Polyamine Tail Research Articles

Polyamine-containing etoposide derivatives as poisons of human type II topoisomerases: Differential effects on topoisomerase IIα and IIβ

Etoposide is an anticancer drug that acts by inducing topoisomerase II-mediated DNA cleavage. Despite its wide use, etoposide is associated with some very serious side-effects including the development of treatment-related acute myelogenous leukemias. Etoposide targets both human topoisomerase IIα and IIβ. However, the contributions of the two enzyme isoforms to the therapeutic vs. leukemogenic properties of the drug are unclear. In order to develop an etoposide-based drug with specificity for cancer cells that express an active polyamine transport system, the sugar moiety of the drug has been replaced with a polyamine tail. To analyze the effects of this substitution on the specificity of hybrid molecules toward the two enzyme isoforms, we analyzed the activity of a series of etoposide-polyamine hybrids toward human topoisomerase IIα and IIβ. All of the compounds displayed an ability to induce enzyme-mediated DNA cleavage that was comparable to or higher than that of etoposide. Relative to the parent drug, the hybrid compounds displayed substantially higher activity toward topoisomerase IIβ than IIα. Modeling studies suggest that the enhanced specificity may result from interactions with Gln778 in topoisomerase IIβ. The corresponding residue in the α isoform is a methionine.

The Aromatic Head Group of Spider Toxin Polyamines Influences Toxicity to Cancer Cells.

Spider venoms constitute incredibly diverse libraries of compounds, many of which are involved in prey capture and defence. Polyamines are often prevalent in the venom and target ionotropic glutamate receptors. Here we show that a novel spider polyamine, PA366, containing a hydroxyphenyl-based structure is present in the venom of several species of tarantula, and has selective toxicity against MCF-7 breast cancer cells. By contrast, a polyamine from an Australian funnel-web spider venom, which contains an identical polyamine tail to PA366 but an indole-based head-group, is only cytotoxic at high concentrations. Our results suggest that the ring structure plays a role in the cytotoxicity and that modification to the polyamine head group might lead to more potent and selective compounds with potential as novel cancer treatments.

Illuminating Ionotropic Glutamate Receptors: Characterization of Fluorescent Antagonists Based on Polyamine Toxins

Ionotropic glutamate (iGlu) receptors, including the NMDA and AMPA subfamilies, mediate fast excitatory synaptic transmission in the mammalian central nervous system. NMDA and AMPA receptors play critical roles in learning and memory and are implicated in multiple neurological and psychiatric diseases. Argiotoxins (exemplified by the prototypical ArgTX-636) are so-called polyamine toxins isolated from the venom of the orb weaver spider Argiope lobata. Argiotoxins consists of an aromatic head group coupled via an amino acid linker to a polyamine moiety and act as open-channel blockers of the iGlu receptor ion channel with nanomolar affinity. Structure-activity studies of synthetic argiotoxin analogs have shown that modification of the polyamine tail can tweak NMDA versus AMPA selectivity (REF NELSON ET AL). Furthermore, the aromatic head group can tolerate substantial modification without loss of affinity.The aim of the present study is to develop NMDA- and AMPA selective argiotoxin analogs containing fluorescent moieties to enable imaging studies of native iGlu receptors and to serve as potential FRET donor or acceptor ligands. Specifically, a series ArgTX-636 analogs with fluorescein, coumarin and boron-dipyrromethene (BODIPY) derived fluorophores incoporated at or replacing the headgroup moiety were synthesized and characterized at recombinant AMPA and NMDA receptors expressed in Xenopus Laevis oocytes (GluA1 and GluN1/2A, respectively). Several analogs were found to posses high-affinity NMDA and/or AMPA receptor binding while maintaining desirable fluorescent properties.

Synthesis, Cytotoxicity, and Cell Death Profile of Polyaminoanthraquinones as Antitumor Agents

Investigation on designed polyaminoanthraquinones revealed that the anthraquinones bearing triamine motifs are generally more potent than their counterparts with diamine or tetramine motifs. Compared with the reference drug mitoxantrone (MTX), 9b and 9c exhibited better inhibitory activity on cancerous HepG2 cells and preferable cell selectivity in the further screen of normal QSG7701 cells, although they were not assimilated by cancer cells via the polyamine transporter. The presence of polyamine motifs elevated the interaction of compounds 9b and 9c with lysosomes and resulted in distinct mode of cell death. 9c and MTX could cause caspases-dependent HepG2 cell apoptotic death involving in mitochondrial membrane potential (MMP) loss, cytochrome c release, and caspase-3 activation. However, 9b, which contained only one less methylene group in the polyamine tail, produced cytotoxicity by necrosis. In conclusion, the modification of anthraquinones with polyamines may furnish potent anticancer drug candidates against hepatocellular carcinoma undergoing distinct cell death mechanisms.

