Discovery of a novel BTK inhibitor S-016 and identification of a new strategy for the treatment of lymphomas including BTK inhibitor-resistant lymphomas.

Bruton's tyrosine kinase (BTK) has emerged as a therapeutic target for B-cell malignancies, which is substantiated by the efficacy of various irreversible or reversible BTK inhibitors. However, on-target BTK mutations facilitating evasion from BTK inhibition lead to resistance that limits the therapeutic efficacy of BTK inhibitors. In this study we employed structure-based drug design strategies based on established BTK inhibitors and yielded a series of BTK targeting compounds. Among them, compound S-016 bearing a unique tricyclic structure exhibited potent BTK kinase inhibitory activity with an IC50 value of 0.5 nM, comparable to a commercially available BTK inhibitor ibrutinib (IC50 = 0.4 nM). S-016, as a novel irreversible BTK inhibitor, displayed superior kinase selectivity compared to ibrutinib and significant therapeutic effects against B-cell lymphoma both in vitro and in vivo. Furthermore, we generated BTK inhibitor-resistant lymphoma cells harboring BTK C481F or A428D to explore strategies for overcoming resistance. Co-culture of these DLBCL cells with M0 macrophages led to the polarization of M0 macrophages toward the M2 phenotype, a process known to support tumor progression. Intriguingly, we demonstrated that SYHA1813, a compound targeting both VEGFR and CSF1R, effectively reshaped the tumor microenvironment (TME) and significantly overcame the acquired resistance to BTK inhibitors in both BTK-mutated and wild-type BTK DLBCL models by inhibiting angiogenesis and modulating macrophage polarization. Overall, this study not only promotes the development of new BTK inhibitors but also offers innovative treatment strategies for B-cell lymphomas, including those with BTK mutations.

Discovery of hematopoietic progenitor kinase 1 inhibitors using machine learning-based screening and free energy perturbation.

Hematopoietic progenitor kinase 1 (HPK1) is a key negative regulator of T-cell receptor (TCR) signaling and a promising target for cancer immunotherapy. The development of novel HPK1 inhibitors is challenging yet promising. In this study, we used a combination of machine learning (ML)-based virtual screening and free energy perturbation (FEP) calculations to identify novel HPK1 inhibitors. ML-based screening yielded 10 potent HPK1 inhibitors (IC50 < 1 µM). The FEP-guided modification of the in-house false-positive hit, DW21302, revealed that a single key atom change could trigger activity cliffs. The resulting DW21302-A was a potent HPK1 inhibitor (IC50 = 2.1 nM) and potently inhibited cellular HPK1 signaling and enhanced T-cell function. Molecular dynamics (MD) simulations and ADME predictions confirmed DW21302-A as candidate compound. This study provides new strategies and chemical scaffolds for HPK1 inhibitor development.Communicated by Ramaswamy H. Sarma.

Discovery of a 1,6-naphthyridin-4-one-based AXL inhibitor with improved pharmacokinetics and enhanced in vivo antitumor efficacy.

The receptor tyrosine kinase AXL has emerged as an attractive target in anticancer drug discovery. Herein, we described the discovery of a new series of 1,6-naphthyridin-4-one derivatives as potent AXL inhibitors. Starting from a low in vivo potency compound 9 which was previously reported by our group, we utilized structure-based drug design and scaffold hopping strategies to discover potent AXL inhibitors. The privileged compound 13c was a highly potent and orally bioavailable AXL inhibitor with an IC50 value of 3.2 ± 0.3 nM. Compound 13c exhibited significantly improved in vivo antitumor efficacy in AXL-driven tumor xenograft mice, causing tumor regression at well-tolerated dose, and demonstrated favorable pharmacokinetic properties (MRT = 16.5 h, AUC0-∞ = 59,815 ng h/mL) in Sprague-Dawley rats. These results suggest that 13c is a promising therapeutic candidate for AXL-targeting cancer treatment.

Preclinical and early clinical studies of a novel compound SYHA1813 that efficiently crosses the blood-brain barrier and exhibits potent activity against glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults and is poorly controlled. Previous studies have shown that both macrophages and angiogenesis play significant roles in GBM progression, and co-targeting of CSF1R and VEGFR is likely to be an effective strategy for GBM treatment. Therefore, this study developed a novel and selective inhibitor of CSF1R and VEGFR, SYHA1813, possessing potent antitumor activity against GBM. SYHA1813 inhibited VEGFR and CSF1R kinase activities with high potency and selectivity and thus blocked the cell viability of HUVECs and macrophages and exhibited anti-angiogenetic effects both in vitro and in vivo. SYHA1813 also displayed potent in vivo antitumor activity against GBM in immune-competent and immune-deficient mouse models, including temozolomide (TMZ) insensitive tumors. Notably, SYHA1813 could penetrate the blood-brain barrier (BBB) and prolong the survival time of mice bearing intracranial GBM xenografts. Moreover, SYHA1813 treatment resulted in a synergistic antitumor efficacy in combination with the PD-1 antibody. As a clinical proof of concept, SYHA1813 achieved confirmed responses in patients with recurrent GBM in an ongoing first-in-human phase I trial. The data of this study support the rationale for an ongoing phase I clinical study (ChiCTR2100045380).

