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A principal component surfactant_map was developed for 91 commonly accessible surfactants for use in surfactant-enabled organic reactions in water, an important approach for sustainable chemical processes. This map was built using 22 experimental and theoretical descriptors relevant to the physicochemical nature of these surfactant-enabled reactions, and advanced principal component analysis algorithms. It is comprised of all classes of surfactants, i.e. cationic, anionic, zwitterionic and neutral surfactants, including designer surfactants. The value of this surfactant_map was demonstrated in activating simple inorganic fluoride salts as effective nucleophiles in water, with the right surfactant. This led to the rapid development (screening 13-15 surfactants) of two fluorination reactions for ß-bromosulfides and sulfonyl chlorides in water. The latter was demonstrated in generating a sulfonyl fluoride with sufficient purity for direct use in labelling of chymotrypsin, under physiological conditions.
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Using N-Myc61-89 as a starting template we showcase the systematic use of truncation and maleimide constraining to develop peptidomimetic inhibitors of the N-Myc/Aurora-A protein-protein interaction (PPI); a potential anticancer drug discovery target. The most promising of these - N-Myc73-94-N85C/G89C-mal - is shown to favour a more Aurora-A compliant binding ensemble in comparison to the linear wild-type sequence as observed through fluorescence anisotropy competition assays, circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Further inâ silico investigation of this peptide in its Aurora-A bound state, by molecular dynamics (MD) simulations, imply (i)â the bound conformation is more stable as a consequence of the constraint, which likely suppresses dissociation and (ii)â the constraint may make further stabilizing interactions with the Aurora-A surface. Taken together this work unveils the first orthosteric N-Myc/Aurora-A inhibitor and provides useful insights on the biophysical properties and thus design of constrained peptides, an attractive therapeutic modality.
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Peptidomiméticos , Peptidomiméticos/farmacología , Proteína Proto-Oncogénica N-Myc , Ciclización , Péptidos/química , Unión ProteicaRESUMEN
Multicellular organisms require carefully orchestrated communication between and within cell types and tissues, and many unicellular organisms also sense their context and environment, sometimes coordinating their responses. This review highlights contributions from chemical biology in discovering and probing mechanisms of cell-cell communication. We focus on chemical tools for labelling proteins in a cellular context and how these can be applied to decipher the target receptor of a signalling molecule, label a receptor of interest in situ to understand its biology, provide a read-out of protein activity or interactions in downstream signalling pathways, or discover protein-protein interactions across cell-cell interfaces.
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Biología , Comunicación CelularRESUMEN
Fluorescent ligands for G-protein coupled receptors (GPCRs) are valuable tools for studying the expression, pharmacology and modulation of these therapeutically important proteins in living cells. Here we report a fluorescent photoaffinity probe for Formyl peptide receptor 1 (FPR1), a critical component of the innate immune response to bacterial infection and a promising target in inflammatory diseases. We demonstrate that the probe binds and covalently crosslinks to FPR1 with good specificity at nanomolar concentrations in living cells and is a useful tool for visualisation and characterisation of this receptor.
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Catalase (CAT) is an extensively studied if somewhat enigmatic enzyme that is at the heart of eukaryotic antioxidant systems with a canonical role in peroxisomal function. The CAT family of proteins exert control over a wide range of plant growth and defence processes. CAT proteins are subject to many types of post-translational modification (PTM), which modify activity, ligand binding, stability, compartmentation and function. The CAT interactome involves many cytosolic and nuclear proteins that appear to be essential for protein functions. Hence, the CAT network of roles extends far beyond those associated with peroxisomal metabolism. Some pathogen effector proteins are able to redirect CAT to the nucleus and recent evidence indicates CAT can traffic to the nucleus in the absence of exogenous proteins. While the mechanisms that target CAT to the nucleus are not understood, CAT activity in the cytosol and nucleus is promoted by interactions with nucleoredoxin. Here we discuss recent findings that have been pivotal in generating a step change in our understanding of CAT functions in plant cells.
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Antioxidantes , Antioxidantes/metabolismo , Catalasa/genética , Catalasa/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citosol/metabolismo , Células Vegetales/enzimologíaRESUMEN
Pseudomonas aeruginosa undergoes diversification during infection of the cystic fibrosis (CF) lung. Understanding these changes requires model systems that capture the complexity of the CF lung environment. We previously identified loss-of-function mutations in the 2-component regulatory system sensor kinase gene pmrB in P. aeruginosa from CF lung infections and from experimental infection of mice. Here, we demonstrate that, while such mutations lowered in vitro minimum inhibitory concentrations for multiple antimicrobial classes, this was not reflected in increased antibiotic susceptibility in vivo. Loss of PmrB impaired aminoarabinose modification of LPS, increasing the negative charge of the outer membrane and promoting uptake of cationic antimicrobials. However, in vivo, this could be offset by increased membrane binding of other positively charged molecules present in lungs. The polyamine spermidine readily coated the surface of PmrB-deficient P. aeruginosa, reducing susceptibility to antibiotics that rely on charge differences to bind the outer membrane and increasing biofilm formation. Spermidine was elevated in lungs during P. aeruginosa infection in mice and during episodes of antimicrobial treatment in people with CF. These findings highlight the need to study antimicrobial resistance under clinically relevant environmental conditions. Microbial mutations carrying fitness costs in vitro may be advantageous during infection, where host resources can be utilized.
