Kinase Inhibition via Small Molecule-Induced Intramolecular Protein Cross-Linking.

Remarkable progress has been made in the development of cysteine-targeted covalent inhibitors. In kinase drug discovery, covalent inhibitors capable of targeting other nucleophilic residues (i.e. lysine, or K) have emerged in recent years. Besides a highly conserved catalytic lysine, almost all human protein kinases possess an equally conserved glutamate/aspartate (e.g. E/D) that forms a K-E/D salt bridge within the enzyme's active site. Electrophilic ynamides were previously used as effective peptide coupling reagents and to develop E/D-targeting covalent protein inhibitors/probes. In the present study, we report the first ynamide-based small-molecule inhibitors capable of inducing intramolecular cross-linking of various protein kinases, leading to subsequent irreversible inhibition of kinase activity. Our strategy took advantage of the close distance between the highly conserved catalytic K and E/D residues in a targeted kinase, thus providing a conceptually general approach to achieve irreversible kinase inhibition with high specificity and desirable cellular potency. Finally, this ynamide-facilitated, ligand-induced mechanism leading to intramolecular kinase cross-linking and inhibition was unequivocally established by using recombinant ABL kinase as a representative.

Prodrug-conjugated tumor-seeking commensals for targeted cancer therapy.

Prodrugs have been explored as an alternative to conventional chemotherapy; however, their target specificity remains limited. The tumor microenvironment harbors a range of microorganisms that potentially serve as tumor-targeting vectors for delivering prodrugs. In this study, we harness bacteria-cancer interactions native to the tumor microbiome to achieve high target specificity for prodrug delivery. We identify an oral commensal strain of Lactobacillus plantarum with an intrinsic cancer-binding mechanism and engineer the strain to enable the surface loading of anticancer prodrugs, with nasopharyngeal carcinoma (NPC) as a model cancer. The engineered commensals show specific binding to NPC via OppA-mediated recognition of surface heparan sulfate, and the loaded prodrugs are activated by tumor-associated biosignals to release SN-38, a chemotherapy compound, near NPC. In vitro experiments demonstrate that the prodrug-loaded microbes significantly increase the potency of SN-38 against NPC cell lines, up to 10-fold. In a mouse xenograft model, intravenous injection of the engineered L. plantarum leads to bacterial colonization in NPC tumors and a 67% inhibition in tumor growth, enhancing the efficacy of SN-38 by 54%.

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.

Global Reactivity Profiling of the Catalytic Lysine in Human Kinome for Covalent Inhibitor Development.

Advances in targeted covalent inhibitors (TCIs) have been made by using lysine-reactive chemistries. Few aminophiles possessing balanced reactivity/stability for the development of cell-active TCIs are however available. We report herein lysine-reactive activity-based probes (ABPs; 2-14) based on the chemistry of aryl fluorosulfates (ArOSO2 F) capable of global reactivity profiling of the catalytic lysine in human kinome from mammalian cells. We concurrently developed reversible covalent ABPs (15/16) by installing salicylaldehydes (SA) onto a promiscuous kinase-binding scaffold. The stability and amine reactivity of these probes exhibited a broad range of tunability. X-ray crystallography and mass spectrometry (MS) confirmed the successful covalent engagement between ArOSO2 F on 9 and the catalytic lysine of SRC kinase. Chemoproteomic studies enabled the profiling of >300 endogenous kinases, thus providing a global landscape of ligandable catalytic lysines of the kinome. By further introducing these aminophiles into VX-680 (a noncovalent inhibitor of AURKA kinase), we generated novel lysine-reactive TCIs that exhibited excellent in vitro potency and reasonable cellular activities with prolonged residence time. Our work serves as a general guide for the development of lysine-reactive ArOSO2 F-based TCIs.

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.

Tumor Metabolism-Rewriting Nanomedicines for Cancer Immunotherapy.

