Characterisation and Outcomes of Patients with Solid Organ Malignancies Admitted to the Intensive Care Unit: Mortality and Impact on Functional Status and Oncological Treatment.

BACKGROUND: Despite the increasing number of ICU admissions among patients with solid tumours, there is a lack of tools with which to identify patients who may benefit from critical support. We aim to characterize the clinical profile and outcomes of patients with solid malignancies admitted to the ICU. METHODS: Retrospective observational study of patients with cancer non-electively admitted to the ICU of the Hospital Clinic of Barcelona (Spain) between January 2019 and December 2019. Data regarding patient and neoplasm characteristics, ICU admission features and outcomes were collected from medical records. RESULTS: 97 ICU admissions of 84 patients were analysed. Lung cancer (22.6%) was the most frequent neoplasm. Most of the patients had metastatic disease (79.5%) and were receiving oncological treatment (75%). The main reason for ICU admission was respiratory failure (38%). Intra-ICU and in-hospital mortality rates were 9.4% and 24%, respectively. Mortality rates at 1, 3 and 6 months were 19.6%, 36.1% and 53.6%. Liver metastasis, gastrointestinal cancer, hypoalbuminemia, elevated basal C-reactive protein, ECOG-PS greater than 2 at ICU admission, admission from ward and an APACHE II score over 14 were related to higher mortality. Functional status was severely affected at discharge, and oncological treatment was definitively discontinued in 40% of the patients. CONCLUSION: Medium-term mortality and functional deterioration of patients with solid cancers non-electively admitted to the ICU are high. Surrogate markers of cachexia, liver metastasis and poor ECOG-PS at ICU admission are risk factors for mortality.

Benzimidazole Derivatives as New and Selective Inhibitors of Arginase from Leishmania mexicana with Biological Activity against Promastigotes and Amastigotes.

Leishmaniasis is a disease caused by parasites of the Leishmania genus that affects 98 countries worldwide, 2 million of new cases occur each year and more than 350 million people are at risk. The use of the actual treatments is limited due to toxicity concerns and the apparition of resistance strains. Therefore, there is an urgent necessity to find new drugs for the treatment of this disease. In this context, enzymes from the polyamine biosynthesis pathway, such as arginase, have been considered a good target. In the present work, a chemical library of benzimidazole derivatives was studied performing computational, enzyme kinetics, biological activity, and cytotoxic effect characterization, as well as in silico ADME-Tox predictions, to find new inhibitors for arginase from Leishmania mexicana (LmARG). The results show that the two most potent inhibitors (compounds 1 and 2) have an I50 values of 52 µM and 82 µM, respectively. Moreover, assays with human arginase 1 (HsARG) show that both compounds are selective for LmARG. According to molecular dynamics simulation studies these inhibitors interact with important residues for enzyme catalysis. Biological activity assays demonstrate that both compounds have activity against promastigote and amastigote, and low cytotoxic effect in murine macrophages. Finally, in silico prediction of their ADME-Tox properties suggest that these inhibitors support the characteristics to be considered drug candidates. Altogether, the results reported in our study suggest that the benzimidazole derivatives are an excellent starting point for design new drugs against leishmanisis.

Design, synthesis, kinetic, molecular dynamics, and hypoglycemic effect characterization of new and potential selective benzimidazole derivatives as Protein Tyrosine Phosphatase 1B inhibitors.

Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling pathway and has been validated as a therapeutic target for type 2 diabetes. A wide variety of scaffolds have been included in the structure of PTP1B inhibitors, one of them is the benzimidazole nucleus. Here, we report the design and synthesis of a new series of di- and tri- substituted benzimidazole derivatives including their kinetic and structural characterization as PTP1B inhibitors and hypoglycemic activity. Results show that compounds 43, 44, 45, and 46 are complete mixed type inhibitors with a Ki of 12.6 µM for the most potent (46). SAR type analysis indicates that a chloro substituent at position 6(5), a ß-naphthyloxy at position 5(6), and a p-benzoic acid attached to the linker 2-thioacetamido at position 2 of the benzimidazole nucleus, was the best combination for PTP1B inhibition and hypoglycemic activity. In addition, molecular dynamics studies suggest that these compounds could be potential selective inhibitors from other PTPs such as its closest homologous TCPTP, SHP-1, SHP-2 and CDC25B. Therefore, the compounds reported here are good hits that provide structural, kinetic, and biological information that can be used to develop novel and selective PTP1B inhibitors based on benzimidazole scaffold.

De novo assembly of the freshwater prawn Macrobrachium carcinus brain transcriptome for identification of potential targets for antibody development.

Crustaceans are major constituents of aquatic ecosystems and, as such, changes in their behavior and the structure and function of their bodies can serve as indicators of alterations in their immediate environment, such as those associated with climate change and anthropogenic contamination. We have used bioinformatics and a de novo transcriptome assembly approach to identify potential targets for developing specific antibodies to serve as nervous system function markers for freshwater prawns of the Macrobrachium spp. Total RNA was extracted from brain ganglia of Macrobrachium carcinus freshwater prawns and Illumina Next Generation Sequencing was performed using an Eel Pond mRNA Seq Protocol to construct a de novo transcriptome. Sequencing yielded 97,202,662 sequences: 47,630,546 paired and 1,941,570 singletons. Assembly with Trinity resulted in 197,898 assembled contigs from which 30,576 were annotated: 9,600 by orthology, 17,197 by homology, and 3,779 by transcript families. We looked for glutamate receptors contigs, due to their main role in crustacean excitatory neurotransmission, and found 138 contigs related to ionotropic receptors, 32 related to metabotropic receptors, and 18 to unidentified receptors. After performing multiple sequence alignments within different biological organisms and antigenicity analysis, we were able to develop antibodies for prawn AMPA ionotropic glutamate receptor 1, metabotropic glutamate receptor 1 and 4, and ionotropic NMDA glutamate receptor subunit 2B, with the expectation that the availability of these antibodies will help broaden knowledge regarding the underlying structural and functional mechanisms involved in prawn behavioral responses to environmental impacts. The Macrobrachium carcinus brain transcriptome can be an important tool for examining changes in many other nervous system molecules as a function of developmental stages, or in response to particular conditions or treatments.

Structure-based identification of a potential non-catalytic binding site for rational drug design in the fructose 1,6-biphosphate aldolase from Giardia lamblia.

Giardia lamblia is the causal agent of giardiasis, one of the most prevalent parasitosis in the world. Even though effective pharmacotherapies against this parasite are available, the disadvantages associated with its use call for the development of new antigiardial compounds. Based on the Giardia dependence on glycolysis as a main energy source, glycolytic enzymes appear to be attractive targets with antiparasitic potential. Among these, fructose 1,6-biphosphate aldolase (GlFBPA) has been highlighted as a promising target for drug design. Current efforts are based on the design of competitive inhibitors of GlFBPA; however, in the kinetic context of metabolic pathways, competitive inhibitors seem to have low potential as therapeutic agents. In this work, we performed an experimental and in silico structure-based approach to propose a non-catalytic binding site which could be used as a hot spot for antigardial drug design. The druggability of the selected binding site was experimentally tested; the alteration of the selected region by site directed mutagenesis disturbs the catalytic properties and the stability of the enzyme. A computational automated search of binding sites supported the potential of this region as functionally relevant. A preliminary docking study was performed, in order to explore the feasibility and type of molecules to be able to accommodate in the proposed binding region. Altogether, the results validate the proposed region as a specific molecular binding site with pharmacological potential.

