Wednesday, 6 January 2016

Antibiotics for the 21st century

The discovery of antibiotics initially by Alexander Fleming, was one of the greatest serendipitous successes of Science in the last century. Its rapid translation into a cost effective treatment by Florey and Chain and then by a consortium of US Pharmaceutical companies,  has been an equally remarkable achievement. [It will be interesting to watch how the promise of Stem Cell Biology and Immunotherapy is realised in a similarly affordable manner in this century.] However, warnings are now being issued that seem to suggest that when it comes to antibiotics, we have had too much of a good thing! In fact it has taken less than 50 years since Fleming's agar plates became contaminated with an airborne strain of the Penicillium mold, for pathogenic bacteria to turn the tide once again on the human race! Health leaders worldwide (Sally Davis, England's Chief Medical Officer is shown below) the are now warning of an impending return to the dark days of untreatable infections in developing countries and in the deep-cleaned wards of UK hospitals! So, let's remind ourselves of just what antibiotics are, how they act, what they act on and what they don't act on, and the molecular and cellular mechanisms underlying this worrying phenomenon of antibiotic resistance.

Image result for sally davisA glossary and some definitions. An antibiotic is, in its simplest terms, a compound (or molecule) that prevents bacterial growth. An effective antibiotic (just like any effective drug), should not impair the normal physiological properties of the individual (or animal, or plant!) taking the antibiotic. That is, it should have no side effects. This is not always easy to achieve and indeed penicillin can be harmful when given to some individuals. Like all medicines, the clinician must weigh the risks of not prescribing an antibiotic to treat an infection, against the possible side effects that might ensue if s/he does prescribe. Pharmaceutical companies today are regulated stringently in respect of the side effects of their products, however (perhaps fortunately, some would say) this wasn't the case in the pioneering stages of penicillin and streptomycin treatment. 

The target of an antibiotic is broadly defined as the cellular process or processes that are inhibited or blocked by the administration of the compound to a patient, or more often in the research and development phase. For example, penicillin (RHS)  acts on the normal assembly of a the bacterial cell wall. In more detail, it acts on one or more of the enzymes needed to complete the biosynthesis of the peptidoglycan barrier that surrounds most bacteria. I shall discuss the range of targets in more detail below. Sometimes clinicians and scientists refer to the mode of action of an antibiotic or drug in general. This is usually a catch-all term to describe a combination of the target and the process(es) that are affected in the bacterium. It should be pointed out that the target is sometimes unknown, sometimes incompletely defined and sometimes there are more than one target molecules. Understanding the mode of action of drugs is generally NOT over when the drugs are approved for clinical use!

Antibiotic resistance is the ability of a bacterium to shrug off exposure to an antibiotic. Some bacteria are naturally sensitive to a particular antibiotic and some are naturally resistant. If a bacterium is sensitive to say penicillin, then its growth will be stalled in the presence of the antibiotic. This inhibition of growth is referred to as a bacteriostatic effect. If the drug goes on to kill the organism, it is said to be bacteriocidal. An understanding of this is at the heart of the recommended dose and the duration of the treatment. These terms and these principles are at the heart of the field of Pharmacology. 

The question you are now asking is how can some bacterial strains be resistant and others sensitive to antibiotics?  Well, there are bacteria found in Nature that can neutralise the effect of antibiotics, and this is one aspect of resistance. However, before I provide some examples, I need to explain something about genes and their intrinsic ability to be altered during every cell division. This process of mutation is a central part of Darwin's theory of evolution by Natural Selection and is the other aspect of antibiotic resistance that we must consider.

Over 200 years ago Charles Darwin articulated the theory of Evolution by Natural Selection. In my view it stands alongside Newton and Einstein's discoveries as one of the greatest scientific achievements ever. However, his ideas were based on visual (at best microscopic) observations. Today, following Watson and Crick's determination of the double helical structure of DNA, and Sanger's development of gene (and now genome) sequencing, we have a pretty good understanding of the molecular mechanisms underlying Evolution. Moreover as those of you who read my Blogs will know, I was particularly delighted to see the award of the 2015 Nobel Prize in Chemistry to three individuals who have been instrumental in elucidating the mechanisms of DNA damage and repair. It is this aspect of molecular evolution that I will try and explain in order to prepare the ground for the discussion on antibiotic resistance.

