Conditional PROTAC: Recent Strategies for Modulating Targeted Protein Degradation.

Proteolysis-targeting chimeras (PROTACs) have emerged as a promising technology for inducing targeted protein degradation by leveraging the intrinsic ubiquitin-proteasome system (UPS). While the potential druggability of PROTACs toward undruggable proteins has accelerated their rapid development and the wide-range of applications across diverse disease contexts, off-tissue effect and side-effects of PROTACs have recently received attentions to improve their efficacy. To address these issues, spatial or temporal target protein degradation by PROTACs has been spotlighted. In this review, we explore chemical strategies for modulating protein degradation in a cell type-specific (spatio-) and time-specific (temporal-) manner, thereby offering insights for expanding PROTAC applications to overcome the current limitations of target protein degradation strategy.

Recent advances in chemical biology tools for protein and RNA profiling of extracellular vesicles.

Extracellular vesicles (EVs) are nano-sized vesicles secreted by cells that contain various cellular components such as proteins, nucleic acids, and lipids from the parent cell. EVs are abundant in body fluids and can serve as circulating biomarkers for a variety of diseases or as a regulator of various biological processes. Considering these characteristics of EVs, analysis of the EV cargo has been spotlighted for disease diagnosis or to understand biological processes in biomedical research. Over the past decade, technologies for rapid and sensitive analysis of EVs in biofluids have evolved, but detection and isolation of targeted EVs in complex body fluids is still challenging due to the unique physical and biological properties of EVs. Recent advances in chemical biology provide new opportunities for efficient profiling of the molecular contents of EVs. A myriad of chemical biology tools have been harnessed to enhance the analytical performance of conventional assays for better understanding of EV biology. In this review, we will discuss the improvements that have been achieved using chemical biology tools.

Regulatory role of Echinochrome A in cancer-associated fibroblast-mediated lung cancer cell migration.

Echinochrome A (Ech A), a marine biosubstance isolated from sea urchins, is a strong antioxidant, and its clinical form, histochrome, is being used to treat several diseases, such as ophthalmic, cardiovascular, and metabolic diseases. Cancer-associated fibroblasts (CAFs) are a component of the tumor stroma and induce phenotypes related to tumor malignancy, including epithelial-mesenchymal transition (EMT) and cancer stemness, through reciprocal interactions with cancer cells. Here, we investigated whether Ech A modulates the properties of CAFs and alleviates CAF-induced lung cancer cell migration. First, we observed that the expression levels of CAF markers, Vimentin and fibroblast-activating protein (FAP), were decreased in Ech A-treated CAF-like MRC5 cells. The mRNA transcriptome analysis revealed that in MRC5 cells, the expression of genes associated with cell migration was largely modulated after Ech A treatment. In particular, the expression and secretion of cytokine and chemokine, such as IL6 and CCL2, stimulating cancer cell metastasis was reduced through the inactivation of STAT3 and Akt in MRC5 cells treated with Ech A compared to untreated MRC5 cells. Moreover, while conditioned medium from MRC5 cells enhanced the migration of non-small cell lung cancer cells, conditioned medium from MRC5 cells treated with Ech A suppressed cancer cell migration. In conclusion, we suggest that Ech A might be a potent adjuvant that increases the efficacy of cancer treatments to mitigate lung cancer progression.

Medium-Sized Ring Expansion Strategies: Enhancing Small-Molecule Library Development.

The construction of a small molecule library that includes compounds with medium-sized rings is increasingly essential in drug discovery. These compounds are essential for identifying novel therapeutic agents capable of targeting "undruggable" targets through high-throughput and high-content screening, given their structural complexity and diversity. However, synthesizing medium-sized rings presents notable challenges, particularly with direct cyclization methods, due to issues such as transannular strain and reduced degrees of freedom. This review presents an overview of current strategies in synthesizing medium-sized rings, emphasizing innovative approaches like ring-expansion reactions. It highlights the challenges of synthesis and the potential of these compounds to diversify the chemical space for drug discovery, underscoring the importance of medium-sized rings in developing new bioactive compounds.

Split Proteins and Reassembly Modules for Biological Applications.

