Development of an Imidazopyridazine-Based MNK1/2 Inhibitor for the Treatment of Lymphoma.

The mitogen-activated protein kinase-interacting protein kinases (MNKs) are the only kinases known to phosphorylate eukaryotic translation initiation factor 4E (eIF4E) at Ser209, which plays a significant role in cap-dependent translation. Dysregulation of the MNK/eIF4E axis has been found in various solid tumors and hematological malignancies, including diffuse large B-cell lymphoma (DLBCL). Herein, structure-activity relationship studies and docking models determined that 20j exhibits excellent MNK1/2 inhibitory activity, stability, and hERG safety. 20j exhibits strong and broad antiproliferative activity against different cancer cell lines, especially GCB-DLBCL DOHH2. 20j suppresses the phosphorylation of eIF4E in Hela cells (IC50 = 90.5 nM) and downregulates the phosphorylation of eIF4E and 4E-BP1 in A549 cells. In vivo studies first revealed that ibrutinib enhances the antitumor effect of 20j without side effects in a DOHH2 xenograft model. This study provided a solid foundation for the future development of a MNK inhibitor for GCB-DLBCL treatment.

Design, Synthesis, and Biological Evaluation of Eukaryotic Initiation Factor 2B (eIF2B) Activators.

The eukaryotic initiation factor 2B (eIF2B) is a key regulator in protein-regulated signaling pathways and is closely related to the function of the central nervous system. Modulating eIF2B could retard the process of neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and vanishing white matter disease (VWM) et al. Here, we designed and synthesized a series of novel eIF2B activators containing oxadiazole fragments. The activating effects of compounds on eIF2B were investigated through testing the inhibition of ATF4 expression. Of all the targeted compounds, compounds 21 and 29 exhibited potent inhibition on ATF4 expression with IC50 values of 32.43 nM and 47.71 nM, respectively, which were stronger than that of ISRIB (IC50=67.90 nM). ATF4 mRNA assay showed that these two compounds could restore ATF4 mRNA to normal levels in thapsigargin-stimulated HeLa cells. Protein Translation assay showed that both compounds were effective in restoring protein synthesis. Compound potency assay showed that both compounds had similar potency to ISRIB with EC50 values of 5.844 and 37.70 nM. Cytotoxicity assay revealed that compounds 21 and 29 had low toxicity and were worth further investigation.

Design, synthesis and biological evaluation of MNK-PROTACs.

Mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) can regulate cellular mRNA translation by controlling the phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E), which plays an important role in tumor initiation, development, and metastasis. Although small-molecule MNK inhibitors have made significant breakthroughs in the treatment of various malignancies, their clinical application can be limited by drug resistance, target selectivity and other factors. The strategy of MNK-PROTACs which selectively degrades MNK kinases provides a new approach for developing small-molecule drugs for related diseases. In this study, DS33059, a small-molecule compound modified based on the ongoing clinical trials drug ETC-206, was chosen as the target protein ligand. A series of novel MNK-PROTACs were designed, synthesized and evaluated biological activity. Several compounds showed good inhibitory activities against MNK1/2. Besides, compounds exhibited moderate to excellent anti-proliferative activity in A549 and TMD-8 cells in vitro. In particular, compound II-5 significantly inhibited A549 (IC50 = 1.79 µM) and TMD-8 (IC50 = 1.07 µM) cells. The protein degradation assay showed that compound II-5 had good capability to degrade MNK1. The MNK-PROTACs strategy represents a new direction in treating tumors and deserves further exploration.

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 inhibitors: Research progress and prospects.

The cancer immunotherapies involved in cGAS-STING pathway have been made great progress in recent years. STING agonists exhibit broad-spectrum anti-tumor effects with strong immune response. As a negative regulator of the cGAS-STING pathway, ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) can hydrolyze extracellular 2', 3'-cGAMP and reduce extracellular 2', 3'-cGAMP concentration. ENPP1 has been validated to play important roles in diabetes, cancers, and cardiovascular disease and now become a promising target for tumor immunotherapy. Several ENPP1 inhibitors under development have shown good anti-tumor effects alone or in combination with other agents in clinical and preclinical researches. In this review, the biological profiles of ENPP1 were described, and the structures and the structure-activity relationships (SAR) of the known ENPP1 inhibitors were summarized. This review also provided the prospects and challenges in the development of ENPP1 inhibitors.

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.

Design, synthesis and biological evaluation of KRASG12C-PROTACs.

