Pharmacophore identification, in silico screening, and virtual library design for inhibitors of the human factor Xa

Factor Xa inhibitors are innovative anticoagulant agents that provide a better safety/efficacy profile compared to other anticoagulative drugs. A chemical feature-based modeling approach was applied to identify crucial pharmacophore patterns from 3D crystal structures of inhibitors bound to human factor Xa (Pdb entries 1fjs, 1kns, 1eqz) using the software LIGANDSCOUT and CATALYST. The complex structures were selected regarding the criteria of high inhibitory potency (i.e. all ligands show K(i) values against factor Xa in the subnanomolar range) and good resolution (i.e. at least 2.2 A) in order to generate selective and high quality pharmacophore models. The resulting chemical-feature based hypotheses were used for virtual screening of commercial molecular databases such as the WDI database. Furthermore, a ligand-based molecular modeling approach was performed to obtain common-feature hypotheses that represent the relevant chemical interactions between 10 bioactive factor Xa inhibitors and the protein, respectively. In a next step a virtual combinatorial library was designed in order to generate new compounds with similar chemical and spatial properties as known inhibitors. The software tool ILIB DIVERSE was used for this procedure in order to provide new scaffolds of this group of anticoagulants. Finally we present the combination of these two techniques, hence virtual screening was performed with selective pharmacophore models in a focused virtual combinatorial database. De novo derived molecular scaffolds that were able to adequately satisfy the pharmacophore criteria are revealed and are promising templates for candidates for further development.     

Strategy for discovering chemical inhibitors of human cyclophilin a: focused library design, virtual screening, chemical synthesis and bioassay

The discovery of cyclophilin A (CypA) inhibitor is now of special interest in the treatment of immunological disorders. In this work, using a strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and bioassay, a series of novel small molecular CypA inhibitors have been discovered. First, using the fragments taken from our previously discovered CypA inhibitors (Bioorg. Med. Chem. 2006, 14, 2209-2224) as building blocks, we designed a focused combinatorial library containing 255 molecules employing the LD1.0 program (J. Comb. Chem. 2005, 7, 398-406) developed by us. Sixteen compounds (1a-e, 2a-b, 3a-b, and 4a-g) were selected by using virtual screening against the X-ray crystal structure of CypA as well as druglike analysis for further synthesis and bioassay. All these sixteen molecules are CypA binders with binding affinities (K(D) values) ranging from 0.076 to 41.0 microM, and five of them (4a, 4c, and 4e-g) are potent CypA inhibitors with PPIase inhibitory activities (IC(50) values) of 0.25-6.43 microM. The hit rates for binders and inhibitors are as high as 100% and 31.25%, respectively. Remarkably, both the binding affinity and inhibitory activity of the most potent compound increase approximately 10 times than that of the most active compound discovered previously. The high hit rate and the high potency of the new CypA inhibitors demonstrated the efficiency of the strategy for focused library design and screening. In addition, the novel chemical entities reported in this study could be leads for discovering new therapies against the CypA pathway.     

Facilitated forward chemical genetics using a tagged triazine library and zebrafish embryo screening

An improved forward chemical genetics approach was successfully demonstrated using a tagged library concept. A small-molecule triazine library with linkers was used to screen for brain/eye developmental phenotypes in a zebrafish embryo system. This approach enabled the rapid isolation of the target proteins by facile affinity matrix preparation and elucidated the first small-molecule inhibitors for several ribosomal accessory proteins or their complex as the target.     

Identification of novel human renin inhibitors through a combined approach of pharmacophore modelling,molecular DFT analysis and in silico screening

Renin is an aspartyl protease of the renin–angiotensin system (RAS) and the first enzyme of the biochemical pathway for the generation of angiotensin II – a potent vasoconstrictor involved in the maintenance of cardiovascular homeostasis and the regulation of blood pressure. High enzymatic specificity of renin and its involvement in the catalysis of the rate-limiting step of the RAS hormone system qualify it as a good target for inhibition of hypertension and other associated diseases. Ligand-based pharmacophore model (Hypo1) was generated from a training set of 24 compounds with renin inhibitory activity. The best hypothesis consisted of one Hydrogen Bond Acceptor (HBA), three Hydrophobic Aliphatic (HY-Al) and one Ring Aromatic (AR) features. This well-validated pharmacophore hypothesis (correlation coefficient 0.95) was further utilized as a 3D query to screen database compounds, which included structures from two natural product repositories. These screened compounds were further analyzed for drug-likeness and ADMET studies. The compounds which satisfied the qualifying criteria were then subjected to molecular docking and Density Functional Theory (DFT) analysis in order to discern their atomic level interactions at the active site of the 3D structure of rennin. The pharmacophore-based modelling that has been used to generate the novel findings of the present study would be an avant-garde approach towards the development of potent inhibitors of renin.     

