Friday, November 16, 2012

On the trail of bosutinib isomers

“Huh, that looks a bit funny.”

The publication of a paper by Nicholas Levinson and Steven Boxer in PLoS ONE in April 2012 hit the biochemistry world like a bombshell.  Its deceptively innocuous title “Structuraland spectroscopic analysis of the kinase inhibitor bosutinib and an isomer of bosutinibbinding to the Abl tyrosine kinase domain” hid a very worrisome discovery: two distinct chemical compounds were being sold by chemical suppliers as “bosutinib.”  In one fell swoop, dozens of research papers and experimental results were cast into doubt.

Bosutinib (developed by Pfizer) is a selective kinase inhibitor, currently in clinical trials as a chemotherapeutic agent.  Levinson, from Stanford University, was working on a crystal structure of bosutinib bound to a tyrosine kinase called Abl.  He noticed a problem with the electron density around the area of the aniline ring – the expected chlorine at the 2 position seemed to be missing from his electron density map, and instead seemed to present at the 3 position.  Somewhat worried about this, Levinson checked the crystal structure of botsutinib bound to serine threonine kinase 10, recently deposited in the Protein DataBank by Stefan Knapp and coworkers from England’s Oxford University.  Upon closer inspection, their electron density data showed that the 2-chloro atom on the aniline ring was missing, and a chlorine atom was instead located in the meta position.  The authors had noted in their title that the compound was “radiation damaged”, but Levinson was now convinced they were afflicted by the same problem he was seeing.

“What is bosutinib?”

Levinson and his supervisor Boxer immediately subjected their “bosutinib” sample to a battery of tests.  Multi-dimensional NMR experiments quickly revealed that not only was the chlorine atom at the 3 position instead of the 2 position, but the other chloro and methoxy group seemed to be switched as well.  What they had been working on was not bosutinib, but in fact a bosutinib isomer.  The difference is subtle – the isomer has the same mass and would give the same elemental analysis results.  It also has some kinase inhibitory activity, so even biological activity assays could be fooled.  The key to distinguishing the isomers is either X-ray, or detailed NMR analysis (preferably 13C NMR) which would reveal the symmetry present in the aniline ring in the bosutinib isomer, but neither analysis is routinely done on reagents purchased commercially.

Levinson and Boxer notified the company who supplied the wrong isomer – LC Laboratories, a subsidiary of PKC Pharmaceuticals – who immediately launched a comprehensive investigation.  What they uncovered was a wide-spread – indeed, world-wide – problem that probably went back as far as 2006.  PKC Pharmaceuticals gathered unequivocal physical, HPLC, TLC, and spectroscopic evidence that at least two different compounds have been, and possibly still are, being offered for sale under the name bosutinib by at least 18 different biochemical suppliers.  What is worse, PKC found some other spectroscopic discrepancies that may indicate the existence of yet a third isomer.  “What is bosutinib?” is now a real and pressing question, as it impacts on many researchers whose results from studies based on the wrong isomer may need repeating.  One thing that does seem certain is that the compound being tested in clinical trials IS the real thing – Pfizer makes it and tests it in house, and they insist that no isomeric material has ever been administered to humans.

“How deep does the rabbit hole go?”

The only reasonable explanation for the production of the isomeric material seems to be if the wrong aniline precursor was used in the synthesis.  This could be due to choosing an incorrect synthesis of the anilinic isomer, purchasing the wrong isomer, or purchasing the right isomer but receiving a wrong compound.  The latter case is probably most worrisome, and indeed, PKC has found that at least one incorrect compound is being sold as the required aniline.  They are currently testing samples of “2,4-dichloro-5-aniline” obtained from 28 worldwide vendors in order to locate a possible company that may be selling the wrong aniline to bosutinib producers.

Overall, instances of incorrect isomers being sold in the marketplace are very rare – bosutinib may be only the second example. Yet the implication that the problem goes back to an incorrect precursor is troubling, not just because many more bosutinib analogues are being generated by the medicinal chemistry community and may be propagating further structural errors.  Other groups may have purchased the incorrect aniline for their own syntheses, leading to structural errors in molecules of a completely different class.  And unfortunately there is no simple mechanism to alert the wider scientific community of this problem.  In the end, the bosutinib saga serves as a warning to researchers never to take the identity of purchased reagents for granted.

This essay was shortlisted for the Royal Society of Chemistry Science Communication Competition, 2012.

