Extending MeCP2 interactome: canonical nucleosomal histones interact with MeCP2.

MeCP2 is a general regulator of transcription involved in the repression/activation of genes depending on the local epigenetic context. It acts as a chromatin regulator and binds with exquisite specificity to gene promoters. The set of epigenetic marks recognized by MeCP2 has been already established (mainly, cytosine modifications in CpG and CpA), as well as many of the constituents of its interactome. We unveil a new set of interactions for MeCP2 with the four canonical nucleosomal histones. MeCP2 interacts with high affinity with H2A, H2B, H3 and H4. In addition, Rett syndrome associated mutations in MeCP2 and histone epigenetic marks modulate these interactions. Given the abundance and the structural/functional relevance of histones and their involvement in epigenetic regulation, this new set of interactions and its modulating elements provide a new addition to the 'alphabet' for this epigenetic reader.

Shedding Light on Dark Chemical Matter: The Discovery of a SARS-CoV-2 Mpro Main Protease Inhibitor through Intensive Virtual Screening and In Vitro Evaluation.

The development of specific antiviral therapies targeting SARS-CoV-2 remains fundamental because of the continued high incidence of COVID-19 and limited accessibility to antivirals in some countries. In this context, dark chemical matter (DCM), a set of drug-like compounds with outstanding selectivity profiles that have never shown bioactivity despite being extensively assayed, appears to be an excellent starting point for drug development. Accordingly, in this study, we performed a high-throughput screening to identify inhibitors of the SARS-CoV-2 main protease (Mpro) using DCM compounds as ligands. Multiple receptors and two different docking scoring functions were employed to identify the best molecular docking poses. The selected structures were subjected to extensive conventional and Gaussian accelerated molecular dynamics. From the results, four compounds with the best molecular behavior and binding energy were selected for experimental testing, one of which presented inhibitory activity with a Ki value of 48 ± 5 µM. Through virtual screening, we identified a significant starting point for drug development, shedding new light on DCM compounds.

The isolated GTPase-activating-protein-related domain of neurofibromin-1 has a low conformational stability in solution.

Neurofibromin-1 (NF1) is a large, multidomain tumour suppressor encoded by the NF1 gene. The gene is mutated in neurofibromatosis type I, a disease characterized by malignant tumours of the nervous system and benign neurofibromas. The best-known activity of NF1 is the down-regulation of the mitogen-activated protein kinase pathway via its three-hundred-residue-long GTPase-activating protein (GAP) domain (the so-called GAP-related domain (NF1-GRD)). The NF1-GRD stimulates Ras GTPase activity in turning off signalling. Despite this activity, NF1-GRD has been demonstrated to bind to other different proteins, such as SPRED1 or MC1R. We have embarked on the biophysical and conformational characterization of NF1-GRD in solution by using several spectroscopic (namely fluorescence and circular dichroism (CD)) and biophysical techniques (namely size exclusion chromatography (SEC) and differential scanning calorimetry (DSC)). This biophysical characterization is crucial in deciphering NF1-GRD interactome and in finding biochemical features, modulating possible protein interactions. The native-like structure of NF1-GRD (as monitored by intrinsic fluorescence and far-UV CD) was strongly pH-dependent showing a pH-titration causing a substantial increase in its helicity. NF1-GRD had a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by intrinsic and ANS fluorescence, and CD. Chemical denaturations showed that NF1-GRD unfolded through an intermediate which has a substantial amount of solvent-exposed hydrophobic patches.

Discovery of Diverse Natural Products as Inhibitors of SARS-CoV-2 Mpro Protease through Virtual Screening.

SARS-CoV-2 is a type of coronavirus responsible for the international outbreak of respiratory illness termed COVID-19 that forced the World Health Organization to declare a pandemic infectious disease situation of international concern at the beginning of 2020. The need for a swift response against COVID-19 prompted to consider different sources to identify bioactive compounds that can be used as therapeutic agents, including available drugs and natural products. Accordingly, this work reports the results of a virtual screening process aimed at identifying antiviral natural product inhibitors of the SARS-CoV-2 Mpro viral protease. For this purpose, ca. 2000 compounds of the Selleck database of Natural Compounds were the subject of an ensemble docking process targeting the Mpro protease. Molecules that showed binding to most of the protein conformations were retained for a further step that involved the computation of the binding free energy of the ligand-Mpro complex along a molecular dynamics trajectory. The compounds that showed a smooth binding free energy behavior were selected for in vitro testing. From the resulting set of compounds, five compounds exhibited an antiviral profile, and they are disclosed in the present work.

