The novel pyridoxal-5′-phosphate derivative PPNDS potently antagonizes activation of P2X1 receptors

doi:10.1016/S0014-2999(99)00834-1

European Journal of Pharmacology

Volume 387, Issue 3, 17 January 2000, Pages R19-R21

https://doi.org/10.1016/S0014-2999(99)00834-1 Get rights and content

Abstract

Pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4′,8′-disulfonate) (PPNDS) potently antagonized P2X₁ receptor-mediated responses in rat vas deferens (pK_B=7.43) and Xenopus laevis oocytes (pIC₅₀=7.84). It showed lower (up to 20,000-fold) inhibitory potency on ecto-nucleotidase in Xenopus oocytes and on P2Y₁ receptors in guinea-pig ileum (pA₂=6.13). PPNDS did not interact with α_1A-adrenoceptors, adenosine A₁ and A_2B, histamine H₁ and muscarinic M₃ receptors. Thus, PPNDS is a novel, specific P2 receptor antagonist and represents the pyridoxal-5′-phosphate derivative with the highest potency at P2X₁ receptors.

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Acknowledgements

The authors gratefully acknowledge the financial support of the Fonds der Chemischen Industrie (Germany) and of the Deutsche Forschungsgemeinschaft (grant numbers: La 350/7-2, Schm 536/2-3, GRK 137/2-98).

