KCNQ1 p.D446E Variant as a Risk Allele for Arrhythmogenic Phenotypes: Electrophysiological Characterization Reveals a Complex Phenotype Affecting the Slow Delayed Rectifier Potassium Current (IKs) Voltage Dependence by Causing a Hyperpolarizing Shift and a Lack of Response to Protein Kinase A Activation.

Genetic testing is crucial in inherited arrhythmogenic channelopathies; however, the clinical interpretation of genetic variants remains challenging. Incomplete penetrance, oligogenic, polygenic or multifactorial forms of channelopathies further complicate variant interpretation. We identified the KCNQ1/p.D446E variant in 2/63 patients with long QT syndrome, 30-fold more frequent than in public databases. We thus characterized the biophysical phenotypes of wildtype and mutant IKs co-expressing these alleles with the ß-subunit minK in HEK293 cells. KCNQ1 p.446E homozygosity significantly shifted IKs voltage dependence to hyperpolarizing potentials in basal conditions (gain of function) but failed to shift voltage dependence to hyperpolarizing potentials (loss of function) in the presence of 8Br-cAMP, a protein kinase A activator. Basal IKs activation kinetics did not differ among genotypes, but in response to 8Br-cAMP, IKs 446 E/E (homozygous) activation kinetics were slower at the most positive potentials. Protein modeling predicted a slower transition of the 446E Kv7.1 tetrameric channel to the stabilized open state. In conclusion, biophysical and modelling evidence shows that the KCNQ1 p.D446E variant has complex functional consequences including both gain and loss of function, suggesting a contribution to the pathogenesis of arrhythmogenic phenotypes as a functional risk allele.

Synergistic effect of compounds directed to triosephosphate isomerase, a combination to develop drug against trichomoniasis.

The development of new drugs is continuous in the world; currently, saving resources (both economic ones and time) and preventing secondary effects have become a necessity for drug developers. Trichomoniasis is the most common nonviral sexually transmitted infection affecting more than 270 million people around the world. In our research group, we focussed on developing a selective and more effective drug against Trichomonas vaginalis, and we previously reported on a compound, called A4, which had a trichomonacidal effect. Later, we determined another compound, called D4, which also had a trichomonacidal effect together with favorable toxicity results. Both A4 and D4 are directed at the enzyme triosephosphate isomerase. Thus, we made combinations between the two compounds, in which we determined a synergistic effect against T. vaginalis, determining the IC50 and the toxicity of the best relationship to obtain the trichomonacidal effect. With these results, we can propose a combination of compounds that represents a promising alternative for the development of a new therapeutic strategy against trichomoniasis.

Leukocyte Nuclear Morphology Alterations in Dilated Cardiomyopathy Caused by a Lamin AC Truncating Mutation (LMNA/Ser431*) Are Modified by the Presence of a LAP2 Missense Polymorphism (TMPO/Arg690Cys).

The clinical phenotype of LMNA-associated dilated cardiomyopathy (DCM) varies even among individuals who share the same mutation. LMNA encodes lamin AC, which interacts with the lamin-associated protein 2 alpha (LAP2α) encoded by the TMPO gene. The LAP2α/Arg690Cys polymorphism is frequent in Latin America and was previously found to disrupt LAP2α-Lamin AC interactions in vitro. We identified a DCM patient heterozygous for both a lamin AC truncating mutation (Ser431*) and the LAP2α/Arg690Cys polymorphism. We performed protein modeling and docking experiments, and used confocal microscopy to compare leukocyte nuclear morphology among family members with different genotype combinations (wild type, LAP2α Arg690Cys heterozygous, lamin AC/Ser431* heterozygous, and LAP2α Arg690Cys/lamin AC Ser431* double heterozygous). Protein modeling predicted that 690Cys destabilizes the LAP2α homodimer and impairs lamin AC-LAP2α docking. Lamin AC-deficient nuclei (Ser431* heterozygous) showed characteristic blebs and invaginations, significantly decreased nuclear area, and increased elongation, while LAP2α/Arg690Cys heterozygous nuclei showed a lower perimeter and higher circularity than wild-type nuclei. LAP2α Arg690Cys apparently attenuated the effect of LMNA Ser431* on the nuclear area and fully compensated for its effect on nuclear circularity. Altogether, the data suggest that LAP2α/Arg690Cys may be one of the many factors contributing to phenotype variation of LMNA-associated DCM.

