Acyldepsipeptides Research Articles

Design and synthesis of Acyldepsipeptide-1 analogues: Antibacterial activity and cytotoxicity screening

Acyldepsipeptides (ADEPs) are receiving more attention as prospective antimicrobial agents due to their unique mode of action and chemical properties. However, their therapeutic potential is limited by their poor pharmacokinetic properties. Chemical modifications have been successful in improving the biocompatibility and bioavailability of ADEPs. In the current study, ADEP1 was modified by introducing a disulphide linkage, replacement of the octa-2,4,6-trienoic acid (OTEA) with either adamantane (Ada) or palmitic acid (Pal), and lastly, comparing the use of D versus L amino acids. The antibacterial effects of the ADEP1 analogues were investigated in Gram-positive and Gram-negative strains using agar well diffusion and microdilution assays. Cytotoxicity was evaluated in human embryonic kidney (HEK)-293 and colon cancer (Caco-2) cells by the MTS assay. Using solid phase peptide synthesis (SPPS), the percentage yield of the synthetic peptides was increased to > 37% with > 96% purity. The anionic ADEP1 analogues demonstrated a broad-spectrum antibacterial activity. Although the ADEP1 analogues did not display the expected antibacterial activity in relation to the parent structure, they were not cytotoxic against the tested cell lines. This study proved that biocompatibility of natural ADEPs can be improved by modifying some of its chemical groups. Further studies are required to enhance the antibacterial activity of the ADEP1 analogues either by structural modifications or through supplementation of these anionic antimicrobial peptides (AMPs) with divalent metal ions.

Acyldepsipeptide Analogues: A Future Generation Antibiotics for Tuberculosis Treatment.

Acyldepsipeptides (ADEPs) are a new class of emerging antimicrobial peptides (AMPs), which are currently explored for treatment of pathogenic infections, including tuberculosis (TB). These cyclic hydrophobic peptides have a unique bacterial target to the conventional anti-TB drugs, and present a therapeutic window to overcome Mycobacterium Tuberculosis (M. tb) drug resistance. ADEPs exerts their antibacterial activity on M. tb strains through activation of the protein homeostatic regulatory protease, the caseinolytic protease (ClpP1P2). ClpP1P2 is normally regulated and activated by the ClpP-ATPases to degrade misfolded and toxic peptides and/or short proteins. ADEPs bind and dysregulate all the homeostatic capabilities of ClpP1P2 while inducing non-selective proteolysis. The uncontrolled proteolysis leads to M. tb cell death within the host. ADEPs analogues that have been tested possess cytotoxicity and poor pharmacokinetic and pharmacodynamic properties. However, these can be improved by drug design techniques. Moreover, the use of nanomaterial in conjunction with ADEPs would yield effective synergistic effect. This new mode of action has potential to combat and eradicate the extensive multi-drug resistance (MDR) problem that is currently faced by the public health pertaining bacterial infections, especially TB.

Structural insights into ClpP protease side exit pore-opening by a pH drop coupled with substrate hydrolysis.

The ClpP serine peptidase is a tetradecameric degradation molecular machine involved in many physiological processes. It becomes a competent ATP-dependent protease when coupled with Clp-ATPases. Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause the dysregulation and activation of ClpP without ATPases and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed its structural details, conformational changes, and activation mechanism. Although product release through side exit pores has been proposed, the detailed driving force for product release remains elusive. Herein, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP-bound states. Cryo-electron microscopy structures of BsClpP revealed various conformational states under different pH conditions. To understand the conformational change required for product release, we investigated the relationship between substrate hydrolysis and the pH-lowering process. The production of hydrolyzed peptides from acidic and basic substrates by proteinase K and BsClpP lowered the pH values. Our data, together with those of previous findings, provide insight into the molecular mechanism of product release by the ClpP self-compartmentalizing protease.

The ClpP peptidase grips protein substrate against external force

All cells spend resources to maintain appropriate protein expression levels and facilitate protein folding, termed proteostasis. Caseinolytic protease P (ClpP) is a self-compartmentalized peptidase that helps maintain proteostasis in prokaryotes and eukaryotic organelles like mitochondria and chloroplasts. To act as a protease in cells, ClpP binds AAA+ unfoldases (ATPases Associated with various cellular Activities) forming a machine capable of unfolding and degrading protein substrates. Additionally, ClpP is activated by a class of natural products called acyldepsipeptides (ADEPs), which activate ClpP in a manner similar to AAA+ binding.

Development of Antibiotics That Dysregulate the Neisserial ClpP Protease.

