Benzimidazoles Containing Piperazine Skeleton at C-2 Position as Promising Tubulin Modulators with Anthelmintic and Antineoplastic Activity.

Benzimidazole anthelmintic drugs hold promise for repurposing as cancer treatments due to their interference with tubulin polymerization and depolymerization, manifesting anticancer properties. We explored the potential of benzimidazole compounds with a piperazine fragment at C-2 as tubulin-targeting agents. In particular, we assessed their anthelmintic activity against isolated Trichinella spiralis muscle larvae and their effects on glioblastoma (U-87 MG) and breast cancer (MDA-MB-231) cell lines. Compound 7c demonstrated exceptional anthelmintic efficacy, achieving a 92.7% reduction in parasite activity at 100 µg/mL after 48 hours. In vitro cytotoxicity analysis of MDA-MB 231 and U87 MG cell lines showed that derivatives 7b, 7d, and 7c displayed lower IC50 values compared to albendazole (ABZ), the control. These piperazine benzimidazoles effectively reduced cell migration in both cell lines, with compound 7c exhibiting the most significant reduction, making it a promising candidate for further study. The binding mode of the most promising compound 7c, was determined using the induced fit docking-molecular dynamics (IFD-MD) approach. Regular docking and IFD were also employed for comparison. The IFD-MD analysis revealed that 7c binds to tubulin in a unique binding cavity near that of ABZ, but the benzimidazole ring was fitted much deeper into the binding pocket. Finally, the absolute free energy of perturbation technique was applied to evaluate the 7c binding affinity, further confirming the observed binding mode.

R346K Mutation in the Mu Variant of SARS-CoV-2 Alters the Interactions with Monoclonal Antibodies from Class 2: A Free Energy Perturbation Study.

The Mu variant of SARS-CoV-2 has been recently classified as a variant of interest (VOI) by the World Health Organization (WHO) but limited data are available at the moment. In particular, special attention was given to the R346K mutation located in the receptor binding domain (RBD). In the current study, we performed free energy perturbation (FEP) calculations to elucidate its possible impact on a set of neutralizing monoclonal antibodies (mAbs) that have been shown to be strong inhibitors of the most other known COVID-19 variants. Our results show that R346K affects class 2 antibodies but its effect is not so significant (0.66 kcal/mol), i.e., it reduces the binding with antibodies by about 3-fold. An identical value was also calculated in the presence of both class 1 and class 2 antibodies (BD-812/836). Further, a similar reduction in the binding (0.4 kcal/mol) was obtained for the BD-821/771 pair of mAbs. For comparison, the addition of the K417N mutation, present in the newly registered Mu variant in July 2021 in the U.K., affected the class 1 mAbs by strongly reducing the binding by 1.29 kcal/mol or about 10-fold. Thus, the resistance effect of the R346K mutation on the Mu variant is possible but not so significant and is due to the additional decrease of antibody neutralization based on the reduced binding of class 2 antibodies.

N501Y and K417N Mutations in the Spike Protein of SARS-CoV-2 Alter the Interactions with Both hACE2 and Human-Derived Antibody: A Free Energy of Perturbation Retrospective Study.

The N501Y and K417N mutations in the spike protein of SARS-CoV-2 and their combination gave rise to questions, but the data on their mechanism of action at the molecular level were limited. In this study, we present free energy perturbation (FEP) calculations, performed at the end of December 2020, for the interactions of the spike S1 receptor-binding domain (RBD) with both the ACE2 receptor and an antibody derived from COVID-19 patients. Our results showed that the S1 RBD-ACE2 interactions were significantly increased whereas those with the STE90-C11 antibody dramatically decreased. The K417N mutation in a combination with N501Y fully abolished the antibody effect. However, Lys417Asn seems to have a compensatory mechanism of action increasing the S1 RBD-ACE2 free energy of binding. This may explain the increased spread of the virus observed in the U.K. and South Africa and also gives rise to an important question regarding the possible human immune response and the success of the already available vaccines. Notably, when the experimental data became available confirming our calculations, it was demonstrated that protein-protein FEP can be a useful tool for providing urgent data to the scientific community.

1H-benzimidazole-2-yl hydrazones as tubulin-targeting agents: Synthesis, structural characterization, anthelmintic activity and antiproliferative activity against MCF-7 breast carcinoma cells and molecular docking studies.

