Exploring Novel Antimalarial Compounds Targeting Plasmodium falciparum Enoyl-ACP Reductase: Computational and Experimental Insights.

Malaria, caused by Plasmodium protozoa with Plasmodium falciparum as the most virulent species, continues to pose significant health challenges. Despite the availability of effective antimalarial drugs, the emergence of resistance has heightened the urgency for developing novel therapeutic compounds. In this study, we investigated the enoyl-ACP reductase enzyme of P. falciparum (PfENR) as a promising target for antimalarial drug discovery. Through a comprehensive analysis, we conducted a comparative evaluation of two lead compounds, LD1 (CID: 44405336, lead compounds 1) and LD2 (CID: 72703246, lead compounds 2), obtained from the PubChem/NCBI ligand database, to serve as reference molecules in the identification of potential derivatives using virtual screening assays. Among the newly identified candidates, Ligand 1 (LG1) and Ligand 2 (LG2) exhibited intriguing characteristics and underwent further investigation through docking and molecular dynamics simulations. Ligand 1 (LG1) demonstrated interactions similar to LD1, including hydrogen bonding with Asp218, while Ligand 2 (LG2) displayed superior binding energy comparable to LD1 and LD2, despite lacking hydrogen bonding interactions observed in the control compounds triclosan and its derivative 7-(4-chloro-2-hydroxyphenoxy)-4-methyl-2H-chromen-2-one (CHJ). Following computational validation using the MM/GBSA method to estimate binding free energy, commercially acquired LG1 and LG2 ligands were subjected to in vitro testing. Inhibition assays were performed to evaluate their potential as PfENR inhibitors alongside triclosan as a control compound. LG1 exhibited no inhibitory effects, while LG2 demonstrated inhibitory effects like triclosan. In conclusion, this study contributes valuable insights into developing novel antimalarial drugs by identifying LG2 as a potential ligand and employing a comprehensive approach integrating computational and experimental methodologies.

Differences in Charge Distribution in Leishmania tarentolae Leishmanolysin Result in a Reduced Enzymatic Activity.

Leishmania tarentolae is a non-pathogenic trypanosomatid isolated from lizards widely used for heterologous protein expression and extensively studied to understand the pathogenic mechanisms of leishmaniasis. The repertoire of leishmanolysin genes was reported to be expanded in L. tarentolae genome, but no proteolytic activity was detected. Here, we analyzed L. tarentolae leishmanolysin proteins from the genome to the structural levels and evaluated the enzymatic activity of the wild-type and overexpressing mutants of leishmanolysin. A total of 61 leishmanolysin sequences were retrieved from the L. tarentolae genome. Five of them were selected for phylogenetic analysis, and for three of them, we built 3D models based on the crystallographic structure of L. major ortholog. Molecular dynamics simulations of these models disclosed a less negative electrostatic potential compared to the template. Subsequently, L. major LmjF.10.0460 and L. tarentolae LtaP10.0650 leishmanolysins were cloned in a pLEXSY expression system into L. tarentolae. Proteins from the wild-type and the overexpressing parasites were submitted to enzymatic analysis. Our results revealed that L. tarentolae leishmanolysins harbor a weak enzymatic activity about three times less abundant than L. major leishmanolysin. Our findings strongly suggest that the less negative electrostatic potential of L. tarentolae leishmanolysin can be the reason for the reduced proteolytic activity detected in this parasite.

pH and non-covalent ligand binding modulate Zika virus NS2B/NS3 protease binding site residues: Discoveries from MD and constant pH MD simulations.

