The endoplasmic reticulum-localized enzyme zDHHC6 mediates S-acylation of short transmembrane constructs from multiple type I and II membrane proteins.

In this study, we investigated the S-acylation of two host cell proteins important for viral infection: TMPRSS2 (transmembrane serine protease 2), which cleaves severe acute respiratory syndrome coronavirus 2 spike to facilitate viral entry, and bone marrow stromal antigen 2, a general viral restriction factor. We found that both proteins were S-acylated by zDHHC6, an S-acyltransferase enzyme localized at the endoplasmic reticulum, in coexpression experiments. Mutagenic analysis revealed that zDHHC6 modifies a single cysteine in each protein, which are in proximity to the transmembrane domains (TMDs). For TMPRSS2, the modified cysteine is positioned two residues into the TMD, whereas the modified cysteine in bone marrow stromal antigen 2 has a cytosolic location two amino acids upstream of the TMD. Cysteine swapping revealed that repositioning the target cysteine of TMPRSS2 further into the TMD substantially reduced S-acylation by zDHHC6. Interestingly, zDHHC6 efficiently S-acylated truncated forms of these proteins that contained only the TMDs and short juxtamembrane regions. The ability of zDHHC6 to modify short TMD sequences was also seen for the transferrin receptor (another type II membrane protein) and for five different type I membrane protein constructs, including cluster of differentiation 4. Collectively, the results of this study show that zDHHC6 can modify diverse membrane proteins (type I and II) and requires only the presence of the TMD and target cysteine for efficient S-acylation. Thus, zDHHC6 may be a broad specificity S-acyltransferase specialized for the modification of a diverse set of transmembrane proteins at the endoplasmic reticulum.

S-acylation of Sprouty and SPRED proteins by the S-acyltransferase zDHHC17 involves a novel mode of enzyme-substrate interaction.

S-acylation is an essential post-translational modification, which is mediated by a family of 23 zDHHC enzymes in humans. Several thousand proteins are modified by S-acylation; however, we lack a detailed understanding of how enzyme-substrate recognition and specificity is achieved. Previous work showed that the ankyrin repeat domain of zDHHC17 (ANK17) recognizes a short linear motif, known as the zDHHC ANK binding motif (zDABM) in substrate protein SNAP25, as a mechanism of substrate recruitment prior to S-acylation. Here, we investigated the S-acylation of the Sprouty and SPRED family of proteins by zDHHC17. Interestingly, although Sprouty-2 (Spry2) contains a zDABM that interacts with ANK17, this mode of binding is dispensable for S-acylation, and indeed removal of the zDABM does not completely ablate binding to zDHHC17. Furthermore, the related SPRED3 protein interacts with and is efficiently S-acylated by zDHHC17, despite lacking a zDABM. We undertook mutational analysis of SPRED3 to better understand the basis of its zDABM-independent interaction with zDHHC17. This analysis found that the cysteine-rich SPR domain of SPRED3, which is the defining feature of all Sprouty and SPRED proteins, interacts with zDHHC17. Surprisingly, the interaction with SPRED3 was independent of ANK17. Our mutational analysis of Spry2 was consistent with the SPR domain of this protein containing a zDHHC17-binding site, and Spry2 also showed detectable binding to a zDHHC17 mutant lacking the ANK domain. Thus, zDHHC17 can recognize its substrates through zDABM-dependent and/or zDABM-independent mechanisms, and some substrates display more than one mode of binding to this enzyme.

Label-Free Screening of Drug-Induced Liver Injury Using Stimulated Raman Scattering Microscopy and Spectral Phasor Analysis.

Hepatic toxicity is a leading cause of the termination of clinical trials and the withdrawal of therapeutics following regulatory approval. The detection of drug-induced liver injury (DILI) is therefore of importance to ensure patient safety and the effectiveness of novel small molecules and drugs. DILI encompasses drug-induced steatosis (DIS) and drug-induced phospholipidosis (DIPL) which involve the accumulation of excess intracellular lipids. Here, we develop hyperspectral stimulated Raman scattering (SRS) microscopy as a label-free methodology for discriminating DIS and DIPL in mammalian cell culture. We demonstrate that hyperspectral SRS imaging in tandem with spectral phasor analysis is capable of discriminating DIS and DIPL based on the nature and distribution of intracellular lipids resulting from each process. To demonstrate the practical application of this methodology, we develop a panel of alkyne-tagged propranolol analogues that display varying DILI effects. Using hyperspectral SRS imaging together with spectral phasor analysis, our label-free methodology corroborated the standard fluorescence-based assay for DILI. As a label-free screening method, it offers a convenient and expedient methodology for visualizing hepatotoxicity in cell cultures which could be integrated into the early stages of the drug development process for screening new chemical entities for DILI.