Design, synthesis, and STING-agonistic activity of benzo[b]thiophene-2-carboxamide derivatives.

STING is an important immune-associated protein that localizes in the endoplasmic reticulum membrane. Upon being activated by its agonists, STING triggers the IRF and NF-κB pathways, which generates type I interferons and proinflammatory cytokines, and ultimately primes the innate immune responses to achieve valid antitumor efficacy. We designed and synthesized a series of benzo[b]thiophene-2-carboxamide derivatives. Through STING-agonistic activity evaluation, compounds 12d and 12e exhibited marginal human STING-activating activities. Western blot analysis demonstrated that both 12d and 12e treatment increased the phosphorylation of the downstream signaling molecules (TBK1 and IRF3) of STING. The proposed binding mode of 12d/12e and STING protein displayed that two canonical hydrogen bonds, a π-π stacking interaction, as well as a π-cation interaction formed between the agonist and the CDN-binding domain of STING protein.

Design, synthesis and pharmacological evaluation of 2,3-dihydrobenzofuran IRAK4 inhibitors for the treatment of diffuse large B-cell lymphoma.

Interleukin-1 receptor associated kinase 4 (IRAK4) is a critical mediator of MYD88 L265P-induced NF-κB activation, indicating it is a promising therapeutic target for diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of 2,3-dihydrobenzofuran IRAK4 inhibitors through structure-based drug design. The representative compound 22 exhibited strong IRAK4 inhibitory potency (IRAK4 IC50 = 8.7 nM), favorable kinase selectivity and high antiproliferative activity against the MYD88 L265P DLBCL cell line (OCI-LY10 IC50 = 0.248 µM). Compound 22 also exhibited the ability to inhibit the activation of IRAK4 signaling pathway and induce apoptosis in MYD88 L265P DLBCL cell line. In combination with Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, 22 showed enhanced apoptosis-inducing effect and antiproliferative potency. The most advanced compound 22 in this inhibitor series holds promise for further development into efficacious and selective IRAK4 inhibitors for the treatment of DLBCL.

Discovery of 10H-Benzo[b]pyrido[2,3-e][1,4]oxazine AXL Inhibitors via Structure-Based Drug Design Targeting c-Met Kinase.

Receptor tyrosine kinase AXL exerts pivotal roles in cancer cell survival, metastasis, and drug resistance. Pharmacologic or genetic targeting of the aberrant AXL signaling has proven preferable antitumor efficacies in both preclinical and clinical studies, which highlights AXL as an attractive antitumor drug target. By conformational restriction of the anilinopyrimidine 10e and systematic structure-activity relationship (SAR) exploration, we discovered 10H-benzo[b]pyrido[2,3-e][1,4]oxazine 16j as a potent and orally bioavailable AXL inhibitor. As a type II AXL inhibitor, compound 16j displayed about 15-fold selectivity for AXL over its highly homologous kinase c-Met. And it significantly blocked cellular AXL signaling, inhibited AXL-mediated cell proliferation, and impaired growth arrest-specific protein 6 (Gas6)/AXL-stimulated cell migration and invasion. Moreover, 16j exhibited significant antitumor efficacy in AXL-driven xenograft model at a well-tolerant dosage, causing tumor stasis or regression.

Structural insights into a shared mechanism of human STING activation by a potent agonist and an autoimmune disease-associated mutation.

Stimulator of interferon gene (STING) is increasingly exploited for the potential in cancer immunotherapy, yet its mechanism of activation remains not fully understood. Herein, we designed a novel STING agonist, designated as HB3089 that exhibits robust and durable anti-tumor activity in tumor models across various cancer types. Cryo-EM analysis reveals that HB3089-bound human STING has structural changes similar to that of the STING mutant V147L, a constitutively activated mutant identified in patients with STING-associated vasculopathy with onset in infancy (SAVI). Both structures highlight the conformational changes of the transmembrane domain (TMD), but without the 180°-rotation of the ligand binding domain (LBD) previously shown to be required for STING activation. Further structure-based functional analysis confirmed a new STING activation mode shared by the agonist and the SAVI-related mutation, in which the connector linking the LBD and the TMD senses the activation signal and controls the conformational changes of the LBD and the TMD for STING activation. Together, our findings lead to a new working model for STING activation and open a new avenue for the rationale design of STING-targeted therapies either for cancer or autoimmune disorders.