In silico identification and in vitro antiviral validation of potential inhibitors against Chikungunya virus

Journal of Computer-Aided Molecular Design - The Chikungunya virus (CHIKV) has become endemic in the Africa, Asia and Indian subcontinent, with its continuous re-emergence causing a significant...     

Design and characterization of a functional library for NMR screening against novel protein targets

In the past few years, NMR has been extensively utilized as a screening tool for drug discovery using various types of compound libraries. The designs of NMR specific chemical libraries that utilize a fragment-based approach based on drug-like characteristics have been previously reported. In this article, a new type of compound library will be described that focuses on aiding in the functional annotation of novel proteins that have been identified from various ongoing genomics efforts. The NMR functional chemical library is comprised of small molecules with known biological activity such as: co-factors, inhibitors, metabolites and substrates. This functional library was developed through an extensive manual effort of mining several databases based on known ligand interactions with protein systems. In order to increase the efficiency of screening the NMR functional library, the compounds are screened as mixtures of 3-4 compounds that avoids the need to deconvolute positive hits by maintaining a unique NMR resonance and function for each compound in the mixture. The functional library has been used in the identification of general biological function of hypothetical proteins identified from the Protein Structure Initiative.     

In silico validation of anti-viral drugs obtained from marine sources as a potential target against SARS-CoV-2 Mpro

COVID-19 caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has threatened the whole world affecting almost 243 million people globally. Originating from China, it has now spread worldwide with USA and India being the two most affected countries which emphasizes the immense potential of the coronaviruses to cause severity in the human population. This study validates the efficacy of some marine antiviral agents to target the viral main protease (Mpro) of SARS-CoV-2 by in silico studies. A total of 14 marine-derived antiviral agents were screened from several databases including PubChem and DrugBank and docked against the crystallised 3D structure of SARS-CoV-2 Mpro. MD simulation of the top two ligands was carried out for 100 ns to validate the protein-ligand stability. Later, their physicochemical, pharmacokinetics, and drug-likeness properties were evaluated and toxicity prediction was performed using eMOLTOX webtool. We found that all the 14 compounds are acting as a good target for Mpro. Among them, avarol and AcDa-1 procured the best docking results with the estimated docking score of −8.05 and −7.74 ​kcal/mol respectively. MD simulation revealed good conformational stability. The docked conformation was visualised and subsequent ligand-amino acid interactions were analysed. Avarol revealed good pharmacokinetic properties with oral bioavailability. The overall finding suggested that these marine compounds may have the potential to be used for the treatment of COVID-19 to tackle this pandemic.     

Selective human serum albumin sensor from the screening of a fluorescent rosamine library

A fluorescent dye library approach for the development of a bioanalyte sensor was sought. The screening of a rosamine dye library against diverse macromolecules led to the discovery of a highly sensitive human serum albumin binder, G13, with approximately 36-fold fluorescence intensity change. G13 showed a highly selective response to HSA over other macromolecules including albumins from other species. The potential use of G13 for the detection of HSA in biofluids is described.     

Development of novel 3D-QSAR combination approach for screening and optimizing B-Raf inhibitors in silico

