Insights into the interaction of potent antimicrobial chalcone triazole analogs with human serum albumin: spectroscopy and molecular docking approaches

Mechanistic insights into the interaction of five previously chemically synthesized triazole-linked chalcone analogs (CTs) with human serum albumin (HSA) were sought using various spectroscopic techniques (UV-visible absorption, fluorescence, and circular dichroism) and molecular docking. The fluorescence quenching experiments performed at three different temperatures (288, 298 and 308 K) revealed the static mode of quenching and the binding constants (K_b ∼ 10^6–9) obtained indicated the strong affinity of these analogs for HSA. Furthermore, significant changes in the secondary structure of HSA in the presence of these analogs were also confirmed by far UV-CD spectroscopy. The thermodynamic properties such as the enthalpy change (ΔH°), Gibbs free energy change (ΔG°) and entropy change (ΔS°) revealed that the binding process was spontaneous and exothermic. Theoretical studies, viz., DFT and molecular docking corroborated the experimental results as these five analogs could bind with HSA through hydrogen bonding and hydrophobic interactions. The present study provides useful information regarding the interaction mechanism of these analogs with HSA, which can provide a new avenue to design more potent chalcone triazole analogs for use in the biomedical field.

Mechanistic insights into the interaction of five previously chemically synthesized triazole-linked chalcone analogs with human serum albumin were analyzed using UV-visible absorption, fluorescence quenching, circular dichroism and molecular docking studies.     

Molecular interaction of silicon quantum dot micelles with plasma proteins: hemoglobin and thrombin

Protein conformational changes are associated with potential cytotoxicity upon interaction with small molecules or nanomaterials. Protein misfolding leads to protein-mediated diseases; thus, it is important to study the conformational changes in proteins using nanoparticles as drug carriers. In this study, the conformational changes in hemoglobin and thrombin were observed using fluorescence spectroscopy, circular dichroism spectroscopy and molecular modelling studies after interaction with non-toxic, water-soluble near-infrared silicon quantum dot micelles. The molecular docking results indicated that the binding affinities of hemoglobin and thrombin with Si QD micelles are good. In addition, molecular dynamics simulations were performed to obtain more detailed information.

Overall graphical representation of 1-decene, F-127, and crystal structures of hemoglobin and thrombin.     

Andrographolide inhibits human serum albumin fibril formations through site-specific molecular interactions

Protein misfolding and fibrillation are the fundamental traits in degenerative diseases like Alzheimer''s, Parkinsonism, and diabetes mellitus. Bioactives such as flavonoids and terpenoids from plant sources are known to express protective effects against an array of diseases including diabetes, Alzheimer''s and obesity. Andrographolide (AG), a labdane diterpenoid is prescribed widely in the Indian and Chinese health care systems for classical efficacy against a number of degenerative diseases. This work presents an in depth study on the effects of AG on protein fibrillating pathophysiology. Thioflavin T fluorescence spectroscopy and DLS results indicated concentration dependent inhibition of human serum albumin (HSA) fibrillation. The results were confirmed by electron microscopy studies. HSA fibril formations were markedly reduced in the presence of AG. Fluorescence studies and UV-Vis experiments confirmed further that AG molecularly interacts with HSA at site. In silico molecular docking studies revealed hydrogen bonding and hydrophobic interactions with HSA in the native state. Thus AG interacts with HSA, stabilizes the native protein structure and inhibits fibrillation. The results demonstrated that the compound possesses anti-amyloidogenic properties and can be promising against some human degenerative diseases.

Andrographolide inhibited HSA protein fibrillation through site specific interactions.     

Interaction mechanism of olaparib binding to human serum albumin investigated with NMR relaxation data and computational methods

The interaction mechanism between olaparib (OLA) and human serum albumin (HSA) has been investigated using experimental and computational techniques. An NMR relaxation approach based on the analysis of proton selective and non-selective spin–lattice relaxation rates at different temperatures can provide quantitative information about the affinity index and the thermodynamic equilibrium constant of the OLA–HSA system. The affinity index and the thermodynamic equilibrium constant decreased as temperature increased, indicating that the interactions between OLA and HSA could be weakened as temperature increased. Molecular docking and dynamics simulations revealed that OLA stably bound to subdomain II (site 1), and OLA could induce the conformational and micro-environmental changes in HSA. CD results suggested that α-helix content decreased after OLA was added, demonstrating that OLA affected the secondary structure of HSA.

The interaction mechanism between olaparib (OLA) and human serum albumin (HSA) has been investigated using experimental and computational techniques.     

