No abstract

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.

Objective: Understanding the heterogeneous pathology in Alzheimer's disease and related tauopathies is one of the most urgent and fundamental challenges facing the discovery of novel disease-modifying therapies. Through monitoring ensembles of toxic and nontoxic tau oligomers spontaneously formed in cells, our biosensor technology can identify tool compounds that modulate tau oligomer structure and toxicity, providing much needed insight into the nature and properties of toxic tau oligomers. Background: Tauopathies are a group of neurodegenerative disorders characterized by pathologic aggregation of the microtubule binding protein tau. Recent studies suggest that tau oligomers are the primary toxic species in tauopathies. New/Updated Hypothesis: We hypothesize that tau biosensors capable of monitoring tau oligomer conformation are able to identify tool compounds that modulate the structure and conformation of these tau assemblies, providing key insight into the unique structural fingerprints of toxic tau oligomers. These fingerprints will provide gravely needed biomarker profiles to improve staging of early tauopathy pathology and generate lead compounds for potential new therapeutics. Our time-resolved fluorescence resonance energy transfer biosensors provide us an exquisitely sensitive technique to monitor minute structural changes in monomer and oligomer conformation. In this proof-ofconcept study, we identified a novel tool compound, MK-886, which directly binds tau, perturbs the conformation of toxic tau oligomers, and rescues tau-induced cytotoxicity. Furthermore, we show that MK-886 alters the conformation of tau monomer at the proline-rich and microtubule binding regions, stabilizing an on-pathway oligomer. Major Challenges for the Hypothesis: Our approach monitors changes in the ensemble of assemblies that are spontaneously formed in cells but does not specifically isolate or enrich unique toxic tau species. However, time-resolved fluorescence resonance energy transfer does not provide highresolution, atomic scale information, requiring additional experimental techniques to resolve the structural features stabilized by different tool compounds. David D. Thomas holds equity in and serves as an executive officer for Photonic Pharma LLC, a company that owns intellectual property related to technology used in part of this project. These relationships have been reviewed and managed by the University of Minnesota in accordance with its conflict-of-interest polices.

Tumor necrosis factor receptor 1 (TNFR1) is a central mediator of the inflammatory pathway and is associated with several autoimmune diseases such as rheumatoid arthritis. A revision to the canonical model of TNFR1 activation suggests that activation involves conformational rearrangements of preassembled receptor dimers. Here, we identified small-molecule allosteric inhibitors of TNFR1 activation and probed receptor dimerization and function. Specifically, we used a fluorescence lifetime–based high-throughput screen and biochemical, biophysical, and cellular assays to identify small molecules that noncompetitively inhibited the receptor without reducing ligand affinity or disrupting receptor dimerization. We also found that residues in the ligand-binding loop that are critical to the dynamic coupling between the extracellular and the transmembrane domains played a key gatekeeper role in the conformational dynamics associated with signal propagation. Last, using a simple structure-activity relationship analysis, we demonstrated that these newly found molecules could be further optimized for improved potency and specificity. Together, these data solidify and deepen the new model for TNFR1 activation.

We have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson’s disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision. This facilitates identification of compounds that cause subtle but significant conformational changes in the ensemble of oligomeric states that are easily missed using intensity-based FRET. We screened a 1280-compound small-molecule library and identified 21 compounds that changed the lifetime by >5 SD. Two of these compounds have nanomolar potency in protecting SH-SY5Y cells from αSN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of αSN pathology (phosphorylation of mouse αSN pre-formed fibrils) and show rescue of pathology for two of them. These hits were further characterized with biophysical and biochemical assays to explore potential mechanisms of action. In vitro αSN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate αSN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block αSN fibril assembly.