Molecular Determinants for Subtype-Selective Ion Channel Block of NMDA Receptors by Argiotoxin Analogs

The NMDA-type of ionotropic glutamate receptors (iGluRs) are involved in excitatory transmission in the mammalian brain and in a range of neurological and psychiatric diseases for which NMDA receptors are considered potential therapeutic drug targets. The majority of NMDA receptor subtypes are comprised of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits (NR2A-D); leading to the existence of four major receptor subtypes with distinct expression patterns and functional properties: GluN1/2A, GluN1/2B, GluN1/2C, and GluN1/2D. While the GluN1/2B subtype can be selectively inhibited by the highly subtype-selective non-competitive antagonist ifenprodile, the lack of inhibitors with similar subtype selectivity for the GluN1/2A, GluN1/2C, and GluN1/2D receptors is a limitation in studies exploring the role of these subtypes in many aspects of brain function and disease.Polyamine toxins isolated from the venom of spiders are open-channel blockers of ion channels, in particular iGluRs. Argiotoxin-636 (ArgTX-636) isolated from the venom of the orb weaver spider Argiope lobata consists of an aromatic amino acid head-group coupled to a polyamine tail. ArgTX-636 is a potent inhibitor of mammalian iGluRs; presumably by binding to the ion channel region in a use- and voltage-dependent manner that prevents ion conduction. We have recently shown1 that modifications within the polyamine tail of ArgTX-636 can control selectivity between the NMDA- and the AMPA-type of iGluRs (1). Further exploration of the analog lead-structures have recently developed a series of analogs of the spider toxin Argiotoxin-636 (ArgTX-636), which display robust selectivity towards GluN1/2A and GluN1/2B over GluN1/2C, and GluN1/2D subtypes of NMDA receptors. In this study we have explorer the molecular determinants for subtype-selectivity of the novel analogs ArgTX-48 and ArgTX-75 within the NMDA receptor ion channel using electrophysiological characterization of chimeric and mutant GluN1/2A and GluN1/2D NMDA receptors.

Cytotoxicity and cell death mechanisms induced by the polyamine-vectorized anti-cancer drug F14512 targeting topoisomerase II

The polyamines transport system (PTS) is usually enhanced in cancer cells and can be exploited to deliver anticancer drugs. The spermine-conjugated epipodophyllotoxin derivative F14512 is a topoisomerase II poison that exploits the PTS to target preferentially tumor cells. F14512 has been characterized as a potent anticancer drug candidate and is currently in phase 1 clinical trials. Here we have analyzed the mechanisms of cell death induced by F14512, compared to the parent drug etoposide lacking the polyamine tail. F14512 proved to be >30-fold more cytotoxic than etoposide against A549 non-small cell lung cancer cells and triggers less but unrecoverable DNA damages. The cytotoxic action of F14512 is extremely rapid (within 3h) and does not lead to a marked accumulation in the S-phase of the cell cycle, unlike etoposide. Interestingly, A549 cells treated with F14512 were less prone to undergo apoptosis (neither caspases-dependent nor caspases-independent pathways) or autophagy but preferentially entered into senescence. Drug-induced senescence was characterized qualitatively and quantitatively by an increased β-galactosidase activity, both by cytochemical staining and by flow cytometry. A morphological analysis by electron microscopy revealed the presence of numerous multi-lamellar and vesicular bodies and large electron-lucent (methuosis-like) vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 compared to etoposide, and this difference may account for their distinct pharmacological profiles and the markedly superior activity of F14512 in vivo. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor activity.

