Comparative Study of Binding Pockets in Human CYP1A2, CYP3A4, CYP3A5, and CYP3A7 with Aflatoxin B1, a Hepato-Carcinogen, by Molecular Dynamics Simulation & Principal Component Analysis.

BACKGROUND: Aflatoxin B1 is a harmful hepatocarcinogen which is metabolized in our body by Cytochrome P450 enzymes, namely CYP1A2, CYP3A4, CYP3A5, and CYP3A7, into toxic (exo-8, 9-epoxide) and nontoxic (AFQ1, endo-epoxide) products. We have found from the literature that due to cooperativity, the rate of metabolic reactions increases in CYP1A2 and CYP3A4 involving more than one site of proteins to form two products at a given time, whereas the interaction of CYP3A5 and CYP3A7 is still unknown. Our work aims to study these four enzymes with AFB1 based on binding site pocket characterization and to find the probable resultant products at each binding site. METHODS: We used computational approaches like homology modeling, molecular docking to form mono and double ligated systems, molecular dynamic simulations to analyze the potential energies (vdW & electrostatic), PCA, RMSF, and residue-wise interactions at the active as well as allosteric sites of these four enzymes. RESULTS: We found that CYP1A2, CYP3A4, and CYP3A5 were more hydrophobic at the first site and may induce epoxidation reaction to form toxic products, whereas the second site would be expected to be more polar and comprising charged interactions, thus enhancing non-toxic hydroxylated products. However, in CYP3A7, the first site favors hydroxylation, whereas the second site is involved in higher hydrophobic interactions. CONCLUSION: Thus, in the fetus where AFB1 is metabolized only by CYP3A7, a lower concentration of toxic metabolites will be expected, while in adults exhibiting CYP1A2, CYP3A4 and CYP3A5 may increase the concentration of the toxic metabolites due to the combined effect of these enzymes, consequently increasing liver toxicity. We believe that AFB1 binding characteristics will be helpful for medicinal chemists in the process of designing a new drug.

Identification of a less toxic vinca alkaloid derivative for use as a chemotherapeutic agent, based on in silico structural insights and metabolic interactions with CYP3A4 and CYP3A5.

Vinca alkaloids are chemotherapeutic agents used in the treatment of both pediatric and adult cancer patients. Cytochrome P450 3A5 (CYP3A5) is 9- to 14-fold more efficient at clearing vincristine than cytochrome P450 3A4 (CYP3A4) is. However, patients who express an inactive form of the polymorphic CYP3A5 enzyme suffer from severe neurotoxicity during vincristine treatment, resulting in chemotherapy failure. Previous studies have found that the addition of new features to the parent drug can enhance its binding affinity to tubulin manyfold and could therefore yield novel anticancer drugs. However, there is no report of any study of the metabolic activities of CYP3A4 and CYP3A5 with respect to vincristine and vinblastine, so we studied the interactions of these two drugs and 15 vinca derivatives with CYP3A4 and CYP3A5 by performing docking studies using GOLD. Six of the vinca derivatives in complexes with CYP3A4 and CYP3A5 were further investigated in 100-ns molecular dynamic simulations. Interaction energies, hydrogen bonds, and linear interaction energies were calculated and principal component analysis was carried out to visualize the binding interface in each complex. The results indicate that the addition of dimethylurea at the C20' position in vincristine may increase its binding affinity and lead to enhanced interactions with the less polymorphic CYP3A4 rather than CYP3A5. Thus, dimethylurea vincristine may be a useful drug in cancer chemotherapy treatment as it should be significantly less likely than vincristine to induce severe neurotoxicity in patients. Graphical Abstract Proposed modification of Vinca alkaloid derivatives to decrease the neurotoxicity level in cancer patients exhibiting CYP3A4 gene rather than polymorphic CYP3A5 gene.

Structural Dynamics Investigation of Human Family 1 & 2 Cystatin-Cathepsin L1 Interaction: A Comparison of Binding Modes.

