Live 3D imaging and mapping of shear stresses within tissues using incompressible elastic beads

To investigate the role of mechanical constraints in morphogenesis and development, we develop a pipeline of techniques based on incompressible elastic sensors. These techniques combine the advantages of incompressible liquid droplets, which have been used as precise in situ shear stress sensors, and of elastic compressible beads, which are easier to tune and to use. Droplets of a polydimethylsiloxane (PDMS) mix, made fluorescent through specific covalent binding to a rhodamin dye, are produced by a microfluidics device. The elastomer rigidity after polymerization is adjusted to the tissue rigidity. Its mechanical properties are carefully calibrated in situ, for a sensor embedded in a cell aggregate submitted to uniaxial compression. Thelocal shear stress tensor is retrieved from the sensor shape, accurately reconstructed through an active contour method. In vitro, within cell aggregates, and in vivo, in the prechordal plate of the Zebrafish embryo during gastrulation,our pipeline of techniques demonstrates its efficiency to directly measure the three dimensional shear stress repartition within a tissue.

Amoxicillin Haptenation of α-Enolase is Modulated by Active Site Occupancy and Acetylation

Allergic reactions to antibiotics are a major concern in the clinic. ß-lactam antibiotics are the class most frequently reported to cause hypersensitivity reactions. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, interest in identifying the corresponding serum and cellular protein targets arises. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α-enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α-enolase using gel-based and activity assays. Moreover, the possible interplay or interference between amoxicillin haptenation and acetylation of α-enolase was studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots after chemical acetylation of the protein, respectively. In addition, the peptide containing lysine 239 was identified by mass spectrometry as the amoxicillin target sequence on α-enolase, thus suggesting a selective haptenation under our conditions. The putative amoxicillin binding site and the surrounding interactions were investigated using the α-enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions.

Cumulus Extracellular Matrix Is an Important Part of Oocyte Microenvironment in Ovarian Follicles: Its Remodeling and Proteolytic Degradation

The extracellular matrix (ECM) is an essential structure with biological activities. It has been shown that the ECM influences gene expression via cytoskeletal components and the gene expression is dependent upon cell interactions with molecules and hormones. The development of ovarian follicles is a hormone dependent process. The surge in the luteinizing hormone triggers ovulatory changes in oocyte microenvironment. In this review, we discuss how proteolytic cleavage affects formation of cumulus ECM following hormonal stimulation; in particular, how the specific proteasome inhibitor MG132 affects gonadotropin-induced cytoskeletal structure, the organization of cumulus ECM, steroidogenesis, and nuclear maturation. We found that after the inhibition of proteolytic cleavage, gonadotropin-stimulated oocyte–cumulus complexes (OCCs) were without any signs of cumulus expansion; they remained compact with preserved cytoskeletal F-actin-rich transzonal projections through the oocyte investments. Concomitantly, a significant decrease was detected in progesterone secretion and in the expression of gonadotropin-stimulated cumulus expansion–related transcripts, such as HAS2 and TNFAIP6. In agreement, the covalent binding between hyaluronan and the heavy chains of serum-derived the inter-alpha-trypsin inhibitor, essential for the organization of cumulus ECM, was missing.

Selection of Promising Novel Fragment Sized S. aureus SrtA Noncovalent Inhibitors Based on QSAR and Docking Modeling Studies

Sortase A (SrtA) of Staphylococcus aureus has been identified as a promising target to a new type of antivirulent drugs, and therefore, the design of lead molecules with a low nanomolar range of activity and suitable drug-like properties is important. In this work, we aimed at identifying new fragment-sized starting points to design new noncovalent S. aureus SrtA inhibitors by making use of the dedicated molecular motif, 5-arylpyrrolidine-2-carboxylate, which has been previously shown to be significant for covalent binding SrtA inhibitors. To this end, an in silico approach combining QSAR and molecular docking studies was used. The known SrtA inhibitors from the ChEMBL database with diverse scaffolds were first employed to derive descriptors and interpret their significance and correlation to activity. Then, the classification and regression QSAR models were built, which were used for rough ranking of the virtual library of the synthetically feasible compounds containing the dedicated motif. Additionally, the virtual library compounds were docked into the “activated” model of SrtA (PDB:2KID). The consensus ranking of the virtual library resulted in the most promising structures, which will be subject to further synthesis and experimental testing in order to establish new fragment-like molecules for further development into antivirulent drugs.

