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Ferreira, R.

Publications and source records attributed to Ferreira, R..

29 records · Page 2Linked to original sources

Structure-based identification of naphthoquinones and derivatives as novel inhibitors of main protease Mpro and papain-like protease PLpro of SARS-CoV-2

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In the present work, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 {micro}M and 66 {micro}M. In addition, eight compounds inhibited PLpro with IC50 ranging from 1.7 {micro}M to 46 {micro}M. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

biochemistry↗

HLA class II mediates type 1 diabetes risk by anti-insulin repertoire selection

Type 1 diabetes (T1D) is a common autoimmune disorder characterized by the destruction of insulin-secreting pancreatic {beta} cells [1], in which polymorphism of the human leukocyte antigen (HLA) class II region is the major genetic risk factor [2, 3, 4]. However, how variation in class II molecules alters T1D risk remains a longstanding question. Here we show how T1D risk due to HLA class II haplotype combinations [5] correlates with the frequency of negatively charged sequences in the CDR3{beta} region of CD4+ T cell receptor (TCR) repertoires purified from peripheral blood. These sequences are known to be common in receptors that bind insulin B:9-23 [6], the primary autoantigen in T1D. We also show the same effect in circulating activated CD4+ T cells from newly-diagnosed T1D cases, and in islet-infiltrating T cells from patients with active T1D. Furthermore, we demonstrate that the proportion of insulin-reactive CD4+ T cells present in islets is predicted by the frequency of these negatively charged CDR3{beta} amino acid sequences. Our results suggest diagnostic uses of T cell repertoire profiling in early detection of insulin autoimmunity, and inform ongoing efforts to improve tolerance induction to insulin and prevention of T1D [7].

genomics↗

Metabolomics, Transcriptomics and Functional Glycomics Reveals Bladder Cancer Cells Plasticity and Enhanced Aggressiveness Facing Hypoxia and Glucose Deprivation

Bladder cancer constitutes one of the deadliest genitourinary diseases, especially when diagnosed at late stages. These tumours harbour microenvironmental niches characterized by low levels of oxygen (hypoxia) and limited glucose supply due to poor vascularization. However, the synergic contribution of these features to disease development is poorly understood. Here, we demonstrated that cells with distinct histopathological and molecular backgrounds responded similarly to such stimuli. Cancer cells arrested proliferation, significantly increased invasive capacity in vitro and enhanced tolerance to cisplatin-based chemotherapy. Reoxygenation and access to glucose restored basal proliferation and invasion levels without triggering stress-induced apoptosis, denoting significant cellular plasticity in adapting to microenvironmental cues. Whole transcriptomics showed major molecular reprogramming, supporting main functional alterations. Metabolomics evidenced fatty acids {beta}-oxidation as main bioenergetic pathway rather than anaerobic glycolysis generally adopted by hypoxic cells. Joint pathway analysis also suggested relevant alterations in mucin-type O-glycan biosynthesis. Glycomics confirmed a major antagonization of O-glycosylation pathways, leading to simple cell glycophenotypes characterized by the accumulation of immature short-chain O-glycans such as Tn and STn antigens at the cell surface. Glycoengineered models reflecting simple cell glycophenotypes were developed and functional studies in vitro and in vivo showed that Tn and STn overexpression decreased proliferation and promoted chemoresistance, reinforcing their close link with tumour aggressiveness. Collectively, we have demonstrated that hypoxia and glucose deprivation trigger more aggressive cell behaviours, in what appears to be an escape mechanism from microenvironmental stress. We propose that, altered glycosylation may be used to target these subpopulations, paving the way for precision oncology.

cancer biology↗

Nuclear stabilisation of p53 requires a functional nucleolar surveillance pathway

The nucleolar surveillance pathway (NSP) monitors nucleolar fidelity and responds to nucleolar stresses (i.e., inactivation of ribosome biogenesis) by mediating the inhibitory binding of ribosomal proteins (RPs) to mouse double minute 2 homolog (MDM2), a nuclear-localised E3 ubiquitin ligase, which results in p53 accumulation. Inappropriate activation of the NSP has been implicated in the pathogenesis of collection of human diseases termed "ribosomopathies", while drugs that selectively activate the NSP are now in trials for cancer. Despite the clinical significance, the precise molecular mechanism(s) regulating the NSP remain poorly understood. Using genome-wide loss of function screens, we demonstrate the ribosome biogenesis (RiBi) axis as the most potent class of genes whose disruption stabilises p53. Furthermore, we identified a novel suite of genes critical for the NSP, including a novel mammalian protein implicated in 5S ribonucleoprotein particle (5S-RNP) biogenesis, HEATR3. By selectively disabling the NSP, we unexpectedly demonstrate that a functional NSP is required for the ability of all nuclear acting stresses tested, including DNA damage, to robustly induce p53 accumulation. Together, our data demonstrates that the NSP has evolved as the dominant central integrator of stresses that regulate nuclear p53 abundance, thus ensuring RiBi is hardwired to cellular proliferative capacity.

cell biology↗

Dissecting cell type-specific metabolism in pancreatic ductal adenocarcinoma

Tumors are composed of many different cell types including cancer cells, fibroblasts, and immune cells. Dissecting functional metabolic differences between various cell types within a mixed population can be limited by the rapid turnover of metabolites relative to the time needed to isolate cells. To overcome this challenge, we traced isotope-labeled nutrients into macromolecules that turn over more slowly than metabolites. This approach was used to assess differences between cancer cell and fibroblast metabolism in pancreatic cancer organoid-fibroblast co-cultures and in pancreatic tumors. In these contexts, we find pancreatic cancer cells exhibit increased pyruvate carboxylation relative to fibroblasts, and that this flux depends on both pyruvate carboxylase and malic enzyme 1 activity. Consequently, expression of both enzymes in cancer cells is necessary for organoid and tumor growth, demonstrating that dissecting the metabolism of specific cell populations within heterogeneous systems can identify dependencies that may not be evident from studying isolated cells in culture or bulk tumor tissue.

