Acylation and deacylation mechanism and kinetics of penicillin G reaction with Streptomyces R61 DD-peptidase.

Two quantum mechanical (QM)-cluster models are built for studying the acylation and deacylation mechanism and kinetics of Streptomyces R61 DD-peptidase with the penicillin G at atomic level detail. DD-peptidases are bacterial enzymes involved in the cross-linking of peptidoglycan to form the cell wall, necessary for bacterial survival. The cross-linking can be inhibited by antibiotic beta-lactam derivatives through acylation, preventing the acyl-enzyme complex from undergoing further deacylation. The deacylation step was predicted to be rate-limiting. Transition state and intermediate structures are found using density functional theory in this study, and thermodynamic and kinetic properties of the proposed mechanism are evaluated. The acyl-enzyme complex is found lying in a deep thermodynamic sink, and deacylation is indeed the severely rate-limiting step, leading to suicide inhibition of the peptidoglycan cross-linking. The usage of QM-cluster models is a promising technique to understand, improve, and design antibiotics to disrupt function of the Streptomyces R61 DD-peptidase.

Structure-Activity Relationships of Novel Tryptamine-Based Inhibitors of Bacterial Transglycosylase.

Penicillin-binding proteins represent well-established, validated, and still very promising targets for the design and development of new antibacterial agents. The transglycosylase domain of penicillin-binding proteins is especially important, as it catalyzes polymerization of glycan chains, using the peptidoglycan precursor lipid II as a substrate. On the basis of the previous discovery of a noncovalent small-molecule inhibitor of transglycosylase activity, we systematically explored the structure-activity relationships of these tryptamine-based inhibitors. The main aim was to reduce the nonspecific cytotoxic properties of the initial hit compound and concurrently to retain the mode of its inhibition. A focused library of tryptamine-based compounds was synthesized, characterized, and evaluated biochemically. The results presented here show the successful reduction of the nonspecific cytotoxicity, and the retention of the inhibition of transglycosylase enzymatic activity, as well as the ability of these compounds to bind to lipid II and to have antibacterial actions.

Cell permeable vanX inhibitors as vancomycin re-sensitizing agents.

VanX is an induced zinc metallo d-Ala-d-Ala dipeptidase involved in the viable remodeling of bacterial cell wall that is essential for the development of VREF. Here we report two cyclic thiohydroxamic acid-based peptide analogs that were designed, synthesized and investigated as vancomycin re-sensitizing agents. These compounds exhibit low micromolar inhibitory activity against vanX, with low cytotoxicity and were shown to increase vancomycin sensitivity against VREF. The improved pharmacological properties of these novel inhibitors over previous transition state mimics should provide an enhanced platform for designing potent vanX inhibitors for overcoming vancomycin resistance.

Inhibition of DD-peptidases by a specific trifluoroketone: crystal structure of a complex with the Actinomadura R39 DD-peptidase.

Inhibitors of bacterial DD-peptidases represent potential antibiotics. In the search for alternatives to ß-lactams, we have investigated a series of compounds designed to generate transition state analogue structures upon reaction with DD-peptidases. The compounds contain a combination of a peptidoglycan-mimetic specificity handle and a warhead capable of delivering a tetrahedral anion to the enzyme active site. The latter includes a boronic acid, two alcohols, an aldehyde, and a trifluoroketone. The compounds were tested against two low-molecular mass class C DD-peptidases. As expected from previous observations, the boronic acid was a potent inhibitor, but rather unexpectedly from precedent, the trifluoroketone [D-α-aminopimelyl(1,1,1-trifluoro-3-amino)butan-2-one] was also very effective. Taking into account competing hydration, we found the trifluoroketone was the strongest inhibitor of the Actinomadura R39 DD-peptidase, with a subnanomolar (free ketone) inhibition constant. A crystal structure of the complex between the trifluoroketone and the R39 enzyme showed that a tetrahedral adduct had indeed formed with the active site serine nucleophile. The trifluoroketone moiety, therefore, should be considered along with boronic acids and phosphonates as a warhead that can be incorporated into new and effective DD-peptidase inhibitors and therefore, perhaps, antibiotics.

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.

Sub-inhibitory cefsulodin sensitization of E. coli to ß-lactams is mediated by PBP1b inhibition.

