ABSTRACT
Rapid clinical progress of hepatitis C virus (HCV) replication inhibitors, including these selecting for resistance in the NS5A region (NS5A inhibitors), promises to revolutionize HCV treatment. Herein, we describe our explorations of diverse spiropyrrolidine motifs in novel NS5A inhibitors and a proposed interaction model. We discovered that the 1,4-dioxa-7-azaspiro[4.4]nonane motif in inhibitor 41H (GSK2236805) supported high potency against genotypes 1a and 1b as well as in genotype 1b L31V and Y93H mutants. Consistent with this, 41H potently suppressed HCV RNA in the 20-day RNA reduction assay. Pharmacokinetic and safety data supported further progression of 41H to the clinic.
Subject(s)
Antiviral Agents/pharmacology , Genotype , Hepacivirus/drug effects , Pyrrolidines/pharmacology , Spiro Compounds/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Drug Design , Hepacivirus/genetics , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Models, Molecular , Mutation , Pyrrolidines/chemistry , Pyrrolidines/pharmacokinetics , RNA, Viral/blood , Spiro Compounds/chemistry , Spiro Compounds/pharmacokineticsABSTRACT
Phosphatidylinositol 4-kinase type IIIα (PI4KA) is a host factor essential for hepatitis C virus replication and hence is a target for drug development. PI4KA has also been linked to endoplasmic reticulum exit sites and generation of plasma membrane phosphoinositides. Here, we developed highly specific and potent inhibitors of PI4KA and conditional knock-out mice to study the importance of this enzyme in vitro and in vivo. Our studies showed that PI4KA is essential for the maintenance of plasma membrane phosphatidylinositol 4,5-bisphosphate pools but only during strong stimulation of receptors coupled to phospholipase C activation. Pharmacological blockade of PI4KA in adult animals leads to sudden death closely correlating with the drug's ability to induce phosphatidylinositol 4,5-bisphosphate depletion after agonist stimulation. Genetic inactivation of PI4KA also leads to death; however, the cause in this case is due to severe intestinal necrosis. These studies highlight the risks of targeting PI4KA as an anti-hepatitis C virus strategy and also point to important distinctions between genetic and pharmacological studies when selecting host factors as putative therapeutic targets.
Subject(s)
Cell Membrane/enzymology , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Animals , COS Cells , Cell Membrane/genetics , Chlorocebus aethiops , Enzyme Activation/genetics , Gene Targeting , HEK293 Cells , Hepatitis C/enzymology , Hepatitis C/genetics , Hepatitis C/therapy , Humans , Mice , Mice, Transgenic , Minor Histocompatibility Antigens , Phosphatidylinositol 4,5-Diphosphate/genetics , Phosphatidylinositol Phosphates/genetics , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Phosphotransferases (Alcohol Group Acceptor)/genetics , Type C Phospholipases/genetics , Type C Phospholipases/metabolismABSTRACT
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 28 that displays high selectivity for PI4KIIIα and potently inhibits HCV replication in vitro.
Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Hepacivirus/drug effects , Animals , Antiviral Agents/chemistry , Drug Discovery , Enzyme Inhibitors/chemistry , Hepacivirus/enzymology , Hepacivirus/physiology , Magnetic Resonance Spectroscopy , Mass Spectrometry , Rats , Structure-Activity Relationship , Virus Replication/drug effectsABSTRACT
Sequence-specific binding in the minor groove of DNA by small molecules is a growing area of research with possible therapeutic relevance. By selectively binding to DNA sequences required by critical transcription factors, these small molecules could potentially modulate the expression levels of disease-causing genes. Precise targeting of a critical transcription factor of a selected gene requires an understanding of the preferred sequence of the DNA binding compound. As new compounds are being synthesized, there is a need to evaluate their DNA recognition profile. We sought to establish a procedure to determine sequence preference of compounds with previously unknown binding properties. A novel procedure for determining the optimal DNA binding sequence of minor groove binding compounds is described here. The assay also allows for determination of the binding affinity to a particular sequence.