Search

Human cathepsin K (CatK) stands out as a promising target for the treatment of osteoporosis, considering its role in degrading the bone matrix. Given the small and shallow S2 subsite of CatK and considering its preference for proline or hydroxyproline, we now propose the rigidification of the leucine fragment found at the P2 position in a dipeptidyl-based inhibitor, generating rigid proline-based analogs. Accordingly, with these new proline-based peptidomimetics inhibitors, we selectively inhibited CatK against other human cathepsins (B, L and S). Among these new ligands, the most active one exhibited a high affinity (pK i  = 7.3 - 50.1 nM) for CatK and no inhibition over the other cathepsins. This specific inhibitor harbors two novel substituents never employed in other CatK inhibitors: the trifluoromethylpyrazole and the 4-methylproline at P3 and P2 positions. These results broaden and advance the path toward new potent and selective inhibitors for CatK., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The enzyme PETase from Ideonella sakaiensis ( Is PETase) strain 201-F6 can catalyze the hydrolysis of polyethylene terephthalate (PET), mainly converting it into mono(2-hydroxyethyl) terephthalic acid (MHET). In this study, we used quantum mechanics/molecular mechanics (QM/MM) simulations to explore the molecular details of the catalytic reaction mechanism of Is PETase in the formation of MHET. The QM region was described with AM1d/PhoT and M06-2 X /6-31+G(d,p) potential. QM/MM simulations unveil the complete enzymatic PET hydrolysis mechanism and identify two possible reaction pathways for acylation and deacylation steps. The barrier obtained at M06-2 X /6-31+G(d,p)/MM potential for the deacylation step corresponds to 20.4 kcal/mol, aligning with the experimental value of 18 kcal/mol. Our findings indicate that deacylation is the rate-limiting step of the process. Furthermore, per-residue interaction energy contributions revealed unfavorable contributions to the transition state of amino acids located at positions 200-230, suggesting potential sites for targeted mutations. These results can contribute to the development of more active and selective enzymes for PET depolymerization.

The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the 'dormant complex' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology., (© 2024 The Protein Society.)

Context: Heparin, one of the drugs reused in studies with antiviral activity, was chosen to investigate a possible blockade of the SARS-CoV-2 spike protein for viral entry through computational simulations and experimental analysis. Heparin was associated to graphene oxide to increase in the binding affinity in biological system. First, the electronic and chemical interaction between the molecules was analyzed through ab initio simulations. Later, we evaluate the biological compatibility of the nanosystems, in the target of the spike protein, through molecular docking. The results show that graphene oxide interacts with the heparin with an increase in the affinity energy with the spike protein, indicating a possible increment in the antiviral activity. Experimental analysis of synthesis and morphology of the nanostructures were carried out, indicating heparin absorption by graphene oxide, confirming the results of the first principle simulations. Experimental tests were conducted on the structure and surface of the nanomaterial, confirming the heparin aggregation on the synthesis with a size between the GO layers of 7.44 Å, indicating a C-O type bond, and exhibiting a hydrophilic surface characteristic (36.2°)., Methods: Computational simulations of the ab initio with SIESTA code, LDA approximations, and an energy shift of 0.05 eV. Molecular docking simulations were performed in the AutoDock Vina software integrated with the AMDock Tools Software using the AMBER force field. GO, GO@2.5Heparin, and GO@5Heparin were synthesized by Hummers and impregnation methods, respectively, and characterized by X-ray diffraction and surface contact angle., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Caffeic acid (CA) exhibits a myriad of biological activities including cardioprotective action, antioxidant, antitumor, anti-inflammatory, and antimicrobial properties. On the other hand, CA presents low water solubility and poor bioavailability, which have limited its use for therapeutic applications. The objective of this study was to develop a nanohybrid of zinc basic salts (ZBS) and chitosan (Ch) containing CA (ZBS-CA/Ch) and evaluate its anti-edematogenic and antioxidant activity in dextran and carrageenan-induced paw edema model. The samples were obtained by coprecipitation method and characterized by X-ray diffraction, Fourier transform infrared (FT-IR), scanning electron microscope (SEM) and UV-visible spectroscopy. The release of caffeate anions from ZBS-CA and ZBS-CA/Ch is pH-dependent and is explained by a pseudo-second order kinetics model, with a linear correlation coefficient of R 2 ≥ 0.99 at pH 4.8 and 7.4. The in vivo pharmacological assays showed excellent anti-edematogenic and antioxidant action of the ZBS-CA/Ch nanoparticle with slowly releases of caffeate anions in the tissue, leading to a prolongation of CA-induced anti-edematogenic and anti-inflammatory activities, as well as improving its inhibition or sequestration antioxidant action toward reactive species. Overall, this study highlighted the importance of ZBS-CA/Ch as an optimal drug carrier.

Trypanosoma cruzi is a parasite that infects about 6-7 million people worldwide, mostly in Latin America, causing Chagas disease. Cruzain, the main cysteine protease of T. cruzi , is a validated target for developing drug candidates for Chagas disease. Thiosemicarbazones are one of the most relevant warheads used in covalent inhibitors targeting cruzain. Despite its relevance, the mechanism of inhibition of cruzain by thiosemicarbazones is unknown. Here, we combined experiments and simulations to unveil the covalent inhibition mechanism of cruzain by a thiosemicarbazone-based inhibitor (compound 1 ). Additionally, we studied a semicarbazone (compound 2 ), which is structurally similar to compound 1 but does not inhibit cruzain. Assays confirmed the reversibility of inhibition by compound 1 and suggested a two-step mechanism of inhibition. The K i was estimated to be 36.3 μM and K i * to be 11.5 μM, suggesting the pre-covalent complex to be relevant for inhibition. Molecular dynamics simulations of compounds 1 and 2 with cruzain were used to propose putative binding modes for the ligands. One-dimensional (1D) quantum mechanics/molecular mechanics (QM/MM) potential of mean force (PMF) and gas-phase energies showed that the attack of Cys25-S - on the C═S or C═O bond yields a more stable intermediate than the attack on the C═N bond of the thiosemicarbazone/semicarbazone. Two-dimensional (2D) QM/MM PMF revealed a putative reaction mechanism for compound 1 , involving the proton transfer to the ligand, followed by the Cys25-S - attack at C═S. The Δ G and energy barrier were estimated to be -1.4 and 11.7 kcal/mol, respectively. Overall, our results shed light on the inhibition mechanism of cruzain by thiosemicarbazones.

