L-Thyroxine and L-thyroxine-based antimicrobials against Streptococcus pneumoniae and other Gram-positive bacteria.

Objectives: The rise of antibiotic-resistant Streptococcus pneumoniae (Sp) poses a significant global health threat, urging the quest for novel antimicrobial solutions. We have discovered that the human hormone l-thyroxine has antibacterial properties. In order to explore its drugability we perform here the characterization of a series of l-thyroxine analogues and describe the structural determinants influencing their antibacterial efficacy. Method: We performed a high-throughput screening of a library of compounds approved for use in humans, complemented with ITC assays on purified Sp-flavodoxin, to pinpoint molecules binding to this protein. Antimicrobial in vitro susceptibility assays of the hit compound (l-thyroxine) as well as of 13 l-thyroxine analogues were done against a panel of Gram-positive and Gram-negative bacteria. Toxicity of compounds on HepG2 cells was also assessed. A combined structure-activity and computational docking analysis was carried out to uncover functional groups crucial for the antimicrobial potency of these compounds. Results: Human l-thyroxine binds to Sp-flavodoxin, forming a 1:1 complex of low micromolar Kd. While l-thyroxine specifically inhibited Sp growth, some derivatives displayed activity against other Gram-positive bacteria like Staphylococcus aureus and Enterococcus faecalis, while remaining inactive against Gram-negative pathogens. Neither l-thyroxine nor some selected derivatives exhibited toxicity to HepG2 cells. Conclusions: l-thyroxine derivatives targeting bacterial flavodoxins represent a new and promising class of antimicrobials.

Energy, water, and protein folding: A molecular dynamics-based quantitative inventory of molecular interactions and forces that make proteins stable.

Protein folding energetics can be determined experimentally on a case-by-case basis but it is not understood in sufficient detail to provide deep control in protein design. The fundamentals of protein stability have been outlined by calorimetry, protein engineering, and biophysical modeling, but these approaches still face great difficulty in elucidating the specific contributions of the intervening molecules and physical interactions. Recently, we have shown that the enthalpy and heat capacity changes associated to the protein folding reaction can be calculated within experimental error using molecular dynamics simulations of native protein structures and their corresponding unfolded ensembles. Analyzing in depth molecular dynamics simulations of four model proteins (CI2, barnase, SNase, and apoflavodoxin), we dissect here the energy contributions to ΔH (a key component of protein stability) made by the molecular players (polypeptide and solvent molecules) and physical interactions (electrostatic, van der Waals, and bonded) involved. Although the proteins analyzed differ in length, isoelectric point and fold class, their folding energetics is governed by the same quantitative pattern. Relative to the unfolded ensemble, the native conformations are enthalpically stabilized by comparable contributions from protein-protein and solvent-solvent interactions, and almost equally destabilized by interactions between protein and solvent molecules. The native protein surface seems to interact better with water than the unfolded one, but this is outweighed by the unfolded surface being larger. From the perspective of physical interactions, the native conformations are stabilized by van de Waals and Coulomb interactions and destabilized by conformational strain arising from bonded interactions. Also common to the four proteins, the sign of the heat capacity change is set by interactions between protein and solvent molecules or, from the alternative perspective, by Coulomb interactions.

Correction: Transcription factor induced conversion of human fibroblasts towards the hair cell lineage.

[This corrects the article DOI: 10.1371/journal.pone.0200210.].

Antimicrobial combinations against Helicobacter pylori including benzoxadiazol-based flavodoxin inhibitors: in vitro characterization.

IMPORTANCE: The antimicrobial resistance of Helicobacter pylori (Hp) currently poses a threat to available treatment regimens. Developing antimicrobial drugs targeting new bacterial targets is crucial, and one such class of drugs includes Hp-flavodoxin (Hp-fld) inhibitors that target an essential metabolic pathway in Hp. Our study demonstrated that combining these new drugs with conventional antibiotics used for Hp infection treatment prevented the regrowth observed with drugs used alone. Hp-fld inhibitors show promise as new drugs to be incorporated into the treatment of Hp infection, potentially reducing the development of resistance and shortening the treatment duration.

