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Diverse computational approaches have been widely used to assist in designing antimicrobial peptides with enhanced activities. This tactic has also been used to address the need for new treatment alternatives to combat resistant bacterial infections. Herein, we have designed eight variants from a natural peptide, pro-adrenomedullin N-terminal 20 peptide (PAMP), using an in silico pattern insertion approach, the Joker algorithm. All the variants show an α-helical conformation, but with differences in the helix percentages according to circular dichroism (CD) results. We found that the C-terminal portion of PAMP may be relevant for its antimicrobial activities, as revealed by the molecular dynamics, CD, and antibacterial results. The analogs showed variable antibacterial potential, but most were not cytotoxic. Nevertheless, PAMP2 exhibited the most potent activities against human and animal-isolated bacteria, showing cytotoxicity only at a substantially higher concentration than its minimal inhibitory concentration (MIC). Our results suggest that the enhanced activity in the profile of PAMP2 may be related to their particular physicochemical properties, along with the adoption of an amphipathic α-helical arrangement with the conserved C-terminus portion. Finally, the peptides designed in this study can constitute scaffolds for the design of improved sequences.
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Adrenomedulina , Dicroísmo Circular , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Humanos , Adrenomedulina/química , Adrenomedulina/farmacologia , Sequência de Aminoácidos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Animais , Simulação por Computador , Precursores de Proteínas/química , Precursores de Proteínas/farmacologia , Precursores de Proteínas/metabolismo , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Estrutura Secundária de ProteínaRESUMO
The search for new therapeutic strategies against cancer has favored the emergence of rationally designed treatments. These treatments have focused on attacking cell plasticity mechanisms to block the transformation of epithelial cells into cancerous cells. The aim of these approaches was to control particularly lethal cancers such as hepatocellular carcinoma. However, they have not been able to control the progression of cancer for unknown reasons. Facing this scenario, emerging areas such as systems biology propose using engineering principles to design and optimize cancer treatments. Beyond the possibilities that this approach might offer, it is necessary to know whether its implementation at a clinical level is viable or not. Therefore, in this paper, we will review the engineering principles that could be applied to rationally design strategies against hepatocellular carcinoma, and discuss whether the necessary elements exist to implement them. In particular, we will emphasize whether these engineering principles could be applied to fight hepatocellular carcinoma.
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Quinoline has been proposed as a privileged molecular framework in medicinal chemistry. Although by itself it has very few applications, its derivatives have diverse biological activities. In this work, 8536 quinoline derivatives, strategically designed using the CADMA-Chem protocol, are presented. This large chemical space was sampled, analyzed and reduced using selection and elimination scores that combine their properties of bioavailability, toxicity and manufacturability. After applying several filters, 25 derivatives were selected to investigate their acid-base, antioxidant and neuroprotective properties. The antioxidant activity was predicted based on the ionization potential and bond dissociation energies, parameters directly related to the transfer of hydrogen atoms and of a single electron, respectively. These two mechanisms are typically involved in the radical scavenging processes. The antioxidant efficiency was compared with reference compounds, and the most promising antioxidants were found to be more efficient than Trolox but less efficient than ascorbate. In addition, based on molecular docking simulations, some derivatives are expected to act as inhibitors of catechol-O methyltransferase (COMT), acetylcholinesterase (AChE) and monoamine oxidase type B (MAO-B) enzymes. Some structural insights about the compounds were found to enhance or decrease the neuroprotection activity. Based on the results, four quinoline derivatives are proposed as candidates to act as multifunctional antioxidants against Alzheimer's (AD) and Parkinson's (PD) diseases.
