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.

LS-106, a novel EGFR inhibitor targeting C797S, exhibits antitumor activities both in vitro and in vivo.

With the wide clinical use of the third-generation epidermal growth factor receptor (EGFR) inhibitor osimertinib for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC), acquired resistance caused by EGFR C797S tertiary mutation has become a concern. Therefore, fourth-generation EGFR inhibitors that could overcome this mutation have gained increasing attention in recent years. Here, we identified LS-106 as a novel EGFR inhibitor against C797S mutation and evaluated its antitumor activity both in vitro and in vivo. In cell-free assay, LS-106 potently inhibited the kinase activities of EGFR19del/T790M/C797S and EGFRL858R/T790M/C797S with IC50 values of 2.4 nmol/L and 3.1 nmol/L, respectively, which was more potent than osimertinib. Meanwhile, LS-106 exhibited comparable kinase inhibitory effect to osimertinib on EGFRL858R/T790M and wild-type EGFR. Results from cellular experiments demonstrated that LS-106 potently blocked the phosphorylation of EGFR C797S triple mutations in the constructed BaF3 cells that highly expressed EGFR19del/T790M/C797S or EGFRL858R/T790M/C797S , and thus inhibited the proliferation of these cells. We also constructed tumor cells harboring EGFR19del/T790M/C797S (named PC-9-OR cells) using the CRISPR/Cas9 system and found that LS-106 markedly suppressed the activation of EGFR19del/T790M/C797S and the proliferation of PC-9-OR cells. Moreover, cells harboring EGFR19del/T790M/C797S underwent remarkable apoptosis upon LS-106 treatment. In vivo experiments further demonstrated that oral administration of LS-106 caused significant tumor regression in a PC-9-OR xenograft model, with a tumor growth inhibition rate (TGI) of 83.5% and 136.6% at doses of 30 and 60 mg/kg, respectively. Taken together, we identified LS-106 as a novel fourth-generation EGFR inhibitor against C797S mutation and confirmed its preclinical antitumor effects in C797S-triple-mutant tumor models.

Discovery of a novel third-generation EGFR inhibitor and identification of a potential combination strategy to overcome resistance.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations initially respond to first-generation EGFR inhibitors; however, the efficacy of these drugs is limited by acquired resistance driven by the EGFR T790M mutation. The discovery of third-generation EGFR inhibitors overcoming EGFR T790M and their new resistance mechanisms have attracted much attention. METHODS: We examined the antitumor activities and potential resistance mechanism of a novel EGFR third-generation inhibitor in vitro and in vivo using ELISA, SRB assay, immunoblotting, flow cytometric analysis, kinase array, qRT-PCR and tumor xenograft models. The clinical effect on a patient was evaluated by computed tomography scan. RESULTS: We identified compound ASK120067 as a novel inhibitor of EGFR T790M, with selectivity over EGFR WT. ASK120067 exhibited potent anti-proliferation activity in tumor cells harboring EGFR T790M (NCI-H1975) and sensitizing mutations (PC-9 and HCC827) while showed moderate or weak inhibition in cells expressing EGFR WT. Oral administration of ASK120067 induced tumor regression in NSCLC xenograft models and in a PDX model harboring EGFR T790M. The treatment of one patient with advanced EGFR T790M-positive NSCLC was described as proof of principle. Moreover, we found that hyperphosphorylation of Ack1 and the subsequent activation of antiapoptotic signaling via the AKT pathway contributed to ASK120067 resistance. Concomitant targeting of EGFR and Ack1 effectively overrode the acquired resistance of ASK120067 both in vitro and in vivo. CONCLUSIONS: Our results idenfity ASK120067 as a promising third-generation EGFR inhibitor and reveal for the first time that Ack1 activation as a novel resistance mechanism to EGFR inhibitors that guide to potential combination strategy.

Design, synthesis and biological evaluation of potent EGFR kinase inhibitors against 19D/T790M/C797S mutation.

