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Targeted Protein Localization by Covalent 14-3-3 Recruitment

ABSTRACT: chemoproteomic profiling of covalent ligands that target 14-3-3

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Liver

SUBMITTER: Daniel Nomura

LAB HEAD: Daniel Nomura

PROVIDER: PXD054696 | Pride | 2024-08-28

REPOSITORIES: Pride

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			Action	DRS
	QS_EN171_T47D_isoDTB_1_1.raw	Raw
	QS_EN171_T47D_isoDTB_1_2.raw	Raw
	QS_EN171_T47D_isoDTB_1_3.raw	Raw
	QS_EN171_T47D_isoDTB_1_4.raw	Raw
	QS_EN171_T47D_isoDTB_1_5.raw	Raw

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Publications

Covalent 14-3-3 Molecular Glues and Heterobifunctional Molecules Against Nuclear Transcription Factors and Regulators.

Shao Qian Q Duong Tuong Nghi TN Park Inji I Nomura Daniel K DK

bioRxiv : the preprint server for biology 20231106

14-3-3 proteins have the unique ability to bind and sequester a multitude of diverse phosphorylated signaling proteins and transcription factors. Many previous studies have shown that 14-3-3 interactions with specific phosphorylated substrate proteins can be enhanced through small-molecule natural product or fully synthetic molecular glue interactions. However, enhancing 14-3-3 interactions with both therapeutically intractable transcription factor substrates as well as potential neo-substrates ...[more]

PMID: 37986959

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Isocyanides inhibit bacterial pathogents by covalent targeting of essential metabolic enzymes

Project description:Isonitrile (also termed isocyanide) natural products exhibit potent antimicrobial activities, but little is known about their molecular targets. Here, we focus on the so far neglected group of monoisonitriles to gain further insights into their antimicrobial mode of action (MoA). Screening a focused monoisonitrile library revealed a potent S. aureus growth inhibitor with a different MoA compared to previously described diisonitrile antibiotics. Chemical proteomics via competitive cysteine reactivity profiling, unraveled covalent modifications of two essential metabolic enzymes involved in the fatty acid biosynthetic process (FabF) and the hexosamine pathway (GlmS) at their active site cysteines. In-depth studies with the recombinant enzymes demonstrated concentration-dependent labeling, covalent binding to the catalytic site and corresponding functional inhibition by the isocyanide. Thermal proteome profiling and full proteome studies of compound-treated S. aureus further highlighted the destabilization and dysregulation of proteins related to the targeted pathways. The here described novel, covalent isocyanide MoA highlights the versatility of the functional group, making it a great tool and out-of-the-box starting point for the development of innovative antibiotics.

2024-06-26 | PXD050437 | Pride

Degrasyn exhibits antibiotic activity against multi-resistant Staphylococcus aureus by modifying several essential cysteines

Project description:In this dataset, we investigate the targets of degrasyn in the methicillin-sensitive S. aureus strain NCTC 8325. This includes data on enrichment studies and competition studies with conventional ABPP using a degrasyn-derived probe, competition studies with residue-specific proteomics using the isoDTB-ABPP method and global analysis of protein expression levels in response to degrasyn treatment.

2020-04-14 | PXD016413 | Pride

Competitive Profiling of Ligandable Cysteines in Staphylococcus aureus with an Organogold Compound

Project description:With the idea of exploiting metal-templated reactions to achieve selective modification of cysteines in proteins for antibacterial applications, an organometallic cyclometalated Au(III) compound was explored in a competitive chemoproteomic approach based on the isoDTB-ABPP (isotopically labelled desthiobiotin azide-activity-based protein profiling) technology in S. aureus cell extracts. In this way, more than 100 ligandable cysteines where identified, of which 10 were close to functional sites of proteins encoded by essential genes indicating potential for antibiotic development. Interestingly, more than 50% of the identified ligandable sites were not engaged by organic alpha-chloroacetamides in a previous study, indicating that organometallic compounds expand the ligandable space in bacteria. A selected interaction identified by isoDTB-ABPP was validated using an enzyme activity assay, and intact protein mass spectrometry showed that cysteine arylation of an unprecedented target occurs with the studied compound. The obtained results constitute the proof-of-concept that this family of organogold compounds has potential for therapeutic protein targeting via selective, covalent modification of cysteine residues in bacteria. Looking more broadly, our study demonstrates that the targets of cyclometalated gold compounds can be studied proteome-wide with competitive residue-specific chemoproteomics enabling the expansion of the known ligandable proteome to sites that can be addressed with this compound class.

