess the lead-like physicochemical properties typically required in a hit compound. A 'deletion approach' on the HTS hit 5 was performed, with the focus on improvement of physicochemical properties, yielding the selective V(1B) antagonist 9f (K(i) 190 nM), with improved druglike characteristics.

The discovery of novel 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamides as vasopressin V1A receptor antagonists.

The discovery of a novel series of 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamide antagonists of the vasopressin V(1A) receptor is disclosed. Compounds 47 and 48 were found to be high affinity, selective vasopressin V(1A) antagonists.

Synthesis and SAR studies of novel 2-(4-oxo-2-aryl-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists.

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) (V(3)) antagonists are described. 2-(4-Oxo-2-aryl-quinazolin-3(4H)-yl)acetamides have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and oxytocin (OT). Optimised compound 12j demonstrates a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.

Differential effect of glucocorticoid receptor antagonists on glucocorticoid receptor nuclear translocation and DNA binding.

The effects of RU486 and S-P, a more selective glucocorticoid receptor antagonist from Schering-Plough, were investigated on glucocorticoid receptor nuclear translocation and DNA binding. In the in vitro study, AtT20 cells were treated with vehicle or with RU486, S-P or corticosterone (3-300 nM) or co-treated with vehicle or glucocorticoid receptor antagonists (3-300 nM) and 30 nM corticosterone. Both glucocorticoid receptor antagonists induced glucocorticoid receptor nuclear translocation but only RU486 induced DNA binding. RU486 potentiated the effect of corticosterone on glucocorticoid receptor nuclear translocation and DNA binding, S-P inhibited corticosterone-induced glucocorticoid receptor nuclear translocation, but not glucocorticoid receptor-DNA binding. In the in vivo study, adrenalectomized rats were treated with vehicle, RU486 (20 mg/kg) and S-P (50 mg/kg) alone or in combination with corticosterone (3 mg/kg). RU486 induced glucocorticoid receptor nuclear translocation in the pituitary, hippocampus and prefrontal cortex and glucocorticoid receptor-DNA binding in the hippocampus, whereas no effect of S-P on glucocorticoid receptor nuclear translocation or DNA binding was observed in any of the areas analysed. These findings reveal differential effects of RU486 and S-P on areas involved in regulation of hypothalamic-pituitary-adrenal axis activity in vivo and they are important in light of the potential use of this class of compounds in the treatment of disorders associated with hyperactivity of the hypothalamic-pituitary-adrenal axis.

Discovery and optimisation of a selective non-steroidal glucocorticoid receptor antagonist.

High-throughput screening of 3.87 million compounds delivered a novel series of non-steroidal GR antagonists. Subsequent rounds of optimisation allowed progression from a non-selective ligand with a poor ADMET profile to an orally bioavailable, selective, stable, glucocorticoid receptor antagonist.

Antiglucocorticoids, neurogenesis and depression.

Recent evidence suggests that antiglucocorticoids, like conventional antidepressants, may recover depressive symptoms by boosting hippocampal neurogenesis. Here, we explore several possible antiglucocorticoid-based antidepressive therapeutic strategies. Firstly, we review specific glucocorticoid receptor/antagonist interactions. Secondly, we discuss a potential new therapeutic target, doublecortin-like kinase, which regulates glucocorticoid signaling in neuronal progenitor cells.

Blockade of the V(1b) receptor reduces ACTH, but not corticosterone secretion induced by stress without affecting basal hypothalamic-pituitary-adrenal axis activity.