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Broad-spectrum non-nucleoside inhibitors for caliciviruses
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Citation Excerpt :
Additionally, PPNDS has been eliminated from other studies due to non-specific, off-target activities (Simeonov et al., 2009), and therefore it lacks the drug-like properties required for further antiviral development. PPNDS was first reported as a potent P2X1 receptor agonist (Lambrecht et al., 2000), but subsequently demonstrated potent inhibition of human NoV and MNV RdRps (Tarantino et al., 2014; Croci et al., 2014b) with IC50 values between 0.45 and 0.88 μM (Tarantino et al., 2014; Croci et al., 2014b). Co-crystallization studies with the human NoV RdRp showed that PPNDS notably interacts with the Site-B amino acids Q414 and R419, amongst others (Tarantino et al., 2014).
Viruses of the Caliciviridae cause significant and sometimes lethal diseases, however despite substantial research efforts, specific antivirals are lacking. Broad-spectrum antivirals could combat multiple viral pathogens, offering a rapid solution when no therapies exist. The RNA-dependent RNA polymerase (RdRp) is an attractive antiviral target as it is essential for viral replication and lacks mammalian homologs. To focus the search for pan-Caliciviridae antivirals, the RdRp was probed with non-nucleoside inhibitors (NNIs) developed against hepatitis C virus (HCV) to reveal both allosteric ligands for structure-activity relationship enhancement, and highly-conserved RdRp pockets for antiviral targeting. The ability of HCV NNIs to inhibit calicivirus RdRp activities was assessed using in vitro enzyme and murine norovirus cell culture assays. Results revealed that three NNIs which bound the HCV RdRp Thumb I (TI) site also inhibited transcriptional activities of six RdRps spanning the Norovirus, Sapovirus and Lagovirus genera of the Caliciviridae. These NNIs included JTK-109 (RdRp inhibition range: IC₅₀ 4.3–16.6 μM), TMC-647055 (IC₅₀ range: 18.8–45.4 μM) and Beclabuvir (IC₅₀ range: 23.8–>100 μM). In silico studies and site-directed mutagenesis indicated the JTK-109 binding site was within the calicivirus RdRp thumb domain, in a pocket termed Site-B, which is highly-conserved within all calicivirus RdRps. Additionally, RdRp inhibition assays revealed that JTK-109 was antagonistic with the previously reported RdRp inhibitor pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4′,8′-disulfonate) tetrasodium salt (PPNDS), that also binds to Site-B. Moreover, like JTK-109, PPNDS was also a potent inhibitor of polymerases from six viruses spanning the three Caliciviridae genera tested (IC₅₀ range: 0.1–2.3 μM). Together, this study demonstrates the potential for de novo development of broad-spectrum antivirals that target the highly-conserved RdRp thumb pocket, Site-B. We also revealed three broad-spectrum HCV NNIs that could be used as antiviral scaffolds for further development against caliciviruses and other viruses.
PPNDS inhibits murine Norovirus RNA-dependent RNA-polymerase mimicking two RNA stacking bases
2014, FEBS Letters
Citation Excerpt :
Since these two molecules display poor membrane permeability [17–19], we focused on Pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4′,8′-disulfonate) tetrasodium salt (PPNDS), a different potential inhibitor identified by an in silico search that focused on a region around the active site of hNV RdRp. PPNDS, a specific P2X1 receptor antagonist [20], hosts a naphthalene disulfonate head (i.e. the Suramin moiety that strongly interacts with the enzyme [16]) and a pyridoxal phosphate group linked to the naphthalene head by an azo bond (Fig. 1). In the human (h) NV-RdRp/PPNDS complex structure, the compound was shown to bind in a region different from that occupied by suramin or by NF023 in their respective complexes [21].
Norovirus (NV) is a major cause of gastroenteritis worldwide. Antivirals against such important pathogens are on demand. Among the viral proteins that orchestrate viral replication, RNA-dependent-RNA-polymerase (RdRp) is a promising drug development target. From an in silico-docking search focused on the RdRp active site, we selected the compound PPNDS, which showed low micromolar IC₅₀ vs. murine NV-RdRp in vitro. We report the crystal structure of the murine NV-RdRp/PPNDS complex showing that two molecules of the inhibitor bind in antiparallel stacking interaction, properly oriented to block exit of the newly synthesized RNA. Such inhibitor-binding mode mimics two stacked nucleotide-bases of the RdRp/ssRNA complex.
Naphthalene-sulfonate inhibitors of human norovirus RNA-dependent RNA-polymerase
2014, Antiviral Research
Citation Excerpt :
NAF2 binding to two distinct sites is also suggested by analysis of the inhibition mechanism which shows a Hill coefficient higher than 1. In order to further characterize the B-site we selected PPNDS, a naphthalene sulfonate-based compound that is a potent and selective P2X1 receptor antagonist (Lambrecht et al., 2000). We demonstrated that PPNDS is able to inhibit the polymerase activity (Hill coefficient ∼1, Fig. S1b) with a potency close to that observed for suramin or NF023.
Noroviruses are members of the Caliciviridae family of positive sense RNA viruses. In humans Noroviruses cause rapid onset diarrhea and vomiting. Currently Norovirus infection is responsible for 21 million gastroenteritis yearly cases in the USA. Nevertheless, despite the obvious public health and socio-economic relevance, no effective vaccines/antivirals are yet available to treat Norovirus infection.
Since the activity of RNA-dependent RNA polymerase (RdRp) plays a key role in genome replication and in the synthesis/amplification of subgenomic RNA, the enzyme is considered a promising target for antiviral drug development. In this context, following the identification of suramin and NF023 as Norovirus RdRp inhibitors, we analyzed the potential inhibitory role of naphthalene di-sulfonate (NAF2), a fragment derived from these two molecules. Although NAF2, tested in enzymatic polymerase inhibition assays, displayed low activity against RdRp (IC₅₀ = 14 μM), the crystal structure of human Norovirus RdRp revealed a thumb domain NAF2 binding site that differs from that characterized for NF023/suramin. To further map the new potential inhibitory site, we focused on the structurally related molecule pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4′,8′-disulfonate) tetrasodium salt (PPNDS). PPNDS displayed below-micromolar inhibitory activity versus human Norovirus RdRp (IC₅₀ = 0.45 μM), similarly to suramin and NF023. Inspection of the crystal structure of the RdRp/PPNDS complex showed that the inhibitor bound to the NAF2 thumb domain site, highlighting the relevance of such new binding site for exploiting Norovirus RdRp inhibitors.