Tuesday, September 18, 2012

Slow-gradient, sample-displacement chromatography

A universal, one-step method for the purification of large quantities of peptides

High performance liquid chromatography (HPLC) is one of the premier chromatographic techniques used world-wide for the purification of a wide variety of chemical compounds. It is essential for the isolation of new natural products, often used in the purification of synthetic molecules, ubiquitous in the peptide and protein synthesis lab, and indispensable for analytical chemistry. 

Figure 1. Schematic 
representation of molecule
elution profiles at different
loading levels: a) full peak
resolution, b) loss of 
resolution due to column
overload, c) displacement

For the most part, peak resolution is required to achieve purification by HPLC, regardless of which mode (e.g. normal phase, reversed phase) is being used.  This means the compound of interest has to be clearly separated from any impurities, as depicted schematically in Figure 1a.  This limits the quantities of compound that can be loaded onto the column, before resolution is lost due to sample overload and peak overlap (see Figure 1b).  Purification of large quantities of compound therefore requires either multiple, repetitive runs, or the use of large (and expensive) prep columns and large quantities of solvent.

Recently, researchers from the Brimble group at The University of Auckland, New Zealand, reported[1] the use of a slow-gradient, sample-displacement chromatography technique pioneered by Hodges et al.[2,3] for the successful purification of a wide variety of peptides.  The key advantage of this approach is that it allows the purification of large quantities (several hundred milligrams) of peptide in a single step.

Displacement chromatography is quite an old technique.  First proposed by Tiselius in 1943 and later developed further by Horváth,[4] it involves loading the sample onto a column, and then displacing it by a constant flow of a ‘displacer’ solution which contains a compound with higher affinity for the stationary phase than any of the components.  As the displacer travels down the column, it pushes the other components downstream giving consecutive areas of highly concentrated pure substances (see Figure 1c).  While this technique allows substantially higher sample loading, the requirement for identification of a suitable displacer and the need for its subsequent removal from the final product provide substantial drawbacks to this method.

Sample displacement chromatography removes these obstacles by using the sample components themselves as displacers. During loading of the sample mixture, which is done under overload conditions, the components compete for adsorption sites on the stationary phase.  The main separation occurs during the column loading phase: the components with higher affinity will compete for sites more successfully than those with lower affinity, which will be displaced further down the column.

Rather than requiring optimization of conditions for every different peptide or molecule, a generic slow gradient of 0.1% organic modifier per minute is used to then elute the components. This approach allows excellent separation and recoveries in a single chromatographic step, and even samples of very low purity, as shown in Figure 2a, can be 'rescued' by this technique.  As described by Harris et al., all 800 mgs of the crude peptide (Figure 2a) were loaded onto a semi-preparative, reversed-phased column and subjected to slow-gradient, sample-displacement purification (Figure 2b).  Remarkably, 70 mgs of pure (>99%) peptide (Figure 2c) were obtained in a single chromatographic step.  The detailed analysis of all the fractions, presented in Figure 2d, shows the impressive separation achieved by this method.

Figure 2. a) Analytical RP-HPLC of the crude peptide. The (*) refers to the desired product. b) Semi-preparative RP-HPLC of the crude peptide under slow-gradient, sample displacement conditions. c) Analytical RP-HPLC of the purified peptide. d) Comparison of all material eluted from the semi-preparative purification.  

A wide range of synthetic peptides, including those with non-natural modifications such as biotin and carboxyfluorescein, have been successfully purified in the Brimble lab using this one-step, universal method.  Harris et al. hope that "given the data presented here, this efficient mode of HPLC purification will be embraced by the wider peptide community."

[1]  Harris, P. W. R.; Lee, D. J.; Brimble, M. A., A slow gradient approach for the purification of synthetic polypeptides by reversed phase high performance liquid chromatographyJ. Pept. Sci. 2012, 18(9), 545-555.
[4]  Horváth, C.; Nahum, A.; Frenz, J. H., High-performance displacement chromatographyJ. Chromatogr., A 1981, 218, 365-393. 

Wednesday, September 12, 2012

100% Chemical Free

This is my winning entry for the 2011 Royal Society of New Zealand Manhire Prize for Creative Science Writing.