Atomistic Mechanisms of Human TRPA1 Activation By Electrophile Irritants Through Molecular Dynamics Simulation and Mutual Information Analysis

The ion channel TRPA1 is a promiscuous chemosensor, with reported response to a wide spectrum of noxious electrophilic irritants, as well as cold, heat, and mechanosensation. It is also implicated in the inception of itch and pain and has hence been investigated as a drug target for novel analgesics. The mechanism of electrophilic activation for TRPA1 is therefore of broad interest. TRPA1 structures with the pore in both open and closed states have recently been published as well as covalent binding modes for electrophile agonists. However, the detailed mechanism of coupling between electrophile binding sites and the pore remains speculative. In addition, while two different cysteine residues (C621 and C665) have been identified as critical for electrophile bonding and activation, the bound geometry has only been resolved at C621. Here, we use molecular dynamics simulations of TRPA1 in both pore-open and pore-closed states to explore the allosteric link between the electrophile binding sites and pore stability. Our simulations reveal that an open pore is structurally stable in the presence of open ‘pockets’ in the C621 / C665 region, but rapidly collapses and closes when these pockets are shut. Binding of electrophiles at either C621 or C665 provides stabilisation of the pore-open state, but molecules bound at C665 are shown to be able to rotate in and out of the pocket, allowing for immediate stabilisation of transient open states. Finally, mutual information analysis of trajectories reveals an informational path linking the electrophile binding site pocket to the pore via the voltage-sensing-like domain, giving a detailed insight into the how the pore is stabilized in the open state.

Characterisation of Zampanolide as a Microtubule-Stabilizing Agent

<p>Microtubule-stabilizing agents (MSAs) are extremely important chemotherapeutic drugs since microtubules (MTs) are one of the most successful cancer drug targets. Currently there are four MSAs that are clinically used for the treatment of cancer. Cancer cells, however, can develop resistance towards these drugs, the most common being over-expression of the P-glycoprotein drug efflux pump. Zampanolide (ZMP), a novel secondary metabolite isolated from a marine sponge consists of a 20-membered macrolide ring with an unusual N-acyl-hemiaminal side chain. It is a potent MSA with similar cellular effects to the clinically relevant MSAs, Taxol®, Taxotere® and Ixempra®. ZMP has a small number of stereogenic centers and therefore is relatively easier to synthesize than other macrolide natural products. Using established cancer cell lines and isolated bovine brain tubulin ZMP in the present study was further characterized as a potential anti-cancer compound and was shown to have significant advantages over currently used MSAs. These studies provided insight into how this important drug class induces MT assembly, suggesting strategies for the development of new generation MSAs for use in the clinic. ZMP and its less active analog dactylolide competed with paclitaxel for binding to MTs and represented a novel MSA chemotype. Unlike traditional taxoid site ligands, ZMP remained significantly more cytotoxic in cell lines with mutations in the taxoid binding site, and behaved in an unusual manner in vitro. This was later found to be due to its mechanism of binding which involved covalent modification of two amino acids in the taxoid binding site, histidine 229 as the major product and asparagine 228 as the minor product. Alkylation of both these luminal site residues was also detected in unassembled tubulin, providing the first direct evidence that the taxoid binding site exists in unassembled tubulin and suggesting that the induction of MT nucleation by MSAs may proceed through an allosteric mechanism. X-ray crystallography data confirmed the presence of this binding site in unassembled tubulin and indicated that covalent modification occurs at C9 of ZMP with the NE2 of the histidine side chain. The potent stabilization of MTs observed with ZMP occurred due to its side chain interaction with the stabilizing M-loop of β-tubulin. In unassembled tubulin the M-loop is unordered. Upon ZMP binding, it is restructured into a short, well-defined helix. It is this restructuring that leads to the potent stabilization by ZMP and most likely other MSAs, including those currently used in the clinic. This information provides a basis for structure-guided drug engineering to design and develop new generation MSAs with potent stabilizing activity. In addition, covalent binding of ZMP means that it is able to avoid drug efflux pumps and thus evade the main mechanism of resistance presented to MSAs in the clinic. It was shown by studying structure-activity relationships that there are a number of key chemical motifs in ZMP responsible for its potent activity. Simpler analog structures that retain significant stabilizing activity could be used as lead compounds for further drug development. Moreover, MSAs have clinically relevant anti-angiogenic and vascular-disrupting properties, and ZMP was also shown to potently inhibit cell migration and thus have possible benefits as a vasculature-targeting compound. It was concluded that ZMP is a potent covalently-binding MSA in both cells and in vitro. Given these promising results, further preclinical development of the compound is warranted.</p>

