Genetic diversity and natural selection of Plasmodium vivax reticulocyte invasion genes in Ecuador.

BACKGROUND: Knowledge of the diversity of invasion ligands in malaria parasites in endemic regions is essential to understand how natural selection influences genetic diversity of these ligands and their feasibility as possible targets for future vaccine development. In this study the diversity of four genes for merozoite invasion ligands was studied in Ecuadorian isolates of Plasmodium vivax. METHODS: Eighty-eight samples from P. vivax infected individuals from the Coast and Amazon region of Ecuador were obtained between 2012 and 2015. The merozoite invasion genes pvmsp-1-19, pvdbpII, pvrbp1a-2 and pvama1 were amplified, sequenced, and compared to the Sal-1 strain. Polymorphisms were mapped and genetic relationships between haplotypes were determined. RESULTS: Only one nonsynonymous polymorphism was detected in pvmsp-1-19, while 44 nonsynonymous polymorphisms were detected in pvdbpII, 56 in pvrbp1a-2 and 33 in pvama1. While haplotypes appeared to be more related within each area of study and there was less relationship between parasites of the coastal and Amazon regions of the country, diversification processes were observed in the two Amazon regions. The highest haplotypic diversity for most genes occurred in the East Amazon of the country. The high diversity observed in Ecuadorian samples is closer to Brazilian and Venezuelan isolates, but lower than reported in other endemic regions. In addition, departure from neutrality was observed in Ecuadorian pvama1. Polymorphisms for pvdbpII and pvama1 were associated to B-cell epitopes. CONCLUSIONS: pvdbpII and pvama1 genetic diversity found in Ecuadorian P. vivax was very similar to that encountered in other malaria endemic countries with varying transmission levels and segregated by geographic region. The highest diversity of P. vivax invasion genes in Ecuador was found in the Amazonian region. Although selection appeared to have small effect on pvdbpII and pvrbp1a-2, pvama1 was influenced by significant balancing selection.

Enzyme Inhibitors from Gorgonians and Soft Corals.

For decades, gorgonians and soft corals have been considered promising sources of bioactive compounds, attracting the interest of scientists from different fields. As the most abundant bioactive compounds within these organisms, terpenoids, steroids, and alkaloids have received the highest coverage in the scientific literature. However, enzyme inhibitors, a functional class of bioactive compounds with high potential for industry and biomedicine, have received much less notoriety. Thus, we revised scientific literature (1974-2022) on the field of marine natural products searching for enzyme inhibitors isolated from these taxonomic groups. In this review, we present representative enzyme inhibitors from an enzymological perspective, highlighting, when available, data on specific targets, structures, potencies, mechanisms of inhibition, and physiological roles for these molecules. As most of the characterization studies for the new inhibitors remain incomplete, we also included a methodological section presenting a general strategy to face this goal by accomplishing STRENDA (Standards for Reporting Enzymology Data) project guidelines.

Canonical or noncanonical? Structural plasticity of serine protease-binding loops in Kunitz-STI protease inhibitors.

The Kunitz-Soybean Trypsin Inhibitor (Kunitz-STI) family is a large family of proteins with most of its members being protease inhibitors. The versatility of the inhibitory profile and the structural plasticity of these proteins, make this family a promising scaffold for designing new multifunctional proteins. Historically, Kunitz-STI inhibitors have been classified as canonical serine protease inhibitors, but new inhibitors with novel inhibition mechanisms have been described in recent years. Different inhibition mechanisms could be the result of different evolutionary pathways. In the present work, we performed a structural analysis of all the crystallographic structures available for Kunitz-STI inhibitors to characterize serine protease-binding loop structural features and locations. Our study suggests a relationship between the conformation of serine protease-binding loops and the inhibition mechanism, their location in the ß-trefoil fold, and the plant source of the inhibitors. The classical canonical inhibitors of this family are restricted to plants from the Fabales order and bind their targets via the ß4-ß5 loop, whereas serine protease-binding loops in inhibitors from other plants lie mainly in the ß5-ß6 and ß9-ß10 loops. In addition, we found that the ß5-ß6 loop is used to inhibit two different families of serine proteases through a steric blockade inhibition mechanism. This work will help to change the general perception that all Kunitz-STI inhibitors are canonical inhibitors and proteins with protease-binding loops adopting noncanonical conformations are exceptions. Additionally, our results will help in the identification of protease-binding loops in uncharacterized or newly discovered inhibitors, and in the design of multifunctional proteins.

