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Abstract According to WHO statistics, breast cancer (BC) disease represents about 2.3 million diagnosed and 685,000 deaths globally. Regarding histological classification of BC, the Estrogen (ER) and Progesterone (PR) receptors negative-expression cancer, named Triple-Negative BC (TNBC), represents the most aggressive type of this disease, making it a challenge for drug discovery. In this context, our research group, applying a well-established Virtual Screening (VS) protocol, in addition to docking and molecular dynamics simulations studies, yielded two ligands identified as 6 and 37 which were chemically synthesized and evaluated on MCF-7 and MDA-MB-231 cancer cell lines. Strikingly, 37 assayed on MDA-MB-231 (a TNBC cell model) depicted an outstanding value of 18.66 μM much lower than 65.67 μM yielded by Gossypol Bcl-2 inhibitor whose main disadvantage is to produce multiple toxic effects. Highlighted above, enforce the premise of the computational tools to find new therapeutic options against the most aggressive forms of breast cancer, as the results herein showed.

IntroductionBreast cancer (BC) is the leading cause of cancer-related deaths among women, with triple-negative breast cancer (TNBC) representing one of the most aggressive and treatment-resistant subtypes. In this study, we aimed to evaluate the antitumor potential of C14 and P8 molecules in both TNBC and radioresistant TNBC cells. These compounds were chosen for their ability to stabilize the complex formed by the overactivated form of K-Ras4BG13D and its membrane transporter (PDE6δ).MethodsThe antitumor potential of C14 and P8 was assessed using TNBC cell lines, MDA-MB-231, and the radioresistant derivative MDA-MB-231RR, both carrying the K-Ras4B> G13D mutation. We investigated the compounds' effects on K-Ras signaling pathways, cell viability, and tumor growth in vivo.ResultsWestern blotting analysis determined the negative impact of C14 and P8 on the activation of mutant K-Ras signaling pathways in MDA-MB-231 and MDA-MB-231RR cells. Proliferation assays demonstrated their efficacy as cytotoxic agents against K-RasG13D mutant cancer cells and in inducing apoptosis. Clonogenic assays proven their ability to inhibit TNBC and radioresistant TNBC cell clonogenicity. In In vivo studies, C14 and P8 inhibited tumor growth and reduced proliferation, angiogenesis, and cell cycle progression markers.DiscussionThese findings suggest that C14 and P8 could serve as promising adjuvant treatments for TNBC, particularly for non-responders to standard therapies. By targeting overactivated K-Ras and its membrane transporter, these compounds offer potential therapeutic benefits against TNBC, including its radioresistant form. Further research and clinical trials are warranted to validate their efficacy and safety as novel TNBC treatments.

A la fecha se han empleado diferentes métodos basados en la estructura para cuantificar las interacciones receptor-ligando, y a partir de estas predecir la energía libre de asociación que proporcionara un estimado de la afinidad de un compuesto por una diana terapéutica. Entre estos métodos está el acoplamiento molecular y las simulaciones de dinámica molecular en conjunto con métodos de cálculo de energía libre de asociación. El acoplamiento molecular, aunque tiene un alto potencial selectivo posee un éxito limitado en la precisión de la estimación de la energía de solvatación y consideración de cambios en la entropía conformacional. Por lo tanto, se ha recurrido a técnicas computacionales más eficientes que predicen la energía libre de unión de una manera más precisa, como lo son los métodos que combinan mecánica molecular con métodos de cálculo de energía. En este contexto, los métodos MMPBSA y MMGBSA permiten predecir la energía libre de unión usando mecánica molecular y modelos continuos de solvatación implícita. Estas técnicas han facilitado la identificación de diferentes compuestos con alta afinidad por una diana farmacológica. En este artículo científico describiremos las bases fundamentales de los métodos MMPBSA y MMGBSA, así como algunos avances relacionados con el empleo de ambos métodos.

