Synthesis and biological evaluation of 1,2,4-trisubstituted imidazoles and 1,3,5-trisubstituted pyrazoles as inhibitors of transforming growth factor β type 1 receptor (ALK5)

Two series of nitrogenous heterocycle compounds—1,2,4-trisubstituted imidazoles and 1,3,5-trisubstituted pyrazoles have been synthesized and evaluated for their ALK5 inhibitory activity and cytotoxicity in TGFβ-Smad2 assay and MTT assay, respectively. The ALK4/5/7 inhibitory activity of some compound was also evaluated in ALK4/5/7 autophosphorylation assays. Compounds 6c and 14c showed relatively potent ALK5 inhibition while weak cytotoxicity. At the same time, compounds 6c and 14c display relatively better ALK5 selectivity versus ALK4/ALK7 (nearly 10-fold) compared with SB431542, a well known ALK5 inhibitor. Compound 6g2 proved to be a moderately selective ALK4 inhibitor versus ALK5 and ALK7 (>10-fold). The binding mode of 14c generated by flexible docking study shows that 14c fits well into the site cavity of ALK5 by forming several tight interactions.     

Binding selectivity studies of PKBα using molecular dynamics simulation and free energy calculations

Designing selective protein kinase B (PKB/Akt) inhibitor is an area of intense research to develop potential anticancer drugs. In the present study, the molecular basis governing PKB-selective inhibition has been investigated using molecular dynamics simulation. The binding free energies calculated by MM/PBSA gave a good correlation with the experimental biological activity and a good explanation of the activity difference of the studied inhibitors. The decomposition of free energies by MM/GBSA indicates that the ethyl group on pyrrolo[2,3-d]pyrimidine ring of inhibitor Lig1 (N-{[(3S)-3-amino-1-(5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrolidin-3-yl]-methyl}-2,4-difluoro-benzamide) is an important contributor to its PKBα selectivity due to its hydrophobic interaction with the side chain of Thr291 in PKBα. The substituted groups on the pyrrolidine ring of Lig1 also show a strong tendency to mediate protein-ligand interactions through the hydrogen bonds formed between the amino or amide groups of Lig1 and the carboxyl O atoms of Glu234, Glu278, and Asp292 of PKBα. It was reported that there are only three key amino acid differences between PKBα (Thr211, Ala230, Met281) and PKA (Val104, Val123, Leu173) within the clefts of ATP-binding sites. These differences propel a drastic conformational change in PKA, weakening its binding interactions with inhibitor. The impact was also confirmed by MD simulated interaction modes of inhibitor binding to PKBα mutants with the in silico mutations of the three key amino acids, respectively. We expect that the results obtained here could be useful for future rational design of specific ATP-competitive inhibitors of PKBα.     

Insight into the binding mode of a novel LSD1 inhibitor by molecular docking and molecular dynamics simulations

Abstract

Lysine-specific demethylase (LSD1) is an important enzyme for histone lysine methylation. Downregulated LSD1 expression has been linked to cancer proliferation, migration and invasion, indicating that it is an important target for anti-cancer medication. In the present study, the binding modes of a recent reported new series of LSD1 inhibitor were analyzed by using molecular docking and molecular dynamics simulations. A binding mode of these inhibitors was proposed based on the results. According to this binding mode, Thr628 can form two important hydrogen bonds with these inhibitors. Moreover, if the inhibitors can form an additional hydrogen bond with hydroxyl group of Ser289, the potency of the inhibitor can be greatly improved, such as the best inhibitor (compound 12d) in this series. Hydrophobic interactions between the inhibitors and LSD1 are also key contributor here, such as the interaction between the hydrophobic groups (benzene rings) of the inhibitors and the hydrophobic residues of LSD1 (including Val288, Val317, Val811, Ala814, Leu659, Trp751 and Tyr761). Based on the results and analysis, it may provide some useful information for future novel LSD1 inhibitor design.     

