From structure to clinic: Design of a muscarinic M1 receptor agonist with potential to treatment of Alzheimer's disease.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Small molecule AZD4635 inhibitor of A2AR signaling rescues immune cell function including CD103+ dendritic cells enhancing anti-tumor immunity.

Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated. METHODS: We report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition. RESULTS: We provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies. CONCLUSION: We provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.

Identification of novel adenosine A(2A) receptor antagonists by virtual screening.

Virtual screening was performed against experimentally enabled homology models of the adenosine A(2A) receptor, identifying a diverse range of ligand efficient antagonists (hit rate 9%). By use of ligand docking and Biophysical Mapping (BPM), hits 1 and 5 were optimized to potent and selective lead molecules (11-13 from 5, pK(I) = 7.5-8.5, 13- to >100-fold selective versus adenosine A(1); 14-16 from 1, pK(I) = 7.9-9.0, 19- to 59-fold selective).

Discovery of 1,2,4-triazine derivatives as adenosine A(2A) antagonists using structure based drug design.

Potent, ligand efficient, selective, and orally efficacious 1,2,4-triazine derivatives have been identified using structure based drug design approaches as antagonists of the adenosine A(2A) receptor. The X-ray crystal structures of compounds 4e and 4g bound to the GPCR illustrate that the molecules bind deeply inside the orthosteric binding cavity. In vivo pharmacokinetic and efficacy data for compound 4k are presented, demonstrating the potential of this series of compounds for the treatment of Parkinson's disease.

Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine.

Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A(2A) receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A(2A) selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins.

The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery.

G protein-coupled receptors (GPCRs) are one of the most important target classes in the central nervous system (CNS) drug discovery, however the fact they are integral membrane proteins and are unstable when purified out of the cell precludes them from a wide range of structural and biophysical techniques that are used for soluble proteins. In this study we demonstrate how protein engineering methods can be used to identify mutations which can both increase the thermostability of receptors, when purified in detergent, as well as biasing the receptor towards a specific physiologically relevant conformational state. We demonstrate this method for the adenosine A(2A) receptor and muscarinic M(1) receptor. The resultant stabilised receptors (known as StaRs) have a pharmacological profile consistent with the inverse agonist conformation. The stabilised receptors can be purified in large quantities, whilst retaining correct folding, thus generating reagents suitable for a broad range of structural and biophysical studies. In the case of the A(2A)-StaR we demonstrate that surface plasmon resonance can be used to profile the association and dissociation rates of a range of antagonists, a technique that can be used to improve the in vivo efficacy of receptor antagonists.

Neonatal enteral feeding tubes as loci for colonisation by members of the Enterobacteriaceae.

BACKGROUND: The objective of this study was to determine whether neonatal nasogastric enteral feeding tubes are colonised by the opportunistic pathogen Cronobacter spp. (Enterobacter sakazakii) and other Enterobacteriaceae, and whether their presence was influenced by the feeding regime. METHODS: One hundred and twenty-nine tubes were collected from two neonatal intensive care units (NICU). A questionnaire on feeding regime was completed with each sample. Enterobacteriaceae present in the tubes were identified using conventional and molecular methods, and their antibiograms determined. RESULTS: The neonates were fed breast milk (16%), fortified breast milk (28%), ready to feed formula (20%), reconstituted powdered infant formula (PIF, 6%), or a mixture of these (21%). Eight percent of tubes were received from neonates who were 'nil by mouth'. Organisms were isolated from 76% of enteral feeding tubes as a biofilm (up to 107 cfu/tube from neonates fed fortified breast milk and reconstituted PIF) and in the residual lumen liquid (up to 107 Enterobacteriaceae cfu/ml, average volume 250 mul). The most common isolates were Enterobacter cancerogenus (41%), Serratia marcescens (36%), E. hormaechei (33%), Escherichia coli (29%), Klebsiella pneumoniae (25%), Raoultella terrigena (10%), and S. liquefaciens (12%). Other organisms isolated included C. sakazakii (2%),Yersinia enterocolitica (1%),Citrobacter freundii (1%), E. vulneris (1%), Pseudomonas fluorescens (1%), and P. luteola (1%). The enteral feeding tubes were in place between < 6 h (22%) to > 48 h (13%). All the S. marcescens isolates from the enteral feeding tubes were resistant to amoxicillin and co-amoxiclav. Of additional importance was that a quarter of E. hormaechei isolates were resistant to the 3rd generation cephalosporins ceftazidime and cefotaxime. During the period of the study, K. pneumoniae and S. marcescens caused infections in the two NICUs. CONCLUSION: This study shows that neonatal enteral feeding tubes, irrespective of feeding regime, act as loci for the bacterial attachment and multiplication of numerous opportunistic pathogens within the Enterobacteriaceae family. Subsequently, these organisms will enter the stomach as a bolus with each feed. Therefore, enteral feeding tubes are an important risk factor to consider with respect to neonatal infections.

