HEC96719 is a small-molecule farnesoid X receptor (FXR) agonist with potential for treating nonalcoholic steatohepatitis (NASH). Herein, we report a robust and scalable kilogram-scale synthesis of compound 9, the central and synthetically challenging spirocyclic benzoxepane-pyridine core of HEC96719. This optimized process features: (i) a protecting-group-enabled nucleophilic acylation, (ii) an industrially practical cyclopropanation using NaOH under phase-transfer conditions, (iii) an ortho-phenol-assisted deoxygenation protocol to access a highly strained methylene intermediate, (iv) a copper-mediated intramolecular Ullmann etherification, and (v) a palladium-catalyzed carbonylation employing a safe CO surrogate to avoid high-pressure carbon monoxide. Collectively, this process enhances operator safety, improves overall efficiency and yield, avoids chromatographic purification, and has been successfully demonstrated on a multikilogram scale, underscoring its potential for industrial application.

RORγt, a member of the nuclear receptor family, plays a central role in directing Th17 cell differentiation and pro-inflammatory function. Dysregulation of this transcription factor contributes significantly to autoimmune conditions such as psoriasis, rheumatoid arthritis, and multiple sclerosis. Consequently, inhibition of RORγt has attracted considerable interest as a therapeutic approach for these disorders. In this study, we designed and synthesized a series of aryl sulfonyl derivatives as RORγt inhibitors. Selected compounds were functionally characterized as inverse agonists, with compound 7 as the initial lead. Through a scaffold-hopping strategy and systematic structure-activity relationship (SAR) studies of key substituents, we identified potent RORγt inhibitors with favorable oral bioavailability. Compound 8v emerged as the most promising candidate. In vivo pharmacokinetic studies demonstrated that 8v possessed good oral absorption. In addition, 8v showed moderate metabolic stability in human and mouse liver microsomes (t₁/₂ = 45 min and 151 min, respectively). Furthermore, in a mouse model of LPS-induced systemic inflammation-where IL-17 elevation is mediated by T-cell activation-and in an IMQ-induced psoriasis-like dermatitis model, 8v significantly inhibited IL-17 secretion and ameliorated disease-related symptoms. Collectively, these results support the therapeutic potential of small-molecule RORγt modulators for the treatment of inflammatory and autoimmune diseases.

Alterations in the FGFR family act as oncogenic drivers for multiple pediatric and adult tumors, leading to the development and approval of several FGFR inhibitors. However, the on-target gatekeeper and "molecular brake" mutations confer clinically acquired resistance to the FDA-approved FGFR inhibitors, which presents a significant unmet medical need. Herein, we report the first novel macrocycle-based FGFR inhibitors targeting both wild-type and clinically acquired variants of the FGFR family. The representative compound 8r potently inhibited FGFR1/2/3 with IC50 values of 10.0, 6.9, and 30.2 nM, respectively. Compound 8r also potently suppressed proliferation of a series of FGFR-driven cancer cell lines with IC50 values of 2.0-13.3 nM. Compared with futibatinib, 8r exhibited superior inhibitory activity toward FGFR1V561M, FGFR2V564F, and FGFR2N549K mutations with IC50 values of 6.8, 0.7, and 0.8 nM, respectively. Moreover, 8r demonstrated favorable antitumor efficacy in an RT112/84 bladder cancer xenograft model. This work provides a promising macrocycle-based lead compound for the treatment of FGFR-driven cancers.

Macrocycles have gained significant attention in drug design owing to their distinctive structural and physicochemical features. Despite the abundance of available experimental data, there remains a need for a centralized resource to support macrocycle-based drug discovery. Here, we present Macrocycle-DB, the most extensive online database dedicated to macrocycles, featuring 45 525 compounds, including 76 approved drugs and 105 clinical candidates that target 2533 proteins. The database offers comprehensive structural information, experimental bioactivity data, and physicochemical properties for each macrocycle, along with co-crystal structures to visualize protein-ligand interactions. Additionally, Macrocycle-DB provides specialized descriptors, scaffold and linker details for synthetic macrocycles, and high-quality downloadable datasets to facilitate computational drug design. Macrocycle-DB is freely accessible at https://macro-db.dpbio.tech/ and https://macro-db.cn/.

FGFR alterations drive multiple cancers. While approved pan-FGFR inhibitors show clinical benefit, their use is limited by FGFR1/4-related toxicity and acquired resistance. We discovered that the selective FGFR2 inhibition described for the covalent inhibitor lirafugratinib is achieved through remote-site allosteric regulation of non-conserved residues FGFR1 V607 and FGFR2 L610, which differentially modulate the conformation of the FGFR1 and FGFR2 DFG motifs influencing covalent warhead accessibility. Guided by this insight, we designed the first reversible macrocyclic FGFR2 selective inhibitor 11c. It exhibits strong selectivity over FGFR1/3/4 (11-, 4.5-and 28.7-fold), potently inhibits FGFR2-driven tumor cells (IC50 = 2.5-15 nM), overcomes FGFR2 gatekeeper and molecular-brake mutations, and shows favorable oral pharmacokinetics and in vivo efficacy without inducing diarrhea or hyperphosphatemia. This work provides a promising macrocyclic lead for FGFR2-driven cancers.

