A Novel Lysosome Targeting Chimera for Targeted Protein Degradation via Split-and-Mix Strategy.

Targeted protein degradation is becoming more and more important in the field of drug development. Compared with proteasomal-based degraders, lysosomal-based degraders have a broader target spectrum of targets, which have been demonstrated to have great potential, especially in degrading undruggable proteins. Recently, we developed a programmable and facile screening PROTAC development platform based on peptide self-assembly termed split-and-mix PROTAC (SM-PROTAC). In this study, we applied this technology for the development of lysosome-based degraders, named a split-and-mix chaperone-mediated autophagy-based degrader (SM-CMAD). We successfully demonstrated SM-CMAD as a universal platform by degrading several targets, including ERα, AR, MEK1/2, and BCR-ABL. Different from other lysosomal-based degraders, SM-CMAD was capable of facile screening with programmable ligand ratios. We believe that our work will promote the development of other multifunctional molecules and clinical translation for lysosomal-based degraders.

Covalent PROTAC design method based on a sulfonyl pyridone probe.

Covalent proteolysis-targeting chimeras (PROTACs) offer enhanced selectivity, prolonged action, and increased efficacy against challenging target proteins. The conventional approach relies on covalent ligands, but our study presents an innovative method employing an N-sulfonyl pyridone warhead to selectively target tyrosine (Tyr) residues. The von Hippel-Lindau (VHL) moiety is transferred from the warhead to the exposed Tyr, allowing us to design a STING degrader (DC50 0.53 µM, Dmax 56.65%). This approach showcases the potential of nucleophilic amino acid labeling probes, particularly for proteins lacking easily accessible cysteine residues, opening new possibilities for covalent PROTAC design and targeted protein degradation therapies.

Covalent Peptide LSD1 Inhibitor Specifically Recognizes Cys360 in the Enzyme-Active Region.

Lysine-specific demethylase 1 (LSD1) is a promising therapeutic target, especially in cancer treatment. Despite several LSD1 inhibitors being discovered for the cofactor pocket, none are FDA-approved. We aimed to develop stabilized peptides for irreversible LSD1 binding, focusing on unique cysteine residue Cys360 in LSD1 and SNAIL1. We created LSD1 C360-targeting peptides, like cyclic peptide S9-CMC1, using our Cysteine-Methionine cyclization strategy. S9-CMC1 effectively inhibited LSD1 at the protein level, as confirmed by MS analysis showing covalent bonding to Cys360. In cells, S9-CMC1 inhibited LSD1 activity, increasing H3K4me1 and H3K4me2 levels, leading to G1 cell cycle arrest and apoptosis and inhibiting cell proliferation. Remarkably, S9-CMC1 showed therapeutic potential in A549 xenograft animal models, regulating LSD1 activity and significantly inhibiting tumor growth with minimal organ damage. These findings suggest LSD1 C360 as a promising site for covalent LSD1 inhibitors' development.

Selective Protein of Interest Degradation through the Split-and-Mix Liposome Proteolysis Targeting Chimera Approach.

Proteolysis Targeting Chimera (PROTAC) technology represents a promising new approach for target protein degradation using a cellular ubiquitin-proteasome system. Recently, we developed a split-and-mix nanoplatform based on peptide self-assembly, which could serve as a self-adjustable platform for multifunctional applications. However, the lower drug efficacy limits further biomedical applications of peptide-based SM-PROTAC. In this study, we develop a novel split-and-mix PROTAC system based on liposome self-assembly (LipoSM-PROTAC), concurrent with modification of FA (folate) to enhance its tumor-targeting capabilities. Estrogen receptors (ERα) were chosen as the protein of interest (POI) to validate the efficacy of Lipo degraders. Results demonstrate that this PROTAC can be efficiently and selectively taken up into the cells by FA receptor-positive cells (FR+) and degrade the POI with significantly reduced concentration. Compared to the peptide-based SM-PROTACs, our designed LipoSM-PROTAC system could achieve therapeutic efficacy with a lower concentration and provide opportunities for clinical translational potential. Overall, the LipoSM-based platform shows a higher drug efficacy, which offers promising potential applications for PROTAC and other biomolecule regulations.

Influence of Pyrolytic Carbon Black Derived from Waste Tires at Varied Temperatures within an Industrial Continuous Rotating Moving Bed System.

