Discovery of a Novel Bifunctional Steroid Analog, YXG-158, as an Androgen Receptor Degrader and CYP17A1 Inhibitor for the Treatment of Enzalutamide-Resistant Prostate Cancer

The androgen/androgen receptor (AR) signaling pathway plays an important role in castration-resistant prostate cancer (CRPC). Bifunctional agents that simultaneously degrade AR and inhibit androgen synthesis are expected to block the androgen/AR signaling pathway more thoroughly, demonstrating the promising therapeutic potential for CRPC, even enzalutamide-resistant CRPC. Herein, a series of steroid analogs were designed, synthesized, and identified as selective AR degraders, among which YXG-158 (23-h) was the most potent antitumor compound with dual functions of AR degradation and CYP17A1 inhibition. In addition, 23-h abrogated the hERG inhibition and exhibited excellent PK profiles. In vivo, 23-h effectively inhibited the growth of hormone-sensitive organs in the Hershberger assay and exhibited robust antitumor efficacy both in enzalutamide-sensitive (LNCaP/AR) and enzalutamide-resistant (C4-2b-ENZ) xenograft models. Thus, 23-h was chosen as a preclinical candidate for the treatment of enzalutamide-resistant prostate cancer

Discovery of a Novel Bifunctional Steroid Analog, YXG-158, as an Androgen Receptor Degrader and CYP17A1 Inhibitor for the Treatment of Enzalutamide-Resistant Prostate Cancer

The androgen/androgen receptor (AR) signaling pathway plays an important role in castration-resistant prostate cancer (CRPC). Bifunctional agents that simultaneously degrade AR and inhibit androgen synthesis are expected to block the androgen/AR signaling pathway more thoroughly, demonstrating the promising therapeutic potential for CRPC, even enzalutamide-resistant CRPC. Herein, a series of steroid analogs were designed, synthesized, and identified as selective AR degraders, among which YXG-158 (23-h) was the most potent antitumor compound with dual functions of AR degradation and CYP17A1 inhibition. In addition, 23-h abrogated the hERG inhibition and exhibited excellent PK profiles. In vivo, 23-h effectively inhibited the growth of hormone-sensitive organs in the Hershberger assay and exhibited robust antitumor efficacy both in enzalutamide-sensitive (LNCaP/AR) and enzalutamide-resistant (C4-2b-ENZ) xenograft models. Thus, 23-h was chosen as a preclinical candidate for the treatment of enzalutamide-resistant prostate cancer

Nifedipine and diazoxide attenuate PA-activated ER-stress.

<p>(A) After MIN6 cells were co-treated with nifedipine and 0.5 mM PA for 48 h, the phosphorylation of eIF2α and the expression of CHOP were detected by western blot. β-actin was used for normalization. (B) The phosphorylation rate of eIF2α was calculated as optical density of phosphorylated-eIF2α (p-eIF2α) divide by total eIF2α (t-eIF2α). * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (C) The expression of CHOP was calculated by optical density. * p<0.05 denote significant difference versus the PA-treated alone group, n = 3. (D) After MIN6 cells were co-treated with diazoxide and 0.5 mM PA for 48 h, the phosphorylation of eIF2α, the expression of CHOP were detected by western blot. (E) The phosphorylation rate of eIF2α was calculated by optical density. * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (F) The expression of CHOP was calculated by optical density. * p < 0.05 denote significant difference versus the PA-treated alone group, n = 3. (G) After treatment, the cells were fixed and stained with CHOP and insulin antibodies. Red fluorescence indicated CHOP expression while green marked insulin. The nuclei were stained with DAPI dye. Scale bar = 50 μm and referred to all panels.</p

Inhibition of Calcium Influx Reduces Dysfunction and Apoptosis in Lipotoxic Pancreatic β-Cells via Regulation of Endoplasmic Reticulum Stress

<div><p>Lipotoxicity plays an important role in pancreatic β-cell failure during the development of type 2 diabetes. Prolonged exposure of β-cells to elevated free fatty acids level could cause deterioration of β-cell function and induce cell apoptosis. Therefore, inhibition of fatty acids-induced β-cell dysfunction and apoptosis might provide benefit for the therapy of type 2 diabetes. The present study examined whether regulation of fatty acids-triggered calcium influx could protect pancreatic β-cells from lipotoxicity. Two small molecule compounds, L-type calcium channel blocker nifedipine and potassium channel activator diazoxide were used to inhibit palmitic acid-induced calcium influx. And whether the compounds could reduce palmitic acid-induced β-cell failure and the underlying mechanism were also investigated. It was found that both nifedipine and diazoxide protected MIN6 pancreatic β-cells and primary cultured murine islets from palmitic acid-induced apoptosis. Meanwhile, the impaired insulin secretion was also recovered to varying degrees by these two compounds. Our results verified that nifedipine and diazoxide could reduce palmitic acid-induced endoplasmic reticulum stress to generate protective effects on pancreatic β-cells. More importantly, it suggested that regulation of calcium influx by small molecule compounds might provide benefits for the prevention and therapy of type 2 diabetes.</p></div

Nifedipine and diazoxide inhibited PA-stimulated Ca²⁺ release.

<p>(A) Pre-incubated of nifedipine dose-dependently inhibited 0.5 mM PA-stimulated Ca^<b>2+</b> release in MIN6 cells. (B) There was no significant change in PA-induced Ca^<b>2+</b> release between diazoxide co-treated and PA-treated alone group. The Ca^<b>2+</b> mobilization buffer did not contain glucose. (C) In the presence of 25.5 mM glucose, pre-incubated of diazoxide dose-dependently inhibited 0.5 mM PA-stimulated Ca^<b>2+</b> release in MIN6 cells. * p<0.05; ** p<0.01; *** p<0.001 denote significant difference versus the PA-treated alone group, n = 6.</p

Hoechst33342 staining analysis in MIN6 cells.

