Structure-based virtual screening (SBVS) has become an indispensable technique for hit identification at the early stage of drug discovery. However, the accuracy of current scoring functions is not high enough to confer success to every target and thus remains to be improved. Previously, we had developed binary pose filters (PFs) using knowledge derived from the protein–ligand interface of a single X-ray structure of a specific target. This novel approach had been validated as an effective way to improve ligand enrichment. Continuing from it, in the present work we attempted to incorporate knowledge collected from diverse protein–ligand interfaces of multiple crystal structures of the same target to build PF ensembles (PFEs). Toward this end, we first constructed a comprehensive data set to meet the requirements of ensemble modeling and validation. This set contains 10 diverse targets, 118 well-prepared X-ray structures of protein–ligand complexes, and large benchmarking actives/decoys sets. Notably, we designed a unique workflow of two-layer classifiers based on the concept of ensemble learning and applied it to the construction of PFEs for all of the targets. Through extensive benchmarking studies, we demonstrated that (1) coupling PFE with Chemgauss4 significantly improves the early enrichment of Chemgauss4 itself and (2) PFEs show greater consistency in boosting early enrichment and larger overall enrichment than our prior PFs. In addition, we analyzed the pairwise topological similarities among cognate ligands used to construct PFEs and found that it is the higher chemical diversity of the cognate ligands that leads to the improved performance of PFEs. Taken together, the results so far prove that the incorporation of knowledge from diverse protein–ligand interfaces by ensemble modeling is able to enhance the screening competence of SBVS scoring functions.

•Firstly designed and synthesized conformationally restricted salinomycin derivatives.•The two conformationally restricted salinomycin showed biological activities in different level.•ROESY analysis of the compounds indicated they adopted different conformations.•The results proved the conformation of salinomycin is crucial to its biological activities.Two conformationally restricted salinomycin derivatives by tethering the hydroxyl groups at C1 and C20 with different chain length were designed and synthesized. The cyclic derivatives showed better biological activities than C1/C20 modified derivatives, indicating the importance of the compact conformation for the ion binding capacity. In addition, the length of the connective chain plays critical role in the biological activities, thus cyclic the derivative 7 preserved some pharmacological activity but derivative 5 with two carbon atom shorter chain showed significantly reduced activity. The conformations of the two cyclic salinomycin derivatives were analyzed by ROESY spectrum in DMSO-d6, indicating derivative 7 may adopt more appropriate conformations for the coordinate with alkali metal ion than derivative 5, which has a closer distance between H3 and H25.Download high-res image (168KB)Download full-size image

Several salinomycin-hydroxamic acid conjugates were designed and synthesized. Most conjugates showed better antiproliferative activities than salinomycin in HT-29 colon cancer, HGC-27 gastric cancer, and especially in MDA-MB-231 triple-negative human breast cancer cells. These conjugates are stable in cell culture media, and they showed much better biological activities than the 1:1 physical mixture with hydroxamic acids and salinomycin. The better membrane permeability and hydrolysis rate of the conjugates may lead to the activity improvements.Salinomycin-hydroxamic acid conjugates showed better antiproliferative activities than salinomycin in HT-29, HGC-27, and especially in MDA-MB-231 cancer cells.Download high-res image (66KB)Download full-size image

Salinomycin diastereoisomers and their benzoylated derivatives were synthesized and evaluated for both antiproliferative activity and neurotoxicity in vitro. The results indicated that the stereoscopic configurations of the spiro C17 and C21 atoms as well as the benzoyl groups of O-20 on the rigid B/C/D spiro-ketal structures are crucial for biological activity and neural toxicity. In general, there are some positive correlations between the antiproliferative activity and neurotoxicity in these salinomycin derivatives, indicating possibly similar mechanisms of action.