HEC96719 is a small-molecule farnesoid X receptor (FXR) agonist with potential for treating nonalcoholic steatohepatitis (NASH). Herein, we report a robust and scalable kilogram-scale synthesis of compound 9, the central and synthetically challenging spirocyclic benzoxepane–pyridine core of HEC96719. This optimized process features: (i) a protecting-group-enabled nucleophilic acylation, (ii) an industrially practical cyclopropanation using NaOH under phase-transfer conditions, (iii) an ortho-phenol-assisted deoxygenation protocol to access a highly strained methylene intermediate, (iv) a copper-mediated intramolecular Ullmann etherification, and (v) a palladium-catalyzed carbonylation employing a safe CO surrogate to avoid high-pressure carbon monoxide. Collectively, this process enhances operator safety, improves overall efficiency and yield, avoids chromatographic purification, and has been successfully demonstrated on a multikilogram scale, underscoring its potential for industrial application.

Abnormal EGFR signaling is considered the cause of the occurrence and development of EGFR-addicted NSCLC. EGFR tyrosine kinase inhibitors (TKIs) have dramatically revolutionized the treatment landscape of NSCLC patients over the past two decades. Most patients with EGFR-activating mutations (such as deletions in exon 19 and the L858R substitution mutation) initially respond to the first-generation of EGFR TKIs, but acquired resistance will inevitably emerge during clinical treatment, most frequently owing to the secondary T790M mutation within the ATP binding site of the receptor. As second-generation TKIs have demonstrated limited clinical efficacy against EGFR T790M-mediated resistance, third-generation TKIs with improved selectivity have spurred intensive research efforts. However, tertiary EGFR C797S mutation-mediated resistance to third-generation of EGFR TKIs remains an unmet clinical need. Moreover, EGFR exon 20 insertion (exon20ins) mutations are insensitive to prior EGFR-TKIs and are associated with poor prognosis; consequently, novel inhibitors targeting these mutations are gaining traction. Moreover, tumor heterogeneity and complexity contribute to EGFR TKIs resistance through mechanisms such as bypass pathway activation, histological transformation, and emerging metabolic reprogramming. Therefore, the development of novel EGFR TKIs and the identification of potential combination therapies to overcome EGFR TKIs resistance have attracted significant attention. Here, we review the historical progress in the development of EGFR TKIs and “on-target”-mediated and EGFR-independent resistance mechanisms, aiming to summarize the current status of and provide future directions for EGFR TKI research.

ABSTRACT Bromodomain and extra‐terminal (BET) proteins are validated therapeutic targets for cancer, but clinical translation of pan‐BET inhibitors is limited by dose‐limiting toxicities from non‐selective inhibition of BD1/BD2 domains. Herein, we report the first‐ever domain‐selective covalent inhibitor, named i p AE1 , of BET BD2 domains by installing an epoxide warhead onto the scaffold of ABBV‐744. i p AE1 was shown to irreversibly modify Glu438 and His437 (to a lesser extent) located within the BRD4(2) binding pocket via a dual‐covalent mechanism, as evidenced by its chemical probe p AE1 . Furthermore, i p AE1 exhibited exceptional potency against BRD4(2) ( K d = 0.096 nM) with > 1900‐fold selectivity over BRD4(1), leading to potent and sustained antiproliferative activity in MV4‐11 cells (GI 50 = 1.5 nM). Subsequent live‐cell proteome‐wide profiling validated BRD4 as the primary cellular target of i p AE1 . Consistent with cellular activities, i p AE1 possessed a significantly enhanced antitumor efficacy in an MV4‐11 xenografted mouse model compared to ABBV‐744, presumably due to its on‐target covalent engagement in vivo. Our study thus establishes for the first time a novel targeted covalent inhibition (TCI) strategy that engages two weakly nucleophilic residues within a single bromodomain, an approach generalizable to other compounds targeting E/H, providing a highly selective platform for future development of next‐generation BET inhibitors.