Allergic rhinitis (AR) is a prevalent inflammatory condition affecting millions globally, with current treatments often associated with significant side effects. To seek safer and more effective alternatives, natural sources like Urtica dioica (UD) are being explored. However, UD’s mechanism of action remains unknown. Therefore, to elucidate it, we conducted an in silico evaluation of UD phytochemicals’ effects on known therapeutic targets of allergic rhinitis: histamine receptor 1 (HR1), neurokinin 1 receptor (NK1R), cysteinyl leukotriene receptor 1 (CLR1), chemoattractant receptor-homologous molecule expressed on type 2 helper T cells (CRTH2), and bradykinin receptor type 2 (BK2R). The docking analysis identified amentoflavone, alpha-tocotrienol, neoxanthin, and isorhamnetin 3-O-rutinoside as possessing a high affinity for all the receptors. Subsequently, molecular dynamics (MD) simulations were used to analyze the key interactions; the free energy of binding was calculated through Generalized Born and Surface Area Solvation (MMGBSA), and the conformational changes were evaluated. Alpha-tocotrienol exhibited a high affinity while also inducing positive conformational changes across all targets. Amentoflavone primarily affected CRTH2, neoxanthin targeted NK1R, CRTH2, and BK2R, and isorhamnetin-3-O-rutinoside acted on NK1R. These findings suggest UD’s potential to treat AR symptoms by inhibiting these targets. Notably, alpha-tocotrienol emerges as a promising multi-target inhibitor. Further in vivo and in vitro studies are needed for validation.

Abstract Background A variety of accessible data, including those of single-nucleotide polymorphisms (SNPs) on the human p53 gene, are made widely available on a global scale. Owing to this, our investigation aimed to deal with the detrimental SNPs in the p53 gene by executing various valid computational tools, including—Filter, SIFT, PredictSNP, Fathmm, UTRScan, ConSurf, SWISS-MODEL, Amber 16 package, Tm-Adjust, I-Mutant, Task Seek, GEPIA2 after practical and basic appraisal, dissolvable openness, atomic progression, analyzing the energy minimization and assessing the gene expression pattern. Results Out of the total 581 p53 SNPs, 420 SNPs were found to be missense or non-synonymous, 435 SNPs were in the three prime UTR, and 112 SNPs were in the five prime UTR from which 16 non-synonymous SNPs (nsSNPs) were predicted to be non-tolerable while PredictSNP package predicted 14. Concentrating on six bioinformatics tools of various dimensions, a combined output was generated, where 14 nsSNPs could exert a deleterious effect. We found 5 missense SNPs in the DNA binding domain's three crucial amino acid positions, using diverse SNP analyzing tools. The underlying discoveries were fortified by microsecond molecular dynamics (MD) simulations, TM-align, I-Mutant, and Project HOPE. The ExPASy-PROSITE tools characterized whether the mutations were located in the functional part of the protein or not. This study provides a decisive outcome, concluding the accessible SNPs' information by recognizing the five unfavorable nsSNPs—rs28934573 (S241F), rs11540652 (R248Q), rs121913342 (R248W), rs121913343 (R273C), and rs28934576 (R273H). By utilizing Heatmapper and GEPIA2, several visualization plots, including heat maps, box plots, and survival plots, were produced. Conclusions These plots disclosed differential expression patterns of the p53 gene in humans. The investigation focused on recognizing the detrimental nsSNPs, which augmented the danger posed by various oncogenesis in patients of different populations, including within the genome-wide studies (GWS).

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged coronavirus responsible for coronavirus disease 2019 (COVID-19); it become a pandemic since March 2020. To date, there have been described three lineages of SARS-CoV-2 circulating worldwide, two of them are found among Mexican population, within these, we observed three mutations of spike (S) protein located at amino acids H49Y, D614G, and T573I. To understand if these mutations could affect the structural behavior of S protein of SARS-CoV-2, as well as the binding with S protein inhibitors (cepharanthine, nelfinavir, and hydroxychloroquine), molecular dynamic simulations and molecular docking were employed. It was found that these punctual mutations affect considerably the structural behavior of the S protein compared to wild type, which also affect the binding of its inhibitors into their respective binding site. Thus, further experimental studies are needed to explore if these affectations have an impact on drug-S protein binding and its possible clinical effect.

