NeoaPred: A deep-learning framework for predicting immunogenic neoantigen based on surface and structural features of peptide-HLA complexes.

MOTIVATION: Neoantigens, derived from somatic mutations in cancer cells, can elicit anti-tumor immune responses when presented to autologous T cells by human leukocyte antigen (HLA). Identifying immunogenic neoantigens is crucial for cancer immunotherapy development. However, the accuracy of current bioinformatic methods remains unsatisfactory. Surface and structural features of peptide-HLA class I (pHLA-I) complexes offer valuable insight into the immunogenicity of neoantigens. RESULTS: We present NeoaPred, a deep-learning framework for neoantigen prediction. NeoaPred accurately constructs pHLA-I complex structures, with 82.37% of the predicted structures showing an RMSD of < 1 Å. Using these structures, NeoaPred integrates differences in surface, structural, and atom group features between the mutant peptide and its wild-type counterpart to predict a foreignness score. This foreignness score is an effective factor for neoantigen prediction, achieving an AUROC of 0.81 and an AUPRC of 0.54 in the test set, outperforming existing methods. AVAILABILITY: The source code is released under an Apache v2.0 license and is available at the GitHub repository https://github.com/Dulab2020/NeoaPred. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Unveiling the immune-modulating power of THz-FEL irradiation.

As terahertz (THz) technology advances, the interaction between THz radiation and the living body, particularly its effects on the immune system, has attracted extensive attention but remains poorly understood. This study firstly elucidated that exposure to 3 THz-FEL radiation markedly suppressed contact hypersensitivity reactions in mice induced by DNFB, as evidenced by a reduction in ear thickness and a discernible recovery in the Th1/Th2 cell balance. 3 THz irradiation led to cellular stress in the irradiated skin locale, increasing the levels of IL-4 and IL-10 and modulating the activity and migration of dendritic cells and mast cells. Furthermore, THz irradiation precipitated a rapid alteration in the skin lipidome, altering several categories of bioactive lipids. These findings offer new insights into the immunomodulatory effects of THz radiation on living organisms and the potential underlying mechanisms, with implications for the development of therapeutic approaches in managing skin allergic diseases.

DNA-DISK: Automated end-to-end data storage via enzymatic single-nucleotide DNA synthesis and sequencing on digital microfluidics.

In the age of information explosion, the exponential growth of digital data far exceeds the capacity of current mainstream storage media. DNA is emerging as a promising alternative due to its higher storage density, longer retention time, and lower power consumption. To date, commercially mature DNA synthesis and sequencing technologies allow for writing and reading of information on DNA with customization and convenience at the research level. However, under the disconnected and nonspecialized mode, DNA data storage encounters practical challenges, including susceptibility to errors, long storage latency, resource-intensive requirements, and elevated information security risks. Herein, we introduce a platform named DNA-DISK that seamlessly streamlined DNA synthesis, storage, and sequencing on digital microfluidics coupled with a tabletop device for automated end-to-end information storage. The single-nucleotide enzymatic DNA synthesis with biocapping strategy is utilized, offering an ecofriendly and cost-effective approach for data writing. A DNA encapsulation using thermo-responsive agarose is developed for on-chip solidification, not only eliminating data clutter but also preventing DNA degradation. Pyrosequencing is employed for in situ and accurate data reading. As a proof of concept, DNA-DISK successfully stored and retrieved a musical sheet file (228 bits) with lower write-to-read latency (4.4 min of latency per bit) as well as superior automation compared to other platforms, demonstrating its potential to evolve into a DNA Hard Disk Drive in the future.

Small Molecule-Induced Post-Translational Acetylation of Catalytic Lysine of Kinases in Mammalian Cells.