Cuidados de Enfermería en pacientes con ventilación mecánica invasiva en la Unidad de Cuidados Intensivos Pediátricos / Nursing Care in patients with invasive mechanical ventilation in the Pediatric Intensive Care Unit

La rehabilitación cardiometabólica es un programa multifactorial útil para el tratamiento de las principales patologías que tienen una relación directa con el sedentarismo, como patologías cardiovasculares, metabólicas y sus complicaciones. En mayo de 2004, durante la 57ª Asamblea Mundial de la Salud, se aprobó "La estrategia mundial de la Organización Mundial de la Salud (OMS) que aborda sobre el régimen alimentario, la actividad física y la salud" con la finalidad de disminuir la inactividad física, los malos hábitos de alimentación que de forma directa llevan al exceso de peso, y que están asociadas al riesgo cardiovascular (RCV), a las enfermedades cardiovasculares, a la diabetes mellitus (DM) del tipo 2 y a otras diferentes enfermedades degenerativas crónicas. En base a la cual se orienta a implementar una correcta prescripción del ejercicio tanto preventiva como terapéutica. Este protocolo de atención clínico-terapeútico va dirigido a profesionales de la salud para el control de enfermedades cardiometabólicas. Los profesionales sanitarios podrán ofertar una terapéutica integral a sus pacientes para dosificar el ejercicio de forma personalizada, a partir del estado de salud, conocimiento de patologías preexistentes, edad y condición física inicial del paciente, así como proporcionar un seguimiento mediante programas individualizados. Cuyo principal fin es mejorar la condición cardiovascular, evitar nuevas complicaciones y reducir la mortalidad.

Cardiometabolic rehabilitation is a multifactorial program useful for the treatment of the main pathologies that have a direct relationship with sedentary lifestyle, such as cardiovascular, metabolic pathologies and their complications. In May 2004, during the 57th World Health Assembly, "The World Strategy of the World Health Organization (WHO) that addresses the diet, physical activity and health" was approved in order to reduce the physical inactivity, poor eating habits that directly lead to excess weight, and that are associated with cardiovascular risk (CVR), cardiovascular diseases, diabetes mellitus type 2 and other different chronic degenerative diseases . Based on which it is oriented to implement a correct prescription of both preventive and therapeutic exercise.This protocol of clinical-therapeutic care is aimed at health professionals for the control of cardiometabolic diseases. Health professionals may offer a comprehensive therapy to their patients to dose the exercise in a personalized way, based on the state of health, knowledge of preexisting pathologies, age and initial physical condition of the patient, as well as providing follow-up through individualized programs. Whose main purpose is to improve the cardiovascular condition, avoid new complications and reduce mortality.

Decontamination Strategies and Bloodstream Infections With Antibiotic-Resistant Microorganisms in Ventilated Patients: A Randomized Clinical Trial.

