Comparison and analysis of the structures and binding modes of antifungal SE and CYP51 inhibitors

With the abuse of clinical broad-spectrum antimicrobial agents, immunosuppressive agents, chemotherapy drugs, the emergence of pathogenic fungi resistance is more and more frequent. However, there is still no effective treatment for the fungal resistance. Squalenee epoxidase (SE) and 14 α-demethylase (CYP51) are important antifungal drug targets. In order to achieve a deeper insight into the structural characteristics and the action modes of SE and CYP51inhibitors, the homology model of SE (Candida albicans) was constructed using monooxygenase of Pseudomonas aeruginosa as template, and the reliability of model was confirmed by Ramachandran plots and Verify 3D. Subsequently, the molecular superimposition and molecular docking were performed, and the pharmacophore model based on the CYP51 receptor structure was constructed. The results indicate that SE and CYP51 inhibitors have common structural feature with two parts of essential fragments, which are mainly composed of aromatic groups. In addition, the fragment structures of inhibitors are combined in the similar hydrophobic pockets through the hydrophobic forces. The present study provides a deeper perspective to understand the characteristics and docking modes of SE and CYP51 inhibitors. It can be used to guide the optimization and design of SE and CYP51 inhibitors. In addition, it also provides the oretical support for the development of dual target antifungal inhibitors (SE and CYP51), which can help us solve the problem of fungi resistance.     

In silico study of subtilisin-like protease 1 (SUB1) from different Plasmodium species in complex with peptidyl-difluorostatones and characterization of potent pan-SUB1 inhibitors

Plasmodium falciparum subtilisin-like protease 1 (SUB1) is a novel target for the development of innovative antimalarials. We recently described the first potent difluorostatone-based inhibitors of the enzyme ((4S)-(N-((N-acetyl-l-lysyl)-l-isoleucyl-l-threonyl-l-alanyl)-2,2-difluoro-3-oxo-4-aminopentanoyl)glycine (1) and (4S)-(N-((N-acetyl-l-isoleucyl)-l-threonyl-l-alanylamino)-2,2-difluoro-3-oxo-4-aminopentanoyl)glycine (2)). As a continuation of our efforts towards the definition of the molecular determinants of enzyme-inhibitor interaction, we herein propose the first comprehensive computational investigation of the SUB1 catalytic core from six different Plasmodium species, using homology modeling and molecular docking approaches. Investigation of the differences in the binding sites as well as the interactions of our inhibitors 1,2 with all SUB1 orthologues, allowed us to highlight the structurally relevant regions of the enzyme that could be targeted for developing pan-SUB1 inhibitors. According to our in silico predictions, compounds 1,2 have been demonstrated to be potent inhibitors of SUB1 from all three major clinically relevant Plasmodium species (P. falciparum, P. vivax, and P. knowlesi). We next derived multiple structure-based pharmacophore models that were combined in an inclusive pan-SUB1 pharmacophore (SUB1-PHA). This latter was validated by applying in silico methods, showing that it may be useful for the future development of potent antimalarial agents.     

In silico and in vitro screening to identify structurally diverse non-azole CYP51 inhibitors as potent antifungal agent

The problem of resistance to azole class of antifungals is a serious cause of concern to the medical fraternity and thus there is an urgent need to identify non-azole scaffolds with high affinity for lanosterol 14α-demethylase (CYP51). In view of this we have attempted to identify novel non-azole CYP51 inhibitors through the application of pharmacophore based virtual screening and in vitro evaluation. A rigorously validated pharmacophore model comprising of 2 hydrogen bond acceptor and 2 hydrophobic features has been developed and used to mine NCI database. Out of 265 retrieved hits, NSC 1215 and 1520 have been chosen on the basis of Lipinski’s rule of five, fit and estimated values. Both the hits were docked into the active site of CYP51. In view of high fit value and CDocker score, NSC 1215 and 1520 have been subjected to in vitro microbiological assay. The result reveals that NSC 1215 and 1520 are active against Candida albicans, Candida parapsilosis, Candida tropicalis, and Aspergillus niger. In addition to this the absorption characteristics of both the hits have also been determined using the rat sac technique and permeation in order of NSC 1520 > NSC 1215 has been observed.     