<p>(A) After the cells were treated with 0.5 mM PA in the presence/absence of nifedipine at indicated concentrations for 48 h, Hoechst33342 staining was performed to detect apoptotic cells. Blue fluorescence indicated all nucleus, the lighter and shrinkage dots were apoptotic cells. Scale bar = 100 μm and referred to all panels. (B) After the cells were treated with 0.5 mM PA in the presence/absence of diazoxide for 48 h, Hoechst33342 staining was performed. (C)(D) The apoptotic rate was calculated as apoptotic cell number divided by total cell number. 10 random sights in each well were selected to count apoptosis, and the data from six duplicated wells were analyzed (n = 6). *** p<0.001 denote significant difference versus the PA-treated alone group.</p

Nifedipine and diazoxide reduced cell apoptosis in PA-impaired islets.

<p>(A) Cultured islets were treated with PA in the presence/absence of different compounds for 48 h, then TUNEL staining was performed. Red fluorescence nuclei indicate apoptotic cells. Blue fluorescence showed all nuclei. Scale bar = 100 μm and referred to all panels. (B) Apoptotic rate was calculated as TUNEL-positive cell number modified by islet area. 10 islets were analyzed from six duplicated wells. * p<0.05; ** p<0.01 denote significant difference versus the PA-treated alone group, n = 6.</p

Halogen Bond: Its Role beyond Drug–Target Binding Affinity for Drug Discovery and Development

Halogen bond has attracted a great deal of attention in the past years for hit-to-lead-to-candidate optimization aiming at improving drug-target binding affinity. In general, heavy organohalogens (i.e., organochlorines, organobromines, and organoiodines) are capable of forming halogen bonds while organofluorines are not. In order to explore the possible roles that halogen bonds could play beyond improving binding affinity, we performed a detailed database survey and quantum chemistry calculation with close attention paid to (1) the change of the ratio of heavy organohalogens to organofluorines along the drug discovery and development process and (2) the halogen bonds between organohalogens and nonbiopolymers or nontarget biopolymers. Our database survey revealed that (1) an obviously increasing trend of the ratio of heavy organohalogens to organofluorines was observed along the drug discovery and development process, illustrating that more organofluorines are worn and eliminated than heavy organohalogens during the process, suggesting that heavy halogens with the capability of forming halogen bonds should have priority for lead optimization; and (2) more than 16% of the halogen bonds in PDB are formed between organohalogens and water, and nearly 20% of the halogen bonds are formed with the proteins that are involved in the ADME/T process. Our QM/MM calculations validated the contribution of the halogen bond to the binding between organohalogens and plasma transport proteins. Thus, halogen bonds could play roles not only in improving drug–target binding affinity but also in tuning ADME/T property. Therefore, we suggest that albeit halogenation is a valuable approach for improving ligand bioactivity, more attention should be paid in the future to the application of the halogen bond for ligand ADME/T property optimization

Nifedipine and diazoxide protects MIN6 cells from PA-induced apoptosis.

<p>(A) After 48 h incubation of nifedipine in the presence/absence of PA, the cell viability was measured by MTT assay. The cell viability was shown as inhibitory ratio (% of control), *** p<0.001 denote significant difference versus the PA-treated alone group, n = 6. (B) After 48 h incubation of diazoxide in the presence/absence of PA, the cell viability was measured by MTT assay. *** p<0.001 denote significant difference versus the PA-treated alone group, n = 6. (C) After the cells were treated with 0.5 mM PA in the presence/absence of nifedipine for 48 h, the expression of cleaved caspase-3 was detected by western blot. (D) After the cells were treated with 0.5 mM PA in the presence/absence of diazoxide for 48 h, the expression of cleaved caspase-3 was detected. (E)(F) Quantitative analysis of western blot. The optical density of each blot band was determined and adjusted by the optical density of β-actin. * p<0.05; ** p<0.01; *** p<0.001 denote significant difference versus the PA-treated alone group, n = 3.</p

Thermodynamics calculation of protein–ligand interactions by QM/MM polarizable charge parameters

<div><p>The calculation of protein–ligand binding free energy (Δ<i>G</i>) is of great importance for virtual screening and drug design. Molecular dynamics (MD) simulation has been an attractive tool to investigate this scientific problem. However, the reliability of such approach is affected by many factors including electrostatic interaction calculation. Here, we present a practical protocol using quantum mechanics/molecular mechanics (QM/MM) calculations to generate polarizable QM protein charge (QMPC). The calculated QMPC of some atoms in binding pockets was obviously different from that calculated by AMBER ff03, which might significantly affect the calculated Δ<i>G.</i> To evaluate the effect, the MD simulations and MM/GBSA calculation with QMPC for 10 protein–ligand complexes, and the simulation results were then compared to those with the AMBER ff03 force field and experimental results. The correlation coefficient between the calculated ΔΔ<i>G</i> using MM/GBSA under QMPC and the experimental data is .92, while that with AMBER ff03 force field is .47 for the complexes formed by streptavidin or its mutants and biotin. Moreover, the calculated ΔΔ<i>G</i> with QMPC for the complexes formed by ERβ and five ligands is positively related to experimental result with correlation coefficient of .61, while that with AMBER ff03 charge is negatively related to experimental data with correlation coefficient of .42. The detailed analysis shows that the electrostatic polarization introduced by QMPC affects the electrostatic contribution to the binding affinity and thus, leads to better correlation with experimental data. Therefore, this approach should be useful to virtual screening and drug design.</p></div