Publications

Lysosomes play a central role in cellular homeostasis and alterations in this compartment associate with many diseases. The most studied example is that of lysosomal storage disorders (LSDs), a group of 60 + maladies due to genetic mutations affecting lysosomal components, mostly enzymes. This leads to aberrant intracellular storage of macromolecules, altering normal cell function and causing multiorgan syndromes, often fatal within the first years of life. Several treatment modalities are available for a dozen LSDs, mostly consisting of enzyme replacement therapy (ERT) strategies. Yet, poor biodistribution to main targets such as the central nervous system, musculoskeletal tissue, and others, as well as generation of blocking antibodies and adverse effects hinder effective LSD treatment. Drug delivery systems are being studied to surmount these obstacles, including polymeric constructs and nanoparticles that consti-tute the focus of this article. We provide an overview of the formulations being tested, the diseases they aim to treat, and the results observed from respective in vitro and in vivo studies. We also discuss the advantages and disadvantages of these strategies, the remaining gaps of knowledge regarding their per-formance, and important items to consider for their clinical translation. Overall, polymeric nanocon-structs hold considerable promise to advance treatment for LSDs.(c) 2023 Elsevier B.V. All rights reserved.

JTD Keywords: cellular and animal models, enzyme replacement therapy, lysosomal storage disorders, nanoemulsions, nanoparticles, Beta-glucuronidase deficiency, Blood-brain-barrier, Cellular and animal models, Central-nervous-system, Drug delivery systems, Enzyme replacement therapy, Feline gm1 gangliosidosis, Human acid sphingomyelinase, Human alpha-galactosidase, Humans, Lysosomal storage diseases, Lysosomal storage disorders, Lysosomes, Mucopolysaccharidosis type-ii, Nanoemulsions, Nanoparticles, Neuronal ceroid-lipofuscinosis, Niemann-pick-disease, Pluripotent stem-cells, Polymer-based drug delivery systems, Polymers, Tissue distribution

The methyl erythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential for malaria parasites and also for several human pathogenic bacteria, thus representing an interesting target for future antimalarials and antibiotics and for diagnostic strategies. We have developed a DNA aptamer (D10) against Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of this metabolic route. D10 binds in vitro to recombinant DXR from P. falciparum and Escherichia coli, showing at 10 mu M a ca. 50% inhibition of the bacterial enzyme. In silico docking analysis indicates that D10 associates with DXR in solvent-exposed regions outside the active center pocket. According to fluorescence confocal microscopy data, this aptamer specifically targets in P. falciparum in vitro cultures the apicoplast organelle where the MEP pathway is localized and is, therefore, a highly specific marker of red blood cells parasitized by Plasmodium vs. naive erythrocytes. D10 is also selective for the detection of MEP+ bacteria (e.g., E. coli and Pseudomonas aeruginosa) vs. those lacking DXR (e.g., Enterococcus faecalis). Based on these results, we discuss the potential of DNA aptamers in the development of ligands that can outcompete the performance of the well-established antibody technology for future therapeutic and diagnostic approaches.

JTD Keywords: 1-deoxy-d-xylulose-5-phosphate reductoisomerase, dna aptamers, plasmodium, 1-deoxy-d-xylulose-5-phosphate reductoisomerase, Apicoplast, Dna aptamers, Drug targets, Evolution, Inhibitors, Isoprenoid biosynthesis, Malaria, Methyl erythritol phosphate pathway, Pathway, Plasmodium, Protein-protein, Web server

Tricyclic chemical structures are the core of many important drugs targeting all neurotransmitter pathways. These medicines enable effective therapies to treat from peptic ulcer disease to psychiatric disorders. However, when administered systemically, they cause serious adverse effects that limit their use. To obtain localized and on-demand pharmacological action using light, we have designed photoisomerizable ligands based on azobenzene that mimic the tricyclic chemical structure and display reversibly controlled activity. Pseudo-analogues of the tricyclic antagonist pirenzepine demonstrate that this is an effective strategy in muscarinic acetylcholine receptors, showing stronger inhibition upon illumination both in vitro and in cardiac atria ex vivo. Despite the applied chemical modifications to make pirenzepine derivatives sensitive to light stimuli, the most potent candidate of the set, cryptozepine-2, maintained a moderate but promising M-1 vs M-2 subtype selectivity. These photoswitchable crypto-azologs of tricyclic drugs might open a general way to spatiotemporally target their therapeutic action while reducing their systemic toxicity and adverse effects.

JTD Keywords: Binding, Dose-response relationship, drug, Drug design, Humans, M1, Molecular structure, Muscarinic antagonists, Pirenzepine, Rat-brain, Receptor, Receptors, muscarinic, Structure-activity relationship

Lateral segregation of cell membranes is accepted as a primary mechanism for cells to regulate a diversity of cellular functions. In this context, lipid rafts have been conceptualized as organizing principle of biological membranes where underlying cholesterol-mediated selective connectivity must exist even at the resting state. However, such a level of nanoscale compositional connectivity has been challenging to prove. Here we used single-molecule near-field scanning optical microscopy to visualize the nanolandscape of raft ganglioside GM1 after tightening by its ligand cholera toxin (CTxB) on intact cell membranes. We show that CTxB tightening of GM1 is sufficient to initiate a minimal raft coalescence unit, resulting in the formation of cholesterol-dependent GM1 nanodomains <120 nm in size. This particular arrangement appeared independent of cell type and GM1 expression level on the membrane. Simultaneous dual color high-resolution images revealed that GPI anchored and certain transmembrane proteins were recruited to regions proximal (<150 nm) to CTxB-GM1 nanodomains without physical intermixing. Together with in silico experiments, our high-resolution data conclusively demonstrate the existence of raft-based interconnectivity at the nanoscale. Such a linked state on resting cell membranes constitutes thus an obligatory step toward the hierarchical evolution of large-scale raft coalescence upon cell activation.

JTD Keywords: Cholera toxin, Membrane heterogeneity, Near-field scanning optical microscopy, Raft ganglioside GM1, Single-molecule detection