Induction of a lasting protective immune response is dependent on presentation of epitopes to patrolling T cells through the HLA complex. While peptide:HLA (pHLA) complex affinity alone is widely ...exploited for epitope selection, we demonstrate that including the pHLA complex stability as a selection parameter can significantly reduce the high false discovery rate observed with predicted affinity. In this study, pHLA complex stability was measured on three common class I alleles and 1286 overlapping 9-mer peptides derived from the SARS-CoV-2 Spike protein. Peptides were pooled based on measured stability and predicted affinity. Strikingly, stability of the pHLA complex was shown to strongly select for immunogenic epitopes able to activate functional CD8
T cells. This result was observed across the three studied alleles and in both vaccinated and convalescent COVID-19 donors. Deconvolution of peptide pools showed that specific CD8
T cells recognized one or two dominant epitopes. Moreover, SARS-CoV-2 specific CD8
T cells were detected by tetramer-staining across multiple donors. In conclusion, we show that stability analysis of pHLA is a key factor for identifying immunogenic epitopes.
In this work, a crystal engineering and thermodynamic based approach has been used aiming at contributing to a deeper knowledge of lamotrigine multicomponent solid forms. Two types of co-molecules ...have been chosen that can give rise to co-crystals with lamotrigine through different supramolecular heterosynthons: the xanthines, theophylline and caffeine, and the three isomeric pyridinecarboxamides. Association with diflunisal, which may result in a salt, was also investigated. Mechanochemistry, differential scanning calorimetry, thermogravimetry, X-ray powder and single crystal diffraction, infrared spectroscopy were the methods used. For all the systems, exploratory neat mechanochemistry experiments, carried out on lamotrigine + co-molecule binary mixtures of different compositions, were not successful in promoting association. From differential scanning calorimetry data and the binary solid-liquid phase diagrams, co-crystals/salts were identified as well as their respective stoichiometry, and a methodology of synthesis was established. For pyridinecarboxamides, molecular recognition is dependent on the position of the amide group in the pyridine ring: co-crystallization did not occur with picolinamide co-former. Both xanthines form co-crystals with lamotrigine, (1:1) with theophylline and (2:1) lamotrigine:caffeine. Additionally, the crystalline structure of a lamotrigine:theophylline 1:1 monohydrate was solved. The (1:1) lamotrigine:theophylline co-crystal converts to this monohydrate in accelerated stability tests. A (1:1) lamotrigine:diflunisal salt was identified, which proved to be stable in accelerated stability assays.
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The demand for sustainable products is increasing worldwide and cosmetic industry is not an exception. Besides exploring nature as source of new ingredients, their production must be sustainable and ...should use environmentally friendly processes. In this work, biogenic silica microparticles were synthesized from sugarcane ash, and their potential application as cosmetic and skincare ingredient was evaluated. For such application, several properties were validated, including cytotoxicity in skin keratinocytes, potential sensitization effect on skin peptides, stimulation of pro-collagen I alpha 1, wound healing capacity, as well as the ingredient stability along a storage period. Biogenic silica showed to be non-cytotoxic on skin keratinocytes, at concentrations up to 5 wt%, and non-skin sensitizer. A positive effect on the stimulation of pro-collagen I alpha 1 suggests a potential anti-ageing activity, while the migration of fibroblasts to a wounded area suggests a regenerative capacity. Under an accelerated stability study, biogenic silica showed an increase on the loss on drying, but no changes were observed on its functional properties, mainly oil absorption capacity, as well the microbiological quality, which was maintained. Overall, novel biogenic silica microparticles produced from a sustainable source are safe, stable over time and have potential to be used as a cosmetic and skincare ingredient.
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•Silica microparticles from sugarcane are not cytotoxicity for skin keratinocytes.•Silica microparticles stimulate pro-collagen I alpha 1 and present wound healing capacity.•Good stability of silica functional properties and on microbiological quality.
