Evidence that pyrophosphate acts as an extracellular signalling molecule to exert direct functional effects in primary cultures of osteoblasts and osteoclasts.

Extracellular pyrophosphate (PPi) is well known for its fundamental role as a physiochemical mineralisation inhibitor. However, information about its direct actions on bone cells remains limited. This study shows that PPi decreased osteoclast formation and resorptive activity by ≤50 %. These inhibitory actions were associated with reduced expression of genes involved in osteoclastogenesis (Tnfrsf11a, Dcstamp) and bone resorption (Ctsk, Car2, Acp5). In osteoblasts, PPi present for the entire (0-21 days) or latter stages of culture (7-21/14-21 days) decreased bone mineralisation by ≤95 %. However, PPi present for the differentiation phase only (0-7/0-14 days) increased bone formation (≤70 %). Prolonged treatment with PPi resulted in earlier matrix deposition and increased soluble collagen levels (≤2.3-fold). Expression of osteoblast (RUNX2, Bglap) and early osteocyte (E11, Dmp1) genes along with mineralisation inhibitors (Spp1, Mgp) was increased by PPi (≤3-fold). PPi levels are regulated by tissue non-specific alkaline phosphatase (TNAP) and ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). PPi reduced NPP1 expression in both cell types whereas TNAP expression (≤2.5-fold) and activity (≤35 %) were increased in osteoblasts. Breakdown of extracellular ATP by NPP1 represents a key source of PPi. ATP release from osteoclasts and osteoblasts was decreased ≤60 % by PPi and by a selective TNAP inhibitor (CAS496014-12-2). Pertussis toxin, which prevents Gαi subunit activation, was used to investigate whether G-protein coupled receptor (GPCR) signalling mediates the effects of PPi. The actions of PPi on bone mineralisation, collagen production, ATP release, gene/protein expression and osteoclast formation were abolished or attenuated by pertussis toxin. Together these findings show that PPi, modulates differentiation, function and gene expression in osteoblasts and osteoclasts. The ability of PPi to alter ATP release and NPP1/TNAP expression and activity indicates that cells can detect PPi levels and respond accordingly. Our data also raise the possibility that some actions of PPi on bone cells could be mediated by a Gαi-linked GPCR.

Successful prednisolone or calcimimetic treatment of acquired hypocalciuric hypercalcemia caused by biased allosteric CaSR autoantibodies.

Biased agonism is a frontier field in GPCR research. Acquired hypocalciuric hypercalcemia (AHH) is a rare disease caused by calcium-sensing receptor (CaSR) autoantibodies, to date, showing either simple blocking or biased properties (i.e., stimulatory or blocking effects on different downstream signaling pathways). This emphasizes the importance of the Gi/o (pertussis toxin-sensitive G proteins, whose ßγ subunits activate multiple signals, including ERK1/2) in regulating parathyroid hormone secretion. We here describe 3 patients with symptomatic AHH who shared characteristics with the 2 cases we previously reported as follows: (a) elderly (74-87 years at diagnosis), (b) male, (c) unexpectedly showed no other autoimmune diseases, (d) showed spontaneously fluctuating Ca levels from approximately normal to near fatally high ranges, (e) acute exacerbations could be successfully treated with prednisolone and/or calcimimetics, (f) the presence of CaSR autoantibodies that operated as biased allosteric modulators of CaSR, and (g) were likely to be conformational (i.e., recognizing and, thereby, stabilizing a unique active conformation of CaSR that activates Gq/11, activating phosphatidylinositol turnover, but not Gi/o). Our observations with these prominent commonalities may provide new insights into the phenotype and characteristics of AHH and the mechanisms by which the biased agonism of GPCRs operate.

A1 Adenosine Receptor Activation Inhibits P2X3 Receptor-Mediated ATP Currents in Rat Dorsal Root Ganglion Neurons.

Purinergic signaling is involved in multiple pain processes. P2X3 receptor is a key target in pain therapeutics, while A1 adenosine receptor signaling plays a role in analgesia. However, it remains unclear whether there is a link between them in pain. The present results showed that the A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA) concentration dependently suppressed P2X3 receptor-mediated and α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents in rat dorsal root ganglion (DRG) neurons. CPA significantly decreased the maximal current response to α,ß-meATP, as shown a downward shift of the concentration-response curve for α,ß-meATP. CPA suppressed ATP currents in a voltage-independent manner. Inhibition of ATP currents by CPA was completely prevented by the A1 adenosine receptor antagonist KW-3902, and disappeared after the intracellular dialysis of either the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, or the cAMP analog 8-Br-cAMP. Moreover, CPA suppressed the membrane potential depolarization and action potential bursts, which were induced by α,ß-meATP in DRG neurons. Finally, CPA relieved α,ß-meATP-induced nociceptive behaviors in rats by activating peripheral A1 adenosine receptors. These results indicated that CPA inhibited the activity of P2X3 receptors in rat primary sensory neurons by activating A1 adenosine receptors and its downstream cAMP signaling pathway, revealing a novel peripheral mechanism underlying its analgesic effect.