Ion Channel Blockade of NMDA Receptors By Argiotoxins: Identification of Structural Determinats For Subtype Selectivity

The NMDA family of ionotropic glutamate receptors are ligand-gated ion channels that mediate the majority excitatory synaptic transmission in the brain. Dysfunction of NMDA receptor signalling is involved in a range of neurological and psychiatric diseases for which NMDA receptors are considered important drug targets. NMDA receptors are tetrameric assemblies comprised of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. Four different GluN2 subunits (GluN2A-D) exist with distinct expression and functional profiles. Identification of subunit-selective NMDA receptor agonists, antagonists, or modulators could prove to be both valuable pharmacological tools as well as potential new therapeutic agents.Polyamine toxins are a group of small molecules found in spiders and wasps that are open-channel blockers of NMDA receptors. Polyamine toxins have found valuable use as pharmacological tools based on their high affinity and selectivity, but have not yet been explored as templates for the development of NMDA receptor drugs. Argiotoxin-636 (ArgTX-636) is a polyamine toxin isolated from the venom of the orb weaver spider Argiope lobata. ArgTX-636 is among the most potent inhibitors of NMDA receptors and blocks these in a use- and voltage-dependent manner, presumably by binding to the ion channel region. We have recently shown that modifications within the polyamine tail of ArgTX-636 can control subtype selectivity. In this study, we explore the rational design of ArgTX-636 analogs to produce NMDA receptor inhibitors with marked improvement in subtype selectivity. Systematic variation of the polyamine tail lead to identification of ArgTX analogs with pronounced subtype selectivity, such as 100-fold differential potency between GluN2A, GluN2B, or GluN2C versus GluN2D containing receptors. To study the structural basis for NMDA subtype selectivity, compounds were docked into models of the NMDA receptor ion channels.

F14512, a Potent Antitumor Agent Targeting Topoisomerase II Vectored into Cancer Cells via the Polyamine Transport System

The polyamine transport system (PTS) is an energy-dependent machinery frequently overactivated in cancer cells with a high demand for polyamines. We have exploited the PTS to selectively deliver a polyamine-containing drug to cancer cells. F14512 combines an epipodophyllotoxin core-targeting topoisomerase II with a spermine moiety introduced as a cell delivery vector. The polyamine tail supports three complementary functions: (a) facilitate formulation of a water-soluble compound, (b) increase DNA binding to reinforce topoisomerase II inhibition, and (c) facilitate selective uptake by tumor cells via the PTS. F14512 is 73-fold more cytotoxic to Chinese hamster ovary cells compared with CHO-MG cells with a reduced PTS activity. A decreased sensitivity of L1210 leukemia cells to F14512 was observed in the presence of putrescine, spermidine, and spermine. In parallel, the spermine moiety considerably enhances the drug-DNA interaction, leading to a reinforced inhibition of topoisomerase II. The spermine tail of F14512 serves as a cell delivery vehicle as well as a DNA anchor, and this property translates at the cellular level into a distinct pharmacologic profile. Twenty-nine human solid or hematologic cell lines were used to characterize the high cytotoxic potential of F14512 (median IC50 of 0.18 micromol/L). Finally, the potent antitumor activity of F14512 in vivo was evidenced with a MX1 human breast tumor xenograft model, with partial and complete tumor regressions. This work supports the clinical development of F14512 as a novel targeted cytotoxic drug and sheds light on the concept of selective delivery of drugs to tumor cells expressing the PTS.

Anthraquinone polyamines: novel channel blockers of N-methyl-d-aspartate receptors

Polyamines, in particular spermine, as well as some natural and synthetic polyamine derivatives have been found to be blockers of N-methyl-D-aspartate receptors. We developed novel, polyamine-based channel blockers to analyze the structure of NMDA receptors. Anthraquinone polyamines block NMDA receptors with some selectivity compared to other glutamate receptors. Results using mutant NR1 and NR2 subunits identified amino acid residues that influence blockade by anthraquinone polyamines. The head group (anthraquinone) may be positioned at the selectivity filter/narrowest constriction of the channel and the polyamine tail penetrates this constriction into the inner vestibule below the level of the selectivity filter. The results are consistent with other work showing that NR1 (Asn616) and NR2B (Asn616), but not NR2B (Asn615), make the narrowest constriction of NMDA channel, and that the M3 segments from the two subunits, which form the outer vestibule, are likely staggered relative to each other in the vertical axis of the channel.