In vitro and in vivo calibration of low affinity genetic Ca2+ indicators.

Calcium is a universal intracellular messenger and proper Ca2+concentrations ([Ca2+]) both in the cytosol and in the lumen of cytoplasmic organelles are essential for cell functions. Ca2+ homeostasis is achieved by a delicate pump/leak balance both at the plasma membrane and at the endomembranes, and improper Ca2+ levels result in malfunction and disease. Selective intraorganellar Ca2+measurements are best achieved by using targeted genetically encoded Ca2+ indicators (GECIs) but to calibrate the luminal fluorescent signals into accurate [Ca2+] is challenging, especially in vivo, due to the difficulty to normalize and calibrate the fluorescent signal in various tissues or conditions. We report here a procedure to calibrate the ratiometric signal of GAP (GFP-Aequorin Protein) targeted to the endo-sarcoplasmic reticulum (ER/SR) into [Ca2+]ER/SR based on imaging of fluorescence after heating the tissue at 50-52 °C, since this value coincides with that obtained in the absence of Ca2+ (Rmin). Knowledge of the dynamic range (Rmax/Rmin) and the Ca2+-affinity (KD) of the indicator permits calculation of [Ca2+] by applying a simple algorithm. We have validated this procedure in vitro using several cell types (HeLa, HEK 293T and mouse astrocytes), as well as in vivo in Drosophila. Moreover, this methodology is applicable to other low Ca2+ affinity green and red GECIs.

Calculation of Protein Folding Thermodynamics Using Molecular Dynamics Simulations.

Despite advances in artificial intelligence methods, protein folding remains in many ways an enigma to be solved. Accurate computation of protein folding energetics could help drive fields such as protein and drug design and genetic interpretation. However, the challenge of calculating the state functions governing protein folding from first-principles remains unaddressed. We present here a simple approach that allows us to accurately calculate the energetics of protein folding. It is based on computing the energy of the folded and unfolded states at different temperatures using molecular dynamics simulations. From this, two essential quantities (ΔH and ΔCp) are obtained and used to calculate the conformational stability of the protein (ΔG). With this approach, we have successfully calculated the energetics of two- and three-state proteins, representatives of the major structural classes, as well as small stability differences (ΔΔG) due to changes in solution conditions or variations in an amino acid residue.

Contribution of Outpatient Ultrasound Transvaginal Biopsy and Puncture in the Diagnosis and Treatment of Pelvic Lesions: A Bicenter Study.

BACKGROUND: The use of transvaginal ultrasound guided biopsy and puncture of pelvic lesions is a minimally invasive technique that allows for accurate diagnosis. It has many advantages compared to other more invasive (lower complication rate) or non-invasive techniques (accurate diagnosis). Furthermore, it offers greater availability, it does not radiate, enables the study of pelvic masses accessible vaginally with ultrasound control in real time, and it is possible to use the colour Doppler avoiding puncturing large vessels among others. The main aim of the work is to describe a standardized ambulatory technique and to determine its usefulness. METHODS: This is a retrospective study of ultrasound transvaginal punctures (core needle biopsies and cytologies) and drainages of pelvic lesions performed on an outpatient basis during the last two years. The punctures were made with local anesthesia, under transvaginal ultrasound guidance with an automatic or semi-automatic 18G biopsy needle with a length of 20-25 cm and a penetration depth of 12 or 22 mm. The material obtained was sent for anatomopathological, cytological and/or microbiological study if necessary. RESULTS: A total of 42 women were recruited in two centers. Fifty procedures (nine punctures, seven drains, and 34 biopsies) were performed. In five cases the punction and drain provided clinical relief in benign pelvic masses. Regarding material of the biopsies performed, 15 were vaginal in women previously histerectomized, finding 10 carcinomas, eight were ovarian tumours in advanced stages or peritoneal carcinomatosis obtaining the appropriate histology in each case, seven were suspicious cervical biopsies finding carcinomas in five of them, three were myometrial biopsies including one breast carcinoma metastasis in the miometrium and a benign placental nodule, and a periurethral biopsy was performed on a woman with a history of endometrial cancer confirming recurrence. The pathological diagnosis was satisfactory in all cases, confirming the nature of the lesion (25 malignant-ten vaginal recurrences of previous gynaecological cancers, eight cases of primary ovarian/peritoneal carcinoma, four new diagnosis of cervical malignant masses, one cervical metastasis of lymphoma, one periurethral recurrence of endometrial carcinoma and one recurrence of breast cancer in the myometrium-and 23 benign). The tolerance was excellent and no complications were detected. CONCLUSION: The ambulatory ultrasound transvaginal puncture and drainage technique is useful for obtaining a sample for pathological and microbiological diagnosis with excellent tolerance that can be used to rule out the recurrence of malignant lesions or progression of the disease, diagnose masses not accessible to gynecological exploration (vaginal vault, myometrium or cervix) and for early histologic diagnosis in cases of advanced peritoneal carcinomatosis or ovarian carcinoma as well as drainage and cytological study of cystic pelvic masses.

