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The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

Funder: Biotechnology and Biological Sciences Research Council, Funder: Wellcome Trust, BACKGROUND: Cystic Fibrosis (CF) is a genetic disorder affecting around 1 in every 3000 newborns. In the most common mutation, F508del, the defective anion channel, CFTR, is prevented from reaching the plasma membrane (PM) by the quality check control of the cell. Little is known about how CFTR pharmacological rescue impacts the cell proteome. METHODS: We used high-resolution mass spectrometry, differential ultracentrifugation, machine learning and bioinformatics to investigate both changes in the expression and localization of the human bronchial epithelium CF model (F508del-CFTR CFBE41o-) proteome following treatment with VX-809 (Lumacaftor), a drug able to improve the trafficking of CFTR. RESULTS: The data suggested no stark changes in protein expression, yet subtle localization changes of proteins of the mitochondria and peroxisomes were detected. We then used high-content confocal microscopy to further investigate the morphological and compositional changes of peroxisomes and mitochondria under these conditions, as well as in patient-derived primary cells. We profiled several thousand proteins and we determined the subcellular localization data for around 5000 of them using the LOPIT-DC spatial proteomics protocol. CONCLUSIONS: We observed that treatment with VX-809 induces extensive structural and functional remodelling of mitochondria and peroxisomes that resemble the phenotype of healthy cells. Our data suggest additional rescue mechanisms of VX-809 beyond the correction of aberrant folding of F508del-CFTR and subsequent trafficking to the PM.

Loss-of-function mutations of the CFTR gene cause cystic fibrosis (CF) through a variety of molecular mechanisms involving altered expression, trafficking, and/or activity of the CFTR chloride channel. The most frequent mutation among CF patients, F508del, causes multiple defects that can be, however, overcome by a combination of three pharmacological agents that improve CFTR channel trafficking and gating, namely, elexacaftor, tezacaftor, and ivacaftor. This study was prompted by the evidence of two CF patients, compound heterozygous for F508del and a minimal function variant, who failed to obtain any beneficial effects following treatment with the triple drug combination. Functional studies on nasal epithelia generated in vitro from these patients confirmed the lack of response to pharmacological treatment. Molecular characterization highlighted the presence of an additional amino acid substitution, L467F, in cis with the F508del variant, demonstrating that both patients were carriers of a complex allele. Functional and biochemical assays in heterologous expression systems demonstrated that the double mutant L467F-F508del has a severely reduced activity, with negligible rescue by CFTR modulators. While further studies are needed to investigate the actual prevalence of the L467F-F508del allele, our results suggest that this complex allele should be taken into consideration as plausible cause in CF patients not responding to CFTR modulators.

The advent of Trikafta (Kaftrio in Europe) (a triple-combination therapy based on two correctors—elexacaftor/tezacaftor—and the potentiator ivacaftor) has represented a revolution for the treatment of patients with cystic fibrosis (CF) carrying the most common misfolding mutation, F508del-CFTR. This therapy has proved to be of great efficacy in people homozygous for F508del-CFTR and is also useful in individuals with a single F508del allele. Nevertheless, the efficacy of this therapy needs to be improved, especially in light of the extent of its use in patients with rare class II CFTR mutations. Using CFBE41o- cells expressing F508del-CFTR, we provide mechanistic evidence that targeting the E1 ubiquitin-activating enzyme (UBA1) by TAK-243, a small molecule in clinical trials for other diseases, boosts the rescue of F508del-CFTR induced by CFTR correctors. Moreover, TAK-243 significantly increases the F508del-CFTR short-circuit current induced by elexacaftor/tezacaftor/ivacaftor in differentiated human primary airway epithelial cells, a gold standard for the pre-clinical evaluation of patients’ responsiveness to pharmacological treatments. This new combinatory approach also leads to an improvement in CFTR conductance on cells expressing other rare CF-causing mutations, including N1303K, for which Trikafta is not approved. These findings show that Trikafta therapy can be improved by the addition of a drug targeting the misfolding detection machinery at the beginning of the ubiquitination cascade and may pave the way for an extension of Trikafta to low/non-responding rare misfolded CFTR mutants.

