Discovery of 2-Aminothiazoles as Potent Antiprion Compounds

Prion diseases are fatal, untreatable neurodegenerative diseases caused by the accumulation of the misfolded, infectious isoform of the prion protein (PrP), termed PrP^Sc. In an effort to identify novel inhibitors of prion formation, we utilized a high-throughput enzyme-linked immunosorbent assay (ELISA) to evaluate PrP^Sc reduction in prion-infected neuroblastoma cell lines (ScN2a). We screened a library of ∼10,000 diverse small molecules in 96-well format and identified 121 compounds that reduced PrP^Sc levels at a concentration of 5 μM. Four chemical scaffolds were identified as potential candidates for chemical optimization based on the presence of preliminary structure-activity relationships (SAR) derived from the primary screening data. A follow-up analysis of a group of commercially available 2-aminothiazoles showed this class as generally active in ScN2a cells. Our results establish 2-aminothiazoles as promising candidates for efficacy studies of animals and validate our drug discovery platform as a viable strategy for the identification of novel lead compounds with antiprion properties.Prion diseases belong to a class of neurodegenerative, protein-conformation disorders whose unifying pathological mechanism is the misprocessing and aggregation of normally benign soluble proteins. These “proteinopathies,” which include Alzheimer''s, Parkinson''s, and Huntington''s diseases, as well as the frontotemporal dementias—including Pick''s disease—and amyotrophic lateral sclerosis (ALS), are uniformly fatal after a period of neurodegeneration, characterized clinically by dementia and motor dysfunction (20). Prion diseases, which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy, are characterized by the formation of “spongiform” vacuolation of the brain (13). Disease onset is caused by the accumulation of a β-sheet-rich, infectious isoform of the prion protein, termed PrP^Sc (1, 16, 19, 20), which is formed from the α-helix-rich cellular prion protein, termed PrP^C. This conversion can occur spontaneously or be induced by the presence of a number of different autosomally dominant mutations of the PrP gene (8, 21). Alternatively, prion disease can result from exogenous exposure to PrP^Sc (20). The infectious nature of prions results from the ability of PrP^Sc to induce its own production by stimulating the alternative folding of PrP^C (18). Although PrP^Sc formation has been demonstrated to be the primary pathogenic event in prion disease, the precise mechanism that features in its formation and the ensuing neurodegeneration remains largely unknown.Despite the lack of a detailed understanding of the cellular mechanism of prion propagation, numerous studies have been directed toward development of therapeutics targeting prions. Screenings utilizing prion-infected cell lines have identified a number of compounds that reduce the level of PrP^Sc in culture. These include pentosan polysulfate (PPS), dextran sulfate (DS), HPA-23, Congo red, suramin, dendritic polyamines, and quinacrine, among others; for a comprehensive review, see reference 22. However, none of these have been shown to be effective against a broad range of prion strains in animal models when administered after clinical signs manifest, and none have been shown to modify the disease course in human clinical studies (22).The current array of antiprion compounds has been discovered mostly by ad hoc, low-throughput screening of small sets of known bioactive compounds. Most antiprion compounds that are active in prion-infected cell lines have failed in vivo, which highlights the need for a sustained, high-throughput drug discovery effort. Such studies need to rapidly screen large libraries of new chemical entities in vitro, analyze the pharmacokinetic and pharmacodynamic properties of the primary hits in vivo, and optimize the chemical properties of lead compounds. In two semi-high-throughput screenings for antiprion compounds, an analysis of ∼2,000-member compound libraries of known drugs and natural products identified sets of 17 and 8 active compounds (10). None of these have been reported to be effective in vivo.In the present study, we assessed the antiprion activity of ∼10,000 compounds encompassing a diverse set of chemical entities and identified a set of 121 compounds that induce the clearance of PrP^Sc. Subsequent analysis of a number of compounds with the 2-aminothiazole scaffold informed the structure-activity relationship (SAR) of this lead class.     

The discovery and structure–activity relationships of pyrano[3,4-b]indole based inhibitors of hepatitis C virus NS5B polymerase

We describe the structure–activity relationship of the C1-group of pyrano[3,4-b]indole based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compound 12.     

Iterative Structure-Based Peptide-Like Inhibitor Design against the Botulinum Neurotoxin Serotype A

The botulinum neurotoxin serotype A light chain (BoNT/A LC) protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs) were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their K_i values are in the nM-range. A co-crystal structure for one of these inhibitors was determined and reveals that the PLM, in accord with the goals of our design strategy, simultaneously involves both ionic interactions via its P1 residue and hydrophobic contacts by means of an aromatic group in the P2′ position. The PLM adopts a helical conformation similar to previously determined co-crystal structures of PLMs, although there are also major differences to these other structures such as contacts with specific BoNT/A LC residues. Our structure further demonstrates the remarkable plasticity of the substrate binding cleft of the BoNT/A LC protease and provides a paradigm for iterative structure-based design and development of BoNT/A LC inhibitors.     

Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy

The noninvasive character of NMR spectroscopy, combined with the sensitivity of the chemical shift, makes it ideally suited to investigate the conformation, binding events and dynamics of macromolecules inside living cells. These 'in-cell NMR' experiments involve labeling the macromolecule of interest with a nonradioactive but NMR-active isotope (15N or 13C). Cellular samples are prepared either by selectively overexpressing the protein in suitable cells (e.g., bacterial cells grown on isotopically labeled media), or by injecting isotopically labeled proteins directly into either cells or cell extracts. Here we provide detailed protocols for in-cell NMR experiments in the prokaryotic organism Escherichia coli, as well as eukaryotic cells and extracts employing Xenopus laevis oocytes or egg extracts. In-cell NMR samples with proteins overexpressed in E. coli can be produced within 13-14 h. Preparing Xenopus oocyte samples for in-cell NMR experiments takes 6-14 h depending on the oocyte preparation scheme and the injection method used.     

Gene-specific countermeasures against Ebola virus based on antisense phosphorodiamidate morpholino oligomers

The filoviruses Marburg virus and Ebola virus (EBOV) quickly outpace host immune responses and cause hemorrhagic fever, resulting in case fatality rates as high as 90% in humans and nearly 100% in nonhuman primates. The development of an effective therapeutic for EBOV is a daunting public health challenge and is hampered by a paucity of knowledge regarding filovirus pathogenesis. This report describes a successful strategy for interfering with EBOV infection using antisense phosphorodiamidate morpholino oligomers (PMOs). A combination of EBOV-specific PMOs targeting sequences of viral mRNAs for the viral proteins (VPs) VP24, VP35, and RNA polymerase L protected rodents in both pre- and post-exposure therapeutic regimens. In a prophylactic proof-of-principal trial, the PMOs also protected 75% of rhesus macaques from lethal EBOV infection. The work described here may contribute to development of designer, "druggable" countermeasures for filoviruses and other microbial pathogens.     

Characterization of a G protein-coupled guanylyl cyclase-B receptor from bovine tracheal smooth muscle

A G protein-coupled natriuretic peptide-guanylyl cyclase receptor-B (NPR-B) located in plasma membranes from bovine tracheal smooth muscle shows complex kinetics and regulation. NPR-B was activated by natriuretic peptides (CNP-53 > ANP-28) at the ligand extracellular domain, stimulated by Gq-protein activators, such as mastoparan, and inhibited by Gi-sensitive chloride, interacting at the juxtamembrane domain. The kinase homology domain was evaluated by the ATP inhibition of Mn2+-activated NPR-B, which was partially reversed by mastoparan. The catalytic domain was studied by kinetics of Mn2+/Mg2+ and GTP, and the catalytic effect with GTP analogues with modifications of the /gamma phosphates and ribose moieties. Most NPR-B biochemical properties remained after detergent solubilization but the mastoparan activation and chloride inhibition of NPR-B disappeared. Our results indicate that NPR-B is a highly regulated nano-machinery with domains acting at cross-talk points with other signal transducing cascades initiated by G protein-coupled receptors and affected by intracellular ligands such as chloride, Mn2+, Mg2+, ATP, and GTP.     

UV measurements in microplates suitable for high-throughput protein determination

An UV spectrophotometric method for protein determination using microplates is described. Using the SPECTRAmax PLUS reader, the UVStar 96- and 384-well microplates and a 96 or 384 parallel channel liquid handling technique, large-scale determinations can be performed with intraassay precision better than 3% CV (coefficient of variation) in the range from 1 to 8000 microg of protein/ml, measuring at 205, 215, and 280 nm and using different volume-dependent light-path lengths. Since the absorbance coefficient at 205 nm is found to be 30 ml/(mgxcm) for eight different proteins with a CV of 5.6% only with the Path Check option of the reader, protein concentration can be determined without any individual calibration. Samples in the volume range of 60-250 microl can be analyzed without time-consuming and expensive treatment and without sample loss. Using a special 96 or 384 parallel dialyzing device, low molecular weight substances which interfere with the analysis by their UV absorbance, such as buffers and detergents, can effectively be removed. Application examples for serum protein separation are also shown in the presence of the strongly UV absorbing detergent Triton X-100.