A systematic analysis of the resistance and sensitivity of HER2YVMA receptor tyrosine kinase mutant to tyrosine kinase inhibitors in HER2-positive lung cancer

Human epidermal growth factor receptor 2 (HER2) has become a well-established target for the treatment of HER2-positive lung cancer. However, a frequently observed in-frame mutation that inserts amino acid quadruplex Tyr776-Val777-Met778-Ala779 at G776 (G776^YVMA) in HER2 kinase domain can cause drug resistance and sensitivity, largely limiting the application of reversible tyrosine kinase inhibitors in lung cancer therapy. A systematic investigation of the intermolecular interactions between the HER2^YVMA mutant and clinical small-molecule inhibitors would help to establish a complete picture of drug response to HER2 G776^YVMA insertion in lung cancer, and to design new tyrosine kinase inhibitors with high potency and selectivity to target the lung cancer-related HER2^YVMA mutant. Here, we combined homology modeling, ligand grafting, structure minimization, molecular simulation and binding affinity analysis to profile a number of tyrosine kinase inhibitors against the G776^YVMA insertion in HER2. It is found that the insertion is far away from HER2 active pocket and thus cannot contact inhibitor ligand directly. However, the insertion is expected to induce marked allosteric effect on some regions around the pocket, including A-loop and hinges connecting between the N- and C-lobes of HER2 kinase domain, which may exert indirect influence to inhibitor binding. Most investigated inhibitors exhibit weak binding strength to both wild-type and mutant HER2, which can be attributed to steric hindrance that impairs ligand compatibility with HER2 active pocket. However, the cognate inhibitor lapatinib and the non-cognate inhibitor bosutinib were predicted to have low affinity for wild-type HER2 but high affinity for HER2^YVMA mutant, which was confirmed by subsequent kinase assay experiments; the inhibitory potencies of bosutinib against wild-type and mutant HER2 were determined to be IC₅₀?>?1000 and =27?nM, respectively, suggesting that the bosutinib might be exploited as a selective inhibitor for mutant over wild-type HER2. Structural examination revealed that formation of additional non-bonded interactions such as hydrogen bonds and hydrophobic contacts with HER2 A-loop region due to G776^YVMA insertion is the primary factor to improve bosutinib affinity upon the mutation.     

Computer-aided molecular design of 1H-imidazole-2,4-diamine derivatives as potential inhibitors of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> DHFR enzyme

Design and discovery of new potential inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR), equally active against both the wild-type and mutant strains, is urgently needed. In this study, a computer-aided molecular design approach that involved ab initio molecular orbital and density functional theory calculations, along with molecular electrostatic potential analysis, and molecular docking studies was employed to design 15 1H-imidazole-2,4-diamine derivatives as potential inhibitors of PfDHFR enzyme. Visual inspection of the binding modes of the compounds demonstrated that they all interact, via H-bond interactions, with key amino acid residues (Asp54, Ileu/Leu164, Asn/Ser108 and Ile14) similar to those of WR99210 (3) in the active site of the enzymes used in the study. These interactions are known to be essential for enzyme inhibition. These compounds showed better or comparable binding affinities to that of the bound ligand (WR99210). In silico toxicity predictions, carried out using TOPKAT software, also indicated that the compounds are non-toxic.     

2D-QSAR study,molecular docking,and molecular dynamics simulation studies of interaction mechanism between inhibitors and transforming growth factor-beta receptor I (ALK5)