Cystatin superfamily is a large group of evolutionarily related proteins involved in numerous physiological activities through their inhibitory activity towards cysteine proteases. Despite sharing the same cystatin fold, and inhibiting cysteine proteases through the same tripartite edge involving highly conserved N-terminal region, L1 and L2 loop; cystatins differ widely in their inhibitory affinity towards C1 family of cysteine proteases and molecular details of these interactions are still elusive. In this study, inhibitory interactions of human family 1 & 2 cystatins with cathepsin L1 are predicted and their stability and viability are verified through protein docking & comparative molecular dynamics. An overall stabilization effect is observed in all cystatins on complex formation. Complexes are mostly dominated by van der Waals interaction but the relative participation of the conserved regions varied extensively. While van der Waals contacts prevail in L1 and L2 loop, N-terminal segment chiefly acts as electrostatic interaction site. In fact the comparative dynamics study points towards the instrumental role of L1 loop in directing the total interaction profile of the complex either towards electrostatic or van der Waals contacts. The key amino acid residues surfaced via interaction energy, hydrogen bonding and solvent accessible surface area analysis for each cystatin-cathepsin L1 complex influence the mode of binding and thus control the diverse inhibitory affinity of cystatins towards cysteine proteases.

Differential Interactions of Cytochrome P450 3A5 and 3A4 with Chemotherapeutic Agent-Vincristine: A Comparative Molecular Dynamics Study.

The chemotherapeutic agent vincristine, used for treatment of acute lymphoblastic leukemia is metabolized preferentially by polymorphic cytochrome P450 3A5 (CYP3A5) with higher clearance rate than cytochrome P450 3A4 (CYP3A4). As a result, CYP3A5 expressers have a reduced amount of vincristine-induced peripheral neuropathy than non-expressers. We modeled the structure of CYP3A5 and its interaction with vincristine, compared with CYP3A4-vincristine complex using molecular docking and simulation studies. This relative study helped us to understand the molecular mechanisms behind the interaction at the atomic level through interaction energy, binding free energy, hydrogen bond and solvent accessible surface area analysis - giving an insight into the binding mode and the main residues involved in this particular interaction. Our results show that the interacting groups get closer in CYP3A5-vincristine complex due to different orientation of vincristine. This leads to higher binding affinity of vincristine towards CYP3A5 compared to CYP3A4 and explains the preferential metabolism of vincristine by CYP3A5. We believe that, the results of the current study will be helpful for future studies on structure-based drug design in this area.

Modelling family 2 cystatins and their interaction with papain.

Cystatins are extensively studied cysteine protease inhibitors, found in wide range of organisms with highly conserved structural folds. S-type of cystatins is well known for their abundance in saliva, high selectivity and poorer activity towards host cysteine proteases in comparison to their immediate ancestor cystatin C. Despite more than 90% sequence similarity, the members of this group show highly dissimilar binding affinity towards papain. Cystatin M/E is a potent inhibitor of legumain and papain like cysteine proteases and recognized for its involvement in skin barrier formation and potential role as a tumor suppressor gene. However, the structures of these proteins and their complexes with papain or legumain are still unknown. In the present study, we have employed computational methods to get insight into the interactions between papain and cystatins. Three-dimensional structures of the cystatins are generated by homology modelling, refined with molecular dynamics simulation, validated through numerous web servers and finally complexed with papain using ZDOCK algorithm in Discovery Studio. A high degree of shape complementarity is observed within the complexes, stabilized by numerous hydrogen bonds (HB) and hydrophobic interactions. Using interaction energy, HB and solvent accessible surface area analyses, we have identified a series of key residues that may be involved in papain-cystatin interaction. Differential approaches of cystatins towards papain are also noticed which are possibly responsible for diverse inhibitory activity within the group. These findings will improve our understanding of fundamental inhibitory mechanisms of cystatin and provide clues for further research.

DB Dehydrogenase: an online integrated structural database on enzyme dehydrogenase.

UNLABELLED: Dehydrogenase enzymes are almost inevitable for metabolic processes. Shortage or malfunctioning of dehydrogenases often leads to several acute diseases like cancers, retinal diseases, diabetes mellitus, Alzheimer, hepatitis B & C etc. With advancement in modern-day research, huge amount of sequential, structural and functional data are generated everyday and widens the gap between structural attributes and its functional understanding. DB Dehydrogenase is an effort to relate the functionalities of dehydrogenase with its structures. It is a completely web-based structural database, covering almost all dehydrogenases [~150 enzyme classes, ~1200 entries from ~160 organisms] whose structures are known. It is created by extracting and integrating various online resources to provide the true and reliable data and implemented by MySQL relational database through user friendly web interfaces using CGI Perl. Flexible search options are there for data extraction and exploration. To summarize, sequence, structure, function of all dehydrogenases in one place along with the necessary option of cross-referencing; this database will be utile for researchers to carry out further work in this field. AVAILABILITY: The database is available for free at http://www.bifku.in/DBD/