“How to create a safe and chemical-free home” advises the Queenstown Lakes district council.  Natural Pools NZ in turn, offers “chemical-free swimming”, and a “chemical-free cosmetic” is touted on our evening news. These are some of the many examples of what Chemical and Engineering News has dubbed as the age of “chemophobia”, or the irrational and unsubstantiated fear of chemistry and chemicals.

read the rest at The Listener

Tuesday, September 11, 2012

The Chemical History of Anaesthesia

Introduction: The Age of Agony

The accounts and recollections of surgery before the discovery of anaesthesia are gruesome and it is difficult to imagine what such surgery was truly like. One of the best descriptions of a pre-anaesthesia medical procedure was provided by Fanny Burney, an English author, in a letter to her sister describing her mastectomy: When the dreadful steel was plunged into the breast - cutting through veins - arteries - flesh - nerves - I needed no injunctions not to restrain my cries. I began a scream that lasted unintermittingly during the whole time of the incision - and I almost marvel that it rings not in my ears still! so excruciating was the agony.[1]

While there were some techniques used to provide a type of primitive anaesthesia that included the barbaric methods of nerve compression, deadly intoxication, exsanguination, refrigeration, carotid compression, and even concussion, ultimately a good surgeon was a fast surgeon.[2]
read the rest at Chemistry in New Zealand.

The Ether Monument, the oldest statue in the
historic Boston Public Garden, possibly the
only statue to a chemical in the world.  It was
erected as an expression of gratitude for the
relief of human suffering occasioned by the
discovery of the anaesthetic properties of
sulphuric ether. It displays the description:
There shall be no more pain.

Monday, September 10, 2012

A new rainbow of colour for bioluminescence imaging

d-luciferin and several luciferin 
analogues synthesized that show 
varying colours of emitted light.
Luciferins are a class of molecules that are oxidized by the luciferase enzymes (for example firefly luciferase), producing oxyluciferin and energy which is released in the form of a photon of light (termed bioluminescence).[1]  Bioluminescence is a very sensitive imaging technique, making it one of the most popular methods for visualizing biological processes in vivo, especially in cancer biology research.[1]  Bioluminescencent imaging is preferred over the fluorescent counterpart because no external light source is required,[2] and the lack of endogenous bioluminescent reactions in mammalian tissue allows for near background-free imaging conditions.[3]  Unfortunately, luciferin-based bioluminescence imaging has been limited to monitoring one cell type or feature at a time, as nearly all the enzymes act on the same substrate (D-luciferin).  Additionally, light of wavelengths below 600 nm is absorbed and scattered by cells, which restricts the application of this technique to only superficial tissue depths.[1]

A range of luciferin analogues have been synthesized which show excellent bioluminescence properties and great potential in cell and tissue imaging. This series of luciferin analogues which absorb at different wavelengths has raised the possibility of multicomponent imaging using multiple colours.  Additionally, several of the analogues display red-shifted emission (>600nm) which give their signal better tissue penetration properties.

Researchers led by Stephen Miller from the University of Massachussets Medical School synthesized four alkylaminoluciferin substrates, which showed red-shifted and more intense light emissions than D-luciferin.[4]  They have also engineered several luciferase mutants that yield improved sustained light emission with aminoluciferins in both lysed and live mammalian cells.[5]

More recently, the Stanford University lab of William Moerner developed an analogue with a selenium atom in place of the native sulfur atom at position 1.  The resulting selenoluciferin emits 55% of its light above 600 nm.[6]

Soon after, Jennifer Prescher and co-workers at the University of California developed two further types of luciferin analogues, replacing the sulfur in either of the two heteroaromatic rings with nitrogen.[7]  One compound specifically shows the highest blue-shift of any luciferins.

Depending on the substitution pattern, the luciferin-emitted light can span a broad range, from deep in the red (>600 nm) up to bright blue (around 460 nm).  This is dependent on the identity and nature of the atoms that are substituted – for example, the more strongly electron-donating nature of the alkylamino group was hypothesized to red-shift the spectral properties.  The polar effect of the selenium atom was also predicted to red-shift the emission maximum; both assumptions turned out to be correct.  

While a palette of luciferin colours has now been developed, many of the analogues are still not ideal substrates.  The alkylaminoluciferins show a significant reduction in light output compared to D-luciferin, consistent with product inhibition and hence lower rate of enzymatic turnover.[4]  The selenocysteine analogue also has reduced light output, partly as a result of lower quantum yield.[6]  Some analogues synthesized displayed very limited or even no bioluminescence, making them of little use for imaging studies.[7]  Further tweaking of their structure will be required before luciferin-based multicomponent imaging is possible.

Luciferases and Fluorescent Proteins: Principles and Advances in Biotechnology and Bioimaging 2007, V. R. Viviani, Y. Ohmiya (eds). Transworld Research Network, 2007.