Fluorescence Liquid Biopsy for Cancer Detection Is Improved by Using Cationic Dendronized Hyperbranched Polymer.

(1) Background: Biophysical techniques applied to serum samples characterization could promote the development of new diagnostic tools. Fluorescence spectroscopy has been previously applied to biological samples from cancer patients and differences from healthy individuals were observed. Dendronized hyperbranched polymers (DHP) based on bis(hydroxymethyl)propionic acid (bis-MPA) were developed in our group and their potential biomedical applications explored. (2) Methods: A total of 94 serum samples from diagnosed cancer patients and healthy individuals were studied (20 pancreatic ductal adenocarcinoma, 25 blood donor, 24 ovarian cancer, and 25 benign ovarian cyst samples). (3) Results: Fluorescence spectra of serum samples (fluorescence liquid biopsy, FLB) in the presence and the absence of DHP-bMPA were recorded and two parameters from the signal curves obtained. A secondary parameter, the fluorescence spectrum score (FSscore), was calculated, and the diagnostic model assessed. For pancreatic ductal adenocarcinoma (PDAC) and ovarian cancer, the classification performance was improved when including DHP-bMPA, achieving high values of statistical sensitivity and specificity (over 85% for both pathologies). (4) Conclusions: We have applied FLB as a quick, simple, and minimally invasive promising technique in cancer diagnosis. The classification performance of the diagnostic method was further improved by using DHP-bMPA, which interacted differentially with serum samples from healthy and diseased subjects. These preliminary results set the basis for a larger study and move FLB closer to its clinical application, providing useful information for the oncologist during patient diagnosis.

Seleno-Functionalization of Quercetin Improves the Non-Covalent Inhibition of Mpro and Its Antiviral Activity in Cells against SARS-CoV-2.

The development of new antiviral drugs against SARS-CoV-2 is a valuable long-term strategy to protect the global population from the COVID-19 pandemic complementary to the vaccination. Considering this, the viral main protease (Mpro) is among the most promising molecular targets in light of its importance during the viral replication cycle. The natural flavonoid quercetin 1 has been recently reported to be a potent Mpro inhibitor in vitro, and we explored the effect produced by the introduction of organoselenium functionalities in this scaffold. In particular, we report here a new synthetic method to prepare previously inaccessible C-8 seleno-quercetin derivatives. By screening a small library of flavonols and flavone derivatives, we observed that some compounds inhibit the protease activity in vitro. For the first time, we demonstrate that quercetin (1) and 8-(p-tolylselenyl)quercetin (2d) block SARS-CoV-2 replication in infected cells at non-toxic concentrations, with an IC50 of 192 µM and 8 µM, respectively. Based on docking experiments driven by experimental evidence, we propose a non-covalent mechanism for Mpro inhibition in which a hydrogen bond between the selenium atom and Gln189 residue in the catalytic pocket could explain the higher Mpro activity of 2d and, as a result, its better antiviral profile.

l-Arginine Improves Solubility and ANTI SARS-CoV-2 Mpro Activity of Rutin but Not the Antiviral Activity in Cells.

The COVID-19 pandemic outbreak prompts an urgent need for efficient therapeutics, and repurposing of known drugs has been extensively used in an attempt to get to anti-SARS-CoV-2 agents in the shortest possible time. The glycoside rutin shows manifold pharmacological activities and, despite its use being limited by its poor solubility in water, it is the active principle of many pharmaceutical preparations. We herein report our in silico and experimental investigations of rutin as a SARS-CoV-2 Mpro inhibitor and of its water solubility improvement obtained by mixing it with l-arginine. Tests of the rutin/l-arginine mixture in a cellular model of SARS-CoV-2 infection highlighted that the mixture still suffers from unfavorable pharmacokinetic properties, but nonetheless, the results of this study suggest that rutin might be a good starting point for hit optimization.

