Dendrimeric Guanidinoneomycin for Cellular Delivery of Bio-macromolecules.

We present the synthesis of polymeric amino- and guanidinoglycosides prepared by tethering neomycin and guanidinoneomycin to PAMAM dendrimers of generations 2 and 4. The ability of these conjugates to promote cellular uptake of high-molecular-weight cargo is discussed, together with their cytotoxicity and mechanisms of entry. We demonstrate that the presence of multiple guanidinoneomycin carriers on the PAMAM surface plays an important role in promoting cellular uptake of the dendrimers, maintaining the heparan sulfate specificity and negligible cytotoxicity typical of monomeric guanidinoglycoside molecular transporters.

Application of Surface Click Reactions to Localized Surface Plasmon Resonance (LSPR) Biosensing.

Localized surface plasmon resonance (LSPR) spectroscopy is an effective tool for sensitive, affordable, and label-free biosensing. LSPR transducers based on nanoparticulate Au films have been applied to biosensing of receptor-analyte interactions, employing primarily thiolated receptors for constructing biorecognition interfaces on nanostructured Au surfaces. This popular method suffers from a major drawback, that is, the need to prepare a thiolated receptor for each system used, which is typically synthetically complex and time-consuming. Herein, we present an alternative approach based on the click reaction between azide and terminal alkyne, which avoids the need to synthesize thiol-derivatized receptors and is applicable to the heterogeneous morphology of LSPR transducers. The receptors are tethered with an alkyne group, which is considerably simpler than thiolation, while producing a stable product. The transducer surface is modified with a layer of a commercial long-chain thiol-azide molecule, then clicked with an alkyne-dertivatized receptor to produce the biorecognition interface. This method is employed for immobilization of four different alkyne-bearing receptor molecules on Au nano-island film based LSPR transducers, followed by testing of their performance in biorecognition of specific analytes using LSPR and FTIR spectroscopies. The results establish the usefulness of click chemistry for the preparation of biorecognition interfaces on nanostructured LSPR transducers.

On the stability of Pt(IV) pro-drugs with haloacetato ligands in the axial positions.

The design of Pt(IV) pro-drugs as anticancer agents is predicated on the assumption that they will not undergo substitution reactions before entering the cancer cell. Attempts to improve the cytotoxic properties of Pt(IV) pro-drugs included the use of haloacetato axial ligands. Herein, we demonstrate that Pt(IV) complexes with trifluoroacetato (TFA) or dichloroacetato (DCA) ligands can be unstable under biologically relevant conditions and readily undergo hydrolysis, which results in the loss of the axial TFA or DCA ligands. The half-lives for Pt(IV) complexes with two TFA or DCA ligands at pH 7 and 37 °C range from 6 to 800 min, which is short relative to the duration of cytotoxicity experiments that last 24-96 h. However, complexes with two monochloroacetato (MCA) or acetato axial ligands are stable under biologically relevant conditions. The loss of the axial ligands depends primarily on the electron-withdrawing strength of the axial ligands, but also upon the nature of the equatorial ligands. We were unable to find obvious correlations between the structures of the Pt(IV) complexes and the rates of decay of the parent compounds. The X-ray crystal structures of the bis-DCA and bis-MCA Pt(IV) derivatives of oxaliplatin did not reveal any significant structural differences that could explain the observed differences in stability.

Macromolecular uptake of alkyl-chain-modified guanidinoglycoside molecular transporters.

Guanidinoglycosides, a family of cellular transporters capable of delivering high Mw biopolymers, have previously been shown to display high selectivity for cell-surface heparan sulfate proteoglycans and promote their clustering. Herein, the internalization mechanism of amphiphilic guanidinoglycoside derivatives was investigated by cell-surface FRET analysis. Unexpectedly, although the heparan sulfate selectivity is maintained, the cellular uptake of these derivatives does not appear to involve clustering of the proteoglycans on the cell surface. This suggests a distinct uptake mechanism when compared to the parent guanidinoglycoside-based carriers.

Relacin, a novel antibacterial agent targeting the Stringent Response.

