Staff member

Blinding diseases due to the degeneration of photoreceptors (PhRs), such as geographic atrophy (GA) secondary to dry age-related macular degeneration and retinitis pigmentosa (RP), leave the rest of the retinal circuitry largely intact, albeit unable to respond to light. Gene therapy has been able to revert PhR degeneration, but it can be applied only to a rare mutation affecting a small subset of RP patients. Alternatively, implanted electronic retinal prostheses aim at a larger population by electrically stimulating surviving neurons. However, the treatment is invasive and costly and provides limited resolution. Photopharmacology can develop photoswitchable small molecules to restore vision impairment by conferring light sensitivity to ion channels that are widely expressed in the remaining inner retinal neurons, and a first-in-human clinical trial is ongoing. Here, we have developed novel photoswitchable small-molecule ligands of metabotropic glutamate 6 (mGlu6) receptors, which are located exclusively at the dendrites of ON bipolar cells (postsynaptic to PhRs) and can leverage a privileged position to mimic physiological signals in the remnant retinal circuit. These photoswitchable ligands (prosthe6) thus act as "molecular prostheses" that can restore the light input to the retina via upstream-targeted control of the circuit after PhR degeneration. Prosthe6 compounds are allosteric, drug-like, water-soluble, and display outstanding in vitro properties including full efficacy, nanomolar potency, fast deactivation in ambient white light, and fast reactivation in the dark. In vivo experiments show that they readily recover the saccadic eye movements of blinded zebrafish larvae and restore the innate light-avoidance behavior in the mouse models of blindness (GA and RP). These effects are mediated by mGlu6 receptors in vivo. In addition, at least two compounds (prosthe6-12 and -15) can restore sight by topical administration and display promising safety properties to become potential drug candidates for sight restoration in patients with degenerative blinding diseases.

JTD Keywords: Allosteric modulators, Bipolar cells, Blind mice, Ectopic expression, Gene-therapy, Human rod, Protein-coupled receptors, Restores visual responses, Retinal degeneration, Zebrafish models

Clathrin-mediated endocytosis (CME) is an essential cellular process, conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of specific pharmacological agents to interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor targeting the AP2-β-adaptin/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP, demonstrating that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts. This is despite the β-adaptin/β-arrestin interaction not being conserved in yeast. Our data indicate that the AP2 α-adaptin is the functional target of activated TL2. We identified as interacting partners for the α-appendage, the Eps15 and epsin homologues Ede1 and Ent1. This demonstrates that endocytic cargo loading and sensing can be executed by conserved molecular interfaces, regardless of the proteins involved.© 2023 The Author(s).

JTD Keywords: adapters, alpha-appendage, azobenzene, cross-linker, mechanism, peptides, proteins, receptor, trafficking, Actin polymerization, Biochemistry, Biological sciences, Cell biology, Molecular biology, Natural sciences

Ratiometric fluorescent nanothermometers with near-infrared emission play an important role in in vivo sensing since they can be used as intracellular thermal sensing probes with high spatial resolution and high sensitivity, to investigate cellular functions of interest in diagnosis and therapy, where current approaches are not effective. Herein, the temperature-dependent fluorescence of organic nanoparticles is designed, synthesized, and studied based on the dual emission, generated by monomer and excimer species, of the tris(2,4,6-trichlorophenyl)methyl radical (TTM) doping organic nanoparticles (TTMd-ONPs), made of optically neutral tris(2,4,6-trichlorophenyl)methane (TTM-αH), acting as a matrix. The excimer emission intensity of TTMd-ONPs decreases with increasing temperatures whereas the monomer emission is almost independent and can be used as an internal reference. TTMd-ONPs show a great temperature sensitivity (3.4% K-1 at 328 K) and a wide temperature response at ambient conditions with excellent reversibility and high colloidal stability. In addition, TTMd-ONPs are not cytotoxic and their ratiometric outputs are unaffected by changes in the environment. Individual TTMd-ONPs are able to sense temperature changes at the nano-microscale. In vivo thermometry experiments in Caenorhabditis elegans (C. elegans) worms show that TTMd-ONPs can locally monitor internal body temperature changes with spatio-temporal resolution and high sensitivity, offering multiple applications in the biological nanothermometry field.© 2023 The Authors. Small published by Wiley-VCH GmbH.