The success story of plant-based medicine has been overlooked during the advent of the modern pharmaceutical industry. Despite the negligence of the multibillion-dollar drug industry, people entirely rely on medicinal plants in some parts of the world. In this study, we have emphasized going back to those traditional medicinal practices to determine their underlying mechanism to move forward on phytochemical-based drug development. We screened Medicinal Plant Database Bangladesh (MPDB2.0) to find traditionally used medicinal plants and their active compounds.Here, mangiferin, extracted from Mangifera indica, has interacted with cell cycle regulator Cyclin-dependent Kinase 4 (CDK4). CDK4 is differentially expressed during Glioblastoma multiforme (GBM), Brain Lower Grade Glioma (LGG), and Sarcoma (SARC). Expression of CDK4 is interlinked to the patients’ survival rate and its consistent expression throughout different stages has provided the advantage to use it as a diagnostic tool and drug target.This study demonstrated that a simple mango tree extracted active compound ‘mangiferin’ may work as a potential anticancer drug, and leveraging the recent advancement of sequencing and gene expression data can accelerate the phytochemical-based drug discovery process.

The heterodimeric complex between retinoic X receptor alpha (RXRα) and peroxisome proliferator-activated receptor gamma (PPARγ) is one of the most important and predominant regulatory systems, controlling lipid metabolism by binding to specific DNA promoter regions. X-ray and molecular dynamics (MD) simulations have revealed the average conformation adopted by the RXRα-PPARγ heterodimer bound to DNA, providing information about how multiple domains communicate to regulate receptor properties. However, knowledge of the energetic basis of the protein-ligand and protein-protein interactions is still lacking. Here we explore the structural and energetic mechanism of RXRα-PPARγ heterodimer bound or unbound to DNA and forming complex with co-crystallized ligands (rosiglitazone and 9-cis-retinoic acid) through microsecond MD simulations, molecular mechanics generalized Born surface area binding free energy calculations, principal component analysis, the free energy landscape, and correlated motion analysis. Our results suggest that DNA binding alters correlated motions and conformational mobility within RXRα–PPARγ system that impact the dimerization and the binding affinity on both receptors. Intradomain correlated motions denotes a stronger correlation map for RXRα-PPARγ-DNA than RXRα-PPARγ, involving residues at the ligand binding site. In addition, our results also corroborated the greater role of PPARγ in regulation of the free and bound DNA state.

The Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a pandemic, resulting in an exponentially increased mortality globally and scientists all over the world are struggling to find suitable solutions to combat it. Multiple repurposed drugs have already been in several clinical trials or recently completed. However, none of them shows any promising effect in combating COVID-19. Therefore, developing an effective drug is an unmet global need. RdRp (RNA dependent RNA polymerase) plays a pivotal role in viral replication. Therefore, it is considered as a prime target of drugs that may treat COVID-19. In this study, we have screened a library of compounds, containing approved RdRp inhibitor drugs that were or in use to treat other viruses (favipiravir, sofosbuvir, ribavirin, lopinavir, tenofovir, ritonavir, galidesivir and remdesivir) and their structural analogues, in order to identify potential inhibitors of SARS-CoV-2 RdRp. Extensive screening, molecular docking and molecular dynamics show that five structural analogues have notable inhibitory effects against RdRp of SARS-CoV-2. Importantly, comparative protein-antagonists interaction revealed that these compounds fit well in the pocket of RdRp. ADMET analysis of these compounds suggests their potency as drug candidates. Our identified compounds may serve as potential therapeutics for COVID-19.