In the present study, fifteen benzimidazolyl-2-hydrazones 7a-7o of fluoro-, hydroxy- and methoxy-substituted benzaldehydes and 1,3-benzodioxole-5-carbaldehyde were synthesized and their structure was identified by IR, NMR, and elemental analysis. The compounds 7j 2-(3-hydroxybenzylidene)-1-(5(6)-methyl-1H-benzimidazol-2-yl)hydrazone and 7i 2-(3-hydroxybenzylidene)-1-(1H-benzimidazol-2-yl)hydrazone have exerted the strongest anthelmintic activity (100% after 24 h incubation period at 37 °C) against isolated muscle larvae of Trichinella spiralis in an in vitro experiment. The in vitro cytotoxicity assay towards MCF-7 breast cancer cells and mouse embryo fibroblasts 3T3 showed that the studied benzimidazolyl-2-hydrazones exhibit low to moderate cytotoxic effects. The ability of the studied benzimidazolyl-2-hydrazones to modulate microtubule polymerization was confirmed and suggested that their anthelmintic action is mediated through inhibition of the tubulin polymerization likewise the other known benzimidazole anthelmitics. It was also shown that the four most promising benzimidazolyl-2-hydrazones do not affect significantly the AChE activity even at high tested concentration, thus indicating that they do not have the potential for neurotoxic effects. The binding mode of compounds 7j and 7n in the colchicine-binding site of tubulin were clarified by molecular docking simulations. Taken together, these results demonstrate that for the synthesized benzimidazole derivatives the anthelmintic activity against T. spiralis and the inhibition of tubulin polymerization are closely related.

Discovery of new AKT1 inhibitors by combination of in silico structure based virtual screening approaches and biological evaluations.

Communicated by Ramaswamy H. Sarma.

An Improved Free Energy Perturbation FEP+ Sampling Protocol for Flexible Ligand-Binding Domains.

Recent improvements to the free energy perturbation (FEP) calculations, especially FEP+ , established their utility for pharmaceutical lead optimization. Herein, we propose a modified version of the FEP/REST (i.e., replica exchange with solute tempering) sampling protocol, based on detail studies on several targets by probing a large number of perturbations with different sampling schemes. Improved FEP+ binding affinity predictions for regular flexible-loop motions and considerable structural changes can be obtained by extending the prior to REST (pre-REST) sampling time from 0.24 ns/λ to 5 ns/λ and 2 × 10 ns/λ, respectively. With this new protocol, much more precise ∆∆G values of the individual perturbations, including the sign of the transformations and decreased error were obtained. We extended the REST simulations from 5 ns to 8 ns to achieve reasonable free energy convergence. Implementing REST to the entire ligand as opposed to solely the perturbed region, and also some important flexible protein residues (pREST region) in the ligand binding domain (LBD) has considerably improved the FEP+ results in most of the studied cases. Preliminary molecular dynamics (MD) runs were useful for establishing the correct binding mode of the compounds and thus precise alignment for FEP+ . Our improved protocol may further increase the FEP+ accuracy.

Discovery of GlyT2 Inhibitors Using Structure-Based Pharmacophore Screening and Selectivity Studies by FEP+ Calculations.

In recent years, mammalian Glycine transporter 2 (GlyT2) has emerged as a promising target for the development of compounds against chronic pain states. In our current work, we discovered a new set of promising hits that inhibit the glycine transporter at nano- and micromolar activity and have excellent selectivity over GlyT1 (as shown by in vitro studies) using a newly designed virtual screening (VS) protocol that combines a structure-based pharmacophore and docking screens with a success rate of 75%. Furthermore, the free energy perturbation calculations and molecular dynamics (MD) studies revealed the GlyT2 amino acid residues critical for the binding and selectivity of both Glycine and our Hit1 compound. The FEP+ results well-matched with the available literature mutational data proving the quality of the generated GlyT2 structure. On the basis of these results, we propose that our hit compounds may lead to new chronic pain agents to address unmet and challenging clinical needs.

Elucidation of the orientation of selected drugs with 2-hydroxylpropyl-ß-cyclodextrin using 2D-NMR spectroscopy and molecular modeling.

