Design Of Novel Penicillins Biology Essay

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The discovery of penicillin in 1928 by Alexandra Fleming was a virtue to medicinal chemistry. It took a decade for a group of chemists including Dr. Abraham, Dr. Heatly, Dr. Chain and Prof. Florey to accomplish purifying the unstable antibiotics. Finally on the 12th of February 1941, penicillin was tested for the first time and its effectiveness in humans was proved

New technologies were implemented for the development of penicillin on a large scale. Many problems related to its fermentation, strain improvement and large scale sterile aerobic submerged fermentation were solved. Ultimately, the domain of penicillin was developed; this includes orally active penicillin, broad spectrum and enzymatically stable penicillin's later creation of beta-lactamase stable penicillin and beta-lactamase inhibitors (Simmonds, 1992).

Ampicillin and Amoxicillin are two drugs which can be administered orally; ampicillin is well absorbed and its oral effectiveness for regular infection can be useful in the preparation of prodrug. Ampicillin however is always linked to drug-induced rash. Clavulanic acid is a beta-lactamase inhibitor which is sometimes combined with penicillin group antibiotics to overcome certain types of antibiotics resistance; it is used to overcome resistance in bacteria that secrete beta-lactamase which otherwise inactivates most penicillin Flucloxacillin has activity against beta-lactamase producing organisms such as Staphylococcus aureus as it is beta-lactamase stable. However this drug is ineffective against Methicillin-resistant Staphylococcus aureus. Like other beta-lactam antibiotics, Flucloxacillin acts by inhibiting the synthesis of the bacteria cell wall (Nagele, 2005, Alhamami, 2003).

Homology or comparative modelling uses experimentally determined protein structures to predict the conformation of another protein that has a similar amino acid sequence. Generally, the process of homology modelling involves four steps - (a) fold assignment, (b) sequence alignment, (c) model building and (d) model refinement. The next stage in the homology modelling process is the model-building phase (Flower, 2002, Bugg & Ealick, 1990).


Although penicillin was discovered in 1928, and is a member of β-lactam antibiotics, it was not until 1942 that the term β-lactam antibiotic was registered in the dictionary of medicinal chemists. The structure of penicillin was eventually isolated, and its clinical effectiveness was first tested on 12th February 1941 in the form of a sodium salt.

Thus, long after antibiotics projected its appearance on the screen of research, the structure of penicillin was determined.

Scheme 1: Foye, Lemke & Williams, 2002 showed the structure of Penicillin.

Penicillin's can be considered as an amido derivative of the 6-aminopenicillanic acid.

Scheme 2: Foye, Lemke & Williams, 2002 showed the structure of 6-Aminopenicillanic acid (6-APA).

In the basic skeleton, a thiazolidine ring is fused with a beta-lactam ring which is a 4 membered cyclic amide. Various penicillin structures differ from each other in antibacterial and pharmacological characteristics due to variation in the structure of acid moiety of the amide side chain at C-6. For example, penicillin G (where R = C6H5CH2-) remains extremely effective after 45 years of clinical use and is the only natural penicillin used clinically. Acylation of 6-APA with an appropriate carboxylic acid resulted in new penicillin's, some of which were broad spectrum antibiotics (Simmonds, 1992).

Structure Activity Relationship:

All beta-lactam antibiotics contain a 4 membered beta-lactam ring which is fused through the nitrogen and tetrahedral carbon atom to a second heterocyclic ring. A difference in the structure of the second heterocyclic ring leads to sub-divisions of beta-lactam antibiotics. For example,

Penicillin consists of β-lactam ring fused with thiazolidine.

Thienamycin consist of β-lactam fused with a pyrroline ring.

Clavulanic acid consists of β-lactam fused with an oxazolidine ring.

A carbonyl group attached to the lactam nitrogen is a common feature of all above classes. In fact, the higher the instability of the molecule, the better is the activity of the drug. Natural penicillin has a relative instability in acidic or basic mediums. Thus natural penicillin is susceptible to degradation under acidic or basic condition; certain strains of microorganism carry β lactamase enzymes that inactivate the drug by hydrolysis. Many analogues have been synthesized in order to overcome these clinical deficiencies prevailing in natural penicillin. The main principles behind this drug design were the manipulation of polar amide side-chains; variations in this moiety resulted in differences in antibiotics potency and in physio-chemical properties including stability.

Introducing chemical inducers in the culture medium can vary the nutritional composition of the growth medium. This change in nutritional composition can induce mutational change in the strain of micro organism. Deliberate mutational changes are one of the few tools employed to increase both quality and quantity of antibiotics, for e.g.

