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The current status of the problem faced by the antibiotics is the frequent rise in the bacterial resistance towards the beta-lactam antibiotics. The functioning of the bacterial resistance assists in damaging the antibiotics by development of beta-lactamases, reduction in the effectiveness of antibiotics and acquiring changes in the completion of novel penicillin-binding proteins (PBP) (Grady et al., 1997).
Alexandra Fleming experiment in 1928 helped in invention and creation of penicillin. But the growth of penicillin as a therapeutic agent was analyzed in 1940 by Florey, Abraham and Chain in Oxford University. The chemical structure of the penicillin is composed of a beta-lactam ring joined to a side-chain and a thiazolidine ring. The main structure of the penicillin is the penicillin nucleus which induces metabolic transformation, biological activity, and reduction in the antibacterial activity by chemically modifying the nucleus (Miranda et al., 1996).
Prohibition of synthesis of peptidoglycan bacterial cell wall is executed by the beta-lactam antibiotics. Beta-lactam antibiotics are basically used for the prevention and treatment of infections induced by the bacteria. It was analyzed that the beta-lactam antibiotics were effective against the Gram-positive bacteria but with the recent development of beta-lactam antibiotics with the broad-spectrum of antibacterial activities it proved to be effective against the Gram-negative bacteria also (Grady et al., 1997).
Thus Penicillin G (benzylpenicillin) and Penicillin V (Phenoxy-methylpenicillin) belongs to this group and they are highly effective against the gram-positive bacteria (Farber et al., 1983). Other type of penicillin such as Ampicillin is an associate of Amoxicillin. Ampicillin is found to be effective against both the bacteria i.e. gram-negative and gram-positive bacteria (Campoli-Richards, 1987). Amoxicillin when clubbed with clavulanic acid is considered to be a beta-lactamase inhibitor it increases the spectrum of penicillin against the bacteria that produces beta-lactamase (Blanca et al., 1988).
Proglumide is a by-product of glutamic acid and operates as a Cholecystokinin (CCK) antagonist. The production of gastric acid is prohibited by proglumide; this activity of prohibition is basically due to interceding of proglumide affinity with the gastrin for the gastric mucosal membrane (Hahne et al., 1981). Thus, with these properties, proglumide will be introduced into the penicillin structure and it could be used as a penicillinase inhibitor to derive novel antibiotics with the broad-spectrum of antibacterial activities.
The four penicillinase enzyme pdb files (3ITA, 2EX8, 3A3F and 1GHP) are selected from the Protein Data Bank. The best pdb files will be then selected on basis of their experimental details. The selected pdb files will be then analyzed by homology modelling.
The major problem for the development of resistance to the beta-lactam antibiotics is the production of beta-lactamases. Before the use of penicillin clinically it was observed that the bacteria such as Staphylococcus aureus can generate resistance to benzylpenicillin not to decrease their intrinsic susceptibility towards the agent, but to lower their capacity. Bacterial enzymes have the ability to access the beta-lactam ring and hence they are called as 'penicillinase' (Grady et al., 1997).
Such types of enzymes proved to be essential as they have the ability to produce resistance in Gram-negative bacteria, the strength of these enzymes can reduce various types of beta-lactam agents. Hence such enzymes are called as beta-lactamases. Thus relevant beta-lactamase can make all the beta-lactams static. Beta-lactamases present in the Gram-positive bacteria are considered to be extracellular enzymes whereas those present in the Gram-negative bacteria are found to be invariably enclosed in periplasmic space, as the movement of larger molecules are inhibited by the outer cell membrane of gram-negative bacteria which results in limited detection of beta-lactamase activity. Thus the calculation of beta-lactamase activity quantitatively in the entire cells is found to be complicated (Grady et al., 1997).