B-Raf is a member of the RAF family of serine/threonine kinases: it mediates cell division, differentiation, and apoptosis signals through the RAS-RAF-MAPK pathway. Thus, B-Raf is of keen interest in cancer therapy, such as melanoma. In this study, we propose the first combination approach to integrate the pharmacophore (PhModel), CoMFA, and CoMSIA models for B-Raf, and this approach could be used for screening and optimizing potential B-Raf inhibitors in silico. Ten PhModels were generated based on the HypoGen BEST algorithm with the flexible fit method and diverse inhibitor structures. Each PhModel was designated to the alignment rule and screening interface for CoMFA and CoMSIA models. Therefore, CoMFA and CoMSIA models could align and recognize diverse inhibitor structures. We used two quality validation methods to test the predication accuracy of these combination models. In the previously proposed combination approaches, they have a common factor in that the number of training set inhibitors is greater than that of testing set inhibitors. In our study, the 189 known diverse series B-Raf inhibitors, which are 7-fold the number of training set inhibitors, were used as a testing set in the partial least-squares validation. The best validation results were made by the CoMFA09 and CoMSIA09 models based on the Hypo09 alignment model. The predictive r(2)(pred) values of 0.56 and 0.56 were derived from the CoMFA09 and CoMSIA09 models, respectively. The CoMFA09 and CoMSIA09 models also had a satisfied predication accuracy of 77.78% and 80%, and the goodness of hit test score of 0.675 and 0.699, respectively. These results indicate that our combination approach could effectively identify diverse B-Raf inhibitors and predict the activity.     

Thermodynamic properties of 9-fluorenone: Mutual validation of experimental and computational results

Measurements leading to the calculation of thermodynamic properties for 9-fluorenone (IUPAC name 9H-fluoren-9-one and Chemical Abstracts registry number [486-25-9]) in the ideal-gas state are reported. Experimental methods were adiabatic heat-capacity calorimetry, inclined-piston manometry, comparative ebulliometry, and combustion calorimetry. Critical properties were estimated. Molar entropies for the ideal-gas state were derived from the experimental studies at selected temperatures T between T = 298.15 K and T = 600 K, and independent statistical calculations were performed based on molecular geometry optimization and vibrational frequencies calculated at the B3LYP/6 − 31 + G(d,p) level of theory. Values derived with the independent methods are shown to be in excellent accord with a scaling factor of 0.975 applied to the calculated frequencies. This same scaling factor was successfully applied in the analysis of results for other polycyclic molecules, as described in recent articles by this research group. All experimental results are compared with property values reported in the literature. Thermodynamic consistency between properties is used to show that several studies in the literature are erroneous.     

An efficient in silico screening method based on the protein-compound affinity matrix and its application to the design of a focused library for cytochrome P450 (CYP) ligands

A new method has been developed to design a focused library based on available active compounds using protein-compound docking simulations. This method was applied to the design of a focused library for cytochrome P450 (CYP) ligands, not only to distinguish CYP ligands from other compounds but also to identify the putative ligands for a particular CYP. Principal component analysis (PCA) was applied to the protein-compound affinity matrix, which was obtained by thorough docking calculations between a large set of protein pockets and chemical compounds. Each compound was depicted as a point in the PCA space. Compounds that were close to the known active compounds were selected as candidate hit compounds. A machine-learning technique optimized the docking scores of the protein-compound affinity matrix to maximize the database enrichment of the known active compounds, providing an optimized focused library.     

In vitro screening and isolation of human aromatase inhibitors from Cicer arietinum by a novel continuous online method combining chromatographic techniques

Ultrafiltration liquid chromatography with mass spectrometry can efficiently and rapidly screen and identify ligands from the seeds of Cicer arietinum for human aromatase. Using this method, we identified 11 major compounds, including organic acids, organic acid glycosides, flavone glycosides, isoflavones, and isoflavone glycosides, as potent human aromatase inhibitors. A continuous online method, including pressurized liquid extraction, countercurrent chromatography, and preparative liquid chromatography, was developed for scaling up the production of these compounds with high purity and efficiency. The bioactivity of the separated compounds was assessed by an in vitro enzyme inhibition assay. This novel approach using a combination of ultrafiltration liquid chromatography with mass spectrometry and pressurized liquid extraction with countercurrent chromatography and preparative liquid chromatography as well as an in vitro enzyme inhibition assay could be applied to efficiently screen and isolate human aromatase inhibitors from complex samples and to the large‐scale production of functional food and nutraceutical ingredients.     

