Development of PD3 and PD3-B for PDEδ inhibition to modulate KRAS activity.

Despite extensive efforts over 40 years, few effective KRAS inhibitors have been developed to date, mainly due to the undruggable features of KRAS proteins. In addition to the direct approach to KRAS via covalent inhibition, modulation of the prenyl-binding protein PDEδ that binds with farnesylated KRAS has emerged as an alternative strategy to abrogate KRAS activity. For the verification of new therapeutic strategies, chemical probes with the dual functions of visualisation and pharmacological inhibition against oncogenic proteins are enormously valuable to understand cellular events related to cancer. Here, we report indolizino[3,2-c]quinoline (IQ)-based fluorescent probes (PD3 and PD3-B) for PDEδ inhibition. By using the unique fluorescent characteristics of the IQ scaffold, a fluorescence polarisation (FP)-based binding assay identified PD3 as the most effective PDEδ probe among the tested PD analogues, with a low Kd value of 0.491 µM and long retention time in the binding site of PDEδ. In particular, a FP-based competition assay using deltarasin verified that PD3 occupies the farnesylation binding site of PDEδ, excluding the possibility that the FP signals resulted from non-specific hydrophobic interactions between the ligand and protein in the assay. We also designed and synthesised PD3-B (5), an affinity-based probe (ABP) from the PD3 structure, which enabled us to pull down PDEδ from bacterial lysates containing a large number of intrinsic bacterial proteins. Finally, KRAS relocalization was verified in PANC-1 cells by treatment with PD3, suggesting its potential as an effective probe to target PDEδ.

Mitochondria-targeting indolizino[3,2-c]quinolines as novel class of photosensitizers for photodynamic anticancer activity.

To achieve efficient photodynamic activity, substantial effort has been dedicated to precise control of the intracellular localization of current photosensitizers (PSs). Given the extremely small radius of action of singlet oxygen, the direct targeting of PSs to the mitochondria is expected to greatly enhance the photodynamic therapy (PDT) activity. Here, we report mitochondria-targeting 6-(furan-2-yl)- and 6-(thiophen-2-yl) indolizino[3,2-c]quinolines (IQs) as novel PSs. IQ derivatives containing 5-membered heterocyclic aromatic rings were synthesized, and their photophysical properties as PSs were characterized. The anticancer potentials of 2a-2f were investigated using various cancer cell lines, and they exhibited dose-dependent and light exposure time-dependent cytotoxicity. Among the synthesized compounds, 2b, which contains a furan ring, showed dual functions as an imaging probe as well as a PS. Real-time confocal fluorescence images revealed the mitochondrial localization of 2b as a primary site of photodamage in live cells. Targeted reactive oxygen species (ROS)-generation capabilities and the photoinduced DNA cleavage of IQs led to mitochondrial dysfunction and photoinduced apoptosis via the intrinsic pathway. 3D RI tomograms of individual live HeLa cells treated with 2b showed that the progress of photoinduced apoptosis was affected by the PS concentration and light irradiation time. The studied IQs (2b, 2d, and 2e) are expected to serve as a new class of heavy-atom-free PSs with low molecular weights less than 350.

Panaxynol, a natural Hsp90 inhibitor, effectively targets both lung cancer stem and non-stem cells.

Cancer stem-like cells (CSCs) contribute to tumor recurrence and chemoresistance. Hence, strategies targeting CSCs are crucial for effective anticancer therapies. Here, we demonstrate the capacities of the non-saponin fraction of Panax ginseng and its active principle panaxynol to inhibit Hsp90 function and viability of both non-CSC and CSC populations of NSCLC in vitro and in vivo. Panaxynol inhibited the sphere forming ability of NSCLC CSCs at nanomolar concentrations, and micromolar concentrations of panaxynol suppressed the viability of NSCLC cells (non-CSCs) and their sublines carrying acquired chemoresistance with minimal effect on normal cells derived from various organs. Orally administered panaxynol significantly reduced lung tumorigenesis in KrasG12D/+ transgenic mice and mice carrying NSCLC xenografts without detectable toxicity. Mechanistically, panaxynol disrupted Hsp90 function by binding to the N-terminal and C-terminal ATP-binding pockets of Hsp90 without increasing Hsp70 expression. These data suggest the potential of panaxynol as a natural Hsp90 inhibitor targeting both the N-terminal and C-terminal of Hsp90 with limited toxicities.

Discovery of highly selective and potent monoamine oxidase B inhibitors: Contribution of additional phenyl rings introduced into 2-aryl-1,3,4-oxadiazin-5(6H)-one.

Monoamine oxidase B (MAO-B) is a flavin adenine dinucleotide (FAD)-containing enzyme that plays a major role in the oxidative deamination of biogenic amines and neurotransmitters. Inhibiting MAO-B activity is a promising approach in the treatment of neurological disorders. Here, we report a series of 2-aryl-1,3,4-oxadiazin-5(6H)-one derivatives as highly selective and potent MAO-B inhibitors. Analysis of the binding sites of hMAO-A and hMAO-B led to design of linear analogs of 2-aryl-1,3,4-oxadiazin-5(6H)-one with an additional phenyl ring. Biological evaluation of the 26 new derivatives resulted in the identification of highly potent and selective inhibitors with optimal physicochemical properties to potentially cross the blood-brain barrier (BBB). Compounds 18a, 18b, 18e and 25b potently inhibited MAO-B, with IC50 values of 4-25 nM and excellent SI over MAO-A (18a > 25000, 18b > 8333 and 18e > 4000 and 25b > 4545). Docking results suggest that an optimal linker between two aromatic rings on the 2-aryl-1,3,4-oxadiazin-5(6H)-one scaffold is a key element in the binding and inhibition of MAO-B.