cancer biology↗

CRISPR-mediated biocontainment

We have exploited the repetitive nature of transposable elements of the human genome to generate synthetic circuits. Transposable elements such as LINE-1 and Alu have successfully replicated in mammalian genomes throughout evolution to reach a copy number ranging from thousands to more than a million. Targeting these repetitive elements with programmable DNA nucleases such as CRISPR-Cas9 rapidly induce extremely high levels of cell death. We use this genotoxic feature to build synthetic biocontainment circuits: CRISPR defense system (CRISPR-DS) capable of preventing CRISPR genome editing, and we introduce the proof-of-concept of CRISPR Safety-Switch, an inducible, stringent and non-leaky kill-switch capable of clearing out cell lines resistant to DNA breaks.

synthetic biology↗

Mechanistic Elucidation of the Antitumor Properties of a Novel Death Receptor 5 Activator

Disulfide bond Disrupting Agents (DDAs) are a new chemical class of agents recently shown to have activity against breast tumors in animal models. However, it is unknown how DDAs trigger cancer cell death without affecting nontransformed cells. As demonstrated here, DDAs are the first compounds identified that upregulate the TRAIL receptor DR5 through both transcriptional and posttranscriptional mechanisms. At the protein level, DDAs alter DR5 disulfide bonding to increase steady-state DR5 levels and oligomerization, leading to downstream Caspase 8 and 3 activation. DDAs and TRAIL synergize to kill cancer cells and are cytotoxic to HER2+ cancer cells with acquired resistance to the EGFR/HER2 tyrosine kinase inhibitor. Investigation of the mechanisms responsible for DDA selectivity for cancer cells reveals that DDA-induced upregulation of DR5 is enhanced in the context of EGFR overexpression, and DDA-induced cytotoxicity is strongly amplified by MYC overexpression. Together, the results demonstrate selective DDA lethality against oncogene-transformed cells, DDA-mediated DR5 upregulation and protein stabilization, and DDAs against drug-resistant and metastatic cancer cells. DDAs thus represent a new therapeutic approach to cancer therapy.

cancer biology↗

Does human P-glycoprotein efflux involve transmembrane alpha helix breakage?

The occluded conformation suggested in a recent article that revealed a new inward-facing conformation for the human P-glycoprotein may not represent the closing of a gate region but instead an artifact derived from lateral compression in a too small sized nanodisc, used to stabilize the transmembrane domains of the transporter.

biophysics↗

Enabling large-scale genome editing by reducing DNA nicking

To extend the frontier of genome editing and enable the radical redesign of mammalian genomes, we developed a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements - ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.\n\nOne Sentence SummaryBase editing with reduced DNA nicking allows for the simultaneous editing of >10,000 loci in human cells.

synthetic biology↗

The plasma biomarker soluble SIGLEC-1 is associated with the type I interferon transcriptional signature, ethnic background and renal disease in systemic lupus erythematosus

The molecular heterogeneity of autoimmune and inflammatory diseases has been one of the main obstacles to the development of safe and specific therapeutic options. Here we have evaluated the diagnostic and clinical value of a robust, inexpensive, immunoassay detecting the circulating soluble form of the monocyte-specific surface receptor sialic acid binding Ig-like lectin 1 (sSIGLEC-1).\n\nWe developed an immunoassay to measure sSIGLEC-1 in small volumes of plasma/serum from systemic lupus erythematosus (SLE) patients and healthy donors. Plasma concentrations of sSIGLEC-1 strongly correlated with expression of SIGLEC-1 on the surface of blood monocytes and with type I interferon (IFN)-regulated gene (IRG) expression in SLE patients. In addition, we identified marked ancestry-related differences in sSIGLEC-1 concentrations in SLE patients, with patients of non-European ancestry showing higher levels compared to patients of European ancestry. Higher sSIGLEC-1 concentrations were associated with lower serum complement component 3 and increased frequency of renal complications in European patients, but not with the SLEDAI clinical score.\n\nOur sSIGLEC-1 immunoassay provides a specific and easily-assayed marker for monocyte-macrophage activation, and interferonopathy in SLE and other diseases. Further studies can extend its clinical associations and its potential use to stratify patients and as a secondary endpoint in trials.

immunology↗

Molecular and functional variation in iPSC-derived sensory neurons

Induced pluripotent stem cells (iPSCs), and cells derived from them, have become key tools to model biological processes and disease mechanisms, particularly in cell types such as neurons that are difficult to access from living donors. Here, we present the first map of regulatory variants in an iPSC-derived cell type. To investigate genetic contributions to human sensory function, we performed 123 differentiations of iPSCs from 103 unique donors to a sensory neuronal fate, and measured gene expression, chromatin accessibility, and neuronal excitability. Compared with primary dorsal root ganglion, where sensory nerves collect near the spinal cord, gene expression was more variable across iPSC-derived neuronal cultures, particularly in genes related to differentiation and nervous system development. Single cell RNA-sequencing revealed that although the majority of cells are neuronal and express the expected marker genes, a substantial fraction have a fibroblast-like expression profile. By applying an allele-specific method we identify 3,778 quantitative trait loci influencing gene expression, 6,318 for chromatin accessibility, and 2,097 for RNA splicing at FDR 10%. A number of these overlap with common disease associations, and suggest candidate causal variants and target genes. These include known causal variants at SNCA for Parkinsons disease and TNFRSF1A for multiple sclerosis, as well as new candidates for migraine, Parkinsons disease, and schizophrenia.

genomics↗