Characterization of an extended-spectrum beta-lactamase Enterobacter hormaechei nosocomial outbreak, and other Enterobacter hormaechei misidentified as Cronobacter (Enterobacter) sakazakii.

Enterobacter hormaechei is a Gram-negative bacterium within the Enterobacter cloacae complex, and has been shown to be of clinical significance by causing nosocomial infections, including sepsis. Ent. hormaechei is spread via horizontal transfer and is often associated with extended-spectrum beta-lactamase production, which increases the challenges associated with treatment by limiting therapeutic options. This report considers 10 strains of Ent. hormaechei (identified by 16S rDNA sequencing) that had originally been identified by phenotyping as Cronobacter (Enterobacter) sakazakii. Seven strains were from different neonates during a nosocomial outbreak in a California hospital. PFGE analysis revealed a clonal relationship among six of the seven isolates and therefore a previously unrecognized Ent. hormaechei outbreak had occurred over a three-month period. Antibiotic-resistance profiles were determined and extended-spectrum beta-lactamase activity was detected. The association of the organism with powdered infant formula, neonatal hosts and Cr. sakazakii suggested that the virulence of these organisms may be similar. Virulence traits were tested and all strains were shown to invade both gut epithelial (Caco-2) and blood-brain barrier endothelial cells (rBCEC4), and to persist in macrophages (U937). Due to misidentification we suggest that Ent. hormaechei may be an under-reported cause of bacterial infection, especially in neonates. Also, its isolation from various sources, including powdered infant milk formula, makes it a cause for concern and merits further investigation.

Virulence studies of Enterobacter sakazakii isolates associated with a neonatal intensive care unit outbreak.

BACKGROUND: In 1994, an outbreak of Enterobacter sakazakii infections in France occurred in a neonatal intensive care unit during which 17 neonates were infected. More than half of the infected neonates had severe clinical symptoms; 7 cases of necrotising enterocolitis (one with abdominal perforation), one case of septicemia, and one case of meningitis. The other 8 neonates were shown to be colonized but remained asymptomatic. There were three deaths. Four distinguishable pulsotypes of E. sakazakii were isolated during the outbreak, and the deaths were attributable to one pulsotype. This paper compares strains, from the four pulsotypes, for attachment and invasion of mammalian intestinal cells, macrophage survival and blood-brain barrier invasion. A fourth death from septic shock also occurred during the E. sakazakii outbreak. This was due to E. cloacae which at the time of the outbreak had been misidentified as E. sakazakii. This isolate has been included in this study. RESULTS: All E. sakazakii strains attached and invaded Caco-2 human epithelial cells, and invaded rat brain capillary endothelial cells. The majority of strains persisted in macrophage cells for 48 h. Two strains from fatal NEC and meningitis cases showed the highest invasion rate of Caco-2 intestinal cells. Their invasion of brain capillary endothelial cells was equivalent or greater than that of the neonatal E. coli meningitis strain K1. These strains also had extended spectrum beta-lactamase activities. E. cloacae differed from E. sakazakii due to the greater attachment and less invasion of epithelial cells, no survival in macrophages, and less invasion of capillary endothelial brain cells. CONCLUSION: While variables such as host factors and treatment strategies determine the outcome of infection, our in vitro studies evaluated the virulence of the isolates associated with this outbreak. It was not possible to directly correlate clinical symptoms and outcomes with in vitro studies. Nevertheless, we have shown the variation in invasive potential of E. sakazakii with intestinal and blood-brain barrier cells between and within pulsotypes from a neonatal intensive care unit outbreak. E. sakazakii strains were able to persist and even replicate for a period within macrophage cells. These traits appear to facilitate host immune evasion and dissemination.

Enterobacter sakazakii invades brain capillary endothelial cells, persists in human macrophages influencing cytokine secretion and induces severe brain pathology in the neonatal rat.

Enterobacter sakazakii is an opportunistic pathogen associated with contaminated powdered infant formula and a rare cause of Gram-negative sepsis that can develop into meningitis and brain abscess formation in neonates. Bacterial pathogenesis remains to be fully elucidated. In this study, the host inflammatory response was evaluated following intracranial inoculation of Ent. sakazakii into infant rats. Infiltrating macrophages and neutrophils composed multiple inflammatory foci and contained phagocytosed bacteria. Several genotypically distinct Ent. sakazakii strains (16S cluster groups 1-4) were shown to invade rat capillary endothelial brain cells (rBCEC4) in vitro. Further, the persistence of Ent. sakazakii in macrophages varied between strains. The presence of putative sod genes and SOD activity may influence the survival of acidic conditions and macrophage oxidase and contribute to Ent. sakazakii intracellular persistence. The influence of macrophage uptake of Ent. sakazakii on immunoregulatory cytokine expression was assessed by ELISA. This demonstrated that the IL-10/IL-12 ratio is high after 24 h. This is suggestive of a type 2 immune response which is inefficient in fighting intracellular infections. These findings may help explain how the diversity in virulence traits among Ent. sakazakii isolates and an unsuccessful immune response contribute to the opportunistic nature of this infection.