Frédéric Sapunaric

Discovery and preliminary SARs of keto-indoles as novel indoleamine 2,3-dioxygenase (IDO) inhibitors

Indoleamine 2,3-dioxygenase (IDO) is an important new therapeutic target for the treatment of cancer. With the aim of discovering novel IDO inhibitors, a virtual screen was undertaken and led to the discovery of the keto-indole derivative 1a endowed with an inhibitory potency in the micromolar range. Detailed kinetics were performed and revealed an uncompetitive inhibition profile. Preliminary SARs were drawn in this series and corroborated the putative binding orientation as suggested by docking.

Indol-2-yl ethanones as novel indoleamine 2,3-dioxygenase (IDO) inhibitors

Indoleamine 2,3-dioxygenase (IDO) is a heme dioxygenase which has been shown to be involved in the pathological immune escape of diseases such as cancer. The synthesis and structure-activity relationships (SAR) of a novel series of IDO inhibitors based on the indol-2-yl ethanone scaffold is described. In vitro and in vivo biological activities have been evaluated, leading to compounds with IC(50) values in the micromolar range in both tests. Introduction of small substituents in the 5- and 6-positions of the indole ring, indole N-methylation and variations of the aromatic side chain are all well tolerated. An iron coordinating group on the linker is a prerequisite for biological activity, thus corroborating the virtual screening results.

The penicillin resistance of Enterococcus faecalis JH2-2r results from an overproduction of the low-affinity penicillin-binding protein PBP4 and does not involve a psr-like gene

A penicillin-resistant mutant, JH2-2r (MIC 75 microg ml(-1)), was isolated from Enterococcus faecalis JH2-2 (MIC 5 microg ml(-1)) by successive passages on plates containing increasing concentrations of benzylpenicillin. A comparison of the penicillin-binding protein (PBP) profiles in the two strains revealed a more intensely labelled PBP4 in JH2-2r. Because the sequences of the JH2-2 and JH2-2r pbp4 genes were strictly identical, even in their promoter regions, this intensive labelling could only be associated with an overproduction of the low-affinity PBP4. No psr gene analogous to that proposed to act as a regulator of PBP5 synthesis in Enterococcus hirae and Enterococcus faecium could be identified in the vicinity of pbp4 in E. faecalis JH2-2 and JH2-2r. However, a psr-like gene distant from pbp4 was identified. The cloning and sequencing of that psr-like gene from both E. faecalis strains indicated that they were identical. It is therefore postulated that the PBP4 overproduction in E. faecalis JH2-2r results from the modification of an as yet unidentified factor.

The Division and Cell Wall Gene Cluster of Enterococcus Hirae S185

A chromosomal 10355-bp segment of Enterococcus hirae S185 contains nine orfs which occur in the same order as the MraW-, FtsL-, PBP3-, MraY-, MurD-, MurG-, FtsQ-, FtsA- and FtsZ-encoding genes of the division and cell wall clusters of Escherichia coli and Bacillus subtilis. The E. hirae DNA segment lacks the genes which in E. coli encode the ligases Ddl, MurC, MurE and MurF and the integral membrane protein FtsW. The encoded E. hirae and E. coli proteins share 25% to 50% identity except FtsL and FtsQ (approximately = 14% identity).

Redefining the role of psr in beta-lactam resistance and cell autolysis of Enterococcus hirae.

The contribution of penicillin-binding protein 5 (PBP5) and the PBP5 synthesis repressor (Psr) to the beta-lactam resistance, growth, and cell autolysis of wild-type strain ATCC 9790 and resistant strain R40 of Enterococcus hirae was investigated by disruption or substitution of the corresponding pbp5 and psr genes by Campbell-type recombination. The resulting modifications were confirmed by hybridization and PCR. The low susceptibility of E. hirae to beta-lactams was confirmed to be largely dependent on the presence of PBP5. However, against all expectations, inactivation of psr in ATCC 9790 or complementation of R40 cells with psr did not modify the susceptibility to benzylpenicillin or the growth and cell autolysis rates. These results indicated that the psr gene does not seem to be involved in the regulation of PBP5 synthesis and consequently in beta-lactam resistance or in the regulation of cell autolysis in E. hirae.

