Targeted Degradation of Cell-Surface Proteins via Chaperone-Mediated Autophagy by Using Peptide-Conjugated Antibodies.

Cell-surface proteins are important drug targets but historically have posed big challenges for the complete elimination of their functions. Herein, we report antibody-peptide conjugates (Ab-CMAs) in which a peptide targeting chaperone-mediated autophagy (CMA) was conjugated with commercially available monoclonal antibodies for specific cell-surface protein degradation by taking advantage of lysosomal degradation pathways. Unique features of Ab-CMAs, including cell-surface receptor- and E3 ligase-independent degradation, feasibility towards different cell-surface proteins (e.g., epidermal growth factor receptor (EGFR), programmed cell death ligand 1 (PD-L1), human epidermal growth factor receptor 2 (HER2)) by a simple change of the antibody, and successful tumor inhibition in vivo, make them attractive protein degraders for biomedical research and therapeutic applications. As the first example employing CMA to degrade proteins from the outside in, our findings may also shed new light on CMA, a degradation pathway typically targeting cytosolic proteins.

Insulin-like Growth Factor 2 (IGF2)-Fused Lysosomal Targeting Chimeras for Degradation of Extracellular and Membrane Proteins.

Targeted degradation of the cell-surface and extracellular proteins via the endogenous lysosomal degradation pathways, such as lysosome-targeting chimeras (LYTACs), has recently emerged as an attractive tool to expand the scope of extracellular chemical biology. Herein, we report a series of recombinant proteins genetically fused to insulin-like growth factor 2 (IGF2), which we termed iLYTACs, that can be conveniently obtained in high yield by standard cloning and bacterial expression in a matter of days. We showed that both type-I iLYTACs, in which IGF2 was fused to a suitable affibody or nanobody capable of binding to a specific protein target, and type-II iLYTAC (or IGF2-Z), in which IGF2 was fused to the IgG-binding Z domain that served as a universal antibody-binding adaptor, could be used for effective lysosomal targeting and degradation of various extracellular and membrane-bound proteins-of-interest. These heterobifunctional iLYTACs are fully genetically encoded and can be produced on a large scale from conventional E. coli expression systems without any form of chemical modification. In the current study, we showed that iLYTACs successfully facilitated the cell uptake, lysosomal localization, and efficient lysosomal degradation of various disease-relevant protein targets from different mammalian cell lines, including EGFR, PD-L1, CD20, and α-synuclein. The antitumor properties of iLYTACs were further validated in a mouse xenograft model. Overall, iLYTACs represent a general and modular strategy for convenient and selective targeted protein degradation, thus expanding the potential applications of current LYTACs and related techniques.

A Q-marker screening strategy based on ADME studies and systems biology for Chinese herbal medicine, taking Qianghuo Shengshi decoction in treating rheumatoid arthritis as an example.

Chinese herbal medicine (CHM) exhibits a broad spectrum of clinical applications and demonstrates favorable therapeutic efficacy. Nonetheless, elucidating the underlying mechanism of action (MOA) of CHM in disease treatment remains a formidable task due to its inherent characteristics of multi-level, multi-linked, and multi-dimensional non-linear synergistic actions. In recent years, the concept of a Quality marker (Q-marker) proposed by Liu et al. has significantly contributed to the monitoring and evaluation of CHM products, thereby fostering the advancement of CHM research. Within this study, a Q-marker screening strategy for CHM formulas has been introduced, particularly emphasising efficacy and biological activities, integrating absorption, distribution, metabolism, and excretion (ADME) studies, systems biology, and experimental verification. As an illustrative case, the Q-marker screening of Qianghuo Shengshi decoction (QHSSD) for treating rheumatoid arthritis (RA) has been conducted. Consequently, from a pool of 159 compounds within QHSSD, five Q-markers exhibiting significant in vitro anti-inflammatory effects have been identified. These Q-markers encompass notopterol, isoliquiritin, imperatorin, cimifugin, and glycyrrhizic acid. Furthermore, by employing an integrated analysis of network pharmacology and metabolomics, several instructive insights into pharmacological mechanisms have been gleaned. This includes the identification of key targets and pathways through which QHSSD exerts its crucial roles in the treatment of RA. Notably, the inhibitory effect of QHSSD on AKT1 and MAPK3 activation has been validated through western blot analysis, underscoring its potential to mitigate RA-related inflammatory responses. In summary, this research demonstrates the proposed strategy's feasibility and provides a practical reference model for the systematic investigation of CHM formulas.