Vasopressin (AVP), produced in parvocellular neurons of the hypothalamic paraventricular nucleus, regulates, together with CRH, pituitary ACTH secretion. The pituitary actions of AVP are mediated through the G protein receptor V(1b) (V(1b)|R). In man, hyperactivity of the hypothalamic-pituitary-adrenal axis has been associated with depression and other stress-related conditions. There are also clinical data suggesting a role for AVP in the dysfunctional HPA axis described in some depressed patients. In this study, we have investigated the effect of a recently synthesised selective antagonist of the V(1b)R both on exogenous AVP-induced ACTH and corticosterone secretion, and on basal and stress-induced pituitary-adrenal activity. Adult male Sprague-Dawley rats treated with the V(1b)R antagonist (Org, 30 mg/kg, s.c.) or vehicle (5% mulgofen in 0.9% saline, 2 ml/kg, s.c.). We found that blockade of the V(1b)R reduced the increase in both ACTH and corticosterone secretion induced by AVP (100 ng, i.v.). The same treatment had no effect either on basal ACTH and corticosterone levels or on the ultradian or diurnal rhythms of corticosterone secretion. Acute administration of the V(1b)R antagonist reduced ACTH secretion following both restraint and lipopolysaccharide, but did not antagonise the ACTH response to noise. The same treatment did not reduce corticosterone secretion in response to any of the three stressors used in this study. Our results confirm that this compound is an antagonist of the V(1b)R in the rat, and that its ability to reduce stress-induced ACTH responses is stressor dependent with differential modulation of pituitary and adrenal responses.

Effect of vasopressin 1b receptor blockade on the hypothalamic-pituitary-adrenal response of chronically stressed rats to a heterotypic stressor.

Exposure to chronic restraint (CR) modifies the hypothalamic-pituitary-adrenal (HPA) axis response to subsequent acute stressors with adaptation of the response to a homotypic and sensitization of the response to a heterotypic stressor. Since vasopressin (AVP) activity has been reported to change during chronic stress, we investigated whether this was an important factor in HPA facilitation. We therefore tested whether vasopressin 1b receptor (AVPR1B) blockade altered the ACTH and corticosterone response to heterotypic stressors following CR stress. Adult male rats were exposed to CR, single restraint, or were left undisturbed in the home cage. Twenty-four hours after the last restraint, rats were injected with either a AVPR1B antagonist (Org, 30 mg/kg, s.c.) or vehicle (5% mulgofen in saline, 0.2/kg, s.c.) and then exposed to either restraint, lipopolysaccharide (LPS) or white noise. CR resulted in the adaptation of the ACTH and corticosterone response to restraint and this effect was not prevented by pretreatment with Org. Although we found no effect of CR on LPS-induced ACTH and corticosterone secretion, both repeated and single episodes of restraint induced the sensitization of the ACTH, but not corticosterone response to acute noise. Pretreatment with Org reduced the exaggerated ACTH response to noise after both single and repeated exposure to restraint.

Glucocorticoid receptor antagonism augments fluoxetine-induced downregulation of the 5-HT transporter.

The effects of combined treatment with a glucocorticoid receptor (GR) antagonist, Org 34850, and a selective serotonin reuptake inhibitor (SSRI), fluoxetine, were investigated on pre- and postsynaptic aspects of 5-HT neurotransmission. Rats were treated for 14 days with Org 34850 (15 mg per kg per day subcutaneously), fluoxetine (10 mg per kg per day intraperitoneally), or a combination of both drugs. [(3)H]-citalopram binding (an index of 5-HT transporter (5-HTT) expression) was only slightly affected by Org 34850 alone: decreased in cortex and midbrain and increased in hippocampus. In contrast, chronic fluoxetine markedly decreased 5-HTT levels in all regions. Importantly, this decrease was significantly enhanced by combined Org 34850/fluoxetine treatment. There were no changes in the expression of 5-HTT mRNA, suggesting these effects were not due to changes in gene transcription. Expression of tryptophan hydroxylase mRNA and both 5-HT(1A) autoreceptor mRNA and protein (assessed using [(3)H]-8-OH-DPAT binding) were unchanged by any treatment. The expression of postsynaptic 5-HT(1A) receptor protein in the forebrain was unaltered by fluoxetine, Org 34850 or the combined Org 34850/fluoxetine treatment. This downregulation of 5-HTT by fluoxetine and its enhancement by Org 34850 can explain our recent observation that GR antagonists augment the SSRI-induced increase in extracellular 5-HT. In addition, these data suggest that the augmentation of forebrain 5-HT does not result in downregulation of forebrain 5-HT(1A) receptor expression. Given the importance of 5-HT(1A) receptor-mediated transmission in the forebrain to the antidepressant response, these data indicate that co-administration of GR antagonists may be effective in augmenting the antidepressant response to SSRI treatment.