N (1)-Fluoroalkyltryptophan Analogues: Synthesis and in vitro Study as Potential Substrates for Indoleamine 2,3-Dioxygenase.

Indoleamine 2,3-dioxygenase (hIDO) is an enzyme that catalyzes the oxidative cleavage of the indole ring of l-tryptophan through the kynurenine pathway, thereby exerting immunosuppressive properties in inflammatory and tumoral tissues. The syntheses of 1-(2-fluoroethyl)-tryptophan (1-FETrp) and 1-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-tryptophan, two N (1)-fluoroalkylated tryptophan derivatives, are described here. In vitro enzymatic assays with these two new potential substrates of hIDO show that 1-FETrp is a good and specific substrate of hIDO. Therefore, its radioactive isotopomer, 1-[(18)F]FETrp, should be a molecule of choice to visualize tumoral and inflammatory tissues and/or to validate new potential inhibitors.

Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

ABSTRACT PER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Amblers scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior.

Interdomain Loop Mutation Asp190Cys of the Tetracycline Efflux Transporter TetA(B) Decreases Affinity for Substrate

TetA(B), the tetracycline efflux protein from Tn10, has 12 predicted α-helices that span the Escherichia coli cytoplasmic membrane (10) and is a member of the major facilitator superfamily (4). TetA(B) and related tetracycline antiporters use the bacterial transmembrane proton motive force to export one metal-tetracycline complex in exchange for one H+ (12), thereby reducing the intracellular tetracycline concentration. In our recent studies of the cytoplasmic interdomain loop connecting transmembrane helices 6 and 7 of TetA(B) (7) and TetA(C) (6), we showed that this loop contains residues implicated in tetracycline efflux activity. Furthermore, we established that residues Asp190, Glu192, and Ser201 of TetA(B) are involved in substrate specificity of the pump (7). Mutations of three adjacent amino acids in the interdomain loop of TetA(A) in two veterinary Salmonella isolates showed an altered substrate specificity with reduced susceptibility to minocycline and glycylcyclines (11). In the present work, we investigated the basis for the effect of the Asp190Cys mutation on tetracycline efflux mediated by TetA(B) in everted membrane vesicles using [H3]tetracycline. The low-copy plasmids (origin of replication pSC101) that specified the Asp190Cys mutation (7) and wild-type TetB (13) were transformed into Escherichia coli DH5α; everted membrane vesicles were prepared as described previously (3). The protein content was quantified using the bicinchoninic acid protein assay (Pierce, Rockford, IL), and vesicle aliquots were stored in 50 mM MOPS (morpholinepropanesulfonic acid), pH 6.6, at −80°C. The antiport activity of vesicles was verified by an acridine orange fluorescence method. The amount of TetA(B) protein in the membranes was also determined by Western immunoblotting using anti-Ct antibody as described previously (7) and showed the same amount in each strain. Graphpad prism 4 software was used to determine the Km and Vmax values by fitting to the Michaelis-Menton equation. The cysteine substitution for aspartate at position 190 showed an average Km value 3.8 times that of the wild type but did not produce any modification in Vmax (Table ​(Table1).1). The Km and Vmax values obtained for the wild type are in agreement with those reported in the literature (3, 8, 9, 14). The lower affinity for tetracycline of the mutant Asp190Cys suggests that the aspartate residue is involved in the substrate interaction. Possibly the negatively charged aspartate interacts with the positively charged divalent metal cation-tetracycline complex which is the substrate. That a substitution causing a change in Km of a protein indicates a position involved in substrate binding has been shown with the β-lactamase TEM1 (1, 2). TABLE 1. Tetracycline resistance levels of E. coli DH5α cells with and without plasmid-borne wild-type and mutant TetA(B) and Km and Vmax for tetracycline uptake by everted membrane vesicles Although Asp190 is not an essential residue for tetracycline efflux (10), we show clearly that changing it to Cys lowered the affinity (higher Km) of the pump for its substrate. The sequence of the approximately 30 amino acids comprising the cytoplasmic interdomain loop is not conserved among the dozen or so related tetracycline efflux pumps (5). This loop has been assumed until recently to be simply a tether holding the two halves of the protein together. Our biochemical results now support previous data in vivo that this loop of TetA(B) has an unexpected role in tetracycline transport.