Zika virus (ZIKV) is a global health concern and has been linked to severe neurological pathologies. Although no medication is available yet, many efforts to develop antivirals and host cell binding inhibitors led to attractive drug-like scaffolds, mainly targeting the nonstructural NS2B/NS3 protease (NS2B/NS3pro). NS2B/NS3pro active site has several titratable residues susceptible to pH changes and ligand binding; hence, understanding these residues' protonation is essential to drug design efforts targeting the active site. Here we use in silico methods to probe non-covalent binding and its effect on pKa shifts of the active site residues on a ligand-free protease and with a non-peptidic competitive inhibitor (Ki=13.5 µM). By applying constant pH molecular dynamics, we found that the catalytic residues of the unbound NS2B/NS3pro achieved the protonation needed for the serine protease mechanism over the pH value of 8.5. Nevertheless, the protease in the holo state achieved this same scenario at lower pH values. Also, non-covalent binding affected the catalytic triad (H51, D75, and S135) by stabilizing their distances and interaction network. Thus, NS2B/NS3pro residues configuration for activity might be both pH-dependent and influenced by ligand binding. However, compound presence within the binding site destabilized the NS2B, interfering with the closed and active conformation necessary for substrate binding and catalysis. Our outcomes provide valuable insights into non-covalent inhibitor behavior and its effect on protease active site residues, impacting optimization and design of novel compounds. Communicated by Ramaswamy H. Sarma.

Guidelines To Predict Binding Poses of Antibody-Integrin Complexes.

Integrins are cell adhesion receptors that transmit bidirectional signals across the plasma membrane. They are noncovalently linked heterodimeric molecules consisting of two subunits and act as biomarkers in several pathologies. Thus, according to the increase of therapeutic antibody production, some efforts have been applied to produce anti-integrin antibodies. Here, we purposed to evaluate methods of generation and identification of the binding pose of integrin-antibody complexes, through protein-protein docking and molecular dynamics simulations, and propose a strategy to assure the confidence of the final model and avoid false-positive poses. The results show that ClusPro and GRAMM-X were the best programs to generate the native pose of integrin-antibody complexes. Furthermore, we were able to recover and to ensure that the selected pose is the native one by using a simple rule. All complexes from ClusPro in which the first model had the lowest energy, at least 5% more negative than the second one, were correctly predicted. Therefore, our methodology seems to be efficient to avoid misranking of wrong poses for integrin-antibody complexes. In cases where the rule is inconclusive, we proposed the use of heated molecular dynamics to identify the native pose characterized by RMSDi <0.5 nm. We believe that the set of methods presented here helps in the rational design of anti-integrin antibodies, giving some insights on the development of new biopharmaceuticals.

Identification of Novel and Recurrent RMRP Variants in a Series of Brazilian Patients with Cartilage-Hair Hypoplasia: McKusick Syndrome.

Cartilage-hair hypoplasia syndrome (CHH) is an autosomal recessive disorder caused by pathogenic variants of the RMRP gene and characterized by metaphyseal bone dysplasia associated with hypotrichosis, immunodeficiency, and predisposition to malignancy. However, the genotype-phenotype correlation in CHH is not well understood. Here, we report a single country cohort of 23 Brazilian patients with clinical and radiological features consistent with CHH. We found 23 different pathogenic variants in the RMRP gene - 12 novel and 11 previously described in the literature. Interestingly, the most frequent Finnish pathogenic variant related to CHH (g.71A>G) was not found in our cohort. In contrast, more than 50% of the patients carried the rare g.196C>T variant suggesting a possible founder effect in the Brazilian population. In silico analysis showed that pathogenic variants occurred either in the regions conserved in mammalian species or within essential domains for the ribonucleoprotein complex. Pathogenicity prediction studies can improve the understanding of how these variants affect RNA.

Studying effects of different protonation states of His11 and His102 in ribose-5-phosphate isomerase of Trypanosoma cruzi: an example of cooperative behavior.

The Trypanosoma cruzi ribose-5-phosphate isomerase B (TcRpiB) is a crucial piece in the pentose phosphate pathway and thus is a potential drug target for treatment of Chagas' disease. TcRpiB residues, such as Cys69, Asp45, Glu149 and Pro47, have confirmed their roles in substrate recognition, catalytic reaction and binding site conformation. However, the joint performance of His11 and His102, in the D-ribose-5-phosphate (R5P) in the catalysis is not well understood. In this work, we probed the influence of different protonation states of His11 and His102 on the behavior of the ligand R5P using molecular dynamics simulations, network analysis and thermodynamic integration. Simulations revealed that a protonated His11 combined with a neutral His102 (His11+‒His102) was able to stabilize the ligand R5P in the binding site. Moreover, calculated relative free energy differences showed that when protonated His11 was coupled to a neutral His102 an exergonic process takes place. On the other hand, neutral His11 combined with a protonated His102 (His11‒His102+), sampled conformations that resembled the catalyzed product D-ribulose-5-phosphate (Ru5P). Network analysis also demonstrated some peculiarities for these systems with some negatively correlated nodes in the binding site for His11‒His102+, and exclusive suboptimal paths for His11+‒His102. Therefore, the combined approach presented in this paper proposes two suitable protonation states for the TcRpiB catalytic mechanism, where an extra proton in either histidines might favor R5P binding or influence isomerization reaction to Ru5P. Our results may guide further in silico drug discovery studies. Communicated by Ramaswamy H. Sarma.

