Chemistry of Antibiotics: Cell Wall Synthesis

Introduction

In this post we will continue our exploration of the chemistry of antibiotics with a look at drugs that interrupt wall synthesis and how bacteria develop resistance to these drugs.

The target of these antibiotics is crucial because it explains how antibiotics can attack bacteria without affecting human cells. Bacterial cells, unlike animal cells, have cell walls. Therefore, a drug that attacks cell walls will not be able to impact human cells. Bacterial cell walls are essentially a peptidoglycan layer that is composed of units of peptides (proteins) and glycans (sugars). This layer is the primary and most important component of the cell wall.

β-lactam Antibiotics

The most common class of antibiotics that interferes with cell wall synthesis are the β-lactam antibiotics, which

include penicillin. These types of antibiotics function by impeding the synthesis of the peptidoglycan of the bacterial cell wall. There are multiple ways to inhibit the synthesis of the peptidoglycan layer but the most common is by means of destroying the enzymes that do so. If the antibiotic does not function by destroying synthetic enzymes, it could also destroy the enzymes that convert the polymers into a layer of the cell wall. β-lactam antibiotics inhibit cell wall synthesis by preventing the assembly of the peptidoglycan layer. This is done when the antibiotic competes with the polymer at the site of binding.

This video gives a general overview of how peptidoglycans are inhibited by the mechanisms of a beta-lactam antibiotic:

 The Chemistry of Penicillin

 

Penicillin is one of the most common β-lactam antibiotics, and is characterized by three structural requirements: one fused β-lactam structure, a free carboxylic acid group, and one or more substituted amino acid side chains. Penicillin’s structure prevents the cross linking of peptide chains – weaker cell walls will allow water to flow into the cell freely and the cell will swell and burst, causing cell death. If you change the side R-group chain on the penicillin, the penicillin can possess different properties, such as acidity or penicillinase resistance.

The β-lactam ring in the penicillin reacts with an enzyme that is used for building the cell wall called DD-transpeptidase. Resemblances between a segment of the penicillin structure and the backbone of a peptide chain of the bacterial cell wall have been used to explain the mechanism of beta-lactam antibiotics.

Bacteria Fighting Back

Bacteria have a number of ways to evolve and beat our human antibiotics. One method that bacteria employ in order to fight drugs that interrupt cell wall synthesis, is changing the structure of their cell walls. One instance of this is mutation of pathogenic bacteria. Bacteria that colonize the mucosal pathways, such as nostrils, lips and eyelids, must undergo changes in order to avoid the antimicrobials of the host. In particular, they must avoid lysozyme, an enzyme that destroys the cell walls of bacteria. The activity of lysozyme is similar to β-lactam antibiotics since they both attack the cell walls of bacteria. Since the muramidase activity of lysozyme leads to hydrolysis of the β-1,4 glycosidic bond between the C-1 carbon of N-acetyl muramic acid (MurNAc) and the C-4 carbon of N-acetylglucosamine (GlcNAc), that is the bond that the bacteria have to change in order to survive.Streptococcus pneumoniae and bacillus anthracis, two lysozyme resistant bacteria, have peptidoglycan cell walls that are N-deacetylated, which means that the composition and structure of the sugar component of the cell wall is changed. It was shown that N-acetylating them caused them to be more susceptible to lysozyme. Therefore, it was concluded that N-deacetylating the caused lysozyme resistance. This shows that through changes in structure, bacteria can evade agents that inhibit their cell wall synthesis. When this happens, we too must evolve and change our tactics. This is why new drugs always need to be developed. Bacteria develop resistance, and we must develop new antibiotics.

Want to know more about different classes of antibiotics that inhibit cell wall synthesis? Check out thislink to learn about glycopeptide antibiotics. Of course that’s not enough to satisfy the hunger of your mind, so here is a website that goes further in-depth regarding the processes of cell wall synthesis.

In this post we will discuss antibiotic resistance and how different ‘superbugs’ are being created because of continued use of antibiotics.

Antibiotic resistance is the ability of bacteria to resist the effects of an antibiotic. This usually occurs when bacteria change in some way that reduces or eliminates the effectiveness of drugs, chemicals, or other agents designed to cure infections. These bacteria survive and continue to multiply, causing more harm. This resistance can cause significant danger for people who have common infections that were once easily treatable with antibiotics. Every time a person takes antibiotics, sensitive bacteria are killed, but resistant germs may be left to grow and multiply.

Here is an interesting TED Talk by Dr. Karl Klose, which describes the ‘superbug’ and how different bacteria evolve into ‘antibiotic-resistant menaces’.

