Adsorption of proteins to thin-films of PDMS and its effect on the adhesion of human endothelial cells

This paper describes a simple and inexpensive procedure to produce thin-films of poly(dimethylsiloxane). Such films were characterized by a variety of techniques (ellipsometry, nuclear magnetic resonance, atomic force microscopy, and goniometry) and used to investigate the adsorption kinetics of three model proteins (fibrinogen, collagen type-I, and bovine serum albumin) under different conditions. The information collected from the protein adsorption studies was then used to investigate the adhesion of human dermal microvascular endothelial cells. The results of these studies suggest that these films can be used to model the surface properties of microdevices fabricated with commercial PDMS. Moreover, the paper provides guidelines to efficiently attach cells in BioMEMS devices.Fil: Chumbimuni Torres, Karin Y.. The University of Texas at San Antonio; Estados UnidosFil: Coronado, Ramon E.. The University of Texas at San Antonio; Estados UnidosFil: Mfuh, Adelphe M.. The University of Texas at San Antonio; Estados UnidosFil: Castro Guerrero, Carlos. The University of Texas at San Antonio; Estados UnidosFil: Silva, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Negrete, George R.. The University of Texas at San Antonio; Estados UnidosFil: Bizios, Rena. The University of Texas at San Antonio; Estados UnidosFil: Garcia, Carlos D.. The University of Texas at San Antonio; Estados Unido

Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs

Many therapeutics elicit cell-type specific polypharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule interactome mapping by photoaffinity labeling (SIM-PAL) and applied it to the in cellulo direct characterization of specific NSAID binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment and isotope-recoding of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, we identified the NSAID interactome consisting of over 1000 significantly enriched proteins and directly characterized nearly 200 conjugated peptides representing direct binding sites of the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-κB) and novel interactions (e.g., AP-2, proteasome). The conjugated peptides revealed direct NSAID binding sites from the cell surface to the nucleus and a specific binding site hotspot for the three photo-NSAIDs on histones H2A and H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal shift assay. Since small molecule stabilization of protein complexes is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest

Scalable, Metal- and Additive-Free, Photoinduced Borylation of Haloarenes and Quaternary Arylammonium Salts

We report herein a simple, metal- and additive-free, photoinduced borylation of haloarenes, including electron-rich fluoroarenes, as well as arylammonium salts directly to boronic acids. This borylation method has a broad scope and functional group tolerance. We show that it can be further extended to boronic esters and carried out on gram scale as well as under flow conditions

Concise Total Synthesis of Trichodermamides A, B, and C Enabled by an Efficient Construction of the 1,2-Oxazadecaline Core

We report herein a facile and efficient method of the construction of the <i>cis</i>-1,2-oxazadecaline system, distinctive of (pre)­trichodermamides, aspergillazine A, gliovirin, and FA-2097. The formation of the 1,2-oxazadecaline core was accomplished by a 1,2-addition of an α<i>C</i>-lithiated <i>O</i>-silyl ethyl pyruvate oxime to benzoquinone, which is followed by an oxa-Michael ring-closure. The method was successfully applied to the concise total synthesis of trichodermamide A (in gram quantities) and trichodermamide B, as well as the first synthesis of trichodermamide C

Cytotoxicity and Mechanism of Action of the Marine-Derived Fungal Metabolite Trichodermamide B and Synthetic Analogues

The trichodermamides are modified dipeptides isolated from a wide variety of fungi, including <i>Trichoderma virens</i>. Previous studies reported that trichodermamide B (<b>2</b>) initiated cytotoxicity in HCT-116 colorectal cancer cells, while trichodermamide A (<b>1</b>) was devoid of activity. We recently developed an efficient total synthesis for the trichodermamides A–C (<b>1</b>–<b>3</b>). Multiple intermediates and analogues were produced, and they were evaluated for biological effects to identify additional structure–activity relationships and the possibility that a simplified analogue would retain the biological effects of <b>2</b>. The antiproliferative effects of 18 compounds were evaluated, and the results show that <b>2</b> and four other compounds are active in HeLa cells, with IC₅₀ values in the range of 1.4–21 μM. Mechanism of action studies of <b>2</b> and the other active analogues revealed different spectra of activity. At the IC₈₅ concentration, <b>2</b> caused S-phase accumulation and cell death in HeLa cells, suggesting response to DNA double-strand breaks. The analogues did not cause S-phase accumulation or induction of DNA damage repair pathways, consistent with an alternate mode of action. The mechanistic differences are hypothesized to be due to the chlorohydrin moiety in <b>2</b>, which is lacking in the analogues, which could form a DNA-reactive epoxide

Additive- and Metal-Free, Predictably 1,2- and 1,3-Regioselective, Photoinduced Dual C–H/C–X Borylation of Haloarenes

We report herein a simple, additive- and metal-free, photoinduced, dual C–H/C–X borylation of chloro-, bromo-, and iodoarenes. The reaction produces 1,2- and 1,3-diborylarenes on gram scales under batch and continuous flow conditions. The regioselectivity of the dual C–H/C–X borylation is determined by the solvent and the substituents in the parent haloarenes

Additive- and Metal-Free, Predictably 1,2- and 1,3-Regioselective, Photoinduced Dual C–H/C–X Borylation of Haloarenes

We report herein a simple, additive- and metal-free, photoinduced, dual C–H/C–X borylation of chloro-, bromo-, and iodoarenes. The reaction produces 1,2- and 1,3-diborylarenes on gram scales under batch and continuous flow conditions. The regioselectivity of the dual C–H/C–X borylation is determined by the solvent and the substituents in the parent haloarenes

Identification of Inhibitors of CD36-Amyloid Beta Binding as Potential Agents for Alzheimer’s Disease

Neuroinflammation is one of the hallmarks of Alzheimer’s disease pathology. Amyloid β has a central role in microglia activation and the subsequent secretion of inflammatory mediators that are associated with neuronal toxicity. The recognition of amyloid β by microglia depends on the expression of several receptors implicated in the clearance of amyloid and in cell activation. CD36 receptor expressed on microglia interacts with fibrils of amyloid inducing the release of proinflammatory cytokines and amyloid internalization. The interruption of the interaction CD36-amyloid β compromises the activation of microglia cells. We have developed and validated a new colorimetric assay to identify potential inhibitors of the binding of amyloid β to CD36. We have found seven molecules, structural analogues of the Trichodermamide family of natural products that interfere with the interaction CD36-amyloid β. By combining molecular docking and dynamics simulations, we suggested the second fatty acids binding site within the large luminal hydrophobic tunnel, present in the extracellular domain of CD36, as the binding pocket of these compounds. Free energy calculations predicted the nonpolar component as the driving force for the binding of these inhibitors. These molecules also inhibited the production of TNF-α, IL-6, and IL-1β by peritoneal macrophages stimulated with fibrils of amyloid β. This work serves as a platform for the identification of new potential anti-inflammatory agents for the treatment of Alzheimer’s disease