Fully automated radiosynthesis of N(1)-[(18)F]fluoroethyl-tryptophan and study of its biological activity as a new potential substrate for indoleamine 2,3-dioxygenase PET imaging.

INTRODUCTION: Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial step in the catabolism of l-tryptophan along the kynurenine pathway and exerts immunosuppressive properties in inflammatory and tumor tissues by blocking locally T-lymphocyte proliferation. Recently, 1-(2-[(19)F]fluoroethyl)-dl-tryptophan (1-[(19)F]FE-dl-Trp) was reported as a good and specific substrate of this enzyme. Herein, the radiosynthesis of its radioactive isotopomer (1-[(18)F]FE-dl-Trp, dl-[(18)F]5) is presented along with in vitro enzymatic and cellular uptake studies. METHODS: The one-pot n.c.a. radiosynthesis of this novel potential PET imaging tracer, including HPLC purification and formulation, has been fully automated on a FASTlab™ synthesizer. Chiral separation of both isomers and their formulation were implemented on a second cassette. In vitro enzymatic and cellular uptake studies were then conducted with the d-, l- and dl-radiotracers. RESULTS: The radiolabeling of the tosylate precursor was performed in DMF (in 5min; RCY: 57% (d.c.), n=3). After hydrolysis, HPLC purification and formulation, dl-[(18)F]5 was obtained with a global radiochemical yield of 18±3% (not decay corrected, n=7, in 80min) and a specific activity of 600±180GBq/µmol (n=5). The subsequent separation of l- and d-enantiomers was performed by chiral HPLC and both were obtained after formulation with an RCY (d.c.) of 6.1% and 5.8%, respectively. In vitro enzymatic assays reveal that l-[(18)F]5 is a better substrate than d-[(18)F]5 for human IDO. In vitro cellular assays show an IDO-specific uptake of the racemate varying from 30% to 50% of that of l-[(18)F]5, and a negligible uptake of d-[(18)F]5. CONCLUSION: In vitro studies show that l-[(18)F]5 is a good and specific substrate of hIDO, while presenting a very low efflux. These results confirm that l-[(18)F]5 could be a very useful PET radiotracer for IDO expressing cells in cancer imaging.

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.

Enantioselective synthesis of α-benzylated lanthionines and related tripeptides for biological incorporation into E. coli peptidoglycan.

The synthesis of modified tripeptides (S)-Ala-γ-(R)-Glu-X, where X = (R,S) or (R,R) diastereomers of α-benzyl or α-(4-azidobenzyl)lanthionine, was carried out. The chemical strategy involved the enantioselective alkylation of a 4-MeO-phenyloxazoline. The reductive opening of the alkylated oxazolines, followed by cyclization and oxidation, led to four PMB-protected sulfamidates. Subsequent PMB removal, Boc protection and regioselective opening with cysteine methyl ester led to protected lanthionines. These compounds were further converted in a one pot process to the corresponding protected tripeptides. After ester and Boc deprotection, the four tripeptides were evaluated as potential analogues of the natural tripeptide (S)-Ala-γ-(R)-Glu-meso-A2pm. These compounds were evaluated for introduction, by means of the biosynthetic recycling pathway, into the peptidoglycan of Escherichia coli. A successful in vitro biosynthesis of UDP-MurNAc-tripeptides from the tripeptides containing α-benzyl lanthionine was achieved using purified murein peptide ligase (Mpl). Bioincorporation into E. coli W7 did not occur under different tested conditions probably due to the bulky benzyl group at the Cα carbon of the C-terminal amino acid.

Stereoselective synthesis of lanthionine derivatives in aqueous solution and their incorporation into the peptidoglycan of Escherichia coli.

