Virtual screening of small molecules databases for discovery of novel PARP-1 inhibitors: combination of in silico and in vitro studies

Poly(ADP-ribose) polymerase-1 (PARP-1) enzyme has critical roles in DNA replication repair and recombination. Thus, PARP-1 inhibitors play an important role in the cancer therapy. In the current study, we have performed combination of in silico and in vitro studies in order to discover novel inhibitors against PARP-1 target. Structure-based virtual screening was carried out for an available small molecules database. A total of 257,951 ligands from Otava database were screened at the binding pocket of PARP-1 using high-throughput virtual screening techniques. Filtered structures based on predicted binding energy results were then used in more sophisticated molecular docking simulations (i.e. Glide/standard precision, Glide/XP, induced fit docking – IFD, and quantum mechanics polarized ligand docking – QPLD). Potential high binding affinity compounds that are predicted by molecular simulations were then tested by in vitro methods. Computationally proposed compounds as PARP-1 inhibitors (Otava Compound Codes: 7111620047 and 7119980926) were confirmed by in vitro studies. In vitro results showed that compounds 7111620047 and 7119980926 have IC₅₀ values of 0.56 and 63 μM against PARP-1 target, respectively. The molecular mechanism analysis, free energy perturbation calculations using long multiple molecular dynamics simulations for the discovered compounds which showed high binding affinity against PARP-1 enzyme, as well as structure-based pharmacophore development (E-pharmacophore) studies were also studied.     

Structure Prediction and Validation of the ERK8 Kinase Domain

Extracellular signal-regulated kinase 8 (ERK8) has been already implicated in cell transformation and in the protection of genomic integrity and, therefore, proposed as a novel potential therapeutic target for cancer. In the absence of a crystal structure, we developed a three-dimensional model for its kinase domain. To validate our model we applied a structure-based virtual screening protocol consisting of pharmacophore screening and molecular docking. Experimental characterization of the hit compounds confirmed that a high percentage of the identified scaffolds was able to inhibit ERK8. We also confirmed an ATP competitive mechanism of action for the two best-performing molecules. Ultimately, we identified an ERK8 drug-resistant “gatekeeper” mutant that corroborated the predicted molecular binding mode, confirming the reliability of the generated structure. We expect that our model will be a valuable tool for the development of specific ERK8 kinase inhibitors.     

Virtual screening and bioassay study of novel inhibitors for dengue virus mRNA cap (nucleoside-2'O)-methyltransferase

We report high-throughput structure-based virtual screening of putative Flavivirus 2′-O-methyltransferase inhibitors together with results from subsequent bioassay tests of selected compounds. Potential inhibitors for the S-adenosylmethionine binding site were explored using 2D similarity searching, pharmacophore filtering and docking. The inhibitory activities of 15 top-ranking compounds from the docking calculations were tested on a recombinant methyltransferase with the RNA substrate ^7MeGpppAC₅. Local and global docking simulations were combined to estimate the ligand selectivity for the target site. The results of the combined computational and experimental screening identified a novel inhibitor, with a previously unknown scaffold, that has an IC₅₀ value of 60 μM.     

Structure-based and shape-complemented pharmacophore modeling for the discovery of novel checkpoint kinase 1 inhibitors

Checkpoint kinase 1 (Chk1), a member of the serine/threonine kinase family, is an attractive therapeutic target for anticancer combination therapy. A structure-based modeling approach complemented with shape components was pursued to develop a reliable pharmacophore model for ATP-competitive Chk1 inhibitors. Common chemical features of the pharmacophore model were derived by clustering multiple structure-based pharmacophore features from different Chk1-ligand complexes in comparable binding modes. The final model consisted of one hydrogen bond acceptor (HBA), one hydrogen bond donor (HBD), two hydrophobic (HY) features, several excluded volumes and shape constraints. In the validation study, this feature-shape query yielded an enrichment factor of 9.196 and performed fairly well at distinguishing active from inactive compounds, suggesting that the pharmacophore model can serve as a reliable tool for virtual screening to facilitate the discovery of novel Chk1 inhibitors. Besides, these pharmacophore features were assumed to be essential for Chk1 inhibitors, which might be useful for the identification of potential Chk1 inhibitors.     