Importance: The effects of chlorhexidine (CHX) mouthwash, selective oropharyngeal decontamination (SOD), and selective digestive tract decontamination (SDD) on patient outcomes in ICUs with moderate to high levels of antibiotic resistance are unknown. Objective: To determine associations between CHX 2%, SOD, and SDD and the occurrence of ICU-acquired bloodstream infections with multidrug-resistant gram-negative bacteria (MDRGNB) and 28-day mortality in ICUs with moderate to high levels of antibiotic resistance. Design, Setting, and Participants: Randomized trial conducted from December 1, 2013, to May 31, 2017, in 13 European ICUs where at least 5% of bloodstream infections are caused by extended-spectrum ß-lactamase-producing Enterobacteriaceae. Patients with anticipated mechanical ventilation of more than 24 hours were eligible. The final date of follow-up was September 20, 2017. Interventions: Standard care was daily CHX 2% body washings and a hand hygiene improvement program. Following a baseline period from 6 to 14 months, each ICU was assigned in random order to 3 separate 6-month intervention periods with either CHX 2% mouthwash, SOD (mouthpaste with colistin, tobramycin, and nystatin), or SDD (the same mouthpaste and gastrointestinal suspension with the same antibiotics), all applied 4 times daily. Main Outcomes and Measures: The occurrence of ICU-acquired bloodstream infection with MDRGNB (primary outcome) and 28-day mortality (secondary outcome) during each intervention period compared with the baseline period. Results: A total of 8665 patients (median age, 64.1 years; 5561 men [64.2%]) were included in the study (2251, 2108, 2224, and 2082 in the baseline, CHX, SOD, and SDD periods, respectively). ICU-acquired bloodstream infection with MDRGNB occurred among 144 patients (154 episodes) in 2.1%, 1.8%, 1.5%, and 1.2% of included patients during the baseline, CHX, SOD, and SDD periods, respectively. Absolute risk reductions were 0.3% (95% CI, -0.6% to 1.1%), 0.6% (95% CI, -0.2% to 1.4%), and 0.8% (95% CI, 0.1% to 1.6%) for CHX, SOD, and SDD, respectively, compared with baseline. Adjusted hazard ratios were 1.13 (95% CI, 0.68-1.88), 0.89 (95% CI, 0.55-1.45), and 0.70 (95% CI, 0.43-1.14) during the CHX, SOD, and SDD periods, respectively, vs baseline. Crude mortality risks on day 28 were 31.9%, 32.9%, 32.4%, and 34.1% during the baseline, CHX, SOD, and SDD periods, respectively. Adjusted odds ratios for 28-day mortality were 1.07 (95% CI, 0.86-1.32), 1.05 (95% CI, 0.85-1.29), and 1.03 (95% CI, 0.80-1.32) for CHX, SOD, and SDD, respectively, vs baseline. Conclusions and Relevance: Among patients receiving mechanical ventilation in ICUs with moderate to high antibiotic resistance prevalence, use of CHX mouthwash, SOD, or SDD was not associated with reductions in ICU-acquired bloodstream infections caused by MDRGNB compared with standard care. Trial Registration: ClinicalTrials.gov Identifier: NCT02208154.

Disulfiram as a novel inactivator of Giardia lamblia triosephosphate isomerase with antigiardial potential.

Giardiasis, the infestation of the intestinal tract by Giardia lamblia, is one of the most prevalent parasitosis worldwide. Even though effective therapies exist for it, the problems associated with its use indicate that new therapeutic options are needed. It has been shown that disulfiram eradicates trophozoites in vitro and is effective in vivo in a murine model of giardiasis; disulfiram inactivation of carbamate kinase by chemical modification of an active site cysteine has been proposed as the drug mechanism of action. The triosephosphate isomerase from G. lamblia (GlTIM) has been proposed as a plausible target for the development of novel antigiardial pharmacotherapies, and chemical modification of its cysteine 222 (C222) by thiol-reactive compounds is evidenced to inactivate the enzyme. Since disulfiram is a cysteine modifying agent and GlTIM can be inactivated by modification of C222, in this work we tested the effect of disulfiram over the recombinant and trophozoite-endogenous GlTIM. The results show that disulfiram inactivates GlTIM by modification of its C222. The inactivation is species-specific since disulfiram does not affect the human homologue enzyme. Disulfiram inactivation induces only minor conformational changes in the enzyme, but substantially decreases its stability. Recombinant and endogenous GlTIM inactivates similarly, indicating that the recombinant protein resembles the natural enzyme. Disulfiram induces loss of trophozoites viability and inactivation of intracellular GlTIM at similar rates, suggesting that both processes may be related. It is plausible that the giardicidal effect of disulfiram involves the inactivation of more than a single enzyme, thus increasing its potential for repurposing it as an antigiardial drug.

Effect of prucalopride on intestinal gas tolerance in patients with functional bowel disorders and constipation.