The three diastereoisomers-(R,R), (S,S) and meso-of lanthionine were synthesized in aqueous solution with high diastereoselectivity (>99%). The (S) and (R) enantiomers of two differently protected sulfamidates were opened by nucleophilic attack of (R) or (S)-cysteine. Acidification and controlled heating liberated the free lanthionines. Using the same chemistry, an α-benzyl lanthionine was also prepared. The proposed method, which avoids the need of enrichment by recrystallization, opens the way to the labelling of these compounds with (35)S. Furthermore, in vivo bioincorporation into Escherichia coli W7 was studied. No incorporation of α-benzyl lanthionine was observed. In contrast, meso-lanthionine can effectively replace meso-diaminopimelic acid in vivo, while in the presence of (R,R)-lanthionine the initial increase of bacterial growth was followed by cell lysis. In the future, meso-[(35)S]lanthionine could be used to study the biosynthesis of peptidoglycan and its turnover in relation to cell growth and division.

Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A.

Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential threonine synthase inhibitors have only been synthesized under enzymatic catalysis. Here we report the chemical stereoselective synthesis of the non-proteinogenic (S,Z)-2-amino-5-phosphonopent-3-enoic acid [(S,Z)-APPA] and its use for the synthesis of Rhizocticin A and Plumbemycin A. In this work, (S,Z)-APPA was synthesized via the Still-Gennari olefination starting from Garner's aldehyde. The Michaelis-Arbuzov reaction was used to form the phosphorus-carbon bond. Oligopeptides were prepared using liquid phase peptide synthesis (LPPS) and were tested against selected bacteria and fungi.

Inhibition of Streptococcus pneumoniae penicillin-binding protein 2x and Actinomadura R39 DD-peptidase activities by ceftaroline.

Although the rate of acylation of a penicillin-resistant form of Streptococcus pneumoniae penicillin-binding protein 2x (PBP2x) by ceftaroline is 80-fold lower than that of its penicillin-sensitive counterpart, it remains sufficiently high (k(2)/K = 12,600 M(-1) s(-1)) to explain the sensitivity of the penicillin-resistant strain to this new cephalosporin. Surprisingly, the Actinomadura R39 DD-peptidase is not very sensitive to ceftaroline.

Development of new drugs for an old target: the penicillin binding proteins.

The widespread use of β-lactam antibiotics has led to the worldwide appearance of drug-resistant strains. Bacteria have developed resistance to β-lactams by two main mechanisms: the production of β-lactamases, sometimes accompanied by a decrease of outer membrane permeability, and the production of low-affinity, drug resistant Penicillin Binding Proteins (PBPs). PBPs remain attractive targets for developing new antibiotic agents because they catalyse the last steps of the biosynthesis of peptidoglycan, which is unique to bacteria, and lies outside the cytoplasmic membrane. Here we summarize the “current state of the art” of non-β-lactam inhibitors of PBPs, which have being developed in an attempt to counter the emergence of β-lactam resistance. These molecules are not susceptible to hydrolysis by β-lactamases and thus present a real alternative to β-lactams. We present transition state analogs such as boronic acids, which can covalently bind to the active serine residue in the catalytic site. Molecules containing ring structures different from the β-lactam-ring like lactivicin are able to acylate the active serine residue. High throughput screening methods, in combination with virtual screening methods and structure based design, have allowed the development of new molecules. Some of these novel inhibitors are active against major pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and thus open avenues new for the discovery of novel antibiotics.

Synthesis and evaluation of boronic acids as inhibitors of Penicillin Binding Proteins of classes A, B and C.

In response to the widespread use of ß-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are potent ß-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2,6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC(50): 1.3 µM). In this work, we synthesized and studied the potential of a number of acylaminomethylboronic acids as inhibitors of PBPs from different classes. Several derivatives inhibited PBPs of classes A, B and C from penicillin sensitive strains. The (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of a class A PBP (PBP1b from Streptococcus pneumoniae, IC(50) = 26 µM), a class B PBP (PBP2xR6 from Streptococcus pneumoniae, IC(50) = 138 µM) and a class C PBP (R39 from Actinomadura sp., IC(50) = 0.6 µM). This work opens new avenues towards the development of molecules that inhibit PBPs, and eventually display bactericidal effects, on distinct bacterial species.