Thermal liquid biopsy for monitoring melanoma patients under surveillance during treatment: A pilot study.

BACKGROUND: Differential Scanning Calorimetry (DSC) is a technique traditionally used to study thermally induced macromolecular transitions, and it has recently been proposed as a novel approach for diagnosis and monitoring of several diseases. We report a pilot study applying Thermal Liquid Biopsy (TLB, DSC thermograms of plasma samples) as a new clinical approach for diagnostic assessment of melanoma patients. METHODS: Multiparametric analysis of DSC thermograms of patient plasma samples collected during treatment and surveillance (63 samples from 10 patients) were compared with clinical and diagnostic imaging assessment to determine the utility of thermograms for diagnostic assessment in melanoma. Nine of the ten patients were stage 2 or 3 melanoma subjects receiving adjuvant therapy after surgical resection of their melanomas. The other patient had unresectable stage 4 melanoma and was treated with immunotherapy. Two reference groups were used: (A) 36 healthy subjects and (B) 13 samples from 8 melanoma patients who had completed successful surgical management of their disease and were determined by continued clinical assessment to have no evidence of disease. RESULTS: Plasma thermogram analysis applied to melanoma patients generally agrees with clinical evaluation determined by physical assessment or diagnostic imaging (~80% agreement). No false negatives were obtained from DSC thermograms. Importantly, this methodology was able to detect changes in disease status before it was identified clinically. CONCLUSIONS: Thermal Liquid Biopsy could be used in combination with current clinical assessment for the earlier detection of melanoma recurrence and metastasis. GENERAL SIGNIFICANCE: TLB offers advantages over current diagnostic techniques (PET/CT imaging), limited in frequency by radiation burden and expense, in providing a minimally-invasive, low-risk, low-cost clinical test for more frequent personalized patient monitoring to assess recurrence and facilitate clinical decision-making.

Direct examination of the relevance for folding, binding and electron transfer of a conserved protein folding intermediate.

Near the minimum free energy basin of proteins where the native ensemble resides, partly unfolded conformations of slightly higher energy can be significantly populated under native conditions. It has been speculated that they play roles in molecular recognition and catalysis, but they might represent contemporary features of the evolutionary process without functional relevance. Obtaining conclusive evidence on these alternatives is difficult because it requires comparing the performance of a given protein when populating and when not populating one such intermediate, in otherwise identical conditions. Wild type apoflavodoxin populates under native conditions a partly unfolded conformation (10% of molecules) whose unstructured region includes the binding sites for the FMN cofactor and for redox partner proteins. We recently engineered a thermostable variant where the intermediate is no longer detectable. Using the wild type and variant, we assess the relevance of the intermediate comparing folding kinetics, cofactor binding kinetics, cofactor affinity, X-ray structure, intrinsic dynamics, redox potential of the apoflavodoxin-cofactor complex (Fld), its affinity for partner protein FNR, and electron transfer rate within the Fld/FNR physiological complex. Our data strongly suggest the intermediate state, conserved in long-chain apoflavodoxins, is not required for the correct assembly of flavodoxin nor does it contribute to shape its electron transfer properties. This analysis can be applied to evaluate other native basin intermediates.

A unified framework based on the binding polynomial for characterizing biological systems by isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) has become the gold-standard technique for studying binding processes due to its high precision and sensitivity, as well as its capability for the simultaneous determination of the association equilibrium constant, the binding enthalpy and the binding stoichiometry. The current widespread use of ITC for biological systems has been facilitated by technical advances and the availability of commercial calorimeters. However, the complexity of data analysis for non-standard models is one of the most significant drawbacks in ITC. Many models for studying macromolecular interactions can be found in the literature, but it looks like each biological system requires specific modeling and data analysis approaches. The aim of this article is to solve this lack of unity and provide a unified methodological framework for studying binding interactions by ITC that can be applied to any experimental system. The apparent complexity of this methodology, based on the binding polynomial, is overcome by its easy generalization to complex systems.

Zinc induced folding is essential for TIM15 activity as an mtHsp70 chaperone.