Eugenol is a natural compound with well-known repellent activity. However, its pharmaceutical and cosmetic applications are limited, since this compound is highly volatile and thermolabile. Nanoencapsulation provides protection, stability, conservation, and controlled release for several compounds. Here, eugenol was included in β-cyclodextrin, and the complex was characterized through X-ray diffraction analysis (XRD) and Fourier-transform infrared spectroscopy (FTIR). Additionally, we used molecular dynamics simulations to explore the eugenol-β-cyclodextrin complex stability with temperature increases. Our computational result demonstrates details of the molecular interactions and conformational changes of the eugenol-β-cyclodextrin complex and explains its stability between temperatures 27°C and 48°C, allowing its use in formulations that are subjected to varied temperatures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Freitas, Costa, da Costa, da Paz, Silva, Alves and Lameira.)

Plasmepsins (Plms) are aspartic proteases involved in the degradation of human hemoglobin by P. falciparum and are essential for the survival and growth of the parasite. Therefore, Plm enzymes are reported as an important antimalarial drug target. Herein, we have applied molecular docking, molecular dynamics (MD) simulations, and binding free energy with the Linear Interaction Energy (LIE) approach to investigate the binding of peptidomimetic PlmIV inhibitors with a particular focus on understanding their selectivity against the human Asp protease cathepsin D (CatD). The residual decomposition analysis results suggest that amino acid differences in the subsite S3 of PlmIV and CatD are responsible for the higher selectivity of the 5a inhibitor. These findings yield excellent agreement with experimental binding data and provide new details regarding van der Waals and electrostatic interactions of subsite residues as well as structural properties of the PlmIV and CatD systems., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (This journal is © The Royal Society of Chemistry.)

Nitriles have broad applications in medicinal chemistry, with more than 60 small molecule drugs on the market containing the cyano functional group. In addition to the well-known noncovalent interactions that nitriles can perform with macromolecular targets, they are also known to improve drug candidates' pharmacokinetic profiles. Moreover, the cyano group can be used as an electrophilic warhead to covalently bind an inhibitor to a target of interest, forming a covalent adduct, a strategy that can present benefits over noncovalent inhibitors. This approach has gained much notoriety in recent years, mainly with diabetes and COVID-19-approved drugs. Nevertheless, the application of nitriles in covalent ligands is not restricted to it being the reactive center, as it can also be employed to convert irreversible inhibitors into reversible ones, a promising strategy for kinase inhibition and protein degradation. In this review, we introduce and discuss the roles of the cyano group in covalent inhibitors, how to tune its reactivity and the possibility of achieving selectivity only by replacing the warhead. Finally, we provide an overview of nitrile-based covalent compounds in approved drugs and inhibitors recently described in the literature., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

We have used molecular dynamics (MD) simulations with hybrid quantum mechanics/molecular mechanics (QM/MM) potentials to investigate the reaction mechanism for covalent inhibition of cathepsin K and assess the reversibility of inhibition. The computed free energy profiles suggest that a nucleophilic attack by the catalytic cysteine on the inhibitor warhead and proton transfer from the catalytic histidine occur in a concerted manner. The results indicate that the reaction is more strongly exergonic for the alkyne-based inhibitors, which bind irreversibly to cathepsin K, than for the nitrile-based inhibitor odanacatib, which binds reversibly. Gas-phase energies were also calculated for the addition of methanethiol to structural prototypes for a number of warheads of interest in cysteine protease inhibitor design in order to assess electrophilicity. The approaches presented in this study are particularly applicable to assessment of novel warheads, and computed transition state geometries can be incorporated into molecular models for covalent docking.

A primary component of all known bacterial cell walls is the peptidoglycan (PG) layer, which is composed of repeating units of sugars connected to short and unusual peptides. The various steps within PG biosynthesis are targets of potent antibiotics as proper assembly of the PG is essential for cellular growth and survival. Synthetic mimics of PG have proven to be indispensable tools to study the bacterial cell structure, growth, and remodeling. Yet, a common component of PG, meso -diaminopimelic acid ( m -DAP) at the third position of the stem peptide, remains challenging to access synthetically and is not commercially available. Here, we describe the synthesis and metabolic processing of a selenium-based bioisostere of m -DAP (selenolanthionine) and show that it is installed within the PG of live bacteria by the native cell wall crosslinking machinery in mycobacterial species. This PG probe has an orthogonal release mechanism that could be important for downstream proteomics studies. Finally, we describe a bead-based assay that is compatible with high-throughput screening of cell wall enzymes. We envision that this probe will supplement the current methods available for investigating PG crosslinking in m -DAP-containing organisms.

The severe acute respiratory syndrome (SARS) coronavirus 2 (CoV-2) variant Omicron spread more rapid than the other variants of SARS-CoV-2 virus. Mutations on the Spike (S) protein receptor-binding domain (RBD) are critical for the antibody resistance and infectivity of the SARS-CoV-2 variants. In this study, we have used accelerated molecular dynamics (aMD) simulations and free energy calculations to present a systematic analysis of the affinity and conformational dynamics along with the interactions that drive the binding between Spike protein RBD and human angiotensin-converting enzyme 2 (ACE2) receptor. We evaluate the impacts of the key mutation that occur in the RBDs Omicron and other variants in the binding with the human ACE2 receptor. The results show that S protein Omicron has stronger binding to the ACE2 than other variants. The evaluation of the decomposition energy per residue shows the mutations N440K, T478K, Q493R and Q498R observed in Spike protein of SARS-CoV-2 provided a stabilization effect for the interaction between the SARS-CoV-2 RBD and ACE2. Overall, the results demonstrate that faster spreading of SARS-CoV-2 Omicron may be correlated with binding affinity of S protein RBD to ACE2 and mutations of uncharged residues to positively charged residues such as Lys and Arg in key positions in the RBD., (© 2022. The Author(s).)