Molecular docking and molecular dynamics simulation studies of Trypanosoma cruzi triosephosphate isomerase inhibitors. Insights into the inhibition mechanism and selectivity

Trypanosoma cruzi (T. cruzi) triosephosphate isomerase (TcTIM) is a glycolytic enzyme essential for parasite survival and has been considered an interesting target for the development of new antichagasic compounds. The homodimeric enzyme is catalytically active only as a dimer. Interestingly, significant differences exist between the human and parasite TIMs interfaces with a sequence identity of 52%. Therefore, compounds able to specifically disrupt TcTIM but not Homo sapiens TIM (hTIM) dimer interface could become selective antichagasic drugs. In the present work, the binding modes of 1,2,4-thiadiazol, phenazine and 1,2,6-thiadiazine derivatives to TcTIM were investigated using molecular docking combined with molecular dynamics (MD) simulations. The results show that phenazine and 1,2,6-thiadiazine derivatives, 2 and 3, act as dimer-disrupting inhibitors of TcTIM having also allosteric effects in the conformation of the active site. On the other hand, the 1,2,4-thiadiazol derivative 1 binds into the active site causing a significant decrease in enzyme mobility in both monomers. The loss of conformational flexibility upon compound 1 binding suggests that this inhibitor could be preventing essential motions of the enzyme required for optimal activity. The lack of inhibitory activity of 1 against hTIM was also investigated and seems to be related with the high mobility of hTIM which would hinder the formation of a stable ligand–enzyme complex. This work has contributed to understand the mechanism of action of this kind of inhibitors and could result of great help for future rational novel drug design.     

神经氨酸酶抑制剂与靶标NA分子结合模式研究

神经氨酸酶(NA)抑制剂作为现阶段治疗流感的主要药物,发挥着重要作用。本文运用Autodock软件,研究了3种上市的NA抑制剂一扎那米韦ZMR.、奥司他韦G39、帕拉米韦BCX与流感病毒H1N1中NA的分子间结合模式。结果指出,3种药剂与靶标NA间相互作用主要包括氢键作用、静电作用和疏水作用。其中氢键结合作用是首要识别靶标的关键因素,主要集中在NA的Site1和Site2;静电作用也主要集中在NA的Site1和Site2;而新颖的疏水作用主要表现在NA的Site4区域,由于G39、BCX中疏水烷基链取代了ZMR中甘油基,导致原有氢键作用消失,使得G39、BCX中烷基链能够自由转动与周围的关键残基形成强烈的疏水结合作用,这成为未来新型NA抑制剂设计的研究热点。期望本研究对基于流感病毒靶标结构进行新型NA抑制剂设计提供理论参考。     

Investigating the binding preferences of small molecule inhibitors of human protein arginine methyltransferase 1 using molecular modelling

Protein arginine methyltransferases (PRMTs) catalyse the methylation of arginine residues of target proteins. PRMTs utilise S-adenosyl methionine (SAM) as the methyl group donor, leading to S-adenosyl homocysteine (SAH) and monomethylarginine (mMA). A combination of homology modelling, molecular docking, Active Site Pressurisation, molecular dynamic simulations and MM-PBSA free energy calculations is used to investigate the binding poses of three PRMT1 inhibitors (ligands 1–3), which target both SAM and substrate arginine binding sites by containing a guanidine group joined by short linkers with the SAM derivative. It was assumed initially that the adenine moieties of the inhibitors would bind in sub-site 1 (PHE44, GLU137, VAL136 and GLU108), the guanidine side chain would occupy sub-site 2 (GLU 161, TYR160, TYR156 and TRP302), with the amino acid side chain occupying sub-site 3 (GLU152, ARG62, GLY86 and ASP84; pose 1). However, the SAH homocysteine moiety does not fully occupy sub-site 3, suggesting another binding pose may exist (pose 2), whereby the adenine moiety binds in sub-site 1, the guanidine side chain occupies sub-site 3, and the amino acid side chain occupies sub-site 2. Our results indicate that ligand 1 (pose 1 or 2), ligand 2 (pose 2) and ligand 3 (pose 1) are the predominant binding poses and we demonstrate for the first time that sub-site 3 contains a large space that could be exploited in the future to develop novel inhibitors with higher binding affinities.     