Design and Synthesis of a Cell-Permeable,Drug-Like Small Molecule Inhibitor Targeting the Polo-Box Domain of Polo-Like Kinase 1

Background

Polo-like kinase-1 (Plk1) plays a crucial role in cell proliferation and the inhibition of Plk1 has been considered as a potential target for specific inhibitory drugs in anti-cancer therapy. Several research groups have identified peptide-based inhibitors that target the polo-box domain (PBD) of Plk1 and bind to the protein with high affinity in in vitro assays. However, inadequate proteolytic resistance and cell permeability of the peptides hinder the development of these peptide-based inhibitors into novel therapeutic compounds.

Methodology/Principal Findings

In order to overcome the shortcomings of peptide-based inhibitors, we designed and synthesized small molecule inhibitors. Among these molecules, bg-34 exhibited a high binding affinity for Plk1-PBD and it could cross the cell membrane in its unmodified form. Furthermore, bg-34-dependent inhibition of Plk1-PBD was sufficient for inducing apoptosis in HeLa cells. Moreover, modeling studies performed on Plk1-PBD in complex with bg-34 revealed that bg-34 can interact effectively with Plk1-PBD.

Conclusion/Significance

We demonstrated that the molecule bg-34 is a potential drug candidate that exhibits anti-Plk1-PBD activity and possesses the favorable characteristics of high cell permeability and stability. We also determined that bg-34 induced apoptotic cell death by inhibiting Plk1-PBD in HeLa cells at the same concentration as PEGylated 4j peptide, which can inhibit Plk1-PBD activity 1000 times more effectively than bg-34 can in in vitro assays. This study may help to design and develop drug-like small molecule as Plk1-PBD inhibitor for better therapeutic activity.     

Pharmacophore modeling and hybrid virtual screening for the discovery of novel IκB kinase 2 (IKK2) inhibitors

IKK2 (IκB kinase 2) inhibitors have been identified as potential drug candidates in the treatment of various immune/inflammatory disorders as well as cancer. So far more than one hundred small molecule inhibitors against IKK2 have been reported publicly. In this investigation, pharmacophore modeling was carried out to clarify the essential structure-activity relationship for the known IKK2 inhibitors. One of the established pharmacophore hypotheses, namely Hypo8, which has the best prediction ability to an external test data set, was suggested as a template for virtual screening. Evaluation of the performances of Hypo8 and a hybrid method (Hypo81docking) in virtual screening indicated that the use of the hybrid virtual screening considerably increased the hit rate and enrichment factor. The hybrid method was therefore adopted for screening several commercially available chemical databases, including Specs, NCI, Maybridge and Chinese Nature Product Database (CNPD), for novel potent IKK2 inhibitors. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five. Finally some of the final hit compounds were selected and suggested for further experimental investigations.     

Discovery of novel EGFR tyrosine kinase inhibitors by structure-based virtual screening

By using of structure-based virtual screening, 13 novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors were discovered from 197,116 compounds in the SPECS database here. Among them, 8 compounds significantly inhibited EGFR kinase activity with IC(50) values lower than 10 μM. 3-{[1-(3-Chloro-4-fluorophenyl)-3,5-dioxo-4-pyrazolidinylidene]methyl}phenyl 2-thiophenecarboxylate (13), particularly, was the most potent inhibitor possessing the IC(50) value of 3.5 μM. The docking studies also provide some useful information that the docking models of the 13 compounds are beneficial to find a new path for designing novel EGFR inhibitors.     

In silico analysis of the effect of mutation on epidermal growth factor receptor in non-small-cell lung carcinoma: from mutational analysis to drug designing

Tyrosine kinase inhibitors (TKI)-resistant mutation in epidermal growth factor receptor’s (EGFR) kinase domain is an important anomaly to look into. Studying the mutations at atomic level using molecular dynamics simulations gave us an insight into the architectural changes happening at the microscopic level. The knowledge was used to design new TKI whose function is devoid of the affect of the mutations in kinase domain. Traditional Chinese medicinal library was used for structure-based drug designing, where virtual screening was followed by ADME/Tox analysis and the shortlisted compounds were docked into the kinase domain of EGFR and simulated there using atomic-level selection of the grid. The shortlisted compounds from molecular docking analysis were subjected to MM-PBSA calculations. The in silico data generated is giving a strong lead compound for further in vitro and in vivo analysis.     