Transforming growth factor type 1 receptor (ALK5) is kinase associated with a wide variety of pathological processes, and inhibition of ALK5 is a good strategy to treat many kinds of cancer and fibrotic diseases. Recently, a series of compounds have been synthesized as ALK5 inhibitors. However, the study of their selectivity against other potential targets remains elusive. In this research, a data-set of ALK5 inhibitors were collected and studied based on the combination of 2D-QSAR, molecular docking and molecular dynamics simulation. The quality of QSAR models were assessed statistically by F, R², and R²_ADJ, proved to be credible. The cross-validations for the models (q²_LOO = 0.571 and 0.629, respectively) showed their robustness, while the external validations (r²_test = 0.703 and 0.764, respectively) showed their predictive power. Besides, the predicted binding free energy results calculated by MM/GBSA method were in accordance with the experimental data, and the van der Waals energy term was the factor that had the most significant impact on ligand binding. What is more, several important residues were found to significantly affect the binding affinity. Finally, based on our analyses above, a proposed series of molecules were designed.     

A common feature-based 3D-pharmacophore model generation and virtual screening: identification of potential PfDHFR inhibitors

A four-feature 3D-pharmacophore model was built from a set of 24 compounds whose activities were reported against the V1/S strain of the Plasmodium falciparum dihydrofolate reductase (PfDHFR) enzyme. This is an enzyme harboring Asn51Ile + Cys59Arg + Ser108Asn + Ile164Leu mutations. The HipHop module of the Catalyst program was used to generate the model. Selection of the best model among the 10 hypotheses generated by HipHop was carried out based on rank and best-fit values or alignments of the training set compounds onto a particular hypothesis. The best model (hypo1) consisted of two H-bond donors, one hydrophobic aromatic, and one hydrophobic aliphatic features. Hypo1 was used as a query to virtually screen Maybridge2004 and NCI2000 databases. The hits obtained from the search were subsequently subjected to FlexX and Glide docking studies. Based on the binding scores and interactions in the active site of quadruple-mutant PfDHFR, a set of nine hits were identified as potential inhibitors.     

Molecular dynamics simulation,binding free energy calculation and molecular docking of human D-amino acid oxidase (DAAO) with its inhibitors

Schizophrenia is a mental illness; most affected people live in developing countries, and neither appropriate treatment nor commercial drugs are currently available. One possibility is to inhibit human-d-amino acid oxidase (h-DAAO). In this study, molecular dynamic simulations of the monomer, dimer and tetramer forms of h-DAAO complexed with the inhibitor 3-hydroxyquinolin-2(1H)-one(2) were performed. Seven residues, Leu51, Gln53, Leu215, Tyr228, Ile230, Arg283 and Gly313, were identified as essential for interacting with the inhibitor. Molecular docking of h-DAAO with pyrrole, quinoline and kojic acid derivatives, representing 69 known or potential h-DAAO inhibitors, was also performed. The results indicated that the activity of the inhibitor can be improved by modifying the compounds to have a substituent group capable of interacting with the side chain of Tyr228. Van der Waals interactions of the inhibitor with the hydrophobic pocket of h-DAAO and electrostatic interactions or H-bonds with Arg283 and Gly313 were important elements in determining the efficiency of the inhibitor. These results provide information on the interaction between h-DAAO and its inhibitors at the molecular level and can aid in the design of novel inhibitors against h-DAAO for new drug development in the treatment of schizophrenia.     

Design,synthesis and docking study of novel coumarin ligands as potential selective acetylcholinesterase inhibitors

New coumaryl-thiazole derivatives with the acetamide moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and in vitro tested as acetylcholinesterase (AChE) inhibitors. 2-(diethylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide (6c, IC₅₀ value of 43?nM) was the best AChE inhibitor with a selectivity index of 4151.16 over BuChE. Kinetic study of AChE inhibition revealed that 6c was a mixed-type inhibitor. Moreover, the result of H4IIE hepatoma cell toxicity assay for 6c showed negligible cell death. Molecular docking studies were also carried out to clarify the inhibition mode of the more active compounds. Best pose of compound 6c is positioned into the active site with the coumarin ring wedged between the residues of the CAS and catalytic triad of AChE. In addition, the coumarin ring is anchored into the gorge of the enzyme by H-bond with Tyr130.     

