Targeting G-rich sequence to regulate the transcription of murine double minute (MDM) genes in triple-negative breast cancers.

Murine double minute 2 (MDM2) and homologous protein murine double minute X (MDMX) are p53 negative regulators that perform significant driving effects in tumorigenesis, and targeting these oncoproteins has became an efficient strategy in treating cancers. However, the definite antitumor activity and significance ordering of each protein in MDM family is still unclear due to the similar structure and complicated regulation. Herein, we identified two G-rich sequences (G1 and G5) located in the promoter that could assemble the G-quadruplex to respectively inhibit and promote the transcription of the MDM2 and MDMX. Based on this target, we designed and synthesized a novel G-quadruplex ligand A3f and achieved the differentiated regulation of MDM protein. In triple-negative breast cancer (TNBC) cells, A3f could induce MDM2-dependent proliferation arrest and exhibit additive therapeutic effect with MDMX inhibitors. Overall, this study provided a novel strategy to regulate the transcription of MDM genes by targeting certain G-rich sequences, and discovered an active antitumor molecule for use in TNBC treatment.

Discovery of novel human acrosin inhibitors by virtual screening.

Human acrosin is an attractive target for the discovery of male contraceptive drugs. For the first time, structure-based drug design was applied to discover structurally diverse human acrosin inhibitors. A parallel virtual screening strategy in combination with pharmacophore-based and docking-based techniques was used to screen the SPECS database. From 16 compounds selected by virtual screening, a total of 10 compounds were found to be human acrosin inhibitors. Compound 2 was found to be the most potent hit (IC(50) = 14 µM) and its binding mode was investigated by molecular dynamics simulations. The hit interacted with human acrosin mainly through hydrophobic and hydrogen-bonding interactions, which provided a good starting structure for further optimization studies.

Three-dimensional model of lanosterol 14 alpha-demethylase from Cryptococcus neoformans: active-site characterization and insights into azole binding.

Cryptococcus neoformans is one of the most important causes of life-threatening fungal infections in immunocompromised patients. Lanosterol 14 alpha-demethylase (CYP51) is the target of azole antifungal agents. This study describes, for the first time, the 3-dimensional model of CYP51 from Cryptococcus neoformans (CnCYP51). The model was further refined by energy minimization and molecular-dynamics simulations. The active site of CnCYP51 was well characterized by multiple-copy simultaneous-search calculations, and four functional regions important for rational drug design were identified. The mode of binding of the natural substrate and azole antifungal agents with CnCYP51 was identified by flexible molecular docking. A G484S substitution mechanism for azole resistance in CnCYP51, which might be important for the conformation of the heme environment, is suggested.

Homology modeling and molecular dynamics simulation of N-myristoyltransferase from protozoan parasites: active site characterization and insights into rational inhibitor design.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is a cytosolic monomeric enzyme that catalyzes the transfer of the myristoyl group from myristoyl-CoA to the N-terminal glycine of a number of eukaryotic cellular and viral proteins. Recent experimental data suggest NMT from parasites could be a promising new target for the design of novel antiparasitic agents with new mode of action. However, the active site topology and inhibitor specificity of these enzymes remain unclear. In this study, three-dimensional models of NMT from Plasmodium falciparum (PfNMT), Leishmania major (LmNMT) and Trypanosoma brucei (TbNMT) were constructed on the basis of the crystal structures of fungal NMTs using homology modeling method. The models were further refined by energy minimization and molecular dynamics simulations. The active sites of PfNMT, LmNMT and TbNMT were characterized by multiple copy simultaneous search (MCSS). MCSS functional maps reveal that PfNMT, LmNMT and TbNMT share a similar active site topology, which is defined by two hydrophobic pockets, a hydrogen-bonding (HB) pocket, a negatively-charged HB pocket and a positively-charged HB pocket. Flexible docking approaches were then employed to dock known inhibitors into the active site of PfNMT. The binding mode, structure-activity relationships and selectivity of inhibitors were investigated in detail. From the results of molecular modeling, the active site architecture and certain key residues responsible for inhibitor binding were identified, which provided insights for the design of novel inhibitors of parasitic NMTs.

[Nandeshi: a powerful inhibitor of human acrosin activity].

