RESUMO
Photoaffinity labeling followed by tandem mass spectrometry is an often used strategy to identify protein targets of small-molecule drugs or drug candidates, which, under ideal conditions, enables the identification of the actual drug binding site. In the case of bioactive peptides, however, identifying the distinct binding site is hampered because of complex fragmentation patterns during tandem mass spectrometry. We here report the development and use of small cleavable photoaffinity reagents that allow functionalization of bioactive peptides for light-induced covalent binding to their protein targets. Upon cleavage of the covalently linked peptide drug, a chemical remnant of a defined mass remains on the bound amino acid, which is then used to unambiguously identify the drug binding site. Applying our approach to known peptide-drug/protein pairs with reported crystal structures, such as the calmodulin-melittin interaction, we were able to validate the identified binding sites based on structural models. Overall, our cleavable photoaffinity labeling strategy represents a powerful tool to enable the identification of protein targets and specific binding sites of a wide variety of bioactive peptides in the future.
RESUMO
Protein kinases, one of the largest enzyme superfamilies, regulate many physiological and pathological processes. They are drug targets for multiple human diseases, including various cancer types. Probes for the photoaffinity labelling of kinases are important research tools for the study of members of this enzyme superfamily. In this review, we discuss the design principles of these probes, which are mainly derived from inhibitors targeting the ATP pocket. Overall, insights from crystal structures guide the placement of photoreactive groups and detection tags. This has resulted in a wide variety of probes, of which we provide a comprehensive overview. We also discuss several areas of application of these probes, including the identification of targets and off-targets of kinase inhibitors, mapping of their binding sites, the development of inhibitor screening assays, the imaging of kinases, and identification of protein binding partners.
Assuntos
Marcadores de Fotoafinidade/química , Proteínas Quinases/química , Sítios de Ligação , Humanos , Proteínas Quinases/metabolismoRESUMO
Covalent chemical probes are important tools in chemical biology. They range from post-translational modification (PTM)-derived metabolic probes, to activity-based probes and photoaffinity labels. Identification of the probe targets is often performed by tandem mass spectrometry-based proteomics methods. In the past fifteen years, cleavable linker technologies have been implemented in these workflows in order to identify probe targets with lower background and higher confidence. In addition, the linkers have enabled identification of modification sites. Overall, this has led to an increased knowledge of PTMs, enzyme function and drug action. This review gives an overview of the different types of cleavable linkers, and their benefits and limitations. Their applicability in target identification is also illustrated by several specific examples.
Assuntos
Marcadores de Fotoafinidade , Processamento de Proteína Pós-Traducional/genética , Proteoma/genética , Proteômica/tendências , Humanos , Espectrometria de Massas em TandemRESUMO
Intramembrane proteases (IMPs) are localized within lipid bilayers of membranes-either the cell membrane or membranes of various organelles. Cleavage of substrates often results in release from the membrane, leading to a downstream biological effect. This mechanism allows different signaling events to happen through intramembrane proteolysis. Over the years, various mechanistically distinct families of IMPs have been discovered, but the research progress has generally been slower than for soluble proteases due to the challenges associated with membrane proteins. In this review we summarize how each mechanistic family of IMPs was discovered, which chemical tools are available for the study of IMPs, and which techniques have been developed for the discovery of IMP substrates. Finally, we discuss the various roles in cellular physiology of some of these IMPs.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Membrana Celular/metabolismo , Cristalização , Humanos , Modelos Moleculares , Nanoestruturas/química , Ligação Proteica , Proteólise , Transdução de SinaisRESUMO
Ligand-directed protein labelling allows the introduction of diverse chemical functionalities onto proteins without the need for genetically encoded tags. Here we report a method for the rapid labelling of a protein using a ruthenium-bipyridyl (Ru(II)(bpy)3)-modified peptide designed to mimic an interacting BH3 ligand within a BCL-2 family protein-protein interactions. Using sub-stoichiometric quantities of (Ru(II)(bpy)3)-modified NOXA-B and irradiation with visible light for 1 min, the anti-apoptotic protein MCL-1 can be photolabelled with a variety of functional tags. In contrast with previous reports on Ru(II)(bpy)3-mediated photolabelling, tandem mass spectrometry experiments reveal that the labelling site is a cysteine residue of MCL-1. MCL-1 can be labelled selectively in mixtures with other proteins, including the structurally related BCL-2 member, BCL-xL. These results demonstrate that proximity-induced photolabelling is applicable to interfaces that mediate protein-protein interactions, and pave the way towards future use of ligand-directed proximity labelling for dynamic analysis of the interactome of BCL-2 family proteins.
RESUMO
Pharmacological inhibition of uncontrolled cell growth with small-molecule inhibitors is a potential strategy for treating glioblastoma multiforme (GBM), the most malignant primary brain cancer. We showed that the synthetic small-molecule KHS101 promoted tumor cell death in diverse GBM cell models, independent of their tumor subtype, and without affecting the viability of noncancerous brain cell lines. KHS101 exerted cytotoxic effects by disrupting the mitochondrial chaperone heat shock protein family D member 1 (HSPD1). In GBM cells, KHS101 promoted aggregation of proteins regulating mitochondrial integrity and energy metabolism. Mitochondrial bioenergetic capacity and glycolytic activity were selectively impaired in KHS101-treated GBM cells. In two intracranial patient-derived xenograft tumor models in mice, systemic administration of KHS101 reduced tumor growth and increased survival without discernible side effects. These findings suggest that targeting of HSPD1-dependent metabolic pathways might be an effective strategy for treating GBM.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Metabolismo Energético , Glioblastoma/metabolismo , Glioblastoma/patologia , Tiazóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chaperonina 60/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Glioblastoma/genética , Glicólise/efeitos dos fármacos , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Invasividade Neoplásica , Estresse Fisiológico/efeitos dos fármacos , Análise de Sobrevida , Transcrição Gênica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
2-Cyanobenzothiazoles (CBTs) are useful building blocks for: 1) luciferin derivatives for bioluminescent imaging; and 2) handles for bioorthogonal ligations. A particularly versatile CBT is 6-amino-2-cyanobenzothiazole (ACBT), which has an amine handle for straight-forward derivatisation. Here we present an economical and scalable synthesis of ACBT based on a cyanation catalysed by 1,4-diazabicyclo[2.2.2]octane (DABCO), and discuss its advantages for scale-up over previously reported routes.