RESUMEN

Azide functionalization of protein and peptide lysine residues allows selective bioorthogonal labeling to introduce new, site selective functionaltiy into proteins. Optimised diazotransfer reactions under mild conditions allow aqueous diazotransfer to occur in just 20 min at pH 8.5 on amino acid, peptide and protein targets. In addition, conditons can be modified to selectively label a single lysine residue in both protein targets investigated. Finally, we demonstrate selective modification of proteins containing a single azidolysine using copper(I)-catalyzed triazole formation.

RESUMEN

Affinity-based probes are valuable tools for detecting binding interactions between small molecules and proteins in complex biological environments. Metalloproteins are a class of therapeutically significant biomolecules which bind metal ions as part of key structural or catalytic domains and are compelling targets for study. However, there is currently a limited range of chemical tools suitable for profiling the metalloproteome. Here, we describe the preparation and application of a novel, photoactivatable affinity-based probe for detection of a subset of previously challenging to engage metalloproteins. The probe, bearing an 8-mercaptoquinoline metal chelator, was anticipated to engage several zinc metalloproteins, including the 26S-proteasome subunit Rpn11. Upon translation of the labelling experiment to mammalian cell lysate and live cell experiments, proteomic analysis revealed that several metalloproteins were competitively enriched. The diazirine probe SMK-24 was found to effectively enrich multiple components of the minichromosome maintenance complex, a zinc metalloprotein assembly with helicase activity essential to DNA replication. Cell cycle analysis experiments revealed that HEK293 cells treated with SMK-24 experienced stalling in G0/G1 phase, consistent with inactivation of the DNA helicase complex. This work represents an important contribution to the library of cell-permeable chemical tools for studying a collection of metalloproteins for which no previous probe existed.

RESUMEN

A series of chemically-modified oligonucleotides for targeting the DNA repair nuclease SNM1A have been designed and synthesised. Each oligonucleotide contains a modified internucleotide linkage designed to both mimic the native phosphodiester backbone and chelate to the catalytic zinc ion(s) in the SNM1A active site. Dinucleoside phosphoramidites containing urea, squaramide, sulfanylacetamide, and sulfinylacetamide linkages were prepared and employed successfully in solid-phase oligonucleotide synthesis. All the modified oligonucleotides were found to interact with SNM1A in a gel electrophoresis-based assay, demonstrating the first examples of inhibition of DNA damage repair enzymes for many of these groups in oligonucleotides. One strand containing a sulfinylacetamide-linkage was found to have the strongest interaction with SNM1A and was further tested in a real-time fluorescence assay. This allowed an IC50 value of 231 nM to be determined, significantly lower than previously reported substrate-mimics targeting this enzyme. It is expected that these modified oligonucleotides will serve as a scaffold for the future development of fluorescent or biotin-labelled probes for the in vivo study of DNA repair processes.

Asunto(s)

Biblioteca de Péptidos , Péptidos , Péptidos/uso terapéutico

RESUMEN

Nucleoside analogs show useful bioactive properties. A versatile solid-phase synthesis that readily enables the diversification of thymine-containing nucleoside analogs is presented. The utility of the approach is demonstrated with the preparation of a library of compounds for analysis with SNM1A, a DNA damage repair enzyme that contributes to cytotoxicity. This exploration provided the most promising nucleoside-derived inhibitor of SNM1A to date with an IC50 of 12.3 µM.

Asunto(s)

Nucleósidos , Timina , Nucleósidos/farmacología , Timina/farmacología , Técnicas de Síntesis en Fase Sólida , Exodesoxirribonucleasas/metabolismo , Reparación del ADN

RESUMEN

We report a two-step validation approach to evaluate the suitability of metal-binding groups for targeting DNA damage-repair metalloenzymes using model enzyme SNM1A. A fragment-based screening approach was first used to identify metal-binding fragments suitable for targeting the enzyme. Effective fragments were then incorporated into oligonucleotides using the copper-catalysed azide-alkyne cycloaddition reaction. These modified oligonucleotides were recognised by SNM1A at >1000-fold lower concentrations than their fragment counterparts. The exonuclease SNM1A is a key enzyme involved in the repair of interstrand crosslinks, a highly cytotoxic form of DNA damage. However, SNM1A and other enzymes of this class are poorly understood, as there is a lack of tools available to facilitate their study. Our novel approach of incorporating functional fragments into oligonucleotides is broadly applicable to generating modified oligonucleotide structures with high affinity for DNA damage-repair enzymes.

