S-acylation of p62 promotes p62 droplet recruitment into autophagosomes in mammalian autophagy.

p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.

Structure, mechanism, and inhibition of Hedgehog acyltransferase.

The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.

Inhibition of vaccinia virus L1 N-myristoylation by the host N-myristoyltransferase inhibitor IMP-1088 generates non-infectious virions defective in cell entry.

We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we examine the importance of N-myristoylation during vaccinia virus (VACV) infection in primate cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors.

Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion.

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of "pseudoschizonts," which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

XerC Is Required for the Repair of Antibiotic- and Immune-Mediated DNA Damage in Staphylococcus aureus.

To survive in the host environment, pathogenic bacteria need to be able to repair DNA damage caused by both antibiotics and the immune system. The SOS response is a key bacterial pathway to repair DNA double-strand breaks and may therefore be a good target for novel therapeutics to sensitize bacteria to antibiotics and the immune response. However, the genes required for the SOS response in Staphylococcus aureus have not been fully established. Therefore, we carried out a screen of mutants involved in various DNA repair pathways to understand which were required for induction of the SOS response. This led to the identification of 16 genes that may play a role in SOS response induction and, of these, 3 that affected the susceptibility of S. aureus to ciprofloxacin. Further characterization revealed that, in addition to ciprofloxacin, loss of the tyrosine recombinase XerC increased the susceptibility of S. aureus to various classes of antibiotics, as well as to host immune defenses. Therefore, the inhibition of XerC may be a viable therapeutic approach to sensitize S. aureus to both antibiotics and the immune response.

Substrate-biased activity-based probes identify proteases that cleave receptor CDCP1.

CUB domain-containing protein 1 (CDCP1) is an oncogenic orphan transmembrane receptor and a promising target for the detection and treatment of cancer. Extracellular proteolysis of CDCP1 by poorly defined mechanisms induces pro-metastatic signaling. We describe a new approach for the rapid identification of proteases responsible for key proteolytic events using a substrate-biased activity-based probe (sbABP) that incorporates a substrate cleavage motif grafted onto a peptidyl diphenyl phosphonate warhead for specific target protease capture, isolation and identification. Using a CDCP1-biased probe, we identify urokinase (uPA) as the master regulator of CDCP1 proteolysis, which acts both by directly cleaving CDCP1 and by activating CDCP1-cleaving plasmin. We show that coexpression of uPA and CDCP1 is strongly predictive of poor disease outcome across multiple cancers and demonstrate that uPA-mediated CDCP1 proteolysis promotes metastasis in disease-relevant preclinical in vivo models. These results highlight CDCP1 cleavage as a potential target to disrupt cancer and establish sbABP technology as a new approach to identify disease-relevant proteases.

Discovery of a Potent Deubiquitinase (DUB) Small-Molecule Activity-Based Probe Enables Broad Spectrum DUB Activity Profiling in Living Cells.

Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, often leading to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto-inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin-derived covalent activity-based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12) as a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs across a range of non-toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes.

A KLK6 Activity-Based Probe Reveals a Role for KLK6 Activity in Pancreatic Cancer Cell Invasion.

Pancreatic cancer has the lowest survival rate of all common cancers due to late diagnosis and limited treatment options. Serine hydrolases are known to mediate cancer progression and metastasis through initiation of signaling cascades and cleavage of extracellular matrix proteins, and the kallikrein-related peptidase (KLK) family of secreted serine proteases have emerging roles in pancreatic ductal adenocarcinoma (PDAC). However, the lack of reliable activity-based probes (ABPs) to profile KLK activity has hindered progress in validation of these enzymes as potential targets or biomarkers. Here, we developed potent and selective ABPs for KLK6 by using a positional scanning combinatorial substrate library and characterized their binding mode and interactions by X-ray crystallography. The optimized KLK6 probe IMP-2352 (kobs/I = 11,000 M-1 s-1) enabled selective detection of KLK6 activity in a variety of PDAC cell lines, and we observed that KLK6 inhibition reduced the invasiveness of PDAC cells that secrete active KLK6. KLK6 inhibitors were combined with N-terminomics to identify potential secreted protein substrates of KLK6 in PDAC cells, providing insights into KLK6-mediated invasion pathways. These novel KLK6 ABPs offer a toolset to validate KLK6 and associated signaling partners as targets or biomarkers across a range of diseases.

UCHL1 as a novel target in breast cancer: emerging insights from cell and chemical biology.

Breast cancer has the highest incidence and death rate among cancers in women worldwide. In particular, metastatic estrogen receptor negative (ER-) breast cancer and triple-negative breast cancer (TNBC) subtypes have very limited treatment options, with low survival rates. Ubiquitin carboxyl terminal hydrolase L1 (UCHL1), a ubiquitin C-terminal hydrolase belonging to the deubiquitinase (DUB) family of enzymes, is highly expressed in these cancer types, and several key reports have revealed emerging and important roles for UCHL1 in breast cancer. However, selective and potent small-molecule UCHL1 inhibitors have been disclosed only very recently, alongside chemical biology approaches to detect regulated UHCL1 activity in cancer cells. These tools will enable novel insights into oncogenic mechanisms driven by UCHL1, and identification of substrate proteins deubiquitinated by UCHL1, with the ultimate goal of realising the potential of UCHL1 as a drug target in breast cancer.

D-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition.

The broad-spectrum antibiotic D-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of D-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and D-Ala:D-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5'-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.

How Structures of Complement Complexes Guide Therapeutic Design.

The complement system is essential for immune defence against infection and modulation of proinflammatory responses. Activation of the terminal pathway of complement triggers formation of the membrane attack complex (MAC), a multi-protein pore that punctures membranes. Recent advances in structural biology, specifically cryo-electron microscopy (cryoEM), have provided atomic resolution snapshots along the pore formation pathway. These structures have revealed dramatic conformational rearrangements that enable assembly and membrane rupture. Here we review the structural basis for MAC formation and show how soluble proteins transition into a giant ß-barrel pore. We also discuss regulatory complexes of the terminal pathway and their impact on structure-guided drug discovery of complement therapeutics.

AWZ1066S, a highly specific anti-Wolbachia drug candidate for a short-course treatment of filariasis.

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

Dynamic Protein Acylation: New Substrates, Mechanisms, and Drug Targets.

Post-translational attachment of lipids to proteins is found in all organisms, and is important for many biological processes. Acylation with myristic and palmitic acids are among the most common lipid modifications, and understanding reversible protein palmitoylation dynamics has become a particularly important goal. Linking acyltransferase enzymes to disease states can be challenging due to a paucity of robust models, compounded by functional redundancy between many palmitoyl transferases; however, in cases such as Wnt or Hedgehog signalling, small molecule inhibitors have been identified, with some progressing to clinical trials. In this review, we present recent developments in our understanding of protein acylation in human health and disease through use of chemical tools, global profiling of acylated proteomes, and functional studies of specific protein targets.

A Suite of Activity-Based Probes To Dissect the KLK Activome in Drug-Resistant Prostate Cancer.

Kallikrein-related peptidases (KLKs) are a family of secreted serine proteases, which form a network (the KLK activome) with an important role in proteolysis and signaling. In prostate cancer (PCa), increased KLK activity promotes tumor growth and metastasis through multiple biochemical pathways, and specific quantification and tracking of changes in the KLK activome could contribute to validation of KLKs as potential drug targets. Herein we report a technology platform based on novel activity-based probes (ABPs) and inhibitors enabling simultaneous orthogonal analysis of KLK2, KLK3, and KLK14 activity in hormone-responsive PCa cell lines and tumor homogenates. Importantly, we identifed a significant decoupling of KLK activity and abundance and suggest that KLK proteolysis should be considered as an additional parameter, along with the PSA blood test, for accurate PCa diagnosis and monitoring. Using selective inhibitors and multiplexed fluorescent activity-based protein profiling (ABPP), we dissect the KLK activome in PCa cells and show that increased KLK14 activity leads to a migratory phenotype. Furthermore, using biotinylated ABPs, we show that active KLK molecules are secreted into the bone microenvironment by PCa cells following stimulation by osteoblasts suggesting KLK-mediated signaling mechanisms could contribute to PCa metastasis to bone. Together our findings show that ABPP is a powerful approach to dissect dysregulation of the KLK activome as a promising and previously underappreciated therapeutic target in advanced PCa.