Aberrant fibroblast growth factor receptor 3 (FGFR3) activation drives bladder carcinogenesis in humans, but currently approved pan-FGFR inhibitors lack FGFR3 isoform selectivity and fail to counter clinically acquired resistance mutations (e.g., FGFR3 V555M/L). Herein, we report the structure-based drug design of 4-(1-methyl-1H-indol-3-yl)pyrimidin-2-amine derivatives as the first covalent FGFR3 selective inhibitors. The representative compound 10s displayed high potency against FGFR3 (IC50 = 6.8 nM) and 5-60-fold selectivity over FGFR1/2/4. It was also effective against the common clinically acquired FGFR3V555M resistance mutation with an IC50 value of 19.2 nM. Furthermore, 10s exhibited strong antiproliferative effects in FGFR3-driven RT112/84 cells (IC50 = 9.2 nM). Structural characterization using MALDI-TOF-MS and X-ray crystallography confirmed covalent binding of 10s to FGFR3. Compound 10s also showed significant antitumor efficacy in the RT112/84 bladder cancer xenograft model, offering a promising compound to address both selectivity and resistance in FGFR3-targeted therapy.

Hydrophobic tags (HyTs) are promising bifunctional protein degraders that mimic misfolded proteins to trigger quality control-mediated target degradation, offering key advantages over traditional heterobifunctional degraders such as proteolysis-targeting chimeras (PROTACs). However, the scope and generality of this targeted protein degradation (TPD) strategy across the human kinome remain unexplored. In this study, we first addressed this gap by developing two general HyTs on the basis of a pan-kinase scaffold capable of large-scale proteome-wide studies of kinase degradation by using quantitative chemoproteomics. We subsequently mapped the degradable kinome landscape by using a HyT-based strategy, leading to the successful identification of 169 HyT-degradable human kinases. Leveraging this comprehensive kinase degradome map, we next rationally designed norbornene-based HyT degraders against ABL and AURKA, obtaining two optimized HyTs with potent degradation capabilities. We further elucidated the detailed mechanistic insight of these novel degraders. To overcome the inherent poor water solubility of HyTs, we next engineered HyT-loaded, tumor microenvironment (TME)-responsive self-assembled nanoparticles (NPs), which showed improved tumor accumulation and therapeutic efficacy in vivo. With key advantages including rapid target identification and a modular NP-based delivery platform, our work herein provides a comprehensive framework for future development of potential kinase therapeutics based on HyT degraders.

HER2 (ErbB2), a ligand-independent HER family member, regulates cell growth, differentiation, and survival. Its overexpression, gene amplification, and activating mutations are oncogenic drivers in multiple malignancies. The past two decades have witnessed transformative advances in HER2-targeted therapeutics, exemplified by tyrosine kinase inhibitors (TKIs), including first-generation reversible pan-HER (e.g., lapatinib), second-generation covalent pan-HER (neratinib, pyrotinib), and novel selective HER2 inhibitors (tucatinib, sevabertinib, zongertinib). Nonetheless, the extensive molecular heterogeneity in HER2 activation (e.g., dimerization, amplification) and mutation profiles (e.g., L755S, exon 20 insertions), combined with tumor-type-specific pathogenic mechanisms, poses significant challenges to precision oncology in clinical practice. Currently, numerous promising inhibitors in preclinical and clinical development hold potential for providing more effective treatment options. This review comprehensively summarizes recent advances in HER2-targeted TKIs and their emergent resistance mechanisms, further analyzing strategies to both mitigate off-target toxicity and overcome resistance through rational design of selective HER2 inhibitors. Collectively, these insights provide a roadmap for developing next-generation precision therapies in HER2-driven cancers.

Replica exchange (REX) is one of the most widely used enhanced sampling methodologies. However, its efficiency is often limited by the requirement for a large number of intermediate temperature replicas. Here, we present Generative Replica Exchange (GREX), an enhanced sampling approach that integrates deep generative models into the REX framework to eliminate the need for this temperature ladder. Drawing inspiration from reservoir replica exchange (res-REX), GREX utilizes trained normalizing flows to generate high-temperature configurations on demand and map them directly to the target distribution. To achieve this, we implement the potential energy as a constraint in GREX, eliminating the need for training data at the target temperature. This approach reduces production simulations to a single replica run at the target temperature while maintaining a Metropolis-filtered acceptance step for generated proposals. We validated GREX on benchmark systems of increasing complexity, highlighting its superior efficiency and practical applicability for molecular simulations. To further demonstrate its applicability to larger biomolecular systems, we applied GREX to bovine pancreatic trypsin inhibitor (BPTI), a 58-residue protein with slow conformational dynamics for which conventional REX and related methods face computational challenges.

