Detection and side-chain specificity of IgE antibodies to flucloxicillin in allergic subjects

Unlike studies on the antigenicity of penicillins in laboratory animals, limited information is available on the allergenicity of penicillins in man, especially with regard to fine structural allergenic differences between the many different penicillins. Inconsistent with the earlier conclusions of others, our studies suggest that side-chain structures to flucloxacillin-reactive IgE antibodies. Quantitative hapten inhibition studies revealed potent inhibition by flucloxacillin and three structurally related penicillins: oxacillin, cloxacillin and dicloxacillin. Analysis of the results showed that the side-chain group of flucloxacillin, 3-2(-chloro-6-fluorophenyl)-5-methyl-4-isoxazolyl, is recognized by some antibodies and that the 5-methyl-3-phenyl-4-isoxazolyl group, with or without halogen substituents, accounts for the reactivity of other antibodies and for the cross-reactions seen with some other penicillins. Since it is the side-chain group that distinguishes the many different types of penicillin, and since IgE antibodies in many of the allergic reactions recognize the side-chain groups, it is becoming clear that specific assays are required for the detection of IgE antibodies to each of the different penicillins.     

A quantitative microbiological determination of 6-aminopenicillanic acid

Several complicated chemical and microbiological methods have been described for the quantitative assay of 6-aminopenicillanic acid in the presence of benzyl- and phenoxymethylpenicillin. In all these methods the penicillins must be removed since they interfere with the assay. A specific microbiological plate assay withSerratia marcescens D 217 (ATCC 27117) is described for estimating even small amounts of 6-APA in benzyl- and phenoxymethylpenicillin samples. The contents of this paper were presented at the Xth International Congress for Microbiology at Mexico City 1970. The author wishes to express his gratitude to Mrs. I. Struyk-Hoette and Messrs. A. P. Struyk and R. G. W. Spickett for their valuable help and discussions.     

Formation of 6-Aminopenicillanic Acid, Penicillins, and Penicillin Acylase by Various Fungi

Several penicillin-producing fungi were examined for ability to produce 6-aminopenicillanic acid (6-APA) and penicillin acylase. 6-APA was found in corn steep liquor fermentations of Trichophyton mentagrophytes, Aspergillus ochraceous, and three strains of Penicillium sp. 6-APA was not detected in fermentations of Epidermophyton floccosum although penicillins were produced. 6-APA formed a large part of the total antibiotic production of T. mentagrophytes. The types of penicillins produced by various fungi were identified by paper chromatography, and it was found that all cultures produced benzylpenicillin. T. mentagrophytes and A. ochraceous showed increased yields of benzylpenicillin and the formation of phenoxymethylpenicillin in response to the addition to the fermentation medium of phenylacetic acid and phenoxyacetic acid, respectively. Washed mycelia of the three Penicillium spp. and two high penicillin-yielding strains of P. chrysogenum possessed penicillin acylase activity against phenoxymethylpenicillin. A. ochraceous, T. mentagrophytes, E. floccosum, and Cephalosporium sp. also had penicillin acylase activity against phenoxymethylpenicillin. Only two of the above fungi, T. mentagrophytes and E. floccosum, showed significant penicillin acylase activity against benzylpenicillin; in both cases it was very low. The acylase activity of A. ochraceous was considerably increased by culturing in the presence of phenoxyacetic acid. It is concluded that 6-APA frequently but not invariably accompanies the formation of penicillin, and that penicillin acylase activity against phenoxymethylpenicillin is present in all penicillin-producing fungi.     

Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans

A study of the final stages of the biosynthesis of the penicillins in Penicillium chrysogenum has revealed two types of enzyme. One hydrolyses phenoxymethyl penicillin to 6-aminopenicillanic acid (6-APA). The other, also obtained from Aspergillus nidulans, transfers a phenylacetyl group from phenylacetyl CoA to 6-APA. The acyltransferase, purified to apparent homogeneity, had a molecular mass of 40 kDa. It also catalyses the conversion of isopenicillin N (IPN) to benzylpenicillin (Pen G) and hydrolyses IPN to 6-APA. In the presence of SDS it dissociates, with loss of activity, into fragments of ca 30 and 10.5 kDa, but activity is regained when these fragments recombine in the absence of SDS.     

Factors affecting the synthesis of penicillins by the immobilized penicillin acylase from Escherichia coli

The effect of pH, temperature, reactant concentration, and reaction time has been investigated for the synthesis of N-benzhydryl-N′-acetamidopiperazyl-6-penicillanic acid and N-benzyl-N′-acetamidopiperazyl-6-penicillanic acid from 6-aminopenicillanic acid by the immobilized penicillin acylase from Escherichia coli. The synthesis of penicillins from carboxylic acids proceeds most rapidly at pH 5; with ethyl ester derivatives of carboxylic acids the pH optimum is higher (6–7). The most rapid synthesis of penicillins was obtained with ethyl ester derivatives of carboxylic acids. The optimum temperatures were 25–35°C.     

Evidence for the involvement of arginyl residue at the active site of penicillin G acylase from Kluyvera citrophila

Penicillin G acylase (PGA) is used for the commercial production of semi-synthetic penicillins. It hydrolyses the amide bond in penicillin producing 6-aminopenicillanic acid and phenylacetate. 6-Aminopenicillanic acid, having the beta-lactam nucleus, is the parent compound for all semi-synthetic penicillins. Penicillin G acylase from Kluyvera citrophila was purified and chemically modified to identify the role of arginine in catalysis. Modification with 20 mM phenylglyoxal and 50 mM 2,3-butanedione resulted in 82% and 78% inactivation, respectively. Inactivation was prevented by protection with benzylpenicillin or phenylacetate at 50 mM. The reaction followed psuedo-first order kinetics and the inactivation kinetics (V(max), K(m), and k(cat)) of native and modified enzyme indicates the essentiality of arginyl residue in catalysis.     

Penicillin Acylase Activity of Penicillium chrysogenum

The penicillin acylase activity of Penicillium chrysogenum was studied. Washed mycelial suspensions of a high penicillin-producing and a nonproducing strain were found to be similar in respect to relative acylase activity on benzylpenicillin, 2-pentenylpenicillin, heptylpenicillin, and phenoxymethylpenicillin. The relative rates for both strains, as determined by 6-aminopenicillanic acid formation, were approximately 1.0, 2.5, 3.5, and 6.0 on the penicillins in the order given. The high producing strain formed both 6-aminopenicillanic acid and "natural" penicillins in fermentations to which no side-chain precursor had been added. Therefore, its demonstrated ability to cleave the natural penicillins, 2-pentenylpenicillin and heptylpenicillin, suggests that at least some of the 6-aminopenicillanic acid produced during such fermentations arises from the hydrolysis of the natural penicillins. At pH 8.5, the mycelial acylase activity of the nonproducing strain was about three times that at pH 6.0; at 35 C, it was about 1.5 times as active as it was at 30 C. When tested on penicillin G or V, no differences in either total or specific penicillin acylase activity were observed among mycelia harvested from cultures of the nonproducer to which penicillin G, penicillin V, or no penicillin had been added. Acetone-dried mycelium from both strains displayed acylase activity, but considerably less than that shown by viable mycelium. Culture filtrates were essentially inactive, although a very low order of activity was detected when culture filtrate from the nonproducer was treated with acetone and the acetone-precipitated material was assayed in a minimal amount of buffer.     

6β-Amidinopenicillanic Acids—a New Group of Antibiotics

SINCE 6-aminopenicillanic acid became available¹, many semisynthetic penicillins carrying an acyl moiety on the 6-amino-group have been prepared. Thereby penicillins with improved oral absorption, resistance to penicillinase and to a lesser degree increased activity towards Gram-negative bacilli have been made available². Many other N-substitutions are possible, however, but these have not so far resulted in useful compounds^{2, 3}. We report here some of our findings on a new type of N-substituted 6-aminopenicillanic acids.     

