Youjun Zhou

Homology Modeling of Lanosterol 14α-Demethylase of Candida albicans and Aspergillus fumigatus and Insights into the Enzyme-Substrate Interactions

Abstract The crystal structure of 14α-sterol demethylase from Mycobacterium tuberculosis(MT_14DM) provides a good template for modeling the three dimensional structure of lanosterol 14α-demethylase, which is the target of azole antifungal agents. Homologous 3D models of lanosterol 14α-demethylase from Candida albicans (CA_14DM) and Aspergillus fumigatus (AF_14DM) were built on the basis of the crystal coordinates of MT_14DM in complex with 4-phenylimidazole and fluconazole. The reliability of the two models was assessed by Ramachandran plots, Profile-3D analysis, and by analyzing the consistency of the two models with the experimental data on the P45014DM. The overall structures of the resulting CA_14DM model and AF_14DM model are similar to those of the template structures. The two models remain the core structure characteristic for cytochrome P450s and most of the insertions and deletions expose the molecular surface. The structurally and functionally important residues such as the heme binding residues, the residues lining the substrate access channel, and residues in active site were identified from the model. To explore the binding mode of the substrate with the two models, 24(28)- methylene-24,25-dihydrolanosterol was docked into the active site of the two models and hydrophobic interaction and hydrogen-bonding were found to play an important role in substrate recognition and orientation. These results provided a basis for experiments to probe structure-function relationships in the P45014DM. Although CA_14DM and AF_14DM shared similar core structural character, the active site of the two models were quite different, thus allowing the rational design of specific inhibitors to the target enzyme and the discovery of novel antifungal agents with broad spectrum.

Design, synthesis, and activity evaluation of broad-spectrum small-molecule inhibitors of anti-apoptotic Bcl-2 family proteins: characteristics of broad-spectrum protein binding and its effects on anti-tumor activity.

On the basis of the comparison of the structure of the Bim BH3: Bcl-x(L) complex and that of the ABT-737: Bcl-x(L) complex, a series of class A compounds were designed. These compounds had the basic skeleton of ABT-737 and the h2 residues of Bim BH3. These residues had shown themselves to be relevant to Bim BH3s broad-spectrum binding properties in saturation mutagenesis assays. Unlike ABT-737, which is a selective inhibitor of anti-apoptotic members of the Bcl-2 protein family, the class A compounds showed broad-spectrum binding activity to target proteins similar to those of Bim BH3 peptide. Then class B compounds were synthesized by modifying the structure of the most effective class A compound, A-4. Most of these class B compounds showed better binding affinity to the target proteins than the class A compounds had. They also showed themselves more effective than ABT-737 at inhibiting growth in multiple tumor cell lines known to express Bcl-x(L), Bcl-2, and Mcl-1 proteins at high levels. Compounds B-11 and B-12 had the strongest anti-tumor activity of any compounds we produced. This study suggests that it is feasible to design small-molecule inhibitors based on the structure of Bim BH3, which shows broad-spectrum binding to Bcl-x(L), Bcl-2, and Mcl-1 proteins. Our results also suggest that the broad-spectrum properties of small-molecule inhibitors binding to target proteins play a critical role in inhibiting the growth of many tumor cells. Finally, our study provides a series of lead compounds that merit further research into anti-cancer therapeutics.

Synthesis and Antifungal Activities in vitro of Novel Pyrazino (2,1-a) Isoquinolin Derivatives

A series of novel pyrazino[2,1-a]isoquinolin compounds were designed and synthesized, and their antifungal activities in vitro were evaluated. The results showed that all of the compounds exhibited antifungal activities. Some of them exhibited stronger antifungal activities than that of lead compounds and among them compound 11b was the most potent one, which showed more potent than that of the active control fluconazole to the four of the five tested fungi. The studies presented here provide a new structural type for the development of novel antifungal agents.

2-Amino-nonyl-6-methoxyl-tetralin muriate inhibits sterol C-14 reductase in the ergosterol biosynthetic pathway

AbstractAim:To investigate the action mechanism of a novel chemical structural aminotetralin derivate, 2-Amino-Nonyl-6-Methoxyl-Tetralin Muriate (10b), against Candida albicans (C albicans) in the ergosterol biosynthetic pathway.Methods:Antifungal susceptibility test of 10b was carried out using broth microdilution method, the action mechanism of 10b against C albicans was investigated by GC-MS spectrometry and real-time RT-PCR assay, and cytotoxicity of 10b in vitro was assessed by MTS/PMS reduction assay.Results:10b reduced the ergosterol content markedly, and the 50% ergosterol content inhibitory concentration (ECIC50 value) was 0.08 μg/mL. Although the sterol composition of 10b-grown cells was completely identical with that of erg24 strain, the content of ergosta-8,14,22-trienol in 10b-grown cells was much higher than that in erg24 strain. Real-time RT-PCR assay revealed a global upregulation of sterol metabolism genes. In addition, the 50% inhibitory concentration (IC50 value) of 10b was 11.30 μg/mL for murine embryonic fibroblasts and 35.70 μg/mL for human normal liver cells.Conclusion:10b possessed a mode of action different from that of azoles and morpholines, whose targets were sterol C-14 reductase (encoded by ERG24 gene) and sterol C-5 desaturase (encoded by ERG3) related enzyme. Although 10b seemed to reduce MTS/PMS reduction in a dose dependent manner, IC50 value for mammalian cells was much higher than 50% minimum inhibitory concentration (MIC50) value for C albicans. This indicates that the formulation is preliminarily safe and warrants further study for possible human applications.