Several small-molecule covalent inhibitors of KRASG12C have made breakthrough progress in the treatment of KRAS mutant cancer. However, the clinical application of KRASG12C small-molecule inhibitors may be limited by adaptive resistance. Emerging PROTAC strategy can achieve complementary advantages with small molecule inhibitors and improve anti-tumor efficacy. Based on AMG-510, a series of novel KRASG12C-PROTACs were designed and synthesized. The protein degradation assay showed that PROTACs I-1, II-1, III-2 and IV-1 had binding and degradation ability to KRASG12C. III-2 and IV-1 showed potent inhibitory effect on downstream p-ERK and were more potent than AMG-510. Mechanistic studies demonstrated that PROTACs exerted degradation effects through the ubiquitin-proteasome pathway. Using cell lines sensitive to KRASG12C, anti-proliferative activities of compounds were assessed. PROTACs tested showed overall anti-proliferative activities. Besides,the structure-activity relationships (SARs) of KRASG12C-PROTACs were summarized. These results supported the use of the PROTAC strategy to degrade oncogene KRASG12C and provided clues for structural optimization of KRASG12C-PROTACs.

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.

Design, synthesis and biological evaluation of isoxazole-containing biphenyl derivatives as small-molecule inhibitors targeting the programmed cell death-1/ programmed cell death-ligand 1 immune checkpoint.

Monoclonal antibodies targeting the programmed cell death-1/ programmed cell death-ligand 1 (PD-1/PD-L1) immune checkpoint have achieved enormous success in cancer immunotherapy. But the antibody-based immunotherapies carry a number of unavoidable deficiencies such as poor pharmacokinetic properties and immunogenicity. Small-molecule PD-1/PD-L1 inhibitors offer the superiority of complementarity with monoclonal antibodies and represent an appealing alternative. A novel series of isoxazole-containing biphenyl compounds were designed, synthesized and evaluated as PD-1/PD-L1 inhibitors in this paper. The structure-activity relationship of the novel synthesized compounds indicated that the ring-closure strategy of introducing isoxazole could be employed and the 3-cyanobenzyl group was significant for the inhibitory activity against the PD-1/PD-L1 protein-protein interactions. Molecular docking studies were performed to help understand the binding mode of the small-molecule inhibitor with the PD-L1 dimer. In particular, compound II-12 was a promising anti-PD-1/PD-L1 inhibitor with the IC50 value of 23.0 nM, providing valuable information for future drug development.

Design, synthesis and biological evaluation of macrocyclic derivatives as TRK inhibitors.

Tropomyosin receptor kinases (TRKA, TRKB, TRKC) are transmembrane receptor tyrosine kinases, which are respectively encoded by NTRK1, NTRK2, and NTRK3 genes. Herein, we reported the design, synthesis and Structure-Activity Relationship (SAR) investigation of a series of macrocyclic derivatives as new TRK inhibitors. Among these compounds, compound 9e exhibited strong kinase inhibitory activity (TRKG595R IC50 = 13.1 nM) and significant antiproliferative activity in the Ba/F3-LMNA-NTRK1 cell line (IC50 = 0.080 µM) and compound 9e has shown a better inhibitory effect (IC50 = 0.646 µM) than control drug LOXO-101 in Ba/F3-LMNA-NTRK1-G595R cell line. These results indicate that compound 9e is a potential TRK inhibitor for further investigation.

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.

Discovery of 1,3,4-oxadiazole derivatives as potential antitumor agents inhibiting the programmed cell death-1/programmed cell death-ligand 1 interaction.

Inhibition of the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction by small-molecule inhibitors is emerging cancer immunotherapy. A series of novel 1,3,4-oxadiazole derivatives were designed, synthesized, and evaluated for their activities in vitro and vivo to find potent inhibitors of the PD-1/PD-L1 interaction. Among them, compoundⅡ-14exhibited outstanding biochemical activity, with an IC50of 0.0380 µM. Importantly, compound II-14, with a TGI value of 35.74 %, had more potent efficacy in a mouse tumor model compared to that in the control group. Surprisingly, when compound II-14 combined with 5-FU in a mouse tumor model having a TGI value of 64.59 %, which showed potential anti-tumor synergistic effects. Furthermore, immunohistochemistry analysis demonstrated thatcompound II-14 activated the immune microenvironment by promoting the infiltration of CD4+ T cells into tumor tissues. These results indicate that compound II-14 is a promising lead compound for further development of small-molecule PD-1/PD-L1 inhibitors for cancer therapy.