BACKGROUND AND AIM: Patients with functional bowel disorders develop gas retention and symptoms in response to intestinal gas loads that are well tolerated by healthy subjects. Stimulation of 5HT-4 receptors in the gut has both prokinetic and antinociceptive effects. The aim of this study is to determine the effect of prucalopride, a highly selective 5HT-4 agonist, on gas transit and tolerance in women with functional bowel disorders complaining of constipation. METHODS: Twenty-four women with functional bowel disorders complaining of constipation were included in the study. Patients were studied twice on separate days in a cross-over design. On each study day, an intestinal gas challenge test was performed. During the five previous days, prucalopride (2 mg/day) or placebo was administered. Abdominal symptoms, stool frequency, and stool consistency were recorded during the treatment period on daily questionnaires. RESULTS: During the gas challenge test, prucalopride did not decrease the volume of gas retained in the subset of patients who had significant gas retention (≥ 200 mL) while on placebo. However, in those patients who had increased symptoms during the gas test (≥ 3 on a 0 to 6 scale) when on placebo, prucalopride did significantly reduce the perception of symptoms (2.3 ± 0.5 mean score vs 3.5 ± 0.3 on placebo; P = 0.045). During the treatment period with prucalopride, patients exhibited an increase in the total number of bowel movements and decreased stool consistency compared with placebo. CONCLUSION: Prucalopride reduces abdominal symptoms without modifying gas retention when patients with functional bowel disorders are challenged with the gas transit and tolerance test. European Clinical Trials Database (EudraCT2011-006354-86).

Effect of Chicory-derived Inulin on Abdominal Sensations and Bowel Motor Function.

GOAL: To determine the effect of a prebiotic chicory-derived inulin-type fructan on the tolerance of intestinal gas. BACKGROUND: Subjects with gas-related complaints exhibit impaired handling of intestinal gas loads and we hypothesized that inulin would have a beneficial effect. STUDY: Placebo-controlled, parallel, randomized, double-blind trial. Subjects with abdominal symptoms and reduced tolerance of intestinal gas (selected by a pretest) received either inulin (8 g/d, n=18) or maltodextrin as a placebo (8 g/d, n=18) for 4 weeks. A gas challenge test (4 h jejunal gas infusion at 12 mL/min while measuring abdominal symptoms and gas retention for 3 h) was performed before and at the end of the intervention phase. Gastrointestinal symptoms and bowel habits (using daily questionnaires for 1 wk) and fecal bifidobacteria counts were measured before and at the end of the intervention. RESULTS: Inulin decreased gas retention during the gas challenge test (by 22%; P=0.035 vs. baseline), while the placebo did not, but the intergroup difference was not statistically significant (P=0.343). Inulin and placebo reduced the perception of abdominal sensations in the gas challenge test to a similar extent (by 52% and 43%, respectively). Participants reported moderate gastrointestinal symptoms and normal bowel habits during baseline examination, and these findings remained unchanged in both groups during the intervention. Inulin led to a higher relative abundance of bifidobacteria counts (P=0.01 vs. placebo). CONCLUSIONS: A daily dose of inulin that promotes bifidobacteria growth and may improve gut function, is well tolerated by subjects with gastrointestinal complaints.

Cloning, Expression and Characterization of Recombinant, NADH Oxidase from Giardia lamblia.

The NADH oxidase family of enzymes catalyzes the oxidation of NADH by reducing molecular O2 to H2O2, H2O or both. In the protozoan parasite Giardia lamblia, the NADH oxidase enzyme (GlNOX) produces H2O as end product without production of H2O2. GlNOX has been implicated in the parasite metabolism, the intracellular redox regulation and the resistance to drugs currently used against giardiasis; therefore, it is an interesting protein from diverse perspectives. In this work, the GlNOX gene was amplified from genomic G. lamblia DNA and expressed in Escherichia coli as a His-Tagged protein; then, the enzyme was purified by immobilized metal affinity chromatography, characterized, and its properties compared with those of the endogenous enzyme previously isolated from trophozoites (Brown et al. in Eur J Biochem 241(1):155-161, 1996). In comparison with the trophozoite-extracted enzyme, which was scarce and unstable, the recombinant heterologous expression system and one-step purification method produce a stable protein preparation with high yield and purity. The recombinant enzyme mostly resembles the endogenous protein; where differences were found, these were attributable to methodological discrepancies or artifacts. This homogenous, pure and functional protein preparation can be used for detailed structural or functional studies of GlNOX, which will provide a deeper understanding of the biology and pathogeny of G. lamblia.