Synthesis and biological evaluation of 1-benzylidene-3,4-dihydronaphthalen-2-one as a new class of microtubule-targeting agents.

A series of 1-benzylidene-3,4-dihydronaphthalen-2-one derivatives were designed and synthesized, and their biological activities in vitro and in vivo were evaluated. The results showed a number of the title compounds exhibiting potent nanomolar activity in several human cancer cell lines. Of these, compound 22b showed the strongest inhibitory activity against human CEM, MDA-MBA-435, and K562 cells (IC(50) = 1 nM), displayed in vitro inhibition of tubulin polymerization (IC(50) = 3.93 μM), and significantly induced cell cycle arrest in G2/M phase. In addition, compound 22b could inhibit the tumor growth in colon nude mouse xenograft tumor model significantly and seemed safer than CA-4 when achieving a similar tumor suppression. This study provided a new molecular scaffold for the further development of antitumor agents that target tubulin.

Synthesis and Biological Evaluation of 1‐Phenyl‐1,2,3,4‐dihydroisoquinoline Compounds as Tubulin Polymerization Inhibitors

A series of 1‐phenyl‐3,4‐dihydroisoquinoline derivatives and several 1‐phenyl‐1,2,3,4‐tetrahydroisoquinoline, 1‐phenyl‐isoquinoline analogues were synthesized, and their cytotoxicity and tubulin polymerization inhibitory activity were evaluated. The 1‐phenyl‐3,4‐dihydroisoquinoline compounds were found to be potential tubulin polymerization inhibitors. Compound 5n, bearing a 3′OH and 4′OCH3 substituted 1‐phenyl B‐ring, was shown to confer optimal bioactivity. The single‐crystal structure of 5n was further determined by X‐ray diffraction, and the binding mode of 5n to tubulin was obtained by molecular docking, which can explain the structure–activity relationships. The studies presented here provide a new structural type for the development of novel antitumor agents.

Synthesis and acrosin inhibitory activity of substituted 4-amino-N-(diaminomethylene) benzenesulfonamide derivatives.

A series of new substituted 4-amino-N-(diaminomethylene) benzenesulfonamides were synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds showed potent acrosin inhibitory activities with compounds 4o and 4p being significantly more potent than the control compound N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK). The compounds provide a new scaffold for the development of acrosin inhibitory agents.

Quantum Chemistry Calculation-Aided Structural Optimization of Combretastatin A-4-like Tubulin Polymerization Inhibitors: Improved Stability and Biological Activity

A potent combretastatin A-4 (CA-4) like tubulin polymerization inhibitor 22b was found with strong antitumor activity previously. However, it easily undergoes cis-trans isomerization under natural light, and the resulting decrease in activity limits its further applications. In this study, we used quantum chemistry calculations to explore the molecular basis of its instability. Aided by the calculations, two rounds of structural optimization of 22b were conducted. Accelerated quantitative light stability testing confirmed that the stability of these designed compounds was significantly improved as predicted. Among them, compounds 1 and 3b displayed more potent inhibitory activity on tumor cell growth than 22b. In addition, the potent in vivo antitumor activity of compound 1 was confirmed. Quantum chemistry calculations were used in the optimization of stilbene-like molecules, providing new insight into stilbenoid optimization and important implications for the future development of novel CA-4-like tubulin polymerization inhibitors.

Synthesis and acrosin inhibitory activities of substituted ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives

A series of novel ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives were designed and synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds exhibited acrosin inhibitory activities. Among them, three compounds (5l, 5n, and 5v) were more potent than that of the control TLCK. These provide a new structural type for the development of novel contraceptive acrosin inhibitory agents.

Luteolin from Flos Chrysanthemi and its derivatives: New small molecule Bcl-2 protein inhibitors.

Over-expression of the Bcl-2 anti-apoptotic proteins is closely related to tumorigenesis and associated with drug resistance. Here we report that luteolin, a main substance found in Flos Chrysanthemi, directly binds to and shows inhibitory activity against the Bcl-2 protein. We studied the binding mode of luteolin and its derivatives with target proteins, their structure-activity relationship, and their effect on the human leukemia cell line HL-60. The results suggest that luteolin and its derivatives with a benzyl group introduced to the B ring, are new small molecule Bcl-2 protein inhibitors, and their anti-tumor activity is likely related to their effect on the Bcl-2 protein.