Introduction: Cathepsin K (CatK) is a lysosomal cysteine protease and the predominant cathepsin expressed in osteoclasts, where it degrades the bone matrix. Hence, CatK is an attractive therapeutic target related to diseases characterized by bone resorption, like osteoporosis., Areas Covered: This review summarizes the patent literature from 2011 to 2021 on CatK inhibitors and their potential use as new treatments for osteoporosis. The inhibitors were classified by their warheads, with the most explored nitrile-based inhibitors. Promising in vivo results have also been disclosed., Expert Opinion: As one of the most potent lysosomal proteins whose primary function is to mediate bone resorption, cathepsin K remains an excellent target for therapeutic intervention. Nevertheless, there is no record of any approved drug that targets CatK. The most notable cases of drug candidates targeting CatK were balicatib and odanacatib, which reached Phase II and III clinical trials, respectively, but did not enter the market. Further developments include exploring new chemical entities beyond the nitrile-based chemical space, with improved ADME and safety profiles. In addition, CatK's role in cancer immunoexpression and its involvement in the pathophysiology of osteo- and rheumatoid arthritis have raised the race to develop activity-based probes with excellent potency and selectivity.

Repellents are compounds that prevent direct contact between the hosts and the arthropods that are vectors of diseases. Several studies have described the repellent activities of natural compounds obtained from essential oils. In addition, these chemical constituents have been pointed out as alternatives to conventional synthetic repellents due to their interesting residual protection and low toxicity to the environment. However, these compounds have been reported with short shelf life, in part, due to their volatile nature. Nanoencapsulation provides protection, stability, conservation, and controlled release for several compounds. Here, we review the most commonly used polymeric/lipid nanosystems applied in the encapsulation of small organic molecules obtained from essential oils that possess repellent activity, and we also explore the theoretical aspects related to the intermolecular interactions, thermal stability, and controlled release of the nanoencapsulated bioactive compounds.

Bacteriocins are antimicrobial peptides expressed by bacteria through ribosomal activity. In this study, we analyzed the diversity of bacteriocin-like genes in the Tucuruí-HPP using a whole-metagenome shotgun sequencing approach. Three layers of the water column were analyzed (photic, aphotic and sediment). Detection of bacteriocin-like genes was performed with blastx using the BAGEL4 database as subject sequences. In order to calculate the abundance of bacteriocin-like genes we also determined the number of 16S rRNA genes using blastn. Taxonomic analysis was performed using RAST server and the metagenome was assembled using IDBA-UD in order to recover the full sequence of a zoocin which had its three-dimensional structure determined. The photic zone presented the highest number of reads affiliated to bacteriocins. The most abundant bacteriocins were sonorensin, Klebicin D , pyocin and colicin. The zoocin model was composed of eight anti-parallel β-sheets and two α-helices with a Zn2+ ion in the active site. This model was considerably stable during 10 ns of molecular dynamics simulation. We observed a high diversity of bacteriocins in the Tucuruí-HPP, demonstrating that the environment is an inexhaustible source for prospecting these molecules. Finally, the zoocin model can be used for further studies of substrate binding and molecular mechanisms involving peptidoglycan degradation.

The inhibition of key enzymes that may contain the viral replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have assumed central importance in drug discovery projects. Nonstructural proteins (nsps) are essential for RNA capping and coronavirus replication since it protects the virus from host innate immune restriction. In particular, nonstructural protein 16 (nsp16) in complex with nsp10 is a Cap-0 binding enzyme. The heterodimer formed by nsp16-nsp10 methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs and thus it is one of the enzymes that is a potential target for antiviral therapy. In this study, we have evaluated the mechanism of the 2'- O methylation of the viral mRNA cap using hybrid quantum mechanics/molecular mechanics (QM/MM) approach. It was found that the calculated free energy barriers obtained at M062X/6-31+G(d,p) is in agreement with experimental observations. Overall, we provide a detailed molecular analysis of the catalytic mechanism involving the 2'- O methylation of the viral mRNA cap and, as expected, the results demonstrate that the TS stabilization is critical for the catalysis.

In SARS-CoV-2 replication complex, the Non-structural protein 9 (Nsp9) is an important RNA binding subunit in the RNA-synthesizing machinery. The dimeric forms of coronavirus Nsp9 increase their nucleic acid binding affinity and the N-finger motif appears to play a critical role in dimerization. Here, we present a structural, lipophilic and energetic study about the Nsp9 dimer of SARS-CoV-2 through computational methods that complement hydrophobicity scales of amino acids with molecular dynamics simulations. Additionally, we presented a virtual N-finger mutation to investigate whether this motif contributes to dimer stability. The results reveal for the native dimer that the N-finger contributes favorably through hydrogen bond interactions and two amino acids bellowing to the hydrophobic region, Leu45 and Leu106, are crucial in the formation of the cavity for potential drug binding. On the other hand, Gly100 and Gly104, are responsible for stabilizing the α-helices and making the dimer interface remain stable in both, native and mutant (without N-finger motif) systems. Besides, clustering results for the native dimer showed accessible cavities to drugs. In addition, the energetic and lipophilic analysis reveal that the higher binding energy in the native dimer can be deduced since it is more lipophilic than the mutant one, increasing non-polar interactions, which is in line with the result of MM-GBSA and SIE approaches where the van der Waals energy term has the greatest weight in the stability of the native dimer. Overall, we provide a detailed study on the Nsp9 dimer of SARS-CoV-2 that may aid in the development of new strategies for the treatment and prevention of COVID-19., (© 2021. The Author(s).)