Reversible lysine acetylation is an important post-translational modification (PTM). This process in cells is typically carried out enzymatically by lysine acetyltransferases and deacetylases. The catalytic lysine in the human kinome is highly conserved and ligandable. Small-molecule strategies that enable post-translational acetylation of the catalytic lysine on kinases in a target-selective manner therefore provide tremendous potential in kinase biology. Herein, we report the first small molecule-induced chemical strategy capable of global acetylation of the catalytic lysine on kinases from mammalian cells. By surveying various lysine-acetylating agents installed on a promiscuous kinase-binding scaffold, Ac4 was identified and shown to effectively acetylate the catalytic lysine of >100 different protein kinases from live Jurkat/K562 cells. In order to demonstrate that this strategy was capable of target-selective and reversible chemical acetylation of protein kinases, we further developed six acetylating compounds on the basis of VX-680 (a noncovalent inhibitor of AURKA). Among them, Ac13/Ac14, while displaying excellent in vitro potency and sustained cellular activity against AURKA, showed robust acetylation of its catalytic lysine (K162) in a target-selective manner, leading to irreversible inhibition of endogenous kinase activity. The reversibility of this chemical acetylation was confirmed on Ac14-treated recombinant AURKA protein, followed by deacetylation with SIRT3 (a lysine deacetylase). Finally, the reversible Ac13-induced acetylation of endogenous AURKA was demonstrated in SIRT3-transfected HCT116 cells. By disclosing the first cell-active acetylating compounds capable of both global and target-selective post-translational acetylation of the catalytic lysine on kinases, our strategy could provide a useful chemical tool in kinase biology and drug discovery.

Structure-Based Drug Design of 2-Amino-[1,1'-biphenyl]-3-carboxamide Derivatives as Selective PKMYT1 Inhibitors for the Treatment of CCNE1-Amplified Breast Cancer.

CCNE1 amplification occurs in breast cancer and currently lacks effective therapies. PKMYT1 as a synthetic lethal target for CCNE1 amplification holds promise for the treatment of CCNE1-amplified breast cancer. Herein, we discover a series of 2-amino-[1,1'-biphenyl]-3-carboxamide derivatives as potent and selective PKMYT1 inhibitors using structure-based drug design. The representative compound 8ma exhibited excellent potency against PKMYT1, while sparing WEE1. It also suppressed proliferation of the CCNE1-amplified HCC1569 breast cancer cell line and showed synergistic cytotoxicity in combination with gemcitabine. PKMYT1 X-ray cocrystallography confirmed that introduction of key binding interactions between the inhibitors and residues Asp251 and Tyr121 of PKMYT1 greatly enhanced the potency and selectivity of the compounds.

Targeting Solvent-Front Mutations for Kinase Drug Discovery: From Structural Basis to Design Strategies.

Solvent-front mutations have emerged as a common mechanism leading to acquired resistance to kinase inhibitors, representing a major challenge in the clinic. Several new-generation kinase inhibitors targeting solvent-front mutations have either been approved or advanced to clinical trials. However, there remains a need to discover effective, new-generation inhibitors. In this Perspective, we systematically summarize the general types of solvent-front mutations across the kinome and describe the development of inhibitors targeting some key solvent-front mutations. Additionally, we highlight the challenges and opportunities for the next generation of kinase inhibitors directed toward overcoming solvent-front mutations.

COA6 promotes the oncogenesis and progression of breast cancer by oxidative phosphorylation pathway.

Mitochondrial oxidative phosphorylation (OXPHOS) has long been considered the primary energy source in breast cancer cells. Cytochrome c oxidase assembly factor 6 (COA6), which functions as a metal chaperone to transport copper to complex Ⅳ during the OXPHOS process, plays a crucial role in the carcinogenesis of lung adenocarcinoma. Nevertheless, its specific function in breast cancer is undefined. The present investigation aimed to clarify COA6's expression profile and regulatory functions in breast cancer, as well as to unveil its underlying mechanisms. Initially, our findings revealed a significant upregulation of COA6 in breast cancer, as evidenced by an analysis of the TCGA database and tissue microarrays. This upregulation correlated with tumor size and histological grade. Additionally, survival analysis revealed that elevated COA6 amounts were correlated with decreased overall survival (OS) in breast cancer. To delve deeper into the functions of COA6, both COA6-overexpressing and COA6-knockdown breast cancer cell models were established. These experiments demonstrated COA6 is pivotal in regulating cell proliferation, apoptosis, migration, and invasion, thereby promoting cancer progression in vitro. Notably, functional enrichment analysis indicated COA6 might be involved in breast cancer progression by modulating oxidative phosphorylation (OXPHOS). Collectively, this study reveals an overt tumorigenic role for COA6 in breast cancer and sheds light on its potential mechanisms, offering valuable therapeutic targets for breast cancer therapy.

Propyl acetate protects intestinal barrier during parenteral nutrition in mice and Caco-2 cells.

