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Tuberculosis, caused by pathogenic M. tuberculosis, remains a global health concern among various infectious diseases. Studies show that ClpB, a major disaggregase, protects the pathogen from various stresses encountered in the host environment. In the present study we have performed a detailed biophysical characterization of M. tuberculosis ClpB followed by a high throughput screening to identify small molecule inhibitors. The sedimentation velocity studies reveal that ClpB oligomerization varies with its concentration and presence of nucleotides. Further, using high throughput malachite green-based screening assay, we identified potential novel inhibitors of ClpB ATPase activity. The enzyme kinetics revealed that the lead molecule inhibits ClpB activity in a competitive manner. These drugs were also able to inhibit ATPase activity associated with E. coli ClpB and yeast Hsp104. The identified drugs inhibited the growth of intracellular bacteria in macrophages. Small angle X-ray scattering based modeling shows that ATP, and not its non-hydrolyzable analogs induce large scale conformational rearrangements in ClpB. Remarkably, the identified small molecules inhibited these ATP inducible conformational changes, suggesting that nucleotide induced shape changes are crucial for ClpB activity. The study broadens our understanding of M. tuberculosis chaperone machinery and provides the basis for designing more potent inhibitors against ClpB chaperone., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation.

The proteolytic complex ClpXP is fundamental to bacterial homeostasis and pathogenesis. Because of its conformational flexibility, the development of potent ClpXP inhibitors is challenging, and novel tools to decipher its intricate regulation are urgently needed. Herein, we present amino acid based phenyl esters as molecular probes to study the activity and oligomerization of the ClpXP complex of S. aureus. Systematic screening of (R)- and (S)-amino acids led to compounds showing potent inhibition, as well as stimulation of ClpXP-mediated proteolysis. Substoichiometric binding of probes arrested ClpXP in an unprecedented heptamer-hexamer assembly, in which the two heptameric ClpP rings are dissociated from each other. At the same time, the affinity between ClpX and ClpP increased, leading to inhibition of both enzymes. This conformational arrest is beneficial for the consolidated shutdown of ClpXP, as well as for the study of the oligomeric state during its catalytic cycle., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Emerging antibiotic resistance among bacterial pathogens has necessitated the development of alternative approaches to combat drug-resistance-associated infection. The abolition of Staphylococcus aureus virulence by targeting multiple-virulence gene products represents a promising strategy for exploration. A multiplex promoter reporter platform using gfp - luxABCDE dual-reporter plasmids with selected promoters from S. aureus -virulence-associated genes was used to identify compounds that modulate the expression of virulence factors. One small-molecule compound, M21, was identified from a chemical library to reverse virulent S. aureus into its nonvirulent state. M21 is a noncompetitive inhibitor of ClpP and alters α-toxin expression in a ClpP-dependent manner. A mouse model of infection indicated that M21 could attenuate S. aureus virulence. This nonantibiotic compound has been shown to suppress the expression of multiple unrelated virulence factors in S. aureus , suggesting that targeting a master regulator of virulence is an effective way to control virulence. Our results illustrate the power of chemical genetics in the modulation of virulence gene expression in pathogenic bacteria., Competing Interests: The authors declare no conflict of interest.

The Staphylococcus aureus ClpXP protease is an important regulator of cell homeostasis and virulence. We utilized a high-throughput screen against the ClpXP complex and identified a specific inhibitor of the ClpX chaperone that disrupts its oligomeric state. Synthesis of 34 derivatives revealed that the molecular scaffold is restrictive for diversification, with only minor changes tolerated. Subsequent analysis of the most active compound revealed strong attenuation of S. aureus toxin production, which was quantified with a customized MS-based assay platform. Transcriptome and whole-proteome studies further confirmed the global reduction of virulence and revealed characteristic signatures of protein expression in the compound-treated cells. Although these partially matched the pattern of ClpX knockout cells, further depletion of toxins was observed, leading to the intriguing perspective that additional virulence pathways may be directly or indirectly addressed by the small molecule., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus , Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA). The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr . On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection.

Mycobacterium tuberculosis is responsible for the greatest number of deaths worldwide due to a bacterial agent. We recently identified bortezomib (Velcade; compound 1) as a promising antituberculosis (anti-TB) compound. We showed that compound 1 inhibits the mycobacterial caseinolytic proteases P1 and P2 (ClpP1P2) and exhibits bactericidal activity, and we established compound 1 and ClpP1P2 as an attractive lead/target couple. However, compound 1 is a human-proteasome inhibitor currently approved for cancer therapy and, as such, exhibits significant toxicity. Selective inhibition of the bacterial protease over the human proteasome is desirable in order to maintain antibacterial activity while reducing toxicity. We made use of structural data in order to design a series of dipeptidyl-boronate derivatives of compound 1. We tested these derivatives for whole-cell ClpP1P2 and human-proteasome inhibition as well as bacterial-growth inhibition and identified compounds that were up to 100-fold-less active against the human proteasome but that retained ClpP1P2 and mycobacterial-growth inhibition as well as bactericidal potency. The lead compound, compound 58, had low micromolar ClpP1P2 and anti- M. tuberculosis activity, good aqueous solubility, no cytochrome P450 liabilities, moderate plasma protein binding, and low toxicity in two human liver cell lines, and despite high clearance in microsomes, this compound was only moderately cleared when administered intravenously or orally to mice. Higher-dose oral pharmacokinetics indicated good dose linearity. Furthermore, compound 58 was inhibitory to only 11% of a panel of 62 proteases. Our work suggests that selectivity over the human proteasome can be achieved with a drug-like template while retaining potency against ClpP1P2 and, crucially, anti- M. tuberculosis activity., (Copyright © 2017 Moreira et al.)