Design, synthesis and biological evaluation of imidazopyridazine derivatives containing isoquinoline group as potent MNK1/2 inhibitors.

Mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) are located at the meeting-point of ERK and p38 MAPK signaling pathways, which can phosphorylate eukaryotic translation initiation factor 4E (eIF4E) at the conserved serine 209 exclusively. MNKs modulate the translation of mRNA involved in tumor-associated signaling pathways. Consequently, selective inhibitors of MNK1/2 could reduce the level of phosphorylated eIF4E. Series of imidazopyrazines, imidazopyridazines and imidazopyridines derivatives were synthesized and evaluated as MNK1/2 inhibitors. Several compounds exhibited great inhibitory activity against MNK1/2 and selected compounds showed moderate to excellent anti-proliferative potency against diffuse large B-cell lymphoma (DLBCL) cell lines. In particular, compound II-5 (MNK1 IC50 = 2.3 nM; MNK2 IC50 = 3.4 nM) exhibited excellent enzymatic inhibitory potency and proved to be the most potent compound against TMD-8 and DOHH-2 cell lines with IC50 value of 0.3896 µM and 0.4092 µM respectively. These results demonstrated that compound II-5 could be considered as a potential MNK1/2 inhibitor for further investigation.

Discovery of 3,5-dimethylisoxazole derivatives as novel, potent inhibitors for bromodomain and extraterminal domain (BET) family.

Bromodomain and extra-terminal (BET) is a promising therapeutic target for various hematologic cancers. We used the BRD4 inhibitor compound 13 as a lead compound to develop a variety of compounds, and we introduced diverse groups into the position of the compound 13 orienting toward the ZA channel. A series of compounds (14-23, 38-41, 43, 47-49) bearing triazolopyridazine motif exhibited remarkable BRD4 protein inhibitory activities. Among them, compound 39 inhibited BRD4(BD1) protein with an IC50 of 0.003 µM was superior to lead compound 13. Meanwhile, compound 39 possess activity, IC50 = 2.1 µM, in antiproliferation activity against U266 cancer cells. On the other hand, compound 39 could arrest tumor cells into the G0/G1 phase and induce apoptosis, which was consistent with its results in inhibiting cell proliferation. Biological and biochemical data suggest that BRD4 protein might be a therapeutic target and that compound 39 is an excellent lead compound for further development.

Insight into the dichotomous regulation of STING activation in immunotherapy.

Cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) signaling pathway (cGAS-STING) is a hub linking innate immunity and adaptive immunity against pathogen infection by inducing the production of type I interferon (IFN-I). It also plays pivotal roles in modulating tumorigenesis by ensuring the antigen presentation, T cell priming, activation, and tumor regression. Given its antitumor immune properties, cGAS-STING has attracted intense focus and several STING agonists have entered into clinical trials. However, some problems still exist when activating STING for use in oncological indications. It is remarkable that multiple downstream cytokines such as TNF-α, IL-6 may lead to inflammatory disease and even tumor metastasis in practical trials. Besides, there is a synergistic effect when STING agonists are combined with other immunotherapies. In this review, we discussed the advanced understanding between STING and anti-tumor immunity, as well as a variety of promising clinical treatment strategies.

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.

Design, synthesis and biological evaluation of imidazolopyridone derivatives as novel BRD4 inhibitors.

Bromodomain containing protein 4 (BRD4) has been demonstrated to play critical roles in cellular proliferation and cell cycle progression. In this study, using the BRD4 inhibitor Fragment 9 as a lead compound, a series of imidazolopyridone derivatives were designed and tested for their inhibitory activity against BRD4 protein in vitro. Among them, HB100-A7 showed excellent BRD4(1) inhibitory activities with an IC50 value of 0.035 µM in amplified luminescent proximity homogeneous assay (Alphascreen). The result of MTT assay showed that HB100-A7 could suppress the proliferation of pancreatic cancer cells. In addition, flow cytometry further illustrated that HB100-A7 treatment resulted in G0/G1 phase arrest and promoted apoptosis of BxPc3 cells. Furthermore, the in vivo study found that HB100-A7 displayed significant tumor growth inhibition in a pancreatic mouse tumor model (Panc-02). Moreover, IHC staining suggested that HB100-A7 induce cell apoptosis in pancreatic cancer tumor tissue. Together, this study revealed, for the first time, HB100-A7 is a promising lead compound for further development as a new generation of small molecule inhibitors targeting the BRD4 protein.