Laccases: structure, function, and potential application in water bioremediation.

The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochemical properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.

KRas4B-PDE6δ complex stabilization by small molecules obtained by virtual screening affects Ras signaling in pancreatic cancer.

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

The small organic molecule C19 binds and strengthens the KRAS4b-PDEδ complex and inhibits growth of colorectal cancer cells in vitro and in vivo.

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

A new nucleocytoplasmic RhoGAP protein contributes to control the pathogenicity of Entamoeba histolytica by regulating EhRacC and EhRacD activity.

Small GTPases are signalling molecules that regulate important cellular processes. GTPases are deactivated by GTPase-activating proteins (GAPs). While human GAPs have been intensively studied, no GAP has yet been characterized in Entamoeba histolytica. In this study, we identified and characterized a novel nucleocytoplasmic RhoGAP in E. histolytica termed EhRhoGAPnc. In silico analyses of the domain structure revealed a previously undescribed peptide region within the carboxy-terminal region of EhRhoGAPnc capable of interacting with phosphatidic acid and phosphatidylinositol 3,5-bisphosphate. The full structural GAP domain showed increase GAP activity compared with the minimum region able to display GAP activity, as analysed both by experimental assays and molecular dynamics simulations. Furthermore, we identified amino acid residues that promote interactions between EhRhoGAPnc and its target GTPases EhRacC and EhRacD. Immunofluorescence studies revealed that EhRhoGAPnc colocalized with EhRacC and EhRacD during uroid formation but not during erythrophagocytosis. Interestingly, during erythrophagocytosis of red blood cells, EhRhoGAPnc colocalized with phosphatidic acid and phosphatidylinositol 3,5-bisphosphate. Overexpression of EhRhoGAPnc in E. histolytica led to inhibition of actin adhesion plate formation, migration, adhesion of E. histolytica to MDCK cells and consequently to an impairment of the cytopathic activity.

Discovery of Entamoeba histolytica hexokinase 1 inhibitors through homology modeling and virtual screening.

Entamoeba histolytica, the parasite which causes amebiasis is responsible for 110,000 deaths a year. Entamoeba histolytica depends on glycolysis to obtain ATP for cellular work. According to metabolic flux studies, hexokinase exerts the highest flux control of this metabolic pathway; therefore, it is an excellent target in the search of new antiamebic drugs. To this end, a tridimensional model of E. histolytica hexokinase 1 (EhHK1) was constructed and validated by homology modeling. After virtual screening of 14,400 small molecules, the 100 with the best docking scores were selected, purchased and assessed in their inhibitory capacity. The results showed that three molecules (compounds 2921, 11275 and 2755) inhibited EhHK1 with an I50 of 48, 91 and 96 µM, respectively. Thus, we found the first inhibitors of EhHK1 that can be used in the search of new chemotherapeutic agents against amebiasis.

Inhibition and biochemical characterization of methicillin-resistant Staphylococcus aureus shikimate dehydrogenase: an in silico and kinetic study.

Methicillin-resistant Staphylococcus auerus (MRSA) strains are having a major impact worldwide, and due to their resistance to all ß-lactams, an urgent need for new drugs is emerging. In this regard, the shikimate pathway is considered to be one of the metabolic features of bacteria and is absent in humans. Therefore enzymes involved in this route, such as shikimate dehydrogenase (SDH), are considered excellent targets for discovery of novel antibacterial drugs. In this study, the SDH from MRSA (SaSDH) was characterized. The results showed that the enzyme is a monomer with a molecular weight of 29 kDa, an optimum temperature of 65 °C, and a maximal pH range of 9-11 for its activity. Kinetic studies revealed that SDH showed Michaelis-Menten kinetics toward both substrates (shikimate and NADP+). Initial velocity analysis suggested that SaSDH catalysis followed a sequential random mechanism. Additionally, a tridimensional model of SaSDH was obtained by homology modeling and validated. Through virtual screening three inhibitors of SaSDH were found (compounds 238, 766 and 894) and their inhibition constants and mechanism were obtained. Flexible docking studies revealed that these molecules make interactions with catalytic residues. The data of this study could serve as starting point in the search of new chemotherapeutic agents against MRSA.