[1] Y.-Q. Sun, J. Liu, P. Wang, J. Zhang, W. Guo. d-Luciferin analogues: a multicolour toolbox for bioluminescence imaging Angew. Chem. Int. Ed. 2012, 51(34), 8428-8430
[2] M. Baker. A broader palette for luciferaseNat. Methods. 2012, 9(3), 225.
[3] D. M. Close, T. Xu, G. S. Sayler, S. Ripp.  In vivo bioluminescent imaging (BLI): noninvasive visualization and interrogation of biological processes in living animalsSensors 2011, 11(1), 180-206.
[4] G. R. Reddy, W. C. Thompson, S. C. Miller.  Robust light emission from cyclic alkylaminoluciferin substrates for firefly luciferaseJ. Am. Chem. Soc. 2010, 132(39), 13586-13587.
[5] K. R. Harwood, D. M. Mofford, G. R. Reddy, S. C. Miller.  Identification of mutant firefly luciferases that efficiently utilize aminoluciferinsChem. Biol. 2011, 18(12), 1649-1657.
[6] N. R. Conley, A. Dragulescu-Andrasi, J. Rao, W. E. Moerner.  A selenium analogue of firefly d-luciferin with red-shifted bioluminescence emissionAngew. Chem. Int. Ed. 2012, 51(14), 3350-3353.
[7] D. C. McCutcheon, M. A. Paley, R. C. Steinhardt, J. A. Prescher.  Expedient synthesis of electronically modified luciferins for bioluminescence imagingJ. Am. Chem. Soc. 2012, 134(18), 7604-7607.

Tuesday, September 4, 2012

Tasty peptides

Taste is simple, right?  Table salt is salty.  Sugar is sweet.  Coffee is bitter.  And lemons are sour.

And of course we mustn't forget umami, the fifth and youngest (most recently agreed on) taste.  Described in 1908 by the Japanese scientist Kikunae Ikeda, it was only accepted quite recently, so that most languages do not even have their own name for it but have adopted the Japanese term.

Yet there is more to taste than simple salts (like NaCl), carbohydrates (like sucrose), alkaloids (like caffeine), and organic acids (like citric acid) or acid salts (monosodium glutamate).  Peptides, despite being subjected to intensive scrutiny of the many biological functions they perform, are rarely considered in light of their taste.  However, peptides essentially cover the whole range of the established tastes, and contribute significantly to the complex flavour of much of the food we eat every day.

In general, and not surprisingly, peptides with acidic residues such as aspartic or glutamic acid tend to have a sour taste.  Sourness is the taste that detects acidity, through the detection of protons (hydrogen ions) that are released when the carboxylic groups of the peptide dissociate.

Salty peptides are few and far between and are often accompanied by a bitter aftertaste. The 1980s saw a flurry of research around the newly discovered L-ornithine-taurine dipeptide, which was reported to have a salty taste without the presence of any sodium. Some controversy erupted over whether the taste was actually due to the peptide, or residual contaminant NaCl, and no salty peptide so far is being used as a salt substitute.

The hydrophobic amino acids phenylalanine, tryptophan, leucine, and tyrosine have bitter tastes, and similarly peptides rich in hydrophobic residues (especially if they are at the C-terminus) are bitter also.  One of the most bitter peptides described is the octapeptide Arg-Arg-Pro-Pro-Pro-Phe-Phe-Phe, with a bitterness comparable to that of strychnine (one of the most bitter molecules known).  While humans have an innate aversion to bitter tasting molecules (as protection from ingestion of poisonous substances, such as strychnine), the rejection of bitter foods is not absolute.  Foods such as beer, tea, and coffee can be highly bitter, yet are beloved world-wide.  In addition, bitter peptides are found in a variety of aged or fermented foodstuffs, including cheese and meaty products such as ham, and other foods containing fermented proteins.

The archetypal umami tastant is of course glutamate, but many peptides have been claimed to be "umami peptides".  The so-called "delicious peptide" - the octapeptide Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala - was suggested to have an umami potency higher than glutamate itself.  Unfortunately, upon re-examination of this peptide and its fragments, no real umami taste could be detected, casting the existence of umami peptides into doubt.  Yet they have not disappeared: recent research describes umami peptides from peanut hydrolysate and soybean paste.  If these results are confirmed, these umami peptides could be very desirable flavour-enhancing alternatives to the sometimes reviled monosodium glutamate.