The big question is how bacteria become resistant to antibiotics. There are two answers to this question. One way that bacteria become resistant to antibiotics is by mutations in DNA. These mutations occur spontaneously and can have two different effects. A change in DNA of bacteria could lead to the prevention of bacterial protein synthesis. In our previous post we discussed how antibiotics target and attack these proteins. However, if the bacteria is active without the protein, there will be nothing for the antibiotics to attack. Mutations causing variations in the shape or size of the protein prevent antibiotics from being able to properly bind to the target protein. Another change in the DNA of bacteria could lead to the overproduction of protein enzymes. This overproduction could lead to various issues. Firstly, a particular dose of antibiotics would not be able to deactivate an overproduction of protein enzymes. Furthermore, some of the proteins produced may be alter the permeability of the cell membrane of cell wall of the bacteria. Aside from spontaneously occurring mutations in the DNA of bacteria, bacteria can become resistant to antibiotics as a result of gene borrowing. According to microbiologist Doctor John Turnidge,  “They’re the original life forms almost, so for thousands of millions of years they’ve had a chance to work out ways to survive and one of those is to borrow genes from other bacteria to survive.” Antibiotic resistance can result from DNA mutations or gene sharing among bacteria. The image below explains how the population of antibiotic resistant bacteria has increased tremendously. For more information regarding the process and causes of antibiotic resistance, check out this website. 

Since the advent of penicillin in the 1940s, antibiotic use has been steadily increasing. Antibiotics are used far more than necessary; they are used as a precautionary measure ‘just in case’ or as a comfort to patients who have viral or fungal infections. According to the CDC, 50 million of the 150 million prescriptions for antibiotics annually are unnecessary. Doctors would rather sign off on a prescription than spend the extra time trying to educate patients on the strengths and weaknesses of antibiotics. In addition to that, increasing use of hand sanitizer and antibacterial cleaning agents also contributes to the development of superbugs. The excessive use of antibiotics has already started to have devastating effects. But why? In the body, antibiotics kill enough bacteria so that the body can finish the job. Shouldn’t more antibiotics just kill more bacteria? It would seem that we should always take antibiotics and heed the adage ‘better safe than sorry’, right? Wrong.

That type of thinking will only drive us towards the edge faster, and here’s why: As soon as researchers develop new antibiotics, there are always already bacteria with the capabilities to defeat them. These resistant bacteria survive the antibiotic agents to multiply and divide, passing on their antibiotic resistanceto their progeny. As a result, the same antibiotics become less effective with time and it becomes harder and harder to develop antibiotics for these so called ‘superbugs’. The most famous strain of bacteria that has risen due to increasing antibiotic use is methicillin-resistant Staphylococcus aureus, or MRSA.

The CDC is currently working to reduce the amount of antibiotics used, because unchecked, it could become a serious public health issue. Already, 2 million people in the US are infected annually by antibiotic resistant bacteria. Should antibiotics continue to be prescribed at such an alarming rate, we could face a post-antibiotic age, in which antibiotics have been rendered useless.

Bringing Antibiotic to the Counter: What Does It Take To Produce Antibiotics?

So far, we’ve talked a lot about antibiotics. But, how did they end up at your neighborhood pharmacy? Well, read further and you’ll learn about what it takes to produce the medication that helps you feel better when you’re not well.

The antibiotic production industry is definitely a lucrative one. Maybe that explains the 10,000+ antibiotics on the market today. Despite their abundance of variety, the production of them is not the simplest. A part of antibiotic productions involves a process called fermentation. However, the processes that occur differ depending on the type of desired antibiotics. It makes sense that there are different processes for antibiotic topical ointment and swallowable tablet antibiotics.

The first step in producing antibiotics is research and testing. This part of production is long-lasting and costly. This is because it requires thousands of organisms. Sometimes, the organism found to produce antibiotic compounds has already been discovered and its back to the drawing board. If the organism being tested produces an original antibiotic compound, a lot of clinical testing and federal regulation and approval is involved.

Fermentation

But, how do we extract this antibiotic compound from the organism on an industrial scale? This is where the fermentation process becomes essential to the production of antibiotics. In sterile conditions, the organism is grown and the antibiotic agent it produces is isolated. Some raw materials are used to create what is referred to as the fermentation broth. This broth is like a bath for the antibiotic producing organisms to grow in. It is composed of some carbon-compound such as molasses or soy meal to act as nutrition for the organism. These compounds are especially significant because they contain both lactose and glucose. Additionally, ammonia is added in order for the organism’s metabolism to run more efficiently. To regulate organism growth, water soluble salts such as zinc, iron, sulfur, copper, phosphorus and magnesium are added. However, another issue arises as the broth is made. The fermentation broth begins to foam. To counteract the foam, compounds containing silicones or lard oil are used.

The figure above puts the entire process into a visual display of fermentation that provides a more coherent idea of the production of antibiotics.