Reduction of butyrate- and methane-producing microorganisms in patients with Irritable Bowel Syndrome.

The pathophysiology of irritable bowel syndrome (IBS) remains unclear. Here we investigated the microbiome of a large cohort of patients to identify specific signatures for IBS subtypes. We examined the microbiome of 113 patients with IBS and 66 healthy controls. A subset of these participants provided two samples one month apart. We analyzed a total of 273 fecal samples, generating more than 20 million 16S rRNA sequences. In patients with IBS, a significantly lower microbial diversity was associated with a lower relative abundance of butyrate-producing bacteria (P = 0.002; q < 0.06), in particular in patients with IBS-D and IBS-M. IBS patients who did not receive any treatment harboured a lower abundance of Methanobacteria compared to healthy controls (P = 0.005; q = 0.05). Furthermore, significant correlations were observed between several bacterial taxa and sensation of flatulence and abdominal pain (P < 0.05). Altogether, our findings showed that IBS-M and IBS-D patients are characterized by a reduction of butyrate producing bacteria, known to improve intestinal barrier function, and a reduction of methane producing microorganisms a major mechanism of hydrogen disposal in the human colon, which could explain excess of abdominal gas in IBS.

Classification of functional bowel disorders by objective physiological criteria based on endoluminal image analysis.

We have previously developed an original method to evaluate small bowel motor function based on computer vision analysis of endoluminal images obtained by capsule endoscopy. Our aim was to demonstrate intestinal motor abnormalities in patients with functional bowel disorders by endoluminal vision analysis. Patients with functional bowel disorders (n = 205) and healthy subjects (n = 136) ingested the endoscopic capsule (Pillcam-SB2, Given-Imaging) after overnight fast and 45 min after gastric exit of the capsule a liquid meal (300 ml, 1 kcal/ml) was administered. Endoluminal image analysis was performed by computer vision and machine learning techniques to define the normal range and to identify clusters of abnormal function. After training the algorithm, we used 196 patients and 48 healthy subjects, completely naive, as test set. In the test set, 51 patients (26%) were detected outside the normal range (P < 0.001 vs. 3 healthy subjects) and clustered into hypo- and hyperdynamic subgroups compared with healthy subjects. Patients with hypodynamic behavior (n = 38) exhibited less luminal closure sequences (41 ± 2% of the recording time vs. 61 ± 2%; P < 0.001) and more static sequences (38 ± 3 vs. 20 ± 2%; P < 0.001); in contrast, patients with hyperdynamic behavior (n = 13) had an increased proportion of luminal closure sequences (73 ± 4 vs. 61 ± 2%; P = 0.029) and more high-motion sequences (3 ± 1 vs. 0.5 ± 0.1%; P < 0.001). Applying an original methodology, we have developed a novel classification of functional gut disorders based on objective, physiological criteria of small bowel function.

Structural effects of protein aging: terminal marking by deamidation in human triosephosphate isomerase.

Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.

Synthesis of nitro(benzo)thiazole acetamides and in vitro antiprotozoal effect against amitochondriate parasites Giardia intestinalis and Trichomonas vaginalis.