Nowadays, waste tires have emerged as one of the most significant sources of environmental pollution. To address this issue, pyrolysis has become a widely adopted method. The continuous rotary kiln reactor has particularly gained popularity in industrial production for pyrolysis due to its suitability. In order to guide the development of new industrial continuous rotary kiln reactors and achieve high-performance pyrolytic carbon black (CBp), this study was conducted to investigate the relationship between the physical and chemical characteristics of CBp and pyrolysis temperature. The elevated-temperature procedure led to a reduction in DBP values from 90 to 70 mL/100 mg, accompanied by a rise in the specific surface area from 63 to 77 m2/g. The augmentation of pyrolysis temperature was noted to induce the agglomeration of CBp particles, thereby negatively impacting their dispersion within polymer matrices. CBp particles at 550 °C exhibited greater structural order, as determined by Raman spectroscopy, which can be attributed to the elevated temperature proximate to the cylinder wall surface. Furthermore, the potential of CBp for reinforcement in natural rubber (NR) was taken into consideration. The pronounced propensity of high-temperature CBps to agglomerate led to uneven dispersion within the polymer, consequently causing heightened heat accumulation and the emergence of the Payne effect. Based on a thorough analysis of the outcomes, the optimal pyrolysis temperature for CBp synthesis within the continuous reactor was ascertained.

Development of Lysine Crotonyl-Mimic Probe to Covalently Identify H3K27Cr Interacting Proteins.

Histone lysine crotonylation (Kcr) is one newly discovered acylation modification and regulates numerous pathophysiological processes. The binding affinity between Kcr and its interacting proteins is generally weak, which makes it difficult to effectively identify Kcr-interacting partners. Changing the amide of crotonyl to an ester increased reactivity with proximal cysteines and retained specificity for Kcr antibody. The probe "H3g27Cr" was designed by incorporating the ester functionality into a H3K27 peptide. Using this probe, multiple Kcr-interacting partners including STAT3 were successfully identified, and this has not been reported previously. Further experiments suggested that STAT3 possibly could form complexes with Histone deacetylase HDACs to downregulate the acetylation and crotonylation of Histone H3K27. Our unique design provided intriguing tools to further explore Kcr-interacting proteins and elucidate their working mechanisms.

Methodology of stable peptide based on propargylated sulfonium.

Peptides can be used as effective molecular tool for covalent modification of proteins and play important roles in ligand directed covalent modification. Tyr-selective protein modifications exert a profound impact on protein functionality. Here, we developed a general strategy that involves nucleophilic addition of alkyne for tyrosine modification. The terminal alkyne of propargyl sulfonium is motivated by the sulfonium center to react with phenolic hydroxyl. This approach provides a straightforward method for tyrosine modification due to its high yield in aqueous solution at physiological temperature. In addition, cyclic peptides could be obtained via adjusting pH to 8.0 from peptides consisting of tyrosine and methionine modified by propargyl bromide, and the resulting cyclic peptides are proved to have better stability, excellent 2-mercaptopyridine resistance and improved cellular uptakes. Furthermore, molecules made from the propargylated sulfonium have the potential to be used as warheads against tyrosine containing biomolecules. Collectively, we develop a direct and uncomplicated technique for modifying tyrosine residues, the strategy concerned can be widely utilized to construct stable peptides and biomolecules imaging.

Bioorthogonal Fluoride-Responsive Azide and Alkynyl Pyridinium Click Cycloaddition in Vitro and in Live Cells.

The copper-free azide-alkyne cycloaddition was broadly applied in numerous research fields. Herein, we report a facile Cu-free click reaction utilizing fluoride-responsive azide and alkynyl pyridinium cycloaddition at ambient temperatures in aqueous media. The reactivity of alkynyl pyridinium was successfully masked by a silyl-protecting group at the alkyne group, and the deprotection could be readily achieved with the addition of F-, which renders the reactivity. The substrates were readily synthesized and proven to be stable at the bench. This bioorthogonal fluoride-responsive click reaction was then successfully employed in peptide modification, protein labeling, and cell imaging, suggesting its potential in various applications.

Preparation of Zinc Oxide with Core-Shell Structure and Its Application in Rubber Products.

Zinc oxide is a crucial component in rubber products, but its excessive usage can lead to environmental damage. As a result, reducing the amount of zinc oxide in products has become a critical issue that many researchers aim to address. This study employs a wet precipitation method to prepare ZnO particles with different nucleoplasmic materials, resulting in ZnO with a core-shell structure. The prepared ZnO underwent XRD, SEM, and TEM analysis, indicating that some of the ZnO particles were loaded onto the nucleosomal materials. Specifically, ZnO with a silica core-shell structure demonstrated 11.9% higher tensile strength, 17.2% higher elongation at break, and 6.9% higher tear strength compared to the indirect method of ZnO preparation. The core-shell structure of ZnO also helps reduce its application in rubber products, thereby achieving the dual objective of protecting the environment and improving the economic efficiency of rubber products.

