Targeted protein degradation in mycobacteria uncovers antibacterial effects and potentiates antibiotic efficacy.

Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit targeted protein degradation (TPD) for antibiotic development remains nascent due to our limited understanding of which bacterial proteins are amenable to a TPD strategy. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in Mycobacterium smegmatis (Msm), a model for the human pathogen M. tuberculosis (Mtb). By integrating experimental screening of 72 protein candidates and machine learning, we find that drug-induced proximity to the bacterial ClpC1P1P2 proteolytic complex leads to the degradation of many endogenous proteins, especially those with disordered termini. Additionally, TPD of essential Msm proteins inhibits bacterial growth and potentiates the effects of existing antimicrobial compounds. Together, our results provide biological principles to select and evaluate attractive targets for future Mtb PROTAC development, as both standalone antibiotics and potentiators of existing antibiotic efficacy.

ClpX Is Essential and Activated by Single-Strand DNA Binding Protein in Mycobacteria.

The ClpP1P2 proteolytic complex is essential in Mycobacterium tuberculosis Proteolysis by ClpP1P2 requires an associated ATPase, either ClpX or ClpC1. Here, we sought to define the unique contributions of the ClpX ATPase to mycobacterial growth. We formally demonstrated that ClpX is essential for mycobacterial growth, and to understand its essential functions, we identified ClpX-His-interacting proteins by pulldown and tandem mass spectrometry. We found an unexpected association between ClpX and proteins involved in DNA replication, and we confirm a physical association between ClpX and the essential DNA maintenance protein single-stranded-DNA binding protein (SSB). Purified SSB is not degraded by ClpXP1P2; instead, SSB enhances ATP hydrolysis by ClpX and degradation of the model substrate GFP-SsrA by ClpXP1P2. This activation of ClpX is mediated by the C-terminal tail of SSB, which had been implicated in the activation of other ATPases associated with DNA replication. Consistent with the predicted interactions, depletion of clpX transcript perturbs DNA replication. These data reveal that ClpX participates in DNA replication and identify the first activator of ClpX in mycobacteria.IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, imposes a major global health burden, surpassing HIV and malaria in annual deaths. The ClpP1P2 proteolytic complex and its cofactor ClpX are attractive drug targets, but their precise cellular functions are unclear. This work confirms ClpX's essentiality and describes a novel interaction between ClpX and SSB, a component of the DNA replication machinery. Further, we demonstrate that a loss of ClpX is sufficient to interrupt DNA replication, suggesting that the ClpX-SSB complex may play a role in DNA replication in mycobacteria.

Pyrazinamide triggers degradation of its target aspartate decarboxylase.

Pyrazinamide is a sterilizing first-line tuberculosis drug. Genetic, metabolomic and biophysical analyses previously demonstrated that pyrazinoic acid, the bioactive form of the prodrug pyrazinamide (PZA), interrupts biosynthesis of coenzyme A in Mycobacterium tuberculosis by binding to aspartate decarboxylase PanD. While most drugs act by inhibiting protein function upon target binding, we find here that pyrazinoic acid is only a weak enzyme inhibitor. We show that binding of pyrazinoic acid to PanD triggers degradation of the protein by the caseinolytic protease ClpC1-ClpP. Thus, the old tuberculosis drug pyrazinamide exerts antibacterial activity by acting as a target degrader, a mechanism of action that has recently emerged as a successful strategy in drug discovery across disease indications. Our findings provide the basis for the rational discovery of next generation PZA.

Development of high throughput screening methods for inhibitors of ClpC1P1P2 from Mycobacteria tuberculosis.