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CONTEXT: Levobunolol is a ß-blocker drug prescribed for the control and prevention of cardiovascular events, such as individuals with cardiac arrhythmia or a history of myocardial infarction. Creating a levobunolol-specific molecularly imprinted polymer (MIP) allows for enhanced selectivity, efficient sample preparation, controlled drug delivery, and improved sensing and detection capabilities. In this sense, the aim of this study was to obtain through DFT calculations the synthesis protocol of a MIP for levobunolol testing different functional monomers (FMs), solvents, and cross-linker agents (CLAs). The analysis of structural and energetic data led to the identification of the optimal MIP synthesis parameters, which involves the use of (trifluoromethyl)-arylic acid (TFMAA) as the functional monomer, toluene and chloroform as the solvents, and pentaerythritol triacrylate (PETRA) as the cross-linking agent. This rational design offers valuable insights for experimentalists seeking to efficiently synthesize a MIP for this important ß-blocker drug. METHODS: DFT calculations were conducted using the B97D functional along with the Pople's split valence 6-31G(d,p) basis set, which includes polarization functions on all atoms (B97D/6-31G(d,p)).
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Levobunolol , Impressão Molecular , Humanos , Polímeros/química , Solventes/química , Sistemas de Liberação de Medicamentos , Polímeros Molecularmente Impressos , Modelos Teóricos , Impressão Molecular/métodosRESUMO
Acylases catalyze the hydrolysis of amide bonds. Penicillin G acylase (PGA) is used for the semi-synthesis of penicillins and cephalosporins. Although protein immobilization increases enzyme stability, the design of immobilized systems is difficult and usually it is empirically performed. We describe a novel application of our strategy for the Rational Design of Immobilized Derivatives (RDID) to produce optimized acylase-based immobilized biocatalysts for enzymatic bioconversion. We studied the covalent immobilization of the porcine kidney aminoacylase-1 onto aldehyde-based supports. Predictions of the RDID1.0 software and the experimental results led to the selection of glyoxyl-Sepharose CL 4B support and pH 10.0. One of the predicted clusters of reactive amino groups generates an enzyme-support configuration with highly accessible active sites, contributing with 82% of the biocatalyst's total activity. For Escherichia coli PGA, the predictions and experimental results show similar maximal amounts of immobilized protein and activity at pH 8.0 and 10.0 on glyoxyl-Sepharose CL 10B. However, thermal stability of the immobilized derivative is higher at pH 10.0 due to an elevated probability of multipoint covalent attachment. In this case, two clusters of amino groups are predicted to be relevant for PGA immobilization in catalytically competent configurations at pH 10.0, showing accessible active sites and contributing with 36% and 44% of the total activity, respectively. Our results support the usefulness of the RDID strategy to model different protein engineering approaches (site-directed mutagenesis or obtainment of fusion proteins) and select the most promising ones, saving time and laboratory work, since the in silico-designed modified proteins could have higher probabilities of success on bioconversion processes.
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Enzimas Imobilizadas , Penicilina Amidase , Animais , Suínos , Enzimas Imobilizadas/metabolismo , Amidoidrolases/metabolismo , Estabilidade Enzimática , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Penicilina Amidase/químicaRESUMO
Heterologous expression of L-asparaginase (L-ASNase) has become an important area of research due to its clinical and food industry applications. This review provides a comprehensive overview of the molecular and metabolic strategies that can be used to optimize the expression of L-ASNase in heterologous systems. This article describes various approaches that have been employed to increase enzyme production, including the use of molecular tools, strain engineering, and in silico optimization. The review article highlights the critical role that rational design plays in achieving successful heterologous expression and underscores the challenges of large-scale production of L-ASNase, such as inadequate protein folding and the metabolic burden on host cells. Improved gene expression is shown to be achievable through the optimization of codon usage, synthetic promoters, transcription and translation regulation, and host strain improvement, among others. Additionally, this review provides a deep understanding of the enzymatic properties of L-ASNase and how this knowledge has been employed to enhance its properties and production. Finally, future trends in L-ASNase production, including the integration of CRISPR and machine learning tools are discussed. This work serves as a valuable resource for researchers looking to design effective heterologous expression systems for L-ASNase production as well as for enzymes production in general.