Antineoplastic effect of compounds C14 and P8 on TNBC and radioresistant TNBC cells by stabilizing the K-Ras4BG13D/PDE6δ complex.

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

Hen lysozyme fibrillogenesis, molten globule intermediate and effect of copper salts.

The amyloid fibres have been related to many diseases. The molten globule intermediate has been proposed to form part of the folding pathway of many proteins. In the present study, we investigated the mechanism of amyloid-fibres formation of hen egg-white lysozyme (HEWL) incubated in a potassium phosphate buffer, pH 11.8, 100 mM, at 37 °C for 30 h, and evaluated the influence of Cu(II) present in two salts (CuSO4 and CuCl2) during fibrillogenesis. Co-incubation and post-incubation of lysozyme with copper salts reduced the fluorescence signal of thioflavin T with an increment in the intrinsic fluorescence of the protein. The ANS fluorescence test showed that incubation of HEWL for 6 h generated a molten globule intermediate state that formed amyloid fibres when incubation was carried out for a 30-h timespan. Dynamic light scattering showed a heterogeneous population of states in samples incubated in the absence or the presence of salts during the fibrillation process. The existence of a reducing potential was verified during the formation of HEWL amyloid fibres with the bathocuproine disulphonate test. Transmission electron microscopy confirmed the presence and absence of fibres in solutions incubated with and without Cu(II). This work demonstrated that lysozyme formed amyloid fibres at 37 °C and copper inhibited its formation.Communicated by Ramaswamy H. Sarma.

Synergistic effect of antagonists to KRas4B/PDE6 molecular complex in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among all human cancers as it is highly resistant to chemotherapy. K-Ras mutations usually trigger the development and progression of PDAC. We hypothesized that compounds stabilizing the KRas4B/PDE6δ complex could serve as PDAC treatments. Using in silico approaches, we identified the small molecules C14 and P8 that reduced K-Ras activation in primary PDAC cells. Importantly, C14 and P8 significantly prevented tumor growth in patient-derived xenotransplants. Combined treatment with C14 and P8 strongly increased cytotoxicity in PDAC cell lines and primary cultures and showed strong synergistic antineoplastic effects in preclinical murine PDAC models that were superior to conventional therapeutics without causing side effects. Mechanistically, C14 and P8 reduced tumor growth by inhibiting AKT and ERK signaling downstream of K-RAS leading to apoptosis, specifically in PDAC cells. Thus, combined treatment with C14 and P8 may be a superior pharmaceutical strategy to improve the outcome of PDAC.

Consequences of two naturally occurring missense mutations in the structure and function of Bruton agammaglobulinemia tyrosine kinase.

Bruton agammaglobulinemia tyrosine kinase (BTK) is a key protein in the B-cell receptor (BCR) signaling pathway and plays an essential role in the differentiation of B lymphocytes. X-linked agammaglobulinemia (XLA) is a primary humoral immunodeficiency caused by mutations in the gene encoding BTK. Previously, we identified two novel variations, L111P and E605G, in BTK; these are localized within the pleckstrin homology and Src homology 1 domains, respectively. In the present study, we evaluated the potential effects of these variations on the structural conformation and the function of BTK. Using in silico methods, we found that the L111P and E650G variations are not located directly in protein-protein interfaces but close to them. They distorted the native structural conformation of the BTK protein, affecting not only its geometry and stability but also its ability for protein recognition and in consequence its functionality. To confirm the results of the in silico assays, WT BTK, L111P, and E650G variants were expressed in the BTK-deficient DT40 cell line. The mutant proteins exhibited an absence of catalytic activity, aberrant redistribution after BCR-crosslinking, and deficient intracellular calcium mobilization. This work demonstrates that L111 and E605 residues are fundamental for the activation and function of BTK.

Conformational dynamics of L-lysine, L-arginine, L-ornithine binding protein reveals ligand-dependent plasticity.