A dual-modal graph learning framework for identifying interaction events among chemical and biotech drugs.

Drug-drug interaction (DDI) identification is essential to clinical medicine and drug discovery. The two categories of drugs (i.e. chemical drugs and biotech drugs) differ remarkably in molecular properties, action mechanisms, etc. Biotech drugs are up-to-comers but highly promising in modern medicine due to higher specificity and fewer side effects. However, existing DDI prediction methods only consider chemical drugs of small molecules, not biotech drugs of large molecules. Here, we build a large-scale dual-modal graph database named CB-DB and customize a graph-based framework named CB-TIP to reason event-aware DDIs for both chemical and biotech drugs. CB-DB comprehensively integrates various interaction events and two heterogeneous kinds of molecular structures. It imports endogenous proteins founded on the fact that most drugs take effects by interacting with endogenous proteins. In the modality of molecular structure, drugs and endogenous proteins are two heterogeneous kinds of graphs, while in the modality of interaction, they are nodes connected by events (i.e. edges of different relationships). CB-TIP employs graph representation learning methods to generate drug representations from either modality and then contrastively mixes them to predict how likely an event occurs when a drug meets another in an end-to-end manner. Experiments demonstrate CB-TIP's great superiority in DDI prediction and the promising potential of uncovering novel DDIs.

The Dawn of a New Era: Targeting the "Undruggables" with Antibody-Based Therapeutics.

The high selectivity and affinity of antibodies toward their antigens have made them a highly valuable tool in disease therapy, diagnosis, and basic research. A plethora of chemical and genetic approaches have been devised to make antibodies accessible to more "undruggable" targets and equipped with new functions of illustrating or regulating biological processes more precisely. In this Review, in addition to introducing how naked antibodies and various antibody conjugates (such as antibody-drug conjugates, antibody-oligonucleotide conjugates, antibody-enzyme conjugates, etc.) work in therapeutic applications, special attention has been paid to how chemistry tools have helped to optimize the therapeutic outcome (i.e., with enhanced efficacy and reduced side effects) or facilitate the multifunctionalization of antibodies, with a focus on emerging fields such as targeted protein degradation, real-time live-cell imaging, catalytic labeling or decaging with spatiotemporal control as well as the engagement of antibodies inside cells. With advances in modern chemistry and biotechnology, well-designed antibodies and their derivatives via size miniaturization or multifunctionalization together with efficient delivery systems have emerged, which have gradually improved our understanding of important biological processes and paved the way to pursue novel targets for potential treatments of various diseases.

Quantitative Analysis of Mitochondrial RNA in Living Cells with a Dual-Color Imaging System.

Accurate quantification and dynamic expression profiling of mitochondrial RNA (mtRNA for short) are critical for illustrating their cellular functions. However, there lack methods for precise detection of mtRNA in situ due to the delivery restrictions and complicated cellular interferences. Herein, a dual-color imaging system featured with signal amplification and normalization capability for quantitative analysis of specific mtRNA is established. As a proof-of-concept example, an enzyme-free hairpin DNA cascade amplifier fine-tailored to specifically recognize mtRNA encoding NADH dehydrogenase subunit 6 (ND6) is employed as the signal output module and integrated into the biodegradable mitochondria-targeting black phosphorus nanosheet (BP-PEI-TPP) to monitor spatial-temporal dynamics of ND6 mtRNA. An internal reference module targeting ß-actin mRNA is sent to the cytoplasm via BP-PEI for signal normalization, facilitating mtRNA quantification inside living cells with a degree of specificity and sensitivity as high as reverse transcription-quantitative polymerase chain reaction (RT-qPCR). With negligible cytotoxicity, this noninvasive "RT-qPCR mimic" can accurately indicate target mtRNA levels across different cells, providing a new strategy for precise analysis of subcellular RNAs in living systems.

