Isabelle Artaud

Structure-activity relationship analysis of the peptide deformylase inhibitor 5-bromo-1H-indole-3-acetohydroxamic acid.

SAR by NMR: A series of indole compounds derived from 5‐bromo‐1H‐indole‐3‐acetohydroxamic acid were synthesized. Their inhibitory activities were evaluated against purified peptide deformylases (PDFs), and their antibacterial activities against B. subtilis, E. coli (wild type and tolC), and a variety of pathogens were also determined. The potency of the best inhibitors was related to the NMR footprints of the respective acids with 15N‐labeled E. coli Ni‐PDF.

A persulfide analogue of the nitrosothiol SNAP: formation, characterization and reactivity.

The proposal of the post‐translational modification “S‐sulfhydration” as a major pathway for H2S‐induced signaling has recently shed light on persulfides. However, the study of these species is hampered by their instability under biologically relevant conditions; this requires generating them in situ immediately prior to use. The current methods to prepare persulfides in aqueous solution suffer from several drawbacks. In particular, their formation requires (or generates) hydrogen sulfide, thus resulting in difficulties in distinguishing polysulfide reactivity from that of H2S. Here we report the H2S‐free formation, characterization, and some biologically relevant reactions of a water‐soluble persulfide analogue of the nitrosothiol SNAP, a widely used nitric oxide donor.

New Biologically Active Hydrogen Sulfide Donors

Generous donors: The dithioperoxyanhydrides (CH3 COS)2 , (PhCOS)2 , CH3 COSSCO2 Me and PhCOSSCO2 Me act as thiol-activated hydrogen sulfide donors in aqueous buffer solution. The most efficient donor (CH3 COS)2 can induce a biological response in cells, and advantageously replace hydrogen sulfide in ex vivo vascular studies.

New antibiotic molecules: bypassing the membrane barrier of gram negative bacteria increases the activity of peptide deformylase inhibitors.

Background Multi-drug resistant (MDR) bacteria have become a major concern in hospitals worldwide and urgently require the development of new antibacterial molecules. Peptide deformylase is an intracellular target now well-recognized for the design of new antibiotics. The bacterial susceptibility to such a cytoplasmic target primarily depends on the capacity of the compound to reach and accumulate in the cytosol. Methodology/Principal Findings To determine the respective involvement of penetration (influx) and pumping out (efflux) mechanisms to peptide deformylase inhibitors (PDF-I) activity, the potency of various series was determined using various genetic contexts (efflux overproducers or efflux-deleted strains) and membrane permeabilizers. Depending on the structure of the tested molecules, two behaviors could be observed: (i) for actinonin the first PDF-I characterized, the AcrAB efflux system was the main parameter involved in the bacterial susceptibility, and (ii), for the lastest PDF-Is such as the derivatives of 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide, the penetration through the membrane was a important limiting step. Conclusions/Significance Our results clearly show that the bacterial membrane plays a key role in modulating the antibacterial activity of PDF-Is. The bacterial susceptibility for these new antibacterial molecules can be improved by two unrelated ways in MDR strains: by collapsing the Acr efflux activity or by increasing the uptake rate through the bacterial membrane. The efficiency of the second method is associated with the nature of the compound.

Hydroxamic Acids as Potent Inhibitors of Fe(II) and Mn(II) E. Coli Methionine Aminopeptidase: Biological Activities and X-Ray Structures of Oxazole Hydroxamate-Ecmetap-Mn Complexes.

New series of acids and hydroxamic acids linked to five‐membered heterocycles including furan, oxazole, 1,2,4‐ or 1,3,4‐oxadiazole, and imidazole were synthesized and tested as inhibitors against the FeII, CoII, and MnII forms of E. coli methionine aminopeptidase (MetAP) and as antibacterial agents against wild‐type and acrAB E. coli strains. 2‐Aryloxazol‐4‐ylcarboxylic acids appeared as potent and selective inhibitors of the CoII MetAP form, with IC50 values in the micromolar range, whereas 5‐aryloxazol‐2‐ylcarboxylic acid regioisomers and 5‐aryl‐1,2,4‐oxadiazol‐3‐ylcarboxylic acids were shown to be inefficient against all forms of EcMetAP. Regardless of the heterocycle, all the hydroxamic acids are highly potent inhibitors and are selective for the MnII and FeII forms, with IC50 values between 1 and 2 μM. One indole hydroxamic acid that we previously reported as a potent inhibitor of E. coli peptide deformylase also demonstrated efficiency against EcMetAP. To gain insight into the positioning of the oxazole heterocycle with reversed substitutions at positions 2 and 5, X‐ray crystal structures of EcMetAP‐Mn complexed with two such oxazole hydroxamic acids were solved. Irrespective of the [metal]/[apo‐MetAP] ratio, the active site consistently contains a dinuclear manganese center, with the hydroxamate as bridging ligand. Asp 97, which adopts a bidentate binding mode to the Mn2 site in the holoenzyme, is twisted in both structures toward the hydroxamate bridging ligand to favor the formation of a strong hydrogen bond. Most of the compounds show weak antibacterial activity against a wild‐type E. coli strain. However, increased antibacterial activity was observed mainly for compounds with a 2‐substituted phenyl group in the presence of the nonapeptide polymyxin B and phenylalanine–arginine–β‐naphthylamide as permeabilizer and efflux pump blocker, respectively, which boost the intracellular uptake of the inhibitors.