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K_i value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development.     

Structure-based discovery of inhibitors of the YycG histidine kinase: New chemical leads to combat Staphylococcus epidermidis infections

Background

Coagulase-negative Staphylococcus epidermidis has become a major frequent cause of infections in relation to the use of implanted medical devices. The pathogenicity of S. epidermidis has been attributed to its capacity to form biofilms on surfaces of medical devices, which greatly increases its resistance to many conventional antibiotics and often results in chronic infection. It has an urgent need to design novel antibiotics against staphylococci infections, especially those can kill cells embedded in biofilm.

Results

In this report, a series of novel inhibitors of the histidine kinase (HK) YycG protein of S. epidermidis were discovered first using structure-based virtual screening (SBVS) from a small molecular lead-compound library, followed by experimental validation. Of the 76 candidates derived by SBVS targeting of the homolog model of the YycG HATPase_c domain of S. epidermidis, seven compounds displayed significant activity in inhibiting S. epidermidis growth. Furthermore, five of them displayed bactericidal effects on both planktonic and biofilm cells of S. epidermidis. Except for one, the compounds were found to bind to the YycG protein and to inhibit its auto-phosphorylation in vitro, indicating that they are potential inhibitors of the YycG/YycF two-component system (TCS), which is essential in S. epidermidis. Importantly, all these compounds did not affect the stability of mammalian cells nor hemolytic activities at the concentrations used in our study.

Conclusion

These novel inhibitors of YycG histidine kinase thus are of potential value as leads for developing new antibiotics against infecting staphylococci. The structure-based virtual screening (SBVS) technology can be widely used in screening potential inhibitors of other bacterial TCSs, since it is more rapid and efficacious than traditional screening technology.     

A Novel Anti-Tumor Inhibitor Identified by Virtual Screen with PLK1 Structure and Zebrafish Assay

Polo-like kinase 1 (PLK1), one of the key regulators of mitosis, is a target for cancer therapy due to its abnormally high activity in several tumors. Plk1 is highly conserved and shares a nearly identical 3-D structure between zebrafish and humans. The initial 10 mitoses of zebrafish embryonic cleavages occur every∼30 minutes, and therefore provide a rapid assay to evaluate mitosis inhibitors including those targeting Plk1. To increase efficiency and specificity, we first performed a computational virtual screen of∼60000 compounds against the human Plk1 3-D structure docked to both its kinase and Polo box domain. 370 candidates with the top free-energy scores were subjected to zebrafish assay and 3 were shown to inhibit cell division. Compared to general screen for compounds inhibiting zebrafish embryonic cleavage, computation increased the efficiency by 11 folds. One of the 3 compounds, named I2, was further demonstrated to effectively inhibit multiple tumor cell proliferation in vitro and PC3 prostate cancer growth in Xenograft mouse model in vivo. Furthermore, I2 inhibited Plk1 enzyme activity in a dose dependent manner. The IC₅₀ values of I2 in these assays are compatible to those of ON-01910, a Plk1 inhibitor currently in Phase III clinic trials. Our studies demonstrate that zebrafish assays coupled with computational screening significantly improves the efficiency of identifying specific regulators of biological targets. The PLK1 inhibitor I2, and its analogs, may have potential in cancer therapeutics.     

Discovery of novel inhibitors of Mycobacterium tuberculosis MurG: homology modelling,structure based pharmacophore,molecular docking,and molecular dynamics simulations

MurG (Rv2153c) is a key player in the biosynthesis of the peptidoglycan layer in Mycobacterium tuberculosis (Mtb). This work is an attempt to highlight the structural and functional relationship of Mtb MurG, the three-dimensional (3D) structure of protein was constructed by homology modelling using Discovery Studio 3.5 software. The quality and consistency of generated model was assessed by PROCHECK, ProSA and ERRAT. Later, the model was optimized by molecular dynamics (MD) simulations and the optimized model complex with substrate Uridine-diphosphate-N-acetylglucosamine (UD1) facilitated us to employ structure-based virtual screening approach to obtain new hits from Asinex database using energy-optimized pharmacophore modelling (e-pharmacophore). The pharmacophore model was validated using enrichment calculations, and finally, validated model was employed for high-throughput virtual screening and molecular docking to identify novel Mtb MurG inhibitors. This study led to the identification of 10 potential compounds with good fitness, docking score, which make important interactions with the protein active site. The 25 ns MD simulations of three potential lead compounds with protein confirmed that the structure was stable and make several non-bonding interactions with amino acids, such as Leu290, Met310 and Asn167. Hence, we concluded that the identified compounds may act as new leads for the design of Mtb MurG inhibitors.     

