Disulfide-rich peptides as next-generation radiopharmaceuticals

Multicyclic peptides that are constrained through covalent cross-linkers can usually maintain stable three-dimensional (3D) structures without the necessity of incorporating noncovalently interacting cores. This configuration allows for a greater utilization of residues for functional purposes compared to larger proteins, rendering multicyclic peptides attractive molecular modalities for the development of chemical tools and therapeutic agents. Even smaller multicyclic peptides, which may lack stable 3D structures due to limited sequence-driven folding capabilities, can still benefit from the specific conformations stabilized by covalent cross-linkers to facilitate target binding. Disulfide-rich peptides (DRPs) are a class of particularly significant multicyclic peptides that are primarily composed of disulfide bonds in their interior. However, the structural diversity of DRPs is limited to a few naturally occurring and designer scaffolds, which significantly impedes the development of multicyclic peptide ligands and therapeutics. To address this issue, we developed a novel method that utilizes disulfide-directing motifs to design and discover DRPs with new structures and functions in random sequence space. Compared with traditional DRPs, these new DRPs that incorporate disulfide-directing motifs exhibit more precise oxidative folding regarding disulfide pairing and demonstrate greater tolerance to sequence manipulations. Thus, we designated these peptides as disulfide-directed multicyclic peptides (DDMPs).Over the past decade, we have developed a new class of multicyclic peptides by leveraging disulfide-directing motifs, including biscysteine motifs such as CPXXC, CPPC, and CXC (C: cysteine; P: proline; X: any amino acid), as well as triscysteine motifs that rationally combine two biscysteine motifs (e.g., CPPCXC and CPXXCXC) to direct the oxidative folding of peptides. This leads to the introduction of a novel concept known as motif-directed oxidative folding, which is valuable for the construction of peptides with multiple disulfide bonds. A large diversity of DDMPs have been designed by simply altering the disulfide-directing motifs, the arrangement of cysteine residues (i.e., cysteine patterns), and the number of random residues separating them. As the oxidative folding of DDMPs is primarily determined by disulfide-directing motifs, these peptides are intrinsically more tolerant of extensive sequence manipulations compared to traditional DRPs. Consequently, multicyclic peptide libraries with an unprecedented high degree of sequence randomization have been developed by utilizing commonly used biological display systems such as phage display. We have validated the applicability of these libraries by successfully discovering DDMPs with unique protein-like 3D structures and high affinity and specificity to various cell-surface receptors, including tumor-associated antigens, immune costimulatory receptors, and G protein-coupled receptors (GPCRs). Currently, multicyclic peptides used in clinical settings are of natural origin or derived from natural DRPs. Our studies have opened up the possibility of developing multicyclic peptides without relying on natural scaffolds, representing a pivotal breakthrough in the field of peptide ligand and drug discovery. Further investigations will facilitate the application of our DDMPs in broader fields such as bioanalysis, chemical biology, and biomedicine.

Spiders are the most successful venomous animals and the most abundant terrestrial predators. Their remarkable success is due in large part to their ingenious exploitation of silk and the evolution of pharmacologically complex venoms that ensure rapid subjugation of prey. Most spider venoms are dominated by disulfide-rich peptides that typically have high affinity and specificity for particular subtypes of ion channels and receptors. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides, making them a valuable resource for drug discovery. Here we review the structure and pharmacology of spider-venom peptides that are being used as leads for the development of therapeutics against a wide range of pathophysiological conditions including cardiovascular disorders, chronic pain, inflammation, and erectile dysfunction.

The present work delineates pairwise interactions underlying the nanomolar affinity of alpha-conotoxin MI (CTx MI) for the alpha-delta site of the muscle acetylcholine receptor (AChR). We mutated all non-cysteine residues in CTx MI, expressed the alpha(2)betadelta(2) pentameric form of the AChR in 293 human embryonic kidney cells, and measured binding of the mutant toxins by competition against the initial rate of (125)I-alpha-bungarotoxin binding. The CTx MI mutations P6G, A7V, G9S, and Y12T all decrease affinity for alpha(2)betadelta(2) pentamers by 10,000-fold. Side chains at these four positions localize to a restricted region of the known three-dimensional structure of CTx MI. Mutations of the AChR reveal major contributions to CTx MI affinity by Tyr-198 in the alpha subunit and by the selectivity determinants Ser-36, Tyr-113, and Ile-178 in the delta subunit. By using double mutant cycles analysis, we find that Tyr-12 of CTx MI interacts strongly with all three selectivity determinants in the delta subunit and that deltaSer-36 and deltaIle-178 are interdependent in stabilizing Tyr-12. We find additional strong interactions between Gly-9 and Pro-6 in CTx MI and selectivity determinants in the delta subunit, and between Ala-7 and Pro-6 and Tyr-198 in the alpha subunit. The overall results reveal the orientation of CTx MI when bound to the alpha-delta interface and show that primarily hydrophobic interactions stabilize the complex.