The molecular basis of multiple ligand binding affinity for amino acids in periplasmic binding proteins (PBPs) and in the homologous domain for class C G-protein coupled receptors is an unsolved question. Here, using unrestrained molecular dynamic simulations, we studied the ligand binding mechanism present in the L-lysine, L-arginine, L-ornithine binding protein. We developed an analysis based on dihedral angles for the description of the conformational changes upon ligand binding. This analysis has an excellent correlation with each of the two main movements described by principal component analysis (PCA) and it's more convenient than RMSD measurements to describe the differences in the conformational ensembles observed. Furthermore, an analysis of hydrogen bonds showed specific interactions for each ligand studied as well as the ligand interaction with the aromatic residues Tyr-14 and Phe-52. Using uncharged histidine tautomers, these interactions are not observed. On the basis of these results, we propose a model in which hydrogen bond interactions place the ligand in the correct orientation to induce a cation-π interaction with Tyr-14 and Phe-52 thereby stabilizing the closed state. Our results also show that this protein adopts slightly different closed conformations to make available specific hydrogen bond interactions for each ligand thus, allowing a single mechanism to attain multiple ligand specificity. These results shed light on the experimental evidence for ligand-dependent conformational plasticity not explained by the previous crystallographic data.

Triosephosphate isomerase as a therapeutic target against trichomoniasis.

Trichomoniasis is the most common non-viral sexually transmitted infection, caused by the protozoan parasite Trichomonas vaginalis, affecting millions of people worldwide. The main treatment against trichomoniasis is metronidazole and other nitroimidazole derivatives, but up to twenty percent of clinical cases of trichomoniasis are resistant to these drugs. In this study, we used high-performance virtual screening to search for molecules that specifically bind to the protein, triosephosphate isomerase from T. vaginalis (TvTIM). By in silico molecular docking analysis, we selected six compounds from a chemical library of almost 500,000 compounds. While none of the six inhibited the enzymatic activity of recombinant triosephosphate isomerase isoforms, one compound (A4; 3,3'-{[4-(4-morpholinyl)phenyl]methylene}bis(4- hydroxy-2H-chromen-2-one) altered their fluorescence emission spectra, suggesting that this chemical might interfere in an important non-glycolytic function of TvTIM. In vitro assays demonstrate that A4 is not cytotoxic but does have trichomonacidal impact on T. vaginalis cultures. With these results, we propose this compound as a potential drug with a new therapeutic target against Trichomonas vaginalis.

Design of thermostable beta-propeller phytases with activity over a broad range of pHs and their overproduction by Pichia pastoris.

Thermostable phytases, which are active over broad pH ranges, may be useful as feed additives, since they can resist the temperatures used in the feed-pelleting process. We designed new beta-propeller phytases, using a structure-guided consensus approach, from a set of amino acid sequences from Bacillus phytases and engineered Pichia pastoris strains to overproduce the enzymes. The recombinant phytases were N-glycosylated, had the correct amino-terminal sequence, showed activity over a pH range of 2.5 to 9, showed a high residual activity after 10 min of heat treatment at 80°C and pH 5.5 or 7.5, and were more thermostable at pH 7.5 than a recombinant form of phytase C from Bacillus subtilis (GenBank accession no. AAC31775). A structural analysis suggested that the higher thermostability may be due to a larger number of hydrogen bonds and to the presence of P257 in a surface loop. In addition, D336 likely plays an important role in the thermostability of the phytases at pH 7.5. The recombinant phytases showed higher thermostability at pH 5.5 than at pH 7.5. This difference was likely due to a different protein total charge at pH 5.5 from that at pH 7.5. The recombinant beta-propeller phytases described here may have potential as feed additives and in the pretreatment of vegetable flours used as ingredients in animal diets.

A plasmid encoding parts of the dengue virus E and NS1 proteins induces an immune response in a mouse model.

A DENV-2 plasmid named pEII*EIII/NS1*,containing sequences encoding portions of the envelope protein that are potentially involved in the induction of neutralizing antibodies and a portion of the NS1 sequence that is involved in protection, is reported in this work. The synthesized subunit protein was recognized by human sera from infected patients and had the predicted size. The immunogenicity of this construct was evaluated using a mouse model in a prime-boost vaccination approach. The priming was performed using the plasmid pEII*EIII/NS1*, followed by a boost with recombinant full-length GST-E and GST-NS1 fusion proteins. The mice showed specific antibody responses to the E and NS1 proteins, as detected by ELISA, compared to the response of animals vaccinated with the parental plasmid. Interestingly, some animals had neutralizing antibodies. These results show that EII*, EIII and NS1* sequences could be considered for the design ofa recombinant subunit vaccine against dengue disease.