2022-04-19 | PXD031708 | Pride

Light-activatable, 2,5-disubstituted tetrazoles for the proteome-wide profiling of aspartates and glutamates in living bacteria

Project description:Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, that do not rely on an enzymatic activity, have almost exclusively been developed to target cysteines. Expanding the scope to other amino acids would be largely facilitated by the ability to globally monitor their engagement by covalent inhibitors. Here, we present the use of light-activatable 2,5-disubstituted tetrazoles that allow quantifying close to 9000 aspartates and glutamates in the bacterial proteome with excellent selectivity. Using these probes, we competitively map the binding sites of two isoxazolium salts and introduce hydrazonyl chlorides as a new class of carboxylic acid-directed covalent protein ligands. As the probes are unreactive prior to activation, they allow global profiling even in living Gram-positive and Gram-negative bacteria. Taken together, this method to monitor aspartates and glutamates proteome-wide will lay the foundation to efficiently develop covalent inhibitors targeting these amino acids.

2020-04-14 | PXD016659 | Pride

Covalent 14-3-3 Molecular Glues and Heterobifunctional Molecules Against Nuclear Transcription Factors and Regulators

Project description:14-3-3 proteins have the unique ability to bind and sequester a multitude of diverse phosphorylated signaling proteins and transcription factors. Many previous studies have shown that 14-3-3 interactions with specific phosphorylated substrate proteins can be enhanced through small-molecule natural product or fully synthetic molecular glue interactions. However, enhancing 14-3-3 interactions with both therapeutically intractable transcription factor substrates as well as potential neo-substrates to sequester and inhibit their function has remained elusive. One of the 14-3-3 proteins, 14-3-3or SFN, has a cysteine C38 at the substrate binding interface near sites where previous 14-3-3 molecular glues have been found to bind. In this study, we screened a fully synthetic cysteine-reactive covalent ligand library to identify molecular glues that enhance interaction of 14-3-3with not only druggable transcription factors such as estrogen receptor (ER), but also challenging oncogenic transcription factors such as YAP and TAZ that are part of the Hippo transducer pathway. We identified a hit EN171 that covalently targets both C38 and C96 on 14-3-3 to enhance 14-3-3 interactions with ERYAP, and TAZ leading to impaired estrogen receptor and Hippo pathway transcriptional activity. We further demonstrate that EN171 could not only be used as a molecular glue to enhance native protein interactions, but also could be used as a covalent 14-3-3 recruiter in heterobifunctional molecules to sequester nuclear neo-substrates such as BRD4 and BLC6 into the cytosol. Overall, our study reveals a covalent ligand that acts as a novel 14-3-3 molecular glue for challenging transcription factors such as YAP and TAZ and demonstrates that these glues can be potentially utilized in heterobifunctional molecules to sequester nuclear neo-substrates out of the nucleus and into the cytosol to enable targeted protein localization.

2024-04-26 | PXD051762 | Pride

A tailored phosphoaspartate probe unravels CprR as a response regulator in Pseudomonas aeruginosa interkingdom signaling

Project description:Pseudomonas aeruginosa is a difficult-to-treat Gram-negative bacterial pathogen causing life-threatening infections. Adaptive resistance (AR) to cationic peptide antibiotics such as polymyxin B impairs the therapeutic success. This self-protection is mediated by two component systems (TCS) consisting of a membrane-bound histidine kinase and an intracellular response regulator (RR). As phosphorylation of the key RR aspartate residue is transient during signaling and hydrolytically unstable, the study of these systems is challenging. Therefore, we applied a tailored reverse polarity chemical proteomic strategy to capture this transient modification and read-out RR phosphorylation in complex proteomes using a nucleophilic probe. An ideal trapping methodology was developed with a recombinant RR demonstrating the importance of fine-tuned acidic pH values to facilitate the attack on the aspartate carbonyl C-atom and prevent unproductive hydrolysis. Analysis of Bacillus subtilis and P. aeruginosa proteomes revealed the detection of multiple phosphoaspartate sites, which closely resembled the conserved RR sequence motif. With this validated strategy we dissected the signaling of dynorphin A, a human peptide stress hormone, which is sensed by P. aeruginosa to mediate AR. Intriguingly, our methodology identified CprR as an unprecedented RR in dynorphin A interkingdom signaling.

