Research paper
Synthesis and evaluation of gallocyanine dyes as potential agents for the treatment of Alzheimer's disease and related neurodegenerative tauopathies

https://doi.org/10.1016/j.ejmech.2015.11.024Get rights and content

Highlights

  • Novel NCI8642 compounds were synthesized as potential LRP/DKK inhibitors.

  • Novel inhibitors of LRPs/DKK1 interactions were identified.

  • Important features for activity of NCI8642 template have been recognized.

  • Gallocyanine dyes exhibit a sufficient physicochemical profile.

  • NCI8642 and derivatives inhibit PGJ2-induced tau phosphorylation at serine 396.

Abstract

In search of safe and effective anti-Alzheimer disease agents a series of gallocyanine dyes have been synthesized and evaluated for their ability to inhibit LRPs/DKK1 interactions. Modulation of the interactions between LRPS and DKK1, regulate Wnt signaling pathway and affect Tau phosphorylation. The current efforts resulted in the identification of potent DKK1 inhibitors which are able to inhibit prostaglandin J2-induced tau phosphorylation at serine 396.

Introduction

Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by excessive accumulation of senile plaques and neurofibrillary tangles (NFT) [1]. Senile plaques are composed mainly of beta-amyloid peptide (Aβ) and neurofibrillary tangles of hyperphosphorylated microtubule-associated Tau protein [2]. Recent findings have established that deregulation of Wnt signaling contributes to the pathophysiology of AD, while activation of the pathway is neuroprotective [3]. According to Hanger and Noble the kinase activity of GSK3β is inhibited following activation of Wnt signaling, implicating this pathway in the hyperphosphorylation of Tau and the formation of NFTs [4].

Wnt signaling is initiated when Wnt proteins simultaneously interact with their receptor and co-receptor, Frizzled (FRZ) and LRP5/6. Through several cytoplasmic components, the signal is transduced to GSK3β with a subsequent stabilization of beta-catenin, which in turn enters the nucleus and forms a complex with TCF/LEFs to activate transcription of target genes [5a]. In the absence of Wnt, phosphorylation of beta-catenin by GSK3β leads to ubiquitination and subsequent proteasomal degradation, resulting in low levels of cytoplasmic beta-catenin [5], [5]b). The canonical Wnt pathway is also negatively modulated by the extracellular protein Dickkopf-1 (DKK1), which binds to LRPs preventing their interaction with Wnts and Frizzled membrane receptor [6]. The Dickkopf family encodes secreted proteins of 255–350 aminoacids and consists of four main members (DKK-1 to -4) in mammals [7]. Sokol and coworkers have reported that the DKK C-terminal domain mainly interacts with the third and fourth propellers of LRP5/6 and these interactions are necessary and sufficient for Wnt inhibition [8].

The expression of DKK1 is increased in brains of AD patients and in AD transgenic mouse models and this induction is dependent on p53 and associated with neurodegeneration [9], [10]. In cultured neurons, DKK1 is able to elicit cell death associated with loss of BCL-2 expression, induction of BAX, and Tau hyperphosphorylation [11]. Along these lines, a recent study demonstrated that DKK1-neutralizing antibodies suppress Aβ-induced synapse loss in mice [12]. Thus, modulation of the interactions between LRPs and DKK1, could regulate Wnt-beta-catenin signaling offering an attractive therapeutic approach for treating AD. In fact the LRP5-DKK1 interaction has been recently targeted with monoclonal anti-DKK1 antibodies [13]. However, the lack of blood brain penetration of the antibodies limits their application for the treatment of CNS diseases.

In this study, a small molecule approach for regulating Wnt signaling has been developed. Based on NCI8642, a recently discovered compound which blocks DKK1 inhibitory activity by disrupting DKK1/LRP6 interactions, a series of NCI8642 derivatives have been synthesized and evaluated. Importantly, this study shows that NCI8642 and related analogues induce inhibition of tau phosphorylation in vivo. Since tau hyperphosphorylation is a hallmark of Alzheimer's disease these results may find application in the treatment of AD and other tauopathies.