Integrating Molecular Docking and Molecular Dynamics Simulations.

Computational methods, applied at the early stages of the drug design process, use current technology to provide valuable insights into the understanding of chemical systems in a virtual manner, complementing experimental analysis. Molecular docking is an in silico method employed to foresee binding modes of small compounds or macromolecules in contact with a receptor and to predict their molecular interactions. Moreover, the methodology opens up the possibility of ranking these compounds according to a hierarchy determined using particular scoring functions. Docking protocols assign many approximations, and most of them lack receptor flexibility. Therefore, the reliability of the resulting protein-ligand complexes is uncertain. The association with the costly but more accurate MD techniques provides significant complementary with docking. MD simulations can be used before docking since a series of "new" and broader protein conformations can be extracted from the processing of the resulting trajectory and employed as targets for docking. They also can be utilized a posteriori to optimize the structures of the final complexes from docking, calculate more detailed interaction energies, and provide information about the ligand binding mechanism. Here, we focus on protocols that offer the docking-MD combination as a logical approach to improving the drug discovery process.

Benzimidazole inhibitors of the major cysteine protease of Trypanosoma brucei.

Aim: Limitations in available therapies for trypanosomiases indicate the need for improved medicines. Cysteine proteases cruzain and rhodesain are validated targets for treatment of Chagas disease and human African trypanosomiasis. Previous studies reported a benzimidazole series as potent cruzain inhibitors. Results & methodology: Considering the high similarity between these proteases, we evaluated 40 benzimidazoles against rhodesain. We describe their structure-activity relationships (SAR), revealing trends similar to those observed for cruzain and features that lead to enzyme selectivity. This series comprises noncovalent competitive inhibitors (best Ki = 0.21 µM against rhodesain) and micromolar activity against Trypanosoma brucei brucei. A cheminformatics analysis confirms scaffold novelty, and the inhibitors described have favorable predicted physicochemical properties. Conclusion: Our results support this series as a starting point for new human African trypanosomiasis medicines.

The role of hydrophobicity in the cold denaturation of proteins under high pressure: A study on apomyoglobin.

An exciting debate arises when microscopic mechanisms involved in the denaturation of proteins at high pressures are explained. In particular, the issue emerges when the hydrophobic effect is invoked, given that hydrophobicity cannot elucidate by itself the volume changes measured during protein unfolding. In this work, we study by the use of molecular dynamics simulations and essential dynamics analysis the relation between the solvation dynamics, volume, and water structure when apomyoglobin is subjected to a hydrostatic pressure regime. Accordingly, the mechanism of cold denaturation of proteins under high-pressure can be related to the disruption of the hydrogen-bond network of water favoring the coexistence of two states, low-density and high-density water, which directly implies in the formation of a molten globule once the threshold of 200 MPa has been overcome.

Thiophenacetamide as a potential modulator to NF-κB: structure and dynamics study using in silico and molecular biology assays.

Nuclear factor kappa B (NF-κB) plays critical roles in the regulation of many pathophysiological processes, including inflammation and immune responses, cell growth and apoptosis. This DNA-binding protein receptor is considered an important molecular target to treat many diseases through host-directed therapy. In this line, several drugs containing thiophene cores have been extensively evaluated due to their ability to interfere on NF-κB translocation to the nucleus. In this work, assays using drug affinity responsive target stability (DARTS) revealed that the parent compound N-(Aryl)-2-thiophen-2-ylacetamide referred to as thiophenacetamide (TAA) specifically binds to the p65 subunit of the NF-κB. Since no experimental binding mode of TAA with p65 is available, we explored TAA within putative sites in silico to gain insights into its possible binding mode and behavior. The binding mode of TAA found in Site 1 formed hydrogen bonds with Lys37 and Asp125 on p65, important residues near DNA-binding region. Molecular dynamics simulations showed the stability of this mode of binding in contrast to the other also tested modes. Our results suggest that TAA binding could occur in regions close to residues responsible for DNA binding, increasing NF-κB protein rigidity and affecting the association between DNA and NF-κB. Communicated by Ramaswamy H. Sarma.

