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

Abstract

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...

An allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR

“…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)(35)(36). More recently, Cediranib, an anticancer drug, was proposed as a novel noncovalent inhibitor of MtClpP1P2 (37).…”

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

“…Interestingly, this conformational change could be reversed and activity partially restored by ADEP binding. More recently Cediranib, an anti-cancer drug, was shown to bind to a hydrophobic pocket at the interface of the head and handle domains of MtClpP1, leading to inhibition of the MtClpP1P2 complex(37).Our study provides an important step towards the goal of manipulating MtClpP1P2 function by providing the structural basis for why this complex is catalytically dead in the absence of activator peptides and why, unlike for many other ClpPs, binding of ADEPs do not activate the protein.The 3.0 Å resolution structure from cryo-EM of the apo-form of MtClpP1P2 shows that the complex is in the compact conformation. The hallmarks of the compact form are truncated handle helices and disordered handle β-strands that lead to an impaired catalytic triad, thereby explaining the lack of activity observed for apo-state of the complex.…”

Biological Calorimetry: Old Friend, New Insights

“…Caseinolytic Protease (ClpP) Caseinolytic protease (ClpP) is a tetradecameric protein considered a bacterial target for drug design (Figure 2). At pH 7.6 (phosphate buffer), ClpP shows a moderate stability (unfolding temperature of 62 • C, which was further increased to 83 • C in the presence of 100 µM bortezomib, a boronic proteasome inhibitor) [24]. It has been reported that ClpP can be activated by inhibitors binding in the active site, a finding difficult to explain.…”