Design and synthesis of tailored human caseinolytic protease P inhibitors

Gronauer, Thomas F.; Mandl, Manuela; Lakemeyer, Markus; Hackl, Mathias W.; Messner, Matthias; Korotkov, Vadim S.; Pachmayr, Johanna; Sieber, Stephan A.

doi:10.1039/c8cc05265d

Cited by 27 publications

(33 citation statements)

References 29 publications

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“…TG42 and TG53 induce apoptosis and decrease cell migration of Huh7 liver cancer cells (Table 2 and Supplementary Fig. 1) 79 . However, further studies are necessary to determine whether these anticancer effects are due to ClpP inhibition or off-target effects as these compounds cross react with multiple human proteases.…”

Section: Small Molecule Clpp Inhibitors-chemical Probes To Understandmentioning

confidence: 99%

“…Through substitutions of the naphtofuran moiety at position-2 of AV167, more potent and selective inhibitors of human mitochondrial ClpP were developed 79 . The modified analogs, termed TG42, TG43, and TG53, preferentially inhibit human ClpP's peptidolytic and proteolytic activities while having a minor effect on S. aureus ClpP (SaClpP) 79 . TG42 and TG53 induce apoptosis and decrease cell migration of Huh7 liver cancer cells (Table 2 and Supplementary Fig.…”

Section: Small Molecule Clpp Inhibitors-chemical Probes To Understandmentioning

confidence: 99%

“…1 ) 78 , suggesting important differences in the active sites between human and bacterial ClpP. Through substitutions of the naphtofuran moiety at position-2 of AV167, more potent and selective inhibitors of human mitochondrial ClpP were developed 79 . The modified analogs, termed TG42, TG43, and TG53, preferentially inhibit human ClpP’s peptidolytic and proteolytic activities while having a minor effect on S. aureus ClpP (SaClpP) 79 .…”

Section: Small Molecule Clpp Inhibitors—chemical Probes To Understandmentioning

confidence: 99%

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Mitochondrial ClpP serine protease-biological function and emerging target for cancer therapy

2020

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Mitochondrial ClpP is a serine protease located in the mitochondrial matrix. This protease participates in mitochondrial protein quality control by degrading misfolded or damaged proteins, thus maintaining normal metabolic function. Mitochondrial ClpP is a stable heptamer ring with peptidase activity that forms a multimeric complex with the ATP-dependent unfoldase ClpX (ClpXP) leading to proteolytic activity. Emerging evidence demonstrates that ClpXP is over-expressed in hematologic malignancies and solid tumors and is necessary for the viability of a subset of tumors. In addition, both inhibition and hyperactivation of ClpXP leads to impaired respiratory chain activity and causes cell death in cancer cells. Therefore, targeting mitochondrial ClpXP could be a novel therapeutic strategy for the treatment of malignancy. Here, we review the structure and function of mitochondrial ClpXP as well as strategies to target this enzyme complex as a novel therapeutic approach for malignancy.

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Section: Small Molecule Clpp Inhibitors-chemical Probes To Understandmentioning

confidence: 99%

Section: Small Molecule Clpp Inhibitors-chemical Probes To Understandmentioning

confidence: 99%

Section: Small Molecule Clpp Inhibitors—chemical Probes To Understandmentioning

confidence: 99%

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Mitochondrial ClpP serine protease-biological function and emerging target for cancer therapy

2020

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“…The potential of exploiting the inherent dynamics and allostery of ClpP to modulate its function has been well recognized (66,(74)(75)(76)(77). One target is the handle region that mediates the interactions between protomers on opposite ClpP rings and anchors the catalytic residues to achieve optimal peptide bond cleavage.…”

Section: Discussionmentioning

confidence: 99%

An allosteric switch regulatesMycobacterium tuberculosisClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR

Vahidi

Ripstein

Juravsky

et al. 2019

Preprint

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Short title: NMR and cryo-EM of M. tuberculosis ClpP1P2The MtClpP1P2 protease is part of the essential protein degradation machinery that helps maintain protein homeostasis in Mycobacterium tuberculosis, the causative agent of TB.Antibiotics that selectively kill both dormant and growing drug-resistant populations of M. tuberculosis by disrupting MtClpP1P2 function have attracted recent attention. Here we characterize a switch that can control MtClpP1P2 activity through binding of small peptides, leading to a concerted conformational change that potentially can be exploited by drug molecules to interfere with MtClpP1P2 function. Overall, this work highlights the power of a combined NMR and cryo-EM approach to provide detailed insights into the structure-dynamics-function relationship of molecular machines critical to human health. the binding of essential AAA+ unfoldases, ClpX or ClpC1, that use the energy of ATP to unfold and translocate substrates into its catalytic chamber for degradation (28). Lassomycin (29), ecumicin (30), and rufomycin (31) are promising antibiotics that selectively kill both dormant and growing drug-resistant populations of M. tuberculosis by binding to ClpC1 and decoupling ATP-dependant protein unfolding from proteolysis. Similarly, ADEPs also kill M. tuberculosis by preventing the binding of AAA+ regulatory unfoldases to MtClpP1P2 (32). MtClpP1P2 reacts with standard inhibitors of serine proteases, such as chloromethyl ketones, which modify the serine and histidine residues at enzyme active sites (33). Peptide boronates have been shown to also directly engage MtClpP1P2 active sites, causing inhibition at low micromolar concentrations and preventing growth of M. tuberculosis (34-36). More recently Cediranib, an anti-cancer drug, was proposed as a novel non-covalent inhibitor of MtClpP1P2 (37).Most bacteria possess a single clpP gene, giving rise to a structure comprising a pair of heptameric rings that are arranged coaxially to form a homotetradecameric barrel-like protein complex enclosing fourteen Asp-His-Ser catalytic triads (11, 12,38). Each of the identical protomers consists of an N-terminal domain that forms gated narrow pores on the apical surface of the barrel, a head domain that generates the main body of the ClpP barrel, and a handle region comprising a helix and a β-sheet that mediate ClpP ring-ring interactions (11,(38)(39)(40). In addition, the handle provides crucial contacts that align the catalytic triad and generates a binding grove for substrate polypeptides (41). Opening of the pores that allow substrate translocation is tightly regulated by AAA+ regulators (42,43). Actinobacteria, the phylum to which mycobacteria such as M. tuberculosis belong, are unique in that they contain two clpP genes, clpP1 and clpP2, that encode for MtClpP1 and MtClpP2, respectively (44,45). Initial structure-function studies concluded that MtClpP1 and SI AppendixAn allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR ...