Enthalpically-driven ligand recognition and cavity solvation of bovine odorant binding protein.

Lipocalins are a widely distributed family of extracellular proteins typically involved in the transport of small hydrophobic molecules. To gain new insights into the molecular basis that governs ligand recognition by this ancient protein family, the binding properties of the domain-swapped dimer bovine odorant binding protein (bOBP) and its monomeric mutant bOBP121G+ were characterized using calorimetric techniques and molecular dynamics simulations. Thermal unfolding profiles revealed that the isolated bOBP subunits behave as a cooperative folding unit. In addition, bOBP and bOBP121G+ exhibited similar ligand binding properties, characterized by a non-classical hydrophobic effect signature. The energetic differences in the binding of bOBP to 1-hexen-3-ol and the physiological ligand 1-octen-3-ol were strikingly larger than those observed for the interaction of other lipocalins with congeneric ligands. MD simulations revealed that the recurrent opening of transient pores in the submicrosecond timescale allows a profuse exchange of water molecules between the protein interior and the surrounding solvent. This picture contrasts with other lipocalins whose ligand-free binding cavities are devoid of solvent molecules. Furthermore, the simulations indicated that internal water molecules solvate the protein cavity suboptimally, forming fewer hydrogen bonds and having lower density and higher potential energy than bulk water molecules. Upon ligand occupation, water molecules were displaced from the binding cavity in an amount that depended on the ligand size. Taken together, calorimetric and MD-simulation results are consistent with a significant contribution of cavity desolvation to the enthalpically-driven interaction of bOBP with its hydrophobic ligands.

Engineering and directed evolution of a Ca2+ binding site A-deficient AprE mutant reveal an essential contribution of the loop Leu75-Leu82 to enzyme activity.

An aprE mutant from B. subtilis 168 lacking the connecting loop Leu(75)-Leu(82) which is predicted to encode a Ca(2+) binding site was constructed. Expression of the mutant gene (aprEDeltaLeu(75)-Leu(82)) produced B. subtilis colonies lacking protease activity. Intrinsic fluorescence analysis revealed spectral differences between wild-type AprE and AprEDeltaL(75)-L(82). An AprEDeltaL(75)-L(82) variant with reestablished enzyme activity was selected by directed evolution. The novel mutations Thr(66)Met/Gly(102)Asp located in positions which are predicted to be important for catalytic activity were identified in this variant. Although these mutations restored hydrolysis, they had no effect with respect to thermal inactivation of AprEDeltaL(75)-L(82) T(66)M G(102)D. These results support the proposal that in addition to function as a calcium binding site, the loop that connects beta-sheet e3 with alpha-helix c plays a structural role on enzyme activity of AprE from B. subtilis 168.

Selective inactivation of triosephosphate isomerase from Trypanosoma cruzi by brevifolin carboxylate derivatives isolated from Geranium bellum Rose.

In the search of molecules that can serve as leads in the design of a new drug for the treatment of Chagas' disease, we found that some brevifolin carboxylate derivatives isolated from Geranium bellum Rose, inactivate triosephosphate isomerase from Trypanosoma cruzi (TcTIM) in a species-specific manner. After spectroscopic characterization, these compounds were identified as methylbrevifolin carboxylate (1), ethylbrevifolin carboxylate (2), butylbrevifolin carboxylate (3) and the methylated derivate methyl tri-O-methylbrevifolin carboxylate (4). The concentrations required to inactivate fifty percent the activity of TcTIM were 6.5, 8 and 14 microM of 1, 2 and 3, respectively, while compound 4 had no inhibitory effect. Molecular docking simulations of 1 on the structure of TcTIM showed that residues of both monomers interact with the compound. These compounds are very selective with respect to the parasite enzyme, since they showed no effect on the activity of human TIM at concentrations as high as 1mM. In conclusion, the brevifolin carboxylate derivatives described here are excellent leads in the search of a new chemotherapy for the treatment of this disease.