6-Aminopenicillanic acid is produced in large quantities with the aid of an amidase from penicillium chrysogenum, and the culture medium is fed with the chemical inducers (e.g. Phenyl acetic acid) in order to achieve the predominance of the desired antibiotic.

The stability of benzyl penicillin can further be increased by substitution of an electron withdrawing group at α-position of benzyl penicillin. E.g. the α-amino benzyl, α-halo benzyl and phenoxy-methylpenicillin are significantly more stable than benzyl penicillin towards acid catalysed hydrolysis.

All of the natural penicillin is strongly dextrorotatory.

The free penicillin is not suitable for oral or parental administration. Penicillin hence utilised in the form of their sodium and potassium salts. For the depot preparations, penicillin's is treated with organic bases such as procaine, benzatine or hydrabamine. These organic salts have limited water solubility and hence can thus release the drug over a long period.

Along with the older ring systems, some new basic skeletons have appeared through fermentation screening programs (Ahluwalia & Chopra, 2008)

Evolution of the series of β-lactam antibiotics:

The β-lactam antibiotics are cell-wall inhibitors towards susceptible bacteria, but cannot kill or inhibit all bacteria; for example penicillin G (Benzyl Penicillin) has fairly narrow antibacterial spectrum. In particular, fungi and many gram-negative bacteria are relatively insensitive to this agent. Beta-lactam is readily hydrolysed by the enzyme penicillinase. The pronounced susceptibility of the β-lactam to penicillinase is to hinder early progress in working with penicillin.

Following the realization that the presence of phenyl acetic acid in the fermentation leads to predominance of the product, benzylpenicillin a wide variety of other acids were added to the growing culture. Thus the second generation, known as semi-synthetic penicillin's were born. The proper design of the side-chains has thus served not only to overcome many of the drawbacks of the early penicillin (like enzyme susceptibility, acid liability and lack of oral activity) but has also helped to develop broad spectrum antibiotics. The involvement of acyl carbon of the side-chain in the hydrolytic cleavage of β-lactam bond was recognised, and improvements in clinical qualities were then achieved by creating steric hindrances to this acyl carbon, thus making it less reactive. The fact was attested by the attachment of an aromatic or heterocyclic ring directly to the amide carbonyl, thus affording antibiotics with increased penicillinase resistance e.g. Methicillin, Oxacillin family and Nafcillin.

One of the most successful penicillin candidates is Ampicillin, and which is a prototype of third generation β-lactam antibiotics. It is characterised by increased oral activity than its precursor. The early observation that drugs acylated by amino acids has a somewhat greater oral activity turned to be an inspiration behind its development. Thus, the addition of an amino group to benzylpenicillin also led to a broadening of spectrum of activity along with an increased oral activity. The next congener is Amoxicillin. The amino penicillin still suffers from the major drawback (i.e. susceptible to β-lactamase enzymes) and are thus ineffective for most staphylococcal infections. In an attempt to improve the pharmacodynamics characteristics further, a prodrug development program of Ampicillin was undertaken. As a result bacampicillin, hetacillin and talampicillin appeared through screening.

The fourth generation of β-lactam add to the list of chemotherapeutic agents in the form of carboxypenicillins. Carboxylic acid group if introduced into primary amine moiety of Ampicillin and other allied skeleton may significantly affect the biological spectrum of the lead, was noted seriously and served as an impulse in the generation of this class. The examples are Carbenicillin family and Ticarcillin.

A parallel observation is also registered stating that the acylation of the amino group of ampicillin broadens the antimicrobial spectrum of the prototype drug. Azlocillin is a front line drug in this series which was named under ureidopenicillins. Recently, new impetus has been added to the chemotherapy by the discovery of a new ring system in fermentation liquors.

New ureidopenicillins derivatives show an expanded gram-negative spectrum and an increased potency against many enteric bacilli. The ureidopenicillins all possess significant stability to β-lactamase. Despite the enormous efforts expanded during the past three decades, the β-lactam antibiotic field still remains a field of severe competition within itself. The new drug coming up differs from benzyl penicillin (natural penicillin) in one or more of four properties: (a) acid sensitivity, (b) susceptibility to inactivation by penicillinase, (c) antibiotic potency and (d) spectrum of antibacterial activity.

When the acid stability of penicillin is increased, the drug is not destroyed by gastric acid and can thus be orally administered. In general, the acid-stable penicillin is a less active agent than the benzylpenicillin. The penicillinase-resistant penicillin is not hydrolysed by the enzymes produced by Staphylococcus aureus, and is hence effectively used to treat infections caused by resistant strains of microorganisms (Foye, Lemke & Williams, 2002).