Split systems, modular entities enabling controlled biological processes, have become instrumental in biological research. This review highlights their utility across applications like gene regulation, protein interaction identification, and biosensor development. Covering significant progress over the last decade, it revisits traditional split proteins such as GFP, luciferase, and inteins, and explores advancements in technologies like Cas proteins and base editors. We also examine reassembly modules and their applications in diverse fields, from gene regulation to therapeutic innovation. This review offers a comprehensive perspective on the recent evolution of split systems in biological research.

Dimethyloxalylglycine Suppresses SREBP1c and Lipogenic Gene Expressions in Hepatocytes Independently of HIF1A.

Dimethyloxalylglycine (DMOG) is a representative inhibitor of the prolyl hydroxylase domain (PHD), which mediates the degradation of hypoxia-inducible factor-1-alpha (HIF1A). DMOG exerts its pharmacological effects via the canonical pathway that involves PHD inhibition; however, it remains unclear whether DMOG affects lipogenic gene expression in hepatocytes. We aimed to elucidate the effects of DMOG on sterol regulatory element-binding protein-1c (SREBP1c), a master regulator of fatty acid synthesis in hepatocytes. DMOG treatment inhibited SREBP1c mRNA and protein expression in HepG2 and AML12 hepatocytes and reduced the transcript levels of SREBP1c-regulated lipogenic genes. A luciferase reporter assay revealed that DMOG inhibited the transcriptional activity of SREBP1c. Moreover, DMOG suppressed SREBP1c expression in mice liver. Mechanistically, treatment with DMOG enhanced the expression of HIF1A and insulin-induced gene 2 (INSIG2), which inhibits the activation of SREBP1c. However, HIF1A or INSIG2 knockdown failed to reverse the inhibitory effect of DMOG on SREBP1c expression, suggesting a redundant role of HIF1A and INSIG2 in terms of repressing SREBP1c. DMOG did not function through the canonical pathway involving inhibition of SREBP1c by PHD, highlighting the presence of non-canonical pathways that mediate its anti-lipogenic effect.

Development of a novel histone deacetylase inhibitor unveils the role of HDAC11 in alleviating depression by inhibition of microglial activation.

Histone deacetylases (HDACs) are key epigenetic regulators and classified into four subtypes. Despite the various roles of each HDAC isoform, the lack of selective HDAC inhibitors has limited the elucidation of their roles in biological systems. HDAC11, the sole class-IV HDAC, is highly expressed in the brain, however, the role of HDAC11 in microglia is not fully understood. Based on the modification of MC1568, we developed a novel HDAC inhibitor, 5. Interestingly, 5 suppresses lipopolysaccharide-induced microglial activation by the initiation of autophagy and subsequent inhibition of nitric oxide production. Furthermore, we demonstrated that 5 significantly alleviates depression-like behavior by inhibiting microglial activation in mouse brain. Our discovery reveals that specific pharmacological regulation of HDAC11 induces autophagy and reactive nitrogen species balance in microglia for the first time, which makes HDAC11 a new therapeutic target for depressive disorder.

Recent Synthetic Biology Approaches for Temperature- and Light-Controlled Gene Expression in Bacterial Hosts.

The expression of genes of interest (GOI) can be initiated by providing external stimuli such as temperature shifts and light irradiation. The application of thermal or light stimuli triggers structural changes in stimuli-sensitive biomolecules within the cell, thereby inducing or repressing gene expression. Over the past two decades, several groups have reported genetic circuits that use natural or engineered stimuli-sensitive modules to manipulate gene expression. Here, we summarize versatile strategies of thermosensors and light-driven systems for the conditional expression of GOI in bacterial hosts.

Recent Advances in Fluorescence Imaging by Genetically Encoded Non-canonical Amino Acids.

Technical innovations in protein labeling with a fluorophore at the specific residue have played a significant role in studying protein dynamics. The genetic code expansion (GCE) strategy enabled the precise installation of fluorophores at the tailored site of proteins in live cells with minimal perturbation of native functions. Considerable advances have been achieved over the past decades in fluorescent imaging using GCE strategies along with bioorthogonal chemistries. In this review, we discuss advances in the GCE-based strategies to site-specifically introduce fluorophore at a defined position of the protein and their bio-imaging applications.

A short ORF-encoded transcriptional regulator.

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.

An orthogonal seryl-tRNA synthetase/tRNA pair for noncanonical amino acid mutagenesis in Escherichia coli.