Delineation of the GPR15 receptor-mediated Gα protein signalling profile in recombinant mammalian cells.

The GPR15 receptor is a G protein-coupled receptor (GPCR), which is activated by an endogenous peptide GPR15L(25-81) and a C-terminal peptide fragment GPR15L(71-81). GPR15 signals through the Gi/o pathway to decrease intracellular cyclic adenosine 3',5'-monophosphate (cAMP). However, the activation profiles of the GPR15 receptor within Gi/o subtypes have not been examined. Moreover, whether the receptor can also couple to Gs , Gq/11 and G12/13 is unclear. Here, GPR15L(25-81) and GPR15L(71-81) are used as pharmacological tool compounds to delineate the GPR15 receptor-mediated Gα protein signalling using a G protein activation assay and second messenger assay conducted on living cells. The results show that the GPR15 receptor preferentially couples to Gi/o rather than other pathways in both assays. Within the Gi/o family, the GPR15 receptor activates all the subtypes (Gi1 , Gi2 , Gi3 , GoA , GoB and Gz ). The Emax and activation rates of Gi1, Gi2 , Gi3, GoA and GoB are similar, whilst the Emax of Gz is smaller and the activation rate is significantly slower. The potencies of both peptides toward each Gi/o subtype have been determined. Furthermore, the GPR15 receptor signals through Gi/o to inhibit cAMP accumulation, which could be blocked by the application of the Gi/o inhibitor pertussis toxin.

Novel Strategies to Inhibit Pertussis Toxin.

Pertussis, also known as whooping cough, is a respiratory disease caused by infection with Bordetella pertussis, which releases several virulence factors, including the AB-type pertussis toxin (PT). The characteristic symptom is severe, long-lasting paroxysmal coughing. Especially in newborns and infants, pertussis symptoms, such as leukocytosis, can become life-threatening. Despite an available vaccination, increasing case numbers have been reported worldwide, including Western countries such as Germany and the USA. Antibiotic treatment is available and important to prevent further transmission. However, antibiotics only reduce symptoms if administered in early stages, which rarely occurs due to a late diagnosis. Thus, no causative treatments against symptoms of whooping cough are currently available. The AB-type protein toxin PT is a main virulence factor and consists of a binding subunit that facilitates transport of an enzyme subunit into the cytosol of target cells. There, the enzyme subunit ADP-ribosylates inhibitory α-subunits of G-protein coupled receptors resulting in disturbed cAMP signaling. As an important virulence factor associated with severe symptoms, such as leukocytosis, and poor outcomes, PT represents an attractive drug target to develop novel therapeutic strategies. In this review, chaperone inhibitors, human peptides, small molecule inhibitors, and humanized antibodies are discussed as novel strategies to inhibit PT.

Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin.

Bordetella pertussis causes the severe childhood disease whooping cough, by releasing several toxins, including pertussis toxin (PT) as a major virulence factor. PT is an AB5-type toxin, and consists of the enzymatic A-subunit PTS1 and five B-subunits, which facilitate binding to cells and transport of PTS1 into the cytosol. PTS1 ADP-ribosylates α-subunits of inhibitory G-proteins (Gαi) in the cytosol, which leads to disturbed cAMP signaling. Since PT is crucial for causing severe courses of disease, our aim is to identify new inhibitors against PT, to provide starting points for novel therapeutic approaches. Here, we investigated the effect of human antimicrobial peptides of the defensin family on PT. We demonstrated that PTS1 enzyme activity in vitro was inhibited by α-defensin-1 and -5, but not ß-defensin-1. The amount of ADP-ribosylated Gαi was significantly reduced in PT-treated cells, in the presence of α-defensin-1 and -5. Moreover, both α-defensins decreased PT-mediated effects on cAMP signaling in the living cell-based interference in the Gαi-mediated signal transduction (iGIST) assay. Taken together, we identified the human peptides α-defensin-1 and -5 as inhibitors of PT activity, suggesting that these human peptides bear potential for developing novel therapeutic strategies against whooping cough.

Four single-basepair mutations in the ptx promoter of Bordetella bronchiseptica are sufficient to activate the expression of pertussis toxin.

Secretion of pertussis toxin (PT) is the preeminent virulence trait of the human pathogen Bordetella pertussis, causing whooping cough. Bordetella bronchiseptica, although it harbors an intact 12-kb ptx-ptl operon, does not express PT due to an inactive ptx promoter (Pptx), which contains 18 SNPs (single nucleotide polymorphisms) relative to B. pertussis Pptx. A systematic analysis of these SNPs was undertaken to define the degree of mutational divergence necessary to activate B. bronchiseptica Pptx. A single change (C-13T), which created a better - 10 element, was capable of activating B. bronchiseptica Pptx sufficiently to allow secretion of low but measureable levels of active PT. Three additional changes in the BvgA-binding region, only in the context of C-13T mutant, raised the expression of PT to B. pertussis levels. These results illuminate a logical evolutionary pathway for acquisition of this key virulence trait in the evolution of B. pertussis from a B. bronchiseptica-like common ancestor.