BACKGROUND: TIM15/Zim17 in yeast and its mammalian ortholog Hep are Zn(2+) finger (Cys4) proteins that assist mtHsp70 in protein import into the mitochondrial matrix. METHODS: Here we characterized the Zn(2+) induced TIM15 folding integrating biophysical and computational approaches. RESULTS: TIM15 folding occurs from an essentially unstructured conformation to a Zn(2+)-coordinated protein in a fast and markedly temperature-dependent process. Moreover, we demonstrate unambiguously that Zn(2+) induced TIM15 folding is essential for its role as mtHsp70 chaperone since in the unstructured apo state TIM15 does not bind to mtHsp70 and is unable to prevent its aggregation. Molecular dynamics simulations help to understand the crucial role of Zn(2+) in promoting a stable and functional 3D architecture in TIM15. It is shown that the metal ion, through its coordinating cysteine residues, can mediate relevant long-range effects with the interaction interface for mtHsp70 coupling thus folding and function. CONCLUSIONS: Zn(2+) induced TIM15 folding is essential for its function and likely occurs in mitochondrial matrix where high concentrations of Zn(2+) were reported. GENERAL SIGNIFICANCE: The combination of experimental and computational approaches presented here provide an integrated structural, kinetic and thermodynamic view of the folding of a mitochondrial zinc finger protein, which might be relevant to understand the organelle import of proteins sharing this fold.

Seizures and dementia in the elderly: Nationwide Inpatient Sample 1999-2008.

OBJECTIVES: This study aimed to examine the association between incidence of admission for a primary diagnosis of "seizure" or "epilepsy" and dementia in a nationally representative database, the Nationwide Inpatient Sample, among the elderly population (55 years of age and above) and to determine whether this relationship is different in individuals with Alzheimer's dementia versus those with non-Alzheimer's dementia. METHODS: Data were obtained from the Nationwide Inpatient Sample using appropriate ICD-9 codes. Frequencies and descriptive analysis adjusting for influence of comorbidities and confounders were utilized. A multivariate logistic regression model adjusted for age, gender, and race was used to further explore the relationship. RESULTS: Those with AD had a higher risk of developing seizures or epilepsy (OR=3.07, 95% CI=2.98-3.16) as compared with cases with NAD (OR=2.20, 95% CI=2.14-2.27). After adjusting for age, the association increased for patients with AD (OR=4.065, 95% CI=3.95-4.17) but not appreciably for patients with NAD (OR=2.68, 95% CI=2.60-2.75). Adding gender and race to the model did not change the relationship for either AD or NAD. Further adjustment for African-American race did not further change the relationship for AD and seizure (OR=3.96, 95% CI=3.854-4.077) as well as for NAD and seizure (OR=2.652, 95% CI=2.575-2.731). Similarly, Hispanic race did not change the relationship significantly for AD (OR=4.1, 95% CI=4.01-4.25) and NAD (OR=2.65, 95% CI=2.56-2.74). CONCLUSION: Patients with AD have a higher prevalence of a seizure compared with patients with NAD. Younger patients with AD were more likely to have seizures. Race, when analyzed as a whole and separately as African-American and Hispanic race, did not alter this relationship.

Snail1 controls bone mass by regulating Runx2 and VDR expression during osteoblast differentiation.

Bone undergoes continuous remodelling throughout adult life, and the equilibrium between bone formation by osteoblasts and bone resorption by osteoclasts defines the final bone mass. Here we show that Snail1 regulates this balance by controlling osteoblast differentiation. Snail1 is necessary for the early steps of osteoblast development, and it must be downregulated for their final differentiation. At the molecular level, Snail1 controls bone mass by repressing the transcription of both the osteoblast differentiation factor Runx2 and the vitamin D receptor (VDR) genes in osteoblasts. Sustained activation of Snail1 in transgenic mice provokes deficient osteoblast differentiation, which, together with the loss of vitamin D signalling in the bone, also impairs osteoclastogenesis. Indeed, the mineralisation of the bone matrix is severely affected, leading to hypocalcemia-independent osteomalacia. Our data show that the impact of Snail1 activity on the osteoblast population regulates the course of bone cells differentiation and ensures normal bone remodelling.

NS3 protease from hepatitis C virus: biophysical studies on an intrinsically disordered protein domain.

The nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) is responsible for processing the non-structural region of the viral precursor polyprotein in infected hepatic cells. NS3 protease activity, located at the N-terminal domain, is a zinc-dependent serine protease. A zinc ion, required for the hydrolytic activity, has been considered as a structural metal ion essential for the structural integrity of the protein. In addition, NS3 interacts with another cofactor, NS4A, an accessory viral protein that induces a conformational change enhancing the hydrolytic activity. Biophysical studies on the isolated protease domain, whose behavior is similar to that of the full-length protein (e.g., catalytic activity, allosteric mechanism and susceptibility to inhibitors), suggest that a considerable global conformational change in the protein is coupled to zinc binding. Zinc binding to NS3 protease can be considered as a folding event, an extreme case of induced-fit binding. Therefore, NS3 protease is an intrinsically (partially) disordered protein with a complex conformational landscape due to its inherent plasticity and to the interaction with its different effectors. Here we summarize the results from a detailed biophysical characterization of this enzyme and present new experimental data.

Bowel Preparation for Colonoscopy Changes Serum Composition as Detected by Thermal Liquid Biopsy and Fluorescence Spectroscopy.

(1) Background: About 50% of prescribed colonoscopies report no pathological findings. A secondary screening test after fecal immunochemical test positivity (FIT+) would be required. Considering thermal liquid biopsy (TLB) as a potential secondary test, the aim of this work was to study possible interferences of colonoscopy bowel preparation on TLB outcome on a retrospective study; (2) Methods: Three groups were studied: 1/514 FIT(+) patients enrolled in a colorectal screening program (CN and CP with normal and pathological colonoscopy, respectively), with blood samples obtained just before colonoscopy and after bowel preparation; 2/55 patients from the CN group with blood sample redrawn after only standard 8-10 h fasting and no bowel preparation (CNR); and 3/55 blood donors from the biobank considered as a healthy control group; (3) Results: The results showed that from the 514 patients undergoing colonoscopy, 247 had CN and 267 had CP. TLB parameters in these two groups were similar but different from those of the blood donors. The resampled patients (with normal colonoscopy and no bowel preparation) had similar TLB parameters to those of the blood donors. TLB parameters together with fluorescence spectra and other serum indicators (albumin and C-reactive protein) confirmed the statistically significant differences between normal colonoscopy patients with and without bowel preparation; (4) Conclusions: Bowel preparation seemed to alter serum protein levels and altered TLB parameters (different from a healthy subject). The diagnostic capability of other liquid-biopsy-based methods might also be compromised. Blood extraction after bowel preparation for colonoscopy should be avoided.

Unexpected thermodynamic signature for the interaction of hydroxymethylated DNA with MeCP2.

Hydroxymethylated cytosine (5hmC) is a stable DNA epigenetic mark recognized by methyl-CpG binding protein 2 (MeCP2), which acts as a transcriptional regulator and a global chromatin-remodeling element. Because 5hmC triggers a gene regulation response markedly different from that produced by methylated cytosine (5mC), both modifications must affect DNA structure and/or DNA interaction with MeCP2 differently. MeCP2 is a six-domain intrinsically disordered protein (IDP) with two domains responsible for dsDNA binding: methyl-CpG binding domain (MBD) and intervening domain (ID). Here we report the detailed thermodynamic characterization of the interaction of hmCpG-DNA with MeCP2. We find that hmCpG-DNA interacts with MeCP2 in a distinctly different mode with a particular thermodynamic signature, compared to methylated or unmethylated DNA. In addition, we find evidence for Rett syndrome-associated mutations altering the interaction of MeCP2 with dsDNA in a cytosine modification-specific manner which may correlate with disease onset time and clinical severity score.

Autochthonous Peruvian Natural Plants as Potential SARS-CoV-2 Mpro Main Protease Inhibitors.

Over 750 million cases of COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), have been reported since the onset of the global outbreak. The need for effective treatments has spurred intensive research for therapeutic agents based on pharmaceutical repositioning or natural products. In light of prior studies asserting the bioactivity of natural compounds of the autochthonous Peruvian flora, the present study focuses on the identification SARS-CoV-2 Mpro main protease dimer inhibitors. To this end, a target-based virtual screening was performed over a representative set of Peruvian flora-derived natural compounds. The best poses obtained from the ensemble molecular docking process were selected. These structures were subjected to extensive molecular dynamics steps for the computation of binding free energies along the trajectory and evaluation of the stability of the complexes. The compounds exhibiting the best free energy behaviors were selected for in vitro testing, confirming the inhibitory activity of Hyperoside against Mpro, with a Ki value lower than 20 µM, presumably through allosteric modulation.