We show that mechanochemically synthesized halide perovskite powders from a ball milling approach can be employed to fabricate a variety of lead halide perovskites with exceptional intrinsic stability. Our MAPbI3 powder exhibits higher thermal stability than conventionally processed thin films, without degradation after more than two and a half years of storage and only negligible degradation after heat treatment at 220 °C for 14 h. We further show facile recovery strategies of nonphase-pure powders by simple remilling or mild heat treatment. Moreover, we demonstrate the mechanochemical synthesis of phase-pure mixed perovskite powders, such as (Cs0.05FA0.95PbI3)0.85(MAPbBr3)0.15, from either the individual metal and organic halides or from readily prepared ternary perovskites, regardless of the precursor phase purity. Adding potassium iodide (KI) to the milling process successfully passivated the powders. We also succeeded in preparing a precursor solution on the basis of the powders and obtained uniform thin films for integration into efficient perovskite solar cells from spin-coating this solution. We find the KI passivation remains in the devices, leading to improved performance and significantly reduced hysteresis. Our work thus demonstrates the potential of mechanochemically synthesized halide perovskite powders for long-time storage and upscaling, further paving the way toward commercialization of perovskite-based optoelectronic devices.

Cadmium sulfide (CdS) persists as the standard electron transport layer (ETL) in high‐efficiency Sb 2 (S,Se) 3 solar cells. However, its narrow bandgap imposes critical performance limitations through parasitic short‐wavelength absorption and interface mismatch. Here, this bottleneck is shattered through an innovative in situ ozone doping strategy. This approach revolutionizes CdS ETLs by simultaneously: 1) Widening the bandgap of CdS ETL to drastically suppress parasitic short‐wavelength photon loss; 2) Triggering a hexagonal‐to‐cubic phase transition that disrupts coherent Sb 2 O 3 impurity growth during Sb 2 (S,Se) 3 deposition; and 3) Engineering an oxygen‐rich, defect‐resistant FTO/CdS interface (DFT‐validated), thereby slashing recombination and enabling superior carrier extraction. As a result, a certified world‐record power conversion efficiency of 9.0% for carbon‐based Sb 2 (S,Se) 3 solar cell is achieved ( V OC of 0.4908 V, J SC of 26.88 mA cm −2 , and FF of 68.19%) using an FTO/CdS:O/Sb 2 (S,Se) 3 /PbS/carbon configuration. This work, beyond setting a new benchmark for carbon‐based Sb 2 (S,Se) 3 solar cells, shows that these devices demonstrate exceptional intrinsic stability under long‐term and extreme damp‐heat conditions without encapsulation, thus propelling Sb 2 (S,Se) 3 technology toward commercial viability.

Identifizierung und Charakterisierung FAP-spezifischer Liganden für die zielgerichtete Diagnostik und Therapie maligner Tumoren

The future significance of peptide radiopharmaceuticals in diagnostic imaging with PET will be dependent on methodological aspects, as well as other requirements such as availability of the radionuclide and cost-effectiveness of its production. The aim of this study was to evaluate whether recent improvements in the modification of peptide pharmacokinetics by carbohydration may open a niche for the use of 11C-labelled peptide receptor binding tracers. A carbohydrated analogue of Tyr3-octreotate was used as a clinically relevant peptide. Oxime-mediated coupling between 4-[11C]methoxy-benzaldehyde and an aminooxy-conjugated peptide precursor provided the 11C-labelled peptide in 21+/-5% decay-corrected yield (n=4) in a synthesis time of about 1 h. In rat pancreas carcinoma xenografted mice, the compound displayed predominant and fast renal clearance combined with high tumour uptake (18.5+/-2.8% ID/g) at 30 min post injection. Corresponding values for kidney, liver and intestine were 18.5+/-2.4% ID/g, 3.2+/-0.5% ID/g and 2.1+/-0.3% ID/g, respectively. In a PET study with xenografted mice, the tumour (0.2-0.3 g) was clearly delineated as early as 20 min after injection. Somatostatin receptor (sstr)-specific uptake was demonstrated by reduction of tumour uptake to 20% of control by co-injection of TOC (0.4 mg/kg; 30 min p.i.). A 11C-labelled octreotate derivative has been prepared which shows suitable pharmacokinetics for in vivo imaging of sstr-overexpressing tumours and thus represents the first proof of principle for the potential of 11C-labelled peptides in tumour imaging.