Designing a New Multi-Epitope Pertussis Vaccine with Highly Population Coverage Based on a Novel Sequence and Structural Filtration Algorithm.

Pertussis vaccine is produced from physicochemically inactivated toxin for many years. Recent advancements in immunoinformatics [N. Tomar and R. K. De, "Immunoinformatics: an integrated scenario," Immunology, vol. 131, no. 2, pp. 153-168, 2010] and structural bioinformatics can provide a new multidisciplinary approach to overcome the concerns including unwanted antibodies and incomplete population coverage. In this study we focused on solving the challenging issues by designing a multi-epitope vaccine (MEV) using rational bioinformatics analyses. The frequencies of All HLA DP, DQ, and DR alleles were evaluated in almost all countries. Strong binder surface epitopes on the pertussis toxin were selected based on our novel filtration strategy. Finally, the population coverage of MEV was determined in the candidate country. Filtration steps yielded 312 strong binder epitopes. Finally, 8 surface strong binder epitopes were selected as candidate epitopes. The population coverage of the MEV in France and the world was 98 and 100 percent, respectively. Our algorithm successfully filtered many unwanted strong binder epitopes. Considering the HLA type of all individuals in a country, we theoretically provided the maximum chance to all humans to be vaccinated efficiently. Application of a MEV would be led to production of highly efficient target specific antibodies, significant reduction of unwanted antibodies, and avoid possible raising of auto-antibodies as well.

T cell and chemokine receptors differentially control CD8 T cell motility behavior in the infected airways immediately before and after virus clearance in a primary infection.

In mice, experimental influenza virus infection stimulates CD8 T cell infiltration of the airways. Virus is cleared by day 9, and between days 8 and 9 there is an abrupt change in CD8 T cell motility behavior transitioning from low velocity and high confinement on day 8, to high velocity with continued high confinement on day 9. We hypothesized that loss of virus and/or antigen signals in the context of high chemokine levels drives the T cells into a rapid surveillance mode. Virus infection induces chemokine production, which may change when the virus is cleared. We therefore sought to examine this period of rapid changes to the T cell environment in the tissue and seek evidence on the roles of peptide-MHC and chemokine receptor interactions. Experiments were performed to block G protein coupled receptor (GPCR) signaling with Pertussis toxin (Ptx). Ptx treatment generally reduced cell velocities and mildly increased confinement suggesting chemokine mediated arrest (velocity <2 µm/min) (Friedman RS, 2005), except on day 8 when velocity increased and confinement was relieved. Blocking specific peptide-MHC with monoclonal antibody unexpectedly decreased velocities on days 7 through 9, suggesting TCR/peptide-MHC interactions promote cell mobility in the tissue. Together, these results suggest the T cells are engaged with antigen bearing and chemokine producing cells that affect motility in ways that vary with the day after infection. The increase in velocities on day 9 were reversed by addition of specific peptide, consistent with the idea that antigen signals become limiting on day 9 compared to earlier time points. Thus, antigen and chemokine signals act to alternately promote and restrict CD8 T cell motility until the point of virus clearance, suggesting the switch in motility behavior on day 9 may be due to a combination of limiting antigen in the presence of high chemokine signals as the virus is cleared.

Discovery of Compounds Inhibiting the ADP-Ribosyltransferase Activity of Pertussis Toxin.

The targeted pathogen-selective approach to drug development holds promise to minimize collateral damage to the beneficial microbiome. The AB5-topology pertussis toxin (PtxS1-S5) is a major virulence factor of Bordetella pertussis, the causative agent of the highly contagious respiratory disease whooping cough. Once internalized into the host cell, PtxS1 ADP-ribosylates α-subunits of the heterotrimeric Gαi-superfamily, thereby disrupting G-protein-coupled receptor signaling. Here, we report the discovery of the first small molecules inhibiting the ADP-ribosyltransferase activity of pertussis toxin. We developed protocols to purify milligram-levels of active recombinant B. pertussis PtxS1 from Escherichia coli and an in vitro high throughput-compatible assay to quantify NAD+ consumption during PtxS1-catalyzed ADP-ribosylation of Gαi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC50-values (3.0 µM and 6.8 µM) were identified in the in vitro NAD+ consumption assay that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gαi. Docking and molecular dynamics simulations identified plausible binding poses of NSC228155 and in particular of NSC29193, most likely owing to the rigidity of the latter ligand, at the NAD+-binding pocket of PtxS1. NSC228155 inhibited the pertussis AB5 holotoxin-catalyzed ADP-ribosylation of Gαi in living human cells with a low micromolar IC50-value (2.4 µM). NSC228155 and NSC29193 might prove to be useful hit compounds in targeted B. pertussis-selective drug development.