The combination of antibiotics is one of the strategies to combat drug-resistant bacteria, though only a handful of such combinations are in use, such as the ß-lactam combinations. In the present study, the efficacy of a specific sub-inhibitory concentration of cefsulodin with other ß-lactams was evaluated against a range of Gram-negative clinical isolates. This approach increased the sensitivity of the isolates, regardless of the ß-lactamase production. The preferred target and mechanism of action of cefsulodin were identified in laboratory strains of Escherichia coli, by examining the effects of deleting the penicillin-binding protein (PBP) 1a and 1b encoding genes individually. Deletion of PBP1b was involved in sensitizing the bacteria to ß-lactam agents, irrespective of its O-antigen status. Moreover, the use of a sub-inhibitory concentration of cefsulodin in combination with a ß-lactam exerted an effect similar to that one obtained for PBP1b gene deletion. We conclude that the identified ß-lactam/cefsulodin combination works by inhibiting PBP1b (at least partially) despite the involvement of ß-lactamases, and therefore could be extended to a broad range of Gram-negative pathogens.

Meropenem inhibits D,D-carboxypeptidase activity in Mycobacterium tuberculosis.

Carbapenems such as meropenem are being investigated for their potential therapeutic utility against highly drug-resistant tuberculosis. These ß-lactams target the transpeptidases that introduce interpeptide cross-links into bacterial peptidoglycan thereby controlling rigidity of the bacterial envelope. Treatment of Mycobacterium tuberculosis (Mtb) with the ß-lactamase inhibitor clavulanate together with meropenem resulted in rapid, polar, cell lysis releasing cytoplasmic contents. In Mtb it has been previously demonstrated that 3-3 cross-linkages [involving two diaminopimelate (DAP) molecules] predominate over 4-3 cross-linkages (involving one DAP and one D-alanine) in stationary-phase cells. We purified and analysed peptidoglycan from Mtb and found that 3-3 cross-linkages predominate throughout all growth phases and the ratio of 4-3/3-3 linkages does not vary significantly under any growth condition. Meropenem treatment was accompanied by a dramatic accumulation of unlinked pentapeptide stems with no change in the tetrapeptide pools, suggesting that meropenem inhibits both a D,D-carboxypeptidase and an L,D-transpeptidase. We purified a candidate D,D-carboxypeptidase DacB2 and showed that meropenem indeed directly inhibits this enzyme by forming a stable adduct at the enzyme active site. These results suggest that the rapid lysis of meropenem-treated cells is the result of synergistically inhibiting the transpeptidases that introduce 3,3-cross-links while simultaneously limiting the pool of available substrates available for cross-linking.

Inhibition of bacterial DD-peptidases (penicillin-binding proteins) in membranes and in vivo by peptidoglycan-mimetic boronic acids.

The DD-peptidases or penicillin-binding proteins (PBPs) catalyze the final steps of bacterial peptidoglycan biosynthesis and are inhibited by the ß-lactam antibiotics. There is at present a question of whether the active site structure and activity of these enzymes is the same in the solubilized (truncated) DD-peptidase constructs employed in crystallographic and kinetics studies as in membrane-bound holoenzymes. Recent experiments with peptidoglycan-mimetic boronic acids have suggested that these transition state analogue-generating inhibitors may be able to induce reactive conformations of these enzymes and thus inhibit strongly. We have now, therefore, measured the dissociation constants of peptidoglycan-mimetic boronic acids from Escherichia coli and Bacillus subtilis PBPs in membrane preparations and, in the former case, in vivo, by means of competition experiments with the fluorescent penicillin Bocillin Fl. The experiments showed that the boronic acids bound measurably (K(i) < 1 mM) to the low-molecular mass PBPs but not to the high-molecular mass enzymes, both in membrane preparations and in whole cells. In two cases, E. coli PBP2 and PBP5, the dissociation constants obtained were very similar to those obtained with the pure enzymes in homogeneous solution. The boronic acids, therefore, are unable to induce tightly binding conformations of these enzymes in vivo. There is no evidence from these experiments that DD-peptidase inhibitors are more or less effective in vivo than in homogeneous solution.

Synthesis and activity study of phosphonamidate dipeptides as potential inhibitors of VanX.