Factors Associated with a Post-Procedure Spontaneous Pregnancy after a Hysterosapingo-Foam-Sonography (HyFoSy): Results from a Multicenter Observational Study.

BACKGROUND: Tubal patency testing constitutes an essential part of infertility work-up. Hysterosalpingo-foam-sonography (HyFoSy) is currently one of the best tests for assessing tubal patency. The objective of our study was to evaluate the post-procedure rate of spontaneous pregnancy among infertile women submitted for an HyFoSy exam with ExEm® foam and the factors associated with this. METHODS: Multicenter, prospective, observational study performed at six Spanish centers for gynecologic sonography and human reproduction. From December 2015 to June 2021, 799 infertile women underwent HyFoSy registration consecutively. The patients' information was collected from their medical records. Multivariable regression analyses were performed, controlling for age, etiology, and time of sterility. The main outcome was to measure post-procedure spontaneous pregnancy rates and the factors associated with the achievement of pregnancy. RESULTS: 201 (26.5%) women got spontaneous conception (SC group), whereas 557 (73.5%) women did not get pregnant (non-spontaneous conception group, NSC). The median time for reaching SC after HyFoSy was 4 months (CI 95% 3.1-4.9), 18.9% of them occurring the same month of the procedure. Couples with less than 18 months of infertility were 93% more likely to get pregnant after HyFoSy (OR 1.93, 95% CI 1.34-2.81; p < 0.001); SC were two times more frequent in women under 35 years with unexplained infertility (OR 2.22, 95% CI 1.07-4.65; P0.033). CONCLUSION: After HyFoSy, one in four patients got pregnant within the next twelve months. Couples with shorter infertility time, unexplained infertility, and women under 35 years are more likely to achieve SC after HyFoSy.

Association between missense variants of uncertain significance in the CHEK2 gene and hereditary breast cancer: a cosegregation and bioinformatics analysis.

Inherited mutations in the CHEK2 gene have been associated with an increased lifetime risk of developing breast cancer (BC). We aim to identify in the study population the prevalence of mutations in the CHEK2 gene in diagnosed BC patients, evaluate the phenotypic characteristics of the tumor and family history, and predict the deleteriousness of the variants of uncertain significance (VUS). A genetic study was performed, from May 2016 to April 2020, in 396 patients diagnosed with BC at the University Hospital Lozano Blesa of Zaragoza, Spain. Patients with a genetic variant in the CHEK2 gene were selected for the study. We performed a descriptive analysis of the clinical variables, a bibliographic review of the variants, and a cosegregation study when possible. Moreover, an in-depth bioinformatics analysis of CHEK2 VUS was carried out. We identified nine genetic variants in the CHEK2 gene in 10 patients (two pathogenic variants and seven VUS). This supposes a prevalence of 0.75% and 1.77%, respectively. In all cases, there was a family history of BC in first- and/or second-degree relatives. We carried out a cosegregation study in two families, being positive in one of them. The bioinformatics analyses predicted the pathogenicity of six of the VUS. In conclusion, CHEK2 mutations have been associated with an increased risk for BC. This risk is well-established for foundation variants. However, the risk assessment for other variants is unclear. The incorporation of bioinformatics analysis provided supporting evidence of the pathogenicity of VUS.