Critical Review of Plant-Derived Compounds as Possible Inhibitors of SARS-CoV-2 Proteases: A Comparison with Experimentally Validated Molecules.

Ever since coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, was declared a pandemic on March 11, 2020, by the WHO, a concerted effort has been made to find compounds capable of acting on the virus and preventing its replication. In this context, researchers have refocused part of their attention on certain natural compounds that have shown promising effects on the virus. Considering the importance of this topic in the current context, this study aimed to present a critical review and analysis of the main reports of plant-derived compounds as possible inhibitors of the two SARS-CoV-2 proteases: main protease (Mpro) and Papain-like protease (PLpro). From the search in the PubMed database, a total of 165 published articles were found that met the search patterns. A total of 590 unique molecules were identified from a total of 122 articles as potential protease inhibitors. At the same time, 114 molecules reported as natural products and with annotation of theoretical support and antiviral effects were extracted from the COVID-19 Help database. After combining the molecules extracted from articles and those obtained from the database, we identified 648 unique molecules predicted as potential inhibitors of Mpro and/or PLpro. According to our results, several of the predicted compounds with higher theoretical confidence are present in many plants used in traditional medicine and even food, such as flavonoids, carboxylic acids, phenolic acids, triterpenes, terpenes phytosterols, and triterpenoids. These are potential inhibitors of Mpro and PLpro. Although the predictions of several molecules against SARS-CoV-2 are promising, little experimental information was found regarding certain families of compounds. Only 45 out of the 648 unique molecules have experimental data validating them as inhibitors of Mpro or PLpro, with the most frequent scaffold present in these 45 compounds being the flavone. The novelty of this work lies in the analysis of the structural diversity of the chemical space among the molecules predicted as inhibitors of SARS-CoV-2 Mpro and PLpro proteases and the comparison to those molecules experimentally validated. This work emphasizes the need for experimental validation of certain families of compounds, preferentially combining classical enzymatic assays with interaction-based methods. Furthermore, we recommend checking the presence of Pan-Assay Interference Compounds (PAINS) and the presence of molecules previously reported as inhibitors of Mpro or PLpro to optimize resources and time in the discovery of new SARS-CoV-2 antivirals from plant-derived molecules.

The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach.

The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. Understanding and manipulating protein stability provide crucial information on the molecular determinants that modulate structure and stability, and expand the applications of de novo proteins. Since the (ß/⍺)8-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel, and subjected them to a thorough folding analysis. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design.

Analysis of organizational power networks through a holistic approach using consensus strategies.

Power is one of the most complex organizational attributes to understand due to the multiple related variables and dimensions in which it appears. The ownership and use and of power are reflected in the interpersonal relationships within an organization, as a result, modeling its structure and interactions can lead to knowledge about the power networks that shape it. The objective of this study was to identify the behavior of organizational networks based on existing sources of power, using a consensual analysis of the different topologies present in these networks. The study was carried out in a private production company in Ecuador, which has representation at a domestic level. To this end, a 12-question personalized questionnaire was designed with the aim of identifying specific networks and was applied to 1190 workers in the company. The results were obtained using organizational network analysis and a consensus strategy to integrate the centralities found in multiple networks into one. This study can serve as a reference to organizations, so they can know the relationships between people within it, as part of their management process. In this way, the identification of people within power networks is useful for knowing the "key" actors in the promotion of organizational changes, as well as for the development of career plans based on the position that people occupy in the organizational system.