In an effort to develop inhibitors of VanX, the phosphonamidate analogs of D-Ala-D-Ala dipeptides, N-[(1-aminoethyl) hydroxyphosphinyl]-glycine (1a), -alanine (1b), -valine (1c), -leucine (1d) and -phenylalanine (1e) were synthesized, characterized and evaluated using recombinant VanX. The crystal structure of the intermediate 6d was obtained (Deposition number: CCDC 839134), and structural analysis revealed that it is orthorhombic with a space group P2(1)2(1)2(1), the bond length of P-N is 1.62Å and angle of C-N-P is 123.6°. Phosphonamidate 1(a-e) showed to be inhibitors of VanX with IC(50) values of 0.39, 0.70, 1.12, 2.82, and 4.13mM, respectively, which revealed that the inhibition activities of the phosphonamidates were dependent on the size of R-substituent of them, with the best inhibitor 1a having the smallest substituent. Also, 1a showed antibacterial activity against Staphylococcus aureus (ATCC 25923) with a MIC value of 0.25 µg/ml.

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.

Crystal structure of a complex between the Actinomadura R39 DD-peptidase and a peptidoglycan-mimetic boronate inhibitor: interpretation of a transition state analogue in terms of catalytic mechanism.

The Actinomadura R39 DD-peptidase is a bacterial low molecular weight class C penicillin-binding protein. It has previously been shown to catalyze hydrolysis and aminolysis of small D-alanyl-D-alanine terminating peptides, especially those with a side chain that mimics the amino terminus of the stem peptide precursor to the bacterial cell wall. This paper describes the synthesis of (D-alpha-aminopimelylamino)-D-1-ethylboronic acid, designed to be a peptidoglycan-mimetic transition state analogue inhibitor of the R39 DD-peptidase. The boronate was found to be a potent inhibitor of the peptidase with a K(i) value of 32 +/- 6 nM. Since it binds some 30 times more strongly than the analogous peptide substrate, the boronate may well be a transition state analogue. A crystal structure of the inhibitory complex shows the boronate covalently bound to the nucleophilic active site Ser 49. The aminopimelyl side chain is bound into the site previously identified as specific for this moiety. One boronate oxygen is held in the oxyanion hole; the other, occupying the leaving group site of acylation or the nucleophile site of deacylation, appears to be hydrogen-bonded to the hydroxyl group of Ser 298. The Ser 49 oxygen appears to be hydrogen bonded to Lys 52. If it is assumed that this structure does resemble a high-energy tetrahedral intermediate in catalysis, it seems likely that Ser 298 participates as part of a proton transfer chain initiated by Lys 52 or Lys 410 as the primary proton donor/acceptor. The structure, therefore, supports a particular class of mechanism that employs this proton transfer device.

Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli.

Drug-resistant bacteria have caused serious medical problems in recent years, and the need for new antibacterial agents is undisputed. Transglycosylase, a multidomain membrane protein essential for cell wall synthesis, is an excellent target for the development of new antibiotics. Here, we determined the X-ray crystal structure of the bifunctional transglycosylase penicillin-binding protein 1b (PBP1b) from Escherichia coli in complex with its inhibitor moenomycin to 2.16-A resolution. In addition to the transglycosylase and transpeptidase domains, our structure provides a complete visualization of this important antibacterial target, and reveals a domain for protein-protein interaction and a transmembrane helix domain essential for substrate binding, enzymatic activity, and membrane orientation.

Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins).

The DD-peptidase enzymes (penicillin-binding proteins) catalyze the final transpeptidation reaction of bacterial cell wall (peptidoglycan) biosynthesis. Although there is now much structural information available about these enzymes, studies of their activity as enzymes lag. It is now established that representatives of two low-molecular-mass classes of DD-peptidases recognize elements of peptidoglycan structure and rapidly react with substrates and inhibitors incorporating these elements. No members of other DD-peptidase classes, including the high-molecular-mass enzymes, essential for bacterial growth, appear to interact strongly with any particular elements of peptidoglycan structure. Rational design of inhibitors for these enzymes is therefore challenging.

Synthesis of 3-Substituted-clavams: Antifungal Properties, DD-Peptidase and beta-Lactamase Inhibition.

The [2+2]cycloaddition of chlorosulfonyl isocyanate to vinyl and (Z)-propenyl ethers derived from the 2-O-sulfonylated (R)- and (S)-1-(furyl-2')-1,2-ethanediols furnished the 4-alkoxy-azetidin-2-ones with a good to moderate stereoselectivity. The intramolecular alkylation of the beta-lactam nitrogen atom led to the corresponding 3-(furyl-2')- and 6-methyl-3-(furyl-2')-clavams. The transformation of the furyl residue into an alkoxycarbonyl group led to clavams related to the natural compounds. The synthesized clavams exhibited moderate inhibitory activities against DD-peptidase 64-575 and beta-lactamase (penase) as well as antifungal activities.