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Ferulic acid has numerous beneficial effects on human health, which are frequently attributed to its antioxidant behavior. In this report, many of them are reviewed, and 185 new ferulic acid derivatives are computationally designed using the CADMA-Chem protocol. Consequently, their chemical space was sampled and evaluated. To that purpose, selection and elimination scores were used, which are built from a set of descriptors accounting for ADME properties, toxicity, and synthetic accessibility. After the first screening, 12 derivatives were selected and further investigated. Their potential role as antioxidants was predicted from reactivity indexes directly related to the formal hydrogen atom transfer and the single electron transfer mechanisms. The best performing molecules were identified by comparisons with the parent molecule and two references: Trolox and α-tocopherol. Their potential as polygenic neuroprotectors was investigated through the interactions with enzymes directly related to the etiologies of Parkinson's and Alzheimer's diseases. These enzymes are acetylcholinesterase, catechol-O-methyltransferase, and monoamine oxidase B. Based on the obtained results, the most promising candidates (FA-26, FA-118, and FA-138) are proposed as multifunctional antioxidants with potential neuroprotective effects. The findings derived from this investigation are encouraging and might promote further investigations on these molecules.
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Peptide engineering has gained attraction as a source of new cationicity-enhanced analogues with high potential for the design of next-generation antibiotics. In this context, cruzioseptin-1 (CZS-1), a peptide identified from Cruziohyla calcarifer, is recognized for its antimicrobial potency. However, this amidated-peptide is moderately hemolytic. In order to reduce toxicity and increase antimicrobial potency, 3 peptide analogues based on cruzioseptin-1 were designed and evaluated. [K4K15]CZS-1, an analogue with increased cationicity and reduced hydrophobicity, showed antibacterial, antifungal and antiproliferative properties. In addition, [K4K15]CZS-1 is less hemolytic than CZS-1. The in silico and scanning electron microscopy analysis reveal that [K4K15]CZS-1 induces a membranolytic effect on bacteria. Overall, these results confirm the potential of CZS-1 as source of inspiration for design new selective antimicrobial analogues useful for development of new therapeutic agents.
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Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Sequência de Aminoácidos , Antibacterianos/farmacologia , Antibacterianos/química , Anti-Infecciosos/química , Antifúngicos/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
In recent years, antimicrobial peptides isolated from amphibian toxins have gained attention as new multifunctional drugs interacting with different molecular targets. We aimed to rationally design a new peptide from temporin-PTa. Hp-MAP3 (NH2-LLKKVLALLKKVL-COOH), net charge (+4), hydrophobicity (0.69), the content of hydrophobic residues (69%), and hydrophobic moment (0.73). For the construction of the analog peptide, the physicochemical characteristics were reorganized into hydrophilic and hydrophobic residues with the addition of lysines and leucines. The minimum inhibitory concentration was 2.7 to 43 µM against the growth of Gram-negative and positive bacteria, and the potential for biofilm eradication was 173.2 µM. Within 20 min, the peptide Hp-MAP3 (10.8 µM) prompted 100% of the damage to E. coli cells. At 43.3 µM, eliminated 100% of S. aureus within 5 min. The effects against yeast species of the Candida genus ranged from 5.4 to 86.6 µM. Hp-MAP3 presents cytotoxic activity against tumor HeLa at a concentration of 21.6 µM with an IC50 of 10.4 µM. Furthermore, the peptide showed hemolytic activity against murine erythrocytes. Structural studies carried out by circular dichroism showed that Hp-MAP3, while in the presence of 50% trifluoroethanol or SDS, an α-helix secondary structure. Finally, Amphipathic Hp-MAP3 building an important model for the design of new multifunctional molecules.