Mechanism of bacterial resistance to β-lactam antibiotics:

It should be noted that penicillin resistance may not always be due to penicillinase production even among the Staphylococci. Certain other mechanism of resistance can be operative such as:

A change in antibiotic target site, which may not be vital for microbial survival, thus resulting into drug resistance.

Inability of the agent to penetrate into the site of action.

A reduction in cellular permeability to the antibiotic.

The antibiotic agent, instead of attacking the microorganisms, may be utilised to antagonise a biochemical intermediate released by microbes.

A sensitive strain may undergo mutational change and thereafter acquire resistance to the antibiotic agent.

Thus penicillin resistance develops into sensitive strains of microbes due to single or combination of mechanisms mentioned above (Foye, Lemke & Williams, 2002).

Penicillinase-Resistant Oral Penicillin:

Isoxazolyl penicillin is produced by replacing isoxazolyl ring for the benzene ring. Methyl and substituted benzene ring is replaced on the other side of the methoxyls of methicillin. They are oxacillin, cloxacillin and dicloxacillin. Chemically they are distinct from each other by number of chlorine atoms present on the benzene ring. Oxacillin, dicloxacillin, cloxacillin efficacy is very less than benzylpenicillin against gram positive microorganisms as they do not create beta-lactamase, but their efficacy is maintained against those that do. As they are stable against acid, they can be administered orally. But isoxazolyl penicillin cannot be used for treatment of septicaemia as they are highly serum protein bound. Microorganisms are resistant against isoxazolyl penicillin as well as methicillin. For treatment of osteomyelitis, septicaemia, endocarditis and CNS infection in Staphylococcus aureus methicillin, naficillin as well as isoxazolyl group is utilized (Jarlsv. et al, 1988).


Flucloxacillin or floxacillin is a narrow spectrum beta-lactam antibiotic of the penicillin class. It is used to treat infection caused by susceptible gram positive bacteria. Unlike other penicillin, flucloxacillin activity against beta-lactamase producing organisms such as Staphylococcus aureus as it is beta-lactamase stable. However it is ineffective against methicillin-resistant Staphylococcus aureus. It is very similar to dicloxacillin as these two agents are considered interchangeable. Like other beta-lactam antibiotics, flucloxacillin acts by inhibiting the synthesis of bacteria cell wall. It inhibits cross-linkage between the linear peptidoglycan polymer chains that make up a major component of the cell wall of gram positive bacteria. Flucloxacillin is insensitive to beta-lactamase enzyme secreted by many penicillin-resistant bacteria. The presence of the isoxazolyl group as the side chain of the penicillin nucleus facilitates the beta-lactamase resistance, since they are relatively intolerant to side chain steric hindrance. Thus it is able to bind to the penicillin binding protein and inhibit peptidoglycan cross-linking, but it is not bound to inactivated beta-lactamase.

Flucloxacillin is more acid-stable than many other penicillin and can be given orally, in addition to parental routes. However like methicillin, it is less potent than benzylpenicillin against non-beta-lactamase producing gram positive bacteria. Flucloxacillin and dicloxacillin has similar pharmacokinetics and anti-bacterial activity and the two agents are considered interchangeable. It is believed to have higher incidence of severe hepatic adverse effects than dicloxacillin but a lower incidence of renal adverse effect (Jarlsv. et al, 1988).

Scheme 3: Foye, Lemke & Williams, 2002 showed the structure of Flucloxacillin.

Penicillinase-Sensitive, Broad-Spectrum, Oral penicillin:


Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infection. It is considered as a part of the aminopenicillin family and is roughly equivalent to amoxicillin in terms of spectrum and level of activity. It can sometime result in non-allergic that range in severity from a rash to potentially lethal anaphylaxis. Belonging to the penicillin group of beta-lactam antibiotics, ampicillin is able to penetrate gram positive and some gram negative bacteria. It differs from penicillin only by the presence of an amino group. That amino group helps the drug penetrate the outer membrane of gram negative bacteria.

Ampicillin acts as a competitive inhibition of the enzyme transpeptidase. Transpeptidase is needed by bacteria to make their cell wall. It inhibits the third and final stage of bacteria cell wall synthesis; which ultimately leads to cell lysis. For infections like pneumococcal, streptococcal and meningococcal ampicillin is used as it is analogous to benzylpenicillin. Ampicillin can be administered orally and it responds well to treatment of many strains of gram negative bacteria like Salmonella, Shigella, Proteus mirabilis, Escherichia coli, Haemophilic influenza and N. Gonorrhoea. Ampicillin is basically used for complex community-acquired urinary tract infections (Alhamami, 2003).