As every cell divides (our own, or those of the many billions of bacteria in our intestines!) it copies its genome (the quota of chromosomes that encode all of the genetic information required to make and maintain an organism). However, during this process mistakes are made. Such mistakes take the form of single nucleotide changes (an adenine (A) becomes a guanine (G) or a T becomes a C, perhaps). Occasionally these changes can inactivate the product of that stretch of DNA (perhaps an enzyme), more often the enzyme (in this example) is largely unaffected and sometimes its activity is modified: the enzyme may become more or less efficient. This is a key element of Darwin's theory and one of the reasons why we all appear different than our siblings. If this process goes unchecked, it can of course jeopardise the normal function of the cell, and this is why we have evolved mechanisms of DNA repair. In some cancers the mistakes are made at too high a frequency that they exceed our repair capacity.

Barbara McClintock, working on the genetics of variation in maize (corn) plants during the 1950s, discovered another important phenomenon of genetic variation that we now refer to as jumping genes, or Transposable Elements (see RHS). Here pieces of DNA literally jump around the genome and make much more dramatic changes to the phenotype (appearance, in this case) of the organism. This is not too dissimilar to the way in which genetic shuffling occurs during sexual reproduction, when offspring emerge, carrying a mixture of genes from mum and dad. This capacity of genes to jump around a genome (and between cells) has give rise to the expression mobile genetic elements. Bacteria have in some cases developed mechanisms for acquiring genetic material from their neighbours. This is called horizontal gene transfer and often the DNA  transferred is called a plasmid (a small circle of DNA that can copy itself many times when taken up by a bacterium). In an extreme case a virus (or bacteriophage) can be considered a mobile genetic element, but in the case of viruses, they possess the means of infecting cells themselves. Both mobile genetic elements and virus genomes carry one or more encoded functions, but are absolutely dependent on their host cell to provide them with the enzymes needed to complete their biological "journey". Think of the HIV virus, it is biologically inert outside of the host cell, but can turn an otherwise normal cell into a lethal "factory" for virus replication, which can, in extreme cases lead to the death of the individual.

To summarise: genomes change naturally as cells divide. The extent of the change is a product of the point mutations and the rearrangements that occur via transposition and recombination. In some cases changes can be acquired by the acquisition of DNA by horizontal transfer, or by viral infection. Importantly, an organism like man has a typical life expectancy of 80-90 years (in the UK), with cells dividing over hours, days and weeks, depending on the tissue. However a bacterial cell replicates every 20-60 minutes. A single pathogenic bacterium in a rich medium (your blood or your stomach) can reach many millions in a very short period. If you are mathematically minded: the numbers every 20 minutes are 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, 4096, 8,192, etc. (students should plot the graph, left: using semi-log graph paper?). 

The natural changes that occur during cell division, can lead to the acquisition of new genes or the modification of existing genes, both of which have the potential to confer resistance on a bacterial cell. The remarkable image on the left shows two bacterial cells caught in the act of gene transfer! Now, imagine a patient with a bacterial chest infection, a very small number of mutations will arise spontaneously (as described above). After administering the antibiotic, such mutants (as they are known) continue to divide and spread through the now dwindling population of sensitive bacteria. After a while, the infection becomes dominated by resistant cells and the infection takes hold. At this point there is  no choice other than to let events take their course, and hope the patient has a robust immune system. Alternatively, the doctor may administer a last resort antibiotic, for which resistance is either very uncommon or has never been observed (more on this later).