Cancer immunotherapy has become an established therapeutic paradigm in oncologic therapy, but its therapeutic efficacy remains unsatisfactory in the majority of cancer patients. Accumulating evidence demonstrates that the metabolically hostile tumor microenvironment (TME), characterized by acidity, deprivation of oxygen and nutrients, and accumulation of immunosuppressive metabolites, promotes the dysfunction of tumor-infiltrating immune cells (TIICs) and thereby compromises the effectiveness of immunotherapy. This indicates the potential role of tumor metabolic intervention in the reinvigoration of antitumor immunity. With the merits of multiple drug codelivery, cell and organelle-specific targeting, controlled drug release, and multimodal therapy, tumor metabolism-rewriting nanomedicines have recently emerged as an attractive strategy to strengthen antitumor immune responses. This review summarizes the current progress in the development of multifunctional tumor metabolism-rewriting nanomedicines for evoking antitumor immunity. A special focus is placed on how these nanomedicines reinvigorate innate or adaptive antitumor immunity by regulating glucose metabolism, amino acid metabolism, lipid metabolism, and nucleotide metabolism at the tumor site. Finally, the prospects and challenges in this emerging field are discussed.

Multitarget inhibitors/probes that target LRRK2 and AURORA A kinases noncovalently and covalently.

Herein, we report a salicylaldehyde-based, reversible covalent inhibitor (A2) that possesses moderate cellular activity against AURKA with a prolonged residence time and shows significant non-covalent inhibition towards LRRK2. Our results indicated that this multitarget kinase inhibitor may be used as the starting point for future development of more potent, selective and dual-targeting covalent kinase inhibitors against AURKA and LRRK2 for mitophagy.

Cell-active, irreversible covalent inhibitors that selectively target the catalytic lysine of EGFR by using fluorosulfate-based SuFEx chemistry.

EGFR signaling is involved in multiple cellular processes including cell proliferation, differentiation and development, making this protein kinase one of the most valuable drug targets for the treatment of non-small cell lung carcinomas (NSCLC). Herein, we describe the design and synthesis of a series of potential covalent inhibitors targeting the catalytically conserved lysine (K745) of EGFR on the basis of Erlotinib, an FDA-approved first-generation EGFR drug. Different amine-reactive electrophiles were introduced at positions on the Erlotinib scaffold proximal to K745 in EGFR. The optimized compound 26 (as well as its close analog 30), possessing a novel arylfluorosulfate group (ArOSO2F), showed excellent in vitro potency (as low as 0.19 nM in independent IC50 determination) and selectivity against EGFR and many of its drug-resistant mutants. Both intact protein mass spectrometry (MS) and site-mapping analysis revealed that compound 26 covalently bound to EGFR at K745 through the formation of a sulfamate. In addition, compound 26 displayed good anti-proliferative potency against EGFR-overexpressing HCC827 cells by inhibiting endogenous EGFR autophosphorylation. The pharmacokinetic studies of compound 26 demonstrated the druggable potential of other ArOSO2F-containing compounds. Finally, competitive activity-based protein profiling (ABPP), cellular thermal shift assay (CETSA), as well as cellular wash-out experiments, all showed compound 26 to be the first cell-active, fluorosulfate-based targeted covalent inhibitor (TCI) of protein kinases capable of covalently engaging the catalytically conserved lysine of its target in live mammalian cells.

Orthogonal Strategies for Profiling Potential Cellular Targets of Anandamide and Cannabidiol.

The human endocannabinoid system regulates a myriad of physiological processes through a complex lipid signaling network involving cannabinoids and their respective receptors, cannabinoid receptor 1 (hCB1 R) and cannabinoid receptor 2 (hCB2 R). Anandamide (AEA) and cannabidiol (CBD) are classical examples of cannabinoids that elicit a variety of effects, both beneficial and detrimental, through these receptors. Mounting evidence suggested the presence of other potential cannabinoid targets that may be responsible for other observable effects. However, prior pharmacological studies on these cannabinoid compounds provided scant evidence of direct engagement to these proposed targets. Moreover, to the best of our knowledge, no chemoproteomic studies have been demonstrated on CBD. Here we showed that, by taking advantage of a recently developed 'label-free' 2D-TPP (2 Dimensional-Thermal Protein Profiling) approach, we have identified several new putative targets of both AEA and CBD. Comparison of these interaction landscapes with those obtained from well-established affinity-based protein profiling (AfBPP) platforms has led to the discovery of both shared and unique protein targets. Subsequent target validation of selected proteins led us to conclude that this 2D-TPP strategy complements well with AfBPP.

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.

A Late-Stage Aryl C-H Olefination Strategy and Its Application Towards Global Proteome Profiling of Δ8 -Tetrahydrocannabinol.