The production of the beta-lactamase is the major cause to generate resistance to the beta-lactam in various species. Thus the prophylaxis from such infections induced by such type of bacteria can be treated with the implementation of labile antibiotics which is able to prohibit the enzyme. But in order to acquire such success, it needs to satisfy certain conditions like; the inhibitor should be effective against the beta-lactamases and the inhibitor features such as absorption, distribution and excretion should be similar to those of the beta-lactam agents when combined and it should not produce toxic effect when it is used. The prohibition of particular beta-lactamases can be initiated by specific semi-synthetic and natural beta-lactam agents, for example Isoxazolyl penicillin exhibit minimal prohibitory action against the enzyme which are of narrow range. Clavulanic acid and carbapenems were introduced when analysis of effective compounds were executed (Grady et al., 1997).
Insignificant anti-microbial actions are observed in beta-lactamase inhibitors when used therapeutically, but when clubbed with beta-lactamase labile penicillins it behaves as a collaborative agent. Penicillins and monobactams are generally antagonistic inhibitors or substrates, as their activity changes regularly and leave the enzyme without any modifications, as they behave as the insignificant substrates which are closely binded to the beta-lactamases and are hydrolysed gradually. Thus, with the discovery of efficient inhibitors lately it proved to be effective, as these inhibitors are constant when combined with enzyme they form a complex where both the enzyme and inhibitors are found to be static. Hence such types of inhibitors are termed as "suicide inhibitors". Clavulanic acid is considered as a beta-lactamase inhibitor (Grady et al., 1997).
History of Penicillin:
In 1929 Alexandra Fleming experiment showed that the mould of Penicillium notatum exhibit effective antibacterial activity which led to the discovery of the penicillin. Until 1940, Florey and his associates were successful in separation of the active compounds in Oxford University. When the chemical nature of the penicillin was analyzed it was observed that the compound obtained from the Penicillium chrysogenum by fermentation had the similar properties but the compound was different in terms of nature of acyl side-chain (Miranda et al., 1996).
While considering the activity, biological properties and commercially availability of the penicillins, Penicillin G was preferred as the chosen one. During the biosynthesis of the mould, acidic side-chain was added as a precursor in fermentation so that it can be adjusted by the mould. Thus, the Penicillin V (phenoxy-methylpenicillin) developed by this method, exhibit to be more influential than the Penicillin G. Penicillin V was developed by inclusion of a precursor i.e. phenoxy acetic acid during fermentation. During the development of semi-synthetic penicillin, different concept was applied by chemical substitution, but the by-products obtained from the synthesis were less active than the Penicillin G (Miranda et al., 1996).
Continuous failures in development of semi-synthetic penicillin lead to the invention of the penicillin nucleus in 1957 with the missing side-chain. For the production of the penicillin nucleus as a natural compound, 6-aminopenicillanic acid (6-APA) was separated in fermentation of Penicillium chrysogenum. Hence it can also be obtained by separation of the side-chain of benzylpenicillin or by chemically (Miranda et al., 1996).
Fig 1: Grady et al., (1997) show the structure of penicillin nucleus.
Thus with the identification of 6-APA, large number of semi-synthetic penicillins were produced since 1959 by addition of acyl side-chain to the 6-amino group. Produced novel penicillins are the by-products of 6-APA and show excellent antibacterial activities, greater stability to beta-lactamases and better pharmacokinetic properties than the Penicillin G and Penicillin V (Miranda et al., 1996).
Benzylpenicillin is considered as the natural penicillin but its absorption is poor when administered orally, so it is given intramuscularly. Benzylpenicillin when combined with oil and waxes it increase the efficacy of the plasma half-life which were found to be short, as the release of the penicillin were limited when administered through intramuscular. Hence in order to improve the circulation of the penicillin, insoluble salts of penicillins were developed, such as procaine penicillin, benethamine penicillin and benzathine penicillin which are considered as the respiratory penicillin and these pencillins are used frequently (Farber et al., 1983).
Fig 2: Grady et al., (1997) show the structure of benzylpenicillin.