BACKGROUND: Gut microbiota dysbiosis induces intestinal barrier damage during parenteral nutrition (PN). However, the underlying mechanisms remain unclear. This study aimed to investigate gut microbiota dysbiosis, luminal short-chain fatty acids, and autophagy in a mouse model and how these short-chain fatty acids regulate autophagy. METHODS: Eight-week-old male specific-pathogen-free mice were randomly divided into a Chow group (standard diet and intravenous normal saline infusion) and a PN group (continuous infusion of PN nutrient solution) for 7 days. Caco-2 cells were also treated with intestinal rinse solutions from Chow and PN mouse models. RESULTS: Compared with the Chow group, the PN group exhibited increased Proteobacteria and decreased Firmicutes, correlating with decreased propyl acetate. In the PN group, intestinal tissue exhibited elevated adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, LC3II protein levels, and Atg3 and Atg7 messenger RNA levels. P62 protein levels were decreased, indicating an increase of autophagy flux in the PN group. In the Caco-2 cell model, cells treated with PN solution plus propyl acetate exhibited increased Claudin-1 and occluding along with decreased interleukin-6 and tumor necrosis factor α compared with those treated with PN solution alone. Propyl acetate addition inhibited the AMPK-mammalian target of rapamycin (mTOR) pathway, mitigating the excessive autophagy induced by the PN intestinal rinse solution in Caco-2 cells. CONCLUSION: PN led to a significant reduction in propyl acetate levels in the intestine, excessive activation of autophagy, and barrier dysfunction. Propyl acetate inhibited excessive autophagy via the AMPK/mTOR signaling pathway and protected the intestinal barrier during PN.

ß-Hydroxybutyrate enhances astrocyte glutamate uptake through EAAT1 expression regulation.

ß-Hydroxybutyrate (BHB) has been reported to exert neuroprotective functions and is considered a promising treatment for neurodegenerative diseases such as Parkinson's and Alzheimer's. Numerous studies have revealed BHB's multifaceted roles, including anti-senescence, anti-oxidative, and anti-inflammatory activities. However, the underlying mechanisms warrant further investigation. Astrocytes, the most abundant glial cells in the central nervous system, play a pivotal role in the development and progression of neurodegenerative diseases. While BHB is known to alter neuronal metabolism and function, its effects on astrocytes remain poorly understood. In this study, we conducted transcriptome sequencing analysis to identify differentially expressed genes induced by BHB in astrocytes and found that the gene Solute carrier family 1 member 3 (Slc1a3), encoding the glutamate transporter EAAT1, was significantly upregulated by BHB treatment. Cellular and animal-based experiments confirmed an increase in EAAT1 protein expression in primary astrocytes and the hippocampus of mice treated with BHB. This upregulation may be due to the activation of the Ca2+/CAMKII pathway by BHB. Furthermore, BHB improved astrocytes' glutamate uptake and partially restored neuronal viability impaired by glutamate-induced excitotoxicity when astrocytes were functionalized. Our results suggest that BHB may alleviate neuronal damage caused by excessive glutamate by enhancing the glutamate absorption and uptake capacity of astrocytes. This study proposes a novel mechanism for the neuroprotective effects of BHB and reinforces its beneficial impact on the central nervous system (CNS).

Non-Thermal Effects of Terahertz Radiation on Gene Expression: Systematic Review and Meta-Analysis.

With the advancement of terahertz technology, unveiling the mysteries of terahertz has had a profound impact on the field of biomedicine. However, the lack of systematic comparisons for gene expression signatures may diminish the effectiveness and efficiency of identifying common mechanisms underlying terahertz effects across diverse research findings. We performed a comprehensive review and meta-analysis to compile patterns of gene expression profiles associated with THz radiation. Thorough bibliographic reviews were conducted, utilizing the PubMed, Embase, Web of Science, and ProQuest databases to extract references from published articles. Raw CEL files were obtained from Gene Expression Omnibus and preprocessed using Bioconductor packages. This systematic review (Registration No. CDR42024502937) resulted in a detailed analysis of 13 studies (14 papers). There are several possible mechanisms and pathways through which THz radiation could cause biological changes. While the established gene expression results are largely associated with immune response and inflammatory markers, other genes demonstrated transcriptional outcomes that may unravel unknown functions. The enrichment of genes primarily found networks associated with broader stress responses. Altogether, the findings showed that THz can induce a distinct transcriptomic profile that is not associated with a microthermal cellular response. However, it is impossible to pinpoint a single gene or family of genes that would accurately and reliably justify the patterns of gene expression response under THz exposure.

High-Throughput Screening for Oligonucleotide Detection by ADE-OPI-MS.