Recently, a bacterium strain of Ideonella sakaiensis was identified with the uncommon ability to degrade the poly(ethylene terephthalate) (PET). The PETase from I. sakaiensis strain 201-F6 (IsPETase) catalyzes the hydrolysis of PET converting it to mono(2-hydroxyethyl) terephthalic acid (MHET), bis(2-hydroxyethyl)-TPA (BHET), and terephthalic acid (TPA). Despite the potential of this enzyme for mitigation or elimination of environmental contaminants, one of the limitations of the use of IsPETase for PET degradation is the fact that it acts only at moderate temperature due to its low thermal stability. Besides, molecular details of the main interactions of PET in the active site of IsPETase remain unclear. Herein, molecular docking and molecular dynamics (MD) simulations were applied to analyze structural changes of IsPETase induced by PET binding. Results from the essential dynamics revealed that the β1-β2 connecting loop is very flexible. This loop is located far from the active site of IsPETase and we suggest that it can be considered for mutagenesis to increase the thermal stability of IsPETase. The free energy landscape (FEL) demonstrates that the main change in the transition between the unbound to the bound state is associated with the β7-α5 connecting loop, where the catalytic residue Asp206 is located. Overall, the present study provides insights into the molecular binding mechanism of PET into the IsPETase structure and a computational strategy for mapping flexible regions of this enzyme, which can be useful for the engineering of more efficient enzymes for recycling plastic polymers using biological systems., (© 2021 Wiley Periodicals LLC.)

Covalent inhibitors are assuming central importance in drug discovery projects, especially in this pandemic scenario. Many research groups have focused their attention on inhibiting viral proteases or human proteases such as cathepsin L (hCatL). The inhibition of these critical enzymes may impair viral replication. However, molecular modeling of covalent ligands is challenging since covalent and noncovalent ligand-bound states must be considered in the binding process. In this work, we evaluated the suitability of free energy perturbation (FEP) calculations as a tool for predicting the binding affinity of reversible covalent inhibitors of hCatL. Our strategy relies on the relative free energy calculated for both covalent and noncovalent complexes and the free energy changes have been compared with experimental data for eight nitrile-based inhibitors, including three new inhibitors of hCatL. Our results demonstrate that the covalent complex can be employed to properly rank the inhibitors. Nevertheless, a comparison of the free energy changes in both noncovalent and covalent states is valuable to interpret the effect triggered by the formation of the covalent bond on the interactions played by functional groups distant from the warhead. Overall, FEP can be employed as a powerful predictor tool in developing and understanding the activity of reversible covalent inhibitors.

Multidrug-resistant organisms contain antibiotic-modifying enzymes that facilitate resistance to a variety of antimicrobial compounds. Particularly, the fosfomycin (FOF) drug can be structurally modified by several FOF-modifying enzymes before it reaches the biological target. Among them, FosB is an enzyme that utilizes l-cysteine or bacillithiol in the presence of a divalent metal to open the epoxide ring of FOF and, consequently, inactivate the drug. Here, we have used hybrid quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulations to explore the mechanism of the reaction involving FosB and FOF. The calculated free-energy profiles show that the cost to open the epoxide ring of FOF at the C2 atom is ∼3.0 kcal/mol higher than that at the C1 atom. Besides, our QM/MM MD results revealed the critical role of conformation change of Cys9 and Asn50 to release the drug from the active site. Overall, the present study provides insights into the mechanism of FOF-resistant proteins., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Natural products are continually explored in the development of new bioactive compounds with industrial applications, attracting the attention of scientific research efforts due to their pharmacophore-like structures, pharmacokinetic properties, and unique chemical space. The systematic search for natural sources to obtain valuable molecules to develop products with commercial value and industrial purposes remains the most challenging task in bioprospecting. Virtual screening strategies have innovated the discovery of novel bioactive molecules assessing in silico large compound libraries, favoring the analysis of their chemical space, pharmacodynamics, and their pharmacokinetic properties, thus leading to the reduction of financial efforts, infrastructure, and time involved in the process of discovering new chemical entities. Herein, we discuss the computational approaches and methods developed to explore the chemo-structural diversity of natural products, focusing on the main paradigms involved in the discovery and screening of bioactive compounds from natural sources, placing particular emphasis on artificial intelligence, cheminformatics methods, and big data analyses., Competing Interests: RB was employed by company InsilicAll Ltda. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Santana, do Nascimento, Lima e Lima, Damasceno, Nahum, Braga and Lameira.)

Allosteric changes modulate the enzymatic activity, leading to activation or inhibition of the molecular target. Understanding the induced fit accommodation mechanism of a ligand in its lowest-free energy state and the subsequent conformational changes induced in the protein are important questions for drug design. In the present study, molecular dynamics (MD) simulations, binding free energy calculations, and principal component analysis (PCA) were applied to analyze the glycerol-3-phosphate dehydrogenase of Leishmania mexicana ( Lm GPDH) conformational changes induced by its cofactor and substrate binding. GPDH is a nicotinamide adenine dinucleotide (NAD)-dependent enzyme, which has been reported as an interesting target for drug discovery and development against leishmaniasis. Despite its relevance for glycolysis and pentose phosphate pathways, the structural flexibility and conformational motions of Lm GPDH in complex with NADH and dihydroxyacetone phosphate (DHAP) remain unexplored. Here, we analyzed the conformational dynamics of the enzyme-NADH complex (cofactor), and the enzyme-NADH-DHAP complex (adduct), mapped the hydrogen-bond interactions for the complexes and pointed some structural determinants of the enzyme that emerge from these contacts to NADH and DHAP. Finally, we proposed a consistent mechanism for the conformational changes on the first step of the reversible redox conversion of dihydroxyacetone phosphate to glycerol 3-phosphate, indicating key residues and interactions that could be further explored in drug discovery.

