The Discovery of Novel PGK1 Activators as Apoptotic Inhibiting and Neuroprotective Agents.

Stroke is the second leading cause of death worldwide and the leading cause of long-term disability that seriously endangers health and quality of human life. Tissue-type fibrinogen activator is currently the only drug approved by FDA for the treatment of ischemic stroke. Neuroprotection is theoretically a common strategy for the treatment of both ischemic and hemorrhagic stroke; therefore, the development of neuroprotective agent has been the focus of research. However, no ideal neuroprotective drug is clinically available. Phosphoglycerate kinase-1 (PGK1) activator has the effect of inhibiting apoptosis and protecting tissue damage, and therefore could be a potential neuroprotective agent. To obtain effective PGK1 activators, we virtually screened a large chemical database and their evaluated the efficacy by the Drosophila oxidative stress model, PGK1 enzymatic activity assay, and oxygen-glucose stripping reperfusion (OGD/R) model. The results showed that compounds 7979989, Z112553128 and AK-693/21087020 are potential PGK1 activators with protective effects against PQ-induced oxidative stress in the Drosophila model and could effectively ameliorate apoptosis induced by OGD/R-induced neuronal cell injury. Additionally, compounds 7979989 and Z112553128 are effective in alleviating LPS-induced cellular inflammation. This study indicated that these compounds are promising lead compounds that provide theoretical and material basis to the neuroprotective drug discovery.

The Discovery of Novel BCR-ABL Tyrosine Kinase Inhibitors Using a Pharmacophore Modeling and Virtual Screening Approach.

Chronic myelogenous leukemia (CML) typically results from a reciprocal translocation between chromosomes 9 and 22 to produce the bcr-abl oncogene that when translated, yields the p210 BCR-ABL protein in more than 90% of all CML patients. This protein has constitutive tyrosine kinase activity that activates numerous downstream pathways that ultimately produces uncontrolled myeloid proliferation. Although the use of the BCR-ABL tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, dasatinib, bosutinib, and ponatinib have increased the overall survival of CML patients, their use is limited by drug resistance and severe adverse effects. Therefore, there is the need to develop novel compounds that can overcome these problems that limit the use of these drugs. Therefore, in this study, we sought to find novel compounds using Hypogen and Hiphip pharmacophore models based on the structures of clinically approved BCR-ABL TKIs. We also used optimal pharmacophore models such as three-dimensional queries to screen the ZINC database to search for potential BCR-ABL inhibitors. The hit compounds were further screened using Lipinski's rule of five, ADMET and molecular docking, and the efficacy of the hit compounds was evaluated. Our in vitro results indicated that compound ZINC21710815 significantly inhibited the proliferation of K562, BaF3/WT, and BaF3/T315I leukemia cells by inducing cell cycle arrest. The compound ZINC21710815 decreased the expression of p-BCR-ABL, STAT5, and Crkl and produced apoptosis and autophagy. Our results suggest that ZINC21710815 may be a potential BCR-ABL inhibitor that should undergo in vivo evaluation.

Identification of key residues determining the binding specificity of human 4-hydroxyphenylpyruvate dioxygenase.

4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is the second enzyme of the tyrosine catabolic pathway. Its physiological function is to catalyze the conversion of 4-hydroxyphenylpyruvic acid to homogentisic acid, which displays different physiological effects in mammals and plants. Insights on the selective inhibition of human HPPD (hHPPD) by triketone inhibitors were furnished by the integrated application of molecular simulation and biological testing. The binding free energy of hHPPD and inhibitors was obtained through molecular dynamics (MD) simulations, and the result was in agreement with the inhibition experiment in vitro. The binding free energy contribution demonstrated that the formation of hHPPD-inhibitor complexes was mainly driven by van der Waals energy. Ser226, Asn241, Gln265, Phe336, Phe359 and Phe364 made great contributions to binding affinities of all the systems. Among the residues involved in the interaction between nitisinone (NTBC) and hHPPD, Tyr221 and Leu224, whose mutation into Ala caused significant decrease of NTBC binding ability, were two key residues in determining the selective binding affinity of inhibitor and hHPPD. This work provides valuable theoretical basis for rational design of highly selective inhibitors targeting hHPPD.

Combined 3D-quantitative structure-activity relationships and topomer technology-based molecular design of human 4-hydroxyphenylpyruvate dioxygenase inhibitors.

Aim: 4-Hydroxyphenylpyruvate dioxygenase (HPPD) has attracted increasing attention as an important target against tyrosinemia type I. This paper aimed to explore the structure-activity relationship of HPPD inhibitors with pyrazole scaffolds and to design novel HPPD inhibitors. Methodology & results: The best 3D-quantitative structure-activity relationships model was established by two different strategies based on 40 pyrazole scaffold-based analogs. Screening of molecular fragments by topomer technology, combined with molecular docking, 14 structures were identified for potential human HPPD inhibitory activity. Molecular dynamics results demonstrated that all the compounds obtained bound to the enzyme and possessed a satisfactory binding free energy. Conclusion: The quantitative structure-activity relationship of HPPD inhibitors of pyrazole scaffolds was clarified and 14 original structures with potential human HPPD inhibitory activity were obtained.