A Mechanistic Investigation of the N-Hydroxyphthalimide Catalyzed Benzylic Oxidation Mediated by Sodium Chlorite.

A detailed investigation into the mechanistic course of N-hydroxyphthalimide catalyzed oxidation of benzylic centers using sodium chlorite as the stoichiometric oxidant is reported. Through a combination of experimental, spectroscopic, and computational techniques, the transformation is interrogated, providing improved reaction conditions and an enhanced understanding of the mechanism. Performing the transformation in the presence of acetic acid or a pH 4.5 buffer leads to extended reaction times but improves the catalyst lifetime, leading to the complete consumption of the starting material. Chlorine dioxide is identified as the active oxidant that is able to oxidize the N-hydroxyphthalimide anion to the phthalimide-N-oxyl radical, the proposed catalytically active species, which is able to abstract a hydrogen atom from the substrate. A second molecule of chlorine dioxide reacts with the resultant radical and, after loss of hypochlorous acid, leads to the observed product. Through a broad variety of techniques including UV/vis, EPR and Raman spectroscopy, isotopic labeling, and the use of radical traps, evidence for the mechanism is presented that is supported through electronic structural calculations.

bbSelect - An Open-Source Tool for Performing a 3D Pharmacophore-Driven Diverse Selection of R-Groups.

The design of compounds during hit-to-lead often seeks to explore a vector from a core scaffold to form additional interactions with the target protein. A rational approach to this is to probe the region of a protein accessed by a vector with a systematic placement of pharmacophore features in 3D, particularly when bound structures are not available. Herein, we present bbSelect, an open-source tool built to map the placements of pharmacophore features in 3D Euclidean space from a library of R-groups, employing partitioning to drive a diverse and systematic selection to a user-defined size. An evaluation of bbSelect against established methods exemplified the superiority of bbSelect in its ability to perform diverse selections, achieving high levels of pharmacophore feature placement coverage with selection sizes of a fraction of the total set and without the introduction of excess complexity. bbSelect also reports visualizations and rationale to enable users to understand and interrogate results. This provides a tool for the drug discovery community to guide their hit-to-lead activities.

Photoaffinity labelling displacement assay using multiple recombinant protein domains.

The development and optimisation of a photoaffinity labelling (PAL) displacement assay is presented, where a highly efficient PAL probe was used to report on the relative binding affinities of compounds to specific binding sites in multiple recombinant protein domains in tandem. The N- and C-terminal bromodomains of BRD4 were used as example target proteins. A test set of 264 compounds annotated with activity against the bromodomain and extra-terminal domain (BET) family in ChEMBL were used to benchmark the assay. The pIC50 values obtained from the assay correlated well with orthogonal TR-FRET data, highlighting the potential of this highly accessible PAL biochemical screening platform.

Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation.

Protein S-acylation is a reversible post-translational modification that modulates the localization and function of many cellular proteins. S-acylation is mediated by a family of zinc finger DHHC (Asp-His-His-Cys) domain-containing (zDHHC) proteins encoded by 23 distinct ZDHHC genes in the human genome. These enzymes catalyze S-acylation in a two-step process involving "autoacylation" of the cysteine residue in the catalytic DHHC motif followed by transfer of the acyl chain to a substrate cysteine. S-acylation is essential for many fundamental physiological processes, and there is growing interest in zDHHC enzymes as novel drug targets for a range of disorders. However, there is currently a lack of chemical modulators of S-acylation either for use as tool compounds or for potential development for therapeutic purposes. Here, we developed and implemented a novel FRET-based high-throughput assay for the discovery of compounds that interfere with autoacylation of zDHHC2, an enzyme that is implicated in neuronal S-acylation pathways. Our screen of >350,000 compounds identified two related tetrazole-containing compounds (TTZ-1 and TTZ-2) that inhibited both zDHHC2 autoacylation and substrate S-acylation in cell-free systems. These compounds were also active in human embryonic kidney 293T cells, where they inhibited the S-acylation of two substrates (SNAP25 and PSD95 [postsynaptic density protein 95]) mediated by different zDHHC enzymes, with some apparent isoform selectivity. Furthermore, we confirmed activity of the hit compounds through resynthesis, which provided sufficient quantities of material for further investigations. The assays developed provide novel strategies to screen for zDHHC inhibitors, and the identified compounds add to the chemical toolbox for interrogating cellular activities of zDHHC enzymes in S-acylation.