Characterization of a novel and selective V1B receptor antagonist.

It has been argued that hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis is a major biological abnormality in patients suffering from psychiatric conditions such as major depression. Both arginine vasopressin (AVP) and corticotrophin releasing factor (CRF) are responsible for stimulating the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary. CRF is thought to be the predominant secretagogue under normal conditions but AVP may play a more important role in situations of aberrant/chronic stress. Studies in patients suffering from melancholic depression indicate a hyper-responsiveness to agonism at the vasopressin receptor type 1B (V(1B)); patients display a heightened ACTH release after challenge with the mixed V(1B)/V(2) (vasopressin receptor type 2) agonist desmopressin in comparison to control subjects. A V(1B) antagonist has been developed which has significant selectivity for the human V(1B) receptor over the other members of the vasopressin receptor sub-family. The compound acts as an effective antagonist at both the human recombinant receptor (stably expressed in Chinese hamster ovary (CHO) cells) and the native rat V(1B) receptor (using isolated anterior pituitary cells), blocking the induction of luciferase and the release of ACTH, respectively. In vivo the compound can block the release of ACTH after challenge with a variety of V(1B) agonists. It can also attenuate the ACTH response to acute stressors in rats. Interestingly, this compound does not modulate the activity of the HPA axis under normal basal conditions.

Effect of the glucocorticoid receptor antagonist Org 34850 on fast and delayed feedback of corticosterone release.

We investigated the effect of the glucocorticoid receptor (GR) antagonist Org 34850 on fast and delayed inhibition of corticosterone secretion in response to the synthetic glucocorticoid methylprednisolone (MPL). Male rats were implanted with a catheter in the right jugular vein, for blood sampling and MPL administration, and with an s.c. cannula for Org 34850 administration. All experiments were conducted at the diurnal hormonal peak in the late afternoon. Rats were connected to an automated sampling system and blood samples were collected every 5 or 10 min. Org 34850 (10 mg/kg, s.c.) or vehicle (5% mulgofen in saline) was injected at 1630 h; 30 min later, rats received an injection of MPL (500 microg/rat, i.v.) or saline (0.1 ml/rat). We found that an acute administration of MPL rapidly decreased the basal corticosterone secretion and this effect was not prevented by acute pretreatment with Org 34850. However, blockade of GR with Org 34850 prevented delayed inhibition of MPL on corticosterone secretion measured between 4 and 12 h after MPL administration. Our data suggest an involvement of GR in modulating delayed, but not fast, inhibition induced by MPL on basal corticosterone secretion.

Innovative approaches for the treatment of depression: targeting the HPA axis.

Altered activity of the hypothalamic pituitary adrenal (HPA) axis is one of the most commonly observed neuroendocrine abnormalities in patients suffering from major depressive disorder (MDD). Altered cortisol secretion can be found in as many as 80% of depressed patients. This observation has led to intensive clinical and preclinical research aiming to better understand the molecular mechanisms which underlie the alteration of the HPA axis responsiveness in depressive illness. Dysfunctional glucocorticoid receptor (GR) mediated negative feedback regulation of cortisol levels and changes in arginine vasopressin (AVP)/vasopressin V1b receptor and corticotrophin-releasing factor/CRF1 receptor regulation of adrenocotricotrophin (ACTH) release have all been implicated in over-activity of the HPA axis. Agents that intervene with the mechanisms involved in (dys)regulation of cortisol synthesis and release are under investigation as possible therapeutic agents. The current status of some of these approaches is described in this review.