Characterization of interaction between scoparone and bovine serum albumin: spectroscopic and molecular docking methods

Scoparone is a major biological active substance derived from the traditional Chinese herbal medicine called Artemisia capillaris. It has been confirmed that scoparone has anti-inflammatory, anti-tumor, hepatoprotective and antioxidant effects. However, the binding interaction of scoparone with bovine serum albumin (BSA) still remains unknown. Therefore, the present study was conducted to clarify the binding interaction of scoparone with BSA under simulated physiological conditions (pH = 7.4) by utilizing spectroscopic and molecular docking methods. The formation of the scoparone–BSA complex was identified by UV-vis absorption spectroscopy experiment results. The fluorescence experiment results revealed that the quenching mechanism was static quenching and the binding procedure was spontaneous mainly driven by hydrophobic interaction. At 310 K, the number of binding sites was approximately equal to 1 and the binding constant was 6.79 × 10⁵ mol L⁻¹. The binding distance (4.81 nm) between scoparone and BSA was determined by Förster''s non-radiative energy transfer theory. Molecular docking and site marker competitive experiment results verified that scoparone was more likely to be located in site I of BSA. In addition, the results of synchronous fluorescence spectroscopy and circular dichroism spectroscopy experiments proved that scoparone slightly changed the conformation of BSA by binding interaction with BSA. These findings would be useful for understanding the pharmacokinetics of scoparone in vivo, including scoparone transport, distribution, metabolism and excretion.

The interaction of scoparone with bovine serum albumin (BSA) was studied by utilizing spectroscopic and molecular docking methodologies.     

The synthesis,characterization, DNA/BSA/HSA interactions,molecular modeling,antibacterial properties,and in vitro cytotoxic activities of novel parent and niosome nano-encapsulated Ho(iii) complexes

Based on the importance of metal-centered complexes that can interact with DNA, this research focused on the synthesis of a new Ho(iii) complex. This complex was isolated and characterized via elemental analysis, and FT-IR, fluorescence, and UV-vis spectroscopy. Additional confirmation of the Ho(iii) complex structure was obtained via single-crystal X-ray diffraction. DNA interaction studies were carried out via circular dichroism (CD) spectroscopy, UV-vis absorption spectroscopy, viscosity measurements and emission spectroscopy; it was proposed that the metal complex acts as an effective DNA binder based on studies in the presence of fish DNA (FS-DNA), showing high binding affinity to DNA in the presence of hydrophobic and electron donating substituents. Also, the interactions of this complex with human (HSA) and bovine serum albumin (BSA) proteins were studied via fluorescence spectroscopy techniques and the obtained results reveal an excellent propensity for binding in both cases. Furthermore, the interactions of the Ho(iii) complex with DNA, BSA and HSA were confirmed via molecular docking analysis. The antimicrobial activities of the Ho(iii) complex were tested against Gram-negative bacteria and Gram-positive bacteria. In addition, a niosome nano-encapsulated Ho(iii) complex was synthesized, and the parent and encapsulated complexes were evaluated as potential antitumor candidates. The main structure of the Ho(iii) complex is maintained after encapsulation using niosome nanoparticles. The MTT method was used to assess the anticancer properties of the Ho(iii) complex and its encapsulated form toward human lung carcinoma and breast cancer cell lines. The anticancer activity in the encapsulated form was more than that of the parent Ho(iii) complex. In conclusion, these compounds could be considered as new antitumor candidates.

A new complex of holmium, [Ho(bpy)(H₂O)₆]Cl₃ has been synthesized, their DNA/BSA/HSA binding, molecular docking, antibacterial activity and MTT assay of niosome nano-encapsulated are investigated.     

Spectroscopic demonstration of sinapic acid methyl ester complexes with serum albumins

The methyl ester of sinapic acid (MESA) is a molecule with confirmed antioxidant properties. It is important to establish whether it can be transported across humans and animals. Therefore, we investigated MESA interactions with serum albumins, namely, human serum albumin (HSA), bovine serum albumin (BSA), rabbit serum albumin (RSA), and sheep serum albumin (SSA). Experiments were performed in a pH range from 5.9 to 10.7 using absorption and fluorescence techniques. It was found that MESA formed complexes with every albumin in the entire pH range under examination, which was confirmed by the appearances of new absorption and fluorescence complex bands. Fluorescence intensities were much higher (up to 20 times) and lifetimes were up to 340 times as compared to those for unbound MESA. The quenching experiments at pH 7.4 showed that the stoichiometry for every albumin was 1 : 1; the binding constant was the highest for HSA, which reached 52 000 M⁻¹. The obtained results suggested that MESA preferred the hydrophobic binding sites in albumins. The analysis of the fluorescence spectra and fluorescence lifetimes showed two possibly different binding sites in BSA, RSA, and SSA as well as three binding sites in HSA.