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis.

Carboxylesterases (CEs) are a class of enzymes that catalyze the hydrolysis of esters in a variety of endogenous and exogenous molecules. CEs play an important role in drug metabolism, in the onset and progression of disease, and can be harnessed for prodrug activation strategies. As such, the regulation of CEs is an important clinical and pharmaceutical consideration. Here, we report the first ratiometric sensor for CE activity using Raman spectroscopy based on a bisarylbutadiyne scaffold. The sensor was shown to be highly sensitive and specific for CE detection and had low cellular cytotoxicity. In hepatocyte cells, the ratiometric detection of esterase activity was possible, and the result was validated by multimodal imaging with standard viability stains used for fluorescence microscopy within the same cell population. In addition, we show that the detection of localized ultraviolet damage in a mixed cell population was possible using stimulated Raman scattering microscopy coupled with spectral phasor analysis. This sensor demonstrates the practical advantages of low molecular weight sensors that are detected using ratiometric Raman imaging and will have applications in drug discovery and biomedical research.

Peracid Oxidation of Unactivated sp3 C-H Bonds: An Important Solvent Effect.

The peracid oxidation of hydrocarbons in chlorinated solvents is a low yielding and poorly selective process. Through a combination of DFT calculations, spectroscopic studies, and kinetic measurement it is shown that the origin of this is electronic in nature and can be influenced through the addition of hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA). Performing the reaction of a cycloalkane with mCPBA in a fluorinated alcohol solvent such as nonafluoro-tert-butanol (NFTB) or hexafluoroisopropanol (HFIP), which act as strong HBD and poor HBA, leads to significantly higher yields and selectivities being observed for the alcohol product. Application of the optimised reaction conditions allows for the selective oxidation of both cyclic and linear alkane substrates delivering the corresponding alcohol in up to 86 % yield. The transformation shows selectivity for tertiary centres over secondary centres and the oxidation of secondary centres is strongly influenced by stereoelectronic effects. Primary centres are not oxidised by this method. A simple computational model developed to understand this transformation provides a powerful tool to reliably predict the influence of substitution and functionality on reaction outcome.

Expanding the Range of Bioorthogonal Tags for Multiplex Stimulated Raman Scattering Microscopy.

Multiplex optical detection in live cells is challenging due to overlapping signals and poor signal-to-noise associated with some chemical reporters. To address this, the application of spectral phasor analysis to stimulated Raman scattering (SRS) microscopy for unmixing three bioorthogonal Raman probes within cells is reported. Triplex detection of a metallacarborane using the B-H stretch at 2480-2650 cm-1 , together with a bis-alkyne and deuterated fatty acid can be achieved within the cell-silent region of the Raman spectrum. When coupled to imaging in the high-wavenumber region of the cellular Raman spectrum, nine discrete regions of interest can be spectrally unmixed from the hyperspectral SRS dataset, demonstrating a new capability in the toolkit of multiplexed Raman imaging of live cells.

Identification of key features required for efficient S-acylation and plasma membrane targeting of sprouty-2.

Sprouty-2 is an important regulator of growth factor signalling and a tumour suppressor protein. The defining feature of this protein is a cysteine-rich domain (CRD) that contains twenty-six cysteine residues and is modified by S-acylation. In this study, we show that the CRD of sprouty-2 is differentially modified by S-acyltransferase enzymes. The high specificity/low activity zDHHC17 enzyme mediated restricted S-acylation of sprouty-2, and cysteine-265 and -268 were identified as key targets of this enzyme. In contrast, the low specificity/high activity zDHHC3 and zDHHC7 enzymes mediated more expansive modification of the sprouty-2 CRD. Nevertheless, S-acylation by all enzymes enhanced sprouty-2 expression, suggesting that S-acylation stabilises this protein. In addition, we identified two charged residues (aspartate-214 and lysine-223), present on opposite faces of a predicted α-helix in the CRD, which are essential for S-acylation of sprouty-2. Interestingly, mutations that perturbed S-acylation also led to a loss of plasma membrane localisation of sprouty-2 in PC12 cells. This study provides insight into the mechanisms and outcomes of sprouty-2 S-acylation, and highlights distinct patterns of S-acylation mediated by different classes of zDHHC enzymes.