Asunto(s)

Proteínas de Ciclo Celular , Exodesoxirribonucleasas , Exodesoxirribonucleasas/metabolismo , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Oligonucleótidos/química

RESUMEN

Certain cancers exhibit upregulation of DNA interstrand crosslink repair pathways, which contributes to resistance to crosslinking chemotherapy drugs and poor prognoses. Inhibition of enzymes implicated in interstrand crosslink repair is therefore a promising strategy for improving the efficacy of cancer treatment. One such target enzyme is SNM1A, a zinc co-ordinating 5'-3' exonuclease. Previous studies have demonstrated the feasibility of inhibiting SNM1A using modified nucleosides appended with zinc-binding groups. In this work, we sought to develop more effective SNM1A inhibitors by exploiting interactions with the phosphate-binding pocket adjacent to the enzyme's active site, in addition to the catalytic zinc ions. A series of nucleoside derivatives bearing phosphate moieties at the 5'-position, as well as zinc-binding groups at the 3'-position, were prepared and tested in gel-electrophoresis and real-time fluorescence assays. As well as investigating novel zinc-binding groups, we found that incorporation of a 5'-phosphate dramatically increased the potency of the inhibitors.

Asunto(s)

Exodesoxirribonucleasas , Nucleósidos , Reparación del ADN , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Nucleósidos/farmacología , Fosfatos , Fosforilación , Zinc/farmacología

RESUMEN

Over the past two decades, activity-based probes have enabled a range of discoveries, including the characterization of new enzymes and drug targets. However, their suitability in some labeling experiments can be limited by nonspecific reactivity, poor membrane permeability, or high toxicity. One method for overcoming these issues is through the development of "inducible" activity-based probes. These probes are added to samples in an unreactive state and require in situ transformation to their active form before labeling can occur. In this Review, we discuss a variety of approaches to inducible activity-based probe design, different means of probe activation, and the advancements that have resulted from these applications. Additionally, we highlight recent developments which may provide opportunities for future inducible activity-based probe innovations.

Asunto(s)

Sondas Moleculares/química , Proteínas/química , Colorantes Fluorescentes/química , Procesos Fotoquímicos , Conformación Proteica , Relación Estructura-Actividad

RESUMEN

SNM1A is a zinc-dependent nuclease involved in the removal of interstrand crosslink lesions from DNA. Inhibition of interstrand crosslink repair enzymes such as SNM1A is a promising strategy for improving the efficacy of crosslinking chemotherapy drugs. Initial studies have demonstrated the feasibility of developing SNM1A inhibitors, but the full potential of this enzyme as a drug target has yet to be explored. Herein, the synthesis of a family of squaramide- and thiosquaramide-bearing nucleoside derivatives and their evaluation as SNM1A inhibitors is reported. A gel electrophoresis assay was used to identify nucleoside derivatives bearing an N-hydroxysquaramide or squaric acid moiety at the 3'-position, and a thymidine derivative bearing a 5'-thiosquaramide, as candidate SNM1A inhibitors. Quantitative IC50 determination showed that a thymidine derivative bearing a 5'-thiosquaramide was the most potent inhibitor, followed by a thymidine derivative bearing a 3'-squaric acid. UV-Vis titrations were carried out to evaluate the binding of the (thio)squaramides to zinc ions, allowing the order of inhibitory potency to be rationalised. The membrane permeability of the active inhibitors was investigated, with several compounds showing promise for future in vivo applications.

Asunto(s)

Enzimas Reparadoras del ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Quinina/análogos & derivados , Enzimas Reparadoras del ADN/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Quinina/síntesis química , Quinina/química , Quinina/farmacología , Relación Estructura-Actividad

RESUMEN

Eosin Y was assessed for its ability to induce a thiol-ene dependent protein-protein reaction in a metal-free, oxygen-tolerant, visible light mediated system. Protein-protein coupling efficiency under these mild conditions was comparable to previously reported UV-dependent conditions. The desired thiol-ene reaction was however limited within more complex biological systems.