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Antiinfecciosos , Fibrosis Quística , Ratones , Animales , Pseudomonas aeruginosa/genética , Poliaminas/metabolismo , Espermidina/metabolismo , Pruebas de Sensibilidad Microbiana , Fibrosis Quística/tratamiento farmacológico , Antiinfecciosos/metabolismoRESUMEN
The leishmaniases, caused by Leishmania species of protozoan parasites, are neglected tropical diseases with millions of cases worldwide. Current therapeutic approaches are limited by toxicity, resistance, and cost. N-Myristoyltransferase (NMT), an enzyme ubiquitous and essential in all eukaryotes, has been validated via genetic and pharmacological methods as a promising anti-leishmanial target. Here we describe a comprehensive structure-activity relationship (SAR) study of a thienopyrimidine series previously identified in a high-throughput screen against Leishmania NMT, across 68 compounds in enzyme- and cell-based assay formats. Using a chemical tagging target engagement biomarker assay, we identify the first inhibitor in this series with on-target NMT activity in leishmania parasites. Furthermore, crystal structure analyses of 12 derivatives in complex with Leishmania major NMT revealed key factors important for future structure-guided optimization delivering IMP-105 (43), a compound with modest activity against Leishmania donovani intracellular amastigotes and excellent selectivity (>660-fold) for Leishmania NMT over human NMTs.
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Aciltransferasas/antagonistas & inhibidores , Antiprotozoarios/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Pirimidinas/farmacología , Tiofenos/farmacología , Aciltransferasas/química , Aciltransferasas/metabolismo , Antiprotozoarios/síntesis química , Antiprotozoarios/metabolismo , Sitios de Unión , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Leishmania donovani/enzimología , Leishmania major/enzimología , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/metabolismoRESUMEN
The dynamic proteome plays numerous roles in the interactions of microbes-whether they are invading pathogens or symbiotic organisms-and their hosts. Host and microbe sense, respond, and manipulate each other's biology via a multitude of mechanisms, resulting in alterations in protein expression or posttranslational modification that influence protein localization, activity, or binding partners. The intrinsic, temporal, and spatial complexity of multispecies systems makes identifying the molecular players challenging. Chemical proteomic approaches apply small molecule chemical tools to interrogate protein function, interactions or modifications. This review highlights recent advances in the application of these methods at the host-microbe interface.
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Biología Computacional/métodos , Interacciones Microbiota-Huesped , Procesamiento Proteico-Postraduccional , Proteoma/análisis , Bibliotecas de Moléculas Pequeñas/análisis , Animales , Humanos , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Plant stem cells are the foundation of plant growth and development. The balance of quiescence and division is highly regulated, while ensuring that proliferating cells are protected from the adverse effects of environment fluctuations that may damage the genome. Redox regulation is important in both the activation of proliferation and arrest of the cell cycle upon perception of environmental stress. Within this context, reactive oxygen species serve as 'pro-life' signals with positive roles in the regulation of the cell cycle and survival. However, very little is known about the metabolic mechanisms and redox-sensitive proteins that influence cell cycle progression. We have identified cysteine residues on known cell cycle regulators in Arabidopsis that are potentially accessible, and could play a role in redox regulation, based on secondary structure and solvent accessibility likelihoods for each protein. We propose that redox regulation may function alongside other known posttranslational modifications to control the functions of core cell cycle regulators such as the retinoblastoma protein. Since our current understanding of how redox regulation is involved in cell cycle control is hindered by a lack of knowledge regarding both which residues are important and how modification of those residues alters protein function, we discuss how critical redox modifications can be mapped at the molecular level.