Drugs and bioactive natural products exert their pharmacological effects by engaging numerous cellular targets in our body. Identification of these protein targets is essential for understanding the mechanism-of-action of these compounds, thus contributing to improved drug design in drug discovery programs. Termed "in situ drug profiling", a common strategy for studying these bioactive compounds centralized on the covalent capture of protein targets along with a reporter tag to facilitate downstream proteomic analyses. Though highly successful, such reliance on innate electrophilic traps to facilitate covalent capture restricted its applications to covalent acting compounds. Late-stage C-H functionalization (LSF) may resolve this by substituting biologically inert C-H bonds with desired electrophilic groups. Herein, we demonstrated this concept by arming a diverse range of electron-rich aromatic drugs and natural products with α,ß-unsaturated esters, via late-stage C-H olefination with an arylthio-based carboxylic acid ligand developed by Ibanez and co-workers. We also showed that covalent probes generated from this LSF approach could be applied for "in situ drug profiling" of Δ8 -THC, as exemplified by the successful target engagement of α-4 db, a Δ8 -THC-based probe, to its native target hCB2 R. In combination with AfBP 7, a photoaffinity-based derivative of Δ8 -THC, we identified several novel putative targets that could account for some of the effects in THC consumption. We anticipate our C-H LSF strategy to be widely adopted for future studies of non-covalent drugs.

Ugi reaction-assisted assembly of covalent PROTACs against glutathione peroxidase 4.

Inducing cell ferroptosis by inactivating glutathione peroxidase 4 (GPX4) is a popular cancer treatment strategy. However, only few GPX4 inhibitors have been developed to date. PROteolysis Targeting Chimera (PROTAC) is a promising approach to provide new opportunities to overcome limitations of traditional therapeutics. Herein, a PROTAC-like activity-based probe PD-Q2 was first assembled using Ugi reaction, consisting of a known GPX4 inhibitor ML-162 homolog to the E3 ligase cereblon ligand-pomalidomide. Pull-down and immunoblotting analysis revealed that GPX4 was a covalent target of PD-Q2, but the degradation efficiency was weak. Therefore, a series of degraders was further synthesized by varying the linkers of heterofunctional PROTACs. Among these degraders, PD-4 and PD-P2 were found to promote effective GPX4 degradation via the ubiquitin-proteasome system and cause lipid ROS accumulation. PD-4 and PD-P2 showed potent inhibitory of colony formation and cell growth. Furthermore, we found that with pomalidomide, the degraders exhibit a high fluorescent signal that is mostly localized in the lysosome, which may affect the effectiveness of anti-cell proliferation. Overall, we provide GPX4 degraders for further exploring therapeutic potential of regulating ferroptosis.

De Novo Designed Self-Assembling Rhodamine Probe for Real-Time, Long-Term and Quantitative Live-Cell Nanoscopy.

Super-resolution imaging provides a powerful approach to image dynamic biomolecule events at nanoscale resolution. An ingenious method involving tuning intramolecular spirocyclization in rhodamine offers an appealing strategy to design cell-permeable fluorogenic probes for super-resolution imaging. Nevertheless, precise control of rhodamine spirocyclization presents a significant challenge. Through detailed study of the structure-activity relationship, we identified that multiple key factors control rhodamime spirocyclization. The findings provide opportunities to create fluorogenic probes with tailored properties. On the basis of our findings, we constructed self-assembling rhodamine probes for no-wash live-cell confocal and super-resolution imaging. The designed self-assembling probe Rho-2CF3 specifically labeled its target proteins and displayed high ring-opening ability, fast labeling kinetics (<1 min), and large turn-on fold (>80 folds), which is very difficult to be realized by the existing methods. Using the probe, we achieved high-contrast super-resolution imaging of nuclei and mitochondria with a spatial resolution of up to 42 nm. The probe also showed excellent photostability and proved ideal for real-time and long-term tracking of mitochondrial fission and fusion events with high spatiotemporal resolution. Furthermore, Rho-2CF3 could resolve the ultrastructure of mitochondrial cristae and quantify their morphological changes under drug treatment at nanoscale. Our strategy thus demonstrates its usefulness in designing self-assembling probes for super-resolution imaging.

