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Structure-Based Design of Fluorogenic Substrates Selective for Human Proteasome Subunits.

ABSTRACT: Proteasomes are established therapeutic targets for hematological cancers and promising targets for autoimmune diseases. In the past, we have designed and synthesized mechanism-based proteasome inhibitors that are selective for the individual catalytic activities of human constitutive proteasomes and immunoproteasomes: ?1c, ?1i, ?2c, ?2i, ?5c and ?5i. We show here that by taking the oligopeptide recognition element and substituting the electrophile for a fluorogenic leaving group, fluorogenic substrates are obtained that report on the proteasome catalytic activity also targeted by the parent inhibitor. Though not generally applicable (?5c and ?2i substrates showing low activity), effective fluorogenic substrates reporting on the individual activity of ?1c, ?1i, ?2c and ?5i subunits in Raji (human B cell) lysates and purified 20S proteasome were identified in this manner. Our work thus adds to the expanding proteasome research toolbox through the identification of new and/or more effective subunit-selective fluorogenic substrates.

SUBMITTER: Maurits E

PROVIDER: S-EPMC7754458 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Structure-Based Design of Fluorogenic Substrates Selective for Human Proteasome Subunits.

Maurits Elmer E Degeling Christian G CG Kisselev Alexei F AF Florea Bogdan I BI Overkleeft Herman S HS

Chembiochem : a European journal of chemical biology 20200729 22

Proteasomes are established therapeutic targets for hematological cancers and promising targets for autoimmune diseases. In the past, we have designed and synthesized mechanism-based proteasome inhibitors that are selective for the individual catalytic activities of human constitutive proteasomes and immunoproteasomes: β1c, β1i, β2c, β2i, β5c and β5i. We show here that by taking the oligopeptide recognition element and substituting the electrophile for a fluorogenic leaving group, fluorogenic su ...[more]

PMID: 32598532

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Project description:The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the ?2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum ?2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this ?2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next-generation anti-malarial agents.

Dataset Information

Structure-Based Design of Fluorogenic Substrates Selective for Human Proteasome Subunits.

Publications

Structure-Based Design of Fluorogenic Substrates Selective for Human Proteasome Subunits.

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