A Simple Fragment of Cyclic Acyldepsipeptides Is Necessary and Sufficient for ClpP Activation and Antibacterial Activity

Abstract

The discovery of new classes of antibacterial agents, particularly those with unique biological targets, is essential to keep pace with emerging drug resistance in pathogenic bacteria. We identified the minimal structural component of the cyclic acyldepsipeptide (ADEP) antibiotics that exhibits antibacterial activity. We found that N-acyldifluorophenylalanine fragments function via the same mechanism of action as ADEPs, as evidenced by the requirement of ClpP for the fragments’ antibacterial activity, the ability of fragments to activate B. subtilis ClpP in vitro, and the capacity of an N-acyldifluorophenylalanine affinity matrix to capture ClpP from B. subtilis cell lysates. N-acyldifluorophenylalanine fragments are much simpler in structure than the full ADEPs and are also highly amenable to structural diversification. Thus, the stage has been set for the development of non-peptide activators of ClpP that can be used as antibacterial agents.

Bacterial proteases, untapped antimicrobial drug targets

“…39,62 Numerous other synthetic chemistry efforts have explored motifs to overcome these limitations, but most have resulted in diminished activity (Figure 4c). 54,[63][64][65][66][67] Given the intimate binding of ADEP in its hydrophobic pocket, this result is perhaps predictable and suggests that modification on the ADEP scaffold may have reached an impasse.…”

Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery

“…Z-Leu-Leu-Nva-CHO was purchased from Boston Biochem. ADEPs and N-E-2-heptenoyldifluorophenylalanine methyl ester were synthesized as described (30,31,33,37 (12) was followed by increases in fluorescence (excitation, 320 nm; emission, 420 nm). GFP-ssrA degradation was assayed by decreases in fluorescence (excitation, 380 nm; emission, 511 nm) in the presence of 2.5 mM ATP (Sigma) and a regeneration system consisting of 16 mM creatine phosphate (MP Biomedicals) and 0.32 mg/mL creatine phosphokinase (Sigma).…”

“…S3B) but were eliminated by conformational rearrangements in the compressed and inactive ClpP1P1 structure, suggesting that an ADEP side chain might bind to an active ClpP1 ring. Thus, we tested ClpP1P2 activation by an ADEP-2B fragment (N-E-2-heptenoyldifluorophenylalanine methyl ester) in which the side chain was appended to a small methoxy group rather than the bulky macrocycle; this fragment activates Bacillus subtilis ClpP and has antibacterial activity (33). In experiments performed in the presence of Z-Ile-Leu agonist, the fragment activated peptide cleavage by ClpP1P2, albeit more weakly and to a lower maximal level than ADEP-2B (Fig.…”

Sclerotiamide: The First Non-Peptide-Based Natural Product Activator of Bacterial Caseinolytic Protease P

“…Much of this limitation arises from lack of activity against Gram-negative pathogens and the susceptibility of ADEPs to active efflux. 27 Structural alteration of ADEPs to address these detriments has been relatively unsuccessful thus far, in part because the N -acylated phenylalanine pharmacophore 38 is the recognized motif for efflux pumps. 39 Efforts to diversify the arsenal of ClpP-activating molecules through screening or structure-based approaches have been extremely limited.…”