In 40–50% of colorectal cancer (CRC) cases, K-Ras gene mutations occur, which induce the expression of the K-Ras4B oncogenic isoform. K-Ras4B is transported by phosphodiesterase-6δ (PDE6δ) to the plasma membrane, where the K-Ras4B–PDE6δ complex dissociates and K-Ras4B, coupled to the plasma membrane, activates signaling pathways that favor cancer aggressiveness. Thus, the inhibition of the K-Ras4B–PDE6δ dissociation using specific small molecules could be a new strategy for the treatment of patients with CRC. This research aimed to perform a preclinical proof-of-concept and a therapeutic potential evaluation of the synthetic I-C19 and 131I-C19 compounds as inhibitors of the K-Ras4B–PDE6δ dissociation. Molecular docking and molecular dynamics simulations were performed to estimate the binding affinity and the anchorage sites of I-C19 in K-Ras4B–PDE6δ. K-Ras4B signaling pathways were assessed in HCT116, LoVo and SW620 colorectal cancer cells after I-C19 treatment. Two murine colorectal cancer models were used to evaluate the I-C19 therapeutic effect. The in vivo biokinetic profiles of I-C19 and 131I-C19 and the tumor radiation dose were also estimated. The K-Ras4B–PDE6δ stabilizer, 131I-C19, was highly selective and demonstrated a cytotoxic effect ten times greater than unlabeled I-C19. I-C19 prevented K-Ras4B activation and decreased its dependent signaling pathways. The in vivo administration of I-C19 (30 mg/kg) greatly reduced tumor growth in colorectal cancer. The biokinetic profile showed renal and hepatobiliary elimination, and the highest radiation absorbed dose was delivered to the tumor (52 Gy/74 MBq). The data support the idea that 131I-C19 is a novel K-Ras4B/PDE6δ stabilizer with two functionalities: as a K-Ras4B signaling inhibitor and as a compound with radiotherapeutic activity against colorectal tumors.

Naltrexone is a potent opioid antagonist with good blood–brain barrier permeability, targeting different endogenous opioid receptors, particularly the mu-opioid receptor (MOR). Therefore, it represents a promising candidate for drug development against drug addiction. However, the details of the molecular interactions of naltrexone and its derivatives with MOR are not fully understood, hindering ligand-based drug discovery. In the present study, taking advantage of the high-resolution X-ray crystal structure of the murine MOR (mMOR), we constructed a homology model of the human MOR (hMOR). A solvated phospholipid bilayer was built around the hMOR and submitted to microsecond (µs) molecular dynamics (MD) simulations to obtain an optimized hMOR model. Naltrexone and its derivatives were docked into the optimized hMOR model and submitted to µs MD simulations in an aqueous membrane system. The MD simulation results were submitted to the molecular mechanics–generalized Born surface area (MMGBSA) binding free energy calculations and principal component analysis. Our results revealed that naltrexone and its derivatives showed differences in protein–ligand interactions; however, they shared contacts with residues at TM2, TM3, H6, and TM7. The binding free energy and principal component analysis revealed the structural and energetic effects responsible for the higher potency of naltrexone compared to its derivatives.

Breast cancer (BC) is the most frequently diagnosed cancer and is the second-most common cause of death in women worldwide. Because of this, the search for new drugs and targeted therapy to treat BC is an urgent and global need. Histone deacetylase 6 (HDAC6) is a promising anti-BC drug target associated with its development and progression. In the present work, the design and synthesis of a new family of dihydropyrazole-carbohydrazide derivatives (DPCH) derivatives focused on HDAC6 inhibitory activity is presented. Computational chemistry approaches were employed to rationalize the design and evaluate their physicochemical and toxic-biological properties. The new family of nine DPCH was synthesized and characterized. Compounds exhibited optimal physicochemical and toxicobiological properties for potential application as drugs to be used in humans. The in silico studies showed that compounds with –Br, –Cl, and –OH substituents had good affinity with the catalytic domain 2 of HDAC6 like the reference compounds. Nine DPCH derivatives were assayed on MCF-7 and MDA-MB-231 BC cell lines, showing antiproliferative activity with IC50 at μM range. Compound 2b showed, in vitro, an IC50 value of 12 ± 3 µM on human HDAC6. The antioxidant activity of DPCH derivatives showed that all the compounds exhibit antioxidant activity similar to that of ascorbic acid. In conclusion, the DPCH derivatives are promising drugs with therapeutic potential for the epigenetic treatment of BC, with low cytotoxicity towards healthy cells and important antioxidant activity.