Evolving antimicrobial resistance has motivated the search for novel targets and alternative therapies. Caseinolytic protease (ClpP) has emerged as an enticing new target since its function is conserved and essential for bacterial fitness, and because its inhibition or dysregulation leads to bacterial cell death. ClpP protease function controls global protein homeostasis and is, therefore, crucial for the maintenance of the bacterial proteome during growth and infection. Previously, acyldepsipeptides (ADEPs) were discovered to dysregulate ClpP, leading to bactericidal activity against both actively growing and dormant Gram-positive pathogens. Unfortunately, these compounds had very low efficacy against Gram-negative bacteria. Hence, we sought to develop non-ADEP ClpP-targeting compounds with activity against Gram-negative species and called these activators of self-compartmentalizing proteases (ACPs). These ACPs bind and dysregulate ClpP in a manner similar to ADEPs, effectively digesting bacteria from the inside out. Here, we performed further ACP derivatization and testing to improve the efficacy and breadth of coverage of selected ACPs against Gram-negative bacteria. We observed that a diverse collection of Neisseria meningitidis and Neisseria gonorrhoeae clinical isolates were exquisitely sensitive to these ACP analogues. Furthermore, based on the ACP-ClpP cocrystal structure solved here, we demonstrate that ACPs could be designed to be species specific. This validates the feasibility of drug-based targeting of ClpP in Gram-negative bacteria.

Acyldepsipeptide Probes Facilitate Specific Detection of Caseinolytic Protease P Independent of Its Oligomeric and Activity State.

Caseinolytic protease P (ClpP) is a tetradecameric peptidase that assembles with chaperones such as ClpX to gain proteolytic activity. Acyldepsipeptides (ADEPs) are small‐molecule mimics of ClpX that bind into hydrophobic pockets on the apical site of the complex, thereby activating ClpP. Detection of ClpP has so far been facilitated with active‐site‐directed probes which depend on the activity and oligomeric state of the complex. To expand the scope of ClpP labeling, we took a stepwise synthetic approach toward customized ADEP photoprobes. Structure–activity relationship studies with small fragments and ADEP derivatives paired with modeling studies revealed the design principles for suitable probe molecules. The derivatives were tested for activation of ClpP and subsequently applied in labeling studies of the wild‐type peptidase as well as enzymes bearing mutations at the active site and an oligomerization sensor. Satisfyingly, the ADEP photoprobes provided a labeling readout of ClpP independent of its activity and oligomeric state.

Pharmacodynamics of ClpP-Activating Antibiotic Combinations against Gram-Positive Pathogens.

It is often difficult to cure endocarditis, osteomyelitis, and device-associated infections caused by Gram-positive pathogens, despite therapy with clinically appropriate antibiotics. This may be due to antibiotic tolerance or resistance development. Acyldepsipeptides (ADEPs) are a class of bactericidal compounds active against a variety of clinically important Gram-positive bacteria, including staphylococci, streptococci, and enterococci. ADEPs activate caseinolytic protease P (ClpP), killing high-density, nondividing cultures of bacteria that are tolerant to approved classes of antibiotics. Acyldepsipeptide analog 4 (ADEP4) was active against a panel of drug-resistant Gram-positive pathogens in MIC assays, with no preexisting resistance detected. Killing of stationary-phase cultures was observed when ADEP4 was combined with multiple classes of approved antibiotics. Additionally, a hollow-fiber infection model was used to assess the effects of ADEP4 antibiotic combinations on bacterial killing and resistance development. These studies were performed on high-density cultures of methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), and vancomycin-resistant Enterococcus faecalis (VRE). None of the approved antibiotics linezolid, ampicillin, and oxacillin tested alone had bactericidal activity under these conditions. ADEP4 initially caused killing, but regrowth of the culture was apparent within 96 h due to resistance. Combinations of ADEP4 with linezolid or oxacillin caused substantially improved killing of MRSA or MSSA cultures, respectively, and no regrowth due to resistance was observed. The combination of ADEP4 and ampicillin eradicated cultures of VRE to the limit of detection within 52 h. These data suggest that combining ClpP activators with traditional antibiotics may be a good strategy to treat complicated Gram-positive infections.

ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores

Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia coli ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.