Characterisation of Zampanolide as a Microtubule-Stabilizing Agent

<p>Microtubule-stabilizing agents (MSAs) are extremely important chemotherapeutic drugs since microtubules (MTs) are one of the most successful cancer drug targets. Currently there are four MSAs that are clinically used for the treatment of cancer. Cancer cells, however, can develop resistance towards these drugs, the most common being over-expression of the P-glycoprotein drug efflux pump. Zampanolide (ZMP), a novel secondary metabolite isolated from a marine sponge consists of a 20-membered macrolide ring with an unusual N-acyl-hemiaminal side chain. It is a potent MSA with similar cellular effects to the clinically relevant MSAs, Taxol®, Taxotere® and Ixempra®. ZMP has a small number of stereogenic centers and therefore is relatively easier to synthesize than other macrolide natural products. Using established cancer cell lines and isolated bovine brain tubulin ZMP in the present study was further characterized as a potential anti-cancer compound and was shown to have significant advantages over currently used MSAs. These studies provided insight into how this important drug class induces MT assembly, suggesting strategies for the development of new generation MSAs for use in the clinic. ZMP and its less active analog dactylolide competed with paclitaxel for binding to MTs and represented a novel MSA chemotype. Unlike traditional taxoid site ligands, ZMP remained significantly more cytotoxic in cell lines with mutations in the taxoid binding site, and behaved in an unusual manner in vitro. This was later found to be due to its mechanism of binding which involved covalent modification of two amino acids in the taxoid binding site, histidine 229 as the major product and asparagine 228 as the minor product. Alkylation of both these luminal site residues was also detected in unassembled tubulin, providing the first direct evidence that the taxoid binding site exists in unassembled tubulin and suggesting that the induction of MT nucleation by MSAs may proceed through an allosteric mechanism. X-ray crystallography data confirmed the presence of this binding site in unassembled tubulin and indicated that covalent modification occurs at C9 of ZMP with the NE2 of the histidine side chain. The potent stabilization of MTs observed with ZMP occurred due to its side chain interaction with the stabilizing M-loop of β-tubulin. In unassembled tubulin the M-loop is unordered. Upon ZMP binding, it is restructured into a short, well-defined helix. It is this restructuring that leads to the potent stabilization by ZMP and most likely other MSAs, including those currently used in the clinic. This information provides a basis for structure-guided drug engineering to design and develop new generation MSAs with potent stabilizing activity. In addition, covalent binding of ZMP means that it is able to avoid drug efflux pumps and thus evade the main mechanism of resistance presented to MSAs in the clinic. It was shown by studying structure-activity relationships that there are a number of key chemical motifs in ZMP responsible for its potent activity. Simpler analog structures that retain significant stabilizing activity could be used as lead compounds for further drug development. Moreover, MSAs have clinically relevant anti-angiogenic and vascular-disrupting properties, and ZMP was also shown to potently inhibit cell migration and thus have possible benefits as a vasculature-targeting compound. It was concluded that ZMP is a potent covalently-binding MSA in both cells and in vitro. Given these promising results, further preclinical development of the compound is warranted.</p>