Asunto(s)

Cisteína Endopeptidasas/química , Enzimas Desubicuitinizantes/química , Eosina Amarillenta-(YS)/química , Sondas Moleculares/química , Alquenos/química , Catálisis/efectos de la radiación , Cisteína/química , Eosina Amarillenta-(YS)/efectos de la radiación , Células HEK293 , Humanos , Luz , Sondas Moleculares/efectos de la radiación

RESUMEN

SNM1A is a nuclease that is implicated in DNA interstrand crosslink repair and, as such, its inhibition is of interest for overcoming resistance to chemotherapeutic crosslinking agents. However, the number and identity of the metal ion(s) in the active site of SNM1A are still unconfirmed, and only a limited number of inhibitors have been reported to date. Herein, we report the synthesis and evaluation of a family of malonate-based modified nucleosides to investigate the optimal positioning of metal-binding groups in nucleoside-derived inhibitors for SNM1A. These compounds include ester, carboxylate and hydroxamic acid malonate derivatives which were installed in the 5'-position or 3'-position of thymidine or as a linkage between two nucleosides. Evaluation as inhibitors of recombinant SNM1A showed that nine of the twelve compounds tested had an inhibitory effect at 1 mM concentration. The most potent compound contains a hydroxamic acid malonate group at the 5'-position. Overall, our studies advance the understanding of requirements for nucleoside-derived inhibitors for SNM1A and indicate that groups containing a negatively charged group in close proximity to a metal chelator, such as hydroxamic acid malonates, are promising structures in the design of inhibitors.

Asunto(s)

Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Exodesoxirribonucleasas/antagonistas & inhibidores , Nucleósidos/farmacología , Compuestos Organometálicos/farmacología , Sitios de Unión/efectos de los fármacos , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Ésteres/química , Ésteres/farmacología , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Malonatos/química , Malonatos/farmacología , Estructura Molecular , Nucleósidos/química , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química

RESUMEN

Deubiquitinating enzymes (DUBs) are known to have numerous important interactions with the ubiquitin cascade and their dysregulation is associated with several diseases, including cancer and neurodegeneration. They are an important class of enzyme, and activity-based probes have been developed as an effective strategy to study them. Existing activity-based probes that target the active site of these enzymes work via nucleophilic mechanisms. We present the development of latent ubiquitin-based probes that target DUBs via a site selective, photoinitiated radical mechanism. This approach differs from existing photocrosslinking probes as it requires a free active site cysteine. In contrast to existing cysteine reactive probes, control over the timing of the enzyme-probe reaction is possible as the alkene warhead is completely inert under ambient conditions, even upon probe binding. The probe's reactivity has been demonstrated against recombinant DUBs and to capture endogenous DUB activity in cell lysate. This allows more finely resolved investigations of DUBs.

RESUMEN

We report the design and optimisation of novel oligonucleotide substrates for a sensitive fluorescence assay for high-throughput screening and functional studies of the DNA repair enzyme, XPF-ERCC1, with a view to accelerating inhibitor and drug discovery.

Asunto(s)

Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Endonucleasas/química , Endonucleasas/genética , Humanos , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Especificidad por Sustrato , Temperatura

RESUMEN

Nine modified nucleosides, incorporating zinc-binding pharmacophores, have been synthesised and evaluated as inhibitors of the DNA repair nuclease SNM1A. The series included oxyamides, hydroxamic acids, hydroxamates, a hydrazide, a squarate ester and a squaramide. A hydroxamic acid-derived nucleoside inhibited the enzyme, offering a novel approach for potential therapeutic development through the use of rationally designed nucleoside derived inhibitors.

