Modulation of Amide Bond Rotamers in 5-Acyl-6,7-dihydrothieno[3,2-c]pyridines

2-Substituted <i>N</i>-acyl-piperidine is a widespread and important structural motif, found in approximately 500 currently available structures, and present in nearly 30 pharmaceutically active compounds. Restricted rotation of the acyl substituent in such molecules can give rise to two distinct chemical environments. Here we demonstrate, using NMR studies and density functional theory modeling of the lowest energy structures of 5-acyl-6,7-dihydrothieno­[3,2-<i>c</i>]­pyridine derivatives, that the amide <i>E</i>:<i>Z</i> equilibrium is affected by non-covalent interactions between the amide oxygen and adjacent aromatic protons. Structural predictions were used to design molecules that promote either the <i>E</i>- or <i>Z</i>-amide conformation, enabling preparation of compounds with a tailored conformational ratio, as proven by NMR studies. Analysis of the available X-ray data of a variety of published <i>N</i>-acyl-piperidine-containing compounds further indicates that these molecules are also clustered in the two observed conformations. This finding emphasizes that directed conformational isomerism has significant implications for the design of both small molecules and larger amide-containing molecular architectures

High-resolution snapshots of human N-myristoyltransferase in action illuminate a mechanism promoting N-terminal Lys and Gly myristoylation

The promising drug target N-myristoyltransferase (NMT) catalyses an essential protein modification thought to occur exclusively at N-terminal glycines (Gly). Here, we present high-resolution human NMT1 structures co-crystallised with reactive cognate lipid and peptide substrates, revealing high-resolution snapshots of the entire catalytic mechanism from the initial to final reaction states. Structural comparisons, together with biochemical analysis, provide unforeseen details about how NMT1 reaches a catalytically competent conformation in which the reactive groups are brought into close proximity to enable catalysis. We demonstrate that this mechanism further supports efficient and unprecedented myristoylation of an N-terminal lysine side chain, providing evidence that NMT acts both as N-terminal-lysine and glycine myristoyltransferase

Characterization of hedgehog acyltransferase inhibitors identifies a small molecule probe for hedgehog signaling by cancer cells

The Sonic Hedgehog (Shh) signaling pathway plays a critical role during embryonic development and cancer progression. N-terminal palmitoylation of Shh by Hedgehog acyltransferase (Hhat) is essential for efficient signaling, raising interest in Hhat as a novel drug target. A recently identified series of dihydrothienopyridines has been proposed to function via this mode of action; however, the lead compound in this series (RUSKI-43) was subsequently shown to possess cytotoxic activity unrelated to canonical Shh signaling. To identify a selective chemical probe for cellular studies, we profiled three RUSKI compounds in orthogonal cell-based assays. We found that RUSKI-43 exhibits off-target cytotoxicity, masking its effect on Hhat-dependent signaling, hence results obtained with this compound in cells should be treated with caution. In contrast, RUSKI-201 showed no off-target cytotoxicity, and quantitative whole-proteome palmitoylation profiling with a bioorthogonal alkyne-palmitate reporter demonstrated specific inhibition of Hhat in cells. RUSKI-201 is the first selective Hhat chemical probe in cells and should be used in future studies of Hhat catalytic function

Bat IFITM3 restriction depends on S-palmitoylation and a polymorphic site within the CD225 domain

Host interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral restriction factors. Of these, IFITM3 potently inhibits viruses that enter cells through acidic endosomes, many of which are zoonotic and emerging viruses with bats (order Chiroptera) as their natural hosts. We previously demonstrated that microbat IFITM3 is antiviral. Here, we show that bat IFITMs are characterized by strong adaptive evolution and identify a highly variable and functionally important site-codon 70-within the conserved CD225 domain of IFITMs. Mutation of this residue in microbat IFITM3 impairs restriction of representatives of four different virus families that enter cells via endosomes. This mutant shows altered subcellular localization and reduced S-palmitoylation, a phenotype copied by mutation of conserved cysteine residues in microbat IFITM3. Furthermore, we show that microbat IFITM3 is S-palmitoylated on cysteine residues C71, C72, and C105, mutation of each cysteine individually impairs virus restriction, and a triple C71A-C72A-C105A mutant loses all restriction activity, concomitant with subcellular re-localization of microbat IFITM3 to Golgi-associated sites. Thus, we propose that S-palmitoylation is critical for Chiropteran IFITM3 function and identify a key molecular determinant of IFITM3 S-palmitoylation