Tuberculosis affects about 100 million people worldwide and causes nearly 2 million deaths annually. It has been estimated that one third of all humans is infected with latent Mycobacterium tuberculosis (Mtb). Moreover, Mtb has become increasingly resistant to available antibiotics. Consequently, it is important to identify and characterize new therapeutic targets in Mtb and to synthesize selective inhibitors. ClpP1, ClpP2 and their associated regulatory ATPases, ClpX and ClpC1 are required for the growth of Mtb and for its virulence during murine infection and are highly attractive drug targets, especially since they are not present in the cytosol of mammalian cells, and they differ markedly from the mitochondrial ClpP complex. The importance of these proteins in Mtb is emphasized by the existence of several natural antibiotics targeting this system. In order to find new inhibitors of ClpC1P1P2 system, we developed an assay based on the ATP-dependent degradation of a fluorescent protein substrate. The hits obtained were further characterized with a set of secondary assays to identify precise targets within a complex. A large library of compounds was screened and led to the identification of a ClpC1 ATPase inhibitor demonstrating that this approach can be used in future searches for anti-TB agents.

An Antibacterial ß-Lactone Kills Mycobacterium tuberculosis by Disrupting Mycolic Acid Biosynthesis.

The spread of antibiotic resistance is a major challenge for the treatment of Mycobacterium tuberculosis infections. In addition, the efficacy of drugs is often limited by the restricted permeability of the mycomembrane. Frontline antibiotics inhibit mycomembrane biosynthesis, leading to rapid cell death. Inspired by this mechanism, we exploited ß-lactones as putative mycolic acid mimics to block serine hydrolases involved in their biosynthesis. Among a collection of ß-lactones, we found one hit with potent anti-mycobacterial and bactericidal activity. Chemical proteomics using an alkynylated probe identified Pks13 and Ag85 serine hydrolases as major targets. Validation through enzyme assays and customized 13 C metabolite profiling showed that both targets are functionally impaired by the ß-lactone. Co-administration with front-line antibiotics enhanced the potency against M. tuberculosis by more than 100-fold, thus demonstrating the therapeutic potential of targeting mycomembrane biosynthesis serine hydrolases.

Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis.

The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, inMycobacterium tuberculosis(Mtb) are essential and, therefore, promising drug targets. TheMtbClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3-4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity ofMtbClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2-3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel ß-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates.

Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease.

The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance and activation mechanism. Whilst most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram-positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl-leucyl-leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP-activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP-dependent protein degradation.

Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity.

The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.

The cyclic peptide ecumicin targeting ClpC1 is active against Mycobacterium tuberculosis in vivo.

Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development.

Lassomycin, a ribosomally synthesized cyclic peptide, kills mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2.

Languishing antibiotic discovery and flourishing antibiotic resistance have prompted the development of alternative untapped sources for antibiotic discovery, including previously uncultured bacteria. Here, we screen extracts from uncultured species against Mycobacterium tuberculosis and identify lassomycin, an antibiotic that exhibits potent bactericidal activity against both growing and dormant mycobacteria, including drug-resistant forms of M. tuberculosis, but little activity against other bacteria or mammalian cells. Lassomycin is a highly basic, ribosomally encoded cyclic peptide with an unusual structural fold that only partially resembles that of other lasso peptides. We show that lassomycin binds to a highly acidic region of the ClpC1 ATPase complex and markedly stimulates its ATPase activity without stimulating ClpP1P2-catalyzed protein breakdown, which is essential for viability of mycobacteria. This mechanism, uncoupling ATPase from proteolytic activity, accounts for the bactericidal activity of lassomycin.

Mycobacterium tuberculosis ClpP1 and ClpP2 function together in protein degradation and are required for viability in vitro and during infection.

In most bacteria, Clp protease is a conserved, non-essential serine protease that regulates the response to various stresses. Mycobacteria, including Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis, unlike most well studied prokaryotes, encode two ClpP homologs, ClpP1 and ClpP2, in a single operon. Here we demonstrate that the two proteins form a mixed complex (ClpP1P2) in mycobacteria. Using two different approaches, promoter replacement, and a novel system of inducible protein degradation, leading to inducible expression of clpP1 and clpP2, we demonstrate that both genes are essential for growth and that a marked depletion of either one results in rapid bacterial death. ClpP1P2 protease appears important in degrading missense and prematurely terminated peptides, as partial depletion of ClpP2 reduced growth specifically in the presence of antibiotics that increase errors in translation. We further show that the ClpP1P2 protease is required for the degradation of proteins tagged with the SsrA motif, a tag co-translationally added to incomplete protein products. Using active site mutants of ClpP1 and ClpP2, we show that the activity of each subunit is required for proteolysis, for normal growth of Mtb in vitro and during infection of mice. These observations suggest that the Clp protease plays an unusual and essential role in Mtb and may serve as an ideal target for antimycobacterial therapy.