But it is probably a sweet peptide that we are most familiar with - the dipeptide aspartame (L-aspartyl-L-phenylalanine methyl ester) is the most used non-caloric sweetener in the world.  Discovered accidentally when a researcher licked his (contaminated) finger to lift a piece of paper, aspartame was found to be 200 times sweeter than sucrose (table sugar).  Following on from this serendipitous discovery, scientists explored many alternatives more rigorously.  They found that Asp cannot be substituted by any other residue, whereas Phe can be replaced by some (but not all) hydrophobic amino acids.  Interestingly, they also found that all the other possible chiral isomers (D-L, L-D, and D-D) are not sweet at all, but quite bitter.  Other modifications, on the other hand, have led to the discovery of super-aspartame molecules, such as neotame.  It has a 3,3-dimethylbutyl group attached to the amino group of the aspartic acid, and is around 10,000 times as sweet as sucrose.  Most jurisdictions have now approved its use in food.

Proteins and peptides make up a large part of the foods we eat every day, and it is clear that they play a significant role in the complex chemical interplay that is taste.  Mostly, the peptides seem to contribute sweet, bitter, and sour tastes, but some evidence suggests salty and umami peptides exist also.  While the taste of the sour and salty peptides is probably simply due to the presence of the charged terminals and side chains, bitter and sweet receptors are clearly activated by specific electronic and conformational features of a specific peptide (as demonstrated by the various isomers of aspartame).  Peptides are therefore extremely useful tools for researching taste receptor function and leading to a better understanding of taste and taste perception.

Wednesday, August 22, 2012

Fraud, greed, and lies: the origin of the anti-vaccination movement

Recently, a good friend of mine had her first baby. He is now in the process of getting his first vaccinations, and so my friend has suddenly become exposed to the anti-vaccination movement. She is, understandably, worried: should she or should she not vaccinate? Is there any truth to what the anti-vaccination proponents are saying? Why are vaccines suddenly seen as the worst thing you could do to your child? How did all this start? Who is Andrew Wakefield?

Vaccination is the administration of an antigenic - antibody generating - material, that is, something that stimulates the immune system to produce antibodies against it.  These antibodies can recognize and neutralize this foreign material, marking it for destruction by other immune cells.  By using material from a killed or weakened pathogen (or sometimes even just a portion of it), the immune system can be primed to deal with this invader.  When an infection then occurs sometime in the future, the immune system is ready to deal with it quickly and efficiently, at the least reducing the severity of the symptoms or the duration of the disease, and in many cases even completely preventing it from developing.

While objections to vaccines have been around as long as vaccines themselves, the beginning of the modern anti-vaccination movement can be pinpointed with accuracy: the publication of a paper in the British medical journal Lancet in February of 1998. It was written by a laboratory researcher, Dr Andrew Wakefield, and co-authored by a dozen other doctors, reporting on the cases of 12 anonymous children with developmental disorders who were admitted to a paediatric bowel unit at the Royal Free hospital in Hampstead, north London, between July 1996 and February 1997. The publication of the paper was followed by a press release and further publicity that received huge media attention and began the concerted attack on vaccinations.

In a 20 minute video press release, later criticised as "science by press conference", Wakefield called for a suspension of the triple measles, mumps, and rubella (MMR) vaccine, suggesting it was linked to the development of autism in children.  A furore around vaccines exploded in the media and on the internet, where the anti-vaccine argument gained significant traction.  The uncritical or at times blatantly irresponsible reporting by numerous media outlets, as well as television talk shows giving vaccine opponents a platform, led to a widespread (and mostly unchallenged) spread of this idea.  As a consequence, vaccination rates - of not just MMR, but all vaccines - in the next decade have fallen, a trend that is world-wide:
  • In the UK, official figures for 2003/2004 have shown a drop in MMR vaccinations to 80%  from a peak coverage of 92% in 1995-6 (just before Wakefield's publication).
  • The American Council on Science and Health warned that childhood vaccination rates against measles, mumps, and rubella (MMR) fell nearly 3 percentage points in 2009 from the year before: almost 10 percent of American children are not vaccinated from serious diseases, which include diphtheria, tetanus, and pertussis.
  • In 2010, Medicare in Australia reports that  the number of youngsters not fully immunised in the national program has doubled over the past five years, and the number of children whose parents have registered as conscientious objectors to vaccinations also rose by 68 per cent in that time.
  • Also in 2010, Switzerland had one of the lowest immunization rates in Europe, with only 71% of children receiving the recommended two doses of the measles vaccine. 
This drop in vaccination is not without consequence.   Herd immunity is the resistance to the spread of infectious disease in a group because susceptible members are few, making transmission from an infected member unlikely.  To achieve herd immunity, 75% to 95% of people must be vaccinated.  The exact level of vaccination required depends on the disease, how contagious it is, and how it is transmitted.  In many places the level of vaccination has dropped below the herd immunity threshold, and the effects are painfully apparent: the incidence of fully preventable (and once considered vanquished!) diseases such as measles and pertussis (whooping cough) is on the rise.