Identification of Tight-Binding Plasmepsin II and Falcipain 2 Inhibitors in Aqueous Extracts of Marine Invertebrates by the Combination of Enzymatic and Interaction-Based Assays.

Natural products from marine origin constitute a very promising and underexplored source of interesting compounds for modern biotechnological and pharmaceutical industries. However, their evaluation is quite challenging and requires specifically designed assays to reliably identify the compounds of interest in a highly heterogeneous and interfering context. In the present study, we describe a general strategy for the confident identification of tight-binding protease inhibitors in the aqueous extracts of 62 Cuban marine invertebrates, using Plasmodium falciparum hemoglobinases Plasmepsin II and Falcipain 2 as model enzymes. To this end, we first developed a screening strategy that combined enzymatic with interaction-based assays and then validated screening conditions using five reference extracts. Interferences were evaluated and minimized. The results from the massive screening of such extracts, the validation of several hits by a variety of interaction-based assays and the purification and functional characterization of PhPI, a multifunctional and reversible tight-binding inhibitor for Plasmepsin II and Falcipain 2 from the gorgonian Plexaura homomalla, are presented.

Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical ß-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop.

Predicting functional residues of the Solanum lycopersicum aspartic protease inhibitor (SLAPI) by combining sequence and structural analysis with molecular docking.

The Solanum lycopersicum aspartic protease inhibitor (SLAPI), which belongs to the STI-Kunitz family, is an effective inhibitor of the aspartic proteases human cathepsin D and Saccharomyces proteinase A. However, in contrast with the large number of studies on the inhibition mechanism of the serine proteases by the STI-Kunitz inhibitors, the structural aspects of the inhibition mechanism of aspartic proteases from this family of inhibitors are poorly understood. In the present study, we have combined sequence and structural analysis methods with protein-protein docking to gain a better understanding of the SLAPI inhibition mechanism of the proteinase A. The results suggest that: i) SLAPI loop L9 may be involved in the inhibitor interaction with the proteinase A´s active site, and ii) the residues I144, V148, L149, P151, F152 and R154 are implicated in the difference in the potency shown previously by SLAPI and another STI-Kunitz inhibitor isolated from Solanum tuberosum to inhibit proteinase A. These results will be useful in the design of site directed mutagenesis experiments to understand more thoroughly the aspartic protease inhibition mechanism of SLAPI and other related STI-Kunitz inhibitors.

Generation of an affinity matrix useful in the purification of natural inhibitors of plasmepsin II, an antimalarial-drug target.

An affinity matrix containing the antimalarial drug target Plm II (plasmepsin II) as ligand was generated. This enzyme belongs to the family of Plasmodium (malarial parasite) aspartic proteinases, known as Plms (plasmepsins). The procedure established to obtain the support has two steps: the immobilization of the recombinant proenzyme of Plm II to NHS (N-hydroxysuccinimide)-activated Sepharose and the activation of the immobilized enzyme by incubation at pH 4.4 and 37 degrees C. The coupling reaction resulted in a high percentage immobilization (95.5%), and the matrices obtained had an average of 4.3 mg of protein/ml of gel. The activated matrices, but not the inactive ones, were able to hydrolyse two different chromogenic peptide substrates and haemoglobin. This ability was completely blocked by the addition of the general aspartic-proteinase inhibitor, pepstatin A, to the reaction mixture. The matrices were useful in the affinity purification of the Plm II inhibitory activity detected in marine invertebrates, such as Xestospongia muta (giant barrel sponge) and the gorgonian (sea-fan coral) Plexaura homomalla (black sea rod), with increases of 10.2- and 5.9-fold in the specific inhibitory activity respectively. The preliminary K(i) values obtained, 46.4 nM (X. muta) and 1.9 nM (P. homomalla), and the concave shapes of the inhibition curves reveal that molecules are reversible tight-binding inhibitors of Plm II. These results validated the use of the affinity matrix for the purification of Plm II inhibitors from complex mixtures and established the presence of Plm II inhibitors in some marine invertebrates.