Understanding Structure-Activity Relationships for Trypanosomal Cysteine Protease Inhibitors by Simulations and Free Energy Calculations.

The protozoan cysteine proteases cruzain in Trypanosoma cruzi and rhodesain in Trypanosoma brucei are therapeutic targets for Chagas disease and Human African Trypanosomiasis (HAT), respectively. A benzimidazole series was previously characterized as potent noncovalent competitive cruzain and rhodesain inhibitors with activity against trypanosomes. Common structure-activity relationships (SAR) trends and structural modifications leading to selectivity against each enzyme were described. However, some of these trends could not be understood based on the reported binding mode of lead compound 1. Therefore, we employed microsecond molecular dynamics simulations and free energy calculations to understand qualitative SAR trends and to quantitatively recapitulate them. Simulations revealed the most stable protein-ligand interactions and provided insights concerning enzyme selectivity. Calculated relative binding free energies of compound 1 analogs exhibited deviations of 1.1 and 2.2 kcal/mol from the experimental values for cruzain and rhodesain, respectively. These data encourage prospective thermodynamic integration (TI) studies to optimize this series and facilitate the prioritization of compounds for synthesis.

Antitumor Effect of Pomolic Acid in Acute Myeloid Leukemia Cells Involves Cell Death, Decreased Cell Growth and Topoisomerases Inhibition.

BACKGROUND: Acute myeloid leukemia (AML) represents the largest number of annual deaths from hematologic malignancy. In the United States, it was estimated that 21.380 individuals would be diagnosed with AML and 49.5% of patients would die in 2017. Therefore, the search for novel compounds capable of increasing the overall survival rate to the treatment of AML cells is urgent. OBJECTIVES: To investigate the cytotoxicity effect of the natural compound pomolic acid (PA) and to explore the mechanism of action of PA in AML cell lines with different phenotypes. METHODS: Three different AML cell lines, HL60, U937 and Kasumi-1 cells with different mechanisms of resistance were used to analyze the effect of PA on the cell cycle progression, on DNA intercalation and on human DNA topoisomerases (hTopo I and IIα) in vitro studies. Theoretical experiments of the inhibition of hTopo I and IIα were done to explore the binding modes of PA. RESULTS: PA reduced cell viability, induced cell death, increased sub-G0/G1 accumulation and activated caspases pathway in all cell lines, altered the cell cycle distribution and inhibited the catalytic activity of both human DNA topoisomerases. CONCLUSION: Finally, this study showed that PA has powerful antitumor activity against AML cells, suggesting that this natural compound might be a potent antineoplastic agent to improve the treatment scheme of this neoplasm.

Pressure effect on micellization of non-ionic surfactant Triton X-100.

Micellar aggregates can be arranged in new types of conformational assemblies when they are isotropically compressed. Thus, the pressure effects in the underlying fundamental interactions leading to self-assembly of micellar aggregates can be represented by changes in the phase boundaries with increasing pressure. In this paper, we have employed molecular dynamics simulations to study the self-assembly of micelles composed of the non-ionic surfactant Triton X-100 at the atomic scale, monitoring the changes in the solvation dynamics when the micelles are subjected to a wide range of hydrostatic pressures. The computational molecular model was capable of self-assembling and forming a non-ionic micelle, which subsequently was coupled to a high-pressure barostat producing a geometric transition of the micelle due to changes in the solvation dynamics. Accordingly, under a high pressure regime, the hydrogen bonds are redistributed, the water density is modified, and water acts as an unstructured liquid, capable of penetrating into the micelle.