Mitochondria-Targeted Gene Silencing Facilitated by Mito-CPDs.

Mitochondrial DNA (mtDNA) plays an essential role in maintaining normal cellular activities. Its heteroplasmic mutations are known to cause various genetic diseases. Current genetic engineering strategies, such as those based on RNA interference (RNAi) and antisense technology, are difficult to genetically alter mtDNA, however, due to the inability of highly negatively charged oligonucleotides to translocate across the double-membrane mitochondria. We report herein a universal mitochondria-targeted gene-delivery approach by using cell-penetrating poly(disulfide)s (CPDs). Novel CPD-based mitochondrial transporters, named Mito-CPDs, were synthesized by using triphenylphosphonium (TPP)-fused propagating monomers containing either disulfide or diselenide backbones. Upon spontaneous complex formation with an oligonucleotide (single- or double-stranded), the resulting nanoscale Mito-CPD@Oligo exhibited excellent properties in common biological media. While the intracellular gene-delivery efficiency of these Mito-CPDs was comparable to that of commercial transfection agents, their unique mitochondria-localized properties enabled effective release of the loaded cargo inside these organelles. Subsequent mitochondrial delivery of siRNA and antisense oligonucleotides against suitable mtDNA-encoded proteins showed successful down-regulation of target protein expression, leading to profound effects on mitochondrial functions. Mito-CPDs thus provide a useful tool for future investigations of mitochondrial biology and treatment of mitochondria-related diseases.

2-Ethynylbenzaldehyde-Based, Lysine-Targeting Irreversible Covalent Inhibitors for Protein Kinases and Nonkinases.

Lysine-targeting irreversible covalent inhibitors have attracted growing interests in recent years, especially in the fields of kinase research. Despite encouraging progress, few chemistries are available to develop inhibitors that are exclusively lysine-targeting, selective, and cell-active. We report herein a 2-ethynylbenzaldehyde (EBA)-based, lysine-targeting strategy to generate potent and selective small-molecule inhibitors of ABL kinase by selectively targeting the conserved catalytic lysine in the enzyme. We showed the resulting compounds were cell-active, capable of covalently engaging endogenous ABL kinase in K562 cells with long-residence time and few off-targets. We further validated the generality of this strategy by developing EBA-based irreversible inhibitors against EGFR (a kinase) and Mcl-1 (a nonkinase) that covalently reacted with the catalytic and noncatalytic lysine within each target.

Multiplex Identification of Post-Translational Modifications at Point-of-Care by Deep Learning-Assisted Hydrogel Sensors.

Multiplex detection of protein post-translational modifications (PTMs), especially at point-of-care, is of great significance in cancer diagnosis. Herein, we report a machine learning-assisted photonic crystal hydrogel (PCH) sensor for multiplex detection of PTMs. With closely-related PCH sensors microfabricated on a single chip, our design achieved not only rapid screening of PTMs at specific protein sites by using only naked eyes/cellphone, but also the feasibility of real-time monitoring of phosphorylation reactions. By taking advantage of multiplex sensor chips and a neural network algorithm, accurate prediction of PTMs by both their types and concentrations was enabled. This approach was ultimately used to detect and differentiate up/down regulation of different phosphorylation sites within the same protein in live mammalian cells. Our developed method thus holds potential for POC identification of various PTMs in early-stage diagnosis of protein-related diseases.

Biodegradable silica nanocapsules enable efficient nuclear-targeted delivery of native proteins for cancer therapy.

Cell nucleus is the desired subcellular organelle of many therapeutic drugs. Although numerous nanomaterial-based methods have been developed which could facilitate nuclear-targeted delivery of small-molecule drugs, few are known to be capable of delivering exogenous native proteins. Herein, we report a convenient and highly robust approach for effective nuclear-targeted delivery of native proteins/antibodies by using biodegradable silica nanocapsules (BSNPs) that were surface-modified with different nuclear localization signals (NLS) peptides. We found that, upon gaining entry to mammalian cells via endocytosis, such nanocapsules (protein@BSNP-NLS) could effectively escape from endolysosomal vesicles with the assistance of an endosomolytic peptide (i.e., L17E), accumulate in cell nuclei and release the encapsulated protein cargo with biological activities. Cloaked with HeLa cell membrane, DNase@BSNP-NLS/L17E-M (with L17E encapsulated) homologously delivered functional proteins to cancer cell nuclei in tumor-xenografted mice. In vitro and in vivo anti-tumor properties, such as long blood circulation time and effective tumor growth inhibition, indicate that the nuclear-targeted cell-membrane-cloaked BSNPs (DNase@BSNP-NLS/L17E-M) platform is a promising therapeutic approach to nuclear related diseases.