Known antioxidant, methyl ester of sinapic acid (MESA) can interact with serum albumins.     

Characterizing the interaction between methyl ferulate and human serum albumin by saturation transfer difference NMR

Methyl ferulate (MF) is an alkyl ferulate ester that widely exists in edible plants and has application value in the food and medicine industries. Thus, its effect on biological macromolecules should be considered. In this study, we exploit saturation transfer difference NMR (STD-NMR) to characterize the interaction of all protons of MF with human serum albumin (HSA) at the molecular level. STD-NMR and K_a (1.298 × 10³ M⁻¹) revealed that protons H1–6 and H8 bound to HSA with a medium affinity. Binding epitope mapping further showed that the aromatic ring played a key role in the HSA–MF interaction. STD-NMR site-marker-displacement experiments and circular dichroism spectroscopy revealed that MF prefered to bind to site II of HSA without changing the basic skeleton of HSA. Computer simulations confirmed these experimental results. Overall, this work elucidates the molecular level interaction of MF with HSA and provides new insights into the possibility of the potential applications of MF in the food and medicine industries.

STD-NMR technique characterized the recognition mechanism of methyl ferulate and human serum albumin qualitatively and quantitatively.     

Insights from a combination of theoretical and experimental methods for probing the biomolecular interactions between human serum albumin and clomiphene

In this study, the interaction of clomiphene (CLO), a non-steroidal and ovulatory stimulant drug employed in the treatment of infertility, with human serum albumin (HSA), the most abundant plasma transport protein, was investigated using spectrofluorometric, FT-IR, UV-Vis, and molecular modeling methods. The obtained results indicated that the binding of CLO to HSA led to intense fluorescence quenching of HSA via a static quenching mechanism, and that the process of CLO binding to HSA was enthalpy driven. By using experimental and theoretical methods, it was confirmed that as a result of binding CLO, slight conformational changes in HSA occurred. Also, the negative ΔH of interaction indicated that the binding of CLO with HSA was mainly enthalpy driven. The experimental and computational results suggested that hydrogen bonds and van der Waals interactions played a major role in the binding, with overall binding constants of K = 3.67 × 10⁹ M⁻¹ at 286 K and 6.52 × 10⁵ mol L⁻¹ at 310 K. Moreover, the results of molecular modeling showed that Asp234, Phe228, Leu327, and Arg209 in HSA had the highest interaction energies with the ligand.

In this study, the interaction of clomiphene with human serum albumin (HSA), the most abundant plasma transport protein, was investigated using spectrofluorometric, FT-IR, UV-Vis, and molecular modeling methods.     

Molecular modeling provides a structural basis for PERK inhibitor selectivity towards RIPK1

Protein kinases are crucial drug targets in cancer therapy. Kinase inhibitors are promiscuous in nature due to the highly conserved nature of the kinase ATP binding pockets. PERK has emerged as a potential therapeutic target in cancer. However, PERK inhibitors GSK2606414 and GSK2656157 also target RIPK1 whereas AMG44 is more specific to PERK. To understand the structural basis for the selectivity of PERK ligands to RIPK1 we have undertaken a detailed in silico analysis using molecular docking followed by molecular dynamics simulations to explore the selectivity profiles of the compounds. Although the binding sites of PERK and RIPK1 are similar, their binding response to small molecules is different. The docking models revealed a common binding mode for GSK2606414 and GSK2656157 in the RIPK1 binding site, similar to its cognate ligand. In contrast, AMG44 had a strikingly different predicted binding profile in the RIPK1 binding site with both rigid docking and induced fit docking settings. Our study shows a molecular mechanism responsible for dual targeting by the GSK ligands. More broadly, this work illustrates the potential of molecular docking to correctly predict the binding towards different kinase structures, and will aid in the design of selective PERK kinase inhibitors.

Molecular modelling explains the lack of selectivity for inhibitors GSK2606414 and GSK2656157, as compared to inhibitor AMG44.     