Benzylpenicillin is effective against the entire Gram-positive bacteria, Gram-negative cocci and few Gram-negative bacteria. Benzylpenicillin undergo some changes and resistance are produced by the strains which develops the beta-lactamase. Staphylococcus aureus were more prone to the benzylpenicillin but eventually almost all their clinical isolates produce beta-lactamase, hence they show resistance towards the antibiotics (Farber et al., 1983).
Benzylpenicillin is weak in acid and it easily gets broken down in stomach. Due to this reaction, when plasma concentration was analyzed after oral administration it was observed to be irregular and weak and it even gets worse when administered orally. Benzylpenicillin is ineffective in removal of pharyngeal caused by N. meningitidis (Farber et al., 1983).
Ampicillin is semi-synthetic penicillin and it can be given orally, whereas only 10% of the ampicillin is soluble in water, but parentally it is given as the soluble sodium salt. Ampicillin is found to be more potent towards the Enterococcus faecalis, but its effectiveness is less compared to the benzylpenicillin against the Gram-positive bacteria. Staphylococcal beta-lactamase has the capacity to break down the activity of the ampicillin so that the Staphylococcus aureus and Staphylococcus epidermis exhibit resistance towards the ampicillin. Strains of Str. pneumoniae are less prone to the benzylpenicillin and more prone to ampicillin (Campoli-Richards, 1987).
Fig 3: Grady et al., (1997) show the structure of ampicillin.
Ampicillin is found to be strong in acid where only 5% reduction in the activity is reported. Absorption of the ampicillin when given orally is observed to be good which results in attaining high peak plasma concentration for doses of 250 and 500g after administration. However when ampicillin is administered together with meals its rate of absorption is poor but its absorption is not damaged when antacids and cimedine is given. Ampicillin when given in pregnancy results in achieving low peak plasma concentration due to rise in body fluid and clearing of renal. Toxicity level is obtained to be less in the ampicillin, whereas only rashes have been observed. The persistent occurring of such reactions may be due frequent dosing. In treatment of urinary tract infection, ampicillin is strongly used (Campoli-Richards, 1987).
Amoxycillin antibacterial spectrum is similar to the ampicillin, but its antibacterial activity is found to be distinct. Beta-lactamase inhibitors provide the considerable barrier against the bacteria that develops the beta-lactamase, as the amoxycillin is not strong against the beta-lactamase. Amoxycillin when combined with clavulanic acid is regarded as an affective agent against the Gram-negative bacteria, Pr. mirabilis, B. fragilis and K. pneumoniae as they are more prone to clavulanic acid. Thus this mixture of clavulanic acid and amoxycillin prohibits the strains of Staphylococcus aureus which produces the beta-lactamase (Blanca et al., 1988).
Fig 4: Grady et al., (1997) show the structure of amoxycillin.
The activity of amoxycillin is retained against the strains which produce the beta-lactamase by introduction of the beta-lactamase inhibitors. Peak concentration for oral absorption of amoxycillin is found to be greater than that of the ampicillin for similar amount of dose. Compared to ampicillin the absorption of amoxycillin is not disturbed by the intake of meals. Side-effects such as rashes are found to similar in amoxycillin compared to the ampicillin, but these side-effects can be bared. Amoxycillin clinical use is similar to that of the ampicillin. Single dose of the amoxycillin assist in treatment of urinary tract infection (Blanca et al., 1988).
Clavulanic acid is considered as the beta-lactamase inhibitor. When introduce with the amoxycillin or ticarcillin it reduces the antibiotics resistance. Clavulanic acid has a broad-spectrum; however its efficacy is observed to be low. With the inclusion of clavulanic acid, explains that such type of strains is more prone to amoxycillin and ticarcillin. Thus, the absorption of clavulanic acid is found to be good in the vicinity of the amoxycillin (Grady et al., 1997).
Fig 5: Grady et al., (1997) show the structure of clavulanic acid.
The peak plasma concentration level is found to be high after 30 minutes of administration. Penetration power of clavulanate into the mucus is observed to be insignificant after oral dosing. Temporary side-effects such as nausea, rashes, diarrhoea and vomiting are observed when co-amoxiclav is given for treatment (Grady et al., 1997).