Quantitative Structure Activity Relationship Studies and Molecular Dynamics Simulations of 2-(Aryloxyacetyl)cyclohexane-1,3-Diones Derivatives as 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a significant enzyme in the biosynthesis of plastoquinone and tocopherol. Moreover, it is also a potential target to develop new herbicide. The technology of computer-aided drug design (CADD) is a useful tool in the efficient discovery of new HPPD inhibitors. Forty-three compounds with known activities were used to generate comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models based on common framework and molecular docking. The structural contribution to the activity was determined, which provided further information for the design of novel inhibitors. Molecular docking was used to explain the changes in activity caused by the binding mode between ligand and protein. The molecular dynamics (MD) results indicated that the electrostatic energy was the major driving force for ligand-protein interaction and the Phe403 made the greatest contribution to the binding. The present work has provided useful information for the rational design of novel HPPD inhibitors with improved activity.

Combination of Virtual Screening Protocol by in Silico toward the Discovery of Novel 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is a potent new bleaching herbicide target. Therefore, in silico structure-based virtual screening was performed in order to speed up the identification of promising HPPD inhibitors. In this study, an integrated virtual screening protocol by combining 3D-pharmacophore model, molecular docking and molecular dynamics (MD) simulation was established to find novel HPPD inhibitors from four commercial databases. 3D-pharmacophore Hypo1 model was applied to efficiently narrow potential hits. The hit compounds were subsequently submitted to molecular docking studies, showing four compounds as potent inhibitor with the mechanism of the Fe(II) coordination and interaction with Phe360, Phe403, and Phe398. MD result demonstrated that nonpolar term of compound 3881 made great contributions to binding affinities. It showed an IC50 being 2.49 µM against AtHPPD in vitro. The results provided useful information for developing novel HPPD inhibitors, leading to further understanding of the interaction mechanism of HPPD inhibitors.

A Molecular Modeling Study of the Hydroxyflutamide Resistance Mechanism Induced by Androgen Receptor Mutations.

Hydroxyflutamide (HF), an active metabolite of the first generation antiandrogen flutamide, was used in clinic to treat prostate cancer targeting androgen receptor (AR). However, a drug resistance problem appears after about one year's treatment. AR T877A is the first mutation that was found to cause a resistance problem. Then W741C_T877A and F876L_T877A mutations were also reported to cause resistance to HF, while W741C and F876L single mutations cannot. In this study, molecular dynamics (MD) simulations combined with the molecular mechanics generalized Born surface area (MM-GBSA) method have been carried out to analyze the interaction mechanism between HF and wild-type (WT)/mutant ARs. The obtained results indicate that AR helix 12 (H12) plays a pivotal role in the resistance of HF. It can affect the coactivator binding site at the activation function 2 domain (AF2, surrounded by H3, H4, and H12). When H12 closes to the AR ligand-binding domain (LBD) like a lid, the coactivator binding site can be formed to promote transcription. However, once H12 is opened to expose LBD, the coactivator binding site will be distorted, leading to invalid transcription. Moreover, per-residue free energy decomposition analyses indicate that N705, T877, and M895 are vital residues in the agonist/antagonist mechanism of HF.

3D Pharmacophore-Based Virtual Screening and Docking Approaches toward the Discovery of Novel HPPD Inhibitors.

p-Hydroxyphenylpyruvate dioxygenase (HPPD) is not only the useful molecular target in treating life-threatening tyrosinemia type I, but also an important target for chemical herbicides. A combined in silico structure-based pharmacophore and molecular docking-based virtual screening were performed to identify novel potential HPPD inhibitors. The complex-based pharmacophore model (CBP) with 0.721 of ROC used for screening compounds showed remarkable ability to retrieve known active ligands from among decoy molecules. The ChemDiv database was screened using CBP-Hypo2 as a 3D query, and the best-fit hits subjected to molecular docking with two methods of LibDock and CDOCKER in Accelrys Discovery Studio 2.5 (DS 2.5) to discern interactions with key residues at the active site of HPPD. Four compounds with top rankings in the HipHop model and well-known binding model were finally chosen as lead compounds with potential inhibitory effects on the active site of target. The results provided powerful insight into the development of novel HPPD inhibitors herbicides using computational techniques.

New Research for Quinazoline-2,4-diones as HPPD Inhibitors Based on 2D-MLR and 3D-QSAR Models.