Cystic fibrosis (CF) is caused by loss of function of the CFTR chloride channel. A substantial number of CF patients carry nonsense mutations in the CFTR gene. These patients cannot directly benefit from pharmacological correctors and potentiators that have been developed for other types of CFTR mutations. We evaluated the efficacy of combinations of drugs targeting at various levels the effects of nonsense mutations: SMG1i to protect CFTR mRNA from nonsense-mediated decay (NMD), G418 and ELX-02 for readthrough, VX-809 and VX-445 to promote protein maturation and function, PTI-428 to enhance CFTR protein synthesis. We found that the extent of rescue and sensitivity to the various agents is largely dependent on the type of mutation, with W1282X and R553X being the mutations most and least sensitive to pharmacological treatments, respectively. In particular, W1282X-CFTR was highly responsive to NMD suppression by SMG1i but also required treatment with VX-445 corrector to show function. In contrast, G542X-CFTR required treatment with readthrough agents and VX-809. Importantly, we never found cooperativity between the NMD inhibitor and readthrough compounds. Our results indicate that treatment of CF patients with nonsense mutations requires a precision medicine approach with the design of specific drug combinations for each mutation.

Deletion of phenylalanine at position 508 (F508del) in the CFTR chloride channel is the most frequent mutation in cystic fibrosis (CF) patients. F508del impairs the stability and folding of the CFTR protein, thus resulting in mistrafficking and premature degradation. F508del-CFTR defects can be overcome with small molecules termed correctors. We investigated the efficacy and properties of VX-445, a newly developed corrector, which is one of the three active principles present in a drug (Trikafta®/Kaftrio®) recently approved for the treatment of CF patients with F508del mutation. We found that VX-445, particularly in combination with type I (VX-809, VX-661) and type II (corr-4a) correctors, elicits a large rescue of F508del-CFTR function. In particular, in primary bronchial epithelial cells of CF patients, the maximal rescue obtained with corrector combinations including VX-445 was close to 60–70% of CFTR function in non-CF cells. Despite this high efficacy, analysis of ubiquitylation, resistance to thermoaggregation, protein half-life, and subcellular localization revealed that corrector combinations did not fully normalize F508del-CFTR behavior. Our study indicates that it is still possible to further improve mutant CFTR rescue with the development of corrector combinations having maximal effects on mutant CFTR structural and functional properties.

A novel class of transmembrane anion carriers, the click-tambjamines, display remarkable anionophoric activities in model liposomes and living cells. The versatility of this building block for the generation of molecular diversity offers promise to develop future drugs based on this design., European Union’s Horizon 2020 research and innovation programme (TAT-CF project, grant agreement 667079), Instituto de Salud Carlos III (Grant PI18/00441) (co-funded by the European Regional Development Fund (ERDF), a way to build Europe) and “La Caixa” Foundation and Caja Burgos Foundation (CAIXAUBU004)

BACKGROUND AND PURPOSE: Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease that originates from the defective function of the CF transmembrane conductance regulator (CFTR) protein, a cAMP‐dependent anion channel involved in fluid transport across epithelium. Because small synthetic transmembrane anion transporters (anionophores) can replace the biological anion transport mechanisms, independent of genetic mutations in the CFTR, such anionophores are candidates as new potential treatments for CF. EXPERIMENTAL APPROACH: In order to assess their effects on cell physiology, we have analysed the transport properties of five anionophore compounds, three prodigiosines and two tambjamines. Chloride efflux was measured in large uni‐lamellar vesicles and in HEK293 cells with chloride‐sensitive electrodes. Iodide influx was evaluated in FRT cells transfected with iodide‐sensitive YFP. Transport of bicarbonate was assessed by changes of pH after a NH(4) (+) pre‐pulse using the BCECF fluorescent probe. Assays were also carried out in FRT cells permanently transfected with wild type and mutant human CFTR. KEY RESULTS: All studied compounds are capable of transporting halides and bicarbonate across the cell membrane, with a higher transport capacity at acidic pH. Interestingly, the presence of these anionophores did not interfere with the activation of CFTR and did not modify the action of lumacaftor (a CFTR corrector) or ivacaftor (a CFTR potentiator). CONCLUSION AND IMPLICATIONS: These anionophores, at low concentrations, transported chloride and bicarbonate across cell membranes, without affecting CFTR function. They therefore provide promising starting points for the development of novel treatments for CF.

Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3β (GSK-3β) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3β inhibitors having the common N-[(1-alkylpiperidin-4-yl)methyl]-1H-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound 5 with GSK-3β. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3β inhibitors with good cell activity. Among the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3β inhibitors as new tools in the development of new treatments for mood disorders.

Anion selective ionophores, anionophores, are small molecules capable of facilitating the transmembrane transport of anions. Inspired in the structure of natural product prodigiosin, four novel anionophores 1a-d, including a 1,2,3-triazole group, were prepared. These compounds proved highly efficient anion exchangers in model phospholipid liposomes. The changes in the hydrogen bond cleft modified the anion transport selectivity exhibited by these compounds compared to prodigiosin and suppressed the characteristic high toxicity of the natural product. Their activity as anionophores in living cells was studied and chloride efflux and iodine influx from living cells mediated by these derivatives was demonstrated. These compounds were shown to permeabilize cellular membranes to halides with efficiencies close to the natural anion channel CFTR at doses that do not compromise cellular viability. Remarkably, optimal transport efficiency was measured in the presence of pH gradients mimicking those found in the airway epithelia of Cystic Fibrosis patients. These results support the viability of developing small molecule anionophores as anion channel protein surrogates with potential applications in the treatment of conditions such as Cystic Fibrosis derived from the malfunction of natural anion transport mechanisms., European Union’s Horizon 2020 research and innovation programme under grant agreement No. 667079, La Marató de TV3 Foundation (20132730), Consejería de Educación de la Junta de Castilla y León (Projects BU340U13 and BU092U16)

© 2015 American Chemical Society. Fatty acid ethanolamides such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are lipid-derived mediators that potently inhibit pain and inflammation by ligating type-α peroxisome proliferator-activated receptors (PPAR-α). These bioactive substances are preferentially degraded by the cysteine hydrolase, N-acylethanolamine acid amidase (NAAA), which is highly expressed in macrophages. Here, we describe a new class of β-lactam derivatives that are potent, selective, and systemically active inhibitors of intracellular NAAA activity. The prototype of this class deactivates NAAA by covalently binding the enzymes catalytic cysteine and exerts profound anti-inflammatory effects in both mouse models and human macrophages. This agent may be used to probe the functions of NAAA in health and disease and as a starting point to discover better anti-inflammatory drugs.

Cl− channels activated by acidic extracellular pH have been observed in various mammalian cells but their molecular identity and mechanisms of regulation are unknown. The aim of this study was to analyse the acid-activated Cl− current (ICl(H)) by elucidating its functional properties and mechanisms of regulation in three different cell types: primary human bronchial epithelial (HBE) cells, neuroblastoma SK-N-MC cells and HEK-293 cells. We found that outward rectification, sensitivity to acidic pH (50% activation at pH5.15), permeability sequence (SCN−>I−>Br−>Cl−>gluconate), voltage dependence and sensitivity to blockers of ICl(H) were identical in all cells. These findings suggest a common molecular basis for ICl(H). We analysed the possible relationship of ICl(H) with members of ClC and TMEM16 protein families. By gene silencing, validated using RT-PCR, we found that ICl(H) is unrelated to ClC-3, ClC-7, TMEM16A, TMEM16D, TMEM16F, TMEM16H and TMEM16K. Analysis of possible mechanisms of regulation indicate that Ca2+, ATP and phosphorylation by PKA or PKC do not seem to be implicated in channel activation. Instead, the inhibition of ICl(H) by genistein and wortmannin suggest regulation by other kinases, possibly a tyrosine kinase and a phosphatidylinositol-3-kinase. Moreover, by using dynasore, the dynamin inhibitor, we found indications that exo/endocytosis is a mechanism responsible for ICl(H) regulation. Our results provide the first evidence about acid-activated Cl− channel regulation and, thus, could open the way for a better understanding of the channel function and for the molecular identification of the underlying protein.