Overcome the problem:
To solve the problem, penicillinase enzyme pdb files will be selected from the Protein Data Bank (http://www.rcsb.orr/pdb). These files will be evaluated according to the molecules, classifications, type of ligands, experimental details, and number of chains present in the protein structure. Taking into consideration all this factors four pdb files (3ITA, 2EX8, 3A3F and 1GHP) was selected.
PDB file: 3ITA
The bacterial cell wall is designed by duplication of disaccharide unit of N-acetyl glucosamine (NAG) and N-acetylmuramic acid (NAM); also NAM possesses a part of a peptide. A mesh-like structure is generated when transglycosylation joins the disaccharides to create the glycan backbone and polymerization of cell wall occurs due to two enzymatic reactions. Penicillin-binding proteins assist in restoring and binding of the bacterial cell wall. Penicillin-binding proteins are categorised in two subgroups, high molecular mass (HMM) and low molecular mass (LMM). The main target for the beta-lactams antibiotics is the penicillin-binding proteins. Hence the beta-lactamase enzyme which exhibit resistance to the beta-lactam antibiotics is the main reason for the medicinal chemistry research (Chen et al., 2009).
DD-Carboxypeptidase is a part of the LMM penicillin-binding proteins which has the ability to break the peptide bond into two ends of D-alanine of muramyl peptide, cross-linking of the cell wall is controlled when the peptidoglycan structure is modified, this activity is executed by transpeptidase. The aim of the DD-carboxypeptidase is to manage the cell morphology and the condition of the bacteria. Hence the X-ray crystallography of the penicillin-binding protein 6 was analyzed, two X-ray structures of penicillin-binding protein 6 were determined with E. coli at 2.1 Å and ampicillin at 1.8 Å. It was observed that these two structures were able to solve the correlation among the beta-lactams and the peptidoglycan substrate as they have the capacity to duplicate (Chen et al., 2009).
The penicillin-binding protein 6 was duplicated so that it can be introduced into the cytoplasm of E. coli and was purified by chromatographic step. Crystallisation of the soluble protein was carried out at pH 4.5. The complex structure was obtained when ampicillin and N-acetylmuramic acid were added to the crystal structure. Hence, both the structures were concluded at 1.8 Å. The entire protein structure has four chains and a single ligand i.e. ampicillin. The four chains in the protein structure share the same backbone structure (Chen et al., 2009).
PDB file: 2EX8
Peptidoglycan assists in formation of the bacteria cell wall, which plays an important role in maintaining the cell growth and its continuity under normal circumstances. Enzymes which interferes with the synthesis of the peptidoglycan are opposite in the biochemistry of the mammalian cell, hence these enzymes shows efficient targets for the invention of novel antibiotics. Various penicillin group and the natural antibiotics compounds manipulates with the survival of the bacteria on the cell wall. The by-products of the penicillins proved to be essential category of the beta-lactam antibiotic, but with the rise in the resistance of the antibiotics have resulted in designing of various categories of antibacterials (Kishida et al., 2006).
Thus, with the detailed studies in designing of new antibacterials it is concluded that the biological functions of different penicillin-binding proteins is present in the periplasm of the gram-negative bacteria, but their functions is still not predictable. Hence this permits the bacteria to expand by utilization of such type of proteins whose objective is to restore, synthesize and hydrolysis of peptidoglycan and retaining a strong cell wall. Penicillin and their by-products objectives are to target the penicillin-binding proteins (Kishida et al., 2006).
Penicillin-binding proteins are categorised in twelve types of penicillin-binding proteins distinguished in E. coli. These penicillin-binding proteins are cleaved into two groups, high-molecular weight (HMW) and low-molecular weight (LMW). High-molecular weight penicillin-binding proteins are PBP1a, PBP1b, PBP2 and PBP3 with DD-transpeptidase and transglycosidase actions. Low-molecular weight penicillin-binding proteins identified in E. coli are observed to be seven, but none of the penicillin-binding proteins are found to be important (Kishida et al., 2006).