Specificity of Penicillin Acylase of Fusarium and of Penicillium chrysogenum

Extracts containing penicillin acylase were obtained by shaking the mycelium of Fusarium avenaceum and of Penicillium chrysogenum in 0.2 M sodium acetate or sodium chloride solution. The optimum pH for conversion of penicillin V into 6-aminopenicillanic acid (6-APA) by the enzyme of Fusarium was about 7.5, and the reaction velocity was increased by a rise in temperature from 27 to 37 C. Penicillin G and penicillins with an aliphatic side chain were cleaved much less readily than was penicillin V. With the enzyme preparation obtained from a nonpenicillin-producing strain of P. chrysogenum, the reaction rate was higher at pH 8.5 than at pH 7.5 and pH 6.5. The acylase of P. chrysogenum hydrolyzes penicillin V more readily than penicillin G. In a series of aliphatic penicillins, the amount of 6-APA formed through the action of this enzyme increased with the number of carbon atoms of the side chain. Penicillins with a glutaryl or an adipyl group as side chain were unaffected by the enzyme of Fusarium and of Penicillium. No reaction was observed upon incubation of penicillin N (with a D-aminoadipyl side chain) or isopenicillin N (with an L-aminoadipyl side chain) with Fusarium and Penicillium extract. When the carboxy group of the side chain of these penicillins was esterified, formation of 6-APA was observed upon incubation with Penicillium extract, whereas no 6-APA or only very small amounts were obtained by acylase of Fusarium.     

THE NEWER PENICILLINS

The newer penicillins give high promise of overcoming some of the few disadvantages of penicillin-G.They fall into three groups: The alpha-phenoxy-penicillins; the penicillinase resistant penicillins; and the penicillins with enhanced activity against gram-negative bacteria.The newer alpha-phenoxy-penicillins offer little over alpha-phenoxy methyl penicillin (penicillin-V). As the length of the side chain is increased, absorption and attainable serum concentration is also increased, but these are questionable benefits and probably not significant for therapeusis.The penicillinase-resistant penicillins have once more brought almost all severe staphylococcal infections within therapeutic range. One of them, methicillin, must be administered parenterally. It is the agent of choice for the treatment of severe, penicillin-G resistant staphylococcal infections, and this is its only clinical indication. Another, oxacillin, which may be administered orally, is partially resistant to gastric acid degradation, but must be given on an empty stomach. It is most useful as prolonged therapy following methicillin, in the treatment of mixed hemolytic streptococcal-penicillin-G resistant staphylococcal infections, and as primary therapy for moderately severe penicillin-G resistant staphylococcal infections.The third group is still mostly in the experimental stage, but some strains of Proteus, E. coli, Salmonella and Shigella are highly vulnerable to their action.Toxic and allergic reactions to the newer penicillins, and crossed allergic reactions with penicillin-G, present unsolved problems.     

Current problems of work hygiene in the production of semisynthetic penicillins

The hygienic pattern of the technological process for production of semisynthetic penicillins is described. It is shown that the chemical factor is the leading one of the environment in manufacture of semisynthetic penicillins unlike production of natural antibiotics. The characteristics of the chemical factor by the conditions of its creation, physicochemical properties and effect on man is presented. A set of hygienic recommendations for safe performance of the technological process for production of semisynthetic penicillins was developed and is now being introduced.     