Identification of novel and selective non-peptide inhibitors targeting the polo-box domain of polo-like kinase 1

A series of non-peptide inhibitors targeting the polo-box domain (PBD) of polo-like kinase 1 (Plk1) was designed based on the potent and selective minimal tripeptide Plk1 PBD inhibitor. Seven compounds were designed, synthesized and evaluated for fluorescence polarization (FP) assay. The most promising compound 10 bound to Plk1 PBD with IC₅₀ of 3.37 μM and had no binding to Plk2 PBD or Plk3 PBD at 100 μM. Molecular docking study was performed and possible binding mode was proposed. MM/GBSA binding free energy calculation were in agreement with the observed experimental results. These novel non-peptide selective Plk1 PBD inhibitors provided new lead compounds for further optimization.     

Ligand‐based pharmacophore modelling and screening of DNA minor groove binders targeting Staphylococcus aureus

The recognition of DNA by small molecules is of special importance in the design of new drugs. Many natural and synthetic compounds have the ability to interact with the minor groove of DNA. In the present study, identification of minor groove binding compounds was attained by the combined approach of pharmacophore modelling, virtual screening and molecular dynamics approach. Experimentally reported 32 minor groove binding compounds were used to develop the pharmacophore model. Based on the fitness score, best three pharmacophore hypotheses were selected and used as template for screening the compounds from drug bank database. This pharmacophore‐based screening provides many compounds with the same pharmacological properties. All these compounds were subjected to four phases of docking protocols with combined Glide‐quantum‐polarized ligand docking approach. Molecular dynamics results indicated that selected compounds are more active and showed good interaction in the binding site of DNA. Based on the scoring parameters and energy values, the best compounds were selected, and antibacterial activity of these compounds was identified using in vitro antimicrobial techniques. Copyright © 2014 John Wiley & Sons, Ltd.     

In silico approaches to identify novel myeloid cell leukemia-1 (Mcl-1) inhibitors for treatment of cancer

Myeloid cell leukemia-1 (Mcl-1) has been a validated and attractive target for cancer therapy. Over-expression of Mcl-1 in many cancers allows cancer cells to evade apoptosis and contributes to the resistance to current chemotherapeutics. Here, we identified new Mcl-1 inhibitors using a multi-step virtual screening approach. First, based on two different ligand-receptor complexes, 20 pharmacophore models were established by simultaneously using ‘Receptor-Ligand Pharmacophore Generation’ method and manual build feature method, and then carefully validated by a test database. Then, pharmacophore-based virtual screening (PB-VS) could be performed by using the 20 pharmacophore models. In addition, docking study was used to predict the possible binding poses of compounds, and the docking parameters were optimized before performing docking-based virtual screening (DB-VS). Moreover, a 3D QSAR model was established by applying the 55 aligned Mcl-1 inhibitors. The 55 inhibitors sharing the same scaffold were docked into the Mcl-1 active site before alignment, then the inhibitors with possible binding conformations were aligned. For the training set, the 3D QSAR model gave a correlation coefficient r² of 0.996; for the test set, the correlation coefficient r² was 0.812. Therefore, the developed 3D QSAR model was a good model, which could be applied for carrying out 3D QSAR-based virtual screening (QSARD-VS). After the above three virtual screening methods orderly filtering, 23 potential inhibitors with novel scaffolds were identified. Furthermore, we have discussed in detail the mapping results of two potent compounds onto pharmacophore models, 3D QSAR model, and the interactions between the compounds and active site residues.     

In silico discovery of 2-amino-4-(2,4-dihydroxyphenyl)thiazoles as novel inhibitors of DNA gyrase B

Cyclothialidines are a class of bacterial DNA gyrase B (GyrB) subunit inhibitors, targeting its ATP-binding site. Starting from the available structural information on cyclothialidine GR122222X (2), an in silico virtual screening campaign was designed combining molecular docking calculations with three-dimensional structure-based pharmacophore information. A novel class of 2-amino-4-(2,4-dihydroxyphenyl)thiazole based inhibitors (5–9) with low micromolar antigyrase activity was discovered.     