Scheme 4: Foye, Lemke & Williams, 2002 showed the structure of Ampicillin.


Amoxicillin is a moderate-spectrum, bacteriolytic, beta-lactam antibiotic used to treat bacterial infections caused by susceptible microorganisms. Amoxicillin is usually the drug of choice within the class because it is better absorbed, following oral administration, than other beta-lactam antibiotics. Amoxicillin acts by inhibiting the synthesis of bacterial cell wall. It inhibits cross-linkage between linear peptidoglycan polymer chains that makes up a major component of the cell wall of gram positive bacteria. Amoxicillin is similar to ampicillin as para-phenolic hydroxyl group is added to the part of phenyl side-chains.

This increases the iso-electric point of the drugs to more acidic value in amoxicillin as its approach somewhat resembles to ampicillin. Thus amoxicillin oral absorptivity is superior (74-92%) than ampicillin and advances to conventionally limited drug-induced diarrhoea. Hence amoxicillin is more accepted in the world as its antimicrobial extent and clinical trials are similar to ampicillin. Amoxicillin is susceptible to degradation by beta-lactamase producing bacteria and so may be given with clavulanic acid to decrease its susceptibility (Nagele, 2005).

Scheme 5: Foye, Lemke & Williams, 2002 showed the structure of Amoxicillin.

Clavulanic acid:

Clavulanic acid is a beta-lactamase inhibitor sometimes combined with penicillin group antibiotics to overcome certain types of antibiotics resistance. It is used to overcome resistance in bacteria that secrete beta-lactamase which otherwise inactivates most penicillin's. In its most common form, the potassium salt clavulanate is combined with amoxicillin or ticarcillin.

Clavulanic acid has negligible intrinsic antimicrobial activity, despite sharing the beta-lactam ring that is the characteristic of beta-lactam antibiotics. However the similarity in chemical structure allows the molecule to act as a competitive inhibitor of beta-lactamase secreted by certain bacteria to give resistance to beta-lactam antibiotics. This inhibition restores the antimicrobial activity of beta-lactam antibiotics against lactamase secreting-resistance bacteria. Despite this, some bacterial strains that are given resistant to such combinations have emerged. Amoxicillin is usually the drug of choice within the class because it is better absorbed, following oral administration, than other beta-lactam antibiotics (Skov, Frimodt-Moller, & Espersen, 2002).

Scheme 6: Foye, Lemke & Williams, 2002 showed the structure of Clavulanic acid.

Recent Literature:

Recently, penicillin was subject to many penicillin binding protein (PBP) analyses. Thus many PDB structures of penicillin's are available in Protein Data Bank database. The selected PDB structures are:

2ex6 - with ampicillin.

2ex8 - with penicillin G.

2ex9 - with penicillin V.

1fxv - with penicillin G in active site.

1pwc - with penicillin G.

Peptidoglycan is important for cell growth and its maintenance under normal condition. Various numbers of validated targets are available for design of antibiotics as the enzyme is engaged in synthesis of peptidoglycan as it is not correlated in mammalian biochemistry. Penicillin and natural bactericidal compounds misuse the survival of bacteria on the cell wall. Penicillin is an essential antibiotic, but the increase in antibiotic resistance has led to create or development of new antibacterial. Thus putting a lot of effort in research of new antibacterial, the biological part of the penicillin binding protein in gram negative bacteria such as Escherichia coli has not been understood yet. Bacteria cell wall can be balanced and maintained by the proteins which are effective in peptidoglycan synthesis, repair and hydrolysis.

Penicillin and beta-lactam are the targets of penicillin binding protein. Part of D-alanyl-D-alanine available on peptidoglycan precursor has identical structure to those drugs which have inhibitory action. Catalytic activity is obstructed when active site serine residue is acylated by beta-lactams. There are total twelve penicillin binding proteins (PBPs) in Escherichia coli, divided in two divisions with high molecular weight and low molecular weight. High molecular weight PBPs has both DD-transpeptidase and transglycosidase activities; while there are seven low molecular weight PBPs but they are not important in Escherichia coli. PBP4s in Escherichia coli is not linked with gram positive bacteria which have similar function to that of PBP5s in Escherichia coli. Genes are absent in Escherichia coli for the entire low molecular weight PBPs, so in order to grow and maintain their survival in rich medium they require PBP1a and PBP1b as they are important in development of peptidoglycan as they have the transpeptidase activity. PBP4s get extremely affected by benzylpenicillin and ampicillin therefore PBP4 active site has to be different from other PBPs. Low molecular weight PBPs are divided into three divisions A, B and C. PBP4 belongs to group C, thus it has three small parts of sequences seen in PBPs and beta-lactamase. PBP4 has no crystallographic model in PBP class determined yet. But crystal structure of PBP from Actinomadura R39 is an analogous to PBP4 structure which is determined as a first model. Hence the first high resolution structure of PBP4 is presented in both, absence of substrate and covalently related to five distinct antibiotics are analyzed with different PBPs (Kishida. et al, 2006).

