Lili Zhu

BACKGROUND: Given the persistently high morbidity and mortality of heart failure (HF), targeting myocardial remodeling, particularly pathological hypertrophy and fibrosis, has become a major therapeutic priority. RhoA (Ras homolog gene family member A), a small GTPase governing cytoskeletal reorganization and cell migration, plays a pivotal role in this process. However, RhoA has long been considered undruggable because of its high-affinity binding to GDP/GTP and the absence of well-defined druggable pockets. METHODS: Structural analyses comparing RhoA-GTP and RhoA-GDP conformations, combined with surface plasmon resonance-based screening, were used to identify a RhoA inhibitor. The underlying mechanism was validated in cultured cells and 3-dimensional myocardial tissue models. Therapeutic efficacy was assessed across multiple species of HF models and supported by multiomics analyses linking RhoA activation to human HF. Key findings were further confirmed by multiplex immunohistochemistry and pulldown assays in human heart specimens. RESULTS: We identified an unrecognized cryptic pocket adjacent to GDP in RhoA. A natural product, AH001, selectively occupied this pocket and interacted with GDP, thereby stabilizing the interaction between RhoA and its endogenous inhibitor, RhoGDIα (Rho GDP-dissociation inhibitor 1). AH001 suppressed downstream signaling by reducing MRTFA (myocardin-related transcription factor A) nuclear translocation and downregulating fibrosis- and hypertrophy-related proteins. Moreover, AH001 disrupted pathological crosstalk between Mrtfa + cardiomyocytes and fibroblasts. Consequently, AH001 markedly attenuated myocardial remodeling in multiple HF animal models, as well as in 3-dimensional myocardial tissue models. CONCLUSIONS: These findings establish pharmacological inhibition of RhoA activation as a viable strategy to mitigate myocardial remodeling in HF and provide a conceptual framework for developing reversible inhibitors against previously undruggable small GTPases.

Sphingosine-1-phosphate receptor 1 is a clinically validated therapeutic target for ulcerative colitis. Substantial clinical evidence indicates that highly selective S1PR3-sparing S1PR1 agonists exhibit improved safety, making selectivity enhancement crucial in drug development. We report the rational design and synthesis of 3-aryl-5-bisaryl-1,2,4-oxadiazole derivatives as novel S1PR1 agonists. Among them, TYY-31 displayed single-digit picomolar activity against S1PR1 (EC₅₀ = 1.13 pM) with minimal activity against S1PR2/3/5 (EC₅₀ > 10 μM) and over 30,000-fold selectivity for S1PR1 compared to S1PR4. TYY-31 efficiently induced S1PR1 internalization (EC₅₀ = 0.73 nM) and blocked receptor recycling. Moreover, TYY-31 demonstrates outstanding pharmacokinetic properties. In vivo, TYY-31 effectively ameliorating dextran sulfate sodium-induced colitis in mice at a dose of 0.3 mg/kg, comparable to Ozanimod (1 mg/kg). Additionally, TYY-31 demonstrated excellent cardiac safety characteristics in zebrafish-based assays. These findings highlight TYY-31 as a highly selective and potent S1PR1 agonist with promising potential as a candidate drug for UC treatment.

Macrocyclic compounds hold great promise as therapeutic agents. However, their structural optimization remains constrained by the limited availability of bioactive candidates, which in turn hampers the systematic exploration of structure-activity relationships. Here we introduce CycleGPT, a generative chemical language model designed specifically to address these challenges. CycleGPT is characterized by a progressive transfer learning paradigm that incrementally transfers knowledge from pre-trained chemical language models to specialized macrocycle generation, thereby overcoming the data shortage issue. Meanwhile, it adopts an innovative probabilistic sampling strategy that effectively improves the structural novelty of generated macrocycles while ensuring domain-specific adaptability. In a prospective drug design based on CycleGPT and a JAK2 activity prediction model, we successfully developed a new JAK2 drug candidate with a good selectivity profile (inhibiting 17 wild-type kinases) and promising potential for treating polycythemia in vivo, demonstrating the practicality of deep learning methods in macrocyclic drug design.

ANO1, a calcium-activated chloride channel, is a newly reported therapeutic target for osteoporosis. Tamsulosin (Tam), an approved α₁-AR antagonist, was previously discovered to be a novel ANO1 allosteric inhibitor. Here, a series of Tam derivatives were designed and synthesized with the aim of developing selective ANO1 inhibitors for osteoporosis treatment. Among them, compound 10 exhibited the best overall activities. The potency of compound 10 against ANO1 was comparable to that of Tam (IC₅₀: 4.57 vs 7.22 μM), but its selectivity over α₁-AR was significantly improved. Compared with Tam, the potency of compound 10 against α_1A-, α_1B-, and α_1D-AR decreased by 118-fold, 642-fold, and 10,000-fold, respectively. In addition, compound 10 exhibited significant inhibitory effects on osteoclast differentiation and function. In the OVX (ovariectomy) mice, compound 10 effectively prevented OVX-induced bone loss. Collectively, these findings highlighted the therapeutic potential of 10 as a novel lead compound for antiosteoporosis by targeting ANO1.