Reactivity of penicillin-binding proteins with peptidoglycan-mimetic beta-lactams: what's wrong with these enzymes?

Beta-lactams exert their antibiotic action through their inhibition of bacterial DD-peptidases (penicillin-binding proteins). Bacteria, in general, carry several such enzymes localized on the outside of their cell membrane to catalyze the final step in cell wall (peptidoglycan) synthesis. They have been classified into two major groups, one of high molecular weight, the other of low. Members of the former group act as transpeptidases in vivo, and their inhibition by beta-lactams leads to cessation of bacterial growth. The latter group consists of DD-carboxypeptidases, and their inhibition by beta-lactams is generally not fatal to bacteria. We have previously shown that representatives of the former group are ineffective at catalyzing the hydrolysis/aminolysis of peptidoglycan-mimetic peptides in vitro [Anderson et al. (2003) Biochem. J. 373, 949-955]. The theme of these experiments is expanded in the present paper where we describe the synthesis of a series of beta-lactams (penicillins and cephalosporins) containing peptidoglycan-mimetic side chains and the kinetics of their inhibition of a panel of penicillin-binding proteins spanning the major classes (Escherichia coli PBP 2 and PBP 5, Streptococcus pneumoniae PBP 1b, PBP 2x and PBP 3, the Actinomadura R39 DD-peptidase, and the Streptomyces R61 DD-peptidase). The results of these experiments mirror and expand the previous results with peptides. Neither peptides nor beta-lactams with appropriate peptidoglycan-mimetic side chains react with the solubilized constructs of membrane-bound penicillin binding proteins (the first five enzymes above) at rates exceeding those of generic analogues. Such peptides and beta-lactams do react at greatly enhanced rates with certain soluble low molecular weight enzymes (R61 and R39 DD-peptidases). The former result is unexpected and interesting. Why do the majority of penicillin-binding proteins not recognize elements of local peptidoglycan structure? Possible answers are discussed. That this question needs to be asked casts fascinating shadows on current studies of penicillin-binding proteins for new drug design.

Using surface plasmon resonance to directly measure slow binding of low-molecular mass inhibitors to a VanX chip.

VanX, a d,d-dipeptidase, is one of five gene products responsible for vancomycin resistance in pathogenic bacteria and is an attractive drug target in circumventing clinical drug resistance. Our previous combinatorial search of VanX substrates in a dipeptide library of d-X(1)-d-X(2) (19(2)=361) forms has led to the discovery of three new compounds (d-Ala-d-Phe, d-Ala-d-Tyr, and d-Ala-d-Trp) having higher k(cat)/K(M) values than those of its natural substrate, d-Ala-d-Ala. Based on structures of newly identified substrates, two representative transition state analogs of substrates, d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) dipeptide phosphonates, used as VanX inhibitor were rationally designed and chemically synthesized. In the synthesis, eight synthetic steps in total were employed for preparing each VanX inhibitor, and their overall isolated yields were 21 and 11% for 6a and 6b, respectively. Binding interactions of d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) with VanX were confirmed unambiguously and measured quantitatively by surface plasmon resonance. The result reveals that both dipeptide phosphonates are slow-binding inhibitors of VanX (for 6a, k(on)=1.18 x 10(3)M(-1)s(-1), k(off)=2.31 x 10(-3) s(-1), K(D)=1.96 microM, chi(2)=0.0737; for 6b, k(on)=1.09 x 10(3)M(-1)s(-1), k(off)=1.80 x 10(-2)s(-1), K(D)=16.5 microM, chi(2)=0.0599). This suggests that only a fraction of the conformers of the inhibitors in solution adopts a conformation best suited for binding interaction with VanX and that the VanX-inhibitor complex may concomitantly undergo a conformational isomerization from an initial but fast weak-binding adduct to slowly convert to a tight-binding complex with a more stable bound geometry. Moreover, in comparison with 6b, an additional aromatic interaction of 6a with the Phe79 residue in the active site of the enzyme, through an energetically favorable face-to-face offset stacked orientation, may account for its higher affinity than 6b to VanX.

Screen for inhibitors of the coupled transglycosylase-transpeptidase of peptidoglycan biosynthesis in Escherichia coli.