Abstract Background The GTPase KRas4B has been utilized as a principal target in the development of anticancer drugs. PDE6δ transports KRas4B to the plasma membrane, where it is released to activate various signaling pathways required for the initiation and maintenance of cancer. Therefore, identifying new small molecules that prevent activation of this GTPase by stabilizing the KRas4B-PDE6δ molecular complex is a practical strategy to fight against cancer. Methods The crystal structure of the KRas4B-PDE6δ heterodimer was employed to locate possible specific binding sites at the protein-protein interface region. Virtual screening of Enamine-database compounds was performed on the located potential binding sites to identify ligands able to simultaneously bind to the KRas4B-PDE6δ heterodimer. A molecular dynamics approach was used to estimate the binding free-energy of the complex. Cell viability and apoptosis were measured by flow cytometry. G-LISA was used to measure Ras inactivation. Western blot was used to measure AKT and ERK activation. MIA PaCa-2 cells implanted subcutaneously into nude mice were treated with D14 or C22 and tumor volumes were recorded. Results According to the binding affinity estimation, D14 and C22 stabilized the protein-protein interaction in the KRas4B-PDE6δ complex based on in vitro evaluation of the 38 compounds showing antineoplastic activity against pancreatic MIA PaCa-2 cancer cells. In this work, we further investigated the antineoplastic cellular properties of two of them, termed D14 and C22, which reduced the viability in the human pancreatic cancer cells lines MIA PaCa-2, PanC-1 and BxPC-3, but not in the normal pancreatic cell line hTERT-HPNE. Compounds D14 and C22 induced cellular death via apoptosis. D14 and C22 significantly decreased Ras-GTP activity by 33% in MIA PaCa-2 cells. Moreover, D14 decreased AKT phosphorylation by 70% and ERK phosphorylation by 51%, while compound C22 reduced AKT phosphorylation by 60% and ERK phosphorylation by 36%. In addition, compounds C22 and D14 significantly reduced tumor growth by 88.6 and 65.9%, respectively, in a mouse xenograft model. Conclusions We identified two promising compounds, D14 and C22, that might be useful as therapeutic drugs for pancreatic ductal adenocarcinoma treatment.

Abstract Background Colorectal cancer is the third most common cancer worldwide; and in 40% of all cases, KRAS4b-activating mutations occur. KRAS4b is transported by phosphodiesterase-6δ (PDEδ) to the plasma membrane, where it gets activated. PDEδ downregulation prevents redistribution and activation of KRAS4b. Thus, targeting the KRAS4b-PDEδ complex is a treatment strategy for colorectal cancer. Methods Using docking and molecular dynamics simulations coupled to molecular mechanics, the generalized born model and solvent accessibility (MMGBSA) approach to explore protein-ligand stability, we found that the compound ((2S)-N-(2,5-diclorofenil)-2-[(3,4-dimetoxifenil)metilamino]-propanamida), termed C19, bound and stabilized the KRAS4b-PDEδ complex. We investigated whether C19 decreases the viability and proliferation of colorectal cancer cells, in addition to knowing the type of cell death that it causes and if C19 decreases the activation of KRAS4b and their effectors. Results C19 showed high cytotoxicity in the colorectal cancer cell lines HCT116 and LoVo, with a stronger effect in KRAS-dependent LoVo cells. Importantly, C19 significantly decreased tumor size in a xenograft mouse model and showed lower side effects than 5-fluorouracil that is currently used as colorectal cancer treatment. Conclusions Mechanistically, the cytotoxic effect was due to increased apoptosis of tumor cells and decreased phosphorylation of Erk and Akt. Therefore, our results suggest that C19 may serve as a promising new treatment for colorectal cancer.