Finding bacterial cellular targets for developing novel antibiotics has become a major challenge in fighting resistant pathogenic bacteria. We present a novel compound, Relacin, designed to inhibit (p)ppGpp production by the ubiquitous bacterial enzyme RelA that triggers the Stringent Response. Relacin inhibits RelA in vitro and reduces (p)ppGpp production in vivo. Moreover, Relacin affects entry into stationary phase in Gram positive bacteria, leading to a dramatic reduction in cell viability. When Relacin is added to sporulating Bacillus subtilis cells, it strongly perturbs spore formation regardless of the time of addition. Spore formation is also impeded in the pathogenic bacterium Bacillus anthracis that causes the acute anthrax disease. Finally, the formation of multicellular biofilms is markedly disrupted by Relacin. Thus, we establish that Relacin, a novel ppGpp analogue, interferes with bacterial long term survival strategies, placing it as an attractive new antibacterial agent.

On guanidinium and cellular uptake.

Guanidinium-rich scaffolds facilitate cellular translocation and delivery of bioactive cargos through biological barriers. Although impressive uptake has been demonstrated for nonoligomeric and nonpept(o)idic guanidinylated scaffolds in cell cultures and animal models, the fundamental understanding of these processes is lacking. Charge pairing and hydrogen bonding with cell surface counterparts have been proposed, but their exact role remains putative. The impact of the number and spatial relationships of the guanidinium groups on delivery and organelle/organ localization is yet to be established.

Facile preparation of mono-, di- and mixed-carboxylato platinum(IV) complexes for versatile anticancer prodrug design.

Facile strategies were developed for the versatile functionalization of platinum(IV) axial sites, allowing for easy accessibility to unsymmetric mono- and mixed-carboxylato, as well as symmetric di-substituted platinum(IV) complexes. The first method involves the direct oxidation and carboxylation of the platinum(II) center using an appropriate peroxide and the carboxylate of choice to firstly yield a monocarboxylato monohydroxido platinum(IV) complex. This platinum(IV) intermediate can undergo further carboxylation to give rise to a mixed-carboxylato platinum(IV) complex. The second method involves the activation of the carboxylate of choice by a common carbodiimide coupling reagent, and its reaction with a dihydroxido platinum(IV) precursor to give the monocarboxylato platinum(IV) complex. Uronium salts can be employed to promote efficient dicarboxylation of the dihydroxido platinum(IV) precursor. Lastly, an axial azide pendant group was demonstrated to be suitable for orthogonal "click" conjugation reactions.

c-di-AMP reports DNA integrity during sporulation in Bacillus subtilis.

The bacterium Bacillus subtilis produces the DNA integrity scanning protein (DisA), a checkpoint protein that delays sporulation in response to DNA damage. DisA scans the chromosome and pauses at sites of DNA lesions. Structural analysis showed that DisA synthesizes the small molecule cyclic diadenosine monophosphate (c-di-AMP). Here, we demonstrate that the intracellular concentration of c-di-AMP rises markedly at the onset of sporulation in a DisA-dependent manner. Furthermore, exposing sporulating cells to DNA-damaging agents leads to a global decrease in the level of this molecule. This drop was associated with stalled DisA complexes that halt c-di-AMP production and with increased levels of the c-di-AMP-degrading enzyme YybT. Reduced c-di-AMP levels cause a delay in sporulation that can be reversed by external supplementation of the molecule. Thus, c-di-AMP acts as a secondary messenger, coupling DNA integrity with progression of sporulation.

Platinum(IV) prodrugs with haloacetato ligands in the axial positions can undergo hydrolysis under biologically relevant conditions.

Losing ligands rapidly: Pt(IV) complexes with haloacetato ligands can hydrolyze rapidly under biological conditions (pH 7 and 37 °C, see scheme) and the rate increases with increasing pH value. Possible mechanisms for this hydrolysis are examined using H2(18)O and ESI-MS analysis.

ppGpp analogues inhibit synthetase activity of Rel proteins from Gram-negative and Gram-positive bacteria.

A prominent feature of the stringent response is the accumulation of two unusual phosphorylated derivatives of GTP and GDP (pppGpp: 5'-triphosphate-3'-diphosphate, and ppGpp: 5'-3'-bis-diphosphate), collectively called (p)ppGpp, within a few seconds after the onset of amino-acid starvation. The synthesis of these 'alarmone' compounds is catalyzed by RelA homologues. Other features of the stringent response include inhibition of stable RNA synthesis and modulation of transcription, replication, and translation. (p)ppGpp accumulation is important for virulence induction, differentiation and antibiotic resistance. We have synthesized a group of (p)ppGpp analogues and tested them as competitive inhibitors of Rel proteins in vitro. 2'-Deoxyguanosine-3'-5'-di(methylene bisphosphonate) [compound (10)] was found as an inhibitor that reduces ppGpp formation in both Gram-negative and Gram-positive bacteria. In silico docking together with competitive inhibition analysis suggests that compound (10) inhibits activity of Rel proteins by competing with GTP/GDP for its binding site. As Rel proteins are completely absent in mammalians, this appears to be a very attractive approach for the development of novel antibacterial agents.