 

Post-Fermentation

After the fermentation process, the broth is allowed to settle for three to five days, as this is when there will be a maximum amount of antibiotics. From here, the broth must be isolated and purified, whether it be through ion-exchange methods for water-soluble antibiotics or solvent extraction methods for oil-soluble antibiotics. At the end of both purification methods, the antibiotic yielded is in a purified powder that can be further refined into different products. For example, they can be made into solutions for IV bags or syringes, solid form for capsules and pills, and even a ground powder for topical ointments.

 Preparation of Antibiotics from Purified Powder

To make the IV bag or syringe, the antibiotic is dissolved into a solution so that it can be administered directly through a vein in the body. Capsules can be created by filling the bottom half of a capsule with the powdered form and closing it off with the top half of the capsule for oral administration. Finally, for ointments and topical medicine, the antibiotic powder itself is mixed with the ointment, whether it be in cream, gel, or lotion. After this final manufacturing, the melting point and pH are tested throughout shipment to ensure quality control and purity.

Neosporin and pills are common forms of antibiotics made from purified powder to create topical ointment and capsules.

Kinetic Instability of Fermented Substances

Although the fermentation process is efficient, the isolation of the antibiotic itself from the chemical solution is very inefficient because the antibiotics are extremely unstable. In fact, the half-life of thienamycin, one of the most potent natural antibiotics known to man, at a pH of 6-8 is approximately 0.3 to 6 hours, varying on the conditions applied to the solution. Additionally, olivanic acids have a chemical degradation half-life ranging from 4 to 27 hours. This kinetic instability makes it quite difficult for this process to run efficiently, but research is being conducted on making this process more efficient and useful for human consumption and the antibiotic industry as a whole.

Future of Antibiotics

So now that we’ve taken a look at how antibiotics are produced today, let’s see what can be done in the future. This really cool journal explains how synthetic biology coupled with rational engineering can help pharmaceutical engineer go beyond the status quo.

Mutagenesis is a process in which organisms’ genetic information is altered to the point where the organism is still living. This alteration results in a mutation because of its exposure to mutagens. These mutations actually work to produce antibiotics more efficiently. No wonder they there are plans to bring back.

This figure shows how the process of industrial antibiotic manufacturing has evolved over time.

Still interested? Of course you are. For some more information on the processes of antibiotic manufacturing check out the following links:

The Synthesis of Antibiotics

Industrial Antibiotic Production (download)

Antibiotic Alternatives

In our group’s blog posts up until now, we have been discussing different aspects of antibiotics from a chemical standpoint. Now we will cover alternatives to antibiotics. Alternatives to antibiotics are important because the overuse of antibiotics is driving us to a post antibiotic age where nearly all bacteria will be resistant to antibiotics, rendering them useless. Continuing to use antibiotics at this rate will only accelerate us faster towards a post antibiotics age. We must look into alternatives so we can avoid a crisis.

Antimicrobial Peptide Chains

In this article, the prospect of using antimicrobial peptide chains is explored. Researchers at the Fraunhofer Institute for Cell Therapy and Immunology have identified 20 of these amino acid chains that are lethal to microbial organisms. They were even successful in inhibiting the growth of methicillin resistant Staphylococcus aureus (MRSA). The polypeptides contain enough cationic amino acids to give them a net positive charge. This net positive charge attracts them to and helps them penetrate through the cell wall of bacteria. These peptides begin to work within minutes and at a concentration 10x less than traditional antibiotics. They do not affect plants and humans because we have cholesterol in our cell membranes. Not only does the cholesterol act as a stabilizing agent, but it lessens the effect of the electrostatic interactions that the peptide chains rely on to enter the cell.

Examples of common antimicrobial peptides.

Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs)

PPMOs are a specific type of peptide chain that has also shown promise as an alternative to traditional antibiotics. A PPMO is a peptide conjugated phosphorodiamidate morpholino oligomer. It is a peptide chain that has been engineered as an analog to a DNA or RNA chain of the target organism. As a result, it has the capability of silencing the expression of certain genes. PPMOs are much more effective than traditional antibiotics and they work in cases where the organisms have become resistant to traditional antibiotics. This is because their mechanism is completely different. Instead of interfering with cellular functions, these antibiotic alternatives strike by going directly for the DNA.

Acyldepsipeptides

While many common antibiotics attempt to interrupt cellular function – inhibit protein synthesis, cell wall synthesis, or even nucleic acid functions – in bacterial cells, research has been done on new techniques of attacking bacteria cells. Researchers at Brown University and Massachusetts Institute of Technology have discovered a way to make the already effective acyldepsipeptides (ADEPs) even more potent by making these ADEPs more rigid. By swapping out many amino acids in the naturally occurring ADEPs, experiments were performed to see which structure would be the most rigid and robust. To test this experiment, researchers placed these molecules in a deuterium solution, or a solution of hydrogen atoms with one extra neutron. The deuterium atoms replace the hydrogen atoms on the ADEP itself (click on this link for more information on the chemical reaction!). A rigid molecule is considered one that has strong intramolecular forces, and if the rate of the deuterium-hydrogen atom reaction was too slow, the intramolecular forces were considered strong, making the molecule rigid.