The efficacy of EGFR inhibitors is frequently affected by acquired resistance. EGFR19D/T790M/C797S mutation is one of the primary reasons for the emergence of resistance after treatment with the third-generation EGFR inhibitors such as AZD9291, CO1686 and Olmutinib. To overcome the resistance mutation 19D/T790M/C797S, we designed and prepared a series of indole derivatives with the terminal hydroxyl of alkyl chain to increase extra interaction with the Asp855 in the conservative DFG site. Activity evaluation, structure-activity relationship and docking analysis were also carried out. Among them, compound 12e displayed significant inhibitory activity against EGFR19D/T790M/C797S (IC50 = 15.3 nM) and good selectivity over EGFR WT (IC50 > 1000 nM), L858R/T790M (IC50, 156.6 nM) and L858R/T790M/C797S (IC50, 218.3 nM) respectively. Furthermore, 12e exhibited good growth inhibition activity, induced G1 phase cell cycle arrest and apoptosis in BaF3/EGFR19D/T790M/C797S cells by suppressing EGFR phosphorylation signaling pathway. In all, our study might provide a novel structural design method and lay the solid foundation for the development of the 4th generation EGFR19D/T790M/C797S inhibitors.

Discovery and biological evaluation of N-(3-(7-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-4-methyl-2-oxo-2H-pyrimido[4,5-d][1,3]oxazin-1(4H)-yl)phenyl)acrylamide as potent Bruton's tyrosine kinase inhibitors.

Bruton's tyrosine kinase (BTK) is a key component of the B cell receptor (BCR) signaling pathway and plays a crucial role in B cell malignancies and autoimmune disorders; thus, it is an attractive target for the treatment of B cell related diseases. Here, we evaluated the BTK inhibitory activity of a series of pyrimido[4,5-d][1,3]oxazin-2-one derivatives. Combining this evaluation with structure-activity relationship (SAR) analysis, we found that compound 2 exhibited potent BTK kinase inhibitory activity, with an IC50 of 7 nM. This derivative markedly inhibited BTK activation in TMD8 B cell lymphoma cells and thus inhibited the in vitro growth of the cells. Further studies revealed that compound 2 dose dependently arrested TMD8 cells at G1 phase, accompanied by decreased levels of Rb, phosphorylated Rb, and cyclin D1. Moreover, following treatment with compound 2, TMD8 cells underwent apoptosis associated with PARP and caspase 3 cleavage. Interestingly, the results of the kinase activity assay on a small panel of 35 kinases showed that the kinase selectivity of compound 2 was superior to that of the first-generation inhibitor ibrutinib, suggesting that compound 2 could be a second-generation inhibitor of BTK. In conclusion, we identified a potent and highly selective BTK inhibitor worthy of further development.

Discovery and Biological evaluation of pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione derivatives as potent Bruton's tyrosine kinase inhibitors.

Aberrant activation of B cell receptor (BCR) signal transduction cascade contributes to the propagation and maintenance of B cell malignancies. The discovery of mall molecules with high potency and selectivity against Bruton's tyrosine kinase (BTK), a key signaling molecule in this cascade, is particularly urgent in modern treatment regimens. Herein, a series of pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione derivatives were reported as potent BTK inhibitors. Compounds 17 and 18 displayed strong BTK inhibitory activities in the enzymatic inhibition assay, with the IC50 values of 1.2 and 0.8 nM, respectively, which were comparable to that of ibrutinib (IC50 = 0.6 nM). Additionally, compound 17 had a more selective profile over EGFR than ibrutinib. According to the putative binding poses, the molecular basis of this series of compounds with respect to potency against BTK and selectivity over EGFR was elucidated. In further experiments at cellular level, compounds 17 and 18 significantly inhibited the proliferation of Ramos and TMD8 cells. And they arrested 75.4% and 75.2% of TMD8 cells in G1 phase, respectively, at the concentration of 1 µM.

Branched-chain amino acid transaminase 1 confers EGFR-TKI resistance through epigenetic glycolytic activation.

