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

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

Ueber Narceïn

n/

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

Acylation-coupled lipophilic induction of polarisation (Acyl-cLIP): a universal assay for lipid transferase and hydrolase enzymes

Posttranslational attachment of lipids to proteins is important for many cellular functions, and the enzymes responsible for these modifications are implicated in many diseases, from cancer to neurodegeneration. Lipid transferases and hydrolases are increasingly tractable therapeutic targets, but present unique challenges for high-throughput biochemical enzyme assays which hinder development of new inhibitors. We present Acylation-coupled Lipophilic Induction of Polarisation (Acyl-cLIP) as the first universally applicable biochemical lipidation assay, exploiting the hydrophobic nature of lipidated peptides to drive a polarised fluorescence readout. Acyl-cLIP allows sensitive, accurate, real-time measurement of S- or N-palmitoylation, N-myristoylation, S-farnesylation or S-geranylgeranylation. Furthermore, it is applicable to transfer and hydrolysis reactions, and we demonstrate its extension to a high-throughput screening format. We anticipate that Acyl-cLIP will greatly expedite future drug discovery efforts against these challenging targets

Photochemical probe identification of a small-molecule inhibitor binding site in Hedgehog acyltransferase (HHAT)

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed via kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (Ki = 38 nM) and a strategy for mapping small molecule interaction sites in MBOATs

Whole proteome profiling of N-Myristoyltransferase activity and inhibition using sortase A

N-myristoylation is the covalent addition of a 14-carbon saturated fatty acid (myristate) to the N-terminal glycine of specific protein substrates by N-myristoyltransferase (NMT) and plays an important role in protein regulation by controlling localization, stability, and interactions. We developed a novel method for whole-proteome profiling of free N-terminal glycines through labeling with S. Aureus sortase A (SrtA) and used it for assessment of target engagement by an NMT inhibitor. Analysis of the SrtA-labeling pattern with an engineered biotinylated depsipeptide SrtA substrate (Biotin-ALPET-Haa, Haa = 2-hydroxyacetamide) enabled whole proteome identification and quantification of de novo generated N-terminal Gly proteins in response to NMT inhibition by nanoLC-MS/MS proteomics, and was confirmed for specific substrates across multiple cell lines by gel-based analyses and ELISA. To achieve optimal signal over background noise we introduce a novel and generally applicable improvement to the biotin/avidin affinity enrichment step by chemically dimethylating commercial NeutrAvidin resin and combining this with two-step LysC on-bead/trypsin off-bead digestion, effectively eliminating avidin-derived tryptic peptides and enhancing identification of enriched peptides. We also report SrtA substrate specificity in whole-cell lysates for the first time, confirming SrtA promiscuity beyond its recognized preference for N-terminal glycine, and its usefulness as a tool for unbiased labeling of N-terminal glycine-containing proteins. Our new methodology is complementary to metabolic tagging strategies, providing the first approach for whole proteome gain-of signal readout for NMT inhibition in complex samples which are not amenable to metabolic tagging