Antibodies are a family of glycoproteins that bind specifically to target molecules (antigens). The antibody‐binding sites are formed by six segments of variable structure (CDRs) supported by a scaffold of essentially invariant architecture (framework regions). Shape complementarity between the contact surfaces (in the case of protein antigens) or close interactions with small antigens (hapten, peptide or others), together with complementation of water molecules, are important to achieve high affinity and specificity. The binding of an antigen to an antibody takes place by the formation of multiple noncovalent bonds between the antigen and the amino acids of the binding site. The increase in van der Waals contacts and/or buried surfaces upon complexation generally correlates well with the binding strength. Hydrogen bonds are in most cases critical to achieve high specificity and affinity for the antigen target. Importantly, antibodies have at least two antigen binding sites, boosting the effective affinity of the antibody for its target by a mechanism termed avidity. Key Concepts The binding between antibodies and antigens is characterised by high specificity and affinity resulting from distinct structural and energetic features. Non‐covalent forces dominate antibody‐antigen interactions. Generally, the recognition of an antigen is driven by the favorable change of enthalpy, and opposed by the entropy term. Multivalency is an important property of antibodies that govern their interaction with antigens in a biological setting. Progress in the fundamental understanding of antigen‐antibody interactions will lead to the rational design of more efficient and potent therapeutic antibodies.

The biological and physical properties of albumin and nitroxides make them attractive candidates as special purpose MRI contrast agents which could be used to study the intravascular compartment or specific targets in tissues. In this study, albumin-nitroxide complexes were prepared by reduction and alkylation of the disulfide bonds of the protein and characterized by electron spin resonance and ultraviolet absorption spectroscopy. An average of six nitroxides were bound covalently to each molecule of human serum albumin. The water proton relaxivity of the protein-bound nitroxide (at 20 MHz and 37 degrees C) was 4-fold greater than that of the free nitroxide. The digestion of the nitroxide-albumin complexes by cells or by trypsin decreased the relaxivity of the nitroxide-protein complex. The rate of reduction of albumin-bound nitroxide by cells was much slower than that of the free nitroxide but still was oxygen-sensitive (2-3-fold increase in the rate of reduction in the absence of oxygen).

Separation under high temperature is a crucial challenge for polymer nanofiltration (NF) membranes. Polyamide NF membranes with favorable compactness and pore size distribution are promising for boosting their thermal stability and high-temperature separation performance. Despite recent strategies to improve thermal stability, the effect of polyamide membrane compactness on structural changes and separation performance at high temperatures remains insufficiently explored. Herein, we propose a straightforward and effective concentration regulation strategy to address this challenge in the classic piperazine-trimesoyl chloride (PIP-TMC) interfacial polymerization system, focusing on the precise tuning of the polyamide network structure and the high-temperature NF performance through the controlled adjustment of monomer concentration and the relative ratios. Polyamide layer fabricated with a PIP/TMC ratio of 30:1.5 exhibits higher cross-linking degree than that with a PIP/TMC ratio of 2:1.5, and the NF membrane exhibits exceptional thermal stability, with MgSO4 rejection remaining >98.5% at 85 °C. Molecular dynamics simulations reveal that denser polyamide network possesses significantly weaker segmental motion, along with smaller changes in pore size distribution and fractional free volume at elevated temperatures. This concentration regulation strategy effectively enhances the intrinsic thermal stability without complex modifications and provides critical insights into polyamide segment evolution at high temperature.

Chapter 19 - Pharmacokinetics of bioactive plant-derived polysaccharides for enhanced drug release, stability, bioavailability and target specificity

Zwitterionic nanogels crosslinked by fluorescent carbon dots for targeted drug delivery and simultaneous bioimaging

Nanobody-as versatile tool emerging in autoimmune diseases

Covalent trapping of DNA-binding proteins via photo-crosslinking is an advantageous method for studying DNA-protein interactions. However, traditional photo-crosslinkers generate highly reactive intermediates that rapidly and non-selectively react with nearby functional groups, resulting in low target-capture yields and high non-target background capture. Herein, we report that photo-caged 2-butene-1,4-dial (PBDA) is an efficient photo-crosslinker for trapping DNA-binding proteins. Photo-irradiation (360 nm) of PBDA-modified DNA generates 2-butene-1,4-dial (BDA), a small, long-lived intermediate that reacts selectively with Lys residues of DNA-binding proteins, leading in minutes to stable DNA-protein crosslinks in up to 70% yield. In addition, BDA exhibits high specificity for target proteins, leading to low non-target background capture. The high photo-crosslinking yield and target specificity make PBDA a powerful tool for studying DNA-protein interactions.

Harnessing anti-idiotypic antibody alternatives to advance biotherapeutic pharmacokinetic assays.