Exploration of the chemical space of novel naphthalene-sulfonamide and anthranilic Acid-based inhibitors of penicillin-binding proteins.

Penicillin-binding proteins are a well established, validated and still a very promising target for the design and development of new antibacterial agents. Based on our previous discovery of several noncovalent small-molecule inhibitor hits for resistant PBPs we decided to additionally explore the chemical space around these compounds. In order to clarify their structure-activity relationships for PBP inhibition two new series of compounds were synthesized, characterized and evaluated biochemically: the derivatives of anthranilic acid and naphthalene-sulfonamide derivatives. The target compounds were tested for their inhibitory activities on three different transpeptidases: PBP2a from methicillin-resistant Staphylococcus aureus (MRSA) strains, PBP5fm from Enterococcus faecium strains, and PBP1b from Streptococcus pneumoniae strains. The most promising results for both of these series of compounds were obtained against the PBP2a enzyme with the IC50 values in the micromolar range. Although these results do not represent a significant breakthrough in the field of noncovalent PBP inhibitors, they do provide useful structure-activity relationship data, and thus a more solid basis for the design of potent and noncovalent inhibitors of resistant PBPs.

Structure-guided design of cell wall biosynthesis inhibitors that overcome ß-lactam resistance in Staphylococcus aureus (MRSA).

ß-Lactam antibiotics have long been a treatment of choice for bacterial infections since they bind irreversibly to Penicillin-Binding Proteins (PBPs), enzymes that are vital for cell wall biosynthesis. Many pathogens express drug-insensitive PBPs rendering ß-lactams ineffective, revealing a need for new types of PBP inhibitors active against resistant strains. We have identified alkyl boronic acids that are active against pathogens including methicillin-resistant S. aureus (MRSA). The crystal structures of PBP1b complexed to 11 different alkyl boronates demonstrate that in vivo efficacy correlates with the mode of inhibitor side chain binding. Staphylococcal membrane analyses reveal that the most potent alkyl boronate targets PBP1, an autolysis system regulator, and PBP2a, a low ß-lactam affinity enzyme. This work demonstrates the potential of boronate-based PBP inhibitors for circumventing ß-lactam resistance and opens avenues for the development of novel antibiotics that target Gram-positive pathogens.

New noncovalent inhibitors of penicillin-binding proteins from penicillin-resistant bacteria.

BACKGROUND: Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs ß-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for ß-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs. METHODOLOGY/PRINCIPAL FINDINGS: Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains. CONCLUSIONS: We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.

Unexpected tricovalent binding mode of boronic acids within the active site of a penicillin-binding protein.

Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and mimicking the transition state of the enzymatic reaction. We have solved the structures of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four amidomethylboronic acids (2,6-dimethoxybenzamidomethylboronic acid, phenylacetamidomethylboronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the boron forms a tricovalent adduct with Oγ of Ser49, Ser298, and the terminal amine group of Lys410, three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents the first tricovalent enzyme-inhibitor adducts observed by crystallography. In two of the five R39-boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent complex from a classical monocovalent complex may involve rotation around the Ser49 Cα-Cß bond to place the boron in a position to interact with Ser298 and Lys410, and a twisting of the side-chain amide such that its carbonyl oxygen is able to hydrogen bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases, and details of the reaction between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. On the basis of the crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed.

Small molecule inhibitors of peptidoglycan synthesis targeting the lipid II precursor.

Bacterial peptidoglycan glycosyltransferases (GTs) of family 51 catalyze the polymerization of the lipid II precursor into linear peptidoglycan strands. This activity is essential to bacteria and represents a validated target for the development of new antibacterials. Application of structure-based virtual screening to the National Cancer Institute library using eHits program and the structure of the glycosyltransferase domain of the Staphylococcus aureus penicillin-binding protein 2 resulted in the identification of two small molecules analogues 5, a 2-[1-[(2-chlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine and 5b, a 2-[1-[(3,4-dichlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine that exhibit antibacterial activity against several Gram-positive bacteria but were less active on Gram-negative bacteria. The two compounds inhibit the activity of five GTs in the micromolar range. Investigation of the mechanism of action shows that the compounds specifically target peptidoglycan synthesis. Unexpectedly, despite the fact that the compounds were predicted to bind to the GT active site, compound 5b was found to interact with the lipid II substrate via the pyrophosphate motif. In addition, this compound showed a negatively charged phospholipid-dependent membrane depolarization and disruption activity. These small molecules are promising leads for the development of more active and specific compounds to target the essential GT step in cell wall synthesis.