The increasing common occurrence of antibiotic-resistant bacteria has become an urgent public health issue. There are currently some infections without any effective treatment, which require new therapeutic strategies. An attractive alternative is the design of compounds capable of disrupting bacterial communication known as quorum sensing (QS). In Gram-negative bacteria, such communication is regulated by acyl-homoserine lactones (AHLs). Triggering of QS after bacteria have reached a high cell density allows them to proliferate before expressing virulence factors. Our group previously reported that hexyloxy phenylimidazoline (9) demonstrated 71% inhibitory activity of QS at 100 μM (IC50 = 90.9 μM) in Chromobacterium violaceum, a Gram-negative bacterium. The aim of the present study was to take 9 as a lead compound to design and synthesize three 2-imidazolines (13–15) and three 2-oxazolines (16–18), to be evaluated as quorum-sensing inhibitors on C. violaceum CV026. We were looking for compounds with a higher affinity towards the Cvi receptor of this bacterium and the ability to inhibit QS. The binding mode of the test compounds on the Cvi receptor was explored with docking studies and molecular dynamics. It was found that 8-pentyloxyphenyl-2-imidazoline (13) reduced the production of violacein (IC50 = 56.38 μM) without affecting bacterial growth, suggesting inhibition of quorum sensing. Indeed, compound 13 is apparently one of the best QS inhibitors known to date. Molecular docking revealed the affinity of compound 13 for the orthosteric site of N-hexanoyl homoserine lactone (C6-AHL) on the CviR protein. Ten amino acid residues in the active binding site of C6-AHL in the Cvi receptor interacted with 13, and 7 of these are the same as those interacting with AHL. Contrarily, 8-octyloxyphenyl-2-imidazoline (14), 8-decyloxyphenyl-2-imidazoline (15), and 9-decyloxyphenyl-2-oxazoline (18) bound only to an allosteric site and thus did not compete with C6-AHL for the orthosteric site.

Repurposing studies have identified several FDA-approved compounds as potential inhibitors of the intracellular domain of epidermal growth factor receptor 1 (EGFR) and human epidermal receptor 2 (HER2). EGFR and HER2 represent important targets for the design of new drugs against different types of cancer, and recently, differences in affinity depending on active or inactive states of EGFR or HER2 have been identified. In this study, we first identified FDA-approved compounds with similar structures in the DrugBank to lapatinib and gefitinib, two known inhibitors of EGFR and HER2. The selected compounds were submitted to docking and molecular dynamics MD simulations with the molecular mechanics generalized Born surface area approach to discover the conformational and thermodynamic basis for the recognition of these compounds on EGFR and HER2. These theoretical studies showed that compounds reached the ligand-binding site of EGFR and HER2, and some of the repurposed compounds did not interact with residues involved in drug resistance. An in vitro assay performed on two different breast cancer cell lines, MCF-7, and MDA-MB-23, showed growth inhibitory activity for these repurposed compounds on tumorigenic cells at micromolar concentrations. These repurposed compounds open up the possibility of generating new anticancer treatments by targeting HER2 and EGFR.