Snail1 is a transcriptional effector of FGFR3 signaling during chondrogenesis and achondroplasias.

Achondroplasias are the most common genetic forms of dwarfism in humans. They are associated with activating mutations in FGFR3, which signal through the Stat and MAPK pathways in a ligand-independent manner to impair chondrocyte proliferation and differentiation. Snail1 has been implicated in chondrocyte differentiation as it represses Collagen II and aggrecan transcription in vitro. Here we demonstrate that Snail1 overexpression in the developing bone leads to achondroplasia in mice. Snail1 acts downstream of FGFR3 signaling in chondrocytes, regulating both Stat and MAPK pathways. Moreover, FGFR3 requires Snail1 during bone development and disease as the inhibition of Snail1 abolishes its signaling even through achondroplastic- and thanatophoric-activating FGFR3 forms. Significantly, Snail1 is aberrantly upregulated in thanatophoric versus normal cartilages from stillborns. Thus, Snail activity may likely be considered a target for achondroplasia therapies.

Snail1 suppresses TGF-beta-induced apoptosis and is sufficient to trigger EMT in hepatocytes.

Although TGF-ß suppresses early stages of tumour development, it later contributes to tumour progression when cells become resistant to its suppressive effects. In addition to circumventing TGF-ß-induced growth arrest and apoptosis, malignant tumour cells become capable of undergoing epithelial-to-mesenchymal transition (EMT), favouring invasion and metastasis. Therefore, defining the mechanisms that allow cancer cells to escape from the suppressive effects of TGF-ß is fundamental to understand tumour progression and to design specific therapies. Here, we have examined the role of Snail1 as a suppressor of TGF-ß-induced apoptosis in murine non-transformed hepatocytes, rat and human hepatocarcinoma cell lines and transgenic mice. We show that Snail1 confers resistance to TGF-ß-induced cell death and that it is sufficient to induce EMT in adult hepatocytes, cells otherwise refractory to this transition upon exposure to TGF-ß. Furthermore, we show that Snail1 silencing prevents EMT and restores the cell death response induced by TGF-ß. As Snail1 is a known target of TGF-ß signalling, our data indicate that Snail1 might transduce the tumour-promoting effects of TGF-ß, namely the EMT concomitant with the resistance to cell death.

Repositioning small molecule drugs as allosteric inhibitors of the BFT-3 toxin from enterotoxigenic Bacteroides fragilis.

Bacteroides fragilis is an abundant commensal component of the healthy human colon. However, under dysbiotic conditions, enterotoxigenic B. fragilis (ETBF) may arise and elicit diarrhea, anaerobic bacteremia, inflammatory bowel disease, and colorectal cancer. Most worrisome, ETBF is resistant to many disparate antibiotics. ETBF's only recognized specific virulence factor is a zinc-dependent metallopeptidase (MP) called B. fragilis toxin (BFT) or fragilysin, which damages the intestinal mucosa and triggers disease-related signaling mechanisms. Thus, therapeutic targeting of BFT is expected to limit ETBF pathogenicity and improve the prognosis for patients. We focused on one of the naturally occurring BFT isoforms, BFT-3, and managed to repurpose several approved drugs as BFT-3 inhibitors through a combination of biophysical, biochemical, structural, and cellular techniques. In contrast to canonical MP inhibitors, which target the active site of mature enzymes, these effectors bind to a distal allosteric site in the proBFT-3 zymogen structure, which stabilizes a partially unstructured, zinc-free enzyme conformation by shifting a zinc-dependent disorder-to-order equilibrium. This yields proBTF-3 incompetent for autoactivation, thus ablating hydrolytic activity of the mature toxin. Additionally, a similar destabilizing effect is observed for the activated protease according to biophysical and biochemical data. Our strategy paves a novel way for the development of highly specific inhibitors of ETBF-mediated enteropathogenic conditions.