We report the development of the orthogonal amber-suppressor pair Archaeoglobus fulgidus seryl-tRNA (Af-tRNASer)/Methanosarcina mazei seryl-tRNA synthetase (MmSerRS) in Escherichia coli. Furthermore, the crystal structure of MmSerRS was solved at 1.45 Å resolution, which should enable structure-guided engineering of its active site to genetically encode small, polar noncanonical amino acids (ncAAs).

Site-Specific Synthesis of Cysteine-Bridged Glycoproteins via Expressed Protein Glycoligation.

Site-specific glycosylation of a functional recombinant protein thioester is reported. The thioester functionalized protein sfGFP-Y151ThioD, prepared by genetic code expansion, underwent native chemical ligation with the cysteine-conjugated glycans H-Cys-NH-GlcNAc and H-Cys-NH-(GlcNAc)2(Man)3 to give the corresponding cysteine-bridged glycoproteins. The intact glycoproteins, which retained their fluorescence, were characterized by top-down mass spectrometry and gel electrophoresis. The bridging cysteine provided a convenient handle for affinity chromatography purification of the glycoproteins via a removable biotin tag. Given the influence that specific glycoforms can have on a protein's function, the ability to attach a homogeneous glycan to an intact protein in a functional group controlled yet sequon-independent manner could find widespread application. These preliminary results set the stage for development of the expressed protein glycoligation (EPG) concept.

2-Sulfonylpyridines as Tunable, Cysteine-Reactive Electrophiles.

The emerging use of covalent ligands as chemical probes and drugs would benefit from an expanded repertoire of cysteine-reactive electrophiles for efficient and diverse targeting of the proteome. Here we use the endogenous electrophile sensor of mammalian cells, the KEAP1-NRF2 pathway, to discover cysteine-reactive electrophilic fragments from a reporter-based screen for NRF2 activation. This strategy identified a series of 2-sulfonylpyridines that selectively react with biological thiols via nucleophilic aromatic substitution (SNAr). By tuning the electrophilicity and appended recognition elements, we demonstrate the potential of the 2-sulfonylpyridine reactive group with the discovery of a selective covalent modifier of adenosine deaminase (ADA). Targeting a cysteine distal to the active site, this molecule attenuates the enzymatic activity of ADA and inhibits proliferation of lymphocytic cells. This study introduces a modular and tunable SNAr-based reactive group for targeting reactive cysteines in the human proteome and illustrates the pharmacological utility of this electrophilic series.

Expanding the genetic code of the human hematopoietic system.

The genetic incorporation of noncanonical amino acids (ncAAs) into proteins has been realized in bacteria, yeast, and mammalian cells, and recently, in multicellular organisms including plants and animals. However, the addition of new building blocks to the genetic code of tissues from human origin has not yet been achieved. To this end, we report a self-replicating Epstein-Barr virus-based episomal vector for the long-term encoding of ncAAs in human hematopoietic stem cells and reconstitution of this genetically engineered hematopoietic system in mice.

A General Strategy for Engineering Noncanonical Amino Acid Dependent Bacterial Growth.

Synthetic auxotrophy in which bacterial viability depends on the presence of a synthetic amino acid provides a robust strategy for the containment of genetically modified organisms and the development of safe, live vaccines. However, a simple, general strategy to evolve essential proteins to be dependent on synthetic amino acids is lacking. Using a temperature-sensitive selection system, we evolved an Escherichia coli (E. coli) sliding clamp variant with an orthogonal protein-protein interface, which contains a Leu273 to p-benzoylphenyl alanine (pBzF) mutation. The E. coli strain with this variant DNA clamp has a very low escape frequency (<10-10), and its growth is strictly dependent on the presence of pBzF. This selection strategy can be generally applied to create ncAA dependence of other organisms with DNA clamp homologues.

Site-Specific Incorporation of a Dithiolane Containing Amino Acid into Proteins.

We have genetically encoded a dithiolane containing amino acid (dtF) in Escherichia coli (E. coli) using a polyspecific aminoacyl-tRNA synthetase (aaRS)/amber suppressor tRNA pair. To demonstrate the utility of dtF for bioapplications, we synthesized gold nanoparticle (AuNP) constructs with a mutant superfolder green fluorescent protein (sfGFP) [sfGFP-AuNP] as a model for the protein-metal conjugation. The resulting sfGFP-AuNP constructs show directional homogeneity and enhanced chemical durability compared to their cysteine analogues toward excess environmental 1,4-dithiothreitol (DTT).