Molecular Basis of ClpP Protease Activation by Small Molecules

Bacterial ClpP is a highly conserved, self‐compartmentalizing, cylindrical serine protease required for maintaining cellular proteostasis. Acyldepsipeptides (ADEPs) and activators of self‐compartmentalized proteases (ACPs) are small molecules that cause the dysregulation and activation of ClpP, which leads to bacterial cell death, highlighting their potential use as novel antibiotics. However, the molecular basis of ClpP activation by small molecules remains undetermined. Structural changes in ClpP from Neisseria meningitidis and Escherichia coli upon binding to novel ACP and ADEP analogs were probed by X‐ray crystallography, methyl‐TROSY NMR, hydrogen‐deuterium exchange, and small angle X‐ray scattering. ACP and ADEP were found to induce distinct conformational changes in the tertiary structure of ClpP. However, ordered N‐terminal axial loops, reorganization of the hydrogen bonding network at the entrance pore of ClpP, and reduced structural heterogeneity of the ClpP cylinder are common features of activator‐bound complexes. Activated ClpP mutants were obtained that recapitulate these effects. Based on our data, we propose a unified mechanism for compound‐based ClpP activation.Support or Funding InformationCanadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acyldepsipeptide Analogs Dysregulate Human Mitochondrial ClpP Protease Activity and Cause Apoptotic Cell Death

Acyldepsipeptides (ADEPs) are potential antibiotics that dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease, leading to bacterial cell death. Here, we identified ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP, causing the protease to non-specifically degrade model substrates. Dysregulation of HsClpP activity by ADEP was found to induce cytotoxic effects via activation of the intrinsic, caspase-dependent apoptosis. ADEP-HsClpP co-crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighboring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a therapeutic potential for ADEPs in cancer treatment.

Dysregulation of Human Mitochondrial ClpP Protease Activity by Acyldepsipeptides Analogs Leads to Apoptotic Cell Death

Acyldepsipeptides (ADEPs) are potential novel antibiotics. They dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease leading to bacterial cell death. Here, we identified several ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP causing the protease to non‐specifically degrade model substrates. Dysregulation of HsClpP activity by these ADEPs was found to have cytotoxic effects in multiple human cell lines as a result of the activation of the intrinsic, caspase‐dependent apoptosis. ADEP‐HsClpP co‐crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighbouring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a novel therapeutic potential for ADEPs in cancer treatment.Support or Funding InformationThis work was funded by Natural Sciences and Engineering Research Council of Canada grant (RGPIN 2014‐05393) to WAHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural basis for the in vitro known acyl-depsipeptide 2 (ADEP2) inhibition to Clp 2 protease from Mycobacterium tuberculosis.

Inhibition of Mycobacterium tuberculosis Clp 2 protease has emerged as an attractive therapeutic option for treatment. Acyldepsipeptides (ADEPs) is known as an inhibitor for Clp 2 protease. Therefore, it is of interest to document its affinity, enzyme activity and ADME profiles. We report the predicted binding affinity of all known Clp 2 inhibitors like IDR-10001 and IDR-10011 against Clp2 protease using MolDock algorithm aided molecular docking. The predicted activity (using Molinspiration server) and ADMET properties (AdmetSAR server) were estimated for these compounds. This data suggest ADEP2 having improved binding features with Mtb Clp 2 having acceptable ADMET properties. This is in agreement with known in vitro data for ADEP2 inhibition with Mtb Clp 2 protease.

Characterization of Gain-of-Function Mutant Provides New Insights into ClpP Structure.

ATP-dependent Clp protease (ClpP), a highly conserved serine protease in vast bacteria, could be converted into a noncontrollable enzyme capable of degrading mature proteins in the presence of acyldepsipeptides (ADEPs). Here, we design such a gain-of-function mutant of Staphylococcus aureus ClpP (SaClpP) capable of triggering the same level of dysfunctional activity that occurs upon ADEPs treatment. The SaClpPY63A mutant degrades FtsZ in vivo and inhibits staphylococcal growth. The crystal structure of SaClpPY63A indicates that Asn42 would be an important domino to fall for further activation of ClpP. Indeed, the SaClpPN42AY63A mutant demonstrates promoted self-activated proteolysis, which is a result of an enlarged entrance pore as observed in cryo-electron microscopy images. In addition, the expression of the engineered clpP allele phenocopies treatment with ADEPs; inhibition of cell division occurs as does showing sterilizing with rifampicin antibiotics. Collectively, we show that the gain-of-function SaClpPN42AY63A mutant becomes a fairly nonspecific protease and kills persisters by degrading over 500 proteins, thus providing new insights into the structure of the ClpP protease.

A Conformationally Constrained Cyclic Acyldepsipeptide Is Highly Effective in Mice Infected with Methicillin-Susceptible and -Resistant Staphylococcus aureus.

