Saturday, 2 August 2014

Personalized medicines, allergy and a renaissance for thalidomide?

This week's announcement that funding will be made available to sequence 100,000 cancer genomes, comes after the announcement two years earlier that 100,000 individuals would have their genomes mapped in Saudi Arabia. The logic is that an knowledge of the complete genome sequences of large cohorts of both healthy and sick individuals will significantly improve clinical decision making in respect of the prescribing of drugs. This approach has become known as personalized medicine. Currently the treatments available for serious illnesses are administered on an empirical basis, using patient assessment procedures that are often viewed as inadequate, with close monitoring of side effects essential in order to evaluate the efficacy of the medication. You will be familiar with the "allergy" that around 10% of the population exhibit towards penicillin: this is one example of how differences between the "genomes" of individuals can impact on the mode of action of a drug. The solution is in this case to prescribe an alternative class of antibiotic.

The most effective antibiotics and antiviral drugs (I wont deal with vaccines here) are targeted at the invading organism or virus or a distinctive feature of their molecular pathology. Thus penicillin interferes with essential steps in bacterial cell wall metabolism, ciprofloxacin targets the terminal stages of genome replication in bacteria and others act to specifically block bacterial protein synthesis. Importantly, these drugs have no effects on the related processes in us. Perhaps the best known antiviral drug is aciclovir (often marketed as Zovirax), used to treat cold sores and chickenpox. This compound acts by inhibiting the replication of the viral genome (targeting the DNA polymerase). However, because antibiotics and antivirals target the infectious agent and not our own physiological processes, they are somewhat special and although the issue of resistance to antibiotics is topical here, I will not discuss these drugs further in this Blog.

Drugs that we take to relieve pain (e.g. aspirin and paracetamol) are considered safe enough to be purchased over the counter (although some individuals can suffer adverse reactions, and paracetamol must be taken at doses that accommodate the rate of its metabolism in the liver). Many of these types of drug are derived from flora that have been found over many years to bring relief to a wide range of illnesses and allergies. It was around 60 years ago that drug discovery became a more "serious" commercial venture and many of you will have heard of the drug Thalidomide. This was a molecule that was launched onto the market for the alleviation of the discomfort (often extreme) associated with the early stages of pregnancy. This kind of medication is referred to as an anti-emetic (i.e. it stops you vomiting). It seems timely to remind you of the Thalidomide story, since an exquisite set of molecular structures have recently been published in the journal Nature, which shed considerable light on the mechanism of action of this somewhat "notorious" drug.

The legacy of thalidomide is somewhat controversial and has recently been
linked to the tragedy of the Nazi inflicted holocaust. There is no doubt that the German company Chemie Grunenthal filed the original patent, but the controversy arises around the similarities between a family of compounds that formed part of the forced human drug trials at a number of Nazi concentration camps. These compounds, it has been suggested, included thalidomide and and the absence of the original "trial" data leave an unpleasant "smoking gun" in the archives. These issues were raised two years ago when the company unveiled a memorial to the victims of their drug. Getting back to the Science, but more specifically the chemistry; this is where the legacy of thalidomide provide key lessons for any future drug development programme. The initial topic was a consideration of the value of personalized medicine. One of the aims of this new approach to therapy is to alleviate (if not eradicate) unpleasant, and the occasional life-threatening side effects of drugs.  Following the dramatic events during the early 1960s, when thalidomide (or in the UK it was marketed by the drug company Distillers as distaval) was linked to birth deformities, investigations began into the cause of these defects. It should not be forgotten that drug companies in Europe, including the UK made a lot of money from sales of thalidomide. However, it was, in large measure the result of a tenacious scientist Frances Oldham Kelsey working for the early stage organisation of drug regulators in the USA, that we now know as the FDA (or Food and Drug Administration), that thalidomide distribution was halted.

The compound taken by pregnant women in the early days was a racemic mixture (the word racemus means a bunch of grapes in Latin, so I can only assume it was applied to chemicals whose optical properties were neutral, since they contained an equal amount of the right and left enantiomers, like a your left and right hands, mirror images or opposites) of the two chiral forms of the drug. You can read more on by linking to the nicely concise and well written description of the chiral centre in thalidomide by Brent Iverson at the University of Texas here. The target (i.e. the protein molecule (in this case) that captures the drug) is a protein called cereblon, which is encoded by the gene CRBN in humans. This protein forms part of a larger protein complex which catalyses the addition of a very small protein called ubiquitin (top LH image). As its name suggests, it is present in all cells in eukaryotes and, when attached to a protein in the appropriate way by the enzyme complex ubuquitin ligase (ligation is the process of tying two things together: a ligature in medicine). Cereblon is one component of the ubiquitin ligase called (sorry about this!) cullin-4-containing E3 ubiquitin ligase complex CUL4-RBX1-DDB1, or CRL4 for short! In short, the addition of ubiquitin can target that protein for degradation, relocation or some other changes in function. 

The work from Nicolas Thoma and colleagues published in the prestigious journal Nature combines X-ray crystallography and a range of other Molecular and Cell Biological experiments. They show not only the site of interaction of the drug thalidomide (and two variants), but they also propose a mechanism for its action. The schematic diagram on the left shows thalidomide occupying the CRBN (cereblon) "active" site. The diagram below it, reveals that this is the site into which the protein MEIS2 binds. The presence of thalidomide blocks the MEIS2 interaction (remember from my blog on dissociation constants that most molecules "exchange" from their binding site, so in this case there will be some MEIS2 bound, but depending on the overall dissociation constant, there will be less MEIS2 bound than in a cell without thalidomide. This type of "inhibition" or blocking is called competitive inhibition: the two molecules compete for the same binding site. Recall that ubiquitin ligation can lead to protein degradation. So the inability of MEIS2 to become ubiquitinylated means it hangs around in the cell too long. This protein is a transcription factor and it is therefore firing genes off inappropriately. Therefore the consequence of administration of thalidomide is a wider de-regulation of gene regulation. The authors go on further to show how other regulators are misfiring in the presence of thalidomide and similar drugs. In summary, thalidomide interferes with a master control system in the cell. This system plays important roles on several occasions in both infant and adult life stages. Unfortunately, by administering thalidomide to pregnant mothers, the drug interferes with a a key set of events that are required to programme the genes that determine the normal development of limbs, hence the disastrous consequences. But there is an up side to thalidomide.

Thalidomide is one of those compounds that crops up now and again that has potent effects on Biological systems and it is proving valuable in the treatment of diseases in later life. It has been used in the treatment of leprosy and in certain myeloma cancers. You can hopefully see how a drug can appear effective if the trials are not conducted correctly. In the 1950s and '60s we had only a rudimentary understanding of the relationship between genes and development. As we embark on the sequencing of 100 000 genomes, let us not forget that it isn't the sequence of the genes alone that is important, but it is our understanding of their encoded function. Moreover, it is vital that we continue to investigate the interactions of the encoded products (proteins and RNA) and the relationship of gene function in different tissues and at different stages in physiological development. These challenges are much more daunting than the sequencing experiments (not they aren't in themselves a considerable challenge!) and require experimental creativity combined with Bioinformatics at levels that transcend today's methodologies. This is what makes the education of young scientists so important.

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