Third-generation EGFR tyrosine kinase inhibitors (TKIs), exemplified by osimertinib, have demonstrated promising clinical efficacy in the treatment of non-small cell lung cancer (NSCLC). Our previous work has identified ASK120067 as a novel third-generation EGFR TKI with remarkable antitumor effects that has undergone New Drug Application (NDA) submission in China. Despite substantial progress, acquired resistance to EGFR-TKIs remains a significant challenge, impeding the long-term effectiveness of therapeutic approaches. In this study, we conducted a comprehensive investigation utilizing high-throughput proteomics analysis on established TKI-resistant tumor models, and found a notable upregulation of branched-chain amino acid transaminase 1 (BCAT1) expression in both osimertinib- and ASK120067-resistant tumors compared with the parental TKI-sensitive NSCLC tumors. Genetic depletion or pharmacological inhibition of BCAT1 impaired the growth of resistant cells and partially re-sensitized tumor cells to EGFR TKIs. Mechanistically, upregulated BCAT1 in resistant cells reprogrammed branched-chain amino acid (BCAA) metabolism and promoted alpha ketoglutarate (α-KG)-dependent demethylation of lysine 27 on histone H3 (H3K27) and subsequent transcriptional derepression of glycolysis-related genes, thereby enhancing glycolysis and promoting tumor progression. Moreover, we identified WQQ-345 as a novel BCAT1 inhibitor exhibiting antitumor activity both in vitro and in vivo against TKI-resistant lung cancer with high BCAT1 expression. In summary, our study highlighted the crucial role of BCAT1 in mediating resistance to third-generation EGFR-TKIs through epigenetic activation of glycolysis in NSCLC, thereby supporting BCAT1 as a promising therapeutic target for the treatment of TKI-resistant NSCLC.

An Efficient Method for Monitoring Birds Based on Object Detection and Multi-Object Tracking Networks.

To protect birds, it is crucial to identify their species and determine their population across different regions. However, currently, bird monitoring methods mainly rely on manual techniques, such as point counts conducted by researchers and ornithologists in the field. This method can sometimes be inefficient, prone to errors, and have limitations, which may not always be conducive to bird conservation efforts. In this paper, we propose an efficient method for wetland bird monitoring based on object detection and multi-object tracking networks. First, we construct a manually annotated dataset for bird species detection, annotating the entire body and head of each bird separately, comprising 3737 bird images. We also built a new dataset containing 11,139 complete, individual bird images for the multi-object tracking task. Second, we perform comparative experiments using a state-of-the-art batch of object detection networks, and the results demonstrated that the YOLOv7 network, trained with a dataset labeling the entire body of the bird, was the most effective method. To enhance YOLOv7 performance, we added three GAM modules on the head side of the YOLOv7 to minimize information diffusion and amplify global interaction representations and utilized Alpha-IoU loss to achieve more accurate bounding box regression. The experimental results revealed that the improved method offers greater accuracy, with mAP@0.5 improving to 0.951 and mAP@0.5:0.95 improving to 0.815. Then, we send the detection information to DeepSORT for bird tracking and classification counting. Finally, we use the area counting method to count according to the species of birds to obtain information about flock distribution. The method described in this paper effectively addresses the monitoring challenges in bird conservation.

ETU-Net: efficient Transformer and convolutional U-style connected attention segmentation network applied to endoscopic image of epistaxis.

Epistaxis is a typical presentation in the otolaryngology and emergency department. When compressive therapy fails, directive nasal cautery is necessary, which strongly recommended operating under the nasal endoscope if it is possible. Limited by the operator's clinical experience, complications such as recurrence, nasal ulcer, and septum perforation may occur due to insufficient or excessive cautery. At present, deep learning technology is widely used in the medical field because of its accurate and efficient recognition ability, but it is still blank in the research of epistaxis. In this work, we first gathered and retrieved the Nasal Bleeding dataset, which was annotated and confirmed by many clinical specialists, filling a void in this sector. Second, we created ETU-Net, a deep learning model that smartly integrated the excellent performance of attention convolution with Transformer, overcoming the traditional model's difficulties in capturing contextual feature information and insufficient sequence modeling skills in picture segmentation. On the Nasal Bleeding dataset, our proposed model outperforms all others models that we tested. The segmentation recognition index, Intersection over Union, and F1-Score were 94.57 and 97.15%. Ultimately, we summarized effective ways of combining artificial intelligence with medical treatment and tested it on multiple general datasets to prove its feasibility. The results show that our method has good domain adaptability and has a cutting-edge reference for future medical technology development.