The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring.

Mycobacterium tuberculosis (Mtb) contains two clpP genes, both of which are essential for viability. We expressed and purified Mtb ClpP1 and ClpP2 separately. Although each formed a tetradecameric structure and was processed, they lacked proteolytic activity. We could, however, reconstitute an active, mixed ClpP1P2 complex after identifying N-blocked dipeptides that stimulate dramatically (>1000-fold) ClpP1P2 activity against certain peptides and proteins. These activators function cooperatively to induce the dissociation of ClpP1 and ClpP2 tetradecamers into heptameric rings, which then re-associate to form the active ClpP1P2 2-ring mixed complex. No analogous small molecule-induced enzyme activation mechanism involving dissociation and re-association of multimeric rings has been described. ClpP1P2 possesses chymotrypsin and caspase-like activities, and ClpP1 and ClpP2 differ in cleavage preferences. The regulatory ATPase ClpC1 was purified and shown to increase hydrolysis of proteins by ClpP1P2, but not peptides. ClpC1 did not activate ClpP1 or ClpP2 homotetradecamers and stimulated ClpP1P2 only when both ATP and a dipeptide activator were present. ClpP1P2 activity, its unusual activation mechanism and ClpC1 ATPase represent attractive drug targets to combat tuberculosis.

Yeast adapt to near-freezing temperatures by STRE/Msn2,4-dependent induction of trehalose synthesis and certain molecular chaperones.

Virtually nothing is known about the biochemical adaptations in eukaryotic cells that may enhance survival at low temperatures or upon freezing. Here we demonstrate an adaptive response in yeast that is activated below 10 degrees C and increases tolerance to low temperatures and freezing. This response involves a dramatic accumulation of the chemical chaperone trehalose and induction of trehalose-synthesizing enzymes (Tps1, Tps2) and certain heat shock proteins (Hsp104, Hsp42, Hsp12, Ssa4). mRNAs for these proteins increase dramatically below 10 degrees C and even at 0 degrees C. Their expression requires Msn2,4 transcription factors but also involves marked mRNA stabilization. Upon return to 30 degrees C, TPS1, TPS2, and HSP104 mRNAs, trehalose levels and tolerance to freezing fall dramatically within minutes. Mutants lacking trehalose or Msn2,4 die more rapidly at 0 degrees C and upon freezing. Thus, below 10 degrees C, yeast show an adaptive response that sustains viability at low or freezing temperatures, which are commonly encountered in natural environments and laboratory refrigerators.

Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures.

Trehalose accumulates dramatically in microorganisms during heat shock and osmotic stress and helps protect cells against thermal injury and oxygen radicals. Here we demonstrate an important role of this sugar in cold-adaptation of bacteria. A mutant Escherichia coli strain unable to produce trehalose died much faster than the wild type at 4 degrees C. Transformation of the mutant with the otsA/otsB genes, responsible for trehalose synthesis, restored trehalose content and cell viability at 4 degrees C. After temperature downshift from 37 degrees C to 16 degrees C ("cold shock"), trehalose levels in wild-type cells increased up to 8-fold. Although this accumulation of trehalose did not influence growth at 16 degrees C, it enhanced cell viability when the temperature fell further to 4 degrees C. Before the trehalose build-up, levels of mRNA encoding OtsA/OtsB increased markedly. This induction required the sigma factor, RpoS, but was independent of the major cold-shock protein, CspA. otsA/B mRNA was much more stable at 16 degrees C than at 37 degrees C and contained a "downstream box," characteristic of cold-inducible mRNAs. Thus, otsA/otsB induction and trehalose synthesis are activated during cold shock (as well as during heat shock) and play an important role in resistance of E. coli (and probably other organisms) to low temperatures.