Structural and energetic insight into the isoform-selective inhibitors of tumour marker Hsp90 against Grp94

The heat shock protein 90 (Hsp90) represents a new avenue for cancer therapy. A novel benzolactam inhibitor, compound 31, showed a great selectivity for Hsp90α and Hsp90β against Grp94. However, the structural basis for the great selectivity of compound 31 for Hsp90α/β versus Grp94 remains poorly understood. In this study, we carried out molecular docking, molecular dynamics simulations and binding free energy calculations (MM-GBSA) to address the isoform selective property. Molecular docking studies indicated the different binding modes of the Hsp90 and Grp94 with compound 31. The MM-GBSA calculations revealed that the hydrophobic interactions between compound 31 and proteins contributed the most to the binding affinity and the Grp94/compound 31 complex could result in a less energy-favourable complex compared with the Hsp90α/compound 31 and the Hsp90β/compound 31 complexes. This may render the weak binding of compound 31 to the Grp94. This study may be helpful for the future design of novel and selective Hsp90 inhibitors.     

Structure-guided discovery of cyclin-dependent kinase inhibitors

CDK2 inhibitors containing the related bicyclic heterocycles pyrazolopyrimidines and imidazopyrazines were discovered through high-throughput screening. Crystal structures of inhibitors with these bicyclic cores and two more related ones show that all but one have a common binding mode featuring two hydrogen bonds (H-bonds) to the backbone of the kinase hinge region. Even though ab initio computations indicated that the imidazopyrazine core would bind more tightly to the hinge, pyrazolopyrimidines gain an advantage in potency through participation of N4 in an H-bond network involving two catalytic residues and bridging water molecules. Further insight into inhibitor/CDK2 interactions was gained from analysis of additional crystal structures. Significant gains in potency were obtained by optimizing the fit of hydrophobic substituents to the gatekeeper region of the ATP binding site. The most potent inhibitors have good selectivity.     

Discovery of 3-(4-sulfamoylnaphthyl)pyrazolo[1,5-a]pyrimidines as potent and selective ALK2 inhibitors

The pyrazolo[1,5-a]pyrimidine LDN-193189 is a potent inhibitor of activin receptor-like kinase 2 (ALK2) but is nonselective for highly homologous ALK3 and shows only modest kinome selectivity. Herein, we describe the discovery of a novel series of potent and selective ALK2 inhibitors by replacing the quinolinyl with a 4-(sulfamoyl)naphthyl, yielding ALK2 inhibitors that exhibit not only excellent discrimination versus ALK3 but also high kinome selectivity. In addition, the optimized compound 23 demonstrates good ADME and in vivo pharmacokinetic properties.     

Effect of guanidine and arginine on protein–ligand interactions in multimodal cation‐exchange chromatography

The addition of fluid phase modifiers provides significant opportunities for increasing the selectivity of multimodal chromatography. In order to optimize this selectivity, it is important to understand the fundamental interactions between proteins and these modifiers. To this end, molecular dynamics (MD) simulations were first performed to study the interactions of guanidine and arginine with three proteins. The simulation results showed that both guanidine and arginine interacted primarily with the negatively charged regions on the proteins and that these regions could be readily predicted using electrostatic potential maps. Protein surface characterization was then carried out using computationally efficient coarse‐grained techniques for a broader set of proteins which exhibited interesting chromatographic retention behavior upon the addition of these modifiers. It was shown that proteins exhibiting an increased retention in the presence of guanidine possessed hydrophobic regions adjacent to negatively charged regions on their surfaces. In contrast, proteins which exhibited a decreased binding in the presence of guanidine did not have hydrophobic regions adjacent to negatively charged patches. These results indicated that the effect of guanidine could be described as a combination of competitive binding, charge neutralization and increased hydrophobic interactions for certain proteins. In contrast, arginine resulted in a significant decrease in protein retention times primarily due to competition for the resin and steric effects, with minimal accompanying increase in hydrophobic interactions. The approach presented in this paper which employs MD simulations to guide the application of coarse‐grained approaches is expected to be extremely useful for methods development in downstream bioprocesses. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:435–447, 2017     