JTD Keywords: dual emission, elegans, excimer emission, fluorescence, in vivo sensing, luminescence, nanoparticles, organic radical nanoparticles, ratiometric nanothermometers, sensors, thermometry, trityl radicals, Caenorhabditis elegans, Excimer emission, In vivo sensing, Intracellular ph, Luminescence, Organic radical nanoparticles, Ratiometric nanothermometers, Trityl radicals

The spatiotemporal control of cellular dynamic processes has great fundamental interest but lacks versatile molecular tools. Dynamin is a key protein in endocytosis and an appealing target to manipulate cell trafficking using patterns of light. We have developed the first photoswitchable small-molecule inhibitors of endocytosis (dynazos), by a stepwise design of the photochromic and pharmacological properties of dynasore, a dynamin inhibitor. We have characterized their photochromism with UV-visible and transient absorption spectroscopy and their biological activity using fluorescence microscopies and flow cytometry. Dynazos are water-soluble, cell permeable, and photostable, and enable fast, single-wavelength photoswitchable inhibition of clathrin-mediated endocytosis at micromolar concentration.

JTD

Although cytotoxic chemotherapy is one of the primary pharmacological treatments for chronic hyperproliferative diseases such as cancer and psoriasis, its efficacy and tolerability are in many cases dramatically limited by off-target toxicity. A promising approach to improve these therapies is to activate the drugs exclusively at their desired place of action. In fact, in those diseases that would benefit from a highly localized treatment, a precise spatiotemporal control over the activity of a chemotherapeutic agent would allow reducing the concentration of active compound outside the targeted region, improving the tolerability of the treatment. Light is a powerful tool in this respect: it offers unparalleled opportunities as a non-invasive regulatory signal for pharmacological applications because it can be delivered with high precision regarding space, time, intensity and wavelength. Photopharmacology represents a new and emerging approach in this regard since the energy of light is used to change the structure of the drug and hence to switch its pharmacological activity on and off on demand. We describe here phototrexate, the first light-regulated inhibitor of the human DHFR. Enzyme and cell viability assays demonstrated that phototrexate behaves as a potent antifolate in its cis configuration, obtained under UVA illumination, and that it is nearly inactive in its dark-relaxed trans form. Experiments in zebrafish confirmed that phototrexate can disrupt folate metabolism in a light-dependent fashion also in vivo. Overall, phototrexate represents a potential candidate towards the development of an innovative photoactivated antifolate chemotherapy.

JTD Keywords: Cancer, Dermatology, Methotrexate, Photoactivated chemotherapy, Photodynamic therapy, Phototherapy, Psoriasis, Rheumatoid arthritis

Bioelectronics moves toward designing nanoscale electronic platforms that allow in vivo determinations. Such devices require interfacing complex biomolecular moieties as the sensing units to an electronic platform for signal transduction. Inevitably, a systematic design goes through a bottom-up understanding of the structurally related electrical signatures of the biomolecular circuit, which will ultimately lead us to tailor its electrical properties. Toward this aim, we show here the first example of bioengineered charge transport in a single-protein electrical contact. The results reveal that a single point-site mutation at the docking hydrophobic patch of a Cu-azurin causes minor structural distortion of the protein blue Cu site and a dramatic change in the charge transport regime of the single-protein contact, which goes from the classical Cu-mediated two-step transport in this system to a direct coherent tunneling. Our extensive spectroscopic studies and molecular-dynamics simulations show that the proteins’ folding structures are preserved in the single-protein junction. The DFT-computed frontier orbital of the relevant protein segments suggests that the Cu center participation in each protein variant accounts for the different observed charge transport behavior. This work is a direct evidence of charge transport control in a protein backbone through external mutagenesis and a unique nanoscale platform to study structurally related biological electron transfer.

JTD

Light-regulated drugs allow remotely photoswitching biological activity and enable plausible therapies based on small molecules. However, only freely diffusible photochromic ligands have been shown to work directly in endogenous receptors and methods for covalent attachment depend on genetic manipulation. Here we introduce a chemical strategy to covalently conjugate and photoswitch the activity of endogenous proteins and demonstrate its application to the kainate receptor channel GluK1. The approach is based on photoswitchable ligands containing a short-lived, highly reactive anchoring group that is targeted at the protein of interest by ligand affinity. These targeted covalent photoswitches (TCPs) constitute a new class of light-regulated drugs and act as prosthetic molecules that photocontrol the activity of GluK1-expressing neurons, and restore photoresponses in degenerated retina. The modularity of TCPs enables the application to different ligands and opens the way to new therapeutic opportunities.

JTD

Control of membrane traffic: Photoswitchable inhibitors of protein-protein interactions were applied to photoregulate clathrin-mediated endocytosis (CME) in living cells. Traffic light (TL) peptides acting as "stop" and "go" signals for membrane traffic can be used to dissect the role of CME in receptor internalization and in cell growth, division, and differentiation.

JTD Keywords: Clathrin-mediated endocytosis, Optopharmacology, Peptides, Photoswitches, Protein-protein interactions