BackgroundCyclic acyldepsipeptides (ADEPs) are a novel class of antibacterial agents, some of which (e.g., ADEP 4) are highly active against Gram-positive bacteria. The focus of these in vivo studies is ADEP B315, a rationally designed compound that has the most potent in vitro activity of any ADEP analog reported to date.MethodsIn vivo efficacy experiments were performed using lethal intraperitoneal mice infection models with a methicillin-sensitive S. aureus (MSSA) and a methicillin-resistant (MRSA) strain. The infected mice were treated with ADEP B315, a des-methyl analog of ADEP 4, vancomycin, or the vehicle used for the ADEPs and their survival was assessed daily. A subset of MSSA-infected mice was sacrificed soon after inoculation and the bacterial burden was measured in their livers and spleens. The toxicity of ADEP B315 was assessed in viability assays using human whole blood cultures.ResultsIn the MSSA experiments, all mice treated with the vehicle succumbed to the infection within 24 hours. All tested compounds were effective in prolonging survival of infected mice (p<0.001). Mice treated with ADEP B315 had a 39% survival rate by 10 days compared to 7% survival in mice treated with a des-methyl ADEP 4 analog (p = 0.017). Survival of the infected mice treated with ADEP B315 was comparable to those treated with vanocmycin (p = 0.12) at the same dose. Further, bacterial burden in the liver and spleen was significantly lower in mice treated with ADEP B315 compared to controls. In the MRSA experiments, ADEP B315 was able to significantly prolong survival compared to mice treated with either the vehicle (p = 0.001) or vancomycin (p = 0.007). ADEP B315 exhibited no significant toxicity in human whole blood cultures at concentrations up to 25 μg/ml.ConclusionsADEP B315 is safe and can cure mice that have lethal infections of methicillin-sensitive and -resistant strains of S. aureus.

Development and Characterization of Potent Cyclic Acyldepsipeptide Analogues with Increased Antimicrobial Activity.

The problem of antibiotic resistance has prompted the search for new antibiotics with novel mechanisms of action. Analogues of the A54556 cyclic acyldepsipeptides (ADEPs) represent an attractive class of antimicrobial agents that act through dysregulation of caseinolytic protease (ClpP). Previous studies have shown that ADEPs are active against Gram-positive bacteria (e.g., MRSA, VRE, PRSP (penicillin-resistant Streptococcus pneumoniae)); however, there are currently few studies examining Gram-negative bacteria. In this study, the synthesis and biological evaluation of 14 novel ADEPs against a variety of pathogenic Gram-negative and Gram-positive organisms is outlined. Optimization of the macrocyclic core residues and N-acyl side chain culminated in the development of 26, which shows potent activity against the Gram-negative species Neisseria meningitidis and Neisseria gonorrheae and improved activity against the Gram-positive organisms Staphylococcus aureus and Enterococcus faecalis in comparison with known analogues. In addition, the co-crystal structure of an ADEP-ClpP complex derived from N. meningitidis was solved.

AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control.

The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA+ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ClpP allosterically control the ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ClpP activity through cooperative binding. ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ClpX to ClpP and, at the same time, partially inhibit ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.

Protein Folding and Binding Characterized by Mass Spectrometry

This presentation will illustrate how the combination of solution phase labeling with mass spectrometry (MS) can elucidate mechanistic aspects of protein behavior. We will focus on two ongoing projects in our laboratory. (A) The initial (submillisecond) stages of protein folding represent a formidable experimental challenge. We have begun to address this issue by using submillisecond mixing with laser-induced oxidative labeling. Apomyoglobin (aMb) serves as a model system for these measurements. Exposure of the protein to a brief pulse of hydroxyl radical (•OH) at different time points during folding introduces covalent modifications at solvent accessible side chains. The extent of labeling is monitored using MS-based peptide mapping, providing spatially-resolved measurements of changes in solvent accessibility. The technique introduced here is capable of providing in-depth structural information on time scales that have thus far been dominated by low resolution spectroscopic probes. (B) The bacterial protease ClpP is a multi-subunit complex with a central degradation chamber that can be accessed via axial pores. In free ClpP these pores are obstructed. Acyldepsipeptides (ADEPs) are antibacterial compounds that bind ClpP and cause the pores to open up. The ensuing uncontrolled degradation of intracellular proteins is responsible for the antibiotic activity of ADEPs. We use hydrogen/deuterium exchange MS to obtain insights into the ClpP behavior with and without ADEP1. Our data point to a mechanism where the pore opening mechanism is mediated primarily by changes in the packing of N-terminal nonpolar side chains. We propose that a “hydrophobic plug” causes pore blockage in ligand-free ClpP. ADEP1 binding provides new hydrophobic anchor points that nonpolar N-terminal residues can interact with. In this way ADEP1 triggers the transition to an open conformation, where nonpolar moieties are clustered around the rim of the pore.

