Genome-wide screens uncover KDM2B as a modifier of protein binding to heparan sulfate.

Heparan sulfate (HS) proteoglycans bind extracellular proteins that participate in cell signaling, attachment and endocytosis. These interactions depend on the arrangement of sulfated sugars in the HS chains generated by well-characterized biosynthetic enzymes; however, the regulation of these enzymes is largely unknown. We conducted genome-wide CRISPR-Cas9 screens with a small-molecule ligand that binds to HS. Screening of A375 melanoma cells uncovered additional genes and pathways impacting HS formation. The top hit was the epigenetic factor KDM2B, a histone demethylase. KDM2B inactivation suppressed multiple HS sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the interaction of HS-binding proteins. KDM2B-deficient cells displayed decreased growth rates, which was rescued by SULF1 inactivation. In addition, KDM2B deficiency altered the expression of many extracellular matrix genes. Thus, KDM2B controls proliferation of A375 cells through the regulation of HS structure and serves as a master regulator of the extracellular matrix.

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.

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.

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.

Bicephalic amphiphile architecture affects antibacterial activity.

A series of cationic amphiphiles, each with an aromatic core, was prepared and investigated for antimicrobial properties. The synthesized amphiphiles in this study are bicephalic (double-headed) in that they each possess two trimethylammonium head groups and a single linear alkoxy tail. Minimum inhibitory and minimum bactericidal concentrations of these amphiphiles were in the low micromolar range. Antimicrobial activities are highly sensitive to the chain length of the hydrophobic region, and modestly reliant on the relative positioning of the head groups on the aromatic core. These trends were more pronounced in time kill assays, wherein longer chain compounds required significantly shorter times to completely kill bacteria. Microscopy suggested that the mode of cell death was lysis. Strong inhibition was observed with both biscationic compounds and monocationic comparisons against Gram-positive bacteria; only biscationic amphiphiles maintained good activity versus the Gram-negative bacteria tested. These observations provide direction for future antimicrobial structural investigations.

Biscationic bicephalic (double-headed) amphiphiles with an aromatic spacer and a single hydrophobic tail.

We report the synthesis and aggregation studies of a homologous family of biscationic "bicephalic" amphiphiles. Each of has a linear alkoxy chain and two trimethylammonium bromide headgroups connected to a benzene ring. Krafft temperatures (T(K)) in water were determined by differential scanning calorimetry (DSC) and conductivity. Critical micelle concentration (cmc) and ionization degree (alpha) values were determined by monitoring the conductivity of aqueous solutions as a function of concentration, and confirmed by monitoring the (1)H NMR chemical shift of the terminal methyl group as a function of concentration. Values for log(cmc) decrease linearly with increasing chain length, with a smaller dependence on chain length than for single-headed amphiphiles, consistent with literature reports on other bicephalic amphiphiles. Comparison to two related amphiphiles, each with a single headgroup reveals that the addition of a second head group results in an increase in cmc and a decrease of T(K). These effects are attributed to greater water solubility due to the incorporation of a second, hydrophilic headgroup. The effect on alpha is also discussed.