Discovery and structure - activity relationship exploration of pyrazolo[1,5-a]pyrimidine derivatives as potent FLT3-ITD inhibitors.

Internal tandem duplications of FLT3 (FLT3-ITD) occur in approximately 25% of all acute myeloid leukemia (AML) cases and confer a poor prognosis. Optimization of the screening hit 1 from our in-house compound library led to the discovery of a series of pyrazolo[1,5-a]pyrimidine derivatives as potent and selective FLT3-ITD inhibitors. Compounds 17 and 19 displayed potent FLT3-ITD activities both with IC50 values of 0.4 nM and excellent antiproliferative activities against AML cell lines. Especially, compounds 17 and 19 inhibited the quizartinib resistance- conferring mutations, FLT3D835Y, both with IC50 values of 0.3 nM. Moreover, western blot analysis indicated that compounds 17 and 19 potently inhibited the phosphorylation of FLT3 and attenuated downstream signaling in AML cells. These results indicated that pyrazolo[1,5-a]pyrimidine derivatives could be promising FLT3-ITD inhibitors for the treatment AML.

Structure-Guided Development of Small-Molecule PRC2 Inhibitors Targeting EZH2-EED Interaction.

Disruption of EZH2-embryonic ectoderm development (EED) protein-protein interaction (PPI) is a new promising cancer therapeutic strategy. We have previously reported the discovery of astemizole, a small-molecule inhibitor targeting the EZH2-EED PPI. Herein, we report the cocrystal structure of EED in complex with astemizole at 2.15 Å. The structure elucidates the detailed binding mode of astemizole to EED and provides a structure-guided design for the discovery of a novel EZH2-EED interaction inhibitor, DC-PRC2in-01, with an affinity Kd of 4.56 µM. DC-PRC2in-01 destabilizes the PRC2 complex, thereby leading to the degradation of PRC2 core proteins and the decrease of global H3K27me3 levels in cancer cells. The proliferation of PRC2-driven lymphomas cells is effectively inhibited, and the cell cycle is arrested in the G0/G1 phase. Together, these data demonstrate that DC-PRC2in-01 could be an effective chemical probe for investigating the PRC2-related physiology and pathology and providing a promising chemical scaffold for further development.

Discovery of pyrrolo[2,3-d]pyrimidine derivatives as potent Axl inhibitors: Design, synthesis and biological evaluation.

Axl has emerged as an attractive target for cancer therapy due to its strong correlation with tumor growth, metastasis, poor survival, and drug resistance. Herein, we report the design, synthesis and structure-activity relationship (SAR) investigation of a series of pyrrolo[2,3-d]pyrimidine derivatives as new Axl inhibitors. Among them, the most promising compound 13b showed high enzymatic and cellular Axl potencies. Furthermore, 13b possessed preferable pharmacokinetic properties and displayed promising therapeutic effect in BaF3/TEL-Axl xenograft tumor model. Compound 13b may serve as a lead compound for new antitumor drug discovery.

Discovery of a Pyrimidinedione Derivative as a Potent and Orally Bioavailable Axl Inhibitor.

The receptor tyrosine kinase Axl plays important roles in promoting cancer progression, metastasis, and drug resistance and has been identified as a promising target for anticancer therapeutics. We used molecular modeling-assisted structural optimization starting with the low micromolar potency compound 9 to discover compound 13c, a highly potent and orally bioavailable Axl inhibitor. Selectivity profiling showed that 13c could inhibit the well-known oncogenic kinase Met with equal potency to its inhibition of Axl superfamily kinases. Compound 13c significantly inhibited cellular Axl and Met signaling, suppressed Axl- and Met-driven cell proliferation, and restrained Gas6/Axl-mediated cancer cell migration or invasion. Furthermore, 13c exhibited significant antitumor efficacy in Axl-driven and Met-driven tumor xenograft models, causing tumor stasis or regression at well-tolerated doses. All these favorable data make 13c a promising therapeutic candidate for cancer treatment.

Design, Synthesis, and Biological Evaluation of IRAK4-Targeting PROTACs.