OBJECTIVE: To evaluate the inhibitory effect of Nandeshi, an acrosin inhibitor, on human acrosin activity. METHODS: We collected sperm samples from 10 healthy fertile men and cultured them with Nandeshi at 30 degrees C for 5 minutes at the concentrations of 0. 100, 0.120, 0.144, 0.173, 0.207, 0.249, 0.299, 0.358 and 0.430 mmol/L, with the controls treated with a well-known acrosin inhibitor N-alpha-p-tosyl-L-lysine chloromethylketone (TLCK) at 150.0, 189.8, 213.6, 240.3, 270.3, 304.1 and 342.1 mmol/L. Then we determined the residual activity of human acrosin by improved Kennedy assay. RESULTS: The residual activity of acrosin was negatively correlated with the Nandeshi concentration, and Nandeshi exhibited an inhibition rate about 800 times that of TLCK. CONCLUSION: Nandeshi has a powerful inhibitory effect on human acrosin, and improved Kennedy assay is a simple, practical and highly sensitive technique for the detection of human acrosin activity.

Synthesis and antifungal activities of novel 2-aminotetralin derivatives.

Novel 2-aminotetralin derivatives were synthesized as antifungal agents. The 2-aminotetralin scaffold was chemically designed to mimic the tetrahydroisoquinoline ring of the lead molecule described before. Their antifungal activities were evaluated in vitro by measuring the minimal inhibitory concentrations (MICs). Compounds 10a, 12a, 12c, 13b, and 13d are more potent than fluconazole against seven testing human fungal pathogens. Compound 10b exhibits much higher antifungal activities against all of the four fluconazole-resistant clinic Candida albicans strains than the control drugs including amphotericin B, terbinafine, ketoconazole, and itraconazole. The mode of action of some compounds to the potential receptor lanosterol 14alpha-demethylase (CYP51) was investigated by molecular docking. The studies presented here provide a new structural type for the development of novel antifungal compounds. Furthermore, 10b was evaluated in vivo by a rat vaginal candidiasis model, and it was found that 10b significantly decreases the number of fungal colony counts.

3D-QSAR and molecular docking studies on benzothiazole derivatives as Candida albicans N-myristoyltransferase inhibitors.

N-Myristoyltransferase has been a promising new target for the design of novel antifungal agents with new mode of action. Molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) methods, CoMFA and CoMSIA, were applied to a set of novel benzothiazole Candida albicans N-myristoyltransferase (CaNmt) inhibitors. The binding mode of the compounds at the active site of CaNmt was explored using flexible docking method and various hydrophobic and hydrogen-bonding interactions were observed between the benzothiazole inhibitors and the target enzyme. The best CoMFA and CoMSIA models had a cross-validated coefficient q(2) of 0.733 and 0.738, respectively, which showed high correlative and predictive abilities on both the test set and training set. The 3D contour maps of CoMFA and CoMSIA provided smooth and interpretable explanation of the structure-activity relationship for the compounds. The analysis of the 3D contour plots permitted interesting conclusions about the effects of different substituent groups at different position of the benzothiazole ring, which will guide the design of novel CaNmt inhibitors with higher activity.

Structure-based optimization of azole antifungal agents by CoMFA, CoMSIA, and molecular docking.

In a continuing effort to develop highly potent azole antifungal agents, the three-dimensional quantitative structure-activity relationship methods, CoMFA and CoMSIA, were applied using a set of novel azole antifungal compounds. The binding mode of the compounds at the active site of lanosterol 14alpha-demethylase was further explored using the flexible docking method. Various hydrophobic, van der Waals, pi-pi stacking, and hydrogen bonding interactions were observed between the azoles and the enzyme. Based on results from the molecular modeling, a receptor-based pharmacophore model was established to guide the rational optimization of the azole antifungal agents. Thus, a total of 57 novel azoles were designed and synthesized by a three-step optimization process. In vitro antifungal assay revealed that the antifungal activities of these novel azoles were greatly improved, which confirmed the reliability of the model from molecular modeling.

Evolutionary trace analysis of eukaryotic DNA topoisomerase I superfamily: identification of novel antitumor drug binding site.