Glycyrrhetinic acid is a bioactive compound extracted from licorice that exhibits inhibition effect on various cancers. However, its hydrophobicity results in low bioavailability that limits ...application. We aim to overcome this barrier, the present research was performed to prepare glycyrrhetinic acid proliposomes mediated mannosylated ligand (mannose-diester lauric diacid-cholesterol, MDC) and to evaluate its physicochemical characterizations, environmental stability and bioactivity. In preliminary optimization studies of glycyrrhetinic acid proliposomes mediated MDC (MDC-GA-PL), four optimum operating parameters, cryoprotectant of glucose and mannitol, the mixed cryoprotectant ratio (glucose/mannitol) of 1:1, a cryoprotectant/egg phosphatidylcholine mass ratio of 10/1, and –60 ℃ pre-freezing temperature, were obtained after investigation. Under the optimum lyophilization conditions, MDC-GA-PL was freeze-dried and reconstituted proliposomes were characterized. These proliposomes showed that MDC-GA-PL were well-dispersible spherical particles with an average particle size of 120.80 nm, a polydispersity index about 0.095, a zeta potential of −33.15 mV, encapsulation efficiency of 85.9% and drug loading of 6.38%. In vitro drug release study showed that glycyrrhetinic acid release of MDC-GA-PL conforms to the Higuchi release model. In addition, these proliposomes were stable during six months at 4 ℃. Moreover, acute toxicity assay revealed no substantial safety concern for MDC-GA-PL. Finally, in vitro bioactivity of proliposomes was evaluated. Cytotoxicity effect and apoptosis efficiency of MDC-GA-PL by HepG2 cells was significantly higher than that of glycyrrhetinic acid proliposomes without MDC, demonstrating that MDC has a desirable effect on liver target. Overall, we have reason to believe that MDC-GA-PL would be a promising target delivery to improve therapeutic against hepatic diseases.
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•Glycyrrhetinic acid proliposomes mediated by MDC (MDC-GA-PL) are prepared.•Physicochemical characterizations of MDC-GA-PL possess desirable.•Storage under low temperatures is a promising approach to increase shelf stability.•MDC-GA-PL enhanced the inhibitory effect and the apoptosis of HepG2 cells.•MDC-GA-PL is a promising target delivery system to combat hepatic diseases.
The development and physicochemical characterization of solid lipid nanoparticles (SLNs) with potential for formulating hair cosmetic products were carried out. SLNs were made from Otoba wax, which ...is native to the tropical Andean region and has a high chemical composition of fatty acids with intermediate chains. SLNs were formulated by preparing wax-in-water dispersions at two internal phase proportions (low = 5% w/w and high = 20% w/w), using the same ratio of surfactant system and preservatives. The coarse dispersions were subjected to ultrahigh pressure homogenization (UHPH), and thermal stability assays for 4 weeks were carried out, where changes in Creaming Index, droplet size, polydispersity, viscosity, zeta potential, conductivity, and pH were evaluated. The results showed that Otoba wax has a required HLB value around 9 and is mainly composed of lauric (~35%) and myristic (~45%), which have been reported to improve the condition of hair loss. Regarding the development on SLNs, it was found that the internal phase concentration did not considerably affect the physicochemical and microbiological properties. Likewise, it was found that UHPH enabled the production of SLNs with particle sizes <200 nm, low polydispersity (<0.3), high zeta potential values, and suitable physical and microbiological stability. Therefore, Otoba wax has potential for the development of SLNs applicable to cosmetic formulations, especially for hair products.
Thermal unfolding methods are commonly used as a predictive technique by tracking the protein's physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can ...help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a 'Gold Standard' for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins.
Soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH) are potential targets for several diseases. Previous studies have reported that concomitant selective inhibition of sEH and FAAH ...produced antinociception effects in an animal model of pain. However, the co-administration of a selective sEH inhibitor and a selective FAAH inhibitor might produce serious side effects due to drug-drug interactions that could complicate drug development in the long term. Thus, discovering dual sEH/FAAH inhibitors, single small molecules that can simultaneously inhibit both sEH and FAAH, would be a significant accomplishment in the medicinal chemistry field. Herein, we report the synthesis and biological evaluation of benzothiazole-phenyl-based analogs as potential dual sEH/FAAH inhibitors. This work represents a follow-up structure-activity relationship (SAR) and metabolic-stability studies of our best dual sEH/FAAH inhibitor identified previously, as well as in vivo evaluation of its effects on voluntary locomotor behavior in rats. Our SAR study indicates that trifluoromethyl groups on the aromatic rings are well tolerated by the targeted enzymes when placed at the ortho and para positions; however, they, surprisingly, did not improve metabolic stability in liver microsomes. Our behavioral studies indicate that doses of dual sEH/FAAH inhibitors that alleviate pain do not depress voluntary behavior in naïve rats, which is a common side effect of currently available analgesic drugs (e.g., opioids). Thus, dual sEH/FAAH inhibitors may be a safe and effective approach to treat pain.
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•Several potent dual sEH/FAAH inhibitors have been discovered.•All new dual inhibitors were active on rat and human sEH, but not on mouse sEH.•The most active dual inhibitor exhibited short half-life in microsomal assay.•Therapeutic doses of lead compound do not impair activity in in vivo evaluation.