Discovery of HCD3514 as a potent EGFR inhibitor against C797S mutation in vitro and in vivo.

Osimertinib (AZD9291), a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), has significantly improved the survival of non-small cell lung cancer (NSCLC) patients with EGFRT790M mutation, the major mechanism of acquired resistance to first-generation EGFR TKI. However, resistance to AZD9291 arises eventually and EGFRC797S mutation was reported to be a major resistance mechanism. Thus, it is highly valuable to develop novel EGFR fourth-generation inhibitors targeting C797S mutation to override the acquired resistance. In this study, we identified HCD3514 as a novel EGFR fourth-generation inhibitors targeting C797S triple mutation. It strongly inhibited EGFRL858R/T790M/C797S and EGFR19del/T790M/C797S mutations with IC50 values of 1.0 and 2.0 nM, respectively. HCD3514 dose-dependently inhibited the activation of EGFR in both engineered BaF3 cells and tumor cells harboring EGFRC797S triple mutant and thus effectively suppressed the proliferation of the cells. Moreover, HCD3514 caused a dose-dependent increase of apoptosis in C797S triple mutant cells accompanied by increased levels of cleaved caspase-3 and cleaved PARP. Furthermore, HCD3514 induced tumor growth inhibition in EGFR19del/T790M/C797S xenograft model as a single oral agent by decreasing the activation of EGFR. In addition to EGFRC797S triple mutations, HCD3514 also potently and selectively inhibited EGFRT790M double mutations (L858R/T790M and 19del/T790M). Collectively, HCD3514 is a highly selective and potent EGFR inhibitor against EGFRC797S triple mutations as well as EGFRT790M double mutations and is confirmed potently anti-tumor activity in preclinical models.

ASK120067 potently suppresses B-cell or T-cell malignancies in vitro and in vivo by inhibiting BTK and ITK.

Hyperactivation of Bruton's tyrosine kinase (BTK) or interleukin-2-inducible T cell kinase (ITK) has been attributed to the pathogenesis of B-cell lymphoma or T-cell leukemia, respectively, which suggests that Bruton's tyrosine kinase and interleukin-2-inducible T cell kinase are critical targets for the treatment of hematological malignancies. We identified a novel third-generation epidermal growth factor receptor (EGFR) inhibitor, ASK120067 (limertinib) in our previous research, which has been applied as a new drug application against non-small cell lung cancer in China. In this work, we found that ASK120067 displayed potent in vitro inhibitory efficacy against Bruton's tyrosine kinase protein and interleukin-2-inducible T cell kinase protein via covalent binding. In cell-based assays, ASK120067 dose-dependently suppressed Bruton's tyrosine kinase phosphorylation and exhibited anti-proliferation potency by inducing apoptosis in numerous B-lymphoma cells. Meanwhile, it caused growth arrest and induced the apoptosis of T-cell leukemia cells by attenuating interleukin-2-inducible T cell kinase activation. Oral administration of ASK120067 led to significant tumor regression in B-cell lymphoma and T-cell leukemia xenograft models by weakening Bruton's tyrosine kinase and interleukin-2-inducible T cell kinase signaling, respectively. Taken together, our studies demonstrated that ASK120067 exerted preclinical anti-tumor activities against B-/T-cell malignancy by targeting BTK/ITK.

Conformational Constrained 4-(1-Sulfonyl-3-indol)yl-2-phenylaminopyrimidine Derivatives as New Fourth-Generation Epidermal Growth Factor Receptor Inhibitors Targeting T790M/C797S Mutations.