A regular acyldepsipeptide (pictured on the left) and the various concentrations of bacteria in solution. A modified acyldepsipeptide (pictured on the right) functionalized with two methyl groups make the molecule much more rigid.

Watch this video for more information on antibiotic resistance!!!

All of these alternatives are very promising and hold a lot of potential for future medications. The common abuse and overuse of antibiotics has taken us to an era in which bacteria are becoming more and more resistant to these antibiotics. These antibiotic alternatives can help us avoid a true bacteria crisis. If we don’t follow certain tips and tricks, common antibiotics will no longer work, and we will have to rely on alternatives similar to antimicrobial peptides, PPMOs, and ADEPs.

Antimetabolites

What are they? Antimetabolites are a class of drugs that interfere with compounds in the cell necessary for metabolism, usually enzymes. They work by tricking the cell into believing they are a different compound found in the cell. The cell will then try to use the antimetabolite for its chemical reactions, however, this gives the antimetabolite a chance to disrupt the cell’s function. The antimetabolite must be similar enough to bind to enzymes of another substrate. The similarity in structure allows the antimetabolite to replace another compound and the differences allow the antimetabolite to either block a reaction entirely, or change its outcome.

And introduction to antimetabolites is provided here.

One of the more common groups of antibacterial antimetabolites are sulfonamides, or sulfa drugs. All sulfa drugs have a sulfonamide functional group bonded to a carbon                                                                                                 ring  bonded to an amine group. This structure are designed to mimic para-aminobenzoic acid. When the process is working correctly, the enzyme dihydropteroate synthetase binds para aminobenzoic acid, or PABA, in a dehydration synthesis to produce an essential compound to bacteria, tetrahydrofolate. Since tetrahydrofolate has no use to humans, it’s an optimal target for an antimetabolite. PABA                                              has an amine group connected to a carbon ring that makes it compatible with dihydropteroate synthetase. The sulfonamide antimetabolite has the same structure, making it compatible with the enzyme. This is how sulfonamides are accepted by the dihydropteroate synthetase. This prevents PABA from binding with the dihydropteroate synthetase. This inhibits bacterial growth, because the bacteria can no longer synthesize a necessary compound, allowing the body’s defense mechanism to take care of the rest.

Sulfonamides consist of the sulfonamide functional group, which is essentially a sulfonyl group connected to an amine group (-S(=O)₂-NH₂). The actual compound of a sulfonamide will have a chemical formula similar to RSO₂NH₂, where R denotes some kind of an organic group (often times using a carbon ring).  Both primary and secondary amines (RNH₂) can be combined with the sulfonyl chloride to create either an N-Substituted sulfonamide or an N,N-Disubstituted sulfonamide.  N-substitution stands for nucleophilic substitution, where an electron nucleophile selectively bonds with or attacks the positive charge of an atom. With a secondary amine, we would have two different instances of this. This difference in electron bonding gives the sulfonamides a wider range of bacteria which they can treat, which only increases the chances of winning the battle against the bacteria.

Antimetabolites, specifically switch antimetabolites, offer interesting insight regarding the metabolic pathways of an organism. When present in a system, antimetabolites can have a profound effect on that system’s chemical kinetics. When switch antimetabolites are ingested though antibiotics, the homeostasis of the body provides positive and negative feedback. The antimetabolite can provide inhibition for positive or negative feedback. The positive feedback of homeostasis refers to the enhancement and acceleration of the process or output which has already been activated whereas the negative feedback of homeostasis refers to the reduction of the process or output to prevent harm to the body.

The chemical kinetics at the site of positive feedback are more thoroughly addressed in this scientific journal. The journal included some surprising results about three different situations.

1. The first and most common occurrence was when the path to synthesis was blocked off at substoichiometric concentration levels of the antimetabolite. In such a reaction with classical metabolites, inhibition would not occur unless a minimum stoichiometric concentration of antimetabolites was present.
2. Second, inhibition was at an “all or nothing” point at specific concentrations of the antimetabolite ranging from substoichiometric to superstoichiometric values. This means the when the antimetabolite is present, the inhibition either occurs fully or not at all at random points whereas in classical metabolites, change in inhibition is continuous meaning that it partially inhibits relatively predictably at various values. However, as concentration of switch antimetabolite rose, the inhibition rate would rise and fall sharply.
3. Finally, the inhibited system remained inhibited even after the antimetabolite was removed from the system. In a classical case, the rate of concentration change of the antimetabolite in a system should not be altered once the inhibitor has been removed.

This experiment exemplified the variations switch metabolites and classical metabolites have on the kinetics of an inhibiting reaction throughout the process of homeostasis.