Comparative Studies of Anthraquinone- and Anthracene-Tetraamines as Blockers ofN-Methyl-d-aspartate Receptors

Anthraquinone spermine [N1-(anthraquinone-2-carbonyl)spermine; AQ343], anthraquinone homospermine [N1-(anthraquinone-2-carbonyl; AQ444], anthracene spermine [N1-(9-anthracenylmethyl)spermine; Ant343], and anthracene homospermine [N1-(9-anthracenylmethyl)homospermine; Ant444] were found to be potent antagonists of recombinant N-methyl-D-aspartate (NMDA) receptors (NRs). The effects of both anthraquinone (AQ)- and anthracene (Ant)-tetraamines were reversible and voltage-dependent. Results of experiments using mutant NR1 and NR2B subunits of NMDA receptor identified residues that influence block by AQ- and Ant-tetraamines. The results indicate that the polyamine tail is crucial for block by AQ- and Ant-tetraamines. Residues in the outer vestibule of the NR1 subunit were more strongly involved in block by AQ-and Ant-tetraamines than residues in the corresponding region of NR2B. Several amino acid residues in the inner vestibule, below the level of the selectivity filter of NR1 and NR2B, affected block by AQ444, Ant343, and Ant444, but they did not affect block by AQ343. AQ-tetraamines could permeate the channel at very negative membrane potentials when the narrowest constriction of the channel was expanded by replacing the Asn residue at Asn616 of NR1 and NR2B with Gly, whereas Ant-tetraamines did not easily pass through the channel, apparently because of differences in the relative position of the head groups on AQ- and Ant-polyamines.

Synthesis and characterization of N(1)-(4-toluenesulfonyl)-N(1)- (9-anthracenemethyl)triamines.

A modular synthetic approach was developed to access triamines with varying tether lengths from commercially available aminoalkanols. Initial N-alkylation via reductive amination with anthracene-9-carbaldehyde provided the secondary amines in good yield. Subsequent ditosylation with excess TsCl yielded the respective bis-N,O-tosylates. The tosylates were reacted with excess putrescine to give the final triamines. X-ray crystallography revealed that the polyamine tail is preferentially oriented over the shielding cone of the anthracene ring.

Argiotoxin636 inhibits NMDA-activated ion channels expressed in Xenopus oocytes

Argiotoxin636, a component of the spider venom of argiope species, was chemically synthesized together with a number of derivatives in order to analyse their blocking activity on mammalian glutamate receptors. Xenopus laevis oocytes injected with rat brain mRNA served as assay system. The results showed that argiotoxin636 had a higher affinity for N-methyl-D-aspartate (NMDA) than for kainate receptors, blocking the corresponding ion channels in a voltage-dependent manner. Modifications of the polyamine tail or the terminal arginine residue strongly reduced the blocking potency. The iodinated monohydroxyl phenylderivatives, however, retained their NMDA-selective binding and could serve as non-competitive antagonists for radioligand binding assays aiding in the biochemical isolation of glutamate receptors.

Inhibition of cation-induced DNA condensation by intercalating dyes.

AbstractSeveral intercalating dyes are shown to inhibit the cation‐induced condensation of λ‐DNA when Co3+(NH3)6 is the condensing agent. The dyes that have been studied are ethidium, propidium, proflavin, quinacrine, and actinomycin D. Earlier work has shown that intercalating dyes inhibit ψ‐DNA condensation. [Lerman, L. S. (1971) Prog. Mol. Subcell. Biol. 2, 382–391; Cheng, S. & Mohr, S. C. (1975) Biopolymers 14, 663–674.] Dye‐induced decondensation of intramolecularly condensed DNA has been studied by making use of conditions in which Co3+(NH3)6 produces intramolecular condensation without significant aggregation. Some aggregation is caused, however, during dye‐induced decondensation. Dye titration curves of DNA decondensation have been measured by excess light scattering to monitor decondensation and by fluorescence to monitor intercalation. All of the dyes studied act as competing cations in displacing the condensing cation Co3+(NH3)6 from the DNA. Competition occurs both in and below the transition zone for condensation. The effectiveness of a dye as a competing cation increases with its net positive charge. Before decondensation begins, no intercalated dye can be detected, suggesting that intercalation might be incompatible with the proper helix packing needed for cation‐induced DNA condensation. To test this last point, methidium–spermine was synthesized: it contains an intercalating methidium head group combined with a polyamine tail. Methidium–spermine is found to cause λ‐DNA condensation, but aggregation accompanies condensation, as has been found earlier for spermine and spermidine. Fluorescence and absorption spectra indicate that the methidium group is intercalated when the DNA is condensed, indicating that intercalation need not be incompatible with DNA condensation. The presence of aggregates among the condensed DNA molecules makes this last conclusion tentative.