Structural determinants stabilizing the axial channel of ClpP for substrate translocation

Acyldepsipeptides (ADEPs) antibiotics bind to Escherichia coli ClpP mimicking the interactions that the IGL/F loops in ClpA or ClpX ATPases establish with the hydrophobic pockets surrounding the axial pore of the tetradecamer that the protease forms. ADEP binding induces opening of the gates blocking the axial channel of ClpP and allowing protein substrates to be translocated and hydrolysed in the degradation chamber. To identify the structural determinants stabilizing the open conformation of the axial channel for efficient substrate translocation, we constructed ClpP variants with amino acid substitutions in the N-terminal region that forms the axial gates. We found that adoption of a β-hairpin loop by this region and the integrity of the hydrophobic cluster at the base of this loop are necessary elements for the axial gate to efficiently translocate protein substrates. Analysis of ClpP variants from Bacillus subtilis suggested that the identified structural requirements of the axial channel for efficient translocation are conserved between Gram-positive and Gram-negative bacteria. These findings provide mechanistic insights into the activation of ClpP by ADEPs as well as the gating mechanism of the protease in the context of the ClpAP and ClpXP complexes.

Activation of ClpP Protease by ADEP Antibiotics: Insights from Hydrogen Exchange Mass Spectrometry

The bacterial protease ClpP consists of 14 subunits that assemble into two stacked heptameric rings. The central degradation chamber can be accessed via axial pores. In free ClpP, these pores are obstructed by the N-terminal regions of the seven subunits at either end of the barrel. Acyldepsipeptides (ADEPs) are antibacterial compounds that bind in hydrophobic clefts surrounding the pore region, causing the pores to open up. The ensuing uncontrolled degradation of intracellular proteins is responsible for the antibiotic activity of ADEPs. Recently published X-ray structures yielded conflicting models regarding the conformation adopted by the N-terminal regions in the open state. Here, we use hydrogen/deuterium exchange (HDX) mass spectrometry to obtain complementary insights into the ClpP behavior with and without ADEP1. Ligand binding causes rigidification of the equatorial belt, accompanied by destabilization in the vicinity of the binding clefts. The N-terminal regions undergo rapid deuteration with only minor changes after ADEP1 binding, revealing a lack of stable H-bonding. Our data point to a mechanism where the pore opening mechanism is mediated primarily by changes in the packing of N-terminal nonpolar side chains. We propose that a “hydrophobic plug” causes pore blockage in ligand-free ClpP. ADEP1 binding provides new hydrophobic anchor points that nonpolar N-terminal residues can interact with. In this way, ADEP1 triggers the transition to an open conformation, where nonpolar moieties are clustered around the rim of the pore. This proposed mechanism helps reconcile the conflicting models that had been put forward earlier.

ClpP: A structurally dynamic protease regulated by AAA+ proteins

Proteolysis is an important process for many aspects of bacterial physiology. Clp proteases carry out a large proportion of protein degradation in bacteria. These enzymes assemble in complexes that combine the protease ClpP and the unfoldase, ClpA or ClpX. ClpP oligomerizes as two stacked heptameric rings enclosing a central chamber containing the proteolytic sites. ClpX and ClpA assemble into hexameric rings that bind both axial surfaces of the ClpP tetradecamer forming a barrel-like complex. ClpP requires association with ClpA or ClpX to unfold and thread protein substrates through the axial pore into the inner chamber where degradation occurs. A gating mechanism regulated by the ATPase exists at the entry of the ClpP axial pore and involves the N-terminal regions of the ClpP protomers. These gating motifs are located at the axial regions of the tetradecamer but in most crystal structures they are not visible. We also lack structural information about the ClpAP or ClpXP complexes. Therefore, the structural details of how the axial gate in ClpP is regulated by the ATPases are unknown. Here, we review our current understanding of the conformational changes that ClpA or ClpX induce in ClpP to open the axial gate and increase substrate accessibility into the degradation chamber. Most of this knowledge comes from the recent crystal structures of ClpP in complex with acyldepsipeptides (ADEP) antibiotics. These small molecules are providing new insights into the gating mechanism of this protease because they imitate the interaction of ClpA/ClpX with ClpP and activate its protease activity.