Schiff base complex conjugates of bovine serum albumin as artificial metalloenzymes for eco-friendly enantioselective sulfoxidation

Artificial metalloenzymes (BSA-ML) have been prepared by non-covalent insertion of transition metal Schiff-base complexes, ML (L = 2-hydroxynaphthalen-1-naphthaldehyde and 3,4-diaminobenzenesulfonic acid; M = Co, Mn, V, Fe, Cr), into bovine serum albumin (BSA) as the host protein and were characterized by UV-visible spectroscopy, ESI-TOF mass spectrometry and molecular docking studies. The catalytic activities of the BSA-ML in the selective oxidation of various prochiral sulfides in aqueous media, using H₂O₂ as oxidant, have been evaluated. During the optimization process, pH and the concentrations of catalyst and oxidant were found to have a remarkable influence on both yield and enantioselectivity. In certain cases, BSA-ML gave satisfactory results in the oxidation of organic sulfides to sulfoxides (up to 100% conversion, 100% chemoselectivity, 96% ee and 500 h⁻¹ turnover frequency).

Artificial metalloenzymes have been prepared by non-covalent insertion of transition metal Schiff-base complexes into bovine serum albumin as the host protein and were characterized by UV-visible spectroscopy, ESI-TOF mass spectrometry and molecular docking studies.     

Characterizing the binding interaction of astilbin with bovine serum albumin: a spectroscopic study in combination with molecular docking technology

Astilbin (ASN) is a flavonoid compound isolated from the rhizome of Smilax china L. (Smilacaceae). It has many bioactivities, such as selective immunosuppression, antioxidant, anti-hepatic injury, etc., and is widely used in traditional Chinese medical treatments. The interaction of ASN with bovine serum albumin (BSA) was studied in a physiological buffer (pH = 7.40) using multi-spectroscopic techniques in combination with molecular docking methods. UV-vis absorption measurements proved that a ASN–BSA complex could be formed. Fluorescence data revealed that ASN could strongly quench the intrinsic fluorescence of BSA in terms of a static quenching procedure. The process of binding was spontaneous and the binding occurred mainly through hydrogen bonding and van der Waals forces. The distance r between donor (BSA) and acceptor (ASN) was calculated to be 4.80 nm based on Förster''s non-radiative energy transfer theory. The binding constant (K_a = 7.31 × 10⁴ mol L⁻¹) and the number of binding sites (n ≈ 1) at 298 K suggested that ASN only occupied one site in BSA with high affinity. Moreover, the results of molecular docking indicated that ASN was more likely to be located in site I (sub-domain IIA) of BSA. The results of synchronous fluorescence and three-dimensional fluorescence spectra showed that ASN induced conformational changes of BSA. The findings would be beneficial for research on the transportation, distribution and some important bioactivities of ASN in the human body.

The interaction of astilbin with bovine serum albumin was confirmed by multi-spectroscopic techniques and molecular docking methods.     

Thermodynamic investigation of the interaction between ionic liquid functionalized gold nanoparticles and human serum albumin for selective determination of glutamine

The excellent biocompatible and monodispersed gold nanoparticles (AuNPs) functionalized by amino based ionic liquid (IL) have been synthesized for the demonstration of their interaction with human serum albumin (HSA). Amino based IL stabilizes the surface of AuNPs and provides a colorimetric sensor platform. The size of synthesized IL–AuNPs was identified by use of transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques. Molecular interaction of functionalized AuNPs with HSA have been investigated using multispectroscopic techniques, such as UV-Vis, fluorescence and Fourier transform infra-red (FT-IR) spectroscopy. The fluorescence and synchronous fluorescent intensity together indicated that IL–AuNPs exhibits a strong ability to quench the intrinsic fluorescence of HSA via a dynamic quenching mechanism. Moreover, the binding constant (K_a), Stern–Volmer quenching constant (K_SV) and different thermodynamic parameters, namely Gibb''s free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) have been evaluated at different temperatures. This interactive study focuses on the nature of surface modification of IL–AuNPs via HSA for selective detection of glutamine (Glu) with a lower limit of detection of 0.67 nM in the linear range of 10–100 nM for Glu.

The excellent biocompatible and monodispersed gold nanoparticles (AuNPs) functionalized by amino based ionic liquid (IL) have been synthesized for the demonstration of their interaction with human serum albumin (HSA).     