We synthesized four 5-nitrothiazole (1-4) and four 6-nitrobenzothiazole acetamides (5-8) using an easy two step synthetic route. All compounds were tested in vitro against amitochondriate parasites Giardia intestinalis and Trichomonas vaginalis, showing excellent antiprotozoal effects. IC50's of the most potent compounds range from nanomolar to low micromolar order, being more active than their drugs of choice. Compound 1 (IC50=122 nM), was 44-times more active than Metronidazole, and 10-fold more effective than Nitazoxanide against G. intestinalis and showed good trichomonicidal activity (IC50=2.24 µM). This compound did not display in vitro cytotoxicity against VERO cells. The in vitro inhibitory effect of compounds 1-8 and Nitazoxanide against G. intestinalis fructose-1,6-biphosphate aldolase (GiFBPA) was evaluated as potential drug target, showing a clear inhibitory effect over the enzyme activity. Molecular docking of compounds 1, 4 and Nitazoxanide into the ligand binding pocket of GiFBPA, revealed contacts with the active site residues of the enzyme. Ligand efficiency metrics of 1 revealed optimal combinations of physicochemical and antiprotozoal properties, better than Nitazoxanide.

Giardial triosephosphate isomerase as possible target of the cytotoxic effect of omeprazole in Giardia lamblia.

Giardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains of Giardia lamblia was evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites. G. lamblia triosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effective in vitro against drug-resistant and drug-susceptible strains of G. lamblia.

The E104D mutation increases the susceptibility of human triosephosphate isomerase to proteolysis. Asymmetric cleavage of the two monomers of the homodimeric enzyme.

The deficiency of human triosephosphate isomerase (HsTIM) generates neurological alterations, cardiomyopathy and premature death. The mutation E104D is the most frequent cause of the disease. Although the wild type and mutant exhibit similar kinetic parameters, it has been shown that the E104D substitution induces perturbation of an interfacial water network that, in turn, reduces the association constant between subunits promoting enzyme inactivation. To gain further insight into the effects of the mutation on the structure, stability and function of the enzyme, we measured the sensitivity of recombinant E104D mutant and wild type HsTIM to limited proteolysis. The mutation increases the susceptibility to proteolysis as consequence of the loss of rigidity of its overall 3-D structure. Unexpectedly, it was observed that proteolysis of wild type HsTIM generated two different stable nicked dimers. One was formed in relatively short times of incubation with proteinase K; as shown by spectrometric and crystallographic data, it corresponded to a dimer containing a nicked monomer and an intact monomer. The formation of the other nicked species requires relatively long incubation times with proteinase K and corresponds to a dimer with two clipped subunits. The first species retains 50% of the original activity, whereas the second species is inactive. Collectively, we found that the E104D mutant is highly susceptible to proteolysis, which in all likelihood contributes to the pathogenesis of enzymopathy. In addition, the proteolysis data on wild type HsTIM illustrate an asymmetric conduct of the two monomers.

Structural and functional perturbation of Giardia lamblia triosephosphate isomerase by modification of a non-catalytic, non-conserved region.

BACKGROUND: We have previously proposed triosephosphate isomerase of Giardia lamblia (GlTIM) as a target for rational drug design against giardiasis, one of the most common parasitic infections in humans. Since the enzyme exists in the parasite and the host, selective inhibition is a major challenge because essential regions that could be considered molecular targets are highly conserved. Previous biochemical evidence showed that chemical modification of the non-conserved non-catalytic cysteine 222 (C222) inactivates specifically GlTIM. The inactivation correlates with the physicochemical properties of the modifying agent: addition of a non-polar, small chemical group at C222 reduces the enzyme activity by one half, whereas negatively charged, large chemical groups cause full inactivation. RESULTS: In this work we used mutagenesis to extend our understanding of the functional and structural effects triggered by modification of C222. To this end, six GlTIM C222 mutants with side chains having diverse physicochemical characteristics were characterized. We found that the polarity, charge and volume of the side chain in the mutant amino acid differentially alter the activity, the affinity, the stability and the structure of the enzyme. The data show that mutagenesis of C222 mimics the effects of chemical modification. The crystallographic structure of C222D GlTIM shows the disruptive effects of introducing a negative charge at position 222: the mutation perturbs loop 7, a region of the enzyme whose interactions with the catalytic loop 6 are essential for TIM stability, ligand binding and catalysis. The amino acid sequence of TIM in phylogenetic diverse groups indicates that C222 and its surrounding residues are poorly conserved, supporting the proposal that this region is a good target for specific drug design. CONCLUSIONS: The results demonstrate that it is possible to inhibit species-specifically a ubiquitous, structurally highly conserved enzyme by modification of a non-conserved, non-catalytic residue through long-range perturbation of essential regions.

