A unique peptide deformylase platform to rationally design and challenge novel active compounds.

Peptide deformylase (PDF) is considered an excellent target to develop antibiotics. We have performed an extensive characterization of a new PDF from the pathogen Streptococcus agalactiae, showing properties similar to other known PDFs. S. agalactiae PDF could be used as PDF prototype as it allowed to get complete sets of 3-dimensional, biophysical and kinetic data with virtually any inhibitor compound. Structure-activity relationship analysis with this single reference system allowed us to reveal distinct binding modes for different PDF inhibitors and the key role of a hydrogen bond in potentiating the interaction between ligand and target. We propose this protein as an irreplaceable tool, allowing easy and relevant fine comparisons between series, to design, challenge and validate novel series of inhibitors. As proof-of-concept, we report here the design and synthesis of effective specific bacterial PDF inhibitors of an oxadiazole series with potent antimicrobial activity against a multidrug resistant clinical isolate.

New peptides with metal binding abilities and their use as drug carriers.

Many new designed molecules that target efficiently in vitro bacterial metalloproteases were completely inactive in cellulo against Gram negative bacteria. Their activities were limited by the severe restriction of the penetration/diffusion rate through the outer membrane barrier. To bypass this limitation, we have assayed the strategy of metallodrugs, to improve the delivery of hydroxamic acid inhibitors to peptide deformylase. In this metal-chaperone, to facilitate bacterial uptake, the ancillary ligand tris(2-pyridylmethyl)amine (TPA) or di(picolyl)amine (DPA) was functionalized by a tetrapeptide analogue of antimicrobial peptide, RWRW(OBn) (AA08 with TPA) and/or an efflux pump modulator PAßN (AA09 with TPA and AA27 with DPA). We prepared Co(III), Zn(II), and Cu(II) metallodrugs. Using a fluorescent hydroxamic acid, we showed that, in contrast to Cu(II) metallodrugs, Co(III) metallodrugs were stable in the Mueller Hinton (MH) broth during the time required for bacterial assays. The antibacterial activities were determined against E. coli strain wild-type (AG100) and E. coli strain deleted from acrAB efflux pump (AG100A). While none of the PDFinhs used in this study (SMP289 with an indole scaffold, AT015 and AT019 built on a 1,2,4-oxadiazole scaffold) displayed activity higher than 128 µM, all the metallodrugs were active with MICs around 8 µM both against AG100 and AG100A. However, compared to the activities of equimolar combinations of PDFinhs and the free chelating peptides (AA08, AA09, or AA27), they showed similar activities. A synergistic association between AT019 and AA08 or AA09 was determined using the fractional inhibitory concentration with AG100 and AG100A. Combinations of peptides lacking the chelating group with PDFinhs were inefficient. LC-MS analyses showed that the chelating peptides bind Zn(II) cation when incubated in MH broth. These results support the in situ formation of a zinc metallodrug, but we failed to detect it by LC-MS in MH. Nevertheless, this chelating peptides metalated with zinc act as permeabilizers which are more efficient than PAßN to facilitate the uptake of PDFinhs by Gram(-) bacteria.

New Peptide-based antimicrobials for tackling drug resistance in bacteria: single-cell fluorescence imaging.

New peptide molecules with metal binding abilities proved to be active against multidrug resistant clinical isolates. One of them labeled with a dansylated lysine has been imaged inside single-multidrug resistant bacteria cells by deep ultraviolet fluorescence, showing a heterogeneous subcellular localization. The fluorescence intensity is clearly related to the accumulation of the drug inside the bacteria, being dependent both on its concentration and on the incubation time with cells.

Characterization of cobalt(III) hydroxamic acid complexes based on a tris(2-pyridylmethyl)amine scaffold: reactivity toward cysteine methyl ester.

Six Co(III) complexes based on unsubstituted or substituted TPA ligands (where TPA is tris(2-pyridylmethyl)amine) and acetohydroxamic acid (A), N-methyl-acetohydroxamic acid (B), or N-hydroxy-pyridinone (C) were prepared and characterized by mass spectrometry, elemental analysis, and electrochemistry: [Co(III)(TPA)(A-2H)](Cl) (1a), [Co(III)((4-Cl(2))TPA)(A-2H)](Cl) (2a), [Co(III)((6-Piva)TPA)(A-2H)](Cl) (3a), [Co(III)((4-Piva)TPA)(A-2H)](Cl) (4a) and [Co(III)(TPA)(B-H)](Cl)(2) (1b), and [Co(III)(TPA)(C-H)](Cl)(2) (1c). Complexes 1a-c and 3a were analyzed by (1)H NMR, using 2D ((1)H, (1)H) COSY and 2D ((1)H, (13)C) HMBC and HSQC, and shown to exist as a mixture of two geometric isomers based on whether the hydroxamic oxygen was trans to a pyridine nitrogen or to the tertiary amine nitrogen. Complex 3a exists as a single isomer that was crystallized. Its crystal structure revealed the presence of an H-bond between the pivaloylamide and the hydroximate oxygen. Complexes 1a, 2a, and 4a are irreversibly reduced beyond -900 mV versus SCE, while complexes 1b and 1c are reduced at less negative values of -330 and -190 mV, respectively. The H-bond in 3a increased the redox potential up to -720 mV. Reaction of complex 1a with L-cysteine methyl ester CysOMe was monitored by (1)H NMR and UV-vis at 2 mM and 0.2 mM in an aqueous buffered solution at pH 7.5. Complex 1a was successively converted into an intermediate [Co(III)(TPA)(CysOMe-H)](2+), 1d, by exchange of the hydroximate with the cysteinate ligand, and further into Co(III)(CysOMe-H)(3), 5. An authentic sample of 1d was prepared and thoroughly characterized. A detailed (1)H NMR analysis showed there was only one isomer, in which the thiolate was trans to the tertiary amine nitrogen.

New peptide deformylase inhibitors and cooperative interaction: a combination to improve antibacterial activity.

OBJECTIVES: Bacterial drug resistance is a worrying public health problem and there is an urgent need for research and development to provide new antibacterial molecules. Peptide deformylase (PDF) is now a well-described intracellular target selected for the design of a new antibiotic group, PDF inhibitors (PDFIs). The initial bacterial susceptibility to an inhibitor of a cytoplasmic target is directly associated with the diffusion of the compound through the membrane barrier of Gram-negative bacteria and with its cytosolic accumulation at the required concentration. METHODS: We have recently demonstrated that the activity of different PDFIs is strongly dependent on the accumulation of the active molecules by using permeabilizing agents, efflux inhibitors or efflux-mutated strains. In this work we assessed various combination protocols using different putative inhibitors (PDFIs, methionine aminopeptidase inhibitors etc.) to improve antibacterial activity against various resistant Gram-negative bacteria. RESULTS: The maximum effect was observed when combining actinonin with a dual inhibitor of methionine aminopeptidase and PDF, this molecule being also able to interact with the target while actinonin is bound to the PDF active site. CONCLUSIONS: Such a combination of inhibitors acting on two tightly associated metabolic steps results in a cooperative effect on bacterial cells and opens an original way to combat multidrug-resistant bacteria.