Study on the binding mode of a pyrrolotriazin derivative with JAK2 by docking and MD simulation

The pyrrolotriazin derivative 2-(4-(4-((7-(3-(N-methylmethylsulfonamido)phenyl)pyrrolo [2,1-f][1,2,4]triazin-2-yl)amino)phenyl)piperidin-1-yl)acetamide (PPA) is a potential Janus kinase 2 (JAK2) inhibitor. The binding mode between PPA and JAK2 was investigated by using a combined method of docking, molecular dynamics (MD) simulation and binding free-energy calculation. The docking calculations preliminarily indicated that there were two possible binding modes 1 and 2; MD simulations and binding free-energy calculations identified that binding mode 1 was more stable and favourable, with the lower MM-PBSA binding free energy of ?34.00?±?0.17?kcal/mol. Moreover, some valuable binding information is revealed as follows: the inhibitor PPA is suitably located at the ATP-binding site of JAK2 and the hydrophobic interaction plays an essential role. PPA not only interacts with residues Leu855, Val863, Ala880, Tyr931, Leu932 and Leu983 via hydrophobic interaction but also interacts with Ser936 and Asp994 by hydrogen bonds. These two factors are advantageous for PPA to strongly bind to JAK2. These results help to understand the action mechanisms and designing new compounds with a higher affinity to JAK2.     

Synthesis,evaluation and molecular modeling of cyclic tetrapeptide histone deacetylase inhibitors as anticancer agents

Histone deacetylase inhibitors (HDACIs) are a promising class of anticancer agents. To examine whether a slight change in the recognition domain could alter their inhibitory activity, we synthesized a series of cyclo(?l ‐Am7(S2Py)‐Aib‐l ‐Phe(n‐Me)‐d ‐Pro)derivatives and evaluated their HDAC inhibitory and anticancer activities. The peptides exhibited potent HDAC inhibitory activity and inhibited three human cancer cell lines with IC₅₀ in the micromolar range. Docking and molecular dynamics simulation were conducted to explore the interaction mechanisms of class I and II HDACs with these inhibitors. It revealed that the zinc ion in the active site coordinated five atoms of HDACs and the sulfur atom of the inhibitor. The metal binding domains of these compounds interacted with HDAC2, and the surface recognition domains of these compounds interacted with HDAC4 through hydrogen bonding. The hydrophobic interactions also provided favorable contributions to stabilize the complexes. The results obtained from this study would be helpful for us to design some novel cyclic tetrapeptides that may act as potent HDACIs. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Discovery of (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phenyl)-amide as potent and isoform selective ROCK2 inhibitors

ROCK1 and ROCK2 are highly homologous isoforms. Accumulated studies indicate that they have distinct different functions, and the development of isoform selective ROCK inhibitors will pave new roads for the treatment of various diseases. In this work, a series of amide-chroman derivatives were synthesized and biologically evaluated in order to develop potent and isoform selective ROCK2 inhibitors. Remarkably, (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phenyl)-amide ((S)-7c) possessed ROCK2 inhibitory activity with an IC₅₀ value of 3?nM and 22.7-fold isoform selectivity (vs. ROCK1). Molecular docking indicated that hydrophobic interactions were the key element for the high potency and isoform selectivity of (S)-7c. The binding free energies predicted by MM/GBSA were in good agreement with the experimental bioactivities, and the analysis of individual energy terms suggested that residue Lys105 in ROCK1 or Lys121 in ROCK2 was the key residue for the isoform selectivity of (S)-7c.     