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Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Oxidación-Reducción , Proteómica , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/genética , Biología Computacional , Estrés Oxidativo/genética , Desarrollo de la Planta/genética , Procesamiento Proteico-Postraduccional/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Infections by protozoan parasites, such as Plasmodium falciparum or Leishmania donovani, have a significant health, social and economic impact and threaten billions of people living in tropical and sub-tropical regions of developing countries worldwide. The increasing range of parasite strains resistant to frontline therapeutics makes the identification of novel drug targets and the development of corresponding inhibitors vital. Post-translational modifications (PTMs) are important modulators of biology and inhibition of protein lipidation has emerged as a promising therapeutic strategy for treatment of parasitic diseases. In this review we summarize the latest insights into protein lipidation in protozoan parasites. We discuss how recent chemical proteomic approaches have delivered the first global overviews of protein lipidation in these organisms, contributing to our understanding of the role of this PTM in critical metabolic and cellular functions. Additionally, we highlight the development of new small molecule inhibitors to target parasite acyl transferases.
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Sistemas de Liberación de Medicamentos/métodos , Leishmania donovani/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Acilación/efectos de los fármacos , Humanos , Leishmania donovani/enzimología , Leishmania donovani/metabolismo , Leishmaniasis/tratamiento farmacológico , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/enzimología , Plasmodium falciparum/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteómica/métodos , Transferasas/antagonistas & inhibidores , Transferasas/efectos de los fármacos , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitologíaRESUMEN
Host-microbe communication via small molecule signals is important for both symbiotic and pathogenic relationships, but is often poorly understood at the molecular level. Under conditions of host stress, levels of the human opioid peptide dynorphin are elevated, triggering virulence in the opportunistic pathogenic bacterium Pseudomonas aeruginosa via an unknown pathway. Here we apply a multilayered chemical biology strategy to unravel the mode of action of this putative interkingdom signal. We designed and applied dynorphin-inspired photoaffinity probes to reveal the protein targets of the peptide in live bacteria via chemical proteomics. ParS, a largely uncharacterized membrane sensor of a two-component system, was identified as the most promising hit. Subsequent full proteome studies revealed that dynorphin(1-13) induces an antimicrobial peptide-like response in Pseudomonas, with specific upregulation of membrane defense mechanisms. No such response was observed in a parS mutant, which was more susceptible to dynorphin-induced toxicity. Thus, P. aeruginosa exploits the ParS sensing machinery to defend itself against the host in response to dynorphin as a signal. This study highlights interkingdom communication as a potential essential strategy not only for induction of P. aeruginosa virulence but also for maintaining viability in the hostile environment of the host.
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Antibacterianos/química , Dinorfinas/química , Sondas Moleculares/química , Proteínas Quinasas/química , Pseudomonas aeruginosa/enzimología , Antibacterianos/metabolismo , Antibacterianos/farmacología , Sitios de Unión , Relación Dosis-Respuesta a Droga , Dinorfinas/metabolismo , Dinorfinas/farmacología , Humanos , Estructura Molecular , Proteínas Quinasas/metabolismo , Proteómica , Pseudomonas aeruginosa/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The enzyme N-myristoyltransferase (NMT) catalyzes the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localization of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labeling with an alkyne-tagged myristic acid analogue, enabling the capture of lipidated proteins in insect and host life stages of T. brucei. We further compare this with a longer chain palmitate analogue to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites. This analysis reveals five ARF family small GTPases, calpain-like proteins, phosphatases, and many uncharacterized proteins as substrates of NMT in the parasite, providing a global view of the scope of this important protein modification and further evidence for the crucial and pleiotropic role of NMT in the cell.
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Biological selection makes natural products promising scaffolds for drug development and the ever growing number of newly identified, structurally diverse molecules helps to fill the gaps in chemical space. Elucidating the function of a small molecule, such as identifying its protein binding partners, its on- and off-targets, is becoming increasingly important. Activity- and affinity-based protein profiling are modern strategies to acquire such molecular-level information. Introduction of a molecular handle (azide, alkyne, biotin) can shed light on the mode of action of small molecules. This Concept article covers central points on synthetic methodology for integrating a terminal alkyne into a molecule of interest.
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Alquinos/química , Productos Biológicos/química , Biotina/química , Azidas/química , Fenómenos Biológicos , Diseño de Fármacos , Modelos Moleculares , Estructura MolecularRESUMEN
N-Myristoyltransferase (NMT) modulates protein function through the attachment of the lipid myristate to the N terminus of target proteins, and is a promising drug target in eukaryotic parasites such as Leishmania donovani. Only a small number of NMT substrates have been characterized in Leishmania, and a global picture of N-myristoylation is lacking. Here, we use metabolic tagging with an alkyne-functionalized myristic acid mimetic in live parasites followed by downstream click chemistry and analysis to identify lipidated proteins in both the promastigote (extracellular) and amastigote (intracellular) life stages. Quantitative chemical proteomics is used to profile target engagement by NMT inhibitors, and to define the complement of N-myristoylated proteins. Our results provide new insight into the multiple pathways modulated by NMT and the pleiotropic effects of NMT inhibition. This work constitutes the first global experimental analysis of protein lipidation in Leishmania, and reveals the extent of NMT-related biology yet to be explored for this neglected human pathogen.
