RNA-Templated Peptide Bond Formation Promotes L-Homochirality.

The world in which we live is homochiral. The ribose units that form the backbone of DNA and RNA are all D-configured and the encoded amino acids that comprise the proteins of all living species feature an all-L-configuration at the α-carbon atoms. The homochirality of α-amino acids is essential for folding of the peptides into well-defined and functional 3D structures and the homochirality of D-ribose is crucial for helix formation and base-pairing. The question of why nature uses only encoded L-α-amino acids is not understood. Herein, we show that an RNA-peptide world, in which peptides grow on RNAs constructed from D-ribose, leads to the self-selection of homo-L-peptides, which provides a possible explanation for the homo-D-ribose and homo-L-amino acid combination seen in nature.

A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha.

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

Discovery of the Hedgehog Pathway Inhibitor Pipinib that Targets PI4KIIIß.

The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh-pathway modulator Pipinib by means of cell-based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4-kinase IIIß (PI4KB) and suppresses GLI-mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl-4-phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling.

Purine analogs as phosphatidylinositol 4-kinase IIIß inhibitors.

We report on an extensive structure-activity relationship study of novel PI4K IIIß inhibitors. The purine derivative of the potent screening hit T-00127-HEV1 has served as a suitable starting point for a thorough investigation of positions 8 and 2. While position 8 of the purine scaffold can only bear a small substituent to maintain the inhibitory activity, position 2 is opened for extensive modification and can accommodate even substituted phenyl rings without the loss of PI4K IIIß inhibitory activity. These empirical observations nicely correlate with the results of our docking study, which suggests that position 2 directs towards solution and can provide the necessary space for the interaction with remote residues of the enzyme, whereas the cavity around position 8 is strictly limited. The obtained compounds have also been subjected to antiviral screening against a panel of (+)ssRNA viruses.

Gradual evolution of a homo-l-peptide world on homo-d-configured RNA and DNA.

Modern life requires the translation of genetic information - encoded by nucleic acids - into proteins, which establishes the essential link between genotype and phenotype. During translation, exclusively l-amino acids are loaded onto transfer RNA molecules (tRNA), which are then connected at the ribosome to give homo-l-proteins. In contrast to the homo-l-configuration of amino acids and proteins, the oligonucleotides involved are all d-configured (deoxy)ribosides. Previously, others and us have shown that if peptide synthesis occurs at homo d-configured oligonucleotides, a pronounced l-amino acid selectivity is observed, which reflects the d-sugar/l-amino acid world that evolved in nature. Here we further explore this astonishing selectivity. We show a peptide-synthesis/recapture-cycle that can lead to a gradual enrichment and hence selection of a homo-l-peptide world. We show that even if peptides with a mixed l/d-stereochemistry are formed, they are not competitive against the homo-l-counterparts. We also demonstrate that this selectivity is not limited to RNA but that peptide synthesis on DNA features the same l-amino acid preference. In total, the data bring us a step closer to an understanding of how homochirality on Earth once evolved.

Step-by-Step Towards Biological Homochirality - from Prebiotic Randomness To Perfect Asymmetry.

The history of life's formation and the origin of its stereochemistry are nearly as multifaceted as the life itself. In this review, we focus on analyzing the step-by-step path leading to what we can define as "life" in parallel to what we know about the emergence of enantiomeric imbalance and subsequent transition to full homochirality. We start at the level of assembly of the building blocks of life from inorganic molecules and build up to the polymerization and formation of nucleic acids and peptides. We report and analyze different theories at various stages of this development and try to elucidate the most plausible theory.

Discovery of Novel Human Constitutive Androstane Receptor Agonists with the Imidazo[1,2-a]pyridine Structure.

The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.

Fluorescent Inhibitors as Tools To Characterize Enzymes: Case Study of the Lipid Kinase Phosphatidylinositol 4-Kinase IIIß (PI4KB).

The lipid kinase phosphatidylinositol 4-kinase IIIß (PI4KB) is an essential host factor for many positive-sense single-stranded RNA (+RNA) viruses including human pathogens hepatitis C virus (HCV), Severe acute respiratory syndrome (SARS), coxsackie viruses, and rhinoviruses. Inhibitors of PI4KB are considered to be potential broad-spectrum virostatics, and it is therefore critical to develop a biochemical understanding of the kinase. Here, we present highly potent and selective fluorescent inhibitors that we show to be useful chemical biology tools especially in determination of dissociation constants. Moreover, we show that the coumarin-labeled inhibitor can be used to image PI4KB in cells using fluorescence-lifetime imaging microscopy (FLIM) microscopy.

Rational Design of Novel Highly Potent and Selective Phosphatidylinositol 4-Kinase IIIß (PI4KB) Inhibitors as Broad-Spectrum Antiviral Agents and Tools for Chemical Biology.

Phosphatidylinositol 4-kinase IIIß (PI4KB) is indispensable for the replication of various positive-sense single stranded RNA viruses, which hijack this cellular enzyme to remodel intracellular membranes of infected cells to set up the functional replication machinery. Therefore, the inhibition of this PI4K isoform leads to the arrest of viral replication. Here, we report on the synthesis of novel PI4KB inhibitors, which were rationally designed based on two distinct structural types of inhibitors that bind in the ATP binding side of PI4KB. These "hybrids" not only excel in outstanding inhibitory activity but also show high selectivity to PI4KB compared to other kinases. Thus, these compounds exert selective nanomolar or even subnanomolar activity against PI4KB as well as profound antiviral effect against hepatitis C virus, human rhinovirus, and coxsackievirus B3. Our crystallographic analysis unveiled the exact position of the side chains and explains their extensive contribution to the inhibitory activity.

Highly Selective Phosphatidylinositol 4-Kinase IIIß Inhibitors and Structural Insight into Their Mode of Action.

Phosphatidylinositol 4-kinase IIIß is a cellular lipid kinase pivotal to pathogenesis of various RNA viruses. These viruses hijack the enzyme in order to modify the structure of intracellular membranes and use them for the construction of functional replication machinery. Selective inhibitors of this enzyme are potential broad-spectrum antiviral agents, as inhibition of this enzyme results in the arrest of replication of PI4K IIIß-dependent viruses. Herein, we report a detailed study of novel selective inhibitors of PI4K IIIß, which exert antiviral activity against a panel of single-stranded positive-sense RNA viruses. Our crystallographic data show that the inhibitors occupy the binding site for the adenine ring of the ATP molecule and therefore prevent the phosphorylation reaction.