Large-scale application of whole-cell glycosylation is still hampered by inefficient UDP-sugar formation. Using a module-based approach, an engineered Escherichia coli strain was constructed to enhance the production of flavonoid glucuronides. The engineered metabolic pathway was partitioned into two modules: an endogenous upstream biosynthetic pathway to produce the sugar donor UDP-glucuronic acid (UDPGA) and a heterologous downstream UDP-dependent glycosyltransferase (UGT) to catalyse the glucuronidation of flavonoids. First, two UGTs (SbUGT-W76 and SbUGT-W112) were isolated from Scutellaria baicalensis Georgi. Enzymatic assays showed that the recombinant SbUGT exhibited regiospecificity towards the C7-OH position of flavonoid substrates, and conflicting results on the specificity of sugar donor and acceptor compared with that of reported SbUBGAT were obtained. Then, the native upstream module of the UDPGA synthetic pathway was strengthened to increase the endogenous level of UDPGA. Using these strategies, up to 797 mg L−1 baicalein-7-O-glucuronide was biosynthesized. Furthermore, systemic engineering of upstream and downstream genes in the glucuronide biosynthetic pathway was conducted for characterization and the capability to biotransform baicalein. The results showed that UDP-glucose 6-dehydrogenase (Ugd) catalysed the rate-determining step for glucuronide production. SbUGT displayed the same catalytic properties both in vivo and in vitro.

20-epi-Salinomycin and six 20-O-acylated analogs were synthesized and tested for their antiproliferative activity. Both the C1-protecting group of the salinomycin and the acidity of the substituted benzoic acid are crucial to the Mitsunobu conversion. 20-epi-Salinomycin showed similar antiproliferative activity as salinomycin, but its 20-O-acylated analogs were 2–10 times more potent. In addition, the 20-epi-20-O-acylated salinomycin derivative 9d and 9e had better selectivities than salinomycin between cancer and neuron cell lines. The spatial configuration of the C20-hydroxyl group has little influence on the activities, but the acyl groups cause an obvious difference by producing possible effects on the stability and permeability of the salinomycin–alkali metal ions complexes.

A novel and efficient approach for the straightforward synthesis of biologically significant acenaphtho[1,2-b]quinoline derivatives in good yields utilizing CuI as a catalyst with a broad array of substrates has been developed. The strategy features as a CuI-catalyzed cascade reaction involving the formation of two new CC bonds and one new CN bond with high atom economy. A proposed mechanism for the reaction is described.A novel and efficient approach for the straightforward synthesis of biologically significant acenaphtho[1,2-b]quinoline derivatives in the presence of CuI as a catalyst in good yield with a broad substrate scope has been developed.

The novel justicidin G analogue 13 and its phosphate ester 15 were synthesized as potential anticancer agents in several steps starting from commercially available methyl gallate and veratraldehyde. The cytotoxicity of the intermediates was tested against HCT-8, BEL-7402, KETR3, HELA, BGC-823, KB and MCF-7 cell lines by the MTT test, and compound 15 exhibited significant cytotoxicity in HELA and KB cell lines.A novel justicidin G analogue and its phosphate ester were synthesized and their cytotoxicity was screened.

Based on the fact that petroselinic acid showed good inhibitory activity (IC50 = 6.99 μmol/L) against protein tyrosine phophatase 1B(PTP1B) in vitro, a series of novel N-(alkoxyphenyl)-aminocarbonylbenzoic acid derivatives were designed and synthesized. The results indicated that most of the derivatives showed more potent activities against PTP1B. Especially, compound 13 had obvious activity with an IC50 of 106 nmol/L in vitro.

Salinomycin diastereoisomers and their benzoylated derivatives were synthesized and evaluated for both antiproliferative activity and neurotoxicity in vitro. The results indicated that the stereoscopic configurations of the spiro C17 and C21 atoms as well as the benzoyl groups of O-20 on the rigid B/C/D spiro-ketal structures are crucial for biological activity and neural toxicity. In general, there are some positive correlations between the antiproliferative activity and neurotoxicity in these salinomycin derivatives, indicating possibly similar mechanisms of action.