Variational autoencoder-based chemical latent space for large molecular structures with 3D complexity.

The structural diversity of chemical libraries, which are systematic collections of compounds that have potential to bind to biomolecules, can be represented by chemical latent space. A chemical latent space is a projection of a compound structure into a mathematical space based on several molecular features, and it can express structural diversity within a compound library in order to explore a broader chemical space and generate novel compound structures for drug candidates. In this study, we developed a deep-learning method, called NP-VAE (Natural Product-oriented Variational Autoencoder), based on variational autoencoder for managing hard-to-analyze datasets from DrugBank and large molecular structures such as natural compounds with chirality, an essential factor in the 3D complexity of compounds. NP-VAE was successful in constructing the chemical latent space from large-sized compounds that were unable to be handled in existing methods, achieving higher reconstruction accuracy, and demonstrating stable performance as a generative model across various indices. Furthermore, by exploring the acquired latent space, we succeeded in comprehensively analyzing a compound library containing natural compounds and generating novel compound structures with optimized functions.

ABPP-CoDEL: Activity-Based Proteome Profiling-Guided Discovery of Tyrosine-Targeting Covalent Inhibitors from DNA-Encoded Libraries.

DNA-encoded chemical library (DEL) has been extensively used for lead compound discovery for decades in academia and industry. Incorporating an electrophile warhead into DNA-encoded compounds recently permitted the discovery of covalent ligands that selectively react with a particular cysteine residue. However, noncysteine residues remain underexplored as modification sites of covalent DELs. Herein, we report the design and utility of tyrosine-targeting DELs of 67 million compounds. Proteome-wide reactivity analysis of tyrosine-reactive sulfonyl fluoride (SF) covalent probes suggested three enzymes (phosphoglycerate mutase 1, glutathione s-transferase 1, and dipeptidyl peptidase 3) as models of tyrosine-targetable proteins. Enrichment with SF-functionalized DELs led to the identification of a series of tyrosine-targeting covalent inhibitors of the model enzymes. In-depth mechanistic investigation revealed their novel modes of action and reactive ligand-accessible hotspots of the enzymes. Our strategy of combining activity-based proteome profiling and covalent DEL enrichment (ABPP-CoDEL), which generated selective covalent binders against a variety of target proteins, illustrates the potential use of this methodology in further covalent drug discovery.

Structure-activity relationship studies on vitamin D-based selective SREBP/SCAP inhibitor KK-052.

Vitamin D3 metabolites block lipid biosynthesis by promoting degradation of the complex of sterol regulatory element-binding protein (SREBP) and SREBP cleavage-activating protein (SCAP) independent of their effects on the vitamin D receptor (VDR). We previously reported the development of KK-052, the first vitamin D-based SREBP inhibitor that mitigates hepatic lipid accumulation without VDR-mediated calcemic action in mice. Herein we extend our previous work to synthesize KK-052 analogues. Various substituents were introduced to the phenyl ring of KK-052, and two KK-052 analogues were found to exhibit more potent SREBP/SCAP inhibitory activity than KK-052, whereas they all lack VDR activity. These new KK-052 analogues may be suited for further development as VDR-silent SREBP/SCAP inhibitors.

Identification of a Self-Assembling Small-Molecule Cancer Vaccine Adjuvant with an Improved Toxicity Profile.

Protein or peptide cancer vaccines usually include immune potentiators, so-called adjuvants. However, it remains challenging to identify structurally simple, chemically accessible synthetic molecules that are effective and safe as vaccine adjuvant. Here, we present cholicamideß (6), a self-assembling small-molecule vaccine adjuvant with an improved toxicity profile and proven efficacy in vivo. We demonstrate that cholicamideß (6), which is less cytotoxic than its parent compound, forms virus-like particles to potently activate dendritic cells with the concomitant secretion of cytokines. When combined with a peptide antigen, cholicamideß (6) potentiated the antigen presentation on dendritic cells to induce antigen-specific T cells. As a therapeutic cancer vaccine adjuvant in mice, a mixture of cholicamideß (6) and a peptide antigen protected mice from the challenges of malignant cancer cells without overt toxicity. Cholicamideß (6) may offer a translational opportunity as an unprecedented class of small-molecule cancer vaccine adjuvants.

