Cholesterol Modification of Smoothened Is Required for Hedgehog Signaling.

Hedgehog (Hh) has been known as the only cholesterol-modified morphogen playing pivotal roles in development and tumorigenesis. A major unsolved question is how Hh signaling regulates the activity of Smoothened (SMO). Here, we performed an unbiased biochemical screen and identified that SMO was covalently modified by cholesterol on the Asp95 (D95) residue through an ester bond. This modification was inhibited by Patched-1 (Ptch1) but enhanced by Hh. The SMO(D95N) mutation, which could not be cholesterol modified, was refractory to Hh-stimulated ciliary localization and failed to activate downstream signaling. Furthermore, homozygous SmoD99N/D99N (the equivalent residue in mouse) knockin mice were embryonic lethal with severe cardiac defects, phenocopying the Smo-/- mice. Together, the results of our study suggest that Hh signaling transduces to SMO through modulating its cholesterylation and provides a therapeutic opportunity to treat Hh-pathway-related cancers by targeting SMO cholesterylation.

Discovery of a potent HMG-CoA reductase degrader that eliminates statin-induced reductase accumulation and lowers cholesterol.

Statins are inhibitors of HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis, and have been clinically used to treat cardiovascular disease. However, a paradoxical increase of reductase protein following statin treatment may attenuate the effect and increase the side effects. Here we present a previously unexplored strategy to alleviate statin-induced reductase accumulation by inducing its degradation. Inspired by the observations that cholesterol intermediates trigger reductase degradation, we identify a potent degrader, namely Cmpd 81, through structure-activity relationship analysis of sterol analogs. Cmpd 81 stimulates ubiquitination and degradation of reductase in an Insig-dependent manner, thus dramatically reducing protein accumulation induced by various statins. Cmpd 81 can act alone or synergistically with statin to lower cholesterol and reduce atherosclerotic plaques in mice. Collectively, our work suggests that inducing reductase degradation by Cmpd 81 or similar chemicals alone or in combination with statin therapy can be a promising strategy for treating cardiovascular disease.

Synthesis and biological evaluation of D-ring fused 1,2,3-thiadiazole dehydroepiandrosterone derivatives as antitumor agents.

A series of D-ring fused 1,2,3-thiadiazole DHEA derivatives were synthesized and investigated for their activity against the growth of various tumor cell lines using the sulforhodamine B (SRB) assay. It is amazing that for these compounds, T47D cell line was much more sensitive than other tumor cell lines. The most potent saturated N-heterocyclic derivatives showed similar antitumor effect with the positive control compound ADM (adriamycin) on T47D cells, that was 44-60 folds more potent than the lead compound DHEA. Most compounds with potent antitumor activity displayed low toxicity on normal human fibroblasts (HAF). Especially compound 25 (CH33) showed an IC50 of 0.058 µM on T47D cells and its selectivity index (SI) between HAF and T47D was 364, which was 214 folds better than ADM (SI = 1.7). The apoptosis, colony formation and transwell migration assays of 25 were performed on T47D cell line. The primary mechanism study showed that 25 caused a dose-dependent induction of apoptosis, and induced phosphorylation of EphA2 and EphB3 in T47D cells. The in vivo antitumor effect of 25 was also observed in T47D tumor-bearing mice without obvious toxicity.

The synthesis and antibacterial activity of pyrazole-fused tricyclic diterpene derivatives.

The diterpenoid compound 5 was identified as an antibacterial lead in our screening of small synthetic natural product-like (NPL) library. A series of novel diterpene derivatives were synthesized and investigated for their activity against Staphylococcus aureus Newman strain and multidrug-resistant strains (NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271). Among the compounds tested, 42 and 43 showed highest activity with a MIC of 1 µg/mL against strain Newman, 45 and 52 showed the most potent activity with MIC values of 0.71-3.12 µg/mL against five multidrug-resistant S. aureus. All high-antimicrobial active compounds showed no obvious toxicity to human fibroblast (HAF) cells at the MIC concentration.

Synthesis of heterocycle-modified betulinic acid derivatives as antitumor agents.

A series of novel heterocycle-modified betulinic acid (BA) derivatives were synthesized and investigated for their activity against the growth of eight non-drug resistant and one multidrug-resistant tumor cell line using a sulforhodamine B (SRB) assay. The most active compound 17 showed an average IC50 1.19 µM, which was about 20 times more potent than the lead compound BA. It is amazing that for most synthetic saturated N-heterocycle derivatives, MCF-7/ADR was the most sensitive tumor cells, especially 17 showed the most potent antitumor activity (IC50 = 0.33 µM) on this multidrug-resistant tumor cell line, that was 117 times more potent than BA. Most of the tested compounds displayed less toxic on human fibroblasts (HAF) in comparison with the tumor cell lines. The cytometry and transwell migration assays were used to test the ability of 17 to induce apoptosis and inhibit metastasis on tumor cell lines respectively.

Synthesis of novel heterocyclic ring-fused 18ß-glycyrrhetinic acid derivatives with antitumor and antimetastatic activity.

Glycyrrhetinic acid (GA) is one of the most important triterpenoic acids shows many pharmacological effects, especially antitumor activity. GA triggers apoptosis in various tumor cell lines. However, the antitumor activity of GA is weak, thus the synthesis of new synthetic analogs with enhanced potency is needed. By introducing various five-member fused heterocyclic rings at C-2 and C-3 positions, 18 novel GA derivatives were obtained. These compounds were evaluated for their inhibitory activity against the growth of eight different tumor cell lines using a SRB assay. The most active compound 37 showed IC50 between 5.19 and 11.72 µm, which was about 11-fold more potent than the lead compound GA. An apoptotic effect of GA and 37 was determined using flow cytometry and trypan blue exclusion assays. We also demonstrated here for the first time that GA and the synthetic derivatives exhibited inhibitory effect on migration of the tested tumor cells, especially 37 which was about 20-fold more potent than GA on antimetastatic activity.