A look at the face of the molten globule: Structural model of the Helicobacter pylori apoflavodoxin ensemble at acidic pH.

Molten globule (MG) is the name given to a compact, non-native conformation of proteins that has stimulated the imagination and work in the protein folding field for more than 40 years. The MG has been proposed to play a central role in the folding reaction and in important cell functions, and to be related to the onset of misfolding diseases. Due to its inherent intractability to high-resolution studies, atomistic structural models have not yet been obtained. We present here an integrative atomistic model of the MG formed at acidic pH by the apoflavodoxin from the human pathogen Helicobacter pylori. This MG has been previously shown to exhibit the archetypical expansion, spectroscopic and thermodynamic features of a molten conformation. To obtain the model, we have analyzed the stability of wild-type and 55 apoflavodoxin mutants to derive experimental equilibrium Φ values that have been used in biased molecular dynamics simulations to convert the native conformation into an MG ensemble. The ensemble has been refined to reproduce the experimental hydrodynamic radius and circular dichroism (CD) spectrum. The refined ensemble, deposited in PDB-Dev, successfully explains the characteristic 1 H-nuclear magnetic resonance (NMR) and near-UV CD spectral features of the MG as well as its solvent-accessible surface area (SASA) change upon unfolding. This integrative model of an MG will help to understand the energetics and roles of these elusive conformations in protein folding and misfolding. Interestingly, the apoflavodoxin MG is structurally unrelated to previously described partly unfolded conformations of this protein, exemplifying that equilibrium MGs need not to reflect the properties of kinetic intermediates.

Protposer: The web server that readily proposes protein stabilizing mutations with high PPV.

Protein stability is a requisite for most biotechnological and medical applications of proteins. As natural proteins tend to suffer from a low conformational stability ex vivo, great efforts have been devoted toward increasing their stability through rational design and engineering of appropriate mutations. Unfortunately, even the best currently used predictors fail to compute the stability of protein variants with sufficient accuracy and their usefulness as tools to guide the rational stabilisation of proteins is limited. We present here Protposer , a protein stabilising tool based on a different approach. Instead of quantifying changes in stability, Protposer uses structure- and sequence-based screening modules to nominate candidate mutations for subsequent evaluation by a logistic regression model, carefully trained to avoid overfitting. Thus, Protposer analyses PDB files in search for stabilization opportunities and provides a ranked list of promising mutations with their estimated success rates (eSR), their probabilities of being stabilising by at least 0.5 kcal/mol. The agreement between eSRs and actual positive predictive values (PPV) on external datasets of mutations is excellent. When Protposer is used with its Optimal kappa selection threshold, its PPV is above 0.7. Even with less stringent thresholds, Protposer largely outperforms FoldX, Rosetta and PoPMusiC. Indicating the PDB file of the protein suffices to obtain a ranked list of mutations, their eSRs and hints on the likely source of the stabilization expected. Protposer is a distinct, straightforward and highly successful tool to design protein stabilising mutations, and it is freely available for academic use at http://webapps.bifi.es/the-protposer.

Alchemical Design of Pharmacological Chaperones with Higher Affinity for Phenylalanine Hydroxylase.

Phenylketonuria (PKU) is a rare metabolic disease caused by variations in a human gene, PAH, encoding phenylalanine hydroxylase (PAH), and the enzyme converting the essential amino acid phenylalanine into tyrosine. Many PKU-causing variations compromise the conformational stability of the encoded enzyme, decreasing or abolishing its catalytic activity, and leading to an elevated concentration of phenylalanine in the blood, which is neurotoxic. Several therapeutic approaches have been developed to treat the more severe manifestations of the disorder, but they are either not entirely effective or difficult to adhere to throughout life. In a search for novel pharmacological chaperones to treat PKU, a lead compound was discovered (compound IV) that exhibited promising in vitro and in vivo chaperoning activity on PAH. The structure of the PAH-IV complex has been reported. Here, using alchemical free energy calculations (AFEC) on the structure of the PAH-IV complex, we design a new generation of compound IV-analogues with a higher affinity for the enzyme. Seventeen novel analogues were synthesized, and thermal shift and isothermal titration calorimetry (ITC) assays were performed to experimentally evaluate their stabilizing effect and their affinity for the enzyme. Most of the new derivatives bind to PAH tighter than lead compound IV and induce a greater thermostabilization of the enzyme upon binding. Importantly, the correspondence between the calculated alchemical binding free energies and the experimentally determined ΔΔGb values is excellent, which supports the use of AFEC to design pharmacological chaperones to treat PKU using the X-ray structure of their complexes with the target PAH enzyme.