Designing of multi-targeted molecules using combination of molecular screening and in silico drug cardiotoxicity prediction approaches

We have previously investigated and reported a set of phenol- and indole-based derivatives at the binding pockets of carbonic anhydrase isoenzymes using in silico and in vitro analyses. In this study, we extended our analysis to explore multi-targeted molecules from this set of compounds. Thus, 26 ligands are screened at the binding sites of 229 proteins from 5 main enzyme family classes using molecular docking algorithms. Derived docking scores are compared with reported results of ligands at carbonic anhydrase I and II isoenzymes. Results showed potency of multi-targeted drugs of a few compounds from investigated ligand set. These promising ligands are then tested in silico for their cardiotoxicity risks. Results of this work can be used to improve the desired effects of these compounds by molecular engineering studies. In addition these results may lead to further investigation of studied molecules by medicinal chemists to explore different therapeutic aims.     

In silico identification and computational analysis of the nucleotide binding site in the C-terminal domain of Hsp90

Hsp90 contains two distinct Nucleotide Binding Sites (NBS), in its N-terminal domain (NTD) and C-terminal domain (CTD), respectively. The NTD site belongs to the GHKL super-family of ATPases and has been the subject of extensive characterization. However, a structure of the nucleotide-bound form of CTD is still unavailable. In this study molecular modeling was employed to incorporate experimental data using partial constructs of the CTD, from work published by many research groups, onto existing structural models of its apo- form. Our attempts to locate potential nucleotide ligand-binding sites or cavities yielded one major candidate—a structurally unconventional site—exhibiting the requisite shape and volume for accommodation of tri-phosphate nucleotides. Its structure was refined by molecular dynamics (MD)-based techniques. We reproducibly docked the Mg²⁺ complexed form of ATP, GTP, CTP, TTP and UTP to this putative NBS. These docking simulations and calculated ligand-binding scores are in general agreement with published data about experimentally measured binding to the CTD. The overall pattern of interactions between residues lining the site and docked nucleotides is conserved and broadly similar to that of other nucleotide-binding sites. Our docking simulations suggest that nucleotide binding stabilizes the only structurally labile region, thereby providing a rationale for the increased resistance to thermal denaturation and proteolysis. The docked nucleotides do not intrude onto the surface of residues involved in dimerization or chaperoning. Our molecular modeling permitted recognition of larger structural changes in the nucleotide-bound CTD dimer, including stabilization of helix-2 in both chains and intra- and inter- chain interactions between three residues (I613, Q617, R620).     

Binding modes of CCR5-targetting HIV entry inhibitors: partial and full antagonists

Since the CC-chemokine receptor 5 (CCR5) was identified as a major co-receptor for human immunodeficiency virus type 1 (HIV-1) entry into a host cell, CCR5-targetting HIV entry inhibitors have been developed and some of them are currently in clinical trials. Most of these inhibitors also inhibit the physiological chemokine reaction function of CCR5, which is so far considered to be safe to patients based on the observation that individuals that naturally lack CCR5 do not show apparent health problems. Nevertheless, to minimize the toxicity and side effects, it would be ideal to preserve the chemokine receptor activity. In this work, we simulated the flexible docking of two small molecule inhibitors to CCR5 in a solvated phospholipid bilayer environment. One of the inhibitors, aplaviroc has a unique feature of preserving two of the natural chemokine ligands binding to CCR5 and subsequent activation whereas the other one, SCH-C fully blocks chemokine-CCR5 interactions. Our results revealed significantly different binding modes of these two inhibitors although both established extensive interaction networks with CCR5. Comparison of the different binding modes suggests that avoiding the deep insertion of inhibitors into the transmembrane helix bundle may be able to preserve chemokine-CCR5 interactions. These results could help design HIV co-receptor activity-specific inhibitors.     