The studies of novel inhibitors of DNA topoisomerase I (Topo I) have already become very promising in cancer chemotherapy. Identifying the new drug-binding residues is playing an important role in the design and optimization of Topo I inhibitors. The designed compounds may have novel scaffolds, thus will be helpful to overcome the toxicities of current camptothecin (CPT) drugs and may provide a solution to cross resistance with these drugs. Multiple sequence alignments were performed on eukaryotic DNA topoisomerase I superfamily and thus the evolutionary tree was constructed. The Evolutionary Trace method was applied to identify functionally important residues of human Topo I. It has been demonstrated that class-specific hydrophobic residues Ala351, Met428, Pro431 are located around the 7,9-position of CPT, indicating suitable substitution of hydrophobic group on CPT will increase antitumor activity. The conservative residue Lys436 in the superfamily is of particular interest and new CPT derivatives designed based on this residue may greatly increase water solubility of such drugs. It has also been demonstrated that the residues Asn352 and Arg364 were conservative in the superfamily, whose mutation will render CPT resistance. As our molecular docking studies demonstrated they did not make any direct interaction with CPT, they are important drug-binding site residues for future design of novel non-camptothecin lead compounds. This work provided a strong basis for the design and synthesis of novel highly potent CPT derivatives and virtual screening for novel lead compounds.

Structure-based de novo design, synthesis, and biological evaluation of non-azole inhibitors specific for lanosterol 14alpha-demethylase of fungi.

The active site of lanosterol 14alpha-demethylase (CYP51) was investigated via MCSS functional group mapping and LUDI calculations. Several non-azole lead molecules were obtained by coupling structure-based de novo design with chemical synthesis and biological evaluation. All of the lead molecules exhibited a strong inhibitory effect on CYP51 of Candida albicans. They occupy the substrate-binding site and interfere with the binding of azole antifungal agents in a competitive manner. The mode of action of the lead molecules was validated by spectrophotomeric analysis and SAR studies. This is the first successful example reported for the inhibitor design of the cytochrome P450 superfamily using the de novo design strategy. Because the affinity of the lead molecules for CYP51 was mainly attributed to their nonbonding interaction with the apoprotein, the studies presented here afford the opportunity to develop novel antifungal agents that specifically interact with the residues in the active site and avoid the serious toxicity arising from coordination binding with the heme of mammalian P450s.

A Three-dimensional Model of Lanosterol 14alpha-demethylase of Candida albicans.

The three-dimensional structure of lanosterol 14alpha demethylase (P450(14DM), P450(51() of Candida albicans was modeled based on crystallographic coordinates of four prokaryotic cytochrome P450(S): P450BM3, P450cam, P450terp and P450eryF. The sequence of P450(51) was aligned to those of known proteins using a knowledge-based alignment method. The main chain coordinates of the structurally conserved regions (SCRs)) were transferred directly from the corresponding coordinates of P450BM3. The side chain conformations of SCRs) were determined based on the equivalent residues of four crystal structures which had the highest homologous scores. The model was then refined using molecular mechanics and molecular dynamics.The rationale of the resulting model were validated by Ramachandran plot,Profile-3K and hydropathy plot analysis. The structure-functionally important residues, such as the heme binding residues, the residues interacting with redox-partner protein and/or involved in electron transfer, the residues lining substrate access channel and the substrate binding residues, were identified from the model. These residues are candidates for further site-directed mutagensis and site-specific antipeptide antibody binding experiments.

Comparative Studies on Crystal Structures of Four Members of Cytochrome P450 Superfamily.

Secondary and steric structures and hydropathy plots of the 4 crystals of P450cam, P450terp, P450eryF and P450BM3 were compared to illustrate the structural conservation of cytochrome P450 superfamily proteins. Although sequence identities of four P450s are generally low (19%-26%), their topology is quite similar. All four structures have 13 alpha-helices and beta1-beta4 sheets in common. Four crystal structures were superimpossed by root-mean-square (RMS) fit of the prophyrin ring carbon atoms of prosthetic group heme to obtain the structure-based sequence alignment of four proteins. The RMS deviations of Calpha distance of each motifs were analyzed by hierarchical cluster analysis. The structural subsets were divided into four categories of structural conservation: the most conserved region, the less conserved region, the less variable region and the variable region. The first two groups (56.9 percentage of the aligned positions that have no gaps) include all the interior structures and active site residues. All four P450 proteins have the common hydrophobic and hydrophilic segments by hydropathy plots analyses. All the comparison results of P450 protein crystal structures provided the basis for structure-based sequence alignment of cytochrome P450 proteins.