Identification of novel inhibitor of protein tyrosine phosphatases delta: structure-based pharmacophore modeling,virtual screening,flexible docking,molecular dynamics simulation,and post-molecular dynamics analysis

Abstract

Owing to their unique functions in regulating the synapse activity of protein tyrosine phosphatases delta (PTPδ) that has drawn special attention for developing drugs to autism spectrum disorders (ASDs). In this study, the PTPδ pharmacophore was first established by the structure-based pharmacophore method. Subsequently, 10 compounds contented Lipinski’s rule of five was acquired by the virtual screening of the PTPδ pharmacophore against ZINC and PubChem databases. Then, the 10 identified molecules were discovered that had better binding affinity than a known PTPδ inhibitors compound SCHEMBL16375396. Two compounds SCHEMBL16375408 and ZINC19796658 with high binding score, low toxicity were gained. They were observed by docking analysis and molecular dynamics simulations that the novel potential inhibitors not only possessed the same function as SCHEMBL16375396 did in inhibiting PTPδ, but also had more favorable conformation to bind with the catalytic active regions. This study provides a new method for identify PTPδ inhibitor for the treatment of ASDs disease.

Communicated by Ramaswamy H. Sarma     

ASK1 pharmacophore model derived from diverse classes of inhibitors

The three-dimensional pharmacophore model of apoptosis signal-regulating kinase 1 (ASK1) inhibitors has been developed with PharmaGist program. The positions of pharmacophore features in the model correspond to conformations of ASK1 highly active inhibitors in which they interact with ATP-binding site of ASK1. The generated pharmacophore model allows accurately predict active and inactive compounds and can be of great use for virtual screening aimed at discovering novel ASK1 inhibitors.     

Identification of novel CDK2 inhibitors by a multistage virtual screening method based on SVM,pharmacophore and docking model

Abstract

Cyclin-dependent kinase 2 (CDK2) is the family of Ser/Thr protein kinases that has emerged as a highly selective with low toxic cancer therapy target. A multistage virtual screening method combined by SVM, protein-ligand interaction fingerprints (PLIF) pharmacophore and docking was utilised for screening the CDK2 inhibitors. The evaluation of the validation set indicated that this method can be used to screen large chemical databases because it has a high hit-rate and enrichment factor (80.1% and 332.83 respectively). Six compounds were screened out from NCI, Enamine and Pubchem database. After molecular dynamics and binding free energy calculation, two compounds had great potential as novel CDK2 inhibitors and they also showed selective inhibition against CDK2 in the kinase activity assay.     

A Combination of Receptor-Based Pharmacophore Modeling & QM Techniques for Identification of Human Chymase Inhibitors

Inhibition of chymase is likely to divulge therapeutic ways for the treatment of cardiovascular diseases, and fibrotic disorders. To find novel and potent chymase inhibitors and to provide a new idea for drug design, we used both ligand-based and structure-based methods to perform the virtual screening(VS) of commercially available databases. Different pharmacophore models generated from various crystal structures of enzyme may depict diverse inhibitor binding modes. Therefore, multiple pharmacophore-based approach is applied in this study. X-ray crystallographic data of chymase in complex with different inhibitors were used to generate four structure–based pharmacophore models. One ligand–based pharmacophore model was also developed from experimentally known inhibitors. After successful validation, all pharmacophore models were employed in database screening to retrieve hits with novel chemical scaffolds. Drug-like hit compounds were subjected to molecular docking using GOLD and AutoDock. Finally four structurally diverse compounds with high GOLD score and binding affinity for several crystal structures of chymase were selected as final hits. Identification of final hits by three different pharmacophore models necessitates the use of multiple pharmacophore-based approach in VS process. Quantum mechanical calculation is also conducted for analysis of electrostatic characteristics of compounds which illustrates their significant role in driving the inhibitor to adopt a suitable bioactive conformation oriented in the active site of enzyme. In general, this study is used as example to illustrate how multiple pharmacophore approach can be useful in identifying structurally diverse hits which may bind to all possible bioactive conformations available in the active site of enzyme. The strategy used in the current study could be appropriate to design drugs for other enzymes as well.