Crystal structure was prepared chemically using the mother liquor (pH 6.5) and PEG 2000 (3-10%). At 20° C crystals evolved, to get the single crystal microseeding was executed to obtain the acceptable size for examination. These crystals were then shifted into cryoprotectant solution and cooled with liquid nitrogen. Penicillin G was then added to this crystal structure by using 100mM substrate for a day. PBP4 structure was concluded at 2.5 Å by using a single wavelength anomalous dispersion procedure. The final structure was estimated by implementation of PROCHECK. The availability of the penicillin G in the crystal structure leads to modifications in the active site. Hence this crystal structure is important in further analysis of PBP4 by manipulating many peptidoglycan fragments with different configurations (Kishida et al., 2006).
PDB file: 3A3F
An important layer is developed inside the bacterial cell wall by peptidoglycan, thus the layer and the peptidoglycan maintain the configuration of the cell and defend from the osmotic shocks. Rebuilding of peptidoglycan occur frequently, at particular locations novel compounds are synthesised during the development of the bacteria. Peptidoglycan is a polymer and it is comprised of sugar and amino acids. The sugar is made up of beta-linked N-acetylglycosamine and N-acetylmuramic acid residues. N-acetylglycosamine and N-acetylmuramic acid are considered as a disaccharide-pentapeptide. Disaccharide-pentapeptide encounters transglycosylation and transpeptidation reactions. Inclusion of novel compounds into the peptidoglycan layer it enhances the peptide by cross-linking of glycan chains. Penicillin-binding proteins (PBPs) are responsible for such reactions to take place. DD-carboxypeptidase does not assist in cross-linking by developing the peptidoglycan by releasing the C-terminal D-alanine residue (Kawai et al., 2009).
The four-membered ring of the beta-lactam antibiotics are considered as a suicide inhibitor substrate of PBPs as it has the ability to duplicate the part of D-alanyl-D-alanine of the stem peptide. Penicillin-binding proteins are isolated into two groups, high-molecular weight (HMW) and low-molecular weight (LMW). The high-molecular weight penicillin-binding proteins are also subdivided into two groups, group A PBPs and group B PBPs. The group A PBPs possess transglycosylase and transpeptidase activity, whereas group B PBPs posses N-terminal but its capacity is unfamiliar and C-terminal with transpeptidase activity. Low-molecular weight PBP4 of E. coli is found to be bifunctional in, in vivo and in vitro. The functions of high-molecular weight PBPs and low-molecular weight PBPs are totally different. Low-molecular weight PBPs are found to be excellent in understanding the shape and structure of the cell as well as in cell division but its functionalities compared to be high-molecular weight PBPs are observed to be poor (Kawai et al., 2009).
To investigate the correlation between the compound and PBP4, the protein structure was crystallised in an apo configuration and antibiotics was added to the crystal structure. This crystal structure was diffracted at 1.6 Å. The final structure was prepared from the pdb model of E. coli protein (2EX6) by molecular replacement. Thus various compounds were developed that resulted in the inhibition of the X-ray diffraction due to activity of the proteins. The effect of phenylalanine residue present in the active site is basically due to the penicillin which covalently joined to the crystal structure and this effect is found to be opposite in other PBPs (Kawai et al., 2009).
PDB file: 1GHP
Bacterial resistance are exhibited by the serine-beta-lactamases to the beta-lactam antibiotics so that the large number of the beta-lactam compounds are reduced by utilization of the hydroxyl group of serine residue. The hydroxyl group of serine residue i.e. balanced glutamic acid residues (GLU 166) initiate a nucleophilic attack on the carbon atom of the beta-lactam antibiotics which results in development of acyl-enzyme intermediate and this acyl bond is separated by the presence of water molecules. Beta-lactamase exhibit a general difference in particular substrate. Serine-beta-lactamase is segmented into three groups A, C and D. Group A serine-beta-lactamase is regarded as a preferred penicillinase enzyme. Recombinant enzyme is utilized in recent studies to analyze the structural dissimilarity between benzylpenicillin and cephaloridine correlated with the beta-lactamase of Staphylococcus aureus (Chen, 2000).