Structure guided development of potent reversibly binding penicillin binding protein inhibitors.

Following from the evaluation of different types of electrophiles, combined modeling and crystallographic analyses are used to generate potent boronic acid based inhibitors of a penicillin binding protein. The results suggest that a structurally informed approach to penicillin binding protein inhibition will be useful for the development of both improved reversibly binding inhibitors, including boronic acids, and acylating inhibitors, such as ß-lactams.

1,6-AnhMurNAc derivatives for assay development of amidase AmiD.

Various peptidoglycan fragments were synthesized from two anhydro-muramic acid derivatives protected with a Bn or a PMB group at the 4th position, in homogenate phase or on a solid support. In order to facilitate HPLC detection, a chromophoric group was attached to the peptide chain. The periplasmic amidase sAmiD of Escherichia coli was used to cleave the amide bond between the lactyl group of the MurNAc and the α-amino group of L-Ala where the peptide chain was at least a dipeptide (L-Ala-γ-D-Glu) amidated by benzylamine on the γ-carboxyl group of D-Glu. In the presence of a tripeptide chain (L-Ala-γ-D-Glu-L-Lys) or a tetrapeptide chain (L-Ala-γ-D-Glu-m-A(2)pm-D-Ala) higher hydrolysis rates were observed. We have also demonstrated that the presence of TNB on the ε-amino group of L-Lys only has a small influence on the hydrolysis capacity of sAmiD.

Specific structural features of the N-acetylmuramoyl-L-alanine amidase AmiD from Escherichia coli and mechanistic implications for enzymes of this family.

AmiD is the fifth identified N-acetylmuramoyl-L-alanine zinc amidase of Escherichia coli. This periplasmic lipoprotein is anchored in the outer membrane and has a broad specificity. AmiD is capable of cleaving the intact peptidoglycan (PG) as well as soluble fragments containing N-acetylmuramic acid regardless of the presence of an anhydro form or not, unlike the four other amidases, AmiA, AmiB, AmiC, and AmpD, which have some specificity. AmiD function is, however, not clearly established but it could be part of the enzymatic machinery involved in the PG turnover in E. coli. We solved three structures of the E. coli zinc amidase AmiD devoid of its lipidic anchorage: the holoenzyme, the apoenzyme in complex with the substrate anhydro-N-acetylmuramic-acid-L-Ala-gamma-d-Glu-L-Lys, and the holoenzyme in complex with the L-Ala-gamma-D-Glu-L-Lys peptide, the product of the hydrolysis of this substrate by AmiD. The AmiD structure shows a relatively flexible N-terminal extension that allows an easy reach of the PG by the enzyme inserted into the outer membrane. The C-terminal domain provides a potential extended geometrical complementarity to the substrate. AmiD shares a common fold with AmpD, the bacteriophage T7 lysozyme, and the PG recognition proteins, which are receptor proteins involved in the innate immune responses of a wide range of organisms. Analysis of the different structures reveals the similarity between the catalytic mechanism of zinc amidases of the AmiD family and the thermolysin-related zinc peptidases.

Structural basis of the inhibition of class A beta-lactamases and penicillin-binding proteins by 6-beta-iodopenicillanate.