[Mutation analysis of the methylmalonyl-CoA mutase gene in ten Mexican patients with methylmalonic acidemia]. / Análisis de mutaciones en el gen de la metilmalonilCoA mutasa en diez pacientes mexicanos con acidemia metilmalónica aislada.

INTRODUCTION: Methylmalonic acidemia (MMA) is a genetically determined human metabolic disease, characterized by deficient activity of the mitochondrial enzyme, methylmalonyl CoA mutase (MCM). This enzyme catalyzes the isomerization of L-methylmalonyl CoA to succinyl CoA and requires adenosylcobalamin as cofactor. Several mutations have been identified in the unique genetic locus encoding the MCM apoenzyme (mut) which causes MMA. AIM: To identify the mutations present in Mexican patients diagnosed with MMA. RESULTS: Complete nucleotide sequencing of mut gene exons of 10 Mexican patients with methylmalonic acidemia (MMA) identified one novel mutation and eight mutations previously reported in the methylmalonyl-CoA mutase (mut) gene. The new mutation c.406G > T (p.V136F) was found in one patient combined with the deletion c.1891delG (p.A631QfsX17). The missense mutation c.322C > T (p.R108C) was found in six non-related patients; in addition, the mutations c.ins671-678dupAATTTATG (p.V227NfsX16), c.682C > T (p.R228X), c1022-1023dupA (p. N341KfsX20), c.1846C > T (p.R616C), c.2080C > T (p.R694W), and c.385+3insTAAGGGT (splice) were found. This work reveals that Mexican patients with MMA have new (p.V136F) as well as worldwide and hispanic reported mutations. The mutation R108C is the most frequent change (40% of total alleles) mainly in patients from León, Guanajuato.

Determining the molecular mechanism of inactivation by chemical modification of triosephosphate isomerase from the human parasite Giardia lamblia: a study for antiparasitic drug design.

Giardiasis, the most prevalent intestinal parasitosis in humans, is caused by Giardia lamblia. Current drug therapies have adverse effects on the host, and resistant strains against these drugs have been reported, demonstrating an urgent need to design more specific antigiardiasic drugs. ATP production in G. lamblia depends mainly on glycolysis; therefore, all enzymes of this pathway have been proposed as potential drug targets. We previously demonstrated that the glycolytic enzyme triosephosphate isomerase from G. lamblia (GlTIM), could be completely inactivated by low micromolar concentrations of thiol-reactive compounds, whereas, in the same conditions, the activity of human TIM (HuTIM) was almost unaltered. We found that the chemical modification (derivatization) of at least one Cys, of the five Cys residues per monomer in GlTIM, causes this inactivation. In this study, structural and functional studies were performed to describe the molecular mechanism of GlTIM inactivation by thiol-reactive compounds. We found that the Cys222 derivatization is responsible for GlTIM inactivation; this information is relevant because HuTIM has a Cys residue in an equivalent position (Cys217). GlTIM inactivation is associated with a decrease in ligand affinity, which affects the entropic component of ligand binding. In summary, this work describes a mechanism of inactivation that has not been previously reported for TIMs from other parasites and furthermore, we show that the difference in reactivity between the Cys222 in GlTIM and the Cys217 in HuTIM, indicates that the surrounding environment of each Cys residue has unique structural differences that can be exploited to design specific antigiardiasic drugs.