Janus kinase 2 with the activating mutation V617F is a potential target for the treatment of myeloproliferative neoplasms. Current JAK2 inhibitors often suffer from multiple pharmacological limitations such as limited selectivity and rapid metabolic clearance. In this study, we describe the design, synthesis, and biological evaluation of a series of macrocyclic 2-amino-4-phenylaminopyrimidine derivatives as JAK2 inhibitors. Among them, compound 11 showed potent inhibitory activity against JAK2 kinase (IC₅₀ = 54.70 nM), as well as JAK2-dependent HEL and SET-2 cells, with IC₅₀ values of 0.57 μM and 1.07 μM, respectively. Besides, compound 11 downregulated the expression of p-STAT3 and p-STAT5 in HEL cells. Importantly, compound 11 also exhibited good bioavailability (F = 54.8%) and potent inhibition of rhEPO-induced extramedullary erythropoiesis and polycythemia vera in vivo. In summary, the macrocyclic design introduces structural innovation and provides a more efficient and selective strategy for JAK2 inhibition.

Macrocyclic compounds have shown great potential as therapeutic agents due to their distinctive structural and pharmacological properties. However, structural optimization of macrocycles—a critical step in the rational design of macrocyclic drugs—remains constrained by the limited availability of bioactive candidates, which in turn hampers the systematic exploration of structure-activity relationships. In this paper, we introduce CycleGPT, a generative chemical language model designed specifically to address these challenges. CycleGPT is characterized by a progressive transfer learning paradigm to incrementally transfer knowledge from pre-trained chemical language models to specialized macrocycle generation to overcome the data shortage issue; in the meantime, it adopts an innovative probabilistic sampling strategy that effectively improves the structural novelty of generated macrocycles while ensuring domain-specific adaptability. In a prospective drug design based on CycleGPT and our custom JAK2 activity predictive model, three synthesized macrocycles exhibited high inhibitory activity against JAK2, with the most potent compound 2 showing an IC₅₀ of 1.17 nM. Moreover, compound 2 exhibited a favorable selectivity profile and demonstrated in vivo efficacy in polycythemia mice model. These novel therapeutic candidates demonstrate the significant potential of CycleGPT for advancing macrocyclic drug discovery.

JAK2 is a promising target for treating myeloproliferative neoplasms (MPNs). However, existing JAK2 inhibitors cannot fully cure these diseases and may induce resistance with prolonged use. Here, we report the design, synthesis, and biological evaluation of a series of highly potent JAK2 degraders based on our previously developed inhibitor WWQ-131. The optimal compound 10i demonstrates a high degradation rate (DR) against JAK2 in SET-2 cells carrying the JAK2 V617F mutation, achieving a DR of 91.32% at 5 μM and a DC₅₀ of 27.35 ± 5.36 nM. Moreover, 10i exhibits more potent antiproliferative activity against SET-2 cells than fedratinib and its parent inhibitor WWQ-131. Mechanistic studies reveal that 10i degrades JAK2 through the ubiquitin-protease pathway. Importantly, 10i suppresses rhEPO-mediated polycythemia and splenomegaly in mice by degrading JAK2 and interfering with the JAK2-STAT signaling pathway. Taken together, the results of this study reveal a promising JAK2 PROTAC degrader for the treatment of MPNs.

TMEM16A, a key calcium-activated chloride channel, is crucial for many physiological and pathological processes such as cancer, hypertension, and osteoporosis, etc. However, the regulatory mechanism of TMEM16A is poorly understood, limiting the discovery of effective modulators. Here, we unveil an allosteric gating mechanism by presenting a high-resolution cryo-EM structure of TMEM16A in complex with a channel inhibitor that we identified, Tamsulosin, which is resolved at 2.93 Å. Tamsulosin wedges itself into a pocket within the extracellular domain of TMEM16A, surrounded by α1-α2, α5-α6, and α9-α10 loops. This binding stabilizes a transient preopen conformation of TMEM16A, which is activated by Ca ²⁺ ions while still preserving a closed pore to prevent Cl ⁻ permeation. Validation of this binding site through computational, electrophysiological, and functional experiments, along with site-directed mutagenesis, confirmed the pivotal roles of the pocket-lining residues R605 and E624 on α5-α6 loop in modulating Tamsulosin binding and pore activity. Tamsulosin induces significant positional shifts in extracellular loops, particularly the α5-α6 loop, which moves toward the extracellular exit of the pore, leading to noticeable structural rearrangements in pore-lining helices. The hinges induced by P595 in α5 and G711 in α7 introduce flexibility to the transmembrane helices, orienting Y593 to collaborate with I641 in effectively gating the preopening pore. Notably, Tamsulosin demonstrates significant antiosteoporotic effects by inhibiting TMEM16A, suggesting potential for its repurposing in new therapeutic indications. Our study not only enhances our understanding of the gating mechanism of TMEM16A inhibition but also facilitates structure-based drug design targeting TMEM16A.