Small-Molecule Drug Repurposing for Counteracting Phototoxic A2E Aggregation.

Despite the well-established role of oxidative stress in the pathogenesis of age-related macular degeneration (AMD), the mechanism underlying phototoxicity remains unclear. Herein, we used a drug repurposing approach to isolate an FDA-approved drug that blocks the aggregation of the photoinducible major fluorophore of lipofuscin, the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E). Our fluorescence-based screening combined with dynamic light scattering (DLS) analysis led to the identification of entacapone as a potent inhibitor of A2E fluorescence and aggregation. The entacapone-mediated inhibition of A2E aggregation blocks its photodegradation and offers photoprotection in A2E-loaded retinal pigment epithelial (RPE) cells exposed to blue light. In-depth mechanistic analysis suggests that entacapone prevents the conversion of toxic aggregates by redirecting A2E into off-pathway oligomers. These findings provide evidence that aggregation contributes to the phototoxicity of A2E.

Xanthine derivatives inhibit FTO in an L-ascorbic acid-dependent manner.

Xanthine derivatives were identified as inhibitors of the N6-methyladenosine (m6A) demethylase activity of fat-mass-and-obesity-associated protein (FTO) by activity-based high-throughput screening using the m6A-sensitive ribonuclease MazF. Pentoxifylline exhibited L-ascorbic acid concentration-dependent inhibitory activity against FTO, an unprecedented mode of inhibition, indicating that L-ascorbic acid is a promising key for designing FTO-specific inhibitors.

Novel gene-intergenic fusion involving ubiquitin E3 ligase UBE3C causes distal hereditary motor neuropathy.

Distal hereditary motor neuropathies (dHMNs) are a group of inherited diseases involving the progressive, length-dependent axonal degeneration of the lower motor neurons. There are currently 29 reported causative genes and four disease loci implicated in dHMN. Despite the high genetic heterogeneity, mutations in the known genes account for less than 20% of dHMN cases, with the mutations identified predominantly being point mutations or indels. We have expanded the spectrum of dHMN mutations with the identification of a 1.35 Mb complex structural variation (SV) causing a form of autosomal dominant dHMN (DHMN1 OMIM %182906). Given the complex nature of SV mutations and the importance of studying pathogenic mechanisms in a neuronal setting, we generated a patient-derived DHMN1 motor neuron model harbouring the 1.35 Mb complex insertion. The DHMN1 complex insertion creates a duplicated copy of the first 10 exons of the ubiquitin-protein E3 ligase gene (UBE3C) and forms a novel gene-intergenic fusion sense transcript by incorporating a terminal pseudo-exon from intergenic sequence within the DHMN1 locus. The UBE3C intergenic fusion (UBE3C-IF) transcript does not undergo nonsense-mediated decay and results in a significant reduction of wild-type full-length UBE3C (UBE3C-WT) protein levels in DHMN1 iPSC-derived motor neurons. An engineered transgenic Caenorhabditis elegans model expressing the UBE3C-IF transcript in GABA-ergic motor neurons shows neuronal synaptic transmission deficits. Furthermore, the transgenic animals are susceptible to heat stress, which may implicate defective protein homeostasis underlying DHMN1 pathogenesis. Identification of the novel UBE3C-IF gene-intergenic fusion transcript in motor neurons highlights a potential new disease mechanism underlying axonal and motor neuron degeneration. These complementary models serve as a powerful paradigm for studying the DHMN1 complex SV and an invaluable tool for defining therapeutic targets for DHMN1.

Interrupting specific hydrogen bonds between ELF3 and MED23 as an alternative drug resistance-free strategy for HER2-overexpressing cancers.