In silico inspired design and synthesis of a novel tubulin-binding anti-cancer drug: folate conjugated noscapine (Targetin)

Our screen for tubulin-binding small molecules that do not depolymerize bulk cellular microtubules, but based upon structural features of well known microtubule-depolymerizing colchicine and podophyllotoxin, revealed tubulin binding anti-cancer property of noscapine (Ye et al. in Proc Natl Acad Sci USA 95:2280–2286, 1998). Guided by molecular modelling calculations and structure–activity relationships we conjugated at C9 of noscapine, a folate group—a ligand for cellular folate receptor alpha (FRα). FRα is over-expressed on some solid tumours such as ovarian epithelial cancers. Molecular docking experiments predicted that a folate conjugated noscapine (Targetin) accommodated well inside the binding cavity (docking score −11.295 kcal/mol) at the interface between α- and β-tubulin. The bulky folate moiety of Targetin is extended toward lumen of microtubules. The binding free energy (ΔG _bind) computed based on molecular mechanics energy minimization was −221.01 kcal/mol that revealed favourable interaction of Targetin with the receptor. Chemical synthesis, tubulin-binding experiments, and anti-cancer activity in vitro corroborate fully well with the molecular modelling experiments. Targetin binds tubulin with a dissociation constant (K _d value) of 149 ± 3.0 μM and decreases the transition frequencies between growth and shortening phases of microtubule assembly dynamics at concentrations that do not alter the total polymer mass. Cancer cells in general were more sensitive to Targetin compared with the founding compound noscapine (IC₅₀ in the range of 15–40 μM). Quite strikingly, ovarian cancer cells (SKOV3 and A2780), known to overexpress FRα, were much more sensitive to targetin (IC₅₀ in the range of 0.3–1.5 μM).     

Synthesis,anticancer screening,and in silico ADME prediction of novel 2-pyridones as Pim inhibitors

A novel series of 26 substituted N-(2-ethylphenyl)-2-oxo-pyridine-3-carbonitriles have been designed and synthesized via one-pot synthesis of various aromatic aldehydes, different aromatic acetophenones, and 2-cyano-N-(2-ethylphenyl)acetamide 1 . Moreover, cytotoxicity of the target compounds was evaluated by NCI, which selected 14 compounds for one-dose screening. Among them, compound 21 was selected for five-dose screening, which confirmed its potency against most of cancer cell lines. This compound elicited selectivity profile against human cell line WI-38. Cell cycle analysis was carried out, revealed that compound 21 is an apoptosis inducer causing cell cycle arrest at G2/M. Further exploration on the mode of action by evaluating its effect against Pim-1, Pim-2, and Pim-3 demonstrated its inhibitory effect on Pim-1 and Pim-3 rather than Pim-2. Molecular docking showed that compound 21 binds with high affinity to the active site of Pim-1 enzyme through three hydrogen bonds and two arene-H bonds.     

Parallel synthesis and antimalarial screening of a 4-aminoquinoline library

Due to growing problems with drug resistance, there is an outstanding need for new, cost-effective drugs for the treatment of malaria. The 4-aminoquinolines have provided a number of useful antimalarials, and Plasmodium falciparum, the causative organism for the most deadly form of human malaria, is generally slow to develop resistance to these drugs. Therefore, diverse screening libraries of quinolines continue to be useful for antimalarial drug discovery. We report herein the development of an efficient method for producing libraries of 4-aminoquinolines variant in the side chain portion of the molecule. The effects of these substitutions were evaluated by screening this library for activity against P. falciparum, revealing four potent compounds active against drug-resistant strains.     

Molecular modeling on pyruvate phosphate dikinase of Entamoeba histolytica and in silico virtual screening for novel inhibitors

Pyruvate phosphate dikinase (PPDK) is the key enzyme essential for the glycolytic pathway in most common and perilous parasite Entamoeba histolytica. Inhibiting the function of this enzyme could control the wide spread of intestinal infections caused by Entamoeba histolytica in humans. With this objective, we modeled the three dimensional structure of the PPDK protein. We used templates with 51% identity and 67% similarity to employ homology-modeling approach. Stereo chemical quality of protein structure was validated by protein structure validation program PROCHECK and VERIFY3D. Experimental proof available in literature along with the in silico studies indicated Lys21, Arg91, Asp323, Glu325 and Gln337 to be the probable active sites in the target protein. Virtual screening was carried out using the genetic docking algorithm GOLD and a consensus scoring function X-Score to substantiate the prediction. The small molecule libraries (ChemDivision database, Diversity dataset, Kinase inhibitor database) were used for screening process. Along with the high scoring results, the interaction studies provided promising ligands for future experimental screening to inhibit the function of PPDK in Entamoeba histolytica. Further, the phylogeny study was carried out to assess the possibility of using the proposed ligands as inhibitors in related pathogens.