PirePred: An Accurate Online Consensus Tool to Interpret Newborn Screening-Related Genetic Variants in Structural Context.

PirePred is a genetic interpretation tool used for a variety of medical conditions investigated in newborn screening programs. The PirePred server retrieves, analyzes, and displays in real time genetic and structural data on 58 genes/proteins associated with medical conditions frequently investigated in the newborn. PirePred analyzes the predictions generated by 15 pathogenicity predictors and applies an optimized majority vote algorithm to classify any possible nonsynonymous single-nucleotide variant as pathogenic, benign, or of uncertain significance. PirePred predictions for variants of clear clinical significance are better than those of any of the individual predictors considered (based on accuracy, sensitivity, and negative predictive value) or are among the best ones (for positive predictive value and Matthews correlation coefficient). PirePred predictions also outperform the comparable in silico predictions offered as supporting evidence, according to American College of Medical Genetics and Genomics guidelines, by VarSome and Franklin. Also, PirePred has very high prediction coverage. To facilitate the molecular interpretation of the missense, nonsense, and frameshift variants in ClinVar, the changing amino acid residue is displayed in its structural context, which is analyzed to provide functional clues. PirePred is an accurate, robust, and easy-to-use tool for clinicians involved in neonatal screening programs and for researchers of related diseases. The server is freely accessible and provides a user-friendly gateway into the structural/functional consequences of genetic variants at the protein level.

Comparison of Electromyographic Activity During Hip Extension Exercises Under Gravitational or Inertial Loading Conditions.

BACKGROUND: Hamstring injury prevention programs include strengthening, especially eccentric exercises using both gravitational and inertial loading. Inertial exercises are characterized by eccentric contractions of high intensity and velocity. This study aimed to analyze the muscular activation of the biceps femoris (BF), semitendinosus (ST), gluteus maximus (GM), and gracilis (GC) muscles during hip extension (HE) exercises performed under both gravitational and inertial loading conditions. HYPOTHESIS: Inertial training would generate a greater activation of HE muscles than gravitational training. STUDY DESIGN: Cross-sectional study. LEVEL OF EVIDENCE: Level 4. METHODS: Fifteen resistance-trained men performed the unilateral straight knee bridge (SKB), 45° of HE, and stiff-leg deadlift (SDL) exercises under gravitational and inertial loading conditions. Concentric and eccentric phases were identified with a linear encoder. Differences between load types, exercises, and their interaction were examined to establish the electromyographic (EMG) activity of each muscle and BF/ST ratio. RESULTS: In the concentric phase, inertial loading showed a higher normalized EMG than gravitational loading for BF, ST, and GM. SKB and HE activated BF and ST between 9.6% and 24.3% more than SDL. In the eccentric phase, the inertial modality achieved greater GM activation than the gravitational form (18.1%). BF activation was increased with HE and SKB as compared with SDL (24.4% and 16.4%, respectively), while ST activation was likewise enhanced with HE as compared with SDL (15.1%). CONCLUSION: Inertial training is more effective than gravitational training for the concentric activation of the hamstring muscles while SDL showed lower hamstring activation than HE and SKB. Therefore, HE and SKB with inertial loading should be taken into account in hamstring training programs. CLINICAL RELEVANCE: Inertial training is more effective than gravitational training for the concentric activation of the hamstring muscles. HE and SKB with inertial loading should be taken into account in hamstring training programs.

A Comparative Analysis of MRI Automated Segmentation of Subcortical Brain Volumes in a Large Dataset of Elderly Subjects.