Synthesis of core-shell material rich in carboxyl groups and its application in the selective adsorption of cationic peptides.

The ability to extract peptides and proteins from biological samples with excellent reusability, high adsorption capacity, and great selectivity is essential in scientific research and medical applications. Inspired by the advantages of core-shell materials, we fabricated a core-shell material using amino-functionalized silica as the core. Benzene-1,3,5-tricarbaldehyde and 3,5-diaminobenzoic acid were used as model organic ligands to construct a shell coating by alternately reacting the two monomers on the surface of silica microspheres. The resultant material featured an outstanding capability for the adsorption of cationic peptides, most likely owing to its porous structure, a large number of carboxylic functional groups, and low mass-transfer resistance. The maximum saturated adsorption capacity reached 833.3 mg/g and the adsorption process took only 20 min. Under optimized adsorption conditions, the core-shell material was used to selectively adsorb cationic peptides from the tryptic digestive solution of lysozyme and bovine serum albumin, Specifically, the analysis results showed seven cationic peptides in the eluate and twenty anionic peptides in the supernatant, which indicates the efficient trap of most cationic peptides in the digestive solution.

PKM2-mediated neuronal hyperglycolysis enhances the risk of Parkinson's disease in diabetic rats.

Epidemiological and animal studies indicate that pre-existing diabetes increases the risk of Parkinson's disease (PD). However, the mechanisms underlying this association remain unclear. In the present study, we found that high glucose (HG) levels in the cerebrospinal fluid (CSF) of diabetic rats might enhance the effect of a subthreshold dose of the neurotoxin 6-hydroxydopamine (6-OHDA) on the development of motor disorders, and the damage to the nigrostriatal dopaminergic neuronal pathway. In vitro, HG promoted the 6-OHDA-induced apoptosis in PC12 cells differentiated to neurons with nerve growth factor (NGF) (NGF-PC12). Metabolomics showed that HG promoted hyperglycolysis in neurons and impaired tricarboxylic acid cycle (TCA cycle) activity, which was closely related to abnormal mitochondrial fusion, thus resulting in mitochondrial loss. Interestingly, HG-induced upregulation of pyruvate kinase M2 (PKM2) combined with 6-OHDA exposure not only mediated glycolysis but also promoted abnormal mitochondrial fusion by upregulating the expression of MFN2 in NGF-PC12 cells. In addition, we found that PKM2 knockdown rescued the abnormal mitochondrial fusion and cell apoptosis induced by HG+6-OHDA. Furthermore, we found that shikonin (SK), an inhibitor of PKM2, restored the mitochondrial number, promoted TCA cycle activity, reversed hyperglycolysis, enhanced the tolerance of cultured neurons to 6-OHDA, and reduced the risk of PD in diabetic rats. Overall, our results indicate that diabetes promotes hyperglycolysis and abnormal mitochondrial fusion in neurons through the upregulation of PKM2, leading to an increase in the vulnerability of dopaminergic neurons to 6-OHDA. Thus, the inhibition of PKM2 and restoration of mitochondrial metabolic homeostasis/pathways may prevent the occurrence and development of diabetic PD.

Diastereo- and enantioselective synthesis of biaryl aldehydes bearing both axial and central chirality.

We describe herein an intriguing method for the synthesis of biaryl aldehydes bearing both axial and central chirality through a desymmetric [3 + 2] cycloaddition reaction of activated isocyanides with prochiral biaryl dialdehydes under silver catalysis. This protocol features excellent enantioselectivity, 100% atom economy, good functional group compatibility, and operational simplicity.

pH-triggered cancer-targeting polymers: From extracellular accumulation to intracellular release.

Stimuli-responsive polymers are promising to achieve targeted delivery, improved stability during circulation, and controlled release of therapeutic and diagnostic agents. Among them, pH-responsive polymeric nanocarriers have attracted significant attention as pH varies in different body fluids (e.g., stomach, intestine, and colon) and intracellular organelles (e.g., endosome, lysosome, and mitochondria) to maintain homeostasis, while distinctive pH changes are also found in certain pathological states. For example, the extracellular environment of the tumor is acidic, which can be employed to drive selective delivery. During the internalization process, since most nanocarriers enter cells upon endocytosis where a drop of pH from 6.5 to 5.0 can occur from endosome to lysosome, pH-sensitive groups have been developed for enhanced cargo release. In this review, both non-covalent and covalent interactions responsive to pH changes are introduced, with a focus on the structure-property relationship and their applications in cancer targeting and endosomal escape.