[Synthesis and antifungal activity of 1-(1,2,4-triazolyl-1H-1-yl)-2-(2,4-diflurophenyl)-3-(4-substituted benzyl-1-piperazinyl)-2-propanols].

AIM: A series of triazole antifungals were synthesized to search for novel triazole antifungals with more potent activity, less toxicity and broader spectrum. METHODS: Nineteen 1-(1,2,4-triazolyl-1H-1-yl)-2-(2,4-diflurophenyl)-3-(4-substituted benzyl-1-piperazinyl)-2-propanols were designed and synthesized, on basis of the three dimensional structure of P450 cytochrome 14 alpha-sterol demethylase (CYP51) and their antifungal activities were also evaluated. RESULTS: All the title compounds were first reported. Results of preliminary biological tests showed that most of the title compounds exhibited high activity against the eight common pathogenic fungi and the activities against deep fungi were higher than that against shallow fungi. CONCLUSION: Most of the title compounds showed higher antifungal activities than Fluconazole and Terbinafine. Compound VIII-1, 10, 12, 17 showed best antifungal activity with broad antifungal spectrum and were chosen for further development.

Evolutionary trace analysis of CYP51 family: implication for site-directed mutagenesis and novel antifungal drug design.

Lanosterol 14alpha-demethylase (CYP51) is an essential enzyme in the fungal life cycle and also an important target for the antifungal drug development. Based on the multiple sequence alignments of CYP51 family, an evolutionary tree of the CYP51 family was constructed by the evolutionary trace (ET) method. The identified trace residues could provide a reliable and rational guide to the design of CYP51 mutations and give more information about the detailed mechanism of substrate (drug) recognition and binding. The reliability of ET analysis to identify residues of functional importance was validated by the reported site-directed mutagenesis studies of CYP51s. Several residues in the active site were also validated by our mutagenesis studies. Mapping the identified trace residues onto the active site of the modeled structure of Candida albicans CYP51 (CACYP51) may provide useful information for the design of novel antifungal agents.

Construction of a three-dimensional pharmacophore for Bcl-2 inhibitors by flexible docking and the multiple copy simultaneous search method.

B-Cell lymphoma-2 (Bcl-2) protein is a new promising target for anticancer drugs. A number of anticancer Bcl-2 inhibitors with diverse chemical structures have been discovered in recent years. In this paper, the flexible docking was performed to determine the binding modes of the representative inhibitors from different structural types. Subsequently, the binding modes of inhibitor were used to construct a primary three- dimensional (3D) pharmacophore model. It proved that this model can effectively disrupt the binding of the BH3 domain of proapoptotic Bcl-2 family members to Bcl-2, and match the structural requirement of a new type of Bcl-2 inhibitors. However, these distances between pharmacophoric points are not optimal due to the fact that not all of individual functional groups are located in the ideal position when inhibitors bind to its receptor. In this paper, we proposed a new idea to improve the quality of the pharmacophore model: the multiple copy simultaneous search (MCSS) method was performed to determine the energetically favorable distribution of functional groups with similar features to these pharmacophoric points in the active site of Bcl-2 first. Then their most energetically favorable minima in the positions near the pharmacophoric points were used to optimize the distances between pharmacophoric points. By examining the binding modes of several inhibitors from the same structural type, it was found that the more potent the inhibitor was, the closer it was to the optimized distances between pharmacophoric points. The optimized 3D pharmacophore model obtained in this paper may provide a good starting point for further rational design of Bcl-2 inhibitors.

Design, synthesis, and antifungal activities in vitro of novel tetrahydroisoquinoline compounds based on the structure of lanosterol 14alpha-demethylase (CYP51) of fungi.

Novel tetrahydroisoquinoline compounds were designed by coupling structure-based de novo design based on the structure of lanosterol 14alpha-demethylase (CYP51). The chemical synthesis and the antifungal activities in vitro of them were reported. The results exhibited that all of the lead compounds showed potent antifungal activities, in which compounds 6 and 7 had equal or stronger antifungal activities against five test fungi than that of fluconazole. The studies presented here provided the antifungal lead compounds. The affinity of the lead molecules for CYP51 was mainly attributed to their non-bonding interaction with the apoprotein, which was different from the azole antifungal agents.