Novel huperzine A based NMDA antagonists: insights from molecular docking,ADME/T and molecular dynamics simulation studies

Huperzine A (HupA) is an alkaloidal natural product and drug isolated from Chinese herb Huperzia serrata, which is a potent selective anticholinesterase inhibitor. HupA has symptomatic, cognitive-enhancing and protective effect on neurons against amyloid beta-induced oxidative injury and antagonizing N-methyl-d-aspartate receptors by blocking the ion channels. The present study aimed to identify the docking, ADME/T and molecular dynamics simulation parameters of a library of 40 analogues which can correlate the binding affinity, conformational stability and selectivity of the ligands towards NMDA receptor through in silico approach. Glide molecular docking analysis was performed for the designed analogues to understand the binding mode and interactions. MD simulations were performed to explain the conformational stability and natural dynamics of the interaction in physiological environmental condition of protein–ligand complex affording a better understanding of chemical-scale interactions between HupA and its analogues with NMDA channel that could potentially benefit the development of new drugs for neurodegenerative diseases involving NMDA receptors.

The in silico study explores the structural behavior and binding affinities of 40 novel analogues of huperzine A. Novel NMDA receptor antagonists have been virtually identified by molecular docking, ADME/T and molecular dynamics simulation studies.     

Interaction mechanism of aloe-emodin with trypsin: molecular structure–affinity relationship and effect on biological activities

The molecular mechanism of interaction between aloe-emodin (AE) and trypsin was investigated, exhibiting remarkable outcomes. To detect the interaction mechanism, the binding of AE with trypsin was examined by a multi-spectroscopy and molecular docking method. Results showed that the binding of AE and trypsin would lead to static quenching and their binding forces were van der Waals forces and hydrogen bonding. The results of simultaneous and three-dimensional fluorescence spectroscopy showed that the combination of AE and trypsin caused changes in the microenvironment around the trypsin fluorophore, which might change the spatial structure of trypsin. FT-IR spectroscopy showed that the contents of α-helix and β-turn in trypsin were decreased and the contents of β-sheet, random coil and antiparallel β-sheet were increased. Moreover, all these experimental results were verified and reasonably explained by molecular docking results. We also investigated the enzyme activity of trypsin and the antioxidant activity of AE. The results showed that both the enzyme activity of trypsin and the antioxidant activity of AE were decreased after interaction between AE and trypsin. The findings outlined in this study should elucidate the molecular mechanisms of interaction between AE and trypsin and contribute to making full use of AE in the food industry.

The mechanism of interaction between AE and trypsin was studied firstly. The biological activity of both decreased after the interaction. These results provide a basis for the development and utilization of AE.     

Spectroscopic characterization of the warfarin drug-binding site of folded and unfolded human serum albumin anchored on gold nanoparticles: effect of bioconjugation on the loading capacity

Protein-conjugated gold nanoparticles (AuNPs) have recently shown promising applications in medicine, owing to their inertness and biocompatibility. Herein, we studied the spectroscopy of 25 nm diameter AuNPs, coated with human serum albumin (HSA) as a model drug carrier. The morphology and coating of the AuNPs were examined using transmission electron microscopy and dynamic light scattering. Resonance energy transfer from the sole tryptophan of HSA (Trp214) to the AuNPs indicates a single layer of protein coverage. Using fluorescein (FL) to probe the warfarin drug-binding site in HSA revealed an increase in the HSA–FL binding by ∼4.5 times when HSA is anchored on the nanoparticle surface, indicating a rise in the loading capacity. Femtosecond transient absorption measurements of the surface plasmonic resonance band of the AuNPs show three ultrafast dynamics that are involved in the relaxation process. The three decay components were assigned to the electron–electron (∼400 fs), electron–phonon (∼2.0 ps) and phonon–phonon (200–250 ps) interactions. These dynamics were not changed upon coating the AuNPs with HSA which indicates the chemical and physical stability of the AuNPs upon bioconjugation. Chemical unfolding of the warfarin binding site with guanidine hydrochloride (GdnHCl) was studied by measuring the spectral shift in the Trp214 fluorescence and the appearance of the Tyr fluorescence. Unfolding was shown to start at [GdnHCl] ≥ 2.0 M and is complete at [GdnHCl] = 6.0 M. HSA anchored onto the nanoparticle surface shows more resistance to the unfolding effect which is attributed to the stability of the native form of HSA on the nanoparticle surface. On the other hand, upon complete unfolding, a larger red shift in the Trp214 fluorescence was observed for the HSA–AuNP complex. This observation indicates that, upon unfolding, the HSA molecule is still anchored on the AuNP surface in which subdomain IIA is facing the outer water molecules in the bulk solution as well as the hydration shell rather than the core of the nanoparticle. The current study is important for a better understanding of the physical and dynamical properties of protein-coated metal nanoparticles, which is expected to help in optimizing their properties for critical applications in nanomedicine.