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Proteínas de Anfíbios , Peptídeos Catiônicos Antimicrobianos , Animais , Humanos , Camundongos , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Dicroísmo Circular , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Ranidae , Staphylococcus aureus/efeitos dos fármacosRESUMO
Antimicrobial peptides (AMPs) are components in the innate immune system of various organisms, and many AMPs can be found in poisons from animals such as spiders, scorpions, and snakes. The peptide Cupiennin-1a is present in the venom of the spider Cupiennius salei and belongs to a group of peptides called cupiennins. The peptide demonstrated high cytotoxic activity against mammalian cells; thus, aiming to solve this problem, seven analogs were designed (R1a, R1b, R2b, R3b, R6b, R8b, and R10b) based on the primary structure of the peptide Cupiennin 1a, reducing its size and substituting some amino acid residues. The antimicrobial results showed that all Cupiennin 1a analogs displayed antimicrobial activity against the tested bacterial and fungal strains. Cytotoxicity tests demonstrated a decrease in the cytotoxic effect of the analogs when compared to the peptide Cupiennin-1a. The antitumor activity against breast adenocarcinoma lines was observed for all the peptides, displaying a better effect against the MCF-7 and MDAMB-231 cell lines. The eight peptides have insecticidal potential, and the original peptide and analogs R6b, R8b, and R10b showed better efficiency even at low concentrations. The rational design of the analogs led to new molecules displaying activities against different cell types and reduced cytotoxicity toward healthy mammalian cells when compared to the original peptide, demonstrating that this was an interesting approach for the development of molecules with biotechnological potential.
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The purpose of this work was to investigate, via DFT calculations, the molecularly imprinted polymer (MIP) for atenolol (ATL) ß-blocker evaluating distinct functional monomers (FMs), solvents, and cross-linker agents (CLAs). As the main result, we could determine from structural and thermodynamic data the best MIP synthesis protocol as being: p-vinyl benzoic acid (APV) as FM, toluene as solvent, and pentaerythritol triacrylate (PETRA) as CLA. We believe this rational design can be very useful for experimentalists in an attempt to perform an efficient synthesis of a MIP for this important ß-blocker drug.
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Atenolol , Polímeros , Polímeros/química , Solventes , TermodinâmicaRESUMO
Single domain antibodies from camelids, or nanobodies, are a unique class of antibody fragments with several advantageous characteristics: small monomeric size, high stability and solubility and easy tailoring for multiple applications. Nanobodies are gaining increasing acceptance as diagnostic tools and promising therapeutic agents in cancer and other diseases. While most nanobodies are obtained from immunized animals of the camelid family, a few synthetic nanobody libraries constructed in recent years have shown the capability of generating high quality nanobodies in terms of affinity and stability. Since this synthetic approach has important advantages over the use of animals, the recent advances are indeed encouraging. Here we review over a dozen synthetic nanobody libraries reported so far and discuss the different approaches followed in their construction and validation, with an emphasis on framework and hypervariable loop design as critical issues defining their potential as high-class nanobody sources.
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Anticorpos de Domínio Único , Animais , AnticorposRESUMO
Several antimicrobial peptides (AMPs) have been reported in amphibian toxins, as temporin-PTa from Hylarana picturata. The amino acid distribution within a helical structure of AMPs favors the design of new bioactive peptides. Therefore, this work reports the rational design of two new synthetic peptides denominated Hp-MAP1 and Hp-MAP2 derived from temporin-PTa. These peptides present an amphipathic helix with positive charges of +4 and +5, hydrophobic moment (<µH>) of 0.66 and 0.72 and hydrophobicity (
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Antibacterianos , Peptídeos Catiônicos Antimicrobianos , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , BiofilmesRESUMO
Genetically encoded FRET sensors for revealing local concentrations of second messengers in living cells have enormously contributed to our understanding of physiological and pathological processes. However, the development of sensors remains an intricate process. Using simulation techniques, we recently introduced a new architecture to measure intracellular concentrations of cAMP named CUTie, which works as a FRET tag for arbitrary targeting domains. Although our method showed quasi-quantitative predictive power in the design of cAMP and cGMP sensors, it remains intricate and requires specific computational skills. Here, we provide a simplified computer-aided protocol to design tailor-made CUTie sensors based on arbitrary cyclic nucleotide-binding domains. As a proof of concept, we applied this method to construct a new CUTie sensor with a significantly higher cAMP sensitivity (EC50 = 460 nM).This simple protocol, which integrates our previous experience, only requires free web servers and can be straightforwardly used to create cAMP sensors adapted to the physicochemical characteristics of known cyclic nucleotide-binding domains.