Identification of potent inhibitors of DNA methyltransferase 1 (DNMT1) through a pharmacophore-based virtual screening approach

DNA methylation is an epigenetic change that results in the addition of a methyl group at the carbon-5 position of cytosine residues. DNA methyltransferase (DNMT) inhibitors can suppress tumour growth and have significant therapeutic value. However, the established inhibitors are limited in their application due to their substantial cytotoxicity. Additionally, the standard drugs for DNMT inhibition are non-selective cytosine analogues with considerable cytotoxic side-effects. In the present study, we have designed a workflow by integrating various ligand-based and structure-based approaches to discover new agents active against DNMT1. We have derived a pharmacophore model with the help of available DNMT1 inhibitors. Utilising this model, we performed the virtual screening of Maybridge chemical library and the identified hits were then subsequently filtered based on the Naïve Bayesian classification model. The molecules that have returned from this classification model were subjected to ensemble based docking. We have selected 10 molecules for the biological assay by inspecting the interactions portrayed by these molecules. Three out of the ten tested compounds have shown DNMT1 inhibitory activity. These compounds were also found to demonstrate potential inhibition of cellular proliferation in human breast cancer MDA-MB-231 cells. In the present study, we have utilized a multi-step virtual screening protocol to identify inhibitors of DNMT1, which offers a starting point to develop more potent DNMT1 inhibitors as anti-cancer agents.     

Computational determination of binding structures and free energies of glucose 6-phosphate dehydrogenase with novel steroid inhibitors

Glucose 6-phosphate dehydrogenase (G6PD), the first and the rate-limiting enzyme in the pentose phosphate pathway (PPP), catalyzes the oxidation of G6P to 6-phosphogluconolactone and the reduction of NADP⁺ to NADPH. Its key role in cancer promotes the development of a potent and selective inhibitor that might increase cancer cell death when combined with radiotherapy. In the present study, we investigated the detailed binding modes and binding free energies for G6PD interacting with a promising series of recently developed inhibitors, i.e., the steroid derivatives, by performing molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations. The docking indicates that the inhibitors occupy the binding sites of both G6P and NADP⁺. The calculated binding free energies on the basis of the MD-simulated enzyme–inhibitor complexes are in good agreement with the experimental activity data for all of the examined inhibitors. The valuable insights into the detailed enzyme–inhibitor binding including the important intermolecular interactions, e.g., the hydrogen bond interaction and the hydrophobic interaction, have been provided. The computational results provide new insights into future rational design of more potent inhibitors of G6PD as a treatment for cancer.     

Comparative modeling and molecular docking analysis of white,brown and soft rot fungal laccases using lignin model compounds for understanding the structural and functional properties of laccases

Extrinsic catalytic properties of laccase enable it to oxidize a wide range of aromatic (phenolic and non-phenolic) compounds which makes it commercially an important enzyme. In this study, we have extensively compared and analyzed the physico-chemical, structural and functional properties of white, brown and soft rot fungal laccases using standard protein analysis software. We have computationally predicted the three-dimensional comparative models of these laccases and later performed the molecular docking studies using the lignin model compounds. We also report a customizable rapid and reliable protein modelling and docking pipeline for developing structurally and functionally stable protein structures. We have observed that soft rot fungal laccases exhibited comparatively higher structural variation (higher random coil) when compared to brown and white rot fungal laccases. White and brown rot fungal laccase sequences exhibited higher similarity for conserved domains of Trametes versicolor laccase, whereas soft rot fungal laccases shared higher similarity towards conserved domains of Melanocarpus albomyces laccase. Results obtained from molecular docking studies showed that aminoacids PRO, PHE, LEU, LYS and GLN were commonly found to interact with the ligands. We have also observed that white and brown rot fungal laccases showed similar docking patterns (topologically monomer, dimer and trimer bind at same pocket location and tetramer binds at another pocket location) when compared to soft rot fungal laccases. Finally, the binding efficiencies of white and brown rot fungal laccases with lignin model compounds were higher compared to the soft rot fungi. These findings can be further applied in developing genetically efficient laccases which can be applied in growing biofuel and bioremediation industries.     

