Discovery of an Oleanolic Acid/Hederagenin-Nitric Oxide Donor Hybrid as an EGFR Tyrosine Kinase Inhibitor for Non-Small-Cell Lung Cancer.

Natural triterpenoids, such as oleanolic acid (OA) and hederagenin, display anti-lung cancer effects, and nitric oxide (NO) is associated with some oncogenic signaling pathways. Accordingly, 17 OA/hederagenin-NO donor hybrids were designed, synthesized, and evaluated against tumor cells. The most potent compound, 13, significantly inhibited the proliferation of five tumor cell lines (IC50 4.6-5.2 µM), while hederagenin inhibited the growth of only A549 tumor cells (IC50 > 10 µM). Furthermore, compound 13 showed stronger inhibitory effects on EGFR-LTC kinase activity (IC50 0.01 µM) than hederagenin (IC50 > 20 µM) and inhibited the proliferation of gefitinib-resistant H1975 (IC50 8.1 µM) and osimertinib-resistant H1975-LTC (IC50 7.6 µM) non-small-cell lung cancer (NSCLC) cells. Moreover, compound 13 produced the most NO in H1975 tumor cells, which indicated that NO may play a synergistic role. Collectively, compound 13, a novel hederagenin-NO donor hybrid with a different chemical structure from those of the current FDA-approved EGFR-targeted anti-NSCLC drugs, may be a promising lead compound for the treatment of NSCLC expressing gefitinib-resistant EGFR with a T790 M mutation or osimertinib-resistant EGFR-LTC with an L858R/T790M/C797S mutation. This work should shed light on the discovery of new anti-NSCLC drugs targeting EGFR from natural products.

Cytotoxicity, Hemolytic Toxicity, and Mechanism of Action of Pulsatilla Saponin D and Its Synthetic Derivatives.

The strong hemolytic toxicity of pulsatilla saponin D (1, HD50 6.3 µM) has hampered its clinical development as an injectable anticancer agent. To combat this challenge, 17 new derivatives of 1 with ring C, C-28, or C-3 modifications were synthesized and evaluated for cytotoxicity against several selected human tumor lines, as well as for hemolytic toxicity against rabbit erythrocytes. Structure-activity relationship (SAR) and structure-toxicity relationship (STR) correlations were also elucidated. Compared to the lead compound 1, the hemolytic activity of all 17 derivatives dropped dramatically. Notably, compound 14 exhibited significant cytotoxicity toward A549 human lung cancer cells (IC50 2.8 µM) in a dose-dependent manner without hemolytic toxicity (HD50 > 500 µM). Molecular studies indicated that 14 induced typical G1 cell cycle arrest and apoptosis in A549 cells, and Western blot assays suggested that both intrinsic and extrinsic apoptosis pathways were activated by 14. Collectively, compound 14 may merit further development as a potential anti-lung cancer agent.

The derivatives of Pulsatilla saponin A, a bioactive compound from Pulsatilla chinensis: Their synthesis, cytotoxicity, haemolytic toxicity and mechanism of action.

The strong haemolytic toxicity of Pulsatilla saponin A (PSA) has hampered its clinical development as an injectable anticancer agent. To circumvent this challenge, twenty PSA derivatives with C ring or C-28 or C-3 modifications were synthesized and evaluated for cytotoxicity against seven selected human tumor lines, as well as for haemolytic toxicity. Structure-activity relationship (SAR) and structure-toxicity relationship (STR) correlations were also elucidated. Compared with PSA, compound 22 showed a better balance between haemolytic toxicity (HD50 > 500 µM) and cytotoxicity toward lung cancer cells A549 (IC50 = 4.68 µM). Molecular studies indicated that 22 was liked to lead to G1 cell cycle arrest and therefore, 22 may be a potent antitumor drug candidate.

Crystallization and preliminary X-ray analysis of the vWA domain of human anthrax toxin receptor 1.

The Gram-positive spore-forming bacterium Bacillus anthracis causes anthrax by secreting anthrax toxin, which consists of protective antigen (PA), lethal factor and oedema factor. Binding of PA to receptors triggers the multi-step process of anthrax toxin entry into target cells. Two distinct cellular receptors, ANTXR1 (also known as tumour endothelial marker 8; TEM8) and ANTXR2 (also known as capillary morphogenesis protein 2; CMG2), for anthrax toxin have been identified. Although the crystal structure of the extracellular von Willebrand factor A (vWA) domain of CMG2 has been reported, the difference between the vWA domains of TEM8 and CMG2 remains unclear because there are no structural data for the TEM8 vWA domain. In this report, the TEM8 vWA domain was expressed, purified and crystallized. X-ray diffraction data were collected to 1.8 Šresolution from a single crystal, which belonged to space group P1 with unit-cell parameters a=65.9, b=66.1, c=74.4 Å, α=63.7, ß=88.2, γ=59.9°.

The structure of tumor endothelial marker 8 (TEM8) extracellular domain and implications for its receptor function for recognizing anthrax toxin.

Anthrax toxin, which is released from the gram-positive bacterium Bacillus anthracis, is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds a receptor on the surface of the target cell and further assembles into a homo-heptameric pore through which EF and LF translocate into the cytosol. Two distinct cellular receptors for anthrax toxin, TEM8/ANTXR1 and CMG2/ANTXR2, have been identified, and it is known that their extracellular domains bind PA with low and high affinities, respectively. Here, we report the crystal structure of the TEM8 extracellular vWA domain at 1.7 A resolution. The overall structure has a typical integrin fold and is similar to that of the previously published CMG2 structure. In addition, using structure-based mutagenesis, we demonstrate that the putative interface region of TEM8 with PA (consisting of residues 56, 57, and 154-160) is responsible for the PA-binding affinity differences between the two receptors. In particular, Leu56 was shown to be a key factor for the lower affinity of TEM8 towards PA compared with CMG2. Because of its high affinity for PA and low expression in normal tissues, an isolated extracellular vWA domain of the L56A TEM8 variant may serve as a potent antitoxin and a potential therapeutic treatment for anthrax infection. Moreover, as TEM8 is often over-expressed in tumor cells, our TEM8 crystal structure may provide new insights into how to design PA mutants that preferentially target tumor cells.