The linker domain of the SNARE protein SNAP25 acts as a flexible molecular spacer that ensures efficient S-acylation.

S-Acylation of the SNARE protein SNAP25 (synaptosome-associated protein of 25 kDa) is mediated by a subset of Golgi zinc finger DHHC-type palmitoyltransferase (zDHHC) enzymes, particularly zDHHC17. The ankyrin repeat domain of zDHHC17 interacts with a short linear motif known as the zDHHC ankyrin repeat-binding motif (zDABM) in SNAP25 (112VVASQP117), which is downstream of its S-acylated, cysteine-rich domain (85CGLCVCPC92). Here, we investigated the importance of a flexible linker region (amino acids 93-111, referred to hereafter as the "mini-linker" region) that separates the zDABM and S-acylated cysteines in SNAP25. Shortening the mini-linker did not affect the SNAP25-zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, but extended and rigid alanine-proline repeats perturbed it. A SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localization as WT SNAP25, indicating that the amino acid composition of the mini-linker is not important for SNAP25 localization. Using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher-affinity zDABM. This mutant interacted with zDHHC17 more strongly but was S-acylated with reduced efficiency in HEK293T cells, implying that a lower-affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. These results show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer that ensures efficient coupling of the SNAP25-zDHHC17 interaction and S-acylation of SNAP25.

Mitokyne: A Ratiometric Raman Probe for Mitochondrial pH.

Mitochondrial pH (pHmito) is intimately related to mitochondrial function, and aberrant values for pHmito are linked to several disease states. We report the design, synthesis, and application of mitokyne 1-the first small molecule pHmito sensor for stimulated Raman scattering (SRS) microscopy. This ratiometric probe can determine subtle changes in pHmito in response to external stimuli and the inhibition of both the electron transport chain and ATP synthase with small molecule inhibitors. In addition, 1 was also used to monitor mitochondrial dynamics in a time-resolved manner with subcellular spatial resolution during mitophagy providing a powerful tool for dissecting the molecular and cell biology of this critical organelle.

One-Step Synthesis of Photoaffinity Probes for Live-Cell MS-Based Proteomics.

We present a one-step Ugi reaction protocol for the expedient synthesis of photoaffinity probes for live-cell MS-based proteomics. The reaction couples an amine affinity function with commonly used photoreactive groups, and a variety of handle functionalities. Using this technology, a series of pan-BET (BET: bromodomain and extra-terminal domain) selective bromodomain photoaffinity probes were obtained by parallel synthesis. Studies on the effects of photoreactive group, linker length and irradiation wavelength on photocrosslinking efficiency provide valuable insights into photoaffinity probe design. Optimal probes were progressed to MS-based proteomics to capture the BET family of proteins from live cells and reveal their potential on- and off-target profiles.

Investigating the effects of the core nitrogen atom configuration on the thermodynamic solubility of 6,5-bicyclic heterocycles.

Physicochemical properties, such as solubility, are important when prioritising compounds for progression on a drug discovery project. There is limited literature around the systematic effects of core changes on thermodynamic solubility. This work details the synthesis of nitrogen containing 6,5-bicyclic heterocyclic cores which are common scaffolds in medicinal chemistry and the analysis of their physicochemical properties, particularly, thermodynamic solubility. Crystalline solids were obtained where possible to enable a robust comparison of the thermodynamic solubility. Other parameters such as pKa, melting point and lipophilicity were also measured to determine the key factors affecting the observed solubility.

Inhibition of the ULK1 protein complex suppresses Staphylococcus-induced autophagy and cell death.