High glucose concentration impairs ATP outflow and immunoglobulin production by human peripheral B lymphocytes: Involvement of P2X7 receptor
2013, Immunobiology
Citation Excerpt :
This compound inhibits with various potency P2X1–6 receptors in concentration range 1–5 μM (reviewed in Coddou et al. 2011). The naphthylazo derivative of PPADS, PPNDS, which is a potent and highly selective antagonist of P2X1 having nanomolar inhibition constant toward P2X1 receptor (Lambrecht et al. 2000) evoked ∼27% decrease of IgM production. Similar effects of PPADS and PPNDS on IgM production by B cells cultured at high and low glucose were observed.
Patients with diabetes are more prone to bacterial infections mostly due to hyperglycemia-induced suppression of immune cells function. B lymphocytes by secreting antibodies inhibit microbial replication, but the impact of high glucose concentration on humoral immune response is not fully resolved. The aim of this work was to investigate the effect of high glucose concentration on B cells response to stimulation with a bacterial antigen and autocrine regulation.
Purified human peripheral blood B cells were cultured at different glucose concentrations and stimulated in vitro with Staphylococus aureus Cowan I (SAC) plus IL-2. B cells proliferation, differentiation and IgM expression were analyzed by flow cytometry. B cell ATP release and involvement of P2 purinergic receptors in regulation of IgM secretion was assessed.
B cells cultured at 25 mM glucose in response to SAC stimulation released significantly less (∼55%) IgM comparing to cells maintained in 5 mM glucose. Under resting and stimulatory conditions B cells released significant quantities of ATP to the culture media, but ATP level decreased when B cells were maintain in high glucose. SAC-induced B cell IgM release was totally blocked by highly selective antagonist (Az11645373) of P2X7 receptor. IgM secretion increased in the presence of potent P2X7 receptor agonist (BzATP), but this effect was abolished by high glucose concentration.
High glucose concentration impairs B cell function by suppression of P2X7 receptor-dependent IgM release in response to in vitro bacterial antigen stimulation. This alteration may greatly contribute to the impaired humoral immune response in diabetics.
Factors affecting guanine nucleotide binding to rat AMPA receptors
2007, Brain Research
Citation Excerpt :
PPNDS and suramin are two of several poly-anionic aromatic sulfonates that have recently been shown to allosterically modulate agonist binding to AMPA receptors, with suramin causing an inhibition and PPNDS an increase in agonist binding (Suzuki et al., 2004). Since PPNDS is thought to competitively antagonize nucleotide binding in other proteins (Lambrecht et al., 2000), we hypothesized that its docking site on the AMPA receptor would overlap with that for guanine nucleotides and involve some of the same residues on the receptor, particularly those that bind phosphate. Fig. 3 shows that this is not the case.
Glutamate receptors are competitively inhibited by guanine nucleotides. Insight into the physiological function of this inhibition would be greatly advanced if nucleotide binding could be eliminated through mutations without altering other aspects of receptor function, or if compounds were discovered that selectively prevent nucleotide binding. It was previously reported that a lysine in the chick kainate binding protein (cKBP) is specifically involved in guanine nucleotide binding. In the present study we mutated the equivalent lysine in the rat AMPA receptor subunit GluR1 flip to alanine (K445A) and assessed changes in nucleotide affinity from the displacement of [³H]fluorowillardiine. As in the cKBP, the affinity for nucleotides was greatly reduced while the binding affinity for agonists remained unchanged. The reduction in affinity was largest for GTP (factor of 5.8) and GDP (4.4) and minor for GMP and guanosine. This suggests that K445 is involved in stabilizing the second phosphate of the nucleotide. Given that bulkier analogs like GDP-fucose are also accommodated at this site, it seems likely that nucleotides bind in such a way that their phosphates project out of the cleft. In excised-patch recordings using short pulses of glutamate, the K445A mutation increased the EC₅₀ for the peak response 1.8-fold and accelerated desensitization and deactivation. This indicates that the effects of this mutation are not as specific as previously suggested. Efforts to selectively eliminate inhibition by nucleotides may therefore depend on mapping out further the docking site. In a first attempt using point mutations we ruled out several amino acids around the cleft as being involved in nucleotide binding. Also, the AMPA receptor modulator PPNDS which competitively inhibits nucleotide binding to purinergic receptors did not affect nucleotide inhibition, suggesting that there are major differences in the topography between purinergic and glutamate receptors. Thus new approaches, including crystallography, may be called for to identify residues uniquely involved in nucleotide binding.
PPADS is a reversible competitive antagonist of the NAADP receptor
2007, Cell Calcium
NAADP has been shown to act as a second messenger in a wide range of systems from plants to mammalian cells. Although it had always been considered as a canonical second messenger, recent work has shown that it is also active when applied extracellularly. It has also been suggested that NAADP might have a direct action on P2 receptors, based on the action of a pharmacological agent, PPADS, on Ca²⁺ signals in response to extracellular NAADP. We have therefore investigated whether PPADS can act directly on the intracellular NAADP-induced Ca²⁺-release system in the well characterised sea urchin egg homogenate system. Indeed, PPADS, and its structural analogue PPNDS were able to compete with [³²P]NAADP for the binding site and binding curves revealed that both compounds display affinities in the low micromolar range. The binding of PPADS was reversible in contrast to that of NAADP. In fluorimetric Ca²⁺-release experiments, PPADS was able to competitively antagonise NAADP-induced Ca²⁺-release with an IC₅₀ of 20 μM, while it did not affect the other Ca²⁺-release channels. This is the first report of a reversible, competitive antagonist of the sea urchin NAADP receptor. Furthermore, PPADS might reveal itself as an invaluable tool to investigate NAADP signalling and is a lead compound for the synthesis of potent and specific antagonists.

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European Journal of Pharmacology

Abstract

Section snippets

Acknowledgements

References (8)

Novel ligands for P2 receptor subtypes in innervated tissues

Prog. Brain Res.

PPADS, a novel functionally selective antagonist of P₂ purinoceptor-mediated responses

Eur. J. Pharmacol.

Effects of P2-purinoceptor antagonists on degradation of adenine nucleotides by ecto-nucleotidases in folliculated oocytes of Xenopus laevis

Biochem. Pharmacol.

A pyridoxine cyclic phosphate and its 6-azoaryl derivative selectively potentiate and antagonize activation of P2X₁ receptors

J. Med. Chem.

Cited by (42)

Broad-spectrum non-nucleoside inhibitors for caliciviruses

PPNDS inhibits murine Norovirus RNA-dependent RNA-polymerase mimicking two RNA stacking bases

Naphthalene-sulfonate inhibitors of human norovirus RNA-dependent RNA-polymerase

High glucose concentration impairs ATP outflow and immunoglobulin production by human peripheral B lymphocytes: Involvement of P2X7 receptor

Factors affecting guanine nucleotide binding to rat AMPA receptors

PPADS is a reversible competitive antagonist of the NAADP receptor

Rapid communicationThe novel pyridoxal-5′-phosphate derivative PPNDS potently antagonizes activation of P2X1 receptors

Abstract

Section snippets

Acknowledgements

Prog. Brain Res.

Eur. J. Pharmacol.

Biochem. Pharmacol.

A pyridoxine cyclic phosphate and its 6-azoaryl derivative selectively potentiate and antagonize activation of P2X1 receptors

J. Med. Chem.

Rapid communication
The novel pyridoxal-5′-phosphate derivative PPNDS potently antagonizes activation of P2X₁ receptors

A pyridoxine cyclic phosphate and its 6-azoaryl derivative selectively potentiate and antagonize activation of P2X₁ receptors