Human cathepsin B (CatB) is an important biological target in cancer therapy. In this work, we performed a knowledge-based design approach and the synthesis of a new set of 19 peptide-like nitrile-based cathepsin inhibitors. Reported compounds were assayed against a panel of human cysteine proteases: CatB, CatL, CatK, and CatS. Three compounds (7h, 7i, and 7j) displayed nanomolar inhibition of CatB and selectivity over CatK and CatL. The selectivity was achieved by using the combination of a para biphenyl ring at P3, halogenated phenylalanine in P2 and Thr-O-Bz group at P1. Likewise, compounds 7i and 7j showed selective CatB inhibition among the panel of enzymes studied. We have also described a successful example of bioisosteric replacement of the amide bond for a sulfonamide one [7e → 6b], where we observed an increase in affinity and selectivity for CatB while lowering the compound lipophilicity (ilogP). Our knowledge-based design approach and the respective structure-activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cathepsins., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The shikimate pathway enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) catalyzes the reaction involved in the production of amino acids essential for plant growth and survival. Thus, EPSPS is the main target of various herbicides, including glyphosate, a broad-spectrum herbicide that acts as a competitive inhibitor of phosphoenolpyruvate (PEP), which is the natural substrate of EPSPS. However, punctual mutations in the EPSPS gene have led to glyphosate resistance in some plants. Here, we investigated the mechanism of EPSPS resistance to glyphosate in mutants of two weed species, Conyza sumatrensis (mutant, P106T) and Eleusine indica (mutant, T102I/P106S), both of which have an economic impact on industrial crops. Molecular dynamics (MD) simulations and binding free energy calculations revealed the influence of the mutations on the affinity of glyphosate in the PEP-binding site. The amino acid residues of the EPSPS protein in both species involved in glyphosate resistance were elucidated as well as other residues that could be useful for protein engineering. In addition, during MD simulations, we identified conformational changes in glyphosate when complexed with resistant EPSPS, related to loss of herbicide activity and binding affinity. Our computational findings are consistent with previous experimental results and clarify the inhibitory activity of glyphosate as well as the structural target-site resistance of EPSPS against glyphosate., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Bacterial cell walls contain peptidoglycan (PG), a scaffold that provides proper rigidity to resist lysis from internal osmotic pressure and a barrier to protect cells against external stressors. It consists of repeating sugar units with a linkage to a stem peptide that becomes cross-linked by cell wall transpeptidases (TP). While synthetic PG fragments containing l-lysine in the third position on the stem peptide are easier to access, those with meso -diaminopimelic acid ( m -DAP) pose a severe synthetic challenge. Herein, we describe a solid phase synthetic scheme based on widely available building blocks to assemble meso -cystine ( m -CYT), which mimics key structural features of m -DAP. To demonstrate proper mimicry of m -DAP, cell wall probes were synthesized with m -CYT in place of m -DAP and evaluated for their metabolic processing in live bacterial cells. We found that m -CYT-based cell wall probes were properly processed by TPs in various bacterial species that endogenously contain m -DAP in their PG. Additionally, we have used hybrid quantum mechanical/molecular mechanical (QM/MM) and molecular dynamics (MD) simulations to explore the influence of m -DAP analogs on the PG cross-linking. The results showed that the cross-linking mechanism of transpeptidases occurred through a concerted process. We anticipate that this strategy, which is based on the use of inexpensive and commercially available building blocks, can be widely adopted to provide greater accessibility of PG mimics for m -DAP containing organisms.

Leishmania mexicana is an obligate intracellular protozoan parasite that causes the cutaneous form of leishmaniasis affecting South America and Mexico. The cysteine protease LmCPB is essential for the virulence of the parasite and therefore, it is an appealing target for antiparasitic therapy. A library of nitrile-based cysteine protease inhibitors was screened against LmCPB to develop a treatment of cutaneous leishmaniasis. Several compounds are sufficiently high-affinity LmCPB inhibitors to serve both as starting points for drug discovery projects and as probes for target validation. A 1.4 Å X ray crystal structure, the first to be reported for LmCPB, was determined for the complex of this enzyme covalently bound to an azadipeptide nitrile ligand. Mapping the structure-activity relationships for LmCPB inhibition revealed superadditive effects for two pairs of structural transformations. Therefore, this work advances our understanding of azadipeptidyl and dipeptidyl nitrile structure-activity relationships for LmCPB structure-based inhibitor design. We also tested the same series of inhibitors on related cysteine proteases cathepsin L and Trypanosoma cruzi cruzain. The modulation of these mammalian and protozoan proteases represents a new framework for targeting papain-like cysteine proteases., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Hence, peptidomimetic cruzipain inhibitors having a reactive group (known as warhead) are subject to continuous studies to discover novel antichagasic compounds. Here, we evaluated how different warheads for a set of structurally similar related compounds could inhibit the activity of cruzipain and, ultimately, their trypanocidal effect. We first investigated in silico the intrinsic reactivity of these compounds by applying the Fukui index to correlate it with the enzymatic affinity. Then, we evaluated their potency against T. cruzi (Y and Tulahuen strains), which revealed the reversible cruzain inhibitor Neq0656 as a better trypanocidal agent (EC Y.strain 50 = 0.1 μM; SI = 58.4) than the current drug benznidazole (EC Y.strain 50 = 5.1 μM; SI > 19.6). We also measured the half-life time by HPLC analysis of three lead compounds in the presence of glutathione and cysteine to experimentally assess their intrinsic reactivity. Results clearly illustrated the reactivity trend for the warheads (azanitrile > aldehyde > nitrile), where the aldehyde displayed an intermediate intrinsic reactivity. Therefore, the aldehyde bearing peptidomimetic compounds should be subject for in-depth evaluation in the drug discovery process., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