NCI8642 (1a, gallocyanine hydrochloride salt), a synthetic blue dyestuff synthesized by Otto in 1888 [14], is a phenoxazinone (see Scheme 1) independently discovered as an inhibitor of DKK1-LRP5/6 interactions by the groups of Remelli [15], Zheng and Wu [16]. Phenoxazines are privileged compounds, present in several bioactive molecules and natural products that display diverse biological activities. Well-known examples are the natural products actinomycins, which are polypeptide antibiotics produced by Streptomyces species that show chemotherapeutic activity in a number of neoplastic diseases [17]. The phenoxazine template is also present in the plectosphaeroic acids isolated from the fungus Plectosphaerella cucumerina, which are inhibitors of indoleamine 2,3-dioxygenase [18]. Simpler phenoxazines and benzophenoxazines have well documented anticancer [19], [20], [21], antiviral [22], antimicrobial [23] and antimalarial [24], [25] properties. Importantly, phenothiazine compounds have also been reported to inhibit heparin-induced assembly of tau protein [26].

Among the known Wnt modulators, NCI8642 is the only DKK1 inhibitor described so far, but its use as a clinically suitable drug is limited, because its association with LRP6 is weak (IC50 of about 3 μM in the inhibition of DKK1 binding). Nevertheless, data show that NCI8642 is effective in vivo as it reduces basal blood-glucose concentrations and improves glucose tolerance in mice [16]. NCI8642 possess both acidic and basic groups and a mesomeric chromophoric system which is depicted in Scheme 2 [27].

Section snippets

Docking studies

In an attempt to gain insight into the structural basis of the observed activity, NCI8642 and related analogues were docked against LRP6. Previous publications have demonstrated that NCI8642 binds to LRP6 and inhibits DKK1-LRP6 binding by using biolayer interferometry and surface plasmon resonance technology (SPR) [15], [16]. Accordingly, the heterotetramer structure 3S8V in the absence of DKK1c module (PDB code: 3S8V) was used in the present studies [28]. The docking results show top scoring

The effect of NCI8642 and related derivatives on the binding of DKK1 to the surface of HEK-293 cells overexpressing LRP6

In order to examine the effect of NCI8642 and related derivatives on the binding of DKK1 on the surface of HEK-293 cells overexpressing LRP6 (HEK-293LRP6), sub-confluent cultures were incubated with DKK1 conditioned medium (DKK1-CM) plus DMSO, 100 μM NCI8642 or 100 μM NCI8642 derivatives for 2 h at 4 °C [15]. Cell surface binding of DKK1 was visualized by immunocytochemistry using a primary anti-DKK1 antibody and fluorescent secondary antibody. Fluorescence images were analyzed using Zen

Conclusion

A series of novel gallocyanine dyes have been synthesized and evaluated for their ability to inhibit LRP/DKK1 interactions. Several compounds have been identified as more potent inhibitors than NCI8642 and important features for activity have been recognized. The present study further showed that NCI8642 and derivatives are able to inhibit prostaglandin J2-induced tau phosphorylation at serine 396. This finding is of particular importance and provides evidence that DKK1 may be a potential

General experimental details

All reactions were carried out under an atmosphere of Ar unless otherwise specified. Commercial reagents of high purity were purchased and used without further purification, unless otherwise noted. Reactions were monitored by TLC and using UV light as a visualizing agent and aqueous ceric sulfate/phosphomolybdic acid, ethanolic p-anisaldehyde solution, potassium permanganate solution, and heat as developing agents. The 1H and 13C NMR spectra were recorded at 500 and 125 MHz, and

Acknowledgment

This programme was implemented within the framework of the Operational Programme «Education and Lifelong Learning» of NSRF: ARISTEIA II. It was co-funded by the European Union and national resources. Furthermore the authors would like to thank Openeye Scientific Software, Inc., Santa Fe, NM, USA; www.eyesopen.com, for providing an academic license of their programs.

References (51)

  • N.M. O'Boyle et al.

    Open babel: an open chemical toolbox

    J. Cheminform.

    (2011)
  • C.A. Lipinski et al.

    Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings

    Adv. Drug Deliv. Rev.

    (2001)
  • Marvin

    Marvin was used for Drawing, Displaying and Characterizing Chemical Structures, Substructures and Reactions, Marvin 14.9.29

    (2014)
  • D.J. Selkoe

    Alzheimer's disease

    Cold Spring Harb. Perspect. Biol.

    (2011)
  • D.P. Hanger et al.

    Functional implications of glycogen synthase kinase-3-mediated tau phosphorylation

    Int. J. Alzheimers Dis.