Progress toward a reduced phage genetic code.

All known living organisms use at least 20 amino acids as the basic building blocks of life. Efforts to reduce the number of building blocks in a replicating system to below the 20 canonical amino acids have not been successful to date. In this work, we use filamentous phage as a model system to investigate the feasibility of removing methionine (Met) from the proteome. We show that all 24 elongation Met sites in the M13 phage genome can be replaced by other canonical amino acids. Most of these changes involve substitution of methionine by leucine (Leu), but in some cases additional compensatory mutations are required. Combining Met substituted sites in the proteome generally led to lower viability/infectivity of the mutant phages, which remains the major challenge in eliminating all methionines from the phage proteome. To date a total of 15 (out of all 24) elongation Mets have been simultaneously deleted from the M13 proteome, providing a useful foundation for future efforts to minimize the genetic code.

Site-Specific Incorporation of a Thioester Containing Amino Acid into Proteins.

Here, we report the site-specific incorporation of a thioester containing noncanonical amino acid (ncAA) into recombinantly expressed proteins. Specifically, we genetically encoded a thioester-activated aspartic acid (ThioD) in bacteria in good yield and with high fidelity using an orthogonal nonsense suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. To demonstrate the utility of ThioD, we used native chemical ligation to label green fluorescent protein with a fluorophore in good yield.

Generation of an Orthogonal Protein-Protein Interface with a Noncanonical Amino Acid.

We have engineered the protein interface of the Escherichia coli chorismate mutase (EcCM) homodimer to be dependent on incorporation of a noncanonical amino acid (ncAA) at residue 72. The large hydrophobic amino acid p-benzoyl phenylalanine (pBzF) was substituted for Tyr72, which led to a catalytically inactive protein. A library of five residues (Leu25', Arg29', Leu76, Ile80' and Asp83') surrounding pBzF72 was generated and subjected to a growth based selection in a chorismate mutase deficient strain. An EcCM variant (Phe25', pBzF72, Thr76, Gly80' and Tyr83') forms a stable homodimer, has catalytic activity similar to the wild type enzyme, and unfolds with a Tm of 53 °C. The X-ray crystal structure reveals a pi-pi stacking and hydrogen bonding interactions that stabilize the new protein interface. The strategy described here should be useful for generating organisms that are dependent on the presence of a ncAA for growth.

Structural basis for differential activities of enantiomeric PPARγ agonists: Binding of S35 to the alternate site.

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily. It functions as a ligand-activated transcription factor and plays important roles in the regulation of adipocyte differentiation, type 2 diabetes mellitus, and inflammation. Many PPARγ agonists bind to the canonical ligand-binding pocket near the activation function-2 (AF-2) helix (i.e., helix H12) of the ligand-binding domain (LBD). More recently, an alternate ligand-binding site was identified in PPARγ LBD; it is located beside the Ω loop between the helices H2' and H3. We reported previously that the chirality of two optimized enantiomeric PPARγ ligands (S35 and R35) differentiates their PPARγ transcriptional activity, binding affinity, and inhibitory activity toward Cdk5 (cyclin-dependent kinase 5)-mediated phosphorylation of PPARγ at Ser245 (in PPARγ1 numbering; Ser273 in PPARγ2 numbering). S35 is a PPARγ phosphorylation inhibitor with promising glucose uptake potential, whereas R35 behaves as a potent conventional PPARγ agonist. To provide a structural basis for understanding the differential activities of these enantiomeric ligands, we have determined crystal structures of the PPARγ LBD in complex with either S35 or R35. S35 and R35 bind to the PPARγ LBD in significantly different manners. The partial agonist S35 occupies the alternate site near the Ω loop, whereas the full agonist R35 binds entirely to the canonical LBP. Alternate site binding of S35 affects the PPARγ transactivation and the inhibitory effect on PPARγ Ser245 phosphorylation. This study provides a useful platform for the development of a new generation of PPARγ ligands as anti-diabetic drug candidates.