A heterodimeric receptor subunit, Integrin αV, often complexed with Integrin β3 plays a vital role in cell signaling to regulate angiogenesis and promote cancer progression. The paramount β-turn ...formed from pentapeptide residues (PPQEE) in the cytoplasmic domain of Integrin αV was previously reported as crucial for cell signaling and its deletion was proved deleterious for protein's cell membrane adhesion and ligand binding properties. This study revealed conformational changes in the Integrin αV subunit upon deletion of PPQEE residues through in silico structural modelling approach followed by analysis of alteration of binding sites. Human Protein Atlas database helped to identify the association of Integrin αV to the unfavourable prognosis of three gastrointestinal cancers: stomach, liver and pancreatic cancers. Molecular modelling and docking techniques were carried out for the necessary complex formations (wild-type and mutant-type). Further comparison was performed for the complexes. The changes in protein's conformation and stability due to PPQEE deletion were observed in both independent subunit and heterodimer. The most noteworthy conformational shift was the disruption of a transmembrane helix into coil, which accounted for protein's impaired cell membrane adhesion, increased solvent accessibility and decreased stability. The deletion also caused a reduction of beta-turn regions, which disrupted ligand binding in the cytoplasmic domain of Integrin αV subunit. This study emphasized on structural basis of how the deletion of PPQEE residues alters stability, ligand binding and signaling activity of Integrin αV subunit highlighting the importance of these residues in maintenance of protein's native structure.
The human Tribbles (TRB)-related pseudokinases are CAMK (calcium/calmodulin-dependent protein kinase)-related family members that have evolved a series of highly unusual motifs in the ...'pseudocatalytic' domain. In canonical kinases, conserved amino acids bind to divalent metal ions and align ATP prior to efficient phosphoryl-transfer to substrates. However, in pseudokinases, atypical residues give rise to diverse and often unstudied biochemical and structural features that are thought to be central to cellular functions. TRB proteins play a crucial role in multiple signalling networks and overexpression confers cancer phenotypes on human cells, marking TRB pseudokinases out as a novel class of drug target. In the present paper, we report that the human pseudokinase TRB2 retains the ability to both bind and hydrolyse ATP weakly in vitro. Kinase activity is metal-independent and involves a catalytic lysine residue, which is conserved in TRB proteins throughout evolution alongside several unique amino acids in the active site. A similar low level of autophosphorylation is also preserved in the closely related human TRB3. By employing chemical genetics, we establish that the nucleotide-binding site of an 'analogue-sensitive' (AS) TRB2 mutant can be targeted with specific bulky ligands of the pyrazolo-pyrimidine (PP) chemotype. Our analysis confirms that TRB2 retains low levels of ATP binding and/or catalysis that is targetable with small molecules. Given the significant clinical successes associated with targeting of cancer-associated kinases with small molecule inhibitors, it is likely that similar approaches will be useful for further evaluating the TRB pseudokinases, with the translation of this information likely to furnish new leads for drug discovery.
Uncoupling protein 1 (UCP1) catalyses mitochondrial proton leak in brown adipose tissue to facilitate nutrient oxidation for heat production, and may combat metabolic disease if activated in humans. ...During the adrenergic stimulation of brown adipocytes, free fatty acids generated from lipolysis activate UCP1 via an unclear interaction. Here, we set out to characterise activator binding to purified UCP1 to clarify the activation process, discern novel activators and the potential to target UCP1.
We assessed ligand binding to purified UCP1 by protein thermostability shift analysis, which unlike many conventional approaches can inform on the binding of hydrophobic ligands to membrane proteins. A detailed activator interaction analysis and screening approach was carried out, supported by investigations of UCP1 activity in liposomes, isolated brown fat mitochondria and UCP1 expression-controlled cell lines.
We reveal that fatty acids and other activators influence UCP1 through a specific destabilising interaction, behaving as transport substrates that shift the protein to a less stable conformation of a transport cycle. Through the detection of specific stability shifts in screens, we identify novel activators, including the over-the-counter drug ibuprofen, where ligand analysis indicates that UCP1 has a relatively wide structural specificity for interacting molecules. Ibuprofen successfully induced UCP1 activity in liposomes, isolated brown fat mitochondria and UCP1-expressing HEK293 cells but not in cultured brown adipocytes, suggesting drug delivery differs in each cell type.
These findings clarify the nature of the activator-UCP1 interaction and demonstrate that the targeting of UCP1 in cells by approved drugs is in principle achievable as a therapeutic avenue, but requires variants with more effective delivery in brown adipocytes.
•UCP1 thermostability shifts can identify fatty acid activator binding.•Activators destabilise UCP1, binding as transport substrates in the mechanism.•UCP1 has a wide ligand specificity for activators.•Screens identify new activators, including the licenced drug ibuprofen.•Drug-targeting UCP1 in cells is viable to increase energy expenditure.
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