The link between recognition and replication is fundamental to the operation of the immune system. In recent years, modeling this process in a format of phagedisplay combinatorial libraries has afforded a powerful tool for obtaining valuable antibodies. However, the ability to readily select and isolate rare catalysts would expand the scope of library technology. A technique in which phage infection controlled the link between recognition and replication was applied to show that chemistry is a selectable process. An antibody that operated by covalent catalysis to form an acyl intermediate restored phage infectivity and allowed selection from a library in which the catalyst constituted 1 in 10 5 members. Three different selection approaches were examined for their convenience and generality. Incorporating these protocols together with well known affinity labels and mechanism-based inactivators should allow the procurement of a wide range of novel catalytic antibodies.Much of the work of the biochemical world is accomplished as a result of protein-ligand binding. In the attempt to mimic proteins found in Nature, selection methods from large libraries of molecules have been extremely valuable. In this regard, the phage-display format is particularly attractive in that it duplicates in vitro the essence of the in vivo immune response by linking the fundamental processes of recognition and replication (1-5). Yet, in both of these in vitro and in vivo selection systems, the recognition event that drives replication is based on noncovalent interactions, wherein the outcome is selection based on binding rather than chemistry. When searching for antibody-antigen interactions, the precise utilization of binding energy is unimportant so long as it is sufficient to confer the function of immunological recognition. In contrast, the selection for function is especially relevant for procuring new enzymes, where a substrate must undergo a chemical transformation upon binding.We recently developed several paradigms for the selection of catalytic antibodies on the basis of chemical reactivity. The direct selection from combinatorial libraries (6, 7) and reactive immunization (8) both afforded a subpopulation of antibodies in which chemistry was installed in the combining site. While investigation of these methods will continue to realize their full potential, the underlying concept could be refined in a design that establishes a more intimate link between chemistry and the replication process. The utilization of phage that are selectively infective (9-11) provides a means to achieve enrichment of antibody catalysts, since selection could be strictly governed by chemistry. In this way, antibodies that carry out a chemical reaction can be identified, isolated, and replicated because the chemical event distinguishes the phage-bearing antibodies from the rest of the population. Herein, we describe the implementation of this approach to select for an antibody that operates by means of covalent catalysis. MATERIALS AND METHODSS...

Semisynthetic combinatorial antibody library methodology in the phage-display format was used to select for a cysteine residue in complementari-determinig regions. Libraries (Fig. 1) of each dilution was plated on a LB (Luria-Bertani) plate/ carbenicillin to measure the output. Carbenicillin (20 pg/ml) was added to the cell culture and shaken at 370C for 1 hr. The concentration of antibiotic was increased to 50 gg/ml and shaken at 370C for another hour. The cell culture was diluted into 100 ml of superbroth that contained carbenicillin at 50 Ag/ml, tetracycline at 10 pg/ml, and 1012 plaque-forming units of VCSM13 helper phage (Stratagene). After shaking 2 hr at 370C, kanamycin (70 Ag/ml) was added and shaken at 370C overnight. The cells were removed by centrifugation, and 4% PEG and 3% NaCl were added to the supernatant. After 30 min on ice, the phage particles were centrifuged (9000 x g, 30 min, 40C). The phage were resuspended with TBS/1% BSA and clarified by centrifugation (16,000 x g, 10 min, 4Q.). The phage solution was ready for further panning.For the next four rounds of panning, the washing procedures were modified as follows: second round (twice with washing buffer, once with acid solution); third round (five times with washing buffer, once with acid solution for 5 min); fourth round (10 times with washing buffer/3% BSA, twice with acid solution for 5 min); fifth round (10 times with washing buffer/3% BSA, twice with acid solution for 5 min). For each round, the bound phage were eluted twice with 50 A4 of 20 mM dithiothreitol for 5 min. The eluted phage were measured and amplified as described above. After the fifth round of panning, the phagemid DNA was purified, digested with Spe I and Nhe I, and purified on agarose gel to remove the DNA of gene III. The 4.7-kb fragment was electroeluted, religated, and transformed into E. coli. The colonies were picked up and grown until the OD600 = 0.8, 1 mM isopropyl 13-Dthiogalactopyranoside was added, and the culture was incubated at 300C overnight. The cells were lysed with freezeand-thaw cycles between -700C and 37c in PBS. The supernatant was tested on ELISA plates coated with the Abbreviation: BSA, bovine serum albumin. *To whom reprint requests should be addressed. 2532The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The classification of multiple sclerosis (MS) has been established by Lublin in 1996 and revised in 2013. The revision includes clinically isolated syndrome, relapsing-remitting, primary progressive and secondary progressive MS, and has added activity (i.e., formation of white matter lesions or clinical relapses) as a qualifier. This allows for the distinction between active and nonactive progression, which has been shown to be of clinical importance. We propose that a logical extension of this classification is the incorporation of additional key pathological processes, such as chronic perilesional inflammation, neuroaxonal degeneration, and remyelination. This will distinguish MS phenotypes that may present as clinically identical but are driven by different combinations of pathological processes. A more precise description of MS phenotypes will improve prognostication and personalized care as well as clinical trial design. Thus, our proposal provides an expanded framework for conceptualizing MS and for guiding development of biomarkers for monitoring activity along the main pathological axes in MS.