Tertiary C797S mutation of epidermal growth factor receptor (EGFR)-mediated resistance in non-small-cell-lung-cancer (NSCLC) patients is still an unmet clinical need. Several classes of adenosine 5'-triphosphate-competitive or allosteric EGFRT790M/C797S inhibitors and degraders have been developed, but none of them have received approval from the regulatory agencies. Herein, we report the structure-based design of conformational constrained 4-(1-ethylsufonyl-3-indolyl)-2-phenylaminopyrimidines as new EGFRT790M/C797S inhibitors by using a macrocyclization strategy. Representative compound 18j potently inhibited EGFR19del/T790M/C797S and EGFRL858R/T790M/C797S mutants with IC50 values of 15.8 and 23.6 nM and suppressed Ba/F3-EGFRL858R/T790M/C797S and Ba/F3-EGFR19del/T790M/C797S cells with IC50 values of 0.036 and 0.052 µM, respectively, which is 10-20-fold more potent than brigatinib. 18j also potently inhibited the EGFR19del/T790M/C797S-mutated PC-9-OR NSCLC cell proliferation with an IC50 value of 0.644 µM but was less potent for parental Ba/F3 and A431 cells. This study provides a new lead compound for drug discovery to combat EGFRC797S-mediated resistance in NSCLC patients.

Optimization of Brigatinib as New Wild-Type Sparing Inhibitors of EGFRT790M/C797S Mutants.

A series of brigatinib derivatives were designed and synthesized as new potent and selective EGFRT790M/C797S inhibitors. One of the most potent and selective compounds 18k strongly suppressed the EGFRL858R/T790M/C797S and EGFR19Del/T790M/C797S kinases with IC50 values of 0.7 and 3.6 nM, respectively, which were over 54-fold more potent than the lead compound. 18k also demonstrated promising EGFRT790M/C797S mutant selectivity, and was 94-fold less potent against the wild type EGFR. A cocrystal structure of EGFRT790M/C797S with a close derivative 18f was solved to provide insight on the inhibitor's binding mode. Moreover, compound 18k was orally bioavailable and demonstrated highly desirable PK properties, making it a promising lead compound for further structural optimization.

Design, synthesis and pharmacological evaluation of bicyclic and tetracyclic pyridopyrimidinone analogues as new KRASG12C inhibitors.

KRAS is the most commonly altered oncogene of the RAS family, especially the G12C mutant (KRASG12C), which has been a promising drug target for many cancers. On the basis of the bicyclic pyridopyrimidinone framework of the first-in-class clinical KRASG12C inhibitor AMG510, a scaffold hopping strategy was conducted including a F-OH cyclization approach and a pyridinyl N-atom working approach leading to new tetracyclic and bicyclic analogues. Compound 26a was identified possessing binding potency of 1.87 µM against KRASG12C and cell growth inhibition of 0.79 µM in MIA PaCa-2 pancreatic cancer cells. Treatment of 26a with NCI-H358 cells resulted in down-regulation of KRAS-GTP levels and reduction of phosphorylation of downstream ERK and AKT dose-dependently. Molecular docking suggested that the fluorophenol moiety of 26a occupies a hydrophobic pocket region thus forming hydrogen bonding to Arg68. These results will be useful to guide further structural modification.

Structure-Based Design of 5-Methylpyrimidopyridone Derivatives as New Wild-Type Sparing Inhibitors of the Epidermal Growth Factor Receptor Triple Mutant (EGFRL858R/T790M/C797S).

Tertiary EGFRC797S mutation induced resistance against osimertinib (1) is an emerging "unmet clinical need" for non-small-cell lung cancer (NSCLC) patients. A series of 5-methylpyrimidopyridone derivatives were designed and synthesized as new selective EGFRL858R/T790M/C797S inhibitors. A representative compound, 8r-B, exhibited an IC50 of 27.5 nM against the EGFRL858R/T790M/C797S mutant, while being a significantly less potent for EGFRWT (IC50 > 1.0 µM). Cocrystallographic structure determination and computational investigation were conducted to elucidate its target selectivity.