Asunto(s)

Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Exodesoxirribonucleasas/antagonistas & inhibidores , Ácidos Hidroxámicos/farmacología , Proteínas de Ciclo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Exodesoxirribonucleasas/metabolismo , Humanos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/química , Estructura Molecular , Relación Estructura-Actividad

RESUMEN

Platelet activators stimulate post-translational modification of signalling proteins to change their activity or their molecular interactions leading to signal propagation. One covalent modification is attachment of the small protein ubiquitin to lysine residues in target proteins. Modification by ubiquitin can either target proteins for degradation by the proteasome or act as a scaffold for other proteins. Pharmacological inhibition of deubiquitylases or the proteasome inhibition of platelet activation by collagen, demonstrating a role for ubiquitylation, but relatively few substrates for ubiquitin have been identified and the molecular basis of inhibition is not established. Here, we report the ubiquitome of human platelets and changes in ubiquitylated proteins following stimulation by collagen-related peptide (CRP-XL). Using platelets from six individuals over three independent experiments, we identified 1,634 ubiquitylated peptides derived from 691 proteins, revealing extensive ubiquitylation in resting platelets. Note that 925 of these peptides show an increase of more than twofold following stimulation with CRP-XL. Multiple sites of ubiquitylation were identified on several proteins including Syk, filamin and integrin heterodimer sub-units. This work reveals extensive protein ubiquitylation during activation of human platelets and opens the possibility of novel therapeutic interventions targeting the ubiquitin machinery.

Asunto(s)

Plaquetas/metabolismo , Glicoproteínas de Membrana Plaquetaria/química , Ubiquitina/química , Ubiquitinación , Plaquetas/efectos de los fármacos , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisina/química , Espectrometría de Masas , Selectina-P/metabolismo , Activación Plaquetaria , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal

RESUMEN

The regulation of ubiquitination status in the cell is controlled by ubiquitin ligases acting in tandem with deubiquitinating enzymes. Ubiquitination controls many key processes in the cell from division to death making its tight regulation key to optimal cell function. Activity based protein profiling has emerged as a powerful technique to study these important enzymes. With around 100 deubiquitinating enzymes and 600 ubiquitin ligases in the human genome targeting a subclass of these enzymes or even a single enzyme is a compelling strategy to unpick this complex system. In this review we will discuss different approaches adopted, including activity-based probes centered around ubiquitin-protein, ubiquitin-peptide and mutated ubiquitin scaffolds. We examine challenges faced and opportunities presented to increase specificity in activity-based protein profiling of the ubiquitin conjugation/deconjugation machinery.

RESUMEN

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

Asunto(s)

Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Proteína 1 Asociada A ECH Tipo Kelch/química , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/agonistas , Factor 2 Relacionado con NF-E2/metabolismo , Succinatos/metabolismo , Alquilación , Animales , Carboxiliasas , Bovinos , Cisteína/química , Cisteína/metabolismo , Citocinas/biosíntesis , Citocinas/inmunología , Retroalimentación Fisiológica , Femenino , Células HEK293 , Humanos , Hidroliasas/biosíntesis , Interferón beta/inmunología , Interferón beta/farmacología , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Proteínas/metabolismo , Ratas , Ratas Wistar , Succinatos/química

RESUMEN

Phosphate groups are often crucial to biological activity and interactions of oligonucleotides, but confer poor membrane permeability. In addition, the group's lability to enzymatic hydrolysis is an obstacle to its use in therapeutics and in biological tools. We present the synthesis of N-oxyamide and squaramide modifications at the 5'-end of oligonucleotides as phosphate replacements and their biological evaluation using the 5'-exonuclease SNM1A. The squaryl diamide modification showed minimal recognition as a 5'-phosphate mimic; however, modest inhibition of SNM1A, postulated to occur through metal coordination at the active site, was observed. Their facile incorporation after solid-phase synthesis and recognition by the exonuclease makes squaryl diamides attractive neutral 5'-phosphate replacements for oligonucleotides. This work is the first example of squaryl diamide modifications at the 5'-terminal position of oligonucleotides and of the potential use of modified oligonucleotides to bind to the metal center of SNM1A.

RESUMEN

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.

Asunto(s)

Piperidinas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Animales , Apoenzimas/antagonistas & inhibidores , Apoenzimas/química , Apoenzimas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Femenino , Humanos , Ratones , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Piperidinas/síntesis química , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Pirazoles/síntesis química , Pirimidinas/síntesis química , Especificidad por Sustrato , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Peptidasa Específica de Ubiquitina 7/química , Peptidasa Específica de Ubiquitina 7/metabolismo , Ubiquitinación/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.

Asunto(s)

Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteína de Replicación A/metabolismo , Proteínas de Ciclo Celular , Humanos , Modelos Biológicos