Interleukin-1 receptor associated kinase 4 (IRAK4) is a promising therapeutic target for diffuse large B-cell lymphoma driven by MYD88 L265P mutant, acting both as a kinase and a scaffolding protein for downstream signaling molecules. While previous efforts to modulate IRAK4 activity with kinase inhibitors alone displayed moderate efficacy, protein degradation may offer a solution to blocking both IRAK4 kinase activity and scaffolding capabilities. To this end, the potent IRAK4 degrader 9 was discovered, and it effectively inhibited the activation of downstream NF-κB signaling and outperformed the parent compound 1. In addition, compound 9 displayed a substantial advantage in reduction of the viability of OCI-LY10 and TMD8 cells over the parent compound 1. These results underline the potential that eliminating both the kinase and scaffolding functions of IRAK4 may result in superior and broader efficacy than inhibiting the kinase activity alone.

Synthesis of triazolotriazine derivatives as c-Met inhibitors.

Receptor tyrosine kinase c-Met is an important antitumor drug target. Triazolotriazine analogues 2-10 were prepared efficiently and evaluated the enzymatic and cellular c-Met activities. Brief structure-activity relationships of triazolotriazine core and CF2-quinoline part were investigated, leading to the discovery of compound 8 with nanomolar enzymatic c-Met activity, and subnanomolar MKN45 and EBC-1 cellular potencies. The proposed binding model of 8 and c-Met unraveled that two canonical hydrogen bonds and a π-π stacking interaction formed between the inhibitor and the ATP binding site of c-Met kinase domain, which accounted for its potent c-Met activities.

DW14383 is an irreversible pan-FGFR inhibitor that suppresses FGFR-dependent tumor growth in vitro and in vivo.

Fibroblast growth factor receptor (FGFR) is a promising anticancer target. Currently, most FGFR inhibitors lack sufficient selectivity and have nonnegligible activity against kinase insert domain receptor (KDR), limiting their feasibility due to the serious side effects. Notably, compensatory activation occurs among FGFR1-4, suggesting the urgent need to develop selective pan-FGFR1-4 inhibitors. Here, we explored the antitumor activity of DW14383, a novel irreversible FGFR1-4 inhibitor. DW14383 exhibited equivalently high potent inhibition against FGFR1, 2, 3 and 4, with IC50 values of less than 0.3, 1.1, less than 0.3, and 0.5 nmol/L, respectively. It is a selective FGFR inhibitor, exhibiting more than 1100-fold selectivity for FGFR1 over recombinant KDR, making it one of the most selective FGFR inhibitors over KDR described to date. Furthermore, DW14383 significantly inhibited cellular FGFR1-4 signaling, inducing G1/S cell cycle arrest, which in turn antagonized FGFR-dependent tumor cell proliferation. In contrast, DW14383 had no obvious antiproliferative effect against cancer cell lines without FGFR aberration, further confirming its selectivity against FGFR. In representative FGFR-dependent xenograft models, DW14383 oral administration substantially suppressed tumor growth by simultaneously inhibiting tumor proliferation and angiogenesis via inhibiting FGFR signaling. In summary, DW14383 is a promising selective irreversible pan-FGFR inhibitor with pan-tumor spectrum potential in FGFR1-4 aberrant cancers, which has the potential to overcome compensatory activation among FGFR1-4.

DW14006 as a Direct AMPKα Activator Ameliorates Diabetic Peripheral Neuropathy in Mice.

Diabetic peripheral neuropathy (DPN) is a long-term complication of diabetes with a complicated pathogenesis. AMP-activated protein kinase (AMPK) senses oxidative stress, and mitochondrial function plays a central role in the regulation of DPN. Here, we reported that DW14006 (2-[3-(7-chloro-6-[2'-hydroxy-(1,1'-biphenyl)-4-yl]-2-oxo-1,2-dihydroquinolin-3-yl)phenyl]acetic acid) as a direct AMPKα activator efficiently ameliorated DPN in both streptozotocin (STZ)-induced type 1 and BKS db/db type 2 diabetic mice. DW14006 administration highly enhanced neurite outgrowth of dorsal root ganglion neurons and improved neurological function in diabetic mice. The underlying mechanisms have been intensively investigated. DW14006 treatment improved mitochondrial bioenergetics profiles and restrained oxidative stress and inflammation in diabetic mice by targeting AMPKα, which has been verified by assay against the STZ-induced diabetic mice injected with adeno-associated virus 8-AMPKα-RNAi. To our knowledge, our work might be the first report on the amelioration of the direct AMPKα activator on DPN by counteracting multiple risk factors including mitochondrial dysfunction, oxidative stress, and inflammation, and DW14006 has been highlighted as a potential leading compound in the treatment of DPN.