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AMP Cíclico , Pedestres , AMP Cíclico/química , GMP Cíclico , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Sistemas do Segundo MensageiroRESUMO
Recently, there has been an increase in the demand for enzymes with modified activity, specificity, and stability. Enzyme engineering is an important tool to meet the demand for enzymes adjusted to different industrial processes. Knowledge of the structure and function of enzymes guides the choice of the best strategy for engineering enzymes. Each enzyme engineering strategy, such as rational design, directed evolution, and semi-rational design, has specific applications, as well as limitations, which must be considered when choosing a suitable strategy. Engineered enzymes can be optimized for different industrial applications by choosing the appropriate strategy. This review features engineered enzymes that have been applied in food, animal feed, pharmaceuticals, medical applications, bioremediation, biofuels, and detergents.
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Biotecnologia , Engenharia de Proteínas , Animais , Biocatálise , Biodegradação Ambiental , Enzimas/química , IndústriasRESUMO
Our novel strategy for the rational design of immobilized derivatives (RDID) is directed to predict the behavior of the protein immobilized derivative before its synthesis, by the usage of mathematic algorithms and bioinformatics tools. However, this approach needs to be validated for each target enzyme. The objective of this work was to validate the RDID strategy for covalent immobilization of the enzyme laccase from Trametes maxima MUCL 44155 on glyoxyl- and monoaminoethyl-N-aminoethyl (MANA)-Sepharose CL 4B supports. Protein surface clusters, more probable configurations of the protein-supports systems at immobilization pHs, immobilized enzyme activity, and protein load were predicted by RDID1.0 software. Afterward, immobilization was performed and predictions were experimentally confirmed. As a result, the laccase-MANA-Sepharose CL 4B immobilized derivative is better than laccase-glyoxyl-Sepharose CL 4B in predicted immobilized derivative activity (63.6% vs. 29.5%). Activity prediction was confirmed by an experimentally expressed enzymatic activity of 68%, using 2,6-dimethoxyphenol as substrate. Experimental maximum protein load matches the estimated value (11.2 ± 1.3 vs. 12.1 protein mg/support mL). The laccase-MANA-Sepharose CL 4B biocatalyst has a high specificity for the acid blue 62 colorant. The results obtained in this work suggest the possibility of using this biocatalyst for wastewater treatment.
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Lacase , Trametes , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Lacase/metabolismo , Polyporaceae , Sefarose/análogos & derivadosRESUMO
Current worldwide challenges are to increase the food production and decrease the environmental contamination by industrial emissions. For this, bacteria can produce plant growth promoter phytohormones and mediate the bioremediation of sewage by heavy metals removal. We developed a Rational Design of Immobilized Derivatives (RDID) strategy, applicable for protein, spore and cell immobilization and implemented in the RDID1.0 software. In this work, we propose new algorithms to optimize the theoretical maximal quantity of cells to immobilize (tMQCell) on solid supports, implemented in the RDIDCell software. The main modifications to the preexisting algorithms are related to the sphere packing theory and exclusive immobilization on the support surface. We experimentally validated the new tMQCell parameter by electrostatic immobilization of ten microbial strains on AMBERJET® 4200 Cl- porous solid support. All predicted tMQCell match the practical maximal quantity of cells to immobilize with a 10% confidence. The values predicted by the RDIDCell software are more accurate than the values predicted by the RDID1.0 software. 3-indolacetic acid (IAA) production by one bacterial immobilized derivative was higher (~ 2.6 µg IAA-like indoles/108 cells) than that of the cell suspension (1.5 µg IAA-like indoles/108 cells), and higher than the tryptophan amount added as indole precursor. Another bacterial immobilized derivative was more active (22 µg Cr(III)/108 cells) than the resuspended cells (14.5 µg Cr(III)/108 cells) in bioconversion of Cr(VI) to Cr(III). Optimized RDID strategy can be used to synthesize bacterial immobilized derivatives with useful biotechnological applications.