Identification of the hot spot residues for pyridine derivative inhibitor CCT251455 and ATP substrate binding on monopolar spindle 1 (MPS1) kinase by molecular dynamic simulation

Protein kinase monopolar spindle 1 plays an important role in spindle assembly checkpoint at the onset of mitosis. Over expression of MPS1 correlated with a wide range of human tumors makes it an attractive target for finding an effective and specific inhibitor. In this work, we performed molecular dynamics simulations of protein MPS1 itself as well as protein bound systems with the inhibitor and natural substrate based on crystal structures. The reported orally bioavailable 1 h-pyrrolo [3,2-c] pyridine inhibitors of MPS1 maintained stable binding in the catalytic site, while natural substrate ATP could not stay. Comparative study of stability and flexibility of three systems reveals position shifting of β-sheet region within the catalytic site, which indicates inhibition mechanism was through stabilizing the β-sheet region. Binding free energies calculated with MM-GB/PBSA method shows different binding affinity for inhibitor and ATP. Finally, interactions between protein and inhibitor during molecular dynamic simulations were measured and counted. Residue Gly605 and Leu654 were suggested as important hot spots for stable binding of inhibitor by molecular dynamic simulation. Our results reveal an important position shifting within catalytic site for non-inhibited proteins. Together with hot spots found by molecular dynamic simulation, the results provide important information of inhibition mechanism and will be referenced for designing novel inhibitors.     

Molecular docking studies on quinazoline antifolate derivatives as human thymidylate synthase inhibitors

We have performed molecular docking on quinazoline antifolates complexed with human thymidylate synthase to gain insight into the structural preferences of these inhibitors. The study was conducted on a selected set of one hundred six compounds with variation in structure and activity. The structural analyses indicate that the coordinate bond interactions, the hydrogen bond interactions, the van der Waals interactions as well as the hydrophobic interactions between ligand and receptor are responsible simultaneously for the preference of inhibition and potency. In this study, fast flexible docking simulations were performed on quinazoline antifolates derivatives as human thymidylate synthase inhibitors. The results indicated that the quinazoline ring of the inhibitors forms hydrophobic contacts with Leu192, Leu221 and Tyr258 and stacking interaction is conserved in complex with the inhibitor and cofactor.     

Pharmacophore based 3D-QSAR modeling and free energy analysis of VEGFR-2 inhibitors

VEGFR-2, a transmembrane tyrosine kinase receptor is responsible for angiogenesis and has been an attractive target in treating cancers. The inhibition mechanism of structurally diverse urea derivatives, reported as VEGFR-2 inhibitors, was explored by pharmacophore modeling, QSAR, and molecular dynamics based free energy analysis.The pharmacophore hypothesis AADRR, resulted in a highly significant atom based 3D-QSAR model (r² = 0.94 and q² = 0.84). Binding free energy analysis of the docked complexes of highly active and inactive compounds, after 7 ns MD simulation, revealed the importance of van der Waals interaction in VEGFR-2 inhibition. The decomposition of binding free energy on a per residue basis disclosed that the residues in hinge region and hydrophobic pocket play a role in discriminating the active and inactive inhibitors. Thus, the present study proposes a pharmacophore hypothesis representing the identified interactions pattern and its further application as a template in screening databases to identify novel VEGFR-2 inhibitor scaffolds.     

Discovery of 7-azaindole based anaplastic lymphoma kinase (ALK) inhibitors: Wild type and mutant (L1196M) active compounds with unique binding mode

We have identified a novel 7-azaindole series of anaplastic lymphoma kinase (ALK) inhibitors. Compounds 7b, 7m and 7n demonstrate excellent potencies in biochemical and cellular assays. X-ray crystal structure of one of the compounds (7k) revealed a unique binding mode with the benzyl group occupying the back pocket, explaining its potency towards ALK and selectivity over tested kinases particularly Aurora-A. This binding mode is in contrast to that of known ALK inhibitors such as Crizotinib and NVP-TAE684 which occupy the ribose binding pocket, close to DFG motif.