Armelle Melet

Proteasome Inhibition Causes Regression of Leukemia and Abrogates BCR-ABL–Induced Evasion of Apoptosis in Part through Regulation of Forkhead Tumor Suppressors

BCR-ABL plays an essential role in the pathogenesis of chronic myeloid leukemia (CML) and some cases of acute lymphocytic leukemia (ALL). Although ABL kinase inhibitors have shown great promise in the treatment of CML, the persistence of residual disease and the occurrence of resistance have prompted investigations into the molecular effectors of BCR-ABL. Here, we show that BCR-ABL stimulates the proteasome-dependent degradation of members of the forkhead family of tumor suppressors in vitro, in an in vivo animal model, and in samples from patients with BCR-ABL-positive CML or ALL. As several downstream mediators of BCR-ABL are regulated by the proteasome degradation pathway, we also show that inhibition of this pathway, using bortezomib, causes regression of CML-like disease. Bortezomib treatment led to inhibition of BCR-ABL-induced suppression of FoxO proteins and their proapoptotic targets, tumor necrosis factor-related apoptosis-inducing ligand and BIM, thereby providing novel insights into the molecular effects of proteasome inhibitor therapy. We additionally show sensitivity of imatinib-resistant BCR-ABL T315I cells to bortezomib. Our data delineate the involvement of FoxO proteins in BCR-ABL-induced evasion of apoptosis and provide evidence that bortezomib is a candidate therapeutic in the treatment of BCR-ABL-induced leukemia.

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 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.

Reversible Detection and Quantification of Hydrogen Sulfide by Fluorescence Using the Hemoglobin I from Lucina pectinata

A new detection system for the endogenous gaseous transmitter and environmental pollutant hydrogen sulfide is presented. It is based on the modulation of the fluorescence spectrum of a coumarin dye by the absorption spectrum of the recombinant hemoglobin I from clam Lucina pectinata upon coordination of the analyte. While we establish that the reported affinity of rHbI for H2S has been overestimated, the association of the protein with an appropriate fluorophore allows fast, easy, and reversible detection and quantification of hydrogen sulfide in buffer as well as biological fluids such as human plasma, with a quantification limit around 200 nM at pH 7.4.