Fibrin-hyaluronic acid hydrogel-based delivery of antisense oligonucleotides for ADAMTS5 inhibition in co-delivered and resident joint cells in osteoarthritis.

To date no disease-modifying drugs for osteoarthritis (OA) are available, with treatment limited to the use of pain killers and prosthetic replacement. The ADAMTS (A Disintegrin and Metallo Proteinase with Thrombospondin Motifs) enzyme family is thought to be instrumental in the loss of proteoglycans during cartilage degeneration in OA, and their inhibition was shown to reverse osteoarthritic cartilage degeneration. Locked Nucleic Acid (LNA)-modified antisense oligonucleotides (gapmers) released from biomaterial scaffolds for specific and prolonged ADAMTS inhibition in co-delivered and resident chondrocytes, is an attractive therapeutic strategy. Here, a gapmer sequence identified from a gapmer screen showed 90% ADAMTS5 silencing in a monolayer culture of human OA chondrocytes. Incorporation of the gapmer in a fibrin-hyaluronic acid hydrogel exhibited a sustained release profile up to 14 days. Gapmers loaded in hydrogels were able to transfect both co-embedded chondrocytes and chondrocytes in a neighboring gapmer-free hydrogel, as demonstrated by flow cytometry and confocal microscopy. Efficient knockdown of ADAMTS5 was shown up to 14 days in both cell populations, i.e. the gapmer-loaded and gapmer-free hydrogel. This work demonstrates the use applicability of a hydrogel as a platform for combined local delivery of chondrocytes and an ADAMTS-targeting gapmer for catabolic gene modulation in OA.

Cellular uptake of modified aminoglycosides.

The uptake of modified amino- and guanidino-glycosides derived from kanamycin, tobramycin and neomycin in native and mutant CHO cells is examined using confocal microscopy and flow cytometry, illustrating the significance of multivalency for mammalian cell internalization of carriers that specifically interact with cell surface heparan sulfate proteoglycans.The Journal of Antibiotics advance online publication, 1 November 2017; doi:10.1038/ja.2017.131.

Delivery of Cargo to Lysosomes Using GNeosomes.

Liposomes have been used to improve the intracellular delivery of a variety of cargos. Encapsulation of cargos in liposomes leads to improved plasma half-lives and minimized degradation. Here, we present a method for improving the selective delivery of liposomes to the lysosomes using a guanidinylated neomycin (GNeo) transporter. The method for synthesizing GNeo-lipids, incorporating them into liposomes, and the enhanced lysosomal delivery of encapsulated cargo are presented. GNeo-liposomes, termed GNeosomes, are capable of delivering a fluorescent dye to the lysosomes of Chinese hamster ovary cells as shown using confocal microscopy. GNeosomes can also be used to deliver therapeutic quantities of lysosomal enzymes to fibroblasts isolated from patients with a lysosomal storage disorder.

Delivery of an active lysosomal enzyme using GNeosomes.

Two methods for assembling guanidinoneomycin-decorated liposomes are presented and their ability to deliver an active enzyme to the lysosomes and restore enzyme function in diseased cells is compared.

Polymyxins Facilitate Entry into Mammalian Cells.

Polymyxin B is an antibiotic used against multi-resistant gram negative infections, despite observed nephrotoxicity. Here we report the synthesis of functionalized derivatives of polymyxin B and its per-guanidinylated derivative in order to further explore the structural requirements necessary to facilitate uptake of the antibiotic into mammalian cells. We also investigate the possibility of using these novel scaffolds as molecular transporters. At nanomolar concentrations, both are capable of delivering large cargo (>300 kDa) into living cells. Their uptake depends exclusively on cell surface heparan sulfate. Mechanistic studies indicate these novel transporters are internalized through caveolae-mediated pathways and confocal microscopy show colocalization with lysosomes. The polymyxin-based transporters demonstrate cytosolic delivery through the delivery of a ribosome-inactivating toxin. Furthermore, the natural polymyxin scaffold can be incorporated into liposomes and enhance their intracellular uptake. In addition to demonstrating the ability of the polymyxin scaffold to facilitate internalization into mammalian cells, these observations suggest the potential use of polymyxin and guanidinopolymyxin for intracellular delivery.