Using RNA Sequence and Structure for the Prediction of Riboswitch Aptamer: A Comprehensive Review of Available Software and Tools.

RNA molecules are essential players in many fundamental biological processes. Prokaryotes and eukaryotes have distinct RNA classes with specific structural features and functional roles. Computational prediction of protein structures is a research field in which high confidence three-dimensional protein models can be proposed based on the sequence alignment between target and templates. However, to date, only a few approaches have been developed for the computational prediction of RNA structures. Similar to proteins, RNA structures may be altered due to the interaction with various ligands, including proteins, other RNAs, and metabolites. A riboswitch is a molecular mechanism, found in the three kingdoms of life, in which the RNA structure is modified by the binding of a metabolite. It can regulate multiple gene expression mechanisms, such as transcription, translation initiation, and mRNA splicing and processing. Due to their nature, these entities also act on the regulation of gene expression and detection of small metabolites and have the potential to helping in the discovery of new classes of antimicrobial agents. In this review, we describe software and web servers currently available for riboswitch aptamer identification and secondary and tertiary structure prediction, including applications.

Essential dynamics of the cold denaturation: pressure and temperature effects in yeast frataxin.

The cold denaturation of globular proteins is a process that can be caused by increasing pressure or decreasing the temperature. Currently, the action mechanism of this process has not been clearly understood, raising an interesting debate on the matter. We have studied the process of cold denaturation using molecular dynamics simulations of the frataxin system Yfh1, which has a dynamic experimental characterization of unfolding at low and high temperatures. The frataxin model here studied allows a comparative analysis using experimental data. Furthermore, we monitored the cold denaturation process of frataxin and also investigated the effect under the high-pressure regime. For a better understanding of the dynamics and structural properties of the cold denaturation, we also analyzed the MD trajectories using essentials dynamic. The results indicate that changes in the structure of water by the effect of pressure and low temperatures destabilize the hydrophobic interaction modifying the solvation and the system volume leading to protein denaturation. Proteins 2016; 85:125-136. © 2016 Wiley Periodicals, Inc.

In Silico Identification of Novel APRIL Peptide Antagonists and Binding Insights by Molecular Modeling and Immunosorbent Assays.

The "A proliferation inducing ligand" protein (APRIL) is a cytokine over-expressed in many transformed and tumoral cells acting onto two distinct receptors of the Tumoral Necrosis Factor B cell maturation antigen (BCMA) and the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). We herein describe, through a detailed computational approach, the molecular interactions between TACI and its ligands APRIL and another structurally similar protein called B-cell activating factor (BAFF) by means of molecular dynamics. Dynamical analysis suggests R84 and D85 residues from TACI as possible mutation candidates, yielding increased affinity between TACI and APRIL. The association of computational simulations, site directed mutagenesis and peptide design could be a powerful tool, driving to better in vitro experiments. Our results contribute to the elucidation of APRIL signaling and help clarify the effects of blocking interaction between APRIL and its receptors through the use of particular peptides.

Dynamic identification of H2 epitopes from Leishmania (Leishmania) amazonensis cysteine proteinase B with potential immune activity during murine infection.

Peptides from the COOH-terminal extension of cysteine proteinase B from Leishmania (Leishmania) amazonensis (cyspep) can modulate immune responses in vertebrate hosts. With this hypothesis as base, we used the online analysis tool SYFPEITHI to predict seven epitopes from this region with potential to bind H2 proteins. We performed proliferation tests and quantified reactive T lymphocytes applying a cytometry analysis, using samples from draining lymph node of lesions from L. (L.) amazonensis-infected mice. To define reactivity of T cells, we used complexes of DimerX (H2 D(b):Ig and H2 L(d):Ig) and the putative epitopes. Additionally, we applied surface plasmon resonance to verify real time interactions between the putative epitopes and DimerX proteins. Five peptides induced blastogenesis in BALB/c cells, while only two presented the same property in C57BL/6 mouse cells. In addition, our data indicate the existence of CD8+ T lymphocyte populations able to recognize each tested peptide in both murine strains. We observed an overlapping of results between the peptides that induced lymphocyte proliferation and those capable of binding to the DimerX in the surface plasmon resonance assays thus indicating that using these recombinant proteins in biosensing analyses is a promising tool to study real time molecular interactions in the context of major histocompatibility complex epitopes. The data gathered in this study reinforce the hypothesis that cyspep-derived peptides are important factors in the murine host infection by L. (L.) amazonensis.