Fragment-derived inhibitors of human N-myristoyltransferase block capsid assembly and replication of the common cold virus

Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections

Cooperative Binding of PhoB(DBD) to Its Cognate DNA Sequence-A Combined Application of Single-Molecule and Ensemble Methods

Ritzefeld M, Walhorn V, Kleineberg C, et al. Cooperative Binding of PhoB(DBD) to Its Cognate DNA Sequence-A Combined Application of Single-Molecule and Ensemble Methods. Biochemistry. 2013;52(46):8177-8186.A combined approach based on isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) experiments, circular dichroism spectroscopy (CD), atomic force microscopy (AFM) dynamic force spectroscopy (DFS), and surface plasmon resonance (SPR) was applied to elucidate the mechanism of protein-DNA complex formation and the impact of protein dimerization of the DNA-binding domain of PhoB (PhoB(DBD)). These insights can be translated to related members of the family of winged helix-turn-helix proteins. One central question was the assembly of the trimeric complex formed by two molecules of PhoB(DBD) and two cognate binding sites of a single oligonucleotide. In addition to the native protein WT-PhoB(DBD), semisynthetic covalently linked dimers with different linker lengths were studied. The ITC, SPR, FRET, and CD results indicate a positive cooperative binding mechanism and a decisive contribution of dimerization on the complex stability. Furthermore, an alanine scan was performed and binding of the corresponding point mutants was analyzed by both techniques to discriminate between different binding types involved in the protein-DNA interaction and to compare the information content of the two methods DFS and SPR. In light of the published crystal structure, four types of contribution to the recognition process of the pho box by the protein PhoB(DBD) could be differentiated and quantified. Consequently, it could be shown that investigating the interactions between DNA and proteins with complementary techniques is necessary to fully understand the corresponding recognition process

Modulation of Cis-Trans Amide Bond Rotamers in 5-Acyl-6,7-dihydrothieno[3,2-c]pyridines

2-Substituted N-acyl-piperidine is a widespread and important structural motif, found in nearly 500 currently available structures, and present in at least 30 pharmaceutically active compounds. Restricted rotation of the acyl substituent in such molecules can give rise to two distinct chemical environments. Here we demonstrate using NMR studies and modelling of the lowest energy structures of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine derivatives that the amide cis-trans equilibrium is affected by intramolecular hydrogen bonding between the amide oxygen and adjacent aromatic protons. Structural predictions were used to design molecules that promote either the cis- or trans-amide conformation; thereby compounds with a tailored conformational ratio were prepared as proven by NMR studies. Analysis of the available X-ray data of a variety of the published N-acyl-piperidine containing compounds further indicates that these molecules are also clustered in the two observed conformations. This finding emphasizes that the reported directed conformational isomerism has significant implications for the design of both small molecules and larger amide-containing molecular architectures.2-Substituted N-acyl-piperidine is a widespread and important structural motif, found in nearly 500 currently available structures, and present in at least 30 pharmaceutically active compounds. Restricted rotation of the acyl substituent in such molecules can give rise to two distinct chemical environments. Here we demonstrate using NMR studies and modelling of the lowest energy structures of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine derivatives that the amide cis-trans equilibrium is affected by intramolecular hydrogen bonding between the amide oxygen and adjacent aromatic protons. Structural predictions were used to design molecules that promote either the cis- or trans-amide conformation; thereby compounds with a tailored conformational ratio were prepared as proven by NMR studies. Analysis of the available X-ray data of a variety of the published N-acyl-piperidine containing compounds further indicates that these molecules are also clustered in the two observed conformations. This finding emphasizes that the reported directed conformational isomerism has significant implications for the design of both small molecules and larger amide-containing molecular architectures.2-Substituted N-acyl-piperidine is a widespread and important structural motif, found in nearly 500 currently available structures, and present in at least 30 pharmaceutically active compounds. Restricted rotation of the acyl substituent in such molecules can give rise to two distinct chemical environments. Here we demonstrate using NMR studies and modelling of the lowest energy structures of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine derivatives that the amide cis-trans equilibrium is affected by intramolecular hydrogen bonding between the amide oxygen and adjacent aromatic protons. Structural predictions were used to design molecules that promote either the cis- or trans-amide conformation; thereby compounds with a tailored conformational ratio were prepared as proven by NMR studies. Analysis of the available X-ray data of a variety of the published N-acyl-piperidine containing compounds further indicates that these molecules are also clustered in the two observed conformations. This finding emphasizes that the reported directed conformational isomerism has significant implications for the design of both small molecules and larger amide-containing molecular architectures