    (2011)
  • K.M. Cadigan et al.

    TCF/LEFs and Wnt signaling in the nucleus

    Cold Spring Harb. Perspect. Biol.

    (2012)
    C. Liu et al.

    β-Trcp couples β-catenin phosphorylation-degradation and regulates Xenopus axis formation

    Proc. Natl. Acad. Sci. U. S. A.

    (1999)
  • A.M. Zorn

    Wnt signalling: antagonistic Dickkopfs

    Curr. Biol.

    (2001)
  • B.K. Brott et al.

    Regulation of Wnt/LRP signaling by distinct domains of Dickkopf proteins

    Mol. Cell Biol.

    (2002)
  • M.C. Rosi et al.

    Increased Dickkopf-1 expression in transgenic mouse models of neurodegenerative disease

    J. Neurochem.

    (2010)
  • A. Caricasole et al.

    Induction of Dickkopf-1, a negative modulator of the Wnt pathway, is associated with neuronal degeneration in Alzheimer's brain

    J. Neurosci.

    (2004)
  • I. Cappuccio et al.

    Induction of Dickkopf-1, a negative modulator of the Wnt pathway, is required for the development of ischemic neuronal death

    J. Neurosci.

    (2005)
  • S.A. Purro et al.

    The secreted Wnt antagonist Dickkopf-1 is required for amyloid β-mediated synaptic loss

    J. Neurosci.

    (2012)
  • H. Glantschnig et al.

    Generation and selection of novel fully human monoclonal antibodies that neutralize Dickkopf-1 (DKK1) inhibitory function in vitro and increase bone mass in vivo

    J. Biol. Chem.

    (2010)
  • S. Iozzi et al.

    Functional characterization of a small-molecule inhibitor of the DKK1-LRP6 interaction

    ISRN Mol. Biol.

    (2012)
  • X. Li et al.

    Chemical and genetic evidence for the involvement of Wnt antagonist Dickkopf2 in regulation of glucose metabolism

    Proc. Natl. Acad. Sci. U. S. A.

    (2012)
  • Cited by (19)

    • The role of DKK1 in Alzheimer's disease: A potential intervention point of brain damage prevention?

      2019, Pharmacological Research
      Citation Excerpt :

      However, there is currently no specific DKK1 inhibitor or activator available on commercial market, and the existing conditions also limit the clinical application. Fortunately, recent studies have found that DKK1 inhibitors could treat AD by inhibiting the phosphorylation of tau protein induced by prostaglandin J2 [46]. In addition, the preliminary results of our ongoing study of the protective effect of EGb761 on DKK1-mediated fluoride-induced cognitive impairment suggested that EGb761 has the potential to inhibit the expression of DKK1 and promote the regeneration of endogenous neurons in rats, thereby alleviating cognitive impairment [47,48].

    • Design and synthesis of gallocyanine inhibitors of DKK1/LRP6 interactions for treatment of Alzheimer's disease

      2018, Bioorganic Chemistry
      Citation Excerpt :

      The Dkk family of secreted proteins includes Dkk-1-4, which bind to LRP5/6, preventing it from interacting with Wnt-Frizzled complexes [14] (see Fig. 1). In previous studies, it is shown that small molecule inhibitors of DKK1/LRP6 interactions affect the Wnt signaling and decrease Tau hyperphosporylation [15,16]. The first identified inhibitor was NCI8642 (gallocyanine) [17,18].

    • Discovery of novel phenoxazinone derivatives as DKK1/LRP6 interaction inhibitors: Synthesis, biological evaluation and structure-activity relationships

      2016, Bioorganic and Medicinal Chemistry
      Citation Excerpt :

      Several physico-chemical (PK) descriptor values were calculated for the above library, by filling their profile as drug candidates (Table S1). Among these are the sp3 factor (Fsp3) providing the compound saturation levels (flatness),30 Lipinski’s rule of five descriptors (where calculated logarithmic lipophilicity ‘c log PMB’ was achieved with the use of program Marvin Beans v. 14.9.29) and spatial characteristics calculated over the web with the freely available Molinspiration property calculation service (www.molinspiration.com). The protein complex, PDB entry 3S8V, was used in this study.20

    View all citing articles on Scopus
    1

    These authors contributed equally.

    View full text