Cysteine proteases (CPs) are involved in a myriad of actions that include not only protein degradation, but also play an essential biological role in infectious and systemic diseases such as cancer. CPs also act as biomarkers and can be reached by active-based probes for diagnostic and mechanistic purposes that are critical in health and disease. In this paper, we present the modulation of a CP panel of parasites and mammals (Trypanosoma cruzi cruzain, LmCPB, CatK, CatL and CatS), whose inhibition by nitrile peptidomimetics allowed the identification of specificity and selectivity for a given CP. The activity cliffs identified at the CP inhibition level are useful for retrieving trends through multiple structure-activity relationships. For two of the cruzain inhibitors (10g and 4e), both enthalpy and entropy are favourable to Gibbs binding energy, thus overcoming enthalpy-entropy compensation (EEC). Group contribution of individual molecular modification through changes in enthalpy and entropy results in a separate partition on the relative differences of Gibbs binding energy (ΔΔG). Overall, this study highlights the role of CPs in polypharmacology and multi-target screening, which represents an imperative trend in the actual drug discovery effort., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Tyrosinase (TYR) is a metalloenzyme classified as a type-3 copper protein, which is involved in the synthesis of melanin through a catalytic process beginning with the conversion of the amino acid l-Tyrosine (l-Tyr) to l-3,4-dihydroxyphenylalanine (l-DOPA). It plays an important role in the mechanism of melanogenesis in various organisms including mammals, plants, and fungi. Herein, we used a combination of computational molecular modeling techniques including molecular dynamic (MD) simulations and the linear interaction energy (LIE) model to evaluate the binding free energy of a set of analogs of kojic acid (KA) in complex with TYR. For the MD simulations, we used a dummy model including the description of the Jahn-Teller effect for Cu 2+ ions in the active site of this enzyme. Our results show that the LIE model predicts the TYR binding affinities of the inhibitor in close agreement to experimental results. Overall, we demonstrate that the classical model provides a suitable description of the main interactions between analogs of KA and Cu 2+ ions in the active site of TYR.

Reversible and irreversible covalent ligands are advanced cysteine protease inhibitors in the drug development pipeline. K777 is an irreversible inhibitor of cruzain, a necessary enzyme for the survival of the Trypanosoma cruzi ( T. cruzi ) parasite, the causative agent of Chagas disease. Despite their importance, irreversible covalent inhibitors are still often avoided due to the risk of adverse effects. Herein, we replaced the K777 vinyl sulfone group with a nitrile moiety to obtain a reversible covalent inhibitor ( Neq0682 ) of cysteine protease. Then, we used advanced experimental and computational techniques to explore details of the inhibition mechanism of cruzain by reversible and irreversible inhibitors. The isothermal titration calorimetry (ITC) analysis shows that inhibition of cruzain by an irreversible inhibitor is thermodynamically more favorable than by a reversible one. The hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) and Molecular Dynamics (MD) simulations were used to explore the mechanism of the reaction inhibition of cruzain by K777 and Neq0682 . The calculated free energy profiles show that the Cys25 nucleophilic attack and His162 proton transfer occur in a single step for a reversible inhibitor and two steps for an irreversible covalent inhibitor. The hybrid QM/MM calculated free energies for the inhibition reaction correspond to -26.7 and -5.9 kcal mol -1 for K777 and Neq0682 at the MP2/MM level, respectively. These results indicate that the Δ G of the reaction is very negative for the process involving K777 , consequently, the covalent adduct cannot revert to a noncovalent protein-ligand complex, and its binding tends to be irreversible. Overall, the present study provides insights into a covalent inhibition mechanism of cysteine proteases.

The catalytic mechanism of SalL chlorinase has been investigated by combining quantum mechanical/molecular mechanical (QM/MM) techniques and umbrella sampling simulations to compute free energy profiles. Our results shed light on the interesting fact that the substitution of chloride with fluorine in SalL chlorinase leads to a loss of halogenase activity. The potential of mean force based on DFTB3/MM analysis shows that fluorination corresponds to a barrier 13.5 kcal·mol -1 higher than chlorination. Additionally, our results present a molecular description of SalL acting as a chlorinase instead of a methyl-halide transferase.

One tactic for cysteine protease inhibition is to form a covalent bond between an electrophilic atom of the inhibitor and the thiol of the catalytic cysteine. In this study, we evaluate the reaction free energy obtained from a hybrid quantum mechanical/molecular mechanical (QM/MM) free energy profile as a predictor of affinity for reversible, covalent inhibitors of rhodesain. We demonstrate that the reaction free energy calculated with the PM6/MM potential is in agreement with the experimental data and suggest that the free energy profile for covalent bond formation in a protein environment may be a useful tool for the inhibitor design.

Background: Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) is an enzyme that isomerizes phosphorylated serine or threonine motifs adjacent to proline residues. Pin1 has important roles in several cellular signaling pathways, consequently impacting the development of multiple types of cancers., Methods: Based on the previously reported inhibitory activity of pentacyclic triterpenoids isolated from the gum resin of Boswellia genus against Pin1, we designed a computational experiment using molecular docking, pharmacophore filtering, and structural clustering allied to molecular dynamics (MD) simulations and binding free energy calculations to explore the inhibitory activity of new triterpenoids against Pin1 structure., Results: Here, we report different computational evidence that triterpenoids from neem (Azadirachta indica A. Juss), such as 6-deacetylnimbinene, 6-Oacetylnimbandiol, and nimbolide, replicate the binding mode of the Pin1 substrate peptide, interacting with high affinity with the binding site and thus destabilizing the Pin1 structure., Conclusions: Our results are supported by experimental data, and provide interesting structural insights into their molecular mechanism of action, indicating that their structural scaffolds could be used as a start point to develop new inhibitors against Pin1., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Odorant-binding proteins (OBPs) are the main olfactory proteins of mosquitoes, and their structures have been widely explored to develop new repellents. In the present study, we combined ligand- and structure-based virtual screening approaches using as a starting point 1633 compounds from 71 botanical families obtained from the Essential Oil Database (EssOilDB). Using as reference the crystallographic structure of N , N -diethyl- meta -toluamide interacting with the OBP1 homodimer of Anopheles gambiae ( Agam OBP1), we performed a structural and pharmacophoric similarity search to select potential natural products from the library . Thymol acetate, 4-(4-methyl phenyl)-pentanal, thymyl isovalerate, and p -cymen-8-yl demonstrated a favorable chemical correlation with DEET and also had high-affinity interactions with the OBP binding pocket that molecular dynamics simulations showed to be stable. To the best of our knowledge, this is the first study to evaluate on a large scale the potentiality of NPs from essential oils as inhibitors of the mosquito OBP1 using in silico approaches. Our results could facilitate the design of novel repellents with improved selectivity and affinity to the protein binding pocket and can shed light on the mechanism of action of these compounds against insect olfactory recognition., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)