In this study, we perform a comparative analysis of automated image segmentation of subcortical structures in the elderly brain. Manual segmentation is very time-consuming and automated methods are gaining importance as a clinical tool for diagnosis. The two most commonly used software libraries for brain segmentation -FreeSurfer and FSL- are put to work in a large dataset of 4,028 magnetic resonance imaging (MRI) scans collected for this study. We find a lack of linear correlation between the segmentation volume estimates obtained from FreeSurfer and FSL. On the other hand, FreeSurfer volume estimates tend to be larger thanFSL estimates of the areas putamen, thalamus, amygdala, caudate, pallidum, hippocampus, and accumbens. The characterization of the performance of brain segmentation algorithms in large datasets as the one presented here is a necessary step towards partially or fully automated end-to-end neuroimaging workflow both in clinical and research settings.

Long-term efficacy of autologous bone marrow mesenchymal stromal cells for treatment of knee osteoarthritis.

Knee osteoarthritis is the most prevalent joint disease and a frequent cause of pain, functional loss and disability. Conventional treatments have demonstrated only modest clinical benefits whereas cell-based therapies have shown encouraging results, but important details, such as dose needed, long-term evolution or number of applications required are scarcely known. Here we have reanalyzed results from two recent pilot trials with autologous bone marrow-derived mesenchymal stromal cells using the Huskisson plot to enhance quantification of efficacy and comparability. We find that cell doses of 10, 40 and 100 million autologous cells per knee provided quite similar healing results and that much of the effect attained 1 year after cell application remained after 2 and 4 years. These results are encouraging because they indicate that, apart from safety and simplicity: (i) the beneficial effect is both significant and sizeable, (ii) it can be achieved with a single injection of cells, and (iii) the effect is perdurable for years.Trial registration: EudraCT 2009-017405-11; NCT02123368. Registered 25 April 2014-Prospectively registered, https://clinicaltrials.gov/ct2/show/NCT02123368?term=02123368&draw=2&rank=1.

Design, synthesis and structure-activity evaluation of novel 2-pyridone-based inhibitors of α-synuclein aggregation with potentially improved BBB permeability.

The treatment of Parkinson's disease (PD), the second most common neurodegenerative human disorder, continues to be symptomatic. Development of drugs able to stop or at least slowdown PD progression would benefit several million people worldwide. SynuClean-D is a low molecular weight 2-pyridone-based promising drug candidate that inhibits the aggregation of α-synuclein in human cultured cells and prevents degeneration of dopaminergic neurons in a Caenorhabditis elegans model of PD. Improving SynuClean-D pharmacokinetic/pharmacodynamic properties, performing structure/activity studies and testing its efficacy in mammalian models of PD requires the use of gr-amounts of the compound. However, not enough compound is on sale, and no synthetic route has been reported until now, which hampers the molecule progress towards clinical trials. To circumvent those problems, we describe here an efficient and economical route that enables the synthesis of SynuClean-D with good yields as well as the synthesis of SynuClean-D derivatives. Structure-activity comparison of the new compounds with SynuClean-D reveals the functional groups of the molecule that can be disposed of without activity loss and those that are crucial to interfere with α-synuclein aggregation. Several of the derivatives obtained retain the parent's compound excellent in vitro anti-aggregative activity, without compromising its low toxicity. Computational predictions and preliminary testing indicate that the blood brain barrier (BBB) permeability of SynuClean-D is low. Importantly, several of the newly designed and obtained active derivatives are predicted to display good BBB permeability. The synthetic route developed here will facilitate their synthesis for BBB permeability determination and for efficacy testing in mammalian models of PD.

Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems.