Effect of Different Silane Coupling Agents In-Situ Modified Sepiolite on the Structure and Properties of Natural Rubber Composites Prepared by Latex Compounding Method.

With the increasing demand for eco-friendly, non-petroleum-based natural rubber (NR) products, sepiolite, a naturally abundant, one-dimensional clay mineral, has been identified as a suitable material for reinforcing NR through the latex compounding method. To create superior NR/sepiolite composites, three silane coupling agents with different functional groups were used to modify sepiolite in situ via grafting or adsorption during the disaggregation and activation of natural sepiolite, which were subsequently mixed with natural rubber latex (NRL) to prepare the composites. The results showed that the modified sepiolite improved the dispersion and interfacial bonding strength with the rubber matrix. VTES-modified sepiolite containing C=C groups slightly improved the performance but retarded the vulcanization of the NR composites, and MPTES and TESPT-modified sepiolites containing -SH and -S4- groups, respectively, effectively accelerated vulcanization, inducing the composites to form a denser crosslink network structure, and exhibiting excellent dynamic and static properties, such as the modulus at a 300% increase from 8.82 MPa to 16.87 MPa, a tear strength increase from 49.6 N·mm-1 to 60.3 N·mm-1, as well as an improved rolling resistance and abrasive resistance of the composites. These findings demonstrate that modified sepiolite can be used to produce high-quality NR/sepiolite composites with enhanced properties.

Targeted Biomolecule Regulation Platform: A Split-and-Mix PROTAC Approach.

The development of bifunction al molecules, which can enable targeted RNA degradation, targeted protein acetylation, or targeted protein degradation, remains a time-consuming process that requires tedious optimization. We propose a split-and-mix nanoplatform that serves as a self-adjustable platform capable of facile screening, programmable ligand ratios, self-optimized biomolecule spatial recognition, and multifunctional applications. Herein, we demonstrate the potential of our proposed nanoplatform by showcasing proteolysis-targeting chimeras (PROTACs), namely, split-and-mix PROTAC (SM-PROTAC). We highlight the scope of our platform through the targeted disruption of intracellular therapeutic targets involving ERα, CDK4/6, AR, MEK1/2, BRD2/4, BCR-ABL, etc. These studies confirm the effectiveness and universality of the SM-PROTAC platform for proximity-induced applications. This platform is programmable, with significant potential applications to biomolecule regulation, including the fields of epigenetics, gene editing, and biomolecule modification regulation.

The thiol-sulfoxonium ylide photo-click reaction for bioconjugation.

Visible-light-mediated methods were heavily studied as a useful tool for cysteine-selective bio-conjugation; however, many current methods suffer from bio-incompatible reaction conditions and slow kinetics. To address these challenges, herein, we report a transition metal-free thiol-sulfoxonium ylide photo-click reaction that enables bioconjugation under bio-compatible conditions. The reaction is highly cysteine-selective and generally finished within minutes with naturally occurring riboflavin derivatives as organic photocatalysts. The catalysts and substrates are readily accessible and bench stable and have satisfactory water solubility. As a proof-of-concept study, the reaction was smoothly applied in chemo-proteomic analysis, which provides efficient tools to explore the druggable content of the human proteome.

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center.

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

A Peptide-Based Ligand-Directed Chemistry Enables Protein Functionalization.

The ligand-directed (LD) chemistry provides powerful tools for site-specific modification of proteins. We utilized a peptide with an appended methionine (Met) as a ligand; then, the Met thioether was modified into sulfonium which enabled a proximity induced group transfer onto protein cysteine in the vicinity upon peptide-target binding. The sulfonium warhead could be easily constructed with unprotected peptides, and the transferable group scope was conducted on model protein PDZ and its ligand peptides. In addition, a living cell labeling was successfully achieved.

Histidine-specific bioconjugation via visible-light-promoted thioacetal activation.