The implementation of chemo- and bioinformatics tools is a crucial step in the design of structure-based drugs, enabling the identification of more specific and effective molecules against cancer without side effects. In this study, three new compounds were designed and synthesized with suitable absorption, distribution, metabolism, excretion and toxicity (ADME-tox) properties and high affinity for the G protein-coupled estrogen receptor (GPER) binding site by in silico methods, which correlated with the growth inhibitory activity tested in a cluster of cancer cell lines. Docking and molecular dynamics (MD) simulations accompanied by a molecular mechanics/generalized Born surface area (MMGBSA) approach yielded the binding modes and energetic features of the proposed compounds on GPER. These in silico studies showed that the compounds reached the GPER binding site, establishing interactions with a phenylalanine cluster (F206, F208 and F278) required for GPER molecular recognition of its agonist and antagonist ligands. Finally, a 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay showed growth inhibitory activity of compounds 4, 5 and 7 in three different cancer cell lines—MIA Paca-2, RCC4-VA and Hep G2—at micromolar concentrations. These new molecules with specific chemical modifications of the GPER pharmacophore open up the possibility of generating new compounds capable of reaching the GPER binding site with potential growth inhibitory activities against nonconventional GPER cell models.

Epitope recognition by major histocompatibility complex II (MHC-II) is essential for the activation of immunological responses to infectious diseases. Several studies have demonstrated that this molecular event takes place in the MHC-II peptide-binding groove constituted by the α and β light chains of the heterodimer. This MHC-II peptide-binding groove has several pockets (P1-P11) involved in peptide recognition and complex stabilization that have been probed through crystallographic experiments and in silico calculations. However, most of these theoretical calculations have been performed without taking into consideration the heavy chains, which could generate misleading information about conformational mobility both in water and in the membrane environment. Therefore, in absence of structural information about the difference in the conformational changes between the peptide-free and peptide-bound states (pMHC-II) when the system is soluble in an aqueous environment or non-covalently bound to a cell membrane, as the physiological environment for MHC-II is. In this study, we explored the mechanistic basis of these MHC-II components using molecular dynamics (MD) simulations in which MHC-II was previously co-crystallized with a small epitope (P7) or coupled by docking procedures to a large (P22) epitope. These MD simulations were performed at 310 K over 100 ns for the water-soluble (MHC-IIw, MHC-II-P(7w), and MHC-II-P(22w)) and 150 ns for the membrane-bound species (MHC-IIm, MHC-II-P(7m), and MHC-II-P(22m)). Our results reveal that despite the different epitope sizes and MD simulation environments, both peptides are stabilized primarily by residues lining P1, P4, and P6-7, and similar noncovalent intermolecular energies were observed for the soluble and membrane-bound complexes. However, there were remarkably differences in the conformational mobility and intramolecular energies upon complex formation, causing some differences with respect to how the two peptides are stabilized in the peptide-binding groove.

Molecular dynamic (MD) simulations have been performed on Tth-MCO, a hyperthermophilic multicopper oxidase from thermus thermophilus HB27, in the apo as well as the holo form, with the aim of exploring the structural dynamic properties common to the two conformational states. According to structural comparison between this enzyme and other MCOs, the substrate in process to electron transfer in an outer-sphere event seems to transiently occupy a shallow and overall hydrophobic cavity near the Cu type 1 (T1Cu). The linker connecting the β-strands 21 and 24 of the second domain (loop (β21-β24)(D2)) has the same conformation in both states, forming a flexible lid at the entrance of the electron-transfer cavity. Loop (β21-β24)(D2) has been tentatively assigned a role occluding the access to the electron-transfer site. The dynamic of the loop (β21-β24)(D2) has been investigated by MD simulation, and results show that the structures of both species have the same secondary and tertiary structure during almost all the MD simulations. In the simulation, loop (β21-β24)(D2) of the holo form undergoes a higher mobility than in the apo form. In fact, loop (β21-β24)(D2) of the holo form experiences a conformational change which enables exposure to the electron-transfer site (open conformation), while in the apo form the opposite effect takes place (closed conformation). To confirm the hypothesis that the open conformation might facilitate the transient electron-donor molecule occupation of the site, the simulation was extended another 40 ns with the electron-donor molecule docked into the protein cavity. Upon electron-donor molecule stabilization, loops near the cavity reduce their mobility. These findings show that coordination between the copper and the protein might play an important role in the general mobility of the enzyme, and that the open conformation seems to be required for the electron transfer process to T1Cu.