Pt(iv) derivatives of cisplatin and oxaliplatin with phenylbutyrate axial ligands are potent cytotoxic agents that act by several mechanisms of action.

Our study demonstrates that Pt(iv) derivative of cisplatin, with two axial PhB ligands, ctc-[Pt(NH3)2(PhB)2Cl2], is a very potent cytotoxic agent against many different human cancer cell lines and is up to 100 fold more potent than cisplatin, and significantly more potent than the Pt(iv) derivatives of cisplatin with either two hydroxido, two acetato or two valproato ligands. The high potency of this compound (and some others) is due to several factors including enhanced internalization, probably driven by "synergistic accumulation" of both the Pt moiety and the phenylbutyrate, that correlates with enhanced DNA binding and cytotoxicity. ctc-[Pt(NH3)2(PhB)2Cl2] inhibits 60-70% HDAC activity in cancer cells, at levels below the IC50 values of PhB, suggesting synergism between Pt and PhB. Mechanistically, ctc-[Pt(NH3)2(PhB)2Cl2] induces activation of caspases (3 and 9) triggering apoptotic signaling via the mitochondrial pathway. Data also suggest that the antiproliferative effect of ctc-[Pt(NH3)2(PhB)2Cl2] may not depend of p53. Pt(iv) derivatives of cisplatin with either two axial PhB or valproate ligands are more potent than their oxaliplatin analogs. ctc-[Pt(NH3)2(PhB)2Cl2] is significantly more potent than its valproate analog ctc-[Pt(NH3)2(VPA)2Cl2]. These compounds combine multiple effects such as efficient uptake of both Pt and PhB with DNA binding, HDAC inhibition and activation of caspases to effectively kill cancer cells.

GNeosomes: Highly Lysosomotropic Nanoassemblies for Lysosomal Delivery.

GNeosomes, lysosomotropic lipid vesicles decorated with guanidinoneomycin, can encapsulate and facilitate the cellular internalization and lysosomal delivery of cargo ranging from small molecules to high molecular weight proteins, in a process that is exclusively dependent on cell surface glycosaminoglycans. Their cellular uptake mechanism and co-localization with lysosomes, as well as the delivery, release, and activity of internalized cargo, are quantified. GNeosomes are proposed as a universal platform for lysosomal delivery with potential as a basic research tool and a therapeutic vehicle.

Design, synthesis and structure-activity relationship of novel Relacin analogs as inhibitors of Rel proteins.

Rel proteins in bacteria synthesize the signal molecules (p)ppGpp that trigger the Stringent Response, responsible for bacterial survival. Inhibiting the activity of such enzymes prevents the Stringent Response, resulting in the inactivation of long-term bacterial survival strategies, leading to bacterial cell death. Herein, we describe a series of deoxyguanosine-based analogs of the Relacin molecule that inhibit in vitro the synthetic activity of Rel proteins from Gram positive and Gram negative bacteria, providing a deeper insight on the SAR for a better understanding of their potential interactions and inhibitory activity. Among the inhibitors evaluated, compound 2d was found to be more effective and potent than our previously reported Relacin.

What do we know about the reduction of Pt(IV) pro-drugs?

Platinum(IV) pro-drugs are an important class of molecules that might improve the pharmacological properties of the platinum(II) anticancer agents that are currently used in the clinic. Their axial ligands that are detached during cellular activation by reductive elimination can be used to confer favorable pharmacological properties to the complexes allowing for potentially lower toxicity and improved efficacy. This manuscript describes the various approaches taken to design and prepare Pt(IV) complexes that will be effective anticancer agents. We review the working hypotheses guiding the researchers in the field while pointing out some more recent results that contradict some of the accepted paradigms in the hope of triggering some rethinking of the existing working hypotheses.

Pt(IV) analogs of oxaliplatin that do not follow the expected correlation between electrochemical reduction potential and rate of reduction by ascorbate.

In contrast to the Pt(IV) derivatives of cisplatin, Pt(IV) derivatives of oxaliplatin do not show the expected correlation between the electrochemical reduction potentials and rates of reduction by ascorbate. This is probably due to the lower ability of the amine and carboxylato ligands to form a bridge with the reducing agents to facilitate electron transfer.