INTRODUCTION: HER2 overexpression induces cancer aggression and frequent recurrences in many solid tumors. Because HER2 overproduction is generally followed by gene amplification, inhibition of protein-protein interaction (PPI) between transcriptional factor ELF3 and its coactivator MED23 has been considered an effective but challenging strategy. OBJECTIVES: This study aimed to determine the hotspot of ELF3-MED23 PPI and further specify the essential residues and their key interactions in the hotspot which are controllable by small molecules with significant anticancer activity. METHODS: Intensive biological evaluation methods including SEAP, fluorescence polarization, LC-MS/MS-based quantitative, biosensor, GST-pull down assays, and in silico structural analysis were performed to determine hotspot of ELF3-MED23 PPI and to elicit YK1, a novel small molecule PPI inhibitor. The effects of YK1 on possible PPIs of MED23 and the efficacy of trastuzumab were assessed using cell culture and tumor xenograft mouse models. RESULTS: ELF3-MED23 PPI was found to be specifically dependent on H-bondings between D400, H449 of MED23 and W138, I140 of ELF3 for upregulating HER2 gene transcription. Employing YK1, we confirmed that interruption on these H-bondings significantly attenuated the HER2-mediated oncogenic signaling cascades and exhibited significant in vitro and in vivo anticancer activity against HER2-overexpressing breast and gastric cancers even in their trastuzumab refractory clones. CONCLUSION: Our approach to develop specific ELF3-MED23 PPI inhibitor without interfering other PPIs of MED23 can finally lead to successful development of a drug resistance-free compound to interrogate HER2 biology in diverse conditions of cancers overexpressing HER2.

Chemoproteomic Identification of Blue-Light-Damaged Proteins.

Visible light, particularly in the blue region of the spectrum, can cause cell dysfunction through the generation of singlet oxygen, contributing to cellular aging and age-related pathologies. Although photooxidation of nucleic acids, lipids, and amino acids has been extensively studied, the magnitude and span of blue-light-induced protein damages within proteome remain largely unknown. Herein we present a chemoproteomic approach to mapping blue-light-damaged proteins in live mammalian cells by exploiting a nucleophilic alkyne chemical probe. A gene ontology enrichment analysis revealed that cell surface proteins are more readily oxidized than other susceptible sets of proteins, including mitochondrial proteins. In particular, the integrin family of cell surface receptors (ITGs) was highly ranked in the mammalian cells tested, including human corneal endothelial cells. The blue-light-oxidized ITGB1 protein was functionally inactive in promoting cell adhesion and proliferation, suggesting that the photodamage of integrins contributes to the blue-light-induced cell dysfunction. Further application of our method to various cells and tissues should lead to a comprehensive analysis of light-sensitive proteins.

Self-assembling small-molecule adjuvants as antigen nano-carriers.

The effective co-delivery of antigens and immune potentiators (adjuvants) and the high degree of antigen presentation have been two major challenges in the development of subunit vaccines. Here, we address these issues by conjugating peptide antigens with cholicamide, a self-assembling small molecule adjuvant. Co-assemblies of the conjugates and cholicamide achieved high levels of both cytokine induction and MHC class II peptide presentation.

Magnetic Control of Cells by Chemical Fabrication of Melanin.

Melanin is an organic material biosynthesized from tyrosine in pigment-producing cells. The present study reports a simple method to generate tailored functional materials in mammalian cells by chemically fabricating intracellular melanin. Our approach exploits synthetic tyrosine derivatives to hijack the melanin biosynthesis pathway in pigment-producing cells. Its application was exemplified by synthesizing and using a paramagnetic tyrosine derivative, m-YR, which endowed melanoma cells with responsiveness to external magnetic fields. The mechanical force generated by the magnet-responsive melanin forced the cells to elongate and align parallel to the magnetic power lines. Critically, even non-pigment cells were similarly remote-controlled by external magnetic fields once engineered to express tyrosinase and treated with m-YR, suggesting the versatility of the approach. The present methodology may potentially provide a new avenue for mechanobiology and magnetogenetic studies and a framework for magnetic control of specific cells.

Small molecule-based detection of non-canonical RNA G-quadruplex structures that modulate protein translation.

Tandem repeats of guanine-rich sequences in RNA often form thermodynamically stable four-stranded RNA structures. Such RNA G-quadruplexes have long been considered to be linked to essential biological processes, yet their physiological significance in cells remains unclear. Here, we report a approach that permits the detection of RNA G-quadruplex structures that modulate protein translation in mammalian cells. The approach combines antibody arrays and RGB-1, a small molecule that selectively stabilizes RNA G-quadruplex structures. Analysis of the protein and mRNA products of 84 cancer-related human genes identified Nectin-4 and CapG as G-quadruplex-controlled genes whose mRNAs harbor non-canonical G-quadruplex structures on their 5'UTR region. Further investigations revealed that the RNA G-quadruplex of CapG exhibits a structural polymorphism, suggesting a possible mechanism that ensures the translation repression in a KCl concentration range of 25-100 mM. The approach described in the present study sets the stage for further discoveries of RNA G-quadruplexes.