Bacterial Clp proteases are important for protein turnover and homeostasis in order to maintain vital cellular functions particularly under stress conditions. Apart from their crucial role in general protein quality control by degrading abnormally folded or otherwise aberrant or malfunctioning proteins, their temporally and spatially precise proteolysis of key regulatory proteins additionally guides several developmental processes like cell motility, genetic competence, cell differentiation, sporulation as well as important aspects of virulence. Due to their apparent relevance for many physiological processes and their conservation among diverse bacterial species including human pathogens, Clp proteases have attracted considerable attention as targets for antibacterial action in recent years. Particularly a novel class of potent acyldepsipeptide antibiotics unleashes ClpP, the uniform proteolytic core unit of the degradative Clp complexes, to bring about bacterial death via uncontrolled proteolysis of proteins that are essential for bacterial viability. In addition, covalent inhibition of the catalytic center of ClpP by another class of small molecule inhibitors is investigated in the context of virulence inhibition. Both antibacterial mechanisms constitute innovative approaches with the potential to control infections caused by multi-resistant bacterial pathogens due to the lack of cross-resistance to established antibiotic classes., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Mycobacterium tuberculosis is a pathogen of major global importance. Validated drug targets are required in order to develop novel therapeutics for drug-resistant strains and to shorten therapy. The Clp protease complexes provide a means for quality control of cellular proteins; the proteolytic activity of ClpP in concert with the ATPase activity of the ClpX/ClpC subunits results in degradation of misfolded or damaged proteins. Thus, the Clp system plays a major role in basic metabolism, as well as in stress responses and pathogenic mechanisms. M. tuberculosis has two ClpP proteolytic subunits. Here we demonstrate that ClpP1 is essential for viability in this organism in culture, since the gene could only be deleted from the chromosome when a second functional copy was provided. Overexpression of clpP1 had no effect on growth in aerobic culture or viability under anaerobic conditions or during nutrient starvation. In contrast, clpP2 overexpression was toxic, suggesting different roles for the two homologs. We synthesized known activators of ClpP protease activity; these acyldepsipeptides (ADEPs) were active against M. tuberculosis. ADEP activity was enhanced by the addition of efflux pump inhibitors, demonstrating that ADEPs gain access to the cell but that export occurs. Taken together, the genetic and chemical validation of ClpP as a drug target leads to new avenues for drug discovery.

Here, we report the synthesis and in depth characterization of a second generation β-lactone derived virulence inhibitors. Based on initial results that emphasized the intriguing possibility to disarm bacteria in their virulence the present study develops this concept further and analyses the potential of this strategy for drug development. We were able to expand the collection of bioactive compounds via an efficient synthetic route. Testing of all compounds revealed several hits with anti-virulence activity. Moreover, we demonstrated that these molecules act solely by reducing virulence but not killing bacteria which is an important prerequisite for preserving the useful microbiome. Finally, incubation of lactones with eukaryotic cell lines indicated a tolerable cytotoxicity which is essential for entering animal studies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

ClpP is a cylindrical protease that is tightly regulated by Clp-ATPases. The activation mechanism of ClpP using acyldepsipeptide antibiotics as mimics of natural activators showed enlargement of the axial entrance pore for easier processing of incoming substrates. However, the elimination of degradation products from inside the ClpP chamber remains unclear since there is no exit pore for releasing these products in all determined ClpP structures. Here we report a new crystal structure of ClpP from Bacillus subtilis, which shows a significantly compressed shape along the axial direction. A portion of the handle regions comprising the heptameric ring-ring contacts shows structural transition from an ordered to a disordered state, which triggers the large conformational change from an extended to an overall compressed structure. Along with this structural change, 14 side pores are generated for product release and the catalytic triad adopts an inactive orientation. We have also determined B. subtilis ClpP inhibited by diisopropylfluoro-phosphate and analyzed the active site in detail. Structural information pertaining to several different conformational steps such as those related to extended, ADEP-activated, DFP-inhibited and compressed forms of ClpP from B. subtilis is available. Structural comparisons suggest that functionally important regions in the ClpP-family such as N-terminal segments for the axial pore, catalytic triads, and handle domains for the product releasing pore exhibit intrinsically dynamic and unique structural features. This study provides valuable insights for understanding the enigmatic cylindrical degradation machinery of ClpP as well as other related proteases such as HslV and the 20S proteasome.

A method to rapidly screen libraries of cyclic peptides in vivo for molecules with biological activity has been developed and used to isolate cyclic peptide inhibitors of the ClpXP protease. Fluorescence activated cell sorting was used in conjunction with a fluorescent reporter to isolate cyclic peptides that inhibit the proteolysis of tmRNA-tagged proteins in Escherichia coli. Inhibitors shared little sequence similarity and interfered with unexpected steps in the ClpXP mechanism in vitro. One cyclic peptide, IXP1, inhibited the degradation of unrelated ClpXP substrates and has bactericidal activity when added to growing cultures of Caulobacter crescentus, a model organism that requires ClpXP activity for viability. The screen used here could be adapted to identify cyclic peptide inhibitors of any enzyme that can be expressed in E. coli in conjunction with a fluorescent reporter.