Microfluidic mobility shift assay for real-time analysis of peptide n-palmitoylation

The Hedgehog pathway is a key developmental signaling pathway but is also implicated in many types of cancer. The extracellular signaling protein Sonic hedgehog (Shh) requires dual lipidation for functional signaling, whereby N-terminal palmitoylation is performed by the enzyme Hedgehog acyltransferase (Hhat). Hhat is an attractive target for small-molecule inhibition to arrest Hedgehog signaling, and methods for assaying Hhat activity are central to understanding its function. However, all existing assays to quantify lipidation of peptides suffer limitations, such as safety hazards, high costs, extensive manual handling, restriction to stopped-assay measurements, or indirect assessment of lipidation. To address these limitations, we developed a microfluidic mobility shift assay (MSA) to analyze Shh palmitoylation. MSA allowed separation of fluorescently labeled Shh amine-substrate and palmitoylated Shh amide-product peptides based on differences in charge and hydrodynamic radius, coupled with online fluorescence intensity measurements for quantification. The MSA format was employed to study Hhat-catalyzed reactions, investigate Hhat kinetics, and determine small-molecule inhibitor IC50 values. Both real-time and stopped assays were performed, with the latter achieved via addition of excess unlabeled Shh peptide. The MSA format therefore allows direct and real-time fluorescence-based measurement of acylation and represents a powerful alternative technique in the study of N-lipidation

Comparative analysis reveals adaptive evolution of bat IFITMs and a novel antiviral determinant

ABSTRACT Host interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral restriction factors. Of these, IFITM3 potently inhibits viruses that enter cells through acidic endosomes, many of which are zoonotic and emerging viruses with bats (order Chiroptera) as natural hosts. We previously demonstrated that microbat IFITM3 is antiviral. Here we show that bat IFITMs are characterized by strong adaptive evolution and identify a highly variable and functionally important site - codon 70 - within the conserved CD225 domain of IFITMs. Mutation of this residue in microbat IFITM3 impairs restriction of four different virus families that enter cells via endosomes. This mutant shows altered subcellular localization and reduced S-palmitoylation, a phenotype copied by mutation of conserved cysteine residues in microbat IFITM3. Furthermore, we show that microbat IFITM3 is S-palmitoylated on cysteine residues C71, C72 and C105, mutation of each cysteine residue individually impairs virus restriction, and a triple C71-C72-C105 mutant loses all restriction, concomitant with subcellular re-localization of microbat IFITM3 to Golgi-associated sites. Thus, we propose that S-palmitoylation is critical for Chiropteran IFITM3 function and identify a key molecular determinant of IFITM3 S-palmitoylation