Sensitive Imaging of Cellular RNA via Cascaded Proximity-Induced Fluorogenic Reactions.

Owing to its important biological functions, RNA has become a promising molecular biomarker of various diseases. With a dynamic change in its expression level and a relatively low amount within the complicated biological matrix, signal amplification detection based on DNA probes has been put forward, which is helpful for early diagnosis and prognostic prediction. However, conventional methods are confined to cell lysates or dead cells and are not only time-consuming in sample preparation but also inaccessible to the spatial-temporal information of target RNAs. To achieve live-cell imaging of specific RNAs, both the detection sensitivity and intracellular delivery issues should be addressed. Herein, a new cascaded fluorogenic system based on the combination of hybridization chain reactions (HCRs) and proximity-induced bioorthogonal chemistry is developed, in which a bioorthogonal reaction pair (a tetrazine-quenched dye and its complementary dienophile) is brought into spatial proximity upon target RNA triggering the HCR to turn on and amplify the fluorescence in one step, sensitively indicating the cellular distribution of RNA with minimal false positive results caused by unspecific degradation. Facilitated by a biodegradable carrier based on black phosphorus with high loading capacity and excellent biocompatibility, the resulting imaging platform allows wash-free tracking of target RNAs inside living cells.

Torsional Strain-Independent Catalytic Enantioselective Synthesis of Biaryl Atropisomers.

Catalytic asymmetric dynamic kinetic resolution of configurationally labile bridged biaryls is emerging as a powerful strategy for atropisomer synthesis. However, the reported examples suffer from an inherent challenge as the reactivity is highly dependent on the torsional strain of the biaryl substrates, which significantly narrows down the scope and hampers the application. Herein, we report our discovery and development of a torsional strain-independent reaction between biaryl thionolactones and activated isocyanides. By employing auto-tandem silver catalysis, a universal synthesis of both tri- and tetra-ortho-substituted thiazole-containing biaryls was realized in high yields with high enantioselectivities. In addition, these products could be facilely converted to a novel type of bridged biaryls bearing an eight-membered lactone. Mechanistic studies were carried out to elucidate the cause of this unusual torsional strain-independent reactivity.

Simultaneous Construction of C-N Axial and Central Chirality via Silver-Catalyzed Desymmetrizative [3 + 2] Cycloaddition of Prochiral N-Aryl Maleimides with Activated Isocyanides.

Herein, we report an unprecedented strategy for the simultaneous construction of a remote C-N stereogenic axis and three contiguous stereogenic carbon centers via silver-catalyzed desymmetrizative [3 + 2] cycloaddition of prochiral N-aryl maleimides with activated isocyanides. This method features operational simplicity, wide substrate scope, high efficiency, and good to excellent stereoselectivity. Notably, it represents the first example of catalytic enantioselective synthesis of C-N atropisomers with the use of activated isocyanides.

Cooperative catalysis-enabled C-N bond cleavage of biaryl lactams with activated isocyanides.

The catalytic reaction of biaryl lactams with activated isocyanides is reported for the first time. By employing a cooperative catalytic system, oxazole-containing axially chiral biaryl anilines were obtained in high yields with excellent enantioselectivities. The key to the success lies in the atroposelective amide C-N bond cleavage with activated isocyanides.

Diastereo- and Enantioselective Silver-Catalyzed [3+3] Cycloaddition and Kinetic Resolution of Azomethine Imines with Activated Isocyanides.

In contrast to the well-established [3+2] cycloaddition reactions, the catalytic enantioselective [3+n] (n≥3) cycloaddition reaction of activated isocyanides for the preparation of six-membered or larger ring systems has remained underdeveloped. Herein, we report the first example of highly diastereo- and enantioselective [3+3] cycloaddition of activated isocyanides with azomethine imines. By employing silver catalysis, a wide range of biologically important bicyclic 1,2,4-triazines were obtained in high yields (up to 99 %) with good to excellent stereoselectivities (up to >20 : 1 dr, 99 % ee). In addition, the same catalytic system could be applied to both the late-stage functionalization of complex bioactive molecules and the kinetic resolution of racemic azomethine imines, further highlighting its versatility and synthetic utility.