Histidine (His, H) undergoes various post-translational modifications (PTMs) and plays multiple roles in protein interactions and enzyme catalyzed reactions. However, compared with other amino acids such as Lys or Cys, His modification is much less explored. Herein we describe a novel visible-light-driven thioacetal activation reaction which enables facile modification on histidine residues. An efficient addition to histidine imidazole N3 under biocompatible conditions was achieved with an electrophilic thionium intermediate. This method allows chemo-selective modification on peptides and proteins with good conversions and efficient histidine-proteome profiling with cell lysates. 78 histidine containing proteins were for the first time found with significant enrichment, most functioning in metal accumulation in brain related diseases. This facile His modification method greatly expands the chemo-selective toolbox for histidine-targeted protein conjugation and helps to reveal histidine's role in protein functions.

Flavin catalyzed desulfurization of peptides and proteins in aqueous media.

A biomimetic method has been established for the chemo-selective desulfurization of cysteinyl peptides and proteins in aqueous media. The derivatives of biocatalytic cofactors, flavins, were found to be efficient photosensitizers in a thiyl-radical-mediated desulfurization of Cys. The reaction was conducted in an ultrafast manner with both polypeptides and proteins.

Low-Toxicity Sulfonium-Based Probes for Cysteine-Specific Profiling in Live Cells.

Despite being a low-abundance amino acid, cysteine plays an essential role in regulating protein function and serves as a satisfactory target of post-translational modifications and drug developments. To comprehensively assess reactive-cysteine-containing proteins, the development of chemical proteomic probes to label cysteine residues in human cells is an important objective. Cysteine modification using sulfonium-based probes is a novel method to identify reactive cysteine residues in proteins. Herein, we reported a set of "cysteine-reactive sulfonium-based (C-Sul)" probes to label the reactive cysteine sites in cellular proteins. Notably, water-soluble C-Sul probes have a significantly enhanced stability and cellular uptakes, displaying a high specificity toward reactive cysteines and compatibility with quantitative proteomic profiling. In comparison to the conventional iodoacetamide-based probe, C-Sul particularly has no inhibitory effects on cell viability, enabling its application in proteomic profiling of reactive cysteine residues under biorelevant conditions. We propose C-Sul probes as optimal tools of cysteine profiling for further broadly basic research.

Sulfonium Triggered Alkyne-Azide Click Cycloaddition.

Herein, we report the first facile Cu-free click reaction between alkynyl sulfonium and azide at ambient temperatures in aqueous media. DFT computations indicate that the sulfonium group is the key factor to gaining reactivity by stabilizing LUMO+1 and influencing the charge distribution of the triple bond. Sulfonium alkynes can be easily synthesized and scaled up, and most of them are biocompatible. We prepared candidate molecules and tested their use in multiple proof-of-concept biological applications.

Electrophilic Sulfonium-Promoted Peptide and Protein Amidation in Aqueous Media.

A novel amidation strategy using electrophilic sulfonium, which is soluble and stable in aqueous conditions, was developed. The sulfoniums could activate thioacid and carboxyl acid to efficiently react with amines to afford amides. This method enables applications in amidation in both aqueous media and solid-phase peptide synthesis, peptide/protein modifications, and reactive lysines of a proteome at pH 10 with activity-based protein profiling. A peptide ligand-directed labeling of the USP7-UBL2 domain was also performed using this method.

5-IP7 is a GPCR messenger mediating neural control of synaptotagmin-dependent insulin exocytosis and glucose homeostasis.

5-diphosphoinositol pentakisphosphate (5-IP7) is a signalling metabolite linked to various cellular processes. How extracellular stimuli elicit 5-IP7 signalling remains unclear. Here we show that 5-IP7 in ß cells mediates parasympathetic stimulation of synaptotagmin-7 (Syt7)-dependent insulin release. Mechanistically, vagal stimulation and activation of muscarinic acetylcholine receptors triggers Gαq-PLC-PKC-PKD-dependent signalling and activates IP6K1, the 5-IP7 synthase. Whereas both 5-IP7 and its precursor IP6 compete with PIP2 for binding to Syt7, Ca2+ selectively binds 5-IP7 with high affinity, freeing Syt7 to enable fusion of insulin-containing vesicles with the cell membrane. ß-cell-specific IP6K1 deletion diminishes insulin secretion and glucose clearance elicited by muscarinic stimulation, whereas mice carrying a phosphorylation-mimicking, hyperactive IP6K1 mutant display augmented insulin release, congenital hyperinsulinaemia and obesity. These phenotypes are absent in mice lacking Syt7. Our study proposes a new conceptual framework for inositol pyrophosphate physiology in which 5-IP7 acts as a GPCR second messenger at the interface between peripheral nervous system and metabolic organs, transmitting Gq-coupled GPCR stimulation to unclamp Syt7-dependent, and perhaps other, exocytotic events.