An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors.

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules that specifically target the receptors, rather than the ligands. Here, we developed a TNFR1 FRET biosensor expressed in living cells to screen compounds from the NIH Clinical Collection. We used an innovative high-throughput fluorescence lifetime screening platform that has exquisite spatial and temporal resolution to identify two small-molecule compounds, zafirlukast and triclabendazole, that inhibit the TNFR1-induced IκBα degradation and NF-κB activation. Biochemical and computational docking methods were used to show that zafirlukast disrupts the interactions between TNFR1 pre-ligand assembly domain (PLAD), whereas triclabendazole acts allosterically. Importantly, neither compound inhibits ligand binding, proving for the first time that it is possible to inhibit receptor activation by targeting TNF receptor-receptor interactions. This strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.

Polyunsaturated chains in asymmetric lipids disorder raft mixtures and preferentially associate with α-Synuclein.

Using molecular dynamics simulations, we have explored the effect of asymmetric lipids-specifically those that contain one polyunsaturated (PUFA) and one saturated fatty acid chain-on phase separation in heterogeneous membranes. These lipids are prevalent in neuronal membranes, particularly in synaptic membranes, where the Parkinson's Disease protein α-Synuclein (αS) is found. We have therefore explored the relationship between asymmetric, PUFA-containing lipids, and αS. The simulations show that asymmetric lipids partition to the liquid disordered (Ld) phase of canonical raft mixtures because of the highly disordered PUFA chain. In the case of a membrane built to mimic the lipid composition of a synaptic vesicle, the PUFA-containing asymmetric lipids completely disrupt phase separation. Because αS is positively charged, we show that it partitions with negatively charged lipids, regardless of the saturation state of the chains. Additionally, αS preferentially associates with the polyunsaturated fatty acid tails of both charged and neutral lipids. This is a consequence of those chains' ability to accommodate the void beneath the amphipathic helix. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

Death Receptor 5 Networks Require Membrane Cholesterol for Proper Structure and Function.

Death receptor 5 (DR5) is an apoptosis-inducing member of the tumor necrosis factor receptor superfamily, whose activity has been linked to membrane cholesterol content. Upon ligand binding, DR5 forms large clusters within the plasma membrane that have often been assumed to be manifestations of receptor co-localization in cholesterol-rich membrane domains. However, we have recently shown that DR5 clusters are more than just randomly aggregated receptors. Instead, these are highly structured networks held together by receptor dimers. These dimers are stabilized by specific transmembrane helix-helix interactions, including a disulfide bond in the long isoform of the receptor. The complex relationships among DR5 network formation, transmembrane helix dimerization, membrane cholesterol, and receptor activity has not been established. It is unknown whether the membrane itself plays an active role in driving DR5 transmembrane helix interactions or in the formation of the networks. We show that cholesterol depletion in cells does not inhibit the formation of DR5 networks. However, the networks that form in cholesterol-depleted cells fail to induce caspase cleavage. These results suggest a potential structural difference between active and inactive networks. As evidence, we show that cholesterol is necessary for the covalent dimerization of DR5 transmembrane domains. Molecular simulations and experiments in synthetic vesicles on the DR5 transmembrane dimer suggest that dimerization is facilitated by increased helicity in a thicker bilayer.

Membrane remodeling and mechanics: Experiments and simulations of α-Synuclein.

We review experimental and simulation approaches that have been used to determine curvature generation and remodeling of lipid bilayers by membrane-bending proteins. Particular emphasis is placed on the complementary approaches used to study α-Synuclein (αSyn), a major protein involved in Parkinson's disease (PD). Recent cellular and biophysical experiments have shown that the protein 1) deforms the native structure of mitochondrial and model membranes; and 2) inhibits vesicular fusion. Today's advanced experimental and computational technology has made it possible to quantify these protein-induced changes in membrane shape and material properties. Collectively, experiments, theory and multi-scale simulation techniques have established the key physical determinants of membrane remodeling and rigidity: protein binding energy, protein partition depth, protein density, and membrane tension. Despite the exciting and significant progress made in recent years in these areas, challenges remain in connecting biophysical insights to the cellular processes that lead to disease. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.