Structure-activity relations and beta-lactamase resistance

beta-Lactam antibiotics resistant to beta-lactamase degradation can be produced by many chemical modifications, but often at the expense of antibacterial activity. Substitution onto several positions in the molecule produces different and often selective resistance; for instance, heavily sterically hindered acyl groups give staphylococcal beta-lactamase resistance to penicillins, and resistance to some enzymes from Gram-negative pathogens to both penicillins and cephalosporins. 6-alpha- or 7-alpha-substituents respectively confer a broad spectrum of resistance (e.g. cefoxitin), but changes at positions 2 or 3 have only a minor influence on enzyme susceptibility. Changes in the ring condensed with the beta-lactam, such as changing ceph-3-em to ceph-2-em may greatly enhance stability. Small improvement can occur when the nuclear sulphur atom is oxidized, but a much better effect is obtained when it is replaced by another atom such as oxygen, as in clavulanic acid. This compound appears to have broad spectrum resistance which is actually due to susceptibility and subsequent produce inhibition.     

Semi-synthetic approaches to novel penicillins

The first part of this account of the discovery of penicillin, published in last month's issue of TIBS, concluded with the detection of 6-beta-amino-penicillanic acid (6-APA), the nucleus of the penicillin structure. The exploitation of 6-APA led to the preparation of a range of clinically important semi-synthetic penicillins, which is described in this article.     

Factors affecting the synthesis of ampicillin and hydroxypenicillins by the cell-bound penicillin acylase of Escherichia coli

1. The effect of pH, temperature, reactant concentration and reaction time has been investigated for the synthesis of benzylpenicillin, dl-alpha-hydroxybenzylpenicillin and d-alpha-aminobenzylpenicillin from 6-aminopenicillanic acid by the penicillin acylase of Escherichia coli. 2. Synthesis of penicillins from carboxylic acids proceeds most rapidly at pH5; with amides the optimum pH is higher (6-7) but the reverse reaction rapidly sets in. This can be counteracted by lowering the pH or adding more amide. Optimum temperatures are 35-40 degrees . 3. Most rapid synthesis of penicillin was obtained with the N-acylglycine and methyl ester derivatives of carboxylic acids. Increasing the amide/6-APA ratio above 1:1 raised the rate of synthesis of penicillins. 4. Preferential synthesis of d-alpha-hydroxybenzylpenicillin takes place in a reaction mixture containing dl-mandelic acid. 5. From d- and l-mandelamide, d- and l-alpha-hydroxybenzylpenicillins were prepared, the former being more bioactive than the latter. p-Hydroxy- and 3,4-dihydroxybenzylpenicillins were also prepared, the latter being more active against some Gram-negative bacteria than benzylpenicillin.     

Early discoveries in the penicillin series.

It is now 50 years since the therapeutic potential of penicillin was first demonstrated. This first antibiotic and the series of compounds derived from it have been of immense importance in modern medicine. This article describes the early search for more potent penicillin derivatives, culminating with the discovery of an intermediate of penicillin biosynthesis, 6-beta-amino penicillanic acid (6-APA). A companion article in next month's TIBS will chart the subsequent exploitation of 6-APA and the preparation of a range of clinically important semi-synthetic penicillins.     

Effect of beta-lactam antibiotics on lipid bilayer membranes. I. Penicillin antibiotics

Interaction between penicillins and model membrane systems, flat black bilayer lipid membranes (BLM) composed of vegetable or bacterial phospholipids was studied with an account of the complicated structure of bacterial cell membranes and possible presence in them of "pure" bilayer lipid areas. By their effect on electroconductivity of the BLM the antibiotics could be divided into three groups: those having no effect on the BLM electroconductivity at the maximum concentrations i.e. benzylpenicillin, carbenicillin, piperacillin (at pH 6.0 and 7.0) and ampicillin (at pH 6.0), those insignificantly changing electroconductivity of the BLM i.e. carfecillin and azlocillin and those having a significant effect on the BLM electroconductivity i.e. ampicillin N-acyl derivatives and 6-APA. The effect of ampicillin on the BLM conductivity markedly depended on the electrolite pH. The penicillins bound to the bilayer and induced changes in the transmembrane potential (evident from the changes in the second harmonic of the capacitive current) and the BLM elasticity-capacitance parameters (evident from the changes in the ratio of the amplitudes of the first and third harmonics). It was shown that all the penicillins penetrated through the BLM composed of either vegetable or bacterial phospholipids. The capacity for the transmembrane transfer without changing of the bilayer conductivity must be connected with the fact that the penetrating antibiotics did not induce any changes in the BLM structure. The effect on the conductivity probably depended in its turn on the form of the molecule and the ratio of the hydrophilic and hydrophobic parts in it.     