Oligonucleotides represent a class of shorter DNA or RNA nucleic acid polymers extensively applied in the biomedical field. Despite progress in detecting and analyzing oligonucleotides, high-throughput analysis of the samples remains challenging. In this work, a high-throughput analysis method for oligonucleotide analysis was developed based on acoustic droplet ejection-open port interface-mass spectrometry (ADE-OPI-MS) technology. This approach was applied to determine the enzymatic activity of terminal deoxynucleotide transferase (TdT) for DNA synthesis, with a rate of 3 s/sample, which enhanced single-sample analysis efficiency approximately 60-fold over the previous gel analysis. After testing approximately 10,000 TdT mutants, we obtained three new variants with higher catalytic activities. Finally, by integrating these mutants, the catalytic activity of TdT was improved about 4 times compared to the starting mutant. Our results successfully established a high-throughput screening method for oligonucleotide analysis, which not only provides a foundation to engineer highly efficient TdT for ab initio synthesis of DNA but also paves the way for the potential application of oligonucleotide analysis in biomedical fields.

Dynamics Playing a Key Role in the Covalent Binding of Inhibitors to Focal Adhesion Kinase.

Covalent kinase inhibitors (CKIs) have recently garnered considerable attention, yet the rational design of CKIs continues to pose a great challenge. In the discovery of CKIs targeting focal adhesion kinase (FAK), it has been observed that the chemical structure of the linkers plays a key role in achieving covalent targeting of FAK. However, the mechanism behind the observation remains elusive. In this work, we employ a comprehensive suite of advanced computational methods to investigate the mechanism of CKIs covalently targeting FAK. We reveal that the linker of an inhibitor influences the contacts between the warhead and residue(s) and the residence time in active conformation, thereby dictating the inhibitor's capability to bind covalently to FAK. This study reflects the complexity of CKI design and underscores the importance of considering the dynamic interactions and residence times for the successful development of covalent drugs.

Therapeutic targeting ERRγ suppresses metastasis via extracellular matrix remodeling in small cell lung cancer.

Small-cell lung cancer (SCLC) is the most aggressive and lethal type of lung cancer, characterized by limited treatment options, early and frequent metastasis. However, the determinants of metastasis in SCLC are poorly defined. Here, we show that estrogen-related receptor gamma (ERRγ) is overexpressed in metastatic SCLC tumors, and is positively associated with SCLC progression. ERRγ functions as an essential activator of extracellular matrix (ECM) remodeling and cell adhesion, two critical steps in metastasis, by directly regulating the expression of major genes involved in these processes. Genetic and pharmacological inhibition of ERRγ markedly reduces collagen production, cell-matrix adhesion, microfilament production, and eventually blocks SCLC cell invasion and tumor metastasis. Notably, ERRγ antagonists significantly suppressed tumor growth and metastasis and restored SCLC vulnerability to chemotherapy in multiple cell-derived and patient-derived xenograft models. Taken together, these findings establish ERRγ as an attractive target for metastatic SCLC and provide a potential pharmacological strategy for treating this lethal disease.

Membrane-mediated modulation of mitochondrial physiology by terahertz waves.

Extensive studies have demonstrated the diverse impacts of electromagnetic waves at gigahertz and terahertz (THz) frequencies on cytoplasmic membrane properties. However, there is little evidence of these impacts on intracellular membranes, particularly mitochondrial membranes crucial for mitochondrial physiology. In this study, human neuroblast-like cells were exposed to continuous 0.1 THz radiation at an average power density of 33 mW/cm2. The analysis revealed that THz exposure significantly altered the mitochondrial ultrastructure. THz waves enhanced the enzymatic activity of the mitochondrial respiratory chain but disrupted supercomplex assembly, compromising mitochondrial respiration. Molecular dynamics simulations revealed altered rates of change in the quantity of hydrogen bonds and infiltration of water molecules in lipid bilayers containing cardiolipin, indicating the specific behavior of cardiolipin, a signature phospholipid in mitochondria, under THz exposure. These findings suggest that THz radiation can significantly alter mitochondrial membrane properties, impacting mitochondrial physiology through a mechanism related to mitochondrial membrane, and provide deeper insight into the bioeffects of THz radiation.

Design, synthesis and biological evaluation of 5-amino-1H-pyrazole-4-carboxamide derivatives as pan-FGFR covalent inhibitors.

