Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from <i>Mycobacterium tuberculosis</i>

Lentz, Christian S.; Ordoñez, Alvaro A.; Kasperkiewicz, Paulina; Greca, Florencia La; O’Donoghue, Anthony J.; Schulze, Christopher J.; Powers, James C.; Craik, Charles S.; Drąg, Marcin; Jain, Sanjay; Bogyo, Matthew

doi:10.1021/acsinfecdis.6b00092

Cited by 45 publications

(58 citation statements)

References 24 publications

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“…1c ). Without knowing the identity of these targets, we screened a library of ~500 compounds that were designed to covalently target serine proteases and hydrolases 24 , 25 using the competition labeling method ( Supplementary Fig. 1a , Supplementary Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP)

et al. 2018

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Serine hydrolases play diverse roles in regulating host-pathogen interactions in a number of organisms, yet few have been characterized in the human pathogen Staphylococcus aureus. Here, we describe a chemical proteomic screen that identified 10 previously uncharacterized S. aureus serine hydrolases that mostly lack human homologues. We termed these enzymes Fluorophosphonate-binding hydrolases (FphA-J). One hydrolase, FphB, can process short fatty acid esters, exhibits increased activity in response to host cell factors, is located predominantly on the bacterial cell surface in a subset of cells, and is concentrated in the division septum. Genetic disruption of the fphB gene confirms that the enzyme is dispensable for bacterial growth in culture but crucial for establishing infection in distinct sites in vivo. A selective small molecule inhibitor of FphB effectively reduces infectivity in vivo, suggesting that it may be a viable therapeutic target for the treatment or management of Staphylococcus infections.

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Section: Resultsmentioning

confidence: 99%

Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP)

et al. 2018

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“… 54 − 60 Accordingly, we focused on a collection of ∼600 compounds bearing vinyl sulfones, epoxy ketones, various Michael acceptors, halomethyl ketones, aldehydes, and acyloxymethyl ketones that had been used in phenotypic screens to find inhibitors of various target enzymes ( Figure 7 A). 61 − 64 We employed a modified version of the cell-based, fluorometric Cresswell assay for ERAD pathway activity. 65 Freeze and co-workers have previously used the Cresswell assay in NGLY1-deficient cell lines as a readout of NGLY1 activity.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity

et al. 2017

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Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This “bounce-back” response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis.

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“…Since HyCoSuL is a new concept in protease research, it is still being validated and improved in order to create a chemical model that covers structural requirements of multiple proteases. However, we have already found HyCoSuL to be versatile tool to profile proteases of different classes, catalytic mechanisms, origins, or activities 18,22,[24][25][26][27] . The library structure allows for detailed exploration of the protease active site, thus the substrate specificity map is much more informative than the classical analysis based on PS-SCL, phage display or natural substrate cleavages.…”

Section: Advantages and Limitations Of The Protocolmentioning

confidence: 99%

Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity

Poręba¹,

Salvesen²,

Drąg³

2017

Nat Protoc

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Many biologically and chemically based approaches have been developed to design highly active and selective protease substrates and probes. It is, however, difficult to find substrate sequences that are truly selective for any given protease, as different proteases can demonstrate a great deal of overlap in substrate specificities. In some cases, better enzyme selectivity can be achieved using peptide libraries containing unnatural amino acids such as the hybrid combinatorial substrate library (HyCoSuL), which uses both natural and unnatural amino acids. HyCoSuL is a combinatorial library of tetrapeptides containing amino acid mixtures at the P4-P2 positions, a fixed amino acid at the P1 position, and an ACC (7-amino-4-carbamoylmethylcoumarin) fluorescent tag occupying the P1' position. Once the peptide is recognized and cleaved by a protease, the ACC is released and produces a readable fluorescence signal. Here, we describe the synthesis and screening of HyCoSuL for human caspases and legumain. We also discuss possible modifications and adaptations of this approach that make it a useful tool for developing highly active and selective reagents for a wide variety of proteolytic enzymes. The protocol can be divided into three major parts: (i) solid-phase synthesis of the fluorescence-labeled HyCoSuL, (ii) screening of protease P4-P2 preferences, and (iii) synthesis of the optimized activity probes equipped with an AOMK (acyloxymethyl ketone) reactive group and a biotin label for easy detection. Beginning with the library design, the entire protocol can be completed in 4-8 weeks (HyCoSuL synthesis: 3-5 weeks; HyCoSuL screening per enzyme: 4-8 d; and activity-based probe synthesis: 1-2 weeks).

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Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis

Cited by 45 publications

References 24 publications

Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP)

Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP)

Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity

Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity

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