ClpP Protease, a Promising Antimicrobial Target

Abstract

The caseinolytic protease proteolytic subunit (ClpP) is a serine protease playing an important role in proteostasis of eukaryotic organelles and prokaryotic cells. Alteration of ClpP function has been proved to affect the virulence and infectivity of a number of pathogens. Increased bacterial resistance to antibiotics has become a global problem and new classes of antibiotics with novel mechanisms of action are needed. In this regard, ClpP has emerged as an attractive and potentially viable option to tackle pathogen fitness without suffering cross-resistance to established antibiotic classes and, when not an essential target, without causing an evolutionary selection pressure. This opens a greater window of opportunity for the host immune system to clear the infection by itself or by co-administration with commonly prescribed antibiotics. A comprehensive overview of the function, regulation and structure of ClpP across the different organisms is given. Discussion about mechanism of action of this protease in bacterial pathogenesis and human diseases are outlined, focusing on the compounds developed in order to target the activation or inhibition of ClpP.

Keywords: ClpP; antibiotics; antivirulence.

Figure 1

Cycle of ATPases associated with diverse cellular activities (AAA+) caseinolytic protease proteolytic subunit (ClpP) complex. (A) Substrate selective binding by the AAA+ unfoldase. (B) Sequence of ATP-driven stretching, leading to a strained structure or alternatively to a substrate release. (C) Successful unfolding leading to translocations and degradation. (D) Initiation of a new cycle.

Figure 2

Structure of the full E. coli ClpP tetradecamer (top: side view, bottom left: top view) and the ClpP monomer (bottom right). Every monomer is shown in a different color in cartoon representation. The monomer is shown to emphasize the active site and the catalytic triad (S172, H122, D171) is shown in grey stick representation. (PDB ID: 1YG6).

Figure 3

Top and side view of E. coli caseinolytic protease subunit A (ClpA) (top) and E. coli ClpX (bottom) hexamers. The ClpA hexamer is shown in grey and wheat surface representation. The structure was obtained through homology modeling, using the B. subtilis caseinolytic protease subunit C (ClpC) monomer as template (PDB ID: 3PXI) in SWISS-MODEL [42]. The subunits in the ClpC hexamer (PDB ID: 3PXG) where then replaced with the ClpA monomers, and the structure was then relaxed through energy minimization. The caseinolytic protease subunit X (ClpX) hexamer is shown in cyan and light cyan surface representation. Missing loops were modeled using Modeller 9.17 [43].

Figure 4

Top view of E. coli ClpP in unbound state (left) (PDB ID: 1YG6) and bound with acyldepsipeptide (ADEP)1 (right) (PDB ID: 3MT6). When bound, an increase in pore size can be observed which defines an active, non-selective conformation. The protein is shown in grey surface representation, with the ADEP1 binding pockets shown in pink. ADEP1 molecules are represented by white spheres.

Figure 5

Chemical structures of ClpP activators. On the left, chemical structures of the mentioned ADEPs are shown. In the color matching their name, consecutive modifications respect to the previous ADEP are highlighted: the original ADEP1 and EnopeptinA (black), ADEP4 (blue), ADEP10c (orange), ADEP1g (green) and ADEP26 (pink). On the right side, alternative cores extracted from screening including ACP1b, Sclerotiamide and D9.

Figure 6

Chemical structures of compounds targeting ATP-unfoldases.

Figure 7

Chemical structure of reported ClpP inhibitors.

Figure 8

(A) Mechanism of action of RKS07 [102], a β-sultam undergoing sulfonylation and subsequent elimination to transform the catalytic serine into an inactive dehydroalanine. (B) Inhibitory mechanism of a diphenyl phosphonate inhibitor with a serine protease. (I) The unreacted inhibitor enters the active site, with the R¹ moiety filling the S1 (recognition) pocket while the phosphonate stays at a reachable distance from the catalytic serine and the oxyanion hole. (II) Nucleophilic attack of the serine to the phosphonate induced by the interaction with the oxyanion hole residues, forming the pentacoordinate transition state. (III) Release of the phenolate group due to the recovery of tetrahedral geometry. (IV) Final configuration leading to irreversible inhibition of the serine protease.