Cancer is one of the leading causes of death worldwide, and the number of patients has only increased each year, despite the considerable efforts and investments in scientific research. Since natural products (NPs) may serve as suitable sources for drug development, the cytotoxicity against cancer cells of 2221 compounds from the Nuclei of Bioassays, Ecophysiology, and Biosynthesis of Natural Products Database (NuBBE DB ) was predicted using CDRUG algorithm. Molecular modeling, chemoinformatics, and chemometric tools were then used to analyze the structural and physicochemical properties of these compounds. We compared the positive NPs with FDA-approved anticancer drugs and predicted the molecular targets involved in the anticancer activity. In the present study, 46 families comprising potential anticancer compounds and at least 19 molecular targets involved in oncogenesis. To the best of our knowledge, this is the first large-scale study conducted to evaluate the potentiality of NPs sourced from Brazilian biodiversity as anticancer agents, using in silico approaches. Our results provided interesting insights about the mechanism of action of these compounds, and also suggested that their structural diversity may aid structure-based optimization strategies for developing novel drugs for cancer therapy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Nitrile reversible covalent inhibitors of human cathepsin L (hCatL) bind covalently to the side chain of the catalytic Cys25 residue in the S1 pocket to form thioimidates. Predicting the binding of reversible covalent inhibitors is essential for their practical application in drug design. In this report, five nitrile-based inhibitors coded Neq0570, Neq0710, Neq0802, Neq0803 and Neq0804 had their hCatL inhibition constants, K i , determined. These analogs of the prototypical Neq0570 are halogenated reversible covalent inhibitors of hCatL, which bear a halogen atom in the meta position of the P3 benzyl ring that can form a halogen bond with the Gly61 of the hCatL. To describe halogen bonding interaction in an inhibitor-hCatL complex, we applied an extra point (EP) of charge to represent the anisotropic distribution of charge on the iodine, bromine and chlorine atoms. Besides, we have used alchemical free energy calculations for evaluating the overall relative binding free energies of these inhibitors using a two-state binding model: noncovalent and covalent bond states. Our results show that free energy perturbation (FEP) can predict the hCatL binding affinities of halogenated reversible covalent inhibitors in close agreement with experiments.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is mainly involved in the regulation of cholesterol biosynthesis. HMGR catalyses the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate at the expense of two NADPH molecules in a two-step reversible reaction. In the present study, we constructed a model of human HMGR ( h HMGR) to explore the conformational changes of HMGR in complex with HMG-CoA and NADPH. In addition, we analysed the complete sequence of the Flap domain using molecular dynamics (MD) simulations and principal component analysis (PCA). The simulations revealed that the Flap domain plays an important role in catalytic site activation and substrate binding. The apo form of h HMGR remained in an open state, while a substrate-induced closure of the Flap domain was observed for holo h HMGR. Our study also demonstrated that the phosphorylation of Ser872 induces significant conformational changes in the Flap domain that lead to a complete closure of the active site, suggesting three principal conformations for the first stage of h HMGR catalysis. Our results were consistent with previous proposed models for the catalytic mechanism of h HMGR. Communicated by Ramaswamy H. Sarma.

The synthase, 3-deoxy-d- manno -octulosonate 8-phosphate (KDO8P), is a key enzyme for the lipopolysaccharide (LPS) biosynthesis of gram-negative bacteria and a potential target for developing new antimicrobial agents. In this study, computational molecular modeling methods were used to determine the complete structure of the KDO8P synthase from Neisseria meningitidis and to investigate the molecular mechanism of its inhibition by three bisphosphate inhibitors: BPH1, BPH2, and BPH3. Our results showed that BPH1 presented a protein-ligand complex with the highest affinity, which is in agreement with experimental data. Furthermore, molecular dynamics (MD) simulations showed that BPH1 is more active due to the many effective interactions, most of which are derived from its phosphoenolpyruvate moiety. Conversely, BPH2 exhibited few hydrogen interactions during the MD simulations with key residues located at the active sites of the KDO8P synthase. In addition, we hydroxylated BPH2 to create the hypothetical molecule named BPH3, to investigate the influence of the hydroxyl groups on the affinity of the bisphosphate inhibitors toward the KDO8P synthase. Overall, we discuss the main interactions between the KDO8P synthase and the bisphosphate inhibitors that are potential starting points for the design of new molecules with significant antibiotic activities.

Mammalian AMP-activated protein kinase (AMPK) is a Ser/Thr protein kinase with a key role as a sensor in cellular energy homeostasis. It has a major role in numerous metabolic disorders, such as type 2 diabetes, obesity, and cancer, and hence it has gained progressive interest as a potential therapeutic target. AMPK is a heterotrimeric enzyme composed by an α-catalytic subunit and two regulatory subunits, β and γ. It is regulated by several mechanisms, including indirect activators such as metformin and direct activators such as compound A-769662. The crystal structure of AMPK bound to A-769662 has been recently reported, suggesting a hypothetical allosteric mechanism of AMPK activation assisted by phosphorylated Ser108 at the β-subunit. Here, we have studied the direct activation mechanism of A-769662 by means of molecular dynamics simulations, suggesting that the activator may act as a glue, coupling the dynamical motion of the β-subunit and the N-terminal domain of the α-subunit, and assisting the preorganization of the ATP-binding site. This is achieved through the formation of an allosteric network that connects the activator and ATP-binding sites, particularly through key interactions formed between αAsp88 and βArg83 and between βpSer108 and αLys29. Overall, these studies shed light into key mechanistic determinants of the allosteric regulation of this cellular energy sensor, and pave the way for the fine-tuning of the rational design of direct activators of this cellular energy sensor.