Beta-lactam is the most essential protection system against bacterial infection. But the bacteria such as D-alanyl-D-alanine transpeptidase destroy beta-lactam. As the bacteria get more prone to antibiotic resistance there is a demand in development of new anti-bacterial drugs. Hence the beta-lactam is unreliable even after years of research. It was suggested that DD-peptidase imitate D-alanyl-D-alanine which is a part of peptidoglycan substrate so beta-lactam prohibit DD-peptidase. Peptidoglycan-mimetic side-chains possessed by beta-lactam were also estimated as an exceptional antibiotic than non-specific counterparts but this research failed due to lack of evidence. Thus two novel beta-lactams were discovered recently, the first is cephalosporin with similar side chain and second is penicillin which possesses glycyl-L-α-amino-ε-pimeyl side chain of Streptomyces strain R61 peptidoglycan which can be used as "perfect penicillin" for organism. Thus the X-ray structure of perfect penicillin was described in non-covalent complex and covalent complex with Streptomyces R61 DD-peptidase. The non-covalent enzyme inhibitor complex with DD-peptidase in crystallography was discovered as a first such complex. However these two covalent complexes were further analyzed by beta-lactams benzylpenicillin and cephalosporin C with appropriate data to prove the peptidoglycan-mimetic side chain can amend beta-lactams as an enhanced inhibitor of DD-peptidase (Silvaggi. et al, 2003).

Penicillin binded to penicillin acylase was examined by X-ray crystallography. The crystals of βN241A penicillin acylase mutant which is in inactive form when saturated with penicillin G, the structure of the enzyme-substrate complex is obtained. Penicillin G sits well into the active site by the help of enzyme when binding of substrates occur which led to a conformational change which allows αF146 and αR145 of side-chain shift from the active site. Thus this result in formation of beta-lactam binding site by side-chains of αF146 and βF71, these side-chains have Van der Waals interactions with thiazolidine ring of penicillin G whereas with the help of two water molecules hydrogen bonding is formed with the side chain of αR145 which is linked to the carboxylate group of ligand. Water molecules act as bridging molecules and form a hydrogen bond between a part of carbonyl oxygen of the phenyl acetic acid and with the backbone oxygen of βQ23. Thus substantial changes in interactions with the beta-lactam substrate are observed. Synthesis of penicillin G from phenylacetamide and 6-aminopenicillanic acid was not successful by αF146Y mutant as it had same similarity for 6-aminopenicillanic acid as the wild type enzyme. The value of αF146 in beta-lactam binding site was analyzed by structural and kinetic studies. This will help in providing enhanced mutants with refined synthetic properties (Alkema. et al, 2000).

Intended Design and Method of investigation:

Homology Modelling:

The majority of drugs available today were discovered either from chance observations or from the screening of synthetic or natural product libraries. The protein structure-based approach relies on an iterative procedure of the initial determination of the structure of the target protein, followed by the prediction of hypothetical ligands for the target proteins from molecular modelling and the subsequent chemical synthesis and biological testing of specific compounds (the structure-based drug design cycle).

Homology or comparative modelling uses experimentally determined protein structures to predict the conformation of another protein that has similar amino acid sequence. The method relies on the observation that in nature the structural conformation of a protein is more highly conserved than its amino acid sequence and that small or medium changes in sequence typically result in only changes in the 3D structure (Flower, 2002).

Generally, the process of homology modelling involves four steps - fold assignment, sequence alignment, model building and model refinement. The assignment process indentifies proteins of known structure (template structure) that are related to the polypeptide sequence of unknown structure (the target sequence; this is not to be mistaken with drug target). Next, a sequence database of proteins with known structures (e.g. the PDB-sequence database) is searched with the target sequence using sequence similarity search algorithms. Following identification of a distinct correlation between the target protein and a protein of know 3D structure, the two protein sequence are aligned to identify the optimum correlation between the residues in the template and target sequences.

The next stage in the homology modelling process is the model-building phase. Here, a model of the target protein is constructed from the substitution of amino acids in 3D structure of the template protein and the insertion or deleting of amino acids according to the sequence alignment. Finally, the constructed model is checked with regard to conformational aspects and is correlated or energy minimized using force field approaches. Homology modelling techniques are dependent on high resolution experimental protein structure data (Bugg & Ealick, 1990).