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“…tuberculosis ClpP1 and ClpP2 (spanning residues 7-200 and 13-214, respectively) was cloned into a pRSF-Duet vector preceded by an N-terminal His6-SUMO tag (Novagen) [11,12]. Full-length EcClpP, SaClpP, PaClpP1 and the hClpP gene without its mitochondrial targeting sequence (residues Met1-Pro57) were also respectively cloned into pRSF-Duet preceded by an N-terminal His6-SUMO tag [33][34][35]. PCR was performed with PrimeSTAR ® max DNA polymerase (Takara) and Ex Taq ® polymerase (Takara).…”

Section: Bacterial Strains and Culture Conditions Escherichia Coli Dmentioning

confidence: 99%

Inhibiting Mycobacterium tuberculosis ClpP1P2 by addressing the equatorial handle domain of ClpP1 subunit

Yang

Zhu

et al. 2019

Preprint

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19Unlike other bacterial ClpP systems, mycobacterial ClpP1P2 complex is essential 20 for mycobacterial survival. The functional details of Mycobacterium tuberculosis (Mtb) 21 ClpP1P2 remains largely elusive and selectively targeting ClpP of different species is a 22 big challenge. In this work, cediranib was demonstrated to significantly decrease the 23 activity of MtbClpP1P2. By solving the crystal structure of cediranib-bound 24 MtbClpP1P2, we found that cediranib dysregulates MtbClpP1P2 by interfering with 25 handle domain of the equatorial region of MtbClpP1, indicating that the inter-ring 26 dynamics are crucial for its function. This finding provides direct evidence for the 27 notion that a conformational switch in the equatorial handle domain is essential for ClpP 28 activity. We also present biochemical data to interpret the distinct interaction pattern 29 and inhibitory properties of cediranib toward MtbClpP1P2. These results suggest that 30 the variable handle domain region is responsible for the species-selectivity of cediranib, 31 which suggests the equatorial handle domain as a potential region for screening 32 pathogen-specific ClpP inhibitors. 33 Introduction 34 Caseinolytic protease ClpP, a widely conserved self-compartmentalizing serine 35 protease in bacteria, plays essential roles in protein metabolism and regulates diverse 36 physiological functions including cell motility, genetic competence, cell differentiation, 37 sporulation, and virulence, and is thus an attractive target for antibiotic development[1-38 8]. Notably, ClpP is an novel drug target for which both inhibition and activation result 39 in an attenuated or lethal phenotype in many pathogens; thus agonists and antagonists 40 Inhibiting ClpP1P2 by addressing the equatorial handle domain of ClpP1 3 / 42 aimed at the ClpP system represent promising drug candidates for further evaluation[9, 41 10]. The ClpP enzyme of Mycobacterium tuberculosis (MTB), MtbClpP1P2, exerts its 42 proteolytic function by a heterotetramer of two protein subunits, ClpP1 and ClpP2[11, 43 12]. Moreover, encoding genes of subunits ClpP1 and ClpP2, have been shown to be 44 essential genes for MTB survival and the deletion of either gene causes bacterial 45 death[13]. A panel of MtbClpP1P2 inhibitors have been reported and they can be 46 classified into two categories depending on their action modes. The first kind of small 47 molecules act on the catalytically active center, covalently modifying the serine 48 residues of the active sites of two subunits of MtbClpP1P2, including bortezomib, 49 boron-containing analogues and beta lactones[14-17]. The second kind of inhibitors act 50 on the chaperone protein (ClpX/ClpC1) binding site that competitively binds to the 51 surface of the ClpP2 subunit. Representative molecules of this category are ADEP and 52 its analogs[12]. Since the chaperones binding L/IGF region of ClpP2 subunit is highly 53 conserved, as with the inhibitor of the MtbClpP1P2 catalytic center[18], the ADEP 54 compounds are nonse...

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Design and synthesis of tailored human caseinolytic protease P inhibitors

Cited by 27 publications

References 29 publications

Mitochondrial ClpP serine protease-biological function and emerging target for cancer therapy

Mitochondrial ClpP serine protease-biological function and emerging target for cancer therapy

An allosteric switch regulatesMycobacterium tuberculosisClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR

Inhibiting Mycobacterium tuberculosis ClpP1P2 by addressing the equatorial handle domain of ClpP1 subunit

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