Activin Receptor-Like Kinase 5 (ALK-5) is related to some types of cancer, such as breast, lung, and pancreas. In this study, we have used molecular docking, molecular dynamics simulations, and free energy calculations in order to explore key interactions between ALK-5 and six bioactive ligands with different ranges of biological activity. The motivation of this work is the lack of crystal structure for inhibitor-protein complexes for this set of ligands. The understanding of the molecular structure and the protein-ligand interaction could give support for the development of new drugs against cancer. The results show that the calculated binding free energy using MM-GBSA, MM-PBSA, and SIE is correlated with experimental data with r 2  = 0.88, 0.80, and 0.94, respectively, which indicates that the calculated binding free energy is in excellent agreement with experimental data. In addition, the results demonstrate that H bonds with Lys232, Glu245, Tyr249, His283, Asp351, and one structural water molecule play an important role for the inhibition of ALK-5. Overall, we discussed the main interactions between ALK-5 and six inhibitors that may be used as starting points for designing new molecules to the treatment of cancer.

Chagas disease affects millions of people in Latin America. This disease is caused by the protozoan parasite Trypanossoma cruzi. The cysteine protease cruzain is a key enzyme for the survival and propagation of this parasite lifecycle. Nitrile-based inhibitors are efficient inhibitors of cruzain that bind by forming a covalent bond with this enzyme. Here, three nitrile-based inhibitors dubbed Neq0409, Neq0410 and Neq0570 were synthesized, and the thermodynamic profile of the bimolecular interaction with cruzain was determined using isothermal titration calorimetry (ITC). The result suggests the inhibition process is enthalpy driven, with a detrimental contribution of entropy. In addition, we have used hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) and Molecular Dynamics (MD) simulations to investigate the reaction mechanism of reversible covalent modification of cruzain by Neq0409, Neq0410 and Neq0570. The computed free energy profile shows that the nucleophilic attack of Cys25 on the carbon C1 of inhibitiors and the proton transfer from His162 to N1 of the dipeptidyl nitrile inhibitor take place in a single step. The calculated free energy of the inhibiton reaction is in agreement with covalent experimental binding. Altogether, the results reported here suggests that nitrile-based inhibitors are good candidates for the development of reversible covalent inhibitors of cruzain and other cysteine proteases.

In Plasmodium falciparum the bifunctional enzyme glucose-6-phosphate dehydrogenase‒6-phosphogluconolactonase (PfG6PD‒6PGL) is involved in the catalysis of the first reaction of the pentose phosphate pathway. Since this enzyme has a key role in parasite development, its unique structure represents a potential target for the discovery of antimalarial drugs. Here we describe the first 3D structural model of the G6PD domain of PfG6PD‒6PGL. Compared to the human enzyme (hG6PD), the 3D model has enabled the identification of a key difference in the substrate-binding site, which involves the replacement of Arg365 in hG6PD by Asp750 in PfG6PD. In a prospective validation of the model, this critical change has been exploited to rationally design a novel family of substrate analog-based inhibitors that can display the necessary selectivity towards PfG6PD. A series of glucose derivatives featuring an α-methoxy group at the anomeric position and different side chains at position 6 bearing distinct basic functionalities has been synthesized, and their PfG6PD and hG6PD inhibitory activities and their toxicity against parasite and mammalian cells have been assessed. Several compounds displayed micromolar affinity (K i up to 23 μM), favorable selectivity (up to > 26-fold), and low cytotoxicity. Phenotypic assays with P. falciparum cultures revealed high micromolar IC 50 values, likely as a result of poor internalization of the compounds in the parasite cell. Overall, these results endorse confidence to the 3D model of PfG6PD, paving the way for the use of target-based drug design approaches in antimalarial drug discovery studies around this promising target., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Inhibition of mushroom tyrosinase was observed with synthetic dihydropyrano[3,2-b]chromenediones. Among them, DHPC04 displayed the most potent tyrosinase inhibitory activity with a K i value of 4 μm, comparable to the reference standard inhibitor kojic acid. A kinetic study suggested that these synthetic heterocyclic compounds behave as competitive inhibitors for the L-DOPA binding site of the enzyme. Furthermore, molecular modeling provided important insight into the mechanism of binding interactions with the tyrosinase copper active site., (© 2017 John Wiley & Sons A/S.)

The Alba superfamily proteins share a common RNA-binding domain. These proteins participate in a variety of regulatory pathways by controlling developmental gene expression. They also interact with ribosomal subunits, translation factors, and other RNA-binding proteins. The Leishmania infantum genome encodes two Alba-domain proteins, LiAlba1 and LiAlba3. In this work, we used homology modeling, protein-protein docking, and molecular dynamics (MD) simulations to explore the details of the Alba1-Alba3-RNA complex from Leishmania infantum at the molecular level. In addition, we compared the structure of LiAlba3 with the human ribonuclease P component, Rpp20. We also mapped the ligand-binding residues on the Alba3 surface to analyze its druggability and performed mutational analyses in Alba3 using alanine scanning to identify residues involved in its function and structural stability. These results suggest that the RGG-box motif of LiAlba1 is important for protein function and stability. Finally, we discuss the function of Alba proteins in the context of pathogen adaptation to host cells. The data provided herein will facilitate further translational research regarding Alba structure and function., (Copyright © 2017 Elsevier B.V. All rights reserved.)