Prediction of Maximum Absorption Wavelength Using Deep Neural Networks.

Fluorescent molecules are important tools in biological detection, and numerous efforts have been made to develop compounds to meet the desired photophysical properties. For example, tuning the wavelength allows an appropriate penetration depth with minimal interference from the autofluorescence/scattering for a better signal-to-noise contrast. However, there are limited guidelines to rationally design or computationally predict the optical properties from first principles, and factors like the solvent effects will make it more complicated. Herein, we established a database (SMFluo1) of 1181 solvated small-molecule fluorophores covering the ultraviolet-visible-near-infrared absorption window and developed new machine learning models based on deep neural networks for accurately predicting photophysical parameters. The optimal system was applied to 120 out-of-sample compounds, and it exhibited remarkable accuracy with a mean relative error of 1.52%. In this new paradigm, a deep learning algorithm is promising to complement conventional theoretical and experimental studies of fluorophores and to greatly accelerate the discovery of new dyes. Due to its simplicity and efficiency, data from newly developed fluorophores can be easily supplemented to this system to further improve the accuracy across various dye families.

In vivo targeted delivery of antibodies into cancer cells with pH-responsive cell-penetrating poly(disulfide)s.

Cell-penetrating poly(disulfide)s (CPDs) are promising vehicles for cytosolic delivery of proteins. However, currently available arginine-rich CPD has rarely been reported for systemic delivery due to its "always" positive charge. Herein, we developed pH-responsive CPDIMD that executes tumor targeting delivery via protonation of imidazole groups within the acidic tumor microenvironment.

Real-time imaging of cell-surface proteins with antibody-based fluorogenic probes.

Cell-surface proteins, working as key agents in various diseases, are the targets for around 66% of approved human drugs. A general strategy to selectively detect these proteins in a real-time manner is expected to facilitate the development of new drugs and medical diagnoses. Although brilliant successes were attained using small-molecule probes, they could cover a narrow range of targets due to the lack of suitable ligands and some of them suffer from selectivity issues. We report herein an antibody-based fluorogenic probe prepared via a two-step chemical modification under physiological conditions, to fulfill the selective recognition and wash-free imaging of membrane proteins, establishing a modular strategy with broad implications for biochemical research and for therapeutics.

Catalytic Atroposelective Dynamic Kinetic Resolution of Biaryl Lactones with Activated Isocyanides.

We report herein an unprecedented atroposelective dynamic kinetic resolution of Bringmann's lactones with C-nucleophiles. By the use of activated isocyanides as the reagent, a wide range of novel axially chiral oxazole-substituted biaryl phenols were accessed in high yields with high enantioselectivities. Key to the success of this process lies in the tandem atroposelective addition of isocyanides to the lactone substrate followed by a rapid cyclization, overcoming the challenge of stereochemical leakage induced by lactol formation.

Isocyanides: Promising Functionalities in Bioorthogonal Labeling of Biomolecules.

Isocyanides have drawn increasing attention in biological applications due to their attractive properties and unique reactivities, which can undergo various reactions, such as multicomponent reactions, α-addition reactions, [4 + 1] cycloaddition reactions, and the reaction scope keeps expanding. In addition to acting as reactants for the preparation of structurally interesting and diverse N-heterocycles or peptidomimetics, this type of functionality may be a good choice in the labeling and modulation of biomolecules due to the high biocompatibility and small size to minimize modifications on the parent molecule. It has been demonstrated that isocyanides can participate in biomolecule labeling through three strategies, including the two-component bioorthogonal reaction, multicomponent reaction, and metal chelation. Among them, the isocyanide-tetrazine reaction has been better studied recently, augmenting the potency of isocyanide as a bioorthogonal handle. This review will focus on the recent progress in isocyanide chemistry for labeling of biomolecules. Meanwhile, methods to introduce isocyano groups into biomacromolecules are also described to facilitate wider applications of this unique functionality.