Virtual mutation and directional evolution of anti-amoxicillin ScFv antibody for immunoassay of penicillins in milk

In this study, an anti-amoxicillin single chain variable fragment (ScFv) antibody was evolved by directional mutagenesis of a contact amino acid residue based on the analysis of virtual mutation. Comparison with its parental ScFv, the mutant showed highly improved affinity for 11 penicillins with up to 6-folds increased sensitivity. Then, its recognition mechanisms for the 11 drugs were studied by using molecular docking. Results showed that the mutant-penicillins intermolecular forces increased and the total binding energies decreased dramatically, which were responsible for the improvement of antibody sensitivity. The ScFv mutant was used to develop an indirect competitive enzyme linked immunosorbent assay for determination of the 11 drugs in milk. The limits of detection were in the range of 0.2–3.0 ng/mL, the crossreactivities were in the range of 31%–132%, and the recoveries from standards fortified blank milk were in the range of 65.7%–92.4%. This is the first study reporting the directional evolution of a ScFv antibody based on virtual mutation and the use of ScFv antibody for determination of penicillins in foods of animal origin.     

'Beta-lactams' as beta-lactamase inhibitors

The application of inhibitors to block the beta-lactamase destruction of penicillins and cephalosporins by resistant bacteria is a potentially useful way of improving the efficacy of established compounds. Certain semi-synthetic penicillins and cephalosporins have been found to be competitive inhibitors of selected beta-lactamases but an examination of streptomycete culture fluids has revealed two new types of beta-lactam compound: clavulanic acid, which is a progressive inactivator of a wide range of beta-lactamases, and the olivanic acids, which are both broad-spectrum antibiotics and potent beta-lactamase inhibitors. Penicillanic acid sulphone and 6-beta-bromopenicillanic acid have been shown to be significant inhibitors of beta-lactamase. The chemotherapeutic application of these compounds is discussed.     

Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases.

Site-saturation mutagenesis was performed on the class A ROB-1 beta-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein-protein and protein-substrate hydrogen bonding. Mutation Thr130 gave a beta-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Km) for ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmax) activity for ampicillin. In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation. Mutation Gly130 gave a beta-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds approximately 15-fold lower than the wild-type, but no detectable activity against cephalosporins. Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate. Modelling the ROB-1 active site was done from the refined 2 A X-ray structure of the homologous Bacillus licheniformis beta-lactamase. Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field. Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral. Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes. The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser130 has a structural as well as a functional role in binding and hydrolysis of penicillins. This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.     

The Pharmacology of Semisynthetic Antibiotics

The chemical structures and reactions of penicillins and cephalosporins are reviewed in relation to their effects upon pharmacodynamic properties. The reactive betalactam ring is common to all penicillins and cephalosporin C analogues. This ring opens during acylation of the bacterial wall-building enzymes, but previous opening of the ring by acid or beta-lactamases destroys antibiotic activity.Semisynthetic substitutions may protect the ring by steric hindrance; this may actually inactivate certain penicillinases, so that resistant penicillins may potentiate penicillin G in some circumstances. However, the protective substitutions reduce the intrinsic activity of the synthetic penicillins themselves. Other properties which are affected include absorption, protein-binding, excretion, and possible allergenicity of the drugs. Effects on antibacterial spectrum may possibly be secondary to alteration of lipid solubility.