Naturally occurring genetic variants of human caspase-1 differ considerably in structure and the ability to activate interleukin-1ß.

Caspase-1 (Interleukin-1 Converting Enzyme, ICE) is a proinflammatory enzyme that plays pivotal roles in innate immunity and many inflammatory conditions such as periodic fever syndromes and gout. Inflammation is often mediated by enzymatic activation of interleukin (IL)-1ß and IL-18. We detected seven naturally occurring human CASP1 variants with different effects on protein structure, expression, and enzymatic activity. Most mutations destabilized the caspase-1 dimer interface as revealed by crystal structure analysis and homology modeling followed by molecular dynamics simulations. All variants demonstrated decreased or absent enzymatic and IL-1ß releasing activity in vitro, in a cell transfection model, and as low as 25% of normal ex vivo in a whole blood assay of samples taken from subjects with variant CASP1, a subset of whom suffered from unclassified autoinflammation. We conclude that decreased enzymatic activity of caspase-1 is compatible with normal life and does not prevent moderate and severe autoinflammation.

Computational modeling of the bHLH domain of the transcription factor TWIST1 and R118C, S144R and K145E mutants.

BACKGROUND: Human TWIST1 is a highly conserved member of the regulatory basic helix-loop-helix (bHLH) transcription factors. TWIST1 forms homo- or heterodimers with E-box proteins, such as E2A (isoforms E12 and E47), MYOD and HAND2. Haploinsufficiency germ-line mutations of the twist1 gene in humans are the main cause of Saethre-Chotzen syndrome (SCS), which is characterized by limb abnormalities and premature fusion of cranial sutures. Because of the importance of TWIST1 in the regulation of embryonic development and its relationship with SCS, along with the lack of an experimentally solved 3D structure, we performed comparative modeling for the TWIST1 bHLH region arranged into wild-type homodimers and heterodimers with E47. In addition, three mutations that promote DNA binding failure (R118C, S144R and K145E) were studied on the TWIST1 monomer. We also explored the behavior of the mutant forms in aqueous solution using molecular dynamics (MD) simulations, focusing on the structural changes of the wild-type versus mutant dimers. RESULTS: The solvent-accessible surface area of the homodimers was smaller on wild-type dimers, which indicates that the cleft between the monomers remained more open on the mutant homodimers. RMSD and RMSF analyses indicated that mutated dimers presented values that were higher than those for the wild-type dimers. For a more careful investigation, the monomer was subdivided into four regions: basic, helix I, loop and helix II. The basic domain presented a higher flexibility in all of the parameters that were analyzed, and the mutant dimer basic domains presented values that were higher than the wild-type dimers. The essential dynamic analysis also indicated a higher collective motion for the basic domain. CONCLUSIONS: Our results suggest the mutations studied turned the dimers into more unstable structures with a wider cleft, which may be a reason for the loss of DNA binding capacity observed for in vitro circumstances.

Structure-based functional inference of hypothetical proteins from Mycoplasma hyopneumoniae.

Enzootic pneumonia caused by Mycoplasma hyopneumoniae is a major constraint to efficient pork production throughout the world. This pathogen has a small genome with 716 coding sequences, of which 418 are homologous to proteins with known functions. However, almost 42% of the 716 coding sequences are annotated as hypothetical proteins. Alternative methodologies such as threading and comparative modeling can be used to predict structures and functions of such hypothetical proteins. Often, these alternative methods can answer questions about the properties of a model system faster than experiments. In this study, we predicted the structures of seven proteins annotated as hypothetical in M. hyopneumoniae, using the structure-based approaches mentioned above. Three proteins were predicted to be involved in metabolic processes, two proteins in transcription and two proteins where no function could be assigned. However, the modeled structures of the last two proteins suggested experimental designs to identify their functions. Our findings are important in diminishing the gap between the lack of annotation of important metabolic pathways and the great number of hypothetical proteins in the M. hyopneumoniae genome.