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

Proteome-wide analysis of protein lipidation using chemical probes: in-gel fluorescence visualisation, identification and quantification of N-myristoylation, N- and S-acylation, Ocholesterylation, S-farnesylation and S-geranylgeranylation

Protein lipidation is one of the most widespread post-translational modifications (PTMs) found in nature, regulating protein function, structure and subcellular localization. Lipid transferases and their substrate proteins are also attracting increasing interest as drug targets because of their dysregulation in many disease states. However, the inherent hydrophobicity and potential dynamic nature of lipid modifications makes them notoriously challenging to detect by many analytical methods. Chemical proteomics provides a powerful approach to identify and quantify these diverse protein modifications by combining bespoke chemical tools for lipidated protein enrichment with quantitative mass spectrometry–based proteomics. Here, we report a robust and proteome-wide approach for the exploration of five major classes of protein lipidation in living cells, through the use of specific chemical probes for each lipid PTM. In-cell labeling of lipidated proteins is achieved by the metabolic incorporation of a lipid probe that mimics the specific natural lipid, concomitantly wielding an alkyne as a bio-orthogonal labeling tag. After incorporation, the chemically tagged proteins can be coupled to multifunctional ‘capture reagents’ by using click chemistry, allowing in-gel fluorescence visualization or enrichment via affinity handles for quantitative chemical proteomics based on label-free quantification (LFQ) or tandem mass-tag (TMT) approaches. In this protocol, we describe the application of lipid probes for N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylation in multiple cell lines to illustrate both the workflow and data obtained in these experiments. We provide detailed workflows for method optimization, sample preparation for chemical proteomics and data processing. A properly trained researcher (e.g., technician, graduate student or postdoc) can complete all steps from optimizing metabolic labeling to data processing within 3 weeks. This protocol enables sensitive and quantitative analysis of lipidated proteins at a proteome-wide scale at native expression levels, which is critical to understanding the role of lipid PTMs in health and disease

Quantum heuristic algorithm for traveling salesman problem

We propose a quantum heuristic algorithm to solve a traveling salesman problem by generalizing Grover search. Sufficient conditions are derived to greatly enhance the probability of finding the tours with extremal costs, reaching almost to unity and they are shown characterized by statistical properties of tour costs. In particular for a Gaussian distribution of the tours along the cost we show that the quantum algorithm exhibits the quadratic speedup of its classical counterpart, similarly to Grover search.Comment: Published versio

Identification of the first structurally validated covalent ligands of the small GTPase RAB27A

Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane via protein–protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket (‘WF-binding pocket’) via a conserved tryptophan–phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets

Rif1 S-acylation mediates DNA double-strand break repair at the inner nuclear membrane

Rif1 is involved in telomere homeostasis, DNA replication timing, and DNA double-strand break (DSB) repair pathway choice from yeast to human. The molecular mechanisms that enable Rif1 to fulfill its diverse roles remain to be determined. Here, we demonstrate that Rif1 is S-acylated within its conserved N-terminal domain at cysteine residues C466 and C473 by the DHHC family palmitoyl acyltransferase Pfa4. Rif1 S-acylation facilitates the accumulation of Rif1 at DSBs, the attenuation of DNA end-resection, and DSB repair by non-homologous end-joining (NHEJ). These findings identify S-acylation as a posttranslational modification regulating DNA repair. S-acylated Rif1 mounts a localized DNA-damage response proximal to the inner nuclear membrane, revealing a mechanism of compartmentalized DSB repair pathway choice by sequestration of a fatty acylated repair factor at the inner nuclear membrane

Targeting the bacterial SOS response for new antimicrobial agents: drug targets, molecular mechanisms and inhibitors

Antimicrobial resistance is a pressing threat to global health, with multidrug-resistant pathogens becoming increasingly prevalent. The bacterial SOS pathway functions in response to DNA damage that occurs during infection, initiating several pro-survival and resistance mechanisms, such as DNA repair and hypermutation. This makes SOS pathway components potential targets that may combat drug-resistant pathogens and decrease resistance emergence. This review discusses the mechanism of the SOS pathway; the structure and function of potential targets AddAB, RecBCD, RecA and LexA; and efforts to develop selective small-molecule inhibitors of these proteins. These inhibitors may serve as valuable tools for target validation and provide the foundations for desperately needed novel antibacterial therapeutics

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

Peroxisomes are a family of organelles which have many unusual features. They can arise de novo from the endoplasmic reticulum by a still poorly characterized process, yet possess a unique machinery for the import of their matrix proteins. As peroxisomes lack DNA, their function, which is highly variable and dependent on developmental and/or environmental conditions, is determined by the post-translational import of specific metabolic enzymes in folded or oligomeric states. The two classes of matrix targeting signals for peroxisomal proteins [PTS1 (peroxisomal targeting signal 1) and PTS2] are recognized by cytosolic receptors [PEX5 (peroxin 5) and PEX7 respectively] which escort their cargo proteins to, or possibly across, the peroxisome membrane. Although the membrane translocation mechanism remains unclear, it appears to be driven by thermodynamically favourable binding interactions. Recycling of the receptors from the peroxisome membrane requires ATP hydrolysis for two linked processes: ubiquitination of PEX5 (and the PEX7 co-receptors in yeast) and the function of two peroxisome-associated AAA (ATPase associated with various cellular activities) ATPases, which play a role in recycling or turnover of the ubiquitinated receptors. This review summarizes and integrates recent findings on peroxisome matrix protein import from yeast, plant and mammalian model systems, and discusses some of the gaps in our understanding of this remarkable protein transport system

Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase

AbstractHedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click–ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click–ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click–ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation