Publications
241 results found
Doll S, Freitas FP, Shah R, et al., 2019, FSP1 is a glutathione-independent ferroptosis suppressor, Nature, Vol: 575, Pages: 693-698, ISSN: 0028-0836
Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.
Panyain N, Godinat A, Lanyon-Hogg T, et al., 2019, Discovery of a Potent and Selective Covalent Inhibitor and Activity-Based Probe for the Deubiquitylating Enzyme UCHL1, with Anti-Fibrotic Activity
<jats:p>Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitylating enzyme which 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, and we show that a previously claimed UCHL1 inhibitor (LDN-57444) fails to engage UCHL1 in cells. We further demonstrate that potent UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting a potential therapeutic role for UCHL1 inhibition and providing a basis for future therapeutic development of selective UCHL1 inhibitors.</jats:p>
Lim C, Ha KP, Clarke R, et al., 2019, Identification of a potent small-molecule inhibitor of bacterial DNA repair that potentiates quinolone antibiotic activity in methicillin-resistant Staphylococcus aureus, Bioorganic and Medicinal Chemistry, Vol: 27, Pages: 1-7, ISSN: 0968-0896
The global emergence of antibiotic resistance is one of the most serious challenges facing modern medicine. There is an urgent need for validation of new drug targets and the development of small molecules with novel mechanisms of action. We therefore sought to inhibit bacterial DNA repair mediated by the AddAB/RecBCD protein complexes as a means to sensitize bacteria to DNA damage caused by the host immune system or quinolone antibiotics. A rational, hypothesis-driven compound optimization identified IMP-1700 as a cell-active, nanomolar potency compound. IMP-1700 sensitized multidrug-resistant Staphylococcus aureus to the fluoroquinolone antibiotic ciprofloxacin, where resistance results from a point mutation in the fluoroquinolone target, DNA gyrase. Cellular reporter assays indicated IMP-1700 inhibited the bacterial SOS-response to DNA damage, and compound-functionalized Sepharose successfully pulled-down the AddAB repair complex. This work provides validation of bacterial DNA repair as a novel therapeutic target and delivers IMP-1700 as a tool molecule and starting point for therapeutic development to address the pressing challenge of antibiotic resistance.
Serwa RA, Sekine E, Brown J, et al., 2019, Analysis of a fully infectious bio-orthogonally modified human virus reveals novel features of virus cell entry, PLoS Pathogens, Vol: 15, ISSN: 1553-7366
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 w
Tapodi A, Clemens DM, Uwineza A, et al., 2019, BFSP1 C-terminal domains released by post-translational processing events can alter significantly the calcium regulation of AQPO water permeability, EXPERIMENTAL EYE RESEARCH, Vol: 185, ISSN: 0014-4835
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Schlott AC, Mayclin S, Reers AR, et al., 2019, Structure-guided identification of resistance breaking antimalarial N-myristoyltransferase inhibitors, Cell Chemical Biology, Vol: 26, Pages: 991-1000.e7, ISSN: 2451-9448
The attachment of myristate to the N-terminal glycine of certain proteins is largely a co-translational modification catalyzed by N-myristoyltransferase (NMT), and involved in protein membrane-localization. Pathogen NMT is a validated therapeutic target in numerous infectious diseases including malaria. In Plasmodium falciparum, NMT substrates are important in essential processes including parasite gliding motility and host cell invasion. Here, we generated parasites resistant to a particular NMT inhibitor series and show that resistance in an in vitro parasite growth assay is mediated by a single amino acid substitution in the NMT substrate-binding pocket. The basis of resistance was validated and analyzed with a structure-guided approach using crystallography, in combination with enzyme activity, stability, and surface plasmon resonance assays, allowing identification of another inhibitor series unaffected by this substitution. We suggest that resistance studies incorporated early in the drug development process help selection of drug combinations to impede rapid evolution of parasite resistance.
Birtley JR, Alomary M, Zanini E, et al., 2019, Inactivating mutations and X-ray crystal structure of the tumor suppressor OPCML reveal cancer-associated functions, Nature Communications, Vol: 10, ISSN: 2041-1723
OPCML, a tumor suppressor gene, is frequently silenced epigenetically in ovarian and other cancers. Here we report, by analysis of databases of tumor sequences, the observation of OPCML somatic missense mutations from various tumor types and the impact of these mutations on OPCML function, by solving the X-ray crystal structure of this glycoprotein to 2.65 Å resolution. OPCML consists of an extended arrangement of three immunoglobulin-like domains and homodimerizes via a network of contacts between membrane-distal domains. We report the generation of a panel of OPCML variants with representative clinical mutations and demonstrate clear phenotypic effects in vitro and in vivo including changes to anchorage-independent growth, interaction with activated cognate receptor tyrosine kinases, cellular migration, invasion in vitro and tumor growth in vivo. Our results suggest that clinically occurring somatic missense mutations in OPCML have the potential to contribute to tumorigenesis in a variety of cancers.
Lanyon-Hogg T, Ritzefeld M, Sefer L, et al., 2019, Acylation-coupled lipophilic induction of polarisation (Acyl-cLIP): a universal assay for lipid transferase and hydrolase enzymes, Chemical Science, Vol: 10, Pages: 8995-9000, ISSN: 2041-6520
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.
Kallemeijn W, Lueg G, Faronato M, et al., 2019, Validation and invalidation of chemical probes for the human N-myristoyltransferases, Cell Chemical Biology, Vol: 26, Pages: 892-900, ISSN: 2451-9456
On-target, cell-active chemical probes are of fundamental importance in both chemical and cell biology, whereas the application of poorly-characterised probes often leads to invalid conclusions.Human N-myristoyltransferase (NMT) has attracted increasing interest as a target in cancer and infectious diseases; here we report an in-depth comparison of five compounds widely applied as human NMT inhibitors, using a combination of quantitative whole-proteome N-myristoylation profiling, biochemical enzyme assays, cytotoxicity, in-cell protein synthesis and cell cycle assays. We find that N-myristoylation is unaffected by 2-hydroxymyristic acid (100 μM), D-NMAPPD (30 μM) or Tris-DBA palladium (10 μM), with the latter compounds causing cytotoxicity through mechanisms unrelated to NMT. In contrast, drug-like inhibitors IMP-366 (DDD85646) and IMP-1088 delivered complete and specific inhibition of N-myristoylation in a range of cell lines at 1 μM and 100 nM, respectively. This study enables the selection of appropriate on-target probes for future studies and suggests the need for reassessment of previous studies which used off-target compounds.
Storck Saha E, Morales Sanfrutos J, Serwa R, et al., 2019, Dual chemical probes enable quantitative system-wide analysis of protein prenylation and prenylation dynamics, Nature Chemistry, Vol: 11, Pages: 552-561, ISSN: 1755-4330
Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.
Jamshidiha M, Pérez-Dorado I, Murray JW, et al., 2019, Coping with strong translational noncrystallographic symmetry and extreme anisotropy in molecular replacement with Phaser: human Rab27a, Acta Crystallographica Section D Structural Biology, Vol: 75, Pages: 342-353, ISSN: 2059-7983
Data pathologies caused by effects such as diffraction anisotropy and translational noncrystallographic symmetry (tNCS) can dramatically complicate the solution of the crystal structures of macromolecules. Such problems were encountered in determining the structure of a mutant form of Rab27a, a member of the Rab GTPases. Mutant Rab27a constructs that crystallize in the free form were designed for use in the discovery of drugs to reduce primary tumour invasiveness and metastasis. One construct, hRab27a<jats:sup>Mut</jats:sup>, crystallized within 24 h and diffracted to 2.82 Å resolution, with a unit cell possessing room for a large number of protein copies. Initial efforts to solve the structure using molecular replacement by <jats:italic>Phaser</jats:italic> were not successful. Analysis of the data set revealed that the crystals suffered from both extreme anisotropy and strong tNCS. As a result, large numbers of reflections had estimated standard deviations that were much larger than their measured intensities and their expected intensities, revealing problems with the use of such data at the time in <jats:italic>Phaser</jats:italic>. By eliminating extremely weak reflections with the largest combined effects of anisotropy and tNCS, these problems could be avoided, allowing a molecular-replacement solution to be found. The lessons that were learned in solving this structure have guided improvements in the numerical analysis used in <jats:italic>Phaser</jats:italic>, particularly in identifying diffraction measurements that convey very little information content. The calculation of information content could also be applied as an alternative to ellipsoidal truncation. The post-mortem analysis also revealed an oversight in accounting for measurement errors in the fast rotation function. While the crystal of mutant Rab27a is not amenable to drug screening, the structure can guide new modifications to obtain more sui
Hong WD, Benayoud F, Nixon GL, et al., 2019, AWZ1066S, a highly specific anti-Wolbachia drug candidate for a short-course treatment of filariasis, Proceedings of the National Academy of Sciences of the United States of America, Vol: 116, Pages: 1414-1419, ISSN: 0027-8424
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.
Kaiser N, Mejuch T, Fedoryshchak R, et al., 2019, Photoactivatable Myristic Acid Probes for UNC119-Cargo Interactions, CHEMBIOCHEM, Vol: 20, Pages: 134-139, ISSN: 1439-4227
Goya Grocin A, Serwa R, Morales Sanfrutos J, et al., 2019, Whole proteome profiling of N-myristoyltransferase activity and inhibition using Sortase A, Molecular and Cellular Proteomics, Vol: 18, Pages: 115-126, ISSN: 1535-9476
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.
Goya Grocin A, Serwa RA, Morales Sanfrutos J, et al., 2019, Whole Proteome Profiling of N-Myristoyltransferase Activity and Inhibition Using Sortase A., Mol Cell Proteomics, Vol: 18, Pages: 115-126
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.
Lovell S, Sutherell CL, Tate EW, 2019, Chemical Probes for Proteins and Networks, MASS SPECTROMETRY-BASED CHEMICAL PROTEOMICS, Pages: 127-158
Furniss RCD, Low WW, Mavridou DAI, et al., 2018, Plasma membrane profiling during enterohemorrhagic E. coli infection reveals that the metalloprotease StcE cleaves CD55 from host epithelial surfaces, Journal of Biological Chemistry, Vol: 293, Pages: 17188-17199, ISSN: 0021-9258
Enterohemorrhagic Escherichia coli (EHEC) is one of several E. coli pathotypes that infect the intestinal tract and cause disease. Formation of the characteristic attaching and effacing (A/E) lesion on the surface of infected cells causes significant remodelling of the host cell surface, however limited information is available about changes at the protein level. Here we employed "plasma membrane profiling", a quantitative cell-surface proteomics technique, to identify host proteins whose cell-surface levels are altered during infection. Using this method, we quantified more than 1100 proteins, 280 of which showed altered cell-surface levels after exposure to EHEC. 22 host proteins were significantly reduced on the surface of infected epithelial cells. These included both known and unknown targets of EHEC infection. The complement decay-accelerating factor CD55 exhibited the greatest reduction in cell surface levels during infection. We showed by flow cytometry and Western blot analysis that CD55 is cleaved from the cell surface by the EHEC-specific protease StcE, and found that StcE-mediated CD55 cleavage results in increased neutrophil adhesion to the apical surface of intestinal epithelial cells. This suggests that StcE alters host epithelial surfaces to depress neutrophil transepithelial migration during infection. This work is the first report of the global manipulation of the epithelial cell surface by a bacterial pathogen and illustrates the power of quantitative cell-surface proteomics in uncovering critical aspects of bacterial infection biology.
Wang Z, Grosskurth SE, Cheung T, et al., 2018, Pharmacological inhibition of PARP6 triggers multipolar spindle formation and demonstrates therapeutic effects in breast cancer, Cancer Research, Vol: 78, Pages: 6691-6702, ISSN: 1538-7445
PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity.
De Vita E, Schuler P, Lovell S, et al., 2018, Depsipeptides Featuring a Neutral P1 Are Potent Inhibitors of Kallikrein-Related Peptidase 6 with On-Target Cellular Activity, JOURNAL OF MEDICINAL CHEMISTRY, Vol: 61, Pages: 8859-8874, ISSN: 0022-2623
Benns HJ, Tate EW, Child MA, 2018, Activity-Based Protein Profiling for the Study of Parasite Biology., Curr Top Microbiol Immunol, Vol: 420, Pages: 155-174, ISSN: 0070-217X
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.
Beard R, Singh N, Grundschober C, et al., 2018, High-yielding 18F radiosynthesis of a novel oxytocin receptor tracer, a probe for nose-to-brain oxytocin uptake in vivo, Chemical Communications, Vol: 54, Pages: 8120-8123, ISSN: 1359-7345
A novel Al18F labelled peptide tracer for PET imaging of oxytocin receptor has been accessed through a high radiochemical yield approach. This tracer showed comparable affinity and higher selectivity and stability compared to oxytocin, and was used to demonstrate direct nose-to-brain uptake following intranasal administration, a common yet controversial delivery route for oxytocin-based therapeutics.
Beard R, Stucki A, Schmitt M, et al., 2018, Building bridges for highly selective, potent and stable oxytocin and vasopressin analogs, Bioorganic and Medicinal Chemistry, Vol: 26, Pages: 3039-3045, ISSN: 0968-0896
Oxytocin (OT) is an exciting potential therapeutic agent, but it is highly sensitive to modification and suffers extensive degradation at elevated temperature and in vivo. Here we report studies towards OT analogs with favorable selectivity, affinity and potency towards the oxytocin receptor (OTR), in addition to improving stability of the peptide by bridging the disulfide region with substituted dibromo-xylene analogs. We found a sensitive structure-activity relationship in which meta-cyclized analogs (dOTmeta) gave highest affinity (50 nM Ki), selectivity (34-fold), and agonist potency (34 nM EC50, 87-fold selectivity) towards OTR. Surprisingly, ortho-cyclized analogs demonstrated OTR and vasopressin V1a receptor subtype affinity (220 nM and 69 nM, respectively) and pharmacological activity (294 nM and 35 nM, respectively). V1a binding and selectivity for ortho-cyclized peptides could be improved 6-fold by substituting a neutral residue at position 8 with a basic amino acid, providing potent antagonists (14 nM IC50) that displayed no activation of the OTR. Furthermore, xylene-bridged analogs demonstrated increased stability compared to OT at elevated temperature, demonstrating promising therapeutic potential for these analogs which warrants further study.
Riviere F, Dian C, Perez-Dorado I, et al., 2018, Mechanistic insight into HsNMT1-mediated acylation, Publisher: WILEY, Pages: 421-422, ISSN: 2211-5463
Tate EW, 2018, Protein N terminal modifications: from chemical biology to drug discovery, Publisher: WILEY, Pages: 72-73, ISSN: 2211-5463
Mousnier A, Bell AS, Swieboda DP, et al., 2018, Fragment-derived inhibitors of human N-myristoyltransferase block capsid assembly and replication of the common cold virus, Nature Chemistry, Vol: 10, Pages: 599-606, ISSN: 1755-4330
Rhinoviruses 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 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. 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 co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, delivering low nanomolar antiviral activity against multiple rhinovirus 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.
Craven G, Affron D, Allen C, et al., 2018, High-throughput kinetic analysis for target-directed covalent ligand discovery, Angewandte Chemie International Edition, Vol: 57, Pages: 5257-5261, ISSN: 1433-7851
Cysteine-reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high-quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan-reactive compounds. Here we describe quantitative irreversible tethering(qIT), a general method for screening cysteine-reactive small moleculesbased upon the maximization of kinetic selectivity. We apply this method prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2-selective allosteric (type IV) kinase inhibitor whose novel mode-of-action could be exploited therapeutically.
Craven GB, Affron DP, Allen CE, et al., 2018, High-throughput kinetic analysis for target-directed covalent ligand discovery, Angewandte Chemie, Vol: 130, Pages: 5355-5359, ISSN: 0044-8249
Cysteine‐reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high‐quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan‐reactive compounds. Quantitative irreversible tethering (qIT), a general method for screening cysteine‐reactive small molecules based upon the maximization of kinetic selectivity, is described. This method was applied prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2‐selective allosteric (type IV) kinase inhibitor whose novel mode‐of‐action could be exploited therapeutically.
Alzahofi N, Robinson CL, Welz T, et al., 2018, Rab27a co-ordinates actin-dependent transport by controlling organelle-associated motors and track assembly proteins
<jats:title>Abstract</jats:title><jats:p>Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that motor myosin-Va, works with dynamic actin tracks, to drive long-range organelle dispersion in microtubule depleted cells. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here we show that SPIRE1/2 and formin-1 (FMN1) proteins generate actin tracks required for myosin-Va-dependent transport in melanocytes. Moreover we show that, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIRE1/2 to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this we suggest a model in which organelles and force generators (motors and track assemblers) are linked forming a cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.</jats:p>
Schlott AC, Holder AA, Tate EW, 2018, N-myristoylation as a drug target in malaria: exploring the role of N-myristoyltransferase substrates in the inhibitor mode of action, ACS Infectious Diseases, Vol: 4, Pages: 449-457, ISSN: 2373-8227
Malaria continues to be a significant cause of death and morbidity worldwide, and there is a need for new antimalarial drugs with novel targets. We have focused as a potential target for drug development on N-myristoyl transferase (NMT), an enzyme that acylates a wide range of substrate proteins. The NMT substrates in Plasmodium falciparum include some proteins that are common to processes in eukaryotes such as secretory transport and others that are unique to apicomplexan parasites. Myristoylation facilitates a protein interaction with membranes that may be strengthened by further lipidation, and the inhibition of NMT results in incorrect protein localization and the consequent disruption of function. The diverse roles of NMT substrates mean that NMT inhibition has a pleiotropic and severe impact on parasite development, growth, and multiplication. To study the mode of action underlying NMT inhibition, it is important to consider the function of proteins upstream and downstream of NMT. In this work, we therefore present our current perspective on the different functions of known NMT substrates as well as compare the inhibition of cotranslational myristoylation to the inhibition of known targets upstream of NMT.
Pollard D, Berger CN, So E, et al., 2018, Broad spectrum regulation of non receptor tyrosine kinases by the bacterial ADP ribosyltransferase EspJ, mBio, Vol: 9, ISSN: 2150-7511
Tyrosine phosphorylation is key for signal transduction fromexogenousstimuli, including the defence against pathogens. Conversely, pathogens cansubvert protein phosphorylation to control hostimmune responsesand facilitateinvasionanddissemination. The bacterial 23effectorsEspJand SeoC areinjected into host cellsthough a type III secretion system by enteropathogenic and enterohaemorrhagic Escherichia coli(EPEC and EHEC), Citrobacter rodentiumand Salmonellaentericawhere they inhibit Src kinase bycoupledamidation andADP-ribosylation. C. rodentium, which is used tomodel EPEC and EHEC infections in human, is a mouse pathogen triggeringcolonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparing the proteomes of intestinal epithelial cells isolated from mice infected with wildtype C.rodentiumor C. rodentiumencoding catalyticallyinactive EspJrevealed that EspJ-induced ADP-ribosylationregulatesmultiple non-receptor tyrosine kinasesin vivo. Investigating the substrate repertoire of EspJ revealed that in HeLa and A549 Src and Csk were significantly targeted; in polarised Caco2 cells EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 EspJ modifiedCsk, the SFKs Hck and Lyn, the Tec family kinases Tec and Btk, and the adapter tyrosine kinase Syk. Furthermore, Abl (HeLa and Caco2) and Lyn (Caco2) were enriched specifically in the EspJ-containing samples. Biochemical assays revealed that EspJ, the only bacterial ADP-ribosyltransferase which targets mammalian kinases,controls immune responses andthe Src/Csk signalling axis.
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