Bifunctional Lipid-Derived Affinity-Based Probes (AfBPs) for Analysis of Lipid-Protein Interactome.

Although lipids are not genetically encoded, they are fundamental building blocks of cell membranes and essential components of cell metabolites. Lipids regulate various biological processes, including energy storage, membrane trafficking, signal transduction, and protein secretion; therefore, their metabolic imbalances cause many diseases. Approximately 47 000 lipid species with diverse structures have been identified, but little is known about their crucial roles in cellular systems. Particularly the structural, metabolic, and signaling functions of lipids often arise from interactions with proteins. Lipids attach to proteins not only by covalent bonds but also through noncovalent interactions, which also influence protein functions and localization. Therefore, it is important to explore this lipid-protein "interactome" to understand its roles in health and disease, which may further provide insight for medicinal development. However, lipid structures are generally quite complicated, rendering the systematic characterization of lipid-protein interactions much more challenging.Chemoproteomics is a well-known chemical biology platform in which small-molecule chemical probes are utilized in combination with high-resolution, quantitative mass spectrometry to study protein-ligand interactions in living cells or organisms, and it has recently been applied to the study of protein-lipid interactions as well. The study of these complicated interactions has been advanced by the development of bifunctional lipid probes, which not only enable probes to form covalent cross-links with lipid-interacting proteins under UV irradiation, but are also capable of enriching these proteins through bioorthogonal reactions.In this Account, we will discuss recent developments in bifunctional lipid-derived, affinity-based probes (AfBP)s that have been developed to investigate lipid-protein interactions in live cell systems. First, we will give a brief introduction of fundamental techniques based on AfBPs which are related to lipid research. Then, we will focus on three aspects, including probes developed on the basis of lipidation, lipid-derived probes with different modification positions (e.g., hydrophobic or hydrophilic parts of a lipid), and, finally, in situ biosynthesis of probes through intrinsic metabolic pathways by using chemically modified building blocks. We will present some case studies to describe these probes' design principles and cellular applications. At the end, we will also highlight key limitations of current approaches so as to provide inspirations for future improvement. The lipid probes that have been constructed are only the tip of the iceberg, and there are still plenty of lipid species that have yet to be explored. We anticipate that AfBP-based chemoproteomics and its further advancement will pave the way for a deep understanding of lipid-protein interactions in the future.

Stimulus-responsive self-assembled prodrugs in cancer therapy.

Small-molecule prodrugs have become the main toolbox to improve the unfavorable physicochemical properties of potential therapeutic compounds in contemporary anti-cancer drug development. Many approved small-molecule prodrugs, however, still face key challenges in their pharmacokinetic (PK) and pharmacodynamic (PD) properties, thus severely restricting their further clinical applications. Self-assembled prodrugs thus emerged as they could take advantage of key benefits in both prodrug design and nanomedicine, so as to maximize drug loading, reduce premature leakage, and improve PK/PD parameters and targeting ability. Notably, temporally and spatially controlled release of drugs at cancerous sites could be achieved by encoding various activable linkers that are sensitive to chemical or biological stimuli in the tumor microenvironment (TME). In this review, we have comprehensively summarized the recent progress made in the development of single/multiple-stimulus-responsive self-assembled prodrugs for mono- and combinatorial therapy. A special focus was placed on various prodrug conjugation strategies (polymer-drug conjugates, drug-drug conjugates, etc.) that facilitated the engineering of self-assembled prodrugs, and various linker chemistries that enabled selective controlled release of active drugs at tumor sites. Furthermore, some polymeric nano-prodrugs that entered clinical trials have also been elaborated here. Finally, we have discussed the bottlenecks in the field of prodrug nanoassembly and offered potential solutions to overcome them. We believe that this review will provide a comprehensive reference for the rational design of effective prodrug nanoassemblies that have clinic translation potential.