Microalgae can produce industrially relevant metabolites using atmospheric CO2 and sunlight as carbon and energy sources, respectively. Developing molecular tools for high-throughput genome engineering could accelerate the generation of tailored strains with improved traits. To this end, we developed a genome editing strategy based on Cas12a ribonucleoproteins (RNPs) and homology-directed repair (HDR) to generate scarless and markerless mutants of the microalga Nannochloropsis oceanica. We also developed an episomal plasmid-based Cas12a system for efficiently introducing indels at the target site. Additionally, we exploited the ability of Cas12a to process an associated CRISPR array to perform multiplexed genome engineering. We efficiently targeted three sites in the host genome in a single transformation, thereby making a major step toward high-throughput genome engineering in microalgae. Furthermore, a CRISPR interference (CRISPRi) tool based on Cas9 and Cas12a was developed for effective downregulation of target genes. We observed up to 85% reduction in the transcript levels upon performing CRISPRi with dCas9 in N. oceanica. Overall, these developments substantially accelerate genome engineering efforts in N. oceanica and potentially provide a general toolbox for improving other microalgal strains.

Selective Targeting of Human and Animal Pathogens of the Helicobacter Genus by Flavodoxin Inhibitors: Efficacy, Synergy, Resistance and Mechanistic Studies.

Antimicrobial resistant (AMR) bacteria constitute a global health concern. Helicobacter pylori is a Gram-negative bacterium that infects about half of the human population and is a major cause of peptic ulcer disease and gastric cancer. Increasing resistance to triple and quadruple H. pylori eradication therapies poses great challenges and urges the development of novel, ideally narrow spectrum, antimicrobials targeting H. pylori. Here, we describe the antimicrobial spectrum of a family of nitrobenzoxadiazol-based antimicrobials initially discovered as inhibitors of flavodoxin: an essential H. pylori protein. Two groups of inhibitors are described. One group is formed by narrow-spectrum compounds, highly specific for H. pylori, but ineffective against enterohepatic Helicobacter species and other Gram-negative or Gram-positive bacteria. The second group includes extended-spectrum antimicrobials additionally targeting Gram-positive bacteria, the Gram-negative Campylobacter jejuni, and most Helicobacter species, but not affecting other Gram-negative pathogens. To identify the binding site of the inhibitors in the flavodoxin structure, several H. pylori-flavodoxin variants have been engineered and tested using isothermal titration calorimetry. An initial study of the inhibitors capacity to generate resistances and of their synergism with antimicrobials commonly used in H. pylori eradication therapies is described. The narrow-spectrum inhibitors, which are expected to affect the microbiota less dramatically than current antimicrobial drugs, offer an opportunity to develop new and specific H. pylori eradication combinations to deal with AMR in H. pylori. On the other hand, the extended-spectrum inhibitors constitute a new family of promising antimicrobials, with a potential use against AMR Gram-positive bacterial pathogens.

Unravelling the Complex Denaturant and Thermal-Induced Unfolding Equilibria of Human Phenylalanine Hydroxylase.

Human phenylalanine hydroxylase (PAH) is a metabolic enzyme involved in the catabolism of L-Phe in liver. Loss of conformational stability and decreased enzymatic activity in PAH variants result in the autosomal recessive disorder phenylketonuria (PKU), characterized by developmental and psychological problems if not treated early. One current therapeutic approach to treat PKU is based on pharmacological chaperones (PCs), small molecules that can displace the folding equilibrium of unstable PAH variants toward the native state, thereby rescuing the physiological function of the enzyme. Understanding the PAH folding equilibrium is essential to develop new PCs for different forms of the disease. We investigate here the urea and the thermal-induced denaturation of full-length PAH and of a truncated form lacking the regulatory and the tetramerization domains. For either protein construction, two distinct transitions are seen in chemical denaturation followed by fluorescence emission, indicating the accumulation of equilibrium unfolding intermediates where the catalytic domains are partly unfolded and dissociated from each other. According to analytical centrifugation, the chemical denaturation intermediates of either construction are not well-defined species but highly polydisperse ensembles of protein aggregates. On the other hand, each protein construction similarly shows two transitions in thermal denaturation measured by fluorescence or differential scanning calorimetry, also indicating the accumulation of equilibrium unfolding intermediates. The similar temperatures of mid denaturation of the two constructions, together with their apparent lack of response to protein concentration, indicate the catalytic domains are unfolded in the full-length PAH thermal intermediate, where they remain associated. That the catalytic domain unfolds in the first thermal transition is relevant for the choice of PCs identified in high throughput screening of chemical libraries using differential scanning fluorimetry.