This work investigates the steady-state and ultrafast spectroscopy of bioconjugated gold nanoparticles and the implications on the protein binding activity and drug-loading capacity.     

Molecular design of anticancer drugs from marine fungi derivatives

Heat shock protein 90 (Hsp90) is one of the most potential targets in cancer therapy. We have demonstrated using a combination of molecular docking and fast pulling of ligand (FPL) simulations that marine fungi derivatives can be possible inhibitors, preventing the biological activity of Hsp90. The computational approaches were validated and compared with previous experiments. Based on the benchmark of available inhibitors of Hsp90, the GOLD docking package using the ChemPLP scoring function was found to be superior over both Autodock Vina and Autodock4 in the preliminary estimation of the ligand-binding affinity and binding pose with the Pearson correlation, R = −0.62. Moreover, FPL calculations were also indicated as a suitable approach to refine docking simulations with a correlation coefficient with the experimental data of R = −0.81. Therefore, the binding affinity of marine fungi derivatives to Hsp90 was evaluated. Docking and FPL calculations suggest that five compounds including 23, 40, 46, 48, and 52 are highly potent inhibitors for Hsp90. The obtained results enhance cancer therapy research.

Five compounds originating from marine fungi species Aspergillus sp. and Penicillium sp. were found to be highly potent inhibitors of cancer therapy target, Hsp90, using molecular docking and FPL calculations.     

Conformational-transited protein corona regulated cell-membrane penetration and induced cytotoxicity of ultrasmall Au nanoparticles

Nanoparticles (NP) in biological fluids almost invariably become coated with proteins to form protein coronas. It is the NP–protein corona rather than the bare nanoparticle that determines the nanoparticle''s bio-behavior. Here, ultrasmall gold nanoparticles (AuNPs) coated by a human serum albumin (HSA) corona were studied by Fourier transform infrared spectroscopy, denature experiments, fluorescence quenching. Moreover, the intracellular fate of AuNPs and the AuNP–HSA corona has also been investigated. The results show that HSA corona undergo a conformational transition (partial β-sheet changed to α-helicity) when they adsorb on AuNPs, which lead to an enhanced thermal stability. Importantly, we observed that the conformation-transited protein corona–AuNP complex could induce cell apoptosis. Meanwhile, for the first time, the conformation-transited HSA on the AuNPs surface are shown to disrupt living cell membranes. The results obtained here not only provide the detailed conformational behavior of HSA molecules on nanoparticles, but also reveal the structure–function relationship of protein corona, which is of utmost importance in the safe application of nanoscale objects in living organisms.

This study demonstrate that the AuNP–HSA corona could penetrate cell membranes and companied by substantial membrane disruption. However, the ultrasmall AuNPs can be internalized by cells without the destruction of cell membranes.     

Importance of protein flexibility in ranking ERK2 Type I1/2 inhibitor affinities: a computational study

Extracellular-regulated kinase (ERK2) has been regarded as an essential target for various cancers, especially melanoma. Recently, pyrrolidine piperidine derivatives were reported as Type I^1/2 inhibitors of ERK2, which occupy both the ATP binding pocket and the allosteric pocket. Due to the dynamic behavior of ERK2 upon the binding of Type I^1/2 inhibitors, it is difficult to predict the binding structures and relative binding potencies of these inhibitors with ERK2 accurately. In this work, the binding mechanism of pyrrolidine piperidines was discussed by using different simulation techniques, including molecular docking, ensemble docking based on multiple receptor conformation, molecular dynamics simulations and free energy calculations. Our computational results show that the traditional docking method cannot predict the relative binding ability of the studied inhibitors with high accuracy, but incorporating ERK2 protein flexibility into docking is an effective method to improve the prediction accuracy. It is worth noting that the binding free energies predicted by MM/GBSA or MM/PBSA based on the MD simulations for the docked poses have the highest correlation with the experimental data, which highlights the importance of protein flexibility for accurately predicting the binding ability of Type I^1/2 inhibitors of ERK2. In addition, the comprehensive analysis of several representative inhibitors indicates that hydrogen bonds and hydrophobic interactions are of significance for improving the binding affinities of the inhibitors. We hope this work will provide valuable information for further design of novel and efficient Type I^1/2 ERK2 inhibitors.

Extracellular-regulated kinase (ERK2) has been regarded as an essential target for various cancers, especially melanoma.