Design and synthesis of Imidazo[1,2-b]pyridazine IRAK4 inhibitors for the treatment of mutant MYD88 L265P diffuse large B-cell lymphoma.

Harboring MYD88 L265P mutation triggers tumors growth through the activation of NF-κB by interleukin-1 receptor associated kinase 4 (IRAK4) in diffuse large B-cell lymphoma (DLBCL), highlighting IRAK4 as a therapeutic target for tumors driven by aberrant MYD88 signaling. Herein, we report the design, synthesis, and structure-activity relationships of imidazo[1,2-b]pyridazines as potent IRAK4 inhibitors. The representative compound 5 exhibited excellent IRAK4 potency (IRAK4 IC50 = 1.3 nM) and favorable kinase selectivity profile. It demonstrated cellular selectivity for activated B cell-like (ABC) subtype DLBCL with MYD88 L265P mutation in cytotoxicity assay. The kinase inhibitory efficiency of compound 5 was further validated by Western blot analysis of phosphorylation of IRAK4 and downstream signaling in OCI-LY10 and TMD8 cells. Besides, combination of compound 5 and BTK inhibitor ibrutinib synergistically reduced the viability of TMD8 cells. These results indicated that compound 5 could be a promising IRAK4 inhibitor for the treatment of mutant MYD88 DLBCL.

Rational Design, synthesis and biological evaluation of novel triazole derivatives as potent and selective PRMT5 inhibitors with antitumor activity.

Protein arginine methyltransferase 5 (PRMT5) is responsible for the mono-methylation and symmetric dimethylation of arginine, and its expression level and methyl transferring activity have been demonstrated to have a close relationship with tumorigenesis, development and poor clinical outcomes of human cancers. Two PRMT5 small molecule inhibitors (GSK3326595 and JNJ-64619178) have been put forward into clinical trials. Here, we describe the design, synthesis and biological evaluation of a series of novel, potent and selective PRMT5 inhibitors with antiproliferative activity against Z-138 mantle cell lymphoma cell line. Among them, compound C_4 exhibited the highest potency with enzymatic and cellular level IC50 values of 0.72 and 2.6 µM, respectively, and displayed more than 270-fold selectivity toward PRMT5 over several other isoenzymes (PRMT1, PRMT4 and PRMT6). Besides, C_4 demonstrated obvious cell apoptotic effect while reduced the cellular symmetric arginine dimethylation levels of SmD3 protein. The potency, small size, and synthetic accessibility of this compound class provide promising hit scaffold for medicinal chemists to further explore this series of PRMT5 inhibitors.

Targeting PRMT5 Activity Inhibits the Malignancy of Hepatocellular Carcinoma by Promoting the Transcription of HNF4α.

Background: Liver cancer stem cells (LCSCs) are responsible for the initiation, progression and chemoresistance of liver cancer. However, no agent targeting LCSC is available in the clinic to date. Here, we investigated the effects of targeting protein arginine methyltransferase 5 (PRMT5), an epigenetic regulator, on LCSCs and HCC using a novel PRMT5 inhibitor DW14800. Methods: Tumor spheroid formation culture was used to enrich LCSCs and assess their self-renewal capability. Human alpha-1-antitrypsin (A1AT) ELISA, acetylated low-density lipoprotein (ac-LDL) uptake, periodic acid-Schiff (PAS) reactions and senescence associated ß-galactosidase (SA-ß-gal) activity assays were performed to examine the differentiation status of HCC cells. The effects of DW14800 on HCC malignancy were assessed in HCC cell lines and on an HCC xenograft model in mice. Chromatin immunoprecipitation was applied to clarify the transcriptional regulation of HNF4α by PRMT5-mediated Histone H4 arginine-3 symmetrical dimethylation (H4R3me2s). Results: Quantitative real-time PCR revealed that the expression of PRMT5 was upregulated in LCSCs. DW14800 specifically decreased the symmetrical dimethylation of arginine residues in HCC cells. Treatment of DW14800 suppressed the self-renewal capacity of LCSCs while re-establishing hepatocyte-specific characteristics in HCC cells. DW14800 displayed antitumor effects in HCC cells in vitro and in xenograft HCC in vivo. Importantly, ChIP assay showed that PRMT5 and H4R3me2s bound to the promoter region of HNF4α gene, and DW14800 increased the expression of HNF4α via reducing the H4R3me2s levels and enhancing the transcription of HNF4α. Conclusions: Our data revealed the significance of targeting PRMT5 activity in LCSC elimination and HCC differentiation, and proposed that DW14800 may represent a promising therapeutic agent for HCC in the clinic.