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.

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 PAβN (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 PAβN to facilitate the uptake of PDFinhs by Gram(-) bacteria.