2021-02-16 | PXD022426 | Pride

Screening privileged alkylguanidinium motifs under host-mimicking conditions reveals a novel antibiotic with an unconventional mode of action

Project description:Screening large molecule libraries against pathogenic bacteria is often challenged by a low hit rate due to limited uptake, underrepresentation of antibiotic structural motifs and assays which do not resemble the infection conditions. To address these limitations, we here screen a library focused on alkylguanidinium compounds, a structural motif associated with antibiotic activity and enhanced uptake, under host-mimicking infection conditions against a panel of disease-associated bacteria. Several hit molecules were obtained with activities against Gram-positive as well as Gram-negative bacteria highlighting the fidelity of the general concept. Based on the best antibiotic activity, we selected one compound (L15) for in-depth mode of action studies. L15 exhibited bactericidal activity against Staphylococcus aureus USA300 Lac (JE2) with a minimum inhibitory concentration of 1.5 µM whereby a structure-activity relationship study with 18 derivatives proved the necessity of the guanidinium motif for antibiotic activity. Electron microscopy studies and depolarization assays at high L15 concentrations showed an impact on the cell membrane and integrity which was absent at the MIC concentration. Sequencing of L15-resistant strains revealed a mutation in an efflux pump but did not provide hints for direct targets. We thus performed affinity-based protein profiling with a L15 probe and identified signal peptidase IB (SpsB) as the most promising hit. Validation by activity assays, binding site identification and molecular docking demonstrated SpsB overactivation by L15, similar to a previously reported activator molecule that dysregulates protein secretion of this essential enzyme. Overall, this study highlights the need for unconventional screening strategies to identify novel antibiotics.

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Chemoproteomics-Enabled Discovery of a Covalent Molecular Glue Degrader

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A chemical probe unravels the reactive proteome of health-associated catechols

Project description:Catechol-containing natural products are common constituents of foods, drinks, and drugs. Natural products carrying this motif are often associated with beneficial biological effects such as anticancer activity and neuroprotection. However, the molecular mode of action behind these properties is poorly understood. Here, we apply a mass spectrometry-based competitive chemical proteomics approach to elucidate the target scope of catechol-containing bioactive molecules from diverse foods and drugs. Inspired by the protein reactivity of catecholamine neurotransmitters, we designed and synthesised a broadly reactive minimalist catechol chemical probe based on dopamine. Initial labelling experiments in live human cells demonstrated broad protein binding by the probe, which was largely outcompeted by its parent compound dopamine. Next, we investigated the competition profile of a selection of biologically relevant catechol-containing compounds. With this approach, we characterised the protein reactivity and the target scope of dopamine and ten catechols. Strikingly, proteins associated with the endoplasmic reticulum (ER) were among the main targets. ER stress assays in the presence of reactive catechols revealed an activation of the IRE1-branch of the unfolded protein response (UPR), whereas unreactive catechols had no effect. As the UPR is an intriguing oncology target, this provides a possible explanation of the health-promoting effects attributed to many catechol natural products.

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Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome

Project description:In the presented datasets, we use newly developed isotopically labeled desthiobiotin azide (isoDTB) tags to residue-specifically quantify cysteines in a variety of bacterial strains and a human cell line with consistently high coverage. We e.g. quantify 59% of all cysteines in essential proteins in Staphylococcus aureus. We discover 88 cysteines with high reactivity, which correlates with functional importance. Furthermore, we identify 268 cysteines that are engaged by covalent ligands. Overall, a comprehensive map of the bacterial cysteinome is obtained.

2019-12-02 | PXD015457 | Pride

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