Benzylpenicillin is hydrolysed by the enzyme hence it is considered as an exceptional substrate of the enzyme. The mutant enzyme N170Q was developed with longer side-chain that can inhibit the entry of water molecules and decrease in deacylation. The crystal structure was prepared chemically by suspending the crystal in 89% of ammonium sulphate with 0.5% of PEG 2000 (pH 8) for 17 hours. Penicillin G was then added to the crystal structure. At 1.7 Å the crystal structure was diffracted for benzylpenicillin (Chen, 2000).
Proglumide is the by-product of glutamic acid and it is consequently used in Europe and Japan for 10 years in treatment of peptic ulcers. Proglumide is also considered as an antagonist of Cholecystokinin (CCK). The successful usage of proglumide is basically due to inhibition of gastric acid production but its mechanism of action is still not determined. Recent investigation predict that the gastrin-stimulated acid secretion and the activation of acid secretion produced by gastrin, histamine, insulin and carbamoyl choline can be prohibited by proglumide but proglumide is unable to show the actions of antihistamine or anticholinergic. Proglumide also prohibits the formation of gastric mucosal membrane and joining of gastric to a variant compound of gastric mucosal cells (Hahne et al., 1981).
Fig 6: Iuchi et al., (1997) show the structure of proglumide.
It is also analyzed that the proglumide has the capacity to bring changes in the activities of caerulin or gallbladder and gastric pyloric muscle and secretion of pancreatic fluid and gastric acid. The prohibitory activities of proglumide is basically for CCK and structurally associated peptides and demonstrate no side-effects when investigated in, in vivo studies. Proglumide availability in market is cheap and found to be efficacious when given orally to humans. Hence proglumide is essential as a therapeutic agent in chemical conditions for inhibition of CCK and for development of advantageous effects (Hahne et al., 1981).
Intended design and method of investigation:
Compound (proglumide) prepared by chemical synthesis:
Proglumide will be initiated into the penicillin structure; it could be used as a penicillinase inhibitor to derive novel antibiotics with broad spectrum of antibacterial activity. Proglumide will be prepared by chemical synthesis by referring to previous research papers. The chemical compound of proglumide will be then binded to the main template of the penicillin structure. The desired chemical compound will be then analyzed for their biological activities in, in vitro cell growth inhibition. Any positive results from the desired compound will be then used to evaluate their toxic level in animals and then in humans before execution of clinical trial.
Preparation of Models:
In these two models will be prepared, the whole structure of the protein will be locked and unlocked at 10-Angstrom spheres (Residues, Waters, and HETs) around and including the substrate. Molecular dynamics will be performed at constant energy (MM3) in water at 300K for 100ps with an equilibration time of 0.5ps. After the completion of molecular dynamics, water molecules will escape from the protein. Thus, in order to keep the water molecules in the protein, molecular dynamics will be performed again but at 200K. Three dimensional projections and trajectories will be observed and will be saved at 50ps. The substrate and water molecules should be within the 8-Angstrom spheres (Residues, Waters, and HETs). The substrate will be deleted from the protein and the remaining residues will be grouped to prepare the protein into an active site. Both the models will be then evaluated in Ramachandran plot to check whether the backbone angles for both the models are present in the allowed region.
Preparation of Ligands:
The sets of different potential ligands will be prepared with their partial negative charge by using the CAChe software programme Version 7.4 and 7.5 (Manufacturer: FUJITSU, Poland).
The project leader mode of this version of CAChe programme will be used to dock the prepared ligands within the active sites of the models. The potential ligands will be then docked into the active sites of the models thrice, to obtain better docking scores and docking poses. The highest low (negative) energy in kilo joules per mole will exhibit a balanced system and expected binding interactions.