用串行ROM和AT89C51实现电子密码锁

本文介绍一款用AT89C51和串行ROM制作的电子密码锁,其主要特点为硬件电路简洁可靠,并可随时更改密码,以下是其硬件和软件部分的设计。     

C—51的BANK模式及代码和数据的混合BANK技术

C-51的BANK编译模式是用来解决程序代码超出51系列单片机最大寻指空间的一种编译模式。它通过硬件设计与软件码中插入切换BANK页面操作相结合，实现页面存储器中不同页中代码的相互调用。本文首先阐述了C－51编译器BANK模式的使用方法。然后介绍了在C－51的BANK模式下代码和数据混合使用BANK的一种技巧。     

Predicting the structures of complexes between phosphoinositide 3-kinase (PI3K) and romidepsin-related compounds for the drug design of PI3K/histone deacetylase dual inhibitors using computational docking and the ligand-based drug design approach

Predictions of the three-dimensional (3D) structures of the complexes between phosphoinositide 3-kinase (PI3K) and two inhibitors were conducted using computational docking and the ligand-based drug design approach. The obtained structures were refined by structural optimizations and molecular dynamics (MD) simulations. The ligands were located deep inside the ligand binding pocket of the p110α subunit of PI3K, and the hydrogen bond formations and hydrophobic effects of the surrounding amino acids were predicted. Although rough structures were obtained for the PI3K–inhibitor complexes before the MD simulations, the refinement of the structures by these simulations clarified the hydrogen bonding patterns of the complexes.     

Binding mode analyses and pharmacophore model development for sulfonamide chalcone derivatives, a new class of alpha-glucosidase inhibitors

Sulfonamide chalcone derivatives are a new class of non-saccharide compounds that effectively inhibit glucosidases which are the major targets in the treatment of Type 2 diabetes and HIV infection. Our aim is to explore their binding mode of interaction at the active site by comparing with the sugar derivatives and to develop a pharmacophore model which would represent the critical features responsible for alpha-glucosidase inhibitory activity. The homology modeled structure of Saccharomyces cerevisiae alpha-glucosidase was built and used for molecular docking of non-sugar/sugar derivatives. The validated docking results projected the crucial role of NH group in the binding of sugar/non-sugar derivatives to the active site. Ligplot analyses revealed that Tyr71, and Phe177 form hydrophobic interactions with sugar/non-sugar derivatives by holding the terminal glycosidic ring mimics. Molecular dynamic (MD) simulation studies were performed for protein alone and with chalcone derivative to prove its binding mechanism as shown by docking/Ligplot results. It would also help to substantiate the homology modeled structure stability. With the knowledge of the crucial interactions between ligand and protein from docking and MD simulation studies, features for pharmacophore model development were chosen. The CATALYST/HipHop was used to generate a five featured pharmacophore model with a training set of five non-sugar derivatives. As validation, all the crucial features of the model were perfectly mapped onto the 3D structures of the sugar derivatives as well as the newly tested non-sugar derivatives. Thus, it can be useful in virtual screening for finding new non-sugar derivatives as alpha-glucosidase inhibitors.     

Prediction of the receptor conformation for iGluR2 agonist binding: QM/MM docking to an extensive conformational ensemble generated using normal mode analysis

Highly flexible proteins constitute a significant challenge in molecular docking within the field of drug design. Depending on the efficacy of the bound ligand, the ligand-binding domain (LBD) of the ionotropic glutamate receptor iGluR2 adopts markedly different degrees of domain closure due to large-scale domain movements. With the purpose of predicting the induced domain closure of five known iGluR2 partial to full agonists we performed a validation study in which normal mode analysis (NMA) was employed to generate a 25-membered ensemble of iGluR2 LBD structures with gradually changing domain closures, followed by accurate QM/MM docking to the ensemble. Based on the docking scores we were able to predict the correct optimal degree of closure for each ligand within 1–3° deviation from the experimental structures. We demonstrate that NMA is a useful tool for reliable ensemble generation and that we are able to predict the ligand induced conformational change of the receptor through docking to such an ensemble. The described protocol expands and improves the information that can be obtained from computational docking when dealing with a flexible receptor.