This project aims to study the nature of interaction and orientation of selected drugs such as dexamethorphan HBr (DXM), diphenhydramine HCl (DPH), and lidocaine HCl (LDC) inclusion complexes with hydroxyl-propyl ß-cyclodextrin (HP-ß-CD) using 1HNMR spectroscopy, 2D-NMR ROESY and molecular-modeling techniques. Freeze-drying technique was used to formulate the inclusion complexes between DXM, DPH and LDC with HP-ß-CD (1:1 M ratio) in solid state. Inclusion complex formation was initially characterized by Fourier transform-infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), X-ray diffraction (XRD) and scanning electron microscopy (SEM) techniques. Further characterization of inclusion complexes to determine the interaction of DXM, DPH and LDC with HP-ß-CD was performed using the 1HNMR spectroscopy, 2D-NMR ROESY and molecular modeling techniques. Inclusion complexes of DXM, DPH and LDC with HP-ß-CD were successfully prepared using the freeze-drying technique. Preliminary studies with FT-IR, DSC, XRD and SEM indicated the formation of inclusion complexes of DXM, DPH and LDC with HP-ß-CD at 1:1 M ratio. 1HNMR study showed a change in proton chemical shift upon complexation. 2D-NMR ROESY (two-dimensional) spectroscopy gave an insight into the spatial arrangement between the host and guest atoms. 2D-ROESY experiments further predicted the direction of orientation of guest molecules, indicating the probability that amino moieties of DXM, DPH and LDC are inside the hydrophobic HP-ß-CD cavity. Cross-peaks of inclusion complexes demonstrated intermolecular nuclear Overhauser effects (NOE) between the amino protons in DXM, DPH and LDC and H-atoms of HP-ß-CD. Molecular modeling studies further confirmed the NMR data, providing a structural basis of the individual complex formations. Microsecond time-level molecular dynamics and metadynamics simulations indicate much stronger binding of DXM to HP-ß-CD and more dynamic behavior for DPH and LDC. In particular, LDC can exhibit multiple binding modes, and even spent some time (∼1-2%) out of the carrier, proving the dynamic nature of the complex. To conclude, 2D-NMR and molecular dynamic simulations elucidate the formation of inclusion complexes and intermolecular interactions of DXM, DPH and LDC with HP-ß-CD.

Prediction of Accurate Binding Modes Using Combination of Classical and Accelerated Molecular Dynamics and Free-Energy Perturbation Calculations: An Application to Toxicity Studies.

Estimating the correct binding modes of ligands in protein-ligand complexes is crucial not only in the drug discovery process but also for elucidating potential toxicity mechanisms. In the current paper, we propose a computational modeling workflow using the combination of docking, classical molecular dynamics (cMD), accelerated molecular dynamics (aMD) and free-energy perturbation (FEP+ protocol) for identification of possible ligand binding modes. It was applied for investigation of selected perfluorocarboxyl acids (PFCAs) in the PPARγ nuclear receptor. Although both regular and induced fit docking failed to reproduce the experimentally determined binding mode of the ligands when docked into a non-native X-ray structure, cMD and aMD simulations successfully identified the most probable binding conformations. Moreover, multiple binding modes were identified for all of these compounds and the shorter-chain PFCAs continuously moved between a few energetically favorable binding conformations. On the basis of MD predictions of binding conformations, we applied the default and also redesigned FEP+ sampling protocols, which accurately reproduced experimental differences in the binding energies. Thus, the preliminary MD simulations can also provide helpful information about correct setup of the FEP+ calculations. These results show that the PFCA binding modes were accurately predicted and that the FEP+ protocol can be used to estimate free energies of binding of flexible ligands that are not typical druglike compounds. Our in silico workflow revealed the specific ligand-residue interactions within the ligand binding domain and the main characteristics of the PFCAs, and it was concluded that these compounds are week PPARγ partial agonists. This work also suggests a common pipeline for identification of ligand binding modes, ligand-protein dynamics description, and relative free-energy calculations.

PPARγ non-covalent antagonists exhibit mutable binding modes with a similar free energy of binding: a case study.

The structural and dynamical properties of PPARγ receptor in a complex with either partial or full agonists have been intensively studied but little is known about the receptor antagonistic conformation. A composition of microsecond accelerated molecular dynamics (aMD) simulation show that like partial agonists a non-covalent PPARγ full antagonist can bind in different modes of similar population size and free energies of binding. Four different and periodically exchanging ligand conformations are detected and described. The studied antagonist interacts with different receptor substructures and affects both the co-activator and the Cdk5 phosphorylation sites and, presumably, the natural complex with the DNA. However, no significant changes in the conformational states of the activation helix 12, and in particular an antagonist orientation, have been recorded. Finally, our results show also that the aMD approach can be successfully used in recovering the possible binding modes, considering fully the receptor flexibility, and is not dependent on the starting conformation.