Efficient systemic CNS delivery of a therapeutic antisense oligonucleotide with a blood-brain barrier-penetrating ApoE-derived peptide.

Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with high selectivity and specificity. However, the poor permeability of ASO across the blood-brain barrier (BBB) diminishes its therapeutic success. Here, we designed and synthesized a series of BBB-penetrating peptides (BPP) derived from either the receptor-binding domain of apolipoprotein E (ApoE) or a transferrin receptor-binding peptide (THR). The BPPs were conjugated to phosphorodiamidate morpholino oligomers (PMO) that are chemically analogous to the 2'-O-(2-methoxyethyl) (MOE)-modified ASO approved by the FDA for treating spinal muscular atrophy (SMA). The BPP-PMO conjugates significantly increased the level of full-length SMN2 in the patient-derived SMA fibroblasts in a concentration-dependent manner with minimal to no toxicity. Furthermore, the systemic administration of the most potent BPP-PMO conjugates significantly increased the expression of full-length SMN2 in the brain and spinal cord of SMN2 transgenic adult mice. Notably, BPP8-PMO conjugate showed a 1.25-fold increase in the expression of full-length functional SMN2 in the brain. Fluorescence imaging studies confirmed that 78% of the fluorescently (Cy7)-labelled BPP8-PMO reached brain parenchyma, with 11% uptake in neuronal cells. Additionally, the BPP-PMO conjugates containing retro-inverso (RI) D-BPPs were found to possess extended half-lives compared to their L-counterparts, indicating increased stability against protease degradation while preserving the bioactivity. This delivery platform based on BPP enhances the CNS bioavailability of PMO targeting the SMN2 gene, paving the way for the development of systemically administered neurotherapeutics for CNS disorders.

Mechanically Induced Switching between Two Discrete Conductance States: A Potential Single-Molecule Variable Resistor.

The fabrication of solid-state single-molecule switches with high on-off conductance ratios has been proposed to advance conventional technology in areas such as molecular electronics. Herein, we employed the scanning tunneling microscope break junction (STM-BJ) technique to modulate conductance in single-molecule junctions using mechanically induced stretching. Compound 1a possesses two dihydrobenzothiophene (DHBT) anchoring groups at the opposite ends linked with rigid alkyne side arms to form a gold-molecule-gold junction, while 1b contains 4-pyridine-anchoring groups. The incorporation of ferrocene into the backbone of each compound allows rotational freedom to the cyclopentadienyl (Cp) rings to give two distinct conductance states (high and low) for each. Various control experiments and suspended junction compression/retraction measurements indicate that these high- and low-conductance plateaus are the results of conformational changes within the junctions (extended and folded states) brought about by mechanically induced stretching. A high-low switching factor of 42 was achieved for 1a, whereas an exceptional conductance ratio in excess of 2 orders of magnitude (205) was observed for 1b. To the best of our knowledge, this is the highest experimental on-off conductance switching ratio for a single-molecule junction exploiting the mechanically induced STM-BJ method. Computational studies indicated that the two disparate conductance states observed for 1a and 1b result from mechanically induced conformational changes due to an interplay between conductance and the dihedral angles associated with the electrode-molecule interfaces. Our study reveals the structure-function relationship that determines conductance in such flexible and dynamic systems and promotes the development of a single-molecule variable resistor with high on-off switching factors.

Unravelling Structural Dynamics within a Photoswitchable Single Peptide: A Step Towards Multimodal Bioinspired Nanodevices.

The majority of the protein structures have been elucidated under equilibrium conditions. The aim herein is to provide a better understanding of the dynamic behavior inherent to proteins by fabricating a label-free nanodevice comprising a single-peptide junction to measure real-time conductance, from which their structural dynamic behavior can be inferred. This device contains an azobenzene photoswitch for interconversion between a well-defined cis, and disordered trans isomer. Real-time conductance measurements revealed three distinct states for each isomer, with molecular dynamics simulations showing each state corresponds to a specific range of hydrogen bond lengths within the cis isomer, and specific dihedral angles in the trans isomer. These insights into the structural dynamic behavior of peptides may rationally extend to proteins. Also demonstrated is the capacity to modulate conductance which advances the design and development of bioinspired electronic nanodevices.