AIM AND OBJECTIVE: 4-Hydroxyphenylpyruvate dioxygenase (HPPD), converting phydroxyphenylpyruvate (HPPA) to homogentisate (HGA), is an important target for treating type I tyrosinemia and synthesizing novel herbicides due to its significant role in tyrosine catabolism. Hence, it is imperative to design novel HPPD inhibitors that can block HPPA-HGA conversion, which leads to the deficiency in isoprenoid redox cofactors such as plastoquinone and tocopherol, and finally caused growth inhibition. This study was undertaken to investigate structural requirements for their HPPD inhibition with better biological activity. MATERIALS AND METHODS: Based on the structure-activity relationships, a series of quinolinone-2,4- diones derivatives were studied using combined of 2D multiple linear regression (2D-MLR) and 3D quantitative structure-activity relationship (3D-QSAR). Firstly, genetic algorithm (GA) was applied and descriptors generated in DRAGON 5.5 software were used for building 2D-MLR models in the QSARINGS. Then CoMFA and CoMSIA models were performed by using alignment of the common framework and the pharmacophore model. The obtained models were validated through internal and external validation to verify predictive abilities. Especially, the CoMFA and CoMSIA contour maps were used to show vital structural characteristics related to HPPD inhibitors activities. RESULTS: The 2D-MLR liner equation and corresponding parameters were listed as follows: pKi = -38.2034Me+22.4078GATS2m-1.4265EEig15r-2.1849Hy+32.9158 ntr=28, npred=6, R2=0.863, Q2LOO=0.787, Q2LMO=0.607, Q2F1=0.780, Q2F2=0.780, Q2F3=0.860, CCCpred=0.920. RMSEtr=0.253, RMSEpred=0.555, F=36.289 The steric contours graph indicated that small and negative electrostatic substitutions at R1 and R2 regions were favorable for the better activity, and hydrogen-bond donors at this region would also increase the activity. Positive electrostatic and bulky substitutions in the R3 position would enhance the activity. The analysis of these models suggested that the steric factor of R4 position was crucial for activity of quinazoline-2,4-diones HPPD inhibitors, bulky substitutions might improve the bioactivity of these inhibitors greatly, meanwhile, hydrogen-bond acceptor groups in this position were required for higher activity. CONCLUSION: In this study, a combined 2D-MLR, CoMFA and CoMSIA models demonstrated satisfying results through internal and external validation, especially good predictive abilities and the CoMFA and CoMSIA contour maps showed vital structural characteristics related to HPPD inhibitors activities.

Metabolomic evaluation for anti-inflammatory effect of volatile oils from different preparations of Angelicae Sinensis Radix / 中国中药杂志

To evaluate the anti-acute inflammation effects of volatile oils from different processed products of Angelicae Sinensis Radix(AS) in the rat model of acute inflammation established by the metabolomic method. Volatile oil of charred AS (C-VOAS), wine-processed AS (J-VOAS), locally processed AS (T-VOAS) and oil-process AS (Y-VOAS) were applied to intervene the rat acute paw swelling inflammation model induced by Carrageenan. Changes in serum HIS, 5-HT, PGE2 and TNF-α content in rats were detected. Gas chromatography-mass spectrometry was used to detect the metabolites in plasma. Potential biomarkers were investigated according to principal component analysis method and partial least-squares discriminant analysis. According to the results, C-VOAS and J-VOAS could significantly inhibit inflammatory mediators Histamine, 5-hydroxytryptamine, prostaglandin-E2 and cytokine tumor necrosis factor-alpha (P<0.01), and T-VOAS and Y-VOAS also showed a significantly inhibitory effect (P<0.05). Compared with the normal group, 14 endogenous metabolite biomarkers showed metabolic disturbance in plasma (P<0.05 or P<0.01). Compared with acute inflammation model group, C-VOAS and J-VOAS could better recover the levels of the endogenous metabolites (P<0.05 or P<0.01) than T-VOAS and Y-VOAS (P<0.05 or P<0.01). This study suggests that C-VOAS and J-VOAS show a better anti-inflammatory effect than T-VOAS and Y-VOAS. Therefore, the metabolomic method could be used to expound the anti-inflammatory mechanism of volatile oils from different processed products of AS, and provide a theoretical basis for clinical application of VOAS.

[Extraction, identification and primary investigation of adhesion specificity of Lactobacillus sp].

Lactobacillus L15, a strain of Lactobacillus sp. isolated from the intestinal tract of healthy Paralichthys olivaceus, and Lactobacillus acidlophilus ATCC4356 were used to investigate their specific adhesions. It was shown that cell surface proteins of two strains could be extracted by 5 mol/L lithium chloride( LiCl). Two surface proteins with molecular weights of 61.8 kDa and 54.6 kDa were identified to mediate the adhesion to intestinal mucus of Paralichthys olivaceus and Common carp respectively by the method of Western blotting, and the two novel proteins were named as MAPPpo1 and MAPPcc. In Lactobacillus acidlophilus ATCC4356, two proteins of 43.0 kDa and 63.3 kDa were identified to mediate the adhesion to intestinal mucus of Paralichthys olivaceus. Nevertheless, only 43.0 kDa protein was identified to mediate the adhesion to intestinal mucus of Common carp. Furthermore, the mucus proteins which participated the adhesion as the bacteria receptors were identified by the same method and it was found that L15 and ATCC4356 had the same receptors.There were 29.7 kDa and 30.3 kDa proteins in the intestinal mucus of Paralichthys olivaceus and only the 26.2 kDa protein was testified in the intestinal mucus of Common carp. It is demonstrated that the adhesion of Lactobacillus to mucus is dependent on the strain specificity, as well as host specificity.