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Aciltransferasas/metabolismo , Leishmania donovani/metabolismo , Proteínas Protozoarias/metabolismo , Aciltransferasas/análisis , Animales , Humanos , Leishmania donovani/química , Leishmaniasis/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Miristatos/análisis , Miristatos/metabolismo , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Proteínas Protozoarias/análisisRESUMEN
Inhibitors of Leishmania N-myristoyltransferase (NMT), a potential target for the treatment of leishmaniasis, obtained from a high-throughput screen, were resynthesized to validate activity. Crystal structures bound to Leishmania major NMT were obtained, and the active diastereoisomer of one of the inhibitors was identified. On the basis of structural insights, enzyme inhibition was increased 40-fold through hybridization of two distinct binding modes, resulting in novel, highly potent Leishmania donovani NMT inhibitors with good selectivity over the human enzyme.
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Aciltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Aciltransferasas/química , Aciltransferasas/metabolismo , Técnicas de Química Sintética , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Leishmania donovani/enzimología , Leishmania major/enzimología , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of >100 N-myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells.
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Proteoma/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Cristalografía por Rayos X , Células HeLa , Humanos , Ácido Mirístico/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma/genéticaRESUMEN
Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria.
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Regulación de la Expresión Génica , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Plasmodium berghei/enzimología , Plasmodium berghei/crecimiento & desarrollo , Animales , Femenino , Técnicas de Inactivación de Genes , Ratones , Plasmodium falciparum/enzimologíaRESUMEN
Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approximately one million deaths per annum worldwide. Chemical validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyses the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, we report an integrated chemical biology approach to explore protein myristoylation in the major human parasite P. falciparum, combining chemical proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-molecule N-myristoyltransferase inhibitors. We demonstrate that N-myristoyltransferase is an essential and chemically tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a critical subcellular organelle in the parasite life cycle. Our studies provide the basis for the development of new antimalarials targeting N-myristoyltransferase.
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Aciltransferasas/antagonistas & inhibidores , Antimaláricos/química , Inhibidores Enzimáticos/química , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Sitios de Unión , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Reacción de Cicloadición , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Malaria/tratamiento farmacológico , Malaria/parasitología , Simulación del Acoplamiento Molecular , Plasmodium falciparum/efectos de los fármacos , Plasmodium vivax/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad por SustratoRESUMEN
Infections caused by protozoan parasites are among the most widespread and intractable transmissible diseases affecting the developing world, with malaria and leishmaniasis being the most costly in terms of morbidity and mortality. Although new drugs are urgently required against both diseases in the face of ever-rising resistance to frontline therapies, very few candidates passing through development pipelines possess a known and novel mode of action. Set in the context of drugs currently in use and under development, we present the evidence for N-myristoyltransferase (NMT), an enzyme that N-terminally lipidates a wide range of specific target proteins through post-translational modification, as a potential drug target in malaria and the leishmaniases. We discuss the limitations of current knowledge regarding the downstream targets of this enzyme in protozoa, and our recent progress towards potent cell-active NMT inhibitors against the most clinically-relevant species of parasite. Finally, we outline the next steps required in terms of both tools to understand N-myristoylation in protozoan parasites, and the generation of potential development candidates based on the output of our recently-reported high-throughput screens.
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Aciltransferasas/metabolismo , Antiprotozoarios/química , Inhibidores Enzimáticos/química , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/metabolismo , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/química , Antiprotozoarios/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Leishmaniasis/tratamiento farmacológico , Malaria/tratamiento farmacológico , Modelos Moleculares , Terapia Molecular Dirigida , Ácido Mirístico/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The phylum Apicomplexa comprises over 5000 intracellular protozoan parasites, including Plasmodium and Toxoplasma, that are clinically important pathogens affecting humans and livestock. Malaria parasites belonging to the genus Plasmodium possess a pellicle comprised of a plasmalemma and inner membrane complex (IMC), which is implicated in parasite motility and invasion. Using live cell imaging and reverse genetics in the rodent malaria model P. berghei, we localise two unique IMC sub-compartment proteins (ISPs) and examine their role in defining apical polarity during zygote (ookinete) development. We show that these proteins localise to the anterior apical end of the parasite where IMC organisation is initiated, and are expressed at all developmental stages, especially those that are invasive. Both ISP proteins are N-myristoylated, phosphorylated and membrane-bound. Gene disruption studies suggest that ISP1 is likely essential for parasite development, whereas ISP3 is not. However, an absence of ISP3 alters the apical localisation of ISP1 in all invasive stages including ookinetes and sporozoites, suggesting a coordinated function for these proteins in the organisation of apical polarity in the parasite.