Analysis of a fully infectious bio-orthogonally modified human virus reveals novel features of virus cell entry.

We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we reveal novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination from primary entering from secondary assembling virions, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, this work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions.

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.

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 by 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.

Discovery of a Potent and Selective Covalent Inhibitor and Activity-Based Probe for the Deubiquitylating Enzyme UCHL1, with Antifibrotic Activity.

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitylating enzyme that is proposed as a potential therapeutic target in neurodegeneration, cancer, and liver and lung fibrosis. Herein we report the discovery of the most potent and selective UCHL1 probe (IMP-1710) to date based on a covalent inhibitor scaffold and apply this probe to identify and quantify target proteins in intact human cells. IMP-1710 stereoselectively labels the catalytic cysteine of UCHL1 at low nanomolar concentration in cells. We further demonstrate that potent and selective UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting the potential of UCHL1 as a potential therapeutic target in fibrotic diseases.

Activity-Based Protein Profiling for the Study of Parasite Biology.

Parasites exist within most ecological niches, often transitioning through biologically and chemically complex host environments over the course of their parasitic life cycles. While the development of technologies for genetic engineering has revolutionised the field of functional genomics, parasites have historically been less amenable to such modification. In light of this, parasitologists have often been at the forefront of adopting new small-molecule technologies, repurposing drugs into biological tools and probes. Over the last decade, activity-based protein profiling (ABPP) has evolved into a powerful and versatile chemical proteomic platform for characterising the function of enzymes. Central to ABPP is the use of activity-based probes (ABPs), which covalently modify the active sites of enzyme classes ranging from serine hydrolases to glycosidases. The application of ABPP to cellular systems has contributed vastly to our knowledge on the fundamental biology of a diverse range of organisms and has facilitated the identification of potential drug targets in many pathogens. In this chapter, we provide a comprehensive review on the different forms of ABPP that have been successfully applied to parasite systems, and highlight key biological insights that have been enabled through their application.

Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells.

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of blood glucose homeostasis. Ligand-specific differences in membrane trafficking of the GLP-1R influence its signalling properties and therapeutic potential in type 2 diabetes. Here, we have evaluated how different factors combine to control the post-endocytic trafficking of GLP-1R to recycling versus degradative pathways. Experiments were performed in primary islet cells, INS-1 832/3 clonal beta cells and HEK293 cells, using biorthogonal labelling of GLP-1R to determine its localisation and degradation after treatment with GLP-1, exendin-4 and several further GLP-1R agonist peptides. We also characterised the effect of a rare GLP1R coding variant, T149M, and the role of endosomal peptidase endothelin-converting enzyme-1 (ECE-1), in GLP1R trafficking. Our data reveal how treatment with GLP-1 versus exendin-4 is associated with preferential GLP-1R targeting towards a recycling pathway. GLP-1, but not exendin-4, is a substrate for ECE-1, and the resultant propensity to intra-endosomal degradation, in conjunction with differences in binding affinity, contributes to alterations in GLP-1R trafficking behaviours and degradation. The T149M GLP-1R variant shows reduced signalling and internalisation responses, which is likely to be due to disruption of the cytoplasmic region that couples to intracellular effectors. These observations provide insights into how ligand- and genotype-specific factors can influence GLP-1R trafficking.