Discovery of a phase-separating small molecule that selectively sequesters tubulin in cells.

Phase-separated membraneless organelles or biomolecular condensates play diverse functions in cells, however recapturing their characteristics using small organic molecules has been a challenge. In the present study, cell-lysate-based screening of 843 self-assembling small molecules led to the discovery of a simple organic molecule, named huezole, that forms liquid droplets to selectively sequester tubulin. Remarkably, this small molecule enters cultured human cells and prevents cell mitosis by forming tubulin-concentrating condensates in cells. The present study demonstrates the feasibility of producing a synthetic condensate out of non-peptidic small molecules for exogenous control of cellular processes. The modular structure of huezole provides a framework for designing a class of organelle-emulating small molecules.

23,25-Dihydroxyvitamin D₃ is liberated as a major vitamin D₃ metabolite in human urine after treatment with ß-glucuronidase: Quantitative comparison with 24,25-dihydroxyvitamin D₃ by LC/MS/MS.

The complete understanding of the excretion of surplus 25-hydroxyvitamin D₃ [25(OH)D₃] in humans remains to be accomplished. In our previous study, 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] 24-glucuronide was identified as a major urinary vitamin D₃ metabolite, while the glucuronide of 23,25-dihydroxyvitamin D₃ [23,25(OH)₂D₃] is another metabolite of interest but has not been sufficiently evaluated. Although the quantitative analysis of 24,25(OH)₂D₃ liberated in urine by the treatment with ß-glucuronidase (GUS) has been conducted, no information was provided about the amount of the glucuronidated 23,25(OH)₂D₃ in the urine. In this study, we first developed and validated a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS)-based method for the simultaneous quantification of 23,25(OH)₂D₃ and 24,25(OH)₂D₃ liberated in urine by GUS. The analysis of the urine samples revealed that the amount of 23,25(OH)₂D₃ was almost as much as that of 24,25(OH)₂D₃, in contrast to the fact that the plasma concentration of 23,25(OH)₂D₃ was much lower than that of 24,25(OH)₂D₃. These results strongly suggested that 23,25(OH)₂D₃ is more susceptible to glucuronidation and more promptly excreted into urine than 24,25(OH)₂D₃. Furthermore, the amount ratios of 23,25(OH)₂D₃ to 24,25(OH)₂D₃ in the urine samples did not markedly vary during the day (morning/evening) and even by a week-long vitamin D₃ supplementation (1000 IU/body/day). We concluded that the C-23 hydroxylation plays a crucial role in the urinary excretion of surplus 25(OH)D₃.

Inhibiting SCAP/SREBP exacerbates liver injury and carcinogenesis in murine nonalcoholic steatohepatitis.

Enhanced de novo lipogenesis mediated by sterol regulatory element-binding proteins (SREBPs) is thought to be involved in nonalcoholic steatohepatitis (NASH) pathogenesis. In this study, we assessed the impact of SREBP inhibition on NASH and liver cancer development in murine models. Unexpectedly, SREBP inhibition via deletion of the SREBP cleavage-activating protein (SCAP) in the liver exacerbated liver injury, fibrosis, and carcinogenesis despite markedly reduced hepatic steatosis. These phenotypes were ameliorated by restoring SREBP function. Transcriptome and lipidome analyses revealed that SCAP/SREBP pathway inhibition altered the fatty acid (FA) composition of phosphatidylcholines due to both impaired FA synthesis and disorganized FA incorporation into phosphatidylcholine via lysophosphatidylcholine acyltransferase 3 (LPCAT3) downregulation, which led to endoplasmic reticulum (ER) stress and hepatocyte injury. Supplementation with phosphatidylcholines significantly improved liver injury and ER stress induced by SCAP deletion. The activity of the SCAP/SREBP/LPCAT3 axis was found to be inversely associated with liver fibrosis severity in human NASH. SREBP inhibition also cooperated with impaired autophagy to trigger liver injury. Thus, excessively strong and broad lipogenesis inhibition was counterproductive for NASH therapy; this will have important clinical implications in NASH treatment.