The aberrant activation of FGFRs plays a critical role in various cancers, leading to the development of several FGFR inhibitors in clinic. However, the emergence of drug resistance, primarily due to gatekeeper mutations in FGFRs, has limited their clinical efficacy. To address the unmet medical need, a series of 5-amino-1H-pyrazole-4-carboxamide derivatives were designed and synthesized as novel pan-FGFR covalent inhibitors targeting both wild-type and the gatekeeper mutants. The representative compound 10h demonstrated nanomolar activities against FGFR1, FGFR2, FGFR3 and FGFR2 V564F gatekeeper mutant in biochemical assays (IC50 = 46, 41, 99, and 62 nM). Moreover, 10h also strongly suppressed the proliferation of NCI-H520 lung cancer cells, SNU-16 and KATO III gastric cancer cells with IC50 values of 19, 59, and 73 nM, respectively. Further X-ray co-crystal structure revealed that 10h irreversibly binds to FGFR1. The study provides a new promising point for anticancer drug development medicated by FGFRs.

Anti-Inflammatory and Immunomodulatory Effects of 0.1 Sub-Terahertz Irradiation in Collagen-Induced Arthritis Mice.

The primary emphasis of photoimmunology is the impact of nonionizing radiation on the immune system. With the development of terahertz (THz) and sub-terahertz (sub-THz) technology, the biological effects of this emerging nonionizing radiation, particularly its influence on immune function, remain insufficiently explored but are progressively attracting attention. Here, we demonstrated that 0.1 sub-THz radiation can modulate the immune system and alleviate symptoms of arthritis in collagen-induced arthritis (CIA) mice through a nonthermal manner. The application of 0.1 sub-THz irradiation led to a decrease in proinflammatory factors within the joints and serum, reducing the levels of blood immune cells and the quantity of splenic CD4+ T cells. Notably, 0.1 sub-THz irradiation restored depleted Treg cells in CIA mice and re-established the Th17/Treg equilibrium. These findings suggested that sub-THz irradiation plays a crucial role in systemic immunoregulation. Further exploration of its immune modulation mechanisms revealed the anti-inflammatory properties of 0.1 sub-THz on LPS-stimulated skin keratinocytes. Through the reduction in NF-κB signaling and NLRP3 inflammasome activation, 0.1 sub-THz irradiation effectively decreased the production of inflammatory factors and immune-active substances, including IL-1ß and PGE2, in HaCaT cells. Consequently, 0.1 sub-THz irradiation mitigated the inflammatory response and contributed to the maintenance of immune tolerance in CIA mice. This research provided significant new evidence supporting the systemic impacts of 0.1 sub-THz radiation, particularly on the immune system. It also enhanced the field of photoimmunology and offered valuable insights into the potential biomedical applications of 0.1 sub-THz radiation for treating autoimmune diseases.

Increased expression of REG3A promotes tumorigenic behavior in triple negative breast cancer cells.

BACKGROUND: Identifying new targets in triple negative breast cancer (TNBC) remains critical. REG3A (regenerating islet-derived protein 3 A), a calcium-dependent lectin protein, was thoroughly investigated for its expression and functions in breast cancer. METHODS: Bioinformatics and local tissue analyses were employed to identify REG3A expression in breast cancer. Genetic techniques were employed to modify REG3A expression, and the resulting effects on the behaviors of breast cancer cells were examined. Subcutaneous xenograft models were established to investigate the involvement of REG3A in the in vivo growth of breast cancer cells. RESULTS: Analysis of the TCGA database uncovered increased REG3A levels in human breast cancer tissues. Additionally, REG3A mRNA and protein levels were elevated in TNBC tissues of locally treated patients, contrasting with low expression in adjacent normal tissues. In primary human TNBC cells REG3A shRNA notably hindered cell proliferation, migration, and invasion while triggering caspase-mediated apoptosis. Similarly, employing CRISPR-sgRNA for REG3A knockout showed significant anti-TNBC cell activity. Conversely, REG3A overexpression bolstered cell proliferation and migration. REG3A proved crucial for activating the Akt-mTOR cascade, as evidenced by decreased Akt-S6K1 phosphorylation upon REG3A silencing or knockout, which was reversed by REG3A overexpression. A constitutively active mutant S473D Akt1 (caAkt1) restored Akt-mTOR activation and counteracted the proliferation inhibition and apoptosis induced by REG3A knockdown in breast cancer cells. Crucially, REG3A played a key role in maintaining mTOR complex integrity. Bioinformatics identified zinc finger protein 680 (ZNF680) as a potential REG3A transcription factor. Knocking down or knocking out ZNF680 reduced REG3A expression, while its overexpression increased it in primary breast cancer cells. Additionally, enhanced binding between ZNF680 protein and the REG3A promoter was observed in breast cancer tissues and cells. In vivo, REG3A shRNA significantly inhibited primary TNBC cell xenograft growth. In REG3A-silenced xenograft tissues, reduced REG3A levels, Akt-mTOR inhibition, and activated apoptosis were evident. CONCLUSION: ZNF680-caused REG3A overexpression drives tumorigenesis in breast cancer possibly by stimulating Akt-mTOR activation, emerging as a promising and innovative cancer target.

