Phenyl Esters Are Potent Inhibitors of Caseinolytic Protease P and Reveal a Stereogenic Switch for Deoligomerization

Abstract

Caseinolytic protease P (ClpP) represents a central bacterial degradation machinery that is involved in cell homeostasis and pathogenicity. The functional role of ClpP has been studied by genetic knockouts and through the use of beta-lactones, which remain the only specific inhibitors of ClpP discovered to date. Beta-lactones have served as chemical tools to manipulate ClpP in several organisms, however, their potency, selectivity and stability is limited. Despite detailed structural insights into the composition and conformational flexibility of the ClpP active site, no rational efforts to design specific non-beta-lactone inhibitors have been reported to date. In this work, an unbiased screen of more than 137,000 compounds was used to identify five phenyl ester compounds as highly potent ClpP inhibitors that were selective for bacterial, but not human ClpP.The potency of phenyl esters largely exceeded that of beta-lactones in ClpP peptidase and protease inhibition assays and displayed unique target selectivity in living S. aureus cells.Analytical studies revealed that while phenyl esters are cleaved like native peptide substrates, they remain covalently trapped as acyl-enzyme intermediates in the active site. The synthesis of 36 derivatives and subsequent structure-activity relationship (SAR) studies provided insights into conserved structural elements that are important for inhibition potency and acylation reactivity.Moreover, the stereochemistry of a methyl-substituent at the alpha position to the ester, resembling amino acid side chains in peptide substrates, impacted ClpP complex stability, causing either dissociation into heptamers or retention of the tetradecameric state. Mechanistic insights into this intriguing stereo switch and the phenyl ester binding mode were obtained by molecular docking experiments.

Insights into ClpXP proteolysis: heterooligomerization and partial deactivation enhance chaperone affinity and substrate turnover in Listeria monocytogenes

“…Select lactones, including E2 and phenyl esters such as AV170 , are known to destabilize tetradecameric ClpP and to induce its dissociation into inactive heptamers. 10,11 In fact, deoligomerization of LmClpP2 could be observed upon E2 binding, while the tetradecameric complex was retained with both the CMKs and D3 (Fig. S4†).…”

“…These studies suggested that steric clash of the inhibitor within the active site triad triggers a rearrangement at the heptamer interface, causing dissociation of the ClpP tetradecamer into two heptameric rings. 11 Recently, the first reversible ClpP inhibitors were reported, which distort the active site catalytic triad through structural rearrangements. 12 However, this inactive state of the ClpP peptidase could be reversed through formation of the ClpXP complex, highlighting the power of conformational control within this dynamic system.…”

PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum

“…Finally, the roles that bacterial Clp proteins play in cell division, stress response and pathogenicity (Frees et al, 2014), have placed them at the center of several drug discovery programs (Böttcher and Sieber, 2008; Brötz-Oesterhelt and Sass, 2014; Conlon et al, 2013; Gersch et al, 2015; Hackl et al, 2015). Our data demonstrate that targeting the P. falciparum plastid-localized Clp proteins is a viable strategy for antimalarial drug development and future work will allow us to repurpose highly active antibacterial compounds as effective anti-malarial agents.…”

Bacterial proteases, untapped antimicrobial drug targets

“…More recently, Sieber and colleagues 49 has discovered a new class of potent ClpP inhibitors, the phenyl esters (AV170; Figure 3a). Discovered using an unbiased screen of 137 000 synthetic compounds in a fluorogenic assay for ClpP activity, phenyl esters act by the same covalent modification of Ser98 as β-lactones.…”

“…Efforts to increase potency revealed a trade-off between stability and reactivity. 49 Although ClpP inhibition shows promise as a mechanism of action, further development and discovery of novel scaffolds is clearly required. Moreover, given recent evidence of drug resistance in certain clpP knockout strains, 34,36 some caution down this path may be warranted.…”