Polo-like kinase 1 (PLK1), a key regulatory protein in cell cycle progression, is a promising target for cancer therapy. In this study, we efficiently identified potential PLK1 inhibitors through a rigorously validated computational-experimental pipeline that included antitumor kinase scaffold prioritization, structure-based docking, molecular dynamics (MD) simulations, and in vitro biochemical and cellular assays. Among the candidates, BDE30671203 and PB4767006058 demonstrated strong binding affinities to PLK1, with calculated binding free energies (ΔGB) of -55.328 ± 0.447 kcal/mol and -55.898 ± 2.035 kcal/mol, respectively. Both compounds exhibited significant antiproliferative effects across seven cancer cell lines, with IC50 values below 10 μM. Notably, BDE30671203 exhibited potent enzymatic inhibition of PLK1 (IC50 = 2.163 ± 0.401 nM). It also effectively induced G2/M phase arrest (88.45%) and apoptosis (17.77%) in HepG2 cells, concomitant with downregulation of key cell cycle regulators (CDC20, CDK1, etc.) and the antiapoptotic gene Bcl-2. Importantly, follow-up kinase selectivity profiling revealed that BDE30671203 is a highly selective PLK1 inhibitor, showing over 450-fold and 1200-fold selectivity against the closely related AURKA and AURKB kinases, respectively. Therefore, this study not only provides promising chemical starting points for PLK1-targeted drug discovery, but more significantly validates a robust screening strategy for kinase inhibitor discovery.

Castration-resistant prostate cancer (CRPC) remains a significant therapeutic challenge with limited effective treatment options. We identified retinoid X receptor γ (RXRγ) as a critical regulator of CRPC cell proliferation, highlighting it as a previously unrecognized and tractable target for therapeutic intervention. However, no RXRγ-selective modulators have been reported. Herein, we utilized the PROTAC approach to develop WCF-598 as a potent RXRγ degrader, which exhibits preferential degradation of RXRγ over RXRα and RXRβ isoforms. WCF-598 promoted efficient RXRγ degradation through the ubiquitin-proteasome system, leading to robust antiproliferative activity in CRPC models. In vivo, WCF-598 induced significant tumor regression in 22Rv1 xenograft-bearing mice without observable toxicity. Notably, WCF-598 also exhibited a secondary activity by degrading androgen receptor splice variant 7 (AR-V7), a clinically relevant driver of therapy resistance in CRPC. These results establish WCF-598 as a specific chemical probe for investigating the function of RXRγ in CRPC and potentially other RXRγ-related diseases.

Due to the lack of effective therapeutic targets for drug development, many diseases remain difficult to treat. To address this issue, phenotypic screening integrated with chemical proteomics has emerged as an efficient strategy to expand the scope of druggable targets. In this study, we constructed a covalent probe library based on diverse covalent kinase inhibitors and natural products containing an α, β-unsaturated ketone electrophilic warhead. Antiproliferation screening revealed that these probes exhibit potent anticancer activity against triple-negative breast cancer (TNBC) and human colon cancer. Subsequent proteomic studies identified a series of novel covalently ligandable targets, including ASNS, AKR1C1, DDX39B, and PRMT5. Functional validation demonstrated that DDX39B may represent a new therapeutic target for TNBC. Moreover, we identified a series of highly selective covalent probes targeting ARK1C1, PDIA1, and ALDH1A1, which could serve as valuable tools for detecting the expression and activity of these critical proteins.

A set of diarylidenyl piperidone-ligated platinum (IV) complexes 8a-8d with chemoimmunotherapy effects was designed and synthesized based on introduction of classic STAT3 inhibitors, diarylidenyl piperidones, into an oxaliplatin (OXA)-based skeleton. 3-(4,5)-Dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide (MTT) assay indicated that complexes 8a-8d exhibited obvious inhibition on T24, MDA-MB-231 and SW480 cell lines compared to OXA, with IC50 values in range of 4.96 +/- 0.14-21.1 +/- 0.35 mu M. SW480 xenograft nude mice assay demonstrated that complexes 8a (2 mg/kg and 4 mg/kg), 8b (4 mg/kg) and 8c (4 mg/kg) exhibited effective inhibition on this model with tumor inhibitory rates (TIR) of 46.06 %, 51.18 %, 48.82 % and 42.16 %, respectively, compared with OXA (2 mg/kg, TIR = 31.89 %/34.31 %) during 21-days treatment, while CT-26 xenograft BALB/C mice assay showed that complexes 8a (10 mg/kg), 8b (5 and 10 mg/kg), 8c (5 and 10 mg/kg), and 8d (5 and 10 mg/kg) exhibited effective inhibition of with TIR values of 56.95 %, 56.28 %, 78.02 %, 47.28 %, 63.80 %, 51.90 % and 70.65 %, respectively, compared with OXA (5 mg/kg, TIR = 69.28 %/67.53 %) during 13-days treatment. The pathology results in SW480 and CT-26 xenograft showed that complexes 8a-8d displayed limited toxicity in comparison with OXA. All these results indicated that complexes 8a-8c may be good chemoimmunotherapeutic agents with potent efficacy and safety profiles. Further mechanistic studies revealed that the representative complex 8b might exert its chemoimmunotherapeutic effect by inhibiting the expression and phosphorylation of STAT3, thus evoking CD4+ and CD8+ T lymphocyte immune responses and inducing ferroptosis and apoptosis.