Structure-Based Optimization of Pyrazinamide-Containing Macrocyclic Derivatives as Fms-like Tyrosine Kinase 3 (FLT3) Inhibitors to Overcome Clinical Mutations.

Secondary mutations in Fms-like tyrosine kinase 3-tyrosine kinase domain (FLT3-TKD) (e.g., D835Y and F691L) have become a major on-target resistance mechanism of FLT3 inhibitors, which present a significant clinical challenge. To date, no effective drugs have been approved to simultaneously overcome clinical resistance caused by these two mutants. Thus, a series of pyrazinamide macrocyclic compounds were first designed and evaluated to overcome the secondary mutations of FLT3. The representative 8v exhibited potent inhibitory activities against FLT3D835Y and FLT3D835Y/F691L with IC50 values of 1.5 and 9.7 nM, respectively. 8v also strongly suppressed the proliferation against Ba/F3 cells transfected with FLT3-ITD, FLT3-ITD-D835Y, FLT3-ITD-F691L, FLT3-ITD-D835Y-F691L, and MV4-11 acute myeloid leukemia (AML) cell lines with IC50 values of 12.2, 10.5, 24.6, 16.9, and 6.8 nM, respectively. Furthermore, 8v demonstrated ideal anticancer efficacy in a Ba/F3-FLT3-ITD-D835Y xenograft model. The results suggested that 8v can serve as a promising macrocycle-based FLT3 inhibitor for the treatment of AML.

0.263 terahertz irradiation induced genes expression changes in Caenorhabditis elegans.

The biosafety of terahertz (THz) waves has emerged as a new area of concern with the gradual application of terahertz radiation. Even though many studies have been conducted to investigate the influence of THz radiation on living organisms, the biological effects of terahertz waves have not yet been fully revealed. In this study, Caenorhabditis elegans (C. elegans) was used to evaluate the biological consequences of whole-body exposure to 0.263 THz irradiation. The integration of transcriptome sequencing and behavioral tests of C. elegans revealed that high-power THz irradiation damaged the epidermal ultrastructures, inhibited the expression of the cuticle collagen genes, and impaired the movement of C. elegans. Moreover, the genes involved in the immune system and the neural system were dramatically down-regulated by high-power THz irradiation. Our findings offer fresh perspectives on the biological impacts of high-power THz radiation that could cause epidermal damage and provoke a systemic response.

Discovery of 6-Formylpyridyl Urea Derivatives as Potent Reversible-Covalent Fibroblast Growth Factor Receptor 4 Inhibitors with Improved Anti-Hepatocellular Carcinoma Activity.

Fibroblast growth factor receptor 4 (FGFR4) has been considered as a potential anticancer target due to FGF19/FGFR4 mediated aberrant signaling in hepatocellular carcinoma (HCC). Several FGFR4 inhibitors have been reported, but none have gained approval. Herein, a series of 5-formyl-pyrrolo[3,2-b]pyridine-3-carboxamides and a series of 6-formylpyridyl ureas were characterized as selective reversible-covalent FGFR4 inhibitors. The representative 6-formylpyridyl urea 8z exhibited excellent potency against FGFR4WT, FGFR4V550L, and FGFR4V550M with IC50 values of 16.3, 12.6, and 57.3 nM, respectively. It also potently suppressed proliferation of Ba/F3 cells driven by FGFR4WT, FGFR4V550L, and FGFR4V550M, and FGFR4-dependent Hep3B and Huh7 HCC cells, with IC50 values of 1.2, 13.5, 64.5, 15.0, and 20.4 nM, respectively. Furthermore, 8z displayed desirable microsomal stability and significant in vivo efficacy in the Huh7 HCC cancer xenograft model in nude mice. The study provides a promising new lead for anticancer drug discovery directed toward overcoming FGFR4 gatekeeper mutation mediated resistance in HCC patients.