Tumor protein D54 binds intracellular nanovesicles via an amphipathic lipid packing sensor (ALPS) motif

Tumor Protein D54 (TPD54) is an abundant cytosolic protein that belongs to the TPD52 family, a family of four proteins (TPD52, 53, 54 and 55) that are overexpressed in several cancer cells. Even though the functions of these proteins remain elusive, recent investigations indicate that TPD54 binds to very small cytosolic vesicles with a diameter of ca. 30 nm, half the size of classical transport vesicles (e.g. COPI and COPII). Here, we investigated the mechanism of intracellular nanovesicle capture by TPD54. Bioinformatical analysis suggests that TPD54 contains a small coiled-coil followed by several amphipathic helices, which could fold upon binding to lipid membranes. One of these helices has the physicochemical features of an Amphipathic Lipid Packing Sensor (ALPS) motif, which, in other proteins, enables membrane binding in a curvature-dependent manner. Limited proteolysis, CD spectroscopy, tryptophan fluorescence and cysteine mutagenesis coupled to covalent binding of a membrane sensitive probe show that binding of TPD54 to small liposomes is accompanied by large structural changes in the amphipathic helix region. TPD54 binding to artificial liposomes is very sensitive to liposome size and to lipid unsaturation but is poorly dependent on lipid charge. Cellular investigations confirmed the key role of the ALPS motif in vesicle targeting. Surprisingly, the vesicles selected by TPD54 poorly overlap with those captured by the golgin GMAP-210, a long vesicle tether at the Golgi apparatus, which displays a dimeric coiled-coil architecture and an N-terminal ALPS motif. We propose that TPD54 recognizes nanovesicles through a combination of ALPS-dependent and -independent mechanisms.

Novel Keap1-Nrf2 Protein-protein interaction inhibitor UBE-1099 Ameliorates Progressive Phenotype in Alport Syndrome mouse model

Background Bardoxolone methyl activates nuclear factor erythroid 2 related factor 2 (Nrf2) via covalent binding and irreversible inhibition of Kelch-like ECH-associated protein-1 (Keap1), the negative regulator of Nrf2. Ongoing clinical trials of Bardoxolone methyl show promising effects for patients with chronic kidney disease (CKD). But the direct inhibition of Keap1-Nrf2 protein-protein interaction (PPI) as an approach to activate Nrf2 is less explored. Methods We developed a non-covalent Nrf2 activator UBE-1099, which highly selectively inhibits Keap1-Nrf2 PPI, and evaluated its efficacy on progressive phenotype in Alport syndrome mouse model (Col4a5-G5X). Results Similar to Bardoxolone methyl, UBE-1099 transiently increased proteinuria and reduced plasma creatinine in Alport mice. Importantly, UBE-1099 improved the glomerulosclerosis, renal inflammation and fibrosis, and prolonged the lifespan of Alport mice. UBE-1099 ameliorated the dysfunction of Nrf2 signaling in renal tissue of Alport mice. Moreover, transcriptome analysis in glomerulus showed that UBE-1099 induced the expression of genes associated with cell cycle and cytoskeleton, which may explain its unique mechanism of improvement such as glomerular morphological change. Conclusions UBE-1099 significantly ameliorates the progressive phenotype in Alport mice. Our results firstly revealed the efficacy of Keap1-Nrf2 PPI inhibitor for glomerulosclerosis and presents a potential therapeutic drug for CKD.