Class A high-molecular-weight penicillin-binding protein 1a (PBP1a) and PBP1b of Escherichia coli have both transglycosylase (TG) and transpeptidase (TP) activity. These enzymes are difficult to assay, since their substrates are difficult to prepare. We show the activity of PBP1a or PBP1b can be measured in membranes by cloning the PBP into an E. coli ponB::Spcr strain. Using this assay, we show that PBP1a is approximately 10-fold more sensitive to penicillin than PBP1b and that the 50% inhibitory concentration (IC50) of moenomycin, a TG inhibitor, is approximately 10-fold higher in the PBP transformants than in wild-type membranes; this increase in IC50 in transformants can be used to test the specificity of test compounds for inhibition of the TG. Alternatively, the coupled TG-TP activity of PBP1b can be directly measured in a two-step microplate assay. In the first step, radiolabeled lipid II, the TG substrate, was made in membranes of the E. coli ponB::Spcr strain by incubation with the peptidoglycan sugar precursors. In the second step, the TG-TP activity was assayed by adding a source of PBP1b to the membranes. The coupled TG-TP activity converts lipid II to cross-linked peptidoglycan, which was specifically captured by wheat germ agglutinin-coated scintillation proximity beads in the presence of 0.2% Sarkosyl (B. Chandrakala et al., Antimicrob. Agents Chemother. 48:30-40, 2004). The TG-TP assay was inhibited by penicillin and moenomycin as expected. Surprisingly, tunicamycin and nisin also inhibited the assay, and paper chromatography analysis revealed that both inhibited the transglycosylase. The assay can be used to screen for novel antibacterial agents.

Transpeptidation reactions of a specific substrate catalyzed by the streptomyces R61 DD-peptidase: characterization of a chromogenic substrate and acyl acceptor design.

The Streptomyces R61 dd-peptidase, a functional model for penicillin-binding proteins, catalyzes the hydrolysis and aminolysis of d-alanyl-d-alanine-terminating peptides by specific amines. In vivo, this reaction completes bacterial cell wall biosynthesis. For in vitro studies of this enzyme to date, various nonspecific acyl-donor substrates have been employed. Recently, however, a peptidoglycan-mimetic peptide substrate, glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl-d-alanine, has been described that is much more specific for this enzyme. In this paper, we describe the synthesis and kinetic characterization of an analogous thiolester substrate, 3-(N-glycyl-l-cysteinyl)-propanoyl-d-alanyl-d-thiolactate, that the enzyme hydrolyzes and aminolyzes very efficiently (k(cat)/K(m) = 1.0 x 10(7) s(-)(1) M(-)(1)). Direct or indirect, by means of a thiol trap, spectrophotometric monitoring of the reactions of this substrate is readily achieved. Deacylation of the enzyme is rate-determining under substrate saturation conditions, and therefore the aminolysis reaction can be directly studied. The results show that d-amino acids and certain Gly-l-Xaa dipeptides and tripeptides may act as acyl acceptors at the active site of the enzyme. d-Phenylalanine and Gly-l-Phe were the most effective d-amino acid and dipeptide acceptors, respectively. On the basis of the dual specificity of the active site for acceptors (d-amino acids and Gly-l-Xaa peptides), "dual function" acceptors were designed and synthesized. Two of these, aminomalon-(N-ethyl)amide and aminomalon-(N-phenethyl)amide, were particularly effective. It did seem, however, that the observed rates of reaction of these very effective acceptors may be limited by some common, possibly physical, step. More extended, peptidoglycan-like, acceptors were found to be essentially unreactive. The reasons for this counterintuitive behavior are discussed.

Inactivation of bacterial DD-peptidase by beta-sultams.

N-Acyl-beta-sultams are time-dependent, irreversible active site-directed inhibitors of Streptomyces R61 DD-peptidase. The rate of inactivation is first order with respect to beta-sultam concentration, and the second-order rate constants show a dependence on pH similar to that for the hydrolysis of a substrate. Inactivation is due to the formation of a stable 1:1 enzyme-inhibitor complex as a result of the active site serine being sulfonylated by the beta-sultam as shown by ESI-MS analysis and by X-ray crystallography. A striking feature of the sulfonyl enzyme is that the inhibitor is not bound to the oxyanion hole but interacts extensively with the "roof" of the active site where the Arg 285 is located.