Autophagy plays multiple roles in host cells challenged with extracellular pathogens. Here, we aimed to explore whether autophagy inhibition could prevent bacterial infections. We first confirmed widely distinct patterns of autophagy responses in host cells infected with Staphylococcus aureus, as compared with Salmonella Only infection with Staphylococcus produced strong accumulation of lipidated autophagy-related protein LC3B (LC3B-II). Infection with virulent Staphylococcus strains induced formation of p62-positive aggregates, suggestive of accumulated ubiquitinated targets. During Salmonella infection, bacteria remain enclosed by lysosomal-associated membrane protein 2 (LAMP2)-positive lysosomes, whereas virulent Staphylococcus apparently exited from enlarged lysosomes and invaded the cytoplasm. Surprisingly, Staphylococcus appeared to escape from the lysosome without generation of membrane-damage signals as detected by galectin-3 recruitment. In contrast, Salmonella infection produced high levels of lysosomal damage, consistent with a downstream antibacterial xenophagy response. Finally, we studied the Unc-51-like autophagy-activating kinase 1 (ULK1) regulatory complex, including the essential subunit autophagy-related protein 13 (ATG13). Infection of cells with either Staphylococcus or Salmonella led to recruitment of ATG13 to sites of cytosolic bacterial cells to promote autophagosome formation. Of note, genetic targeting of ATG13 suppressed autophagy and the ability of Staphylococcus to infect and kill host cells. Two different ULK1 inhibitors also prevented Staphylococcus intracellular replication and host cell death. Interestingly, inhibition of the ULK1 pathway had the opposite effect on Salmonella, sensitizing cells to the infection. Our results suggest that ULK1 inhibitors may offer a potential strategy to impede cellular infection by S. aureus.

A new class of ratiometric small molecule intracellular pH sensors for Raman microscopy.

Intracellular pH (pHi) homeostasis is intertwined with a myriad of normal cellular behaviors as well as pathological processes. As such, small molecule probes for the measurement of pHi are invaluable tools for chemical biology, facilitating the study of the role of pH in cellular function and disease. The field of small molecule pHi sensors has traditionally been dominated with probes based on fluorescent scaffolds. In this study, a series of low molecular weight (<260) oligoyne compounds have been developed which exhibit pH sensitive alkyne stretching frequencies (νalkyne) in Raman spectroscopy. The modular design of the compounds enabled tuneability of their pKa(H) through simple structural modification, such that continuous pH sensitivity is achieved over the range 2-10. Alkyne stretching bands reside in the 'cell-silent' region of the Raman spectrum (1800-2600 cm-1) and are readily detectable in a cellular environment with subcellular spatial resolution. This enabled the application of a pH sensitive oligoyne compound to the ratiometric sensing of pHi in prostate cancer (PC3) cells in response to drug treatment. We propose that probes based on Alkyne Tag Raman Imaging offer an entirely new platform for the sensing of pHi, complementary to fluorescence microscopy.

Molecular basis of fatty acid selectivity in the zDHHC family of S-acyltransferases revealed by click chemistry.

S-acylation is a major posttranslational modification, catalyzed by the zinc finger DHHC domain containing (zDHHC) enzyme family. S-acylated proteins can be modified by different fatty acids; however, very little is known about how zDHHC enzymes contribute to acyl chain heterogeneity. Here, we used fatty acid-azide/alkyne labeling of mammalian cells, showing their transformation into acyl-CoAs and subsequent click chemistry-based detection, to demonstrate that zDHHC enzymes have marked differences in their fatty acid selectivity. This difference in selectivity was apparent even for highly related enzymes, such as zDHHC3 and zDHHC7, which displayed a marked difference in their ability to use C18:0 acyl-CoA as a substrate. Furthermore, we identified isoleucine-182 in transmembrane domain 3 of zDHHC3 as a key determinant in limiting the use of longer chain acyl-CoAs by this enzyme. This study uncovered differences in the fatty acid selectivity profiles of cellular zDHHC enzymes and mapped molecular determinants governing this selectivity.

A Scalable Metal-, Azide-, and Halogen-Free Method for the Preparation of Triazoles.

A scalable metal-, azide-, and halogen-free method for the synthesis of substituted 1,2,3-triazoles has been developed. The reaction proceeds through a 3-component coupling of α-ketoacetals, tosyl hydrazide, and a primary amine. The reaction shows outstanding functional-group tolerance with respect to both the α-ketoacetal and amine coupling partners, providing access to 4-, 1,4-, 1,5-, and 1,4,5-substituted triazoles in excellent yield. This robust method results in densely functionalised 1,2,3-triazoles that remain challenging to prepare by azide-alkyne cycloaddition (AAC, CuAAC, RuAAC) methods and can be scaled in either batch or flow reactors. Methods for the chemoselective reaction of either aliphatic amines or anilines are also described, revealing some of the potential of this novel and highly versatile transformation.

A Photoaffinity-Based Fragment-Screening Platform for Efficient Identification of Protein Ligands.

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment-screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.