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Biodegradação Ambiental , Células Imobilizadas/metabolismo , Biologia Computacional/métodos , Algoritmos , Bactérias/metabolismo , Biomassa , Poluentes Ambientais , Metais Pesados/metabolismo , Software , Eletricidade EstáticaRESUMO
Human African Trypanosomiasis (HAT), Chagas disease or American Trypanosomiasis (CD), and leishmaniases are protozoan infections produced by trypanosomatid parasites belonging to the kinetoplastid order and they constitute an urgent global health problem. In fact, there is an urgent need of more efficient and less toxic chemotherapy for these diseases. Medicinal inorganic chemistry currently offers an attractive option for the rational design of new drugs and, in particular, antiparasitic ones. In this sense, one of the main strategies for the design of metal-based antiparasitic compounds has been the coordination of an organic ligand with known or potential biological activity, to a metal centre or an organometallic core. Classical metal coordination complexes or organometallic compounds could be designed as multifunctional agents joining, in a single molecule, different chemical species that could affect different parasitic targets. This review is focused on the rational design of palladium(II) and platinum(II) compounds with bioactive ligands as prospective drugs against trypanosomatid parasites that has been conducted by our group during the last 20 years.
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Evolutionarily related proteins can present similar structures but very dissimilar sequences. Hence, understanding the role of the inter-residues contacts for the protein structure has been the target of many studies. Contacts comprise non-covalent interactions, which are essential to stabilize macromolecular structures such as proteins. Here we show VTR, a new method for the detection of analogous contacts in protein pairs. The VTR web tool performs structural alignment between proteins and detects interactions that occur in similar regions. To evaluate our tool, we proposed three case studies: we 1) compared vertebrate myoglobin and truncated invertebrate hemoglobin; 2) analyzed interactions between the spike protein RBD of SARS-CoV-2 and the cell receptor ACE2; and 3) compared a glucose-tolerant and a non-tolerant ß-glucosidase enzyme used for biofuel production. The case studies demonstrate the potential of VTR for the understanding of functional similarities between distantly sequence-related proteins, as well as the exploration of important drug targets and rational design of enzymes for industrial applications. We envision VTR as a promising tool for understanding differences and similarities between homologous proteins with similar 3D structures but different sequences. VTR is available at http://bioinfo.dcc.ufmg.br/vtr.
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The SARS-CoV-2 spread quickly across the globe. The World Health Organization (WHO) on March 11 declared COVID-19 a pandemic. The mortality rate, hospital disorders and incalculable economic and social damages, besides the unproven efficacy of the treatments evaluated against COVID-19, raised the need for immediate control of this disease. Therefore, the current study employed in silico tools to rationally identify new possible SARS-CoV-2 main protease (Mpro) inhibitors. That is an enzyme conserved among the coronavirus species; hence, the identification of an Mpro inhibitor is to make it a broad-spectrum drug. Molecular docking studies described the binding sites and the interaction energies of 74 Mpro-ligand complexes deposited in the Protein Data Bank (PDB). A structural similarity screening was carried out in order to identify possible Mpro ligands that show additional pharmacological properties against COVID-19. We identified 59 hit compounds and among them, melatonin stood out due to its prominent immunomodulatory and anti-inflammatory activities; it can reduce oxidative stress, defence cell mobility and efficiently combat the cytokine storm and sepsis. In addition, melatonin is an inhibitor of calmodulin, an essential intracellular component to maintain angiotensin-converting enzyme 2 (ACE-2) on the cell surface. Interestingly, one of the most promising hits in our docking study was melatonin. It revealed better interaction energy with Mpro compared to ligands in complexes from PDB. Consequently, melatonin can have response potential in early stages for its possible effects on ACE-2 and Mpro, although it is also promising in more severe stages of the disease for its action against hyper-inflammation. These results definitely do not confirm antiviral activity, but can rather be used as a basis for further preclinical and clinical trials.