The role of phosphodiesterase 12 (PDE12) as a negative regulator of the innate immune response and the discovery of antiviral inhibitors

2',5'-Oligoadenylate synthetase (OAS) enzymes and RNase-L constitute a major effector arm of interferon (IFN)-mediated antiviral defense. OAS produces a unique oligonucleotide second messenger, 2',5'-oligoadenylate (2-5A), that binds and activates RNase-L. This pathway is down-regulated by virus- and host-encoded enzymes that degrade 2-5A. Phosphodiesterase 12 (PDE12) was the first cellular 2-5A- degrading enzyme to be purified and described at a molecular level. Inhibition of PDE12 may up-regulate the OAS/RNase-L pathway in response to viral infection resulting in increased resistance to a variety of viral pathogens. We generated a PDE12-null cell line, HeLaΔPDE12, using transcription activator-like effector nuclease-mediated gene inactivation. This cell line has increased 2-5A levels in response to IFN and poly(I-C), a double-stranded RNA mimic compared with the parental cell line. Moreover, HeLaΔPDE12 cells were resistant to viral pathogens, including encephalomyocarditis virus, human rhinovirus, and respiratory syncytial virus. Based on these results, we used DNA-encoded chemical library screening to identify starting points for inhibitor lead optimization. Compounds derived from this effort raise 2-5A levels and exhibit antiviral activity comparable with the effects observed with PDE12 gene inactivation. The crystal structure of PDE12 complexed with an inhibitor was solved providing insights into the structure-activity relationships of inhibitor potency and selectivity

Georgia marble at the Minnesota State Capitol The Effects of Mineralogy and Climate on Durability

Based on visual observations and tests made of Georgia marble units that have been in service at the historic Minnesota State Capitol for 110 years, a fielddiscernible correlation exists between marble unit mineralogical composition and unit weathering performance. Field studies noted over the course of a five-year restoration project of the capitol exterior were used to categorize the original marble cladding into four perceptible types, which were evaluated by field testing that suggested a correlation between the textural and mineralogical differences within the marble and its long-term durability. To test this theory, field-issued repair assignments made during an exterior marble restoration project at the capitol were examined statistically. The nature and frequency of assigned repairs were compared to the type of marble from which they were originally fabricated to determine whether a correlation existed. The results of this comparison provided convincing statistical verification of the link between mineralogy and durability. This hypothesis was then verified through detailed petrographic analysis in a laboratory setting, which determined that marble consisting of a smaller, more interlocked grain configuration that was more dolomitic than calcitic in its basic chemistry consistently proved to be more durable in weathering performance over time. The paper describes the process followed by the authors to categorize and study the distinct varieties of Georgia marble present on the Minnesota State Capitol, and how this information was used to guide the restoration process with the intent of maximizing the anticipated service life of new marble replacements

Discovery of Selective Small Molecule Type III Phosphatidylinositol 4‑Kinase Alpha (PI4KIIIα) Inhibitors as Anti Hepatitis C (HCV) Agents

Hepatitis C virus (HCV) assembles many host cellular proteins into unique membranous replication structures as a prerequisite for viral replication, and PI4KIIIα is an essential component of these replication organelles. RNA interference of PI4KIIIα results in a breakdown of this replication complex and cessation of HCV replication in Huh-7 cells. PI4KIIIα is a lipid kinase that interacts with the HCV nonstructural 5A protein (NS5A) and enriches the HCV replication complex with its product, phosphoinositol 4-phosphate (PI4P). Elevated levels of PI4P at the endoplasmic reticulum have been linked to HCV infection in the liver of HCV infected patients. We investigated if small molecule inhibitors of PI4KIIIα could inhibit HCV replication in vitro. The synthesis and structure–activity relationships associated with the biological inhibition of PI4KIIIα and HCV replication are described. These efforts led directly to identification of quinazolinone <b>28</b> that displays high selectivity for PI4KIIIα and potently inhibits HCV replication in vitro

Imidazo[1,2-<i>a</i>]pyridines That Directly Interact with Hepatitis C NS4B: Initial Preclinical Characterization

A series of imidazo­[1,2-<i>a</i>]­pyridines which directly bind to HCV Non-Structural Protein 4B (NS4B) is described. This series demonstrates potent <i>in vitro</i> inhibition of HCV replication (EC₅₀ < 10 nM), direct binding to purified NS4B protein (IC₅₀ < 20 nM), and an HCV resistance pattern associated with NS4B (H94N/R, V105L/M, F98L) that are unique among reported HCV clinical assets, suggestive of the potential for additive or synergistic combination with other small molecule inhibitors of HCV replication

Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315

IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,β-unsaturated enone <b>1</b> makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design

Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315

IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,β-unsaturated enone <b>1</b> makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design