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.

Synthesis, Characterization, and Reactivity of Alkyldisulfanido Zinc Complexes

The alkyldisulfanido zinc complexes Tp(iPr,iPr)Zn(SSR) and Tp(Ph,Me)Zn(SSR) where Tp(iPr,iPr) is hydridotris-((3,5-isopropyl)pyrazolyl)borate, Tp(Ph,Me) is hydridotris-((3-phenyl,5-methyl)pyrazolyl)borate, and (SSR) is tert-butyldisulfanido or triphenylmethanedisulfanido were synthesized by reaction between the corresponding hydroxo complexes TpZn(OH) and the synthetic persulfide RSSH. All the complexes were characterized by elemental analysis and (1)H NMR spectroscopy, and representative members of the class were also structurally characterized. The reactivity of the alkyldisulfanido TpZn(SSR) complexes with thiols was studied. In the absence of base, a simple exchange reaction between the alkyldisulfanido ligand and the thiol was observed in dichloromethane; when in the presence of base, the corresponding hydrogen(sulfido) complexes TpZn(SH) were obtained. The mechanism of the latter reaction has been studied and does not involve the coordinated alkyldisulfanido group. Reaction of the hydrogen(sulfido) complexes Tp(iPr,iPr)Zn(SH) with the thiosulfonate PhCH(2)S-SO(2)CF(3) did not yield the expected alkyldisulfanido complex but benzyltrisulfide and a new complex tentatively assigned as Tp(iPr,iPr)Zn(O(2)SCF(3)).

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 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.

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.

Reductive Metalation of Cyclic and Acyclic Pseudopeptidic Bis-Disulfides and Back Conversion of the Resulting Diamidato/Dithiolato Complexes to Bis-Disulfides

Cyclic and acyclic pseudopeptidic bis-disulfides built on an o-phenylene diamine scaffold were prepared: (N(2)H(2)S(2))(2), 1a, N(2)H(2)(S-SCH(3))(2), 1b, and N(2)H(2)(S-StBu)(2), 1c. Reductive metalation of these disulfides with (PF(6))[Cu(CH(3)CN)(4)] in the presence of Et(4)NOH as a base, or with (Et(4)N)[Fe(SEt)(4)] and Et(4)NCl, yields the corresponding diamidato/dithiolato copper(III) or iron(III) complex, (Et(4)N)[Cu(N(2)S(2))], 2, or (Et(4)N)(2)[Fe(N(2)S(2))Cl], 5. These complexes display characteristics similar to those previously described in the literature. The mechanism of the metalation with copper has been investigated by X-band electron paramagnetic resonance (EPR) spectroscopy at 10 K. After metalation of the bis-disulfide 1c and deprotonation of the amide nitrogens, the reductive cleavage of the S-S bonds occurs by two one-electron transfers leading to the intermediate formation of a copper(II) complex and a thyil radical. Complexes 2 and 5 can be converted back to the cyclic bis-disulfide 1a with iodine in an 80% yield. Reaction of 5 with iodine in the presence of CH(3)S-SCH(3) affords a 1/1 mixture of the acyclic N(2)H(2)(S-SCH(3))(2) disulfide 1b and cyclic bis-disulfide 1a. From 2, the reaction was monitored by (1)H NMR and gives 1b as major product. While there is no reaction of 2 or 5 with tBuS-StBu and iodine, reaction with an excess of tBuSI affords quantitatively the di-tert-butyl disulfide 1c. To assess the role of the Cu(III) oxidation state, control experiments were carried out under strictly anaerobic conditions with the copper(II) complex, (Et(4)N)(2)[Cu(N(2)S(2))], 6. Complex 6 is oxidized to 2 by iodine, and it reacts with an excess of tBuSI, yielding 1c as final product, through the intermediate formation of complex 2.