Glucose as a Protein-Condensing Cellular Solute.

The present study reports a surprising protein-condensing effect of glucose, prompted by our accidental observation during chemical library screening under a high-glucose condition. We noticed "glucosing-out" of certain compounds, in which physiological concentrations of glucose induced compound aggregation. Adapting the "glucosing-out" concept to proteins, our proteomic analysis identified three cellular proteins (calmodulin, rho guanine nucleotide exchange factor 40, and polyubiquitin-C) that displayed robust glucose-dependent precipitation. One of these proteins, calmodulin, formed glucose-dependent condensates that control cellular glycogenolysis in hepatic cells. Our findings suggest that glucose is a heretofore underappreciated driver of protein phase separation that may have profound effects on cellular homeostasis.

Discovery of Non-Cysteine-Targeting Covalent Inhibitors by Activity-Based Proteomic Screening with a Cysteine-Reactive Probe.

Covalent inhibitors of enzymes are increasingly appreciated as pharmaceutical seeds, yet discovering non-cysteine-targeting inhibitors remains challenging. Herein, we report an intriguing experience during our activity-based proteomic screening of 1601 reactive small molecules, in which we monitored the ability of library molecules to compete with a cysteine-reactive iodoacetamide probe. One epoxide molecule, F8, exhibited unexpected enhancement of the probe reactivity for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a rate-limiting glycolysis enzyme. In-depth mechanistic analysis suggests that F8 forms a covalent adduct with an aspartic acid in the active site to displace NAD+, a cofactor of the enzyme, with concomitant enhancement of the probe reaction with the catalytic cysteine. The mechanistic underpinning permitted the identification of an optimized aspartate-reactive GAPDH inhibitor. Our findings exemplify that activity-based proteomic screening with a cysteine-reactive probe can be used for discovering covalent inhibitors that react with non-cysteine residues.

Controlled lipid ß-oxidation and carnitine biosynthesis by a vitamin D metabolite.

Lactone-vitamin D3 is a major metabolite of vitamin D3, a lipophilic vitamin biosynthesized in numerous life forms by sunlight exposure. Although lactone-vitamin D3 was discovered 40 years ago, its biological role remains largely unknown. Chemical biological analysis of its photoaffinity probe identified the hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA), a mitochondrial enzyme that catalyzes ß-oxidation of long-chain fatty acids, as its selective binding protein. Intriguingly, the interaction of lactone-vitamin D3 with HADHA does not affect the HADHA enzymatic activity but instead limits biosynthesis of carnitine, an endogenous metabolite required for the transport of fatty acids into the mitochondria for ß-oxidation. Lactone-vitamin D3 dissociates the protein-protein interaction of HADHA with trimethyllysine dioxygenase (TMLD), thereby impairing the TMLD enzyme activity essential in carnitine biosynthesis. These findings suggest a heretofore undescribed role of lactone-vitamin D3 in lipid ß-oxidation and carnitine biosynthesis, and possibly in sunlight-dependent shifts of lipid metabolism in animals.

Chemical Genetics Reveals a Role of Squalene Synthase in TGFß Signaling and Cardiomyogenesis.

KY02111 is a widely used small molecule that boosts cardiomyogenesis of the mesoderm cells derived from pluripotent stem cells, yet its molecular mechanism of action remains elusive. The present study resolves the initially perplexing effects of KY02111 on Wnt signaling and subsequently identifies squalene synthase (SQS) as a molecular target of KY02111 and its optimized version, KY-I. By disrupting the interaction of SQS with cardiac ER-membrane protein TMEM43, KY02111 impairs TGFß signaling, but not Wnt signaling, and thereby recapitulates the clinical mutation of TMEM43 that causes arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited heart disease that involves a substitution of myocardium with fatty tissue. These findings reveal a heretofore undescribed role of SQS in TGFß signaling and cardiomyogenesis. KY02111 may find its use in ARVC modeling as well as serve as a chemical tool for studying TGFß/SMAD signaling.

Asian Chemical Biology Initiative (ACBI): Bringing Together Chemical Biologists Within Asia.

Asian Chemical Biology Initiative (ACBI) has been influential in promoting chemical biology research and education throughout the Asian region. This editorial will walk you through the unprecedented journey the ACBI has taken in the past 10 years.