6-Beta-halogenopenicillanates are powerful, irreversible inhibitors of various beta-lactamases and penicillin-binding proteins. Upon acylation of these enzymes, the inhibitors are thought to undergo a structural rearrangement associated with the departure of the iodide and formation of a dihydrothiazine ring, but, to date, no structural evidence has proven this. 6-Beta-iodopenicillanic acid (BIP) is shown here to be an active antibiotic against various bacterial strains and an effective inhibitor of the class A beta-lactamase of Bacillus subtilis BS3 (BS3) and the D,D-peptidase of Actinomadura R39 (R39). Crystals of BS3 and of R39 were soaked with a solution of BIP and their structures solved at 1.65 and 2.2 A, respectively. The beta-lactam and the thiazolidine rings of BIP are indeed found to be fused into a dihydrothiazine ring that can adopt two stable conformations at these active sites. The rearranged BIP is observed in one conformation in the BS3 active site and in two monomers of the asymmetric unit of R39, and is observed in the other conformation in the other two monomers of the asymmetric unit of R39. The BS3 structure reveals a new mode of carboxylate interaction with a class A beta-lactamase active site that should be of interest in future inhibitor design.

Synthesis and evaluation of 3-(dihydroxyboryl)benzoic acids as D,D-carboxypeptidase R39 inhibitors.

Penicillin binding proteins (PBPs) catalyze steps in the biosynthesis of bacterial cell walls and are the targets for the beta-lactam antibiotics. Non-beta-lactam based antibiotics that target PBPs are of interest because bacteria have evolved resistance to the beta-lactam antibiotics. Boronic acids have been developed as inhibitors of the mechanistically related serine beta-lactamases and serine proteases; however, they have not been explored extensively as PBP inhibitors. Here we report aromatic boronic acid inhibitors of the D,D-carboxypeptidase R39 from Actinomadura sp. strain. Analogues of an initially identified inhibitor [3-(dihydroxyboryl)benzoic acid 1, IC(50) 400 microM] were prepared via routes involving pinacol boronate esters, which were deprotected via a two-stage procedure involving intermediate trifluorborate salts that were hydrolyzed to provide the free boronic acids. 3-(Dihydroxyboryl)benzoic acid analogues containing an amide substituent in the meta, but not ortho position were up to 17-fold more potent inhibitors of the R39 PBP and displayed some activity against other PBPs. These compounds may be useful for the development of even more potent boronic acid based PBP inhibitors with a broad spectrum of antibacterial activity.

Discovery of new inhibitors of resistant Streptococcus pneumoniae penicillin binding protein (PBP) 2x by structure-based virtual screening.

Penicillin binding proteins (PBPs) are involved in the biosynthesis of the peptidoglycan layer constitutive of the bacterial envelope. They have been targeted for more than half a century by extensively derived molecular scaffolds of penicillins and cephalosporins. Streptococcus pneumoniae resists the antibiotic pressure by inducing highly mutated PBPs that can no longer bind the beta-lactam containing agents. To find inhibitors of PBP2x from Streptococcus pneumoniae (spPBP2x) with novel chemical scaffold so as to circumvent the resistance problems, a hierarchical virtual screening procedure was performed on the NCI database containing approximately 260000 compounds. The calculations involved ligand-based pharmacophore mapping studies and molecular docking simulations in a homology model of spPBP2x from the highly resistant strain 5204. A total of 160 hits were found, and 55 were available for experimental tests. Three compounds harboring two novel chemical scaffolds were identified as inhibitors of the resistant strain 5204-spPBP2x at the micromolar range.

Characterization of the proteins encoded by the Bacillus subtilis yoxA-dacC operon.

In Bacillus subtilis, the yoxA and dacC genes were proposed to form an operon. The yoxA gene was overexpressed in Escherichia coli and its product fused to a polyhistidine tag was purified. An aldose-1-epimerase or mutarotase activity was measured with the YoxA protein that we propose to rename as GalM by analogy with its counterpart in E. coli. The peptide D-Glu-delta-m-A(2)pm-D-Ala-m-A(2)pm-D-Ala mimicking the B. subtilis and E. coli interpeptide bridge was synthesized and incubated with the purified dacC product, the PBP4a. A clear dd-endopeptidase activity was obtained with this penicillin-binding protein, or PBP. The possible role of this class of PBP, present in almost all bacteria, is discussed.