Let's now look at the targets of antibiotics in a little more detail. Antibiotics can 
be classified in many ways. One of the most common ways is to refer to their chemical structure. Penicillin and its derivatives are referred to as beta lactams (see my molecule of the month here), owing to the central "ring" element at the core of the molecule. Similarly the class of drugs that includes ciprafloxicin (RHS), are referred to as "quinolones" again, a consequence of their structure. Sometimes the class of drug relates to the target. Penicillins generally inhibit cell wall biosynthesis and quinolones, for example inhibit the enzyme DNA Gyrase, which normally allows bacteria to complete chromosome replication and segregation, just before cells divide. The key to understanding antibiotics is to recognise that they selectively inhibit essential molecular processes in bacteria, without interfering with related processes in man. For example, mammalian cells don't possess peptidoglycan cell walls, but for bacteria these are essential for cell viability. The bacterial ribosome, like its human counterpart is the site of protein synthesis, however, despite considerable biochemical similarities, there are some key differences that enable compounds like tetracycline and chloramphenicol to block bacterial protein synthesis, leaving human protein synthesis unaffected.  
The diagram above taken from an educational resource for orthopaedic clinicians captures the targets of many of the common antibiotics in use today. As you can see there are three broad modes of action of antibiotics. If we look in a little more detail the cell wall synthesis category, which comprises beta lactams and cyclic peptides (vancomycin and the polymyxins). The beta lactam group act via a completely different mechanism to, say, polymyxins. The latter seem to combine a cell wall synthesis inhibitory action with a detergent-like effect, that involves disruption of the cell membrane which in turn involves a metal ion displacement action. I mention the poymyxins since they include colistin, and recently I commented on the emergence of resistance to colistin, which is considered to be a last resort antibiotic. Prescriptions for polymyxins have been kept in close check since the 1980s, in order to retain their value in treating patients in particularly challenging situations. However,  the emergence of colistin resistance via uncontrolled use in the Chinese farming community, threatens to exacerbate the problem. [Resistance mechanisms are less likely to arise if a drug (like colistin) acts on multiple molecular targets].

What does the future hold for the development of new antibiotics? The three key targets for current antibiotics are relatively easy to rationalise: they are all fundamental processes that underpin cell division: no cells without a cell wall, a genome or proteins! Importantly the molecules in bacteria that catalyse these functions are sufficiently different to our own enzymes, that we are largely unaffected by antibiotics (see above). Let us consider the highly selective targeting of genome replication a process that in bacteria is dependent on the DNA gyrase enzyme. This is the site of action of the quinolone antibiotic ciprofloxacin (top LHS is an image of DNA gyrase with the drug bound in green and the DNA in orange). If the gene encoding DNA gyrase acquires a random mutation that changes a key amino acid in the ciprofloxacin interaction site, it will only be a matter of time before this (now resistant) bacterial strain spreads through the now, global route of transmission; and the problem of resistance becomes suddenly much worse . One strategy might be to design a new generation of antibiotics with multiple targets (along the lines of colistin). The probability of mutations arising simultaneously in two or more genes that confer resistance is much lower (but not impossible). 

Slava Epstein, right, talks with PhD students, from left, Brittany Berdy and Violetta Medik in Epstein's lab at Northeastern University.Unfortunately, however, one of the major difficulties in developing new antibiotics is our own fundamental ignorance of the molecular basis of life! Fortunately, however, it is clear that microbes themselves are capable of destroying other bacteria. Indeed, have any antibiotics emerged from what we call "rational drug design"? Not to my knowledge: they are all based on "Natural Products" from microbes (including fungi). We may of course, be able to extend their usage by making small chemical modifications to the natural beta lactams say, or we can go back to Nature and look harder. You may have read about the recent excitement over the "hope" that non-culturable (or non-cultivable) microbes might yield new classes of antibiotics. I particularly enjoyed the article from the popular medical news forum STAT, who sent a reporter to Professor Slava Epstein's lab (above) at North Eastern University in Boston. If you want to see what a patent in this area looks like and how Epstein views the successes, failures and prospects for antibiotic discovery take a look here. I think he summarises the issues, albeit at a high level, very clearly and Science have made his original 2002 publication freely available online. It seems to me that we literally can't live without antibiotics and so we need "all hands to the pumps" to find some new ones. And moreover, I don't believe this would require an unreasonable level of government and industry backed investment to re-ignite this vital area of research.

Of course, apart from the scientific challenge of generating a "pipeline" of new antibiotics, there is also the economic challenge. If you would like to read more about this, a recent review of the current state of the AMR challenge (the abbreviation increasingly used by the press, for antimicrobial resistance) for the Pharmaceutical sector is presented here, with some recommendations. There are some similarities to the challenges that fac the development of  new Tropical Diesease drugs, and the Longitude Prize initiative is an attempt to perhaps mimic the success of the Gates Foundation in overcoming the "Big Pharma Inertia" to investment in such (less profitable) areas of drug discovery. I hope this has been of interest and if you are interested in reading more about antibiotics I suggest you get up early, get comfortable and simply type antibiotics into Google!

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