Short Photoswitchable Antibacterial Peptides.

Three photoswitchable tetrapeptides, based on a known synthetic antibacterial, were designed and synthesized to determine activity against Staphylococcus aureus. Each peptide contains an azobenzene photoswitch incorporated into either the N-terminal side chain (1), C-terminal side chain (2), or the C-terminus (3) to allow reversible switching between cis- and trans-enriched photostationary states. Biological assays revealed that the C-terminus azobenzene (3) possessed the most potent antibacterial activity, with an MIC of 1 µg/mL. In this study, net positive charge, hydrophobicity, position of the azobenzene, secondary structure, and amphiphilicity were all found to contribute to antibacterial activity, with each of these factors likely facilitating the peptide to disrupt the negatively charged bacterial lipid membrane. Hence, these short photoswitchable antibacterial tetrapeptides provide insights for the future design and synthesis of antibiotics targeting S. aureus.

A hypoxia-activated antibacterial prodrug.

A prodrug based on a known antibacterial compound is reported to target Staphylococcus aureus and Escherichia coli under reductive conditions. The prodrug was prepared by masking the N-terminus and side chain amines of a component lysine residue as 4-nitrobenzyl carbamates. Activation to liberate the antibacterial was demonstrated on treatment with a model reductant, tin(II) chloride. The bioactivity of 1 was confirmed in antibacterial susceptibility assays whereas prodrug 2 was inactive.

Photopharmacological Control of Cyclic Antimicrobial Peptides.

Gramicidin S is a naturally occurring antimicrobial cyclic peptide. Herein, we present a series of cyclic peptides based on gramicidin S that contain an azobenzene photoswitch to reversibly control secondary structure and, hence, antimicrobial activity. 1 H NMR spectroscopy and density functional theory calculations revealed a ß-sheet/ß-turn secondary structure for the cis configuration of each peptide, and an ill-defined conformation for all associated trans structures. The cis-enriched and trans-enriched photostationary states (PSSs) for peptides 1-3 were assayed against Staphylococcus aureus to reveal a clear relationship between well-defined secondary structure, amphiphilicity and optimal antimicrobial activity. Most notably, peptides 2 a and 2 b exhibited a fourfold difference in antimicrobial activity in the cis-enriched PSS over the trans-enriched equivalent. This photopharmacological approach allows antimicrobial activity to be regulated through photochemical control of the azobenzene photoswitch, thereby opening new avenues in the design and synthesis of future antibiotics.

Peptides as Bio-inspired Molecular Electronic Materials.

Understanding the electronic properties of single peptides is not only of fundamental importance to biology, but it is also pivotal to the realization of bio-inspired molecular electronic materials. Natural proteins have evolved to promote electron transfer in many crucial biological processes. However, their complex conformational nature inhibits a thorough investigation, so in order to study electron transfer in proteins, simple peptide models containing redox active moieties present as ideal candidates. Here we highlight the importance of secondary structure characteristic to proteins/peptides, and its relevance to electron transfer. The proposed mechanisms responsible for such transfer are discussed, as are details of the electrochemical techniques used to investigate their electronic properties. Several factors that have been shown to influence electron transfer in peptides are also considered. Finally, a comprehensive experimental and theoretical study demonstrates that the electron transfer kinetics of peptides can be successfully fine tuned through manipulation of chemical composition and backbone rigidity. The methods used to characterize the conformation of all peptides synthesized throughout the study are outlined, along with the various approaches used to further constrain the peptides into their geometric conformations. The aforementioned sheds light on the potential of peptides to one day play an important role in the fledgling field of molecular electronics.