1,4-Thiazepines (TZEPs) featuring enyne modifications represent promising candidates in cancer therapy. We synthesized novel TZEP derivatives and assessed their cytotoxicity, apoptosis induction, EGFR inhibition, and molecular interactions. TZEPs exhibited cytotoxic effects against cancer cell lines, with compounds TZEP6 and TZEP7 showing significant activity. Flow cytometry analysis revealed TZEP7-induced apoptosis across various cancer types. RT-qPCR analysis demonstrated downregulation of antiapoptotic Bcl-2, upregulation of pro-apoptotic Bax, and increased caspase levels following TZEP7 treatment. Additionally, TZEP7 inhibited EGFR kinase activity in cancer cells, with molecular docking confirming strong binding affinities to EGFRWT and mutant EGFRT790M. AdmetSAR analysis indicated favorable pharmacokinetic properties for TZEP7. These findings underscore the potential of enyne-modified TZEPs as selective cytotoxic agents with apoptotic and EGFR inhibitory activities, highlighting their significance in cancer therapy.
© 2024 The Authors. Published by American Chemical Society.

Cancer, characterized by uncontrolled cell proliferation, remains a global health challenge. Despite advancements in cancer treatment, drug resistance and adverse effects on normal cells remain challenging. The epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase protein, is crucial in controlling cell proliferation and is implicated in various cancers. Here, the cytotoxic and apoptotic potential of 21 newly synthesized spiro-pyrrolopyridazine (SPP) derivatives was investigated on breast (MCF-7), lung (H69AR), and prostate (PC-3) cancer cells. XTT assay was used for cytotoxicity assessment. Flow cytometry and western blot (WB) analyses were conducted for apoptosis detection. Additionally, the EGFR inhibitory potential of these derivatives was evaluated via a homogeneous time-resolved fluorescence (HTRF) assay, and WB and molecular docking studies were conducted to analyze the binding affinities of SPP10 with EGFR. SPPs, especially SPP10, exhibit significant cytotoxicity across MCF-7, H69AR, and PC-3 cancer cells with IC50 values of 2.31 ± 0.3, 3.16 ± 0.8, and 4.2 ± 0.2 μM, respectively. Notably, SPP10 demonstrates selective cytotoxicity against cancer cells with a low impact on nontumorigenic cells (IC50 value: 26.8 ± 0.4 μM). Flow cytometric analysis demonstrated the potent induction of apoptotic cell death by SPP10 in all of the tested cancer cells. Western blot analysis revealed the involvement of key apoptotic proteins, with SPP10 notably inhibiting antiapoptotic Bcl-2 while inducing pro-apoptotic Bax and cytochrome c. SPP10 exhibited significant EGFR kinase inhibitory activity, surpassing the efficacy of the reference drug erlotinib. Molecular docking studies support these findings, revealing strong binding affinities of SPP10 with both wild-type and mutated EGFR. The study underscores the significance of heterocyclic compounds, particularly spiro-class heterocyclic molecules, in advancing cancer research. Overall, SPP10 emerges as a promising candidate for further investigations in cancer treatment, combining potent cytotoxicity, apoptotic induction, and targeted EGFR inhibition.
© 2024 The Authors. Published by American Chemical Society.

Lung cancer is commonly caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric kinase inhibitors are unaffected by common ATP-site resistance mutations and represent a promising therapeutic strategy for targeting drug-resistant EGFR variants. However, allosteric inhibitors are antagonized by kinase dimerization, and understanding this phenomenon has been limited to cellular experiments. To facilitate the study of allosteric inhibitor pharmacology, we designed and purified a constitutive EGFR kinase dimer harboring the clinically relevant L858R/T790M mutations. Kinetic characterization revealed that the EGFR kinase dimer is more active than monomeric EGFR(L858R/T790M) kinase and has the same Km,ATP Biochemical profiling of a large panel of ATP-competitive and allosteric EGFR inhibitors showed that allosteric inhibitor potency decreased by >500-fold in the kinase dimer compared with monomer, yielding IC50 values that correlate well with Ba/F3 cellular potencies. Thus, this readily purifiable constitutive asymmetric EGFR kinase dimer represents an attractive tool for biochemical evaluation of EGFR inhibitor pharmacology, in particular for allosteric inhibitors. SIGNIFICANCE STATEMENT: Drugs targeting epidermal growth factor receptor (EGFR) kinase are commonly used to treat lung cancers but are affected by receptor dimerization. Here, we describe a locked kinase dimer that can be used to study EGFR inhibitor pharmacology.
Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.

Epidermal growth factor receptor (EGFR) C797S mutation leads to Osimertinib drug resistance by disturbing the covalent biding of Michael acceptor group to the Cys797 residue in the ATP biding cleft. In this manuscript, a class of 2-amine-4-oxyphosaniline pyrimidine derivatives were designed, synthesized and evaluated as new noncovalent reversible EGFR inhibitors against L858R/T790M/C797S (CTL) triple mutant. The kinases inhibitiory activity evaluation showed that four compounds exhibited significant inhibitory activities against CTL (IC50 < 30 nM). In particularly, the most promising compound 7a showed excellent enzymatic inhibitory activity against CTL with IC50 value of 9.9 nM, which was more potent than control compound Osimertinib. Moreover, cell proliferation assays indicated that 7a effectively inhibited H1975-EGFR L858R/T790M/C797S with IC50 value of 0.33 µM. Furthermore, compound 7a displayed good metabolic stabilities in human, rat and mouse liver microsomes, and the putative biding mode of compound 7a with ATP was revealed by molecular docking study. These findings strongly indicated that compound 7a was a promising L858R/T790M/C797S mutant EGFR inhibitor.

B-cell antigen receptor (BCR) signaling is required to maintain the physiological functions of normal B cells and plays an important pathogenic role in B-cell malignancies. Bruton tyrosine kinase (BTK), a critical mediator of BCR signaling, is an attractive target for the treatment of B-cell malignancies. This study aimed to identify a highly potent and selective BTK inhibitor.
Homogeneous time-resolved fluorescence assays were used to screen BTK inhibitors. Typhoon fluorescence imaging and Western blot analysis were used to confirm the effects of SY-1530 on the BCR signaling pathway. Additionally, the anti-tumor activities of SY-1530 were evaluated in TMD8 xenografts and spontaneous canine B-cell lymphoma.
We found a novel irreversible and non-competitive inhibitor of BTK, SY-1530, which provided dose-dependent and time-dependent inhibition. SY-1530 selectively bound to BTK rather than inducible T-cell kinase; consequently, it did not significantly affect T-cell receptor signaling and caused limited off-target effects. SY-1530 blocked the BCR signaling pathway through down-regulation of BTK activity, thus leading to impaired phosphorylation of BTK and its downstream kinases. Moreover, SY-1530 induced apoptosis in a caspase-dependent manner and efficaciously inhibited tumor growth in mouse xenograft models of B-cell malignancy (P < 0.001). SY-1530 also induced positive clinical responses in spontaneous canine B-cell lymphoma.
SY-1530 is an irreversible and selective BTK inhibitor that shows inhibitory effects on B-cell malignancies by blocking the BCR signaling pathway. Therefore, it may be a promising therapeutic approach for the treatment of B-cell malignancies.
Copyright © 2021 Cancer Biology & Medicine.