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1.
Nat Commun ; 15(1): 2005, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38443338

RESUMO

Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance.


Assuntos
Mycobacterium smegmatis , Mycobacterium tuberculosis , Proteólise , Dimerização , Descoberta de Drogas
2.
Cell ; 186(10): 2176-2192.e22, 2023 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-37137307

RESUMO

The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.


Assuntos
Antituberculosos , Mycobacterium tuberculosis , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Choque Térmico/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Proteostase
3.
Cell ; 185(13): 2338-2353.e18, 2022 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-35662409

RESUMO

Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, so far, it has not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, drug susceptibility and degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, allowing selective targeting of endogenous proteins via fusion to an established degron. In addition to guiding antibiotic discovery, the BacPROTAC technology presents a versatile research tool enabling the inducible degradation of bacterial proteins.


Assuntos
Proteínas de Bactérias , Chaperonas Moleculares , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Proteólise
4.
J Med Chem ; 65(6): 4893-4908, 2022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-35293761

RESUMO

Ohmyungsamycin A and ecumicin are structurally related cyclic depsipeptide natural products that possess activity against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). Herein, we describe the design and synthesis of a library of analogues of these two natural products using an efficient solid-phase synthesis and late-stage macrolactamization strategy. Lead analogues possessed potent activity against Mtb in vitro (minimum inhibitory concentration 125-500 nM) and were shown to inhibit protein degradation by the mycobacterial ClpC1-ClpP1P2 protease with an associated enhancement of ClpC1 ATPase activity. The most promising analogue from the series exhibited rapid bactericidal killing activity against Mtb, capable of sterilizing cultures after 7 days, and retained bactericidal activity against hypoxic non-replicating Mtb. This natural product analogue was also active in an in vivo zebrafish model of infection.


Assuntos
Produtos Biológicos , Depsipeptídeos , Mycobacterium tuberculosis , Animais , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Produtos Biológicos/farmacologia , Depsipeptídeos/farmacologia , Chaperonas Moleculares , Mycobacterium tuberculosis/metabolismo , Peptídeos Cíclicos , Peixe-Zebra/metabolismo
5.
Proteomics ; 18(13): e1700436, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29775240

RESUMO

SugarQb (www.imba.oeaw.ac.at/sugarqb) is a freely available collection of computational tools for the automated identification of intact glycopeptides from high-resolution HCD MS/MS datasets in the Proteome Discoverer environment. We report the migration of SugarQb to the latest and free version of Proteome Discoverer 2.1, and apply it to the analysis of PNGase F-resistant N-glycopeptides from mouse embryonic stem cells. The analysis of intact glycopeptides highlights unexpected technical limitations to PNGase F-dependent glycoproteomic workflows at the proteome level, and warrants a critical reinterpretation of seminal datasets in the context of N-glycosylation-site prediction.


Assuntos
Células-Tronco Embrionárias/metabolismo , Glicopeptídeos/análise , Glicoproteínas/análise , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Proteoma/análise , Software , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Camundongos , Proteoma/química , Especificidade por Substrato
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