California reported a whooping cough epidemic in 2010 that may have been the worst in 50 years.  Measles is on the rise in Europe, and England reported a 10-fold rise in cases in 2011.  Most recently, southern Alberta, Canada, is in the grips of an ugly whooping cough outbreak.  So is New Zealand.

These diseases are not innocuous.  Measles complications can affect one in 15 children infected, and can include bronchitis, seizures, and encephalitis (which may be fatal). Pertussis is especially risky for very young babies - newborns under two months have a 1 in 100 change of dying from the disease, and children up to 12 months have a 1 in 200 chance. Serious complications can include pneumonia, encephalopathy, seizures, and failure to thrive. Pertussis can also cause severe paroxysm-induced cerebral hypoxia and apnea (when the baby is coughing so hard it can't breathe and the brain is starved of oxygen, leading to brain damage).

So what is the science behind Wakefield's claim to stop vaccinating children?

An investigation into Wakefield's research by the journalist Brian Deer, of the Sunday Times, revealed an astounding scandal. As Wakefield was warning parents to avoid MMR, and publishing papers claiming a link between vaccines and autism, he was in fact being funded by Richard Barr, a solicitor hoping to raise a class action law suit against the company manufacturing the vaccine. After an initial payment of £55,000, Barr paid Wakefield out of the UK legal aid fund (run to give poorer people access to justice) a sum eventually totalling £435,643 (more than eight times Wakefield's reported annual salary).

The children themselves, supposedly routine patients at the Royal Free Hospital, turned out to have been recruited through MMR campaign groups, and their parents were contacts or clients of the lawyer Barr. In addition, nine months before the publication of the paper, Wakefield filed a series of patents, including one for a single - supposedly safer - measles vaccine, "which only stood any prospect of success if confidence in MMR was damaged."
None of these substantial conflicts of interest were reported in the paper, or declared to the journal Lancet.

The conflict of interest was just the beginning, as further investigation revealed worrying ethical issues as well.  Research on human subjects is strictly governed by national and international standards, including the Declaration of Helsinki which is a set of ethical principles regarding human experimentation developed for the medical community by the World Medical Association (WMA).  No reputable hospital review board would have endorsed Wakefield's proposed "fishing expedition" - which included a battery of invasive and distressing procedures like lumbar punctures and colonoscopies.  Without approval, however, no reputable medical journal would publish the findings.  So Wakefield simply lied in the paper, stating ethical approval had been obtained.

In addition, he showed further unethical behaviour by buying (for £5) blood from children at a birthday party, completely ignoring the inappropriateness of the setting. Later, he would go on to describe this event in a public forum, making a joke out of children fainting and vomiting, showing callous disregard for any distress or pain to the children.

As if the blatant conflict of interest, and the entirely unethical way this "research" was conducted wasn't enough, the actual data presented in the Lancet paper - the article that started the whole vaccination and autism debate - was in fact to a large degree manufactured by Wakefield himself.  In most of the 12 cases reported, the actual hospital and GP records differed to what was described in the Lancet.  The research paper claimed autism-like symptoms began within days of the vaccination, but in all cases but one such concerns were raised significantly before vaccination.  The majority of cases were presented in the Lancet as having an abnormal gut; hospital pathologists had previously declared them fully normal.

So, let's be clear: this supposed link between vaccines and autism was completely fabiracted by Wakefield in order to profit from the fallout. Numerous studies have tried to replicate his findings, but have always failed to find any link. A number of health authorities (including the American Academy of PediatricsCenters for Disease Control, the World Health Organization, and the Institute of Medicine of the National Academies), have reviewed all the available evidence, and every single time the conclusion is the same:

The body of epidemiological evidence does NOT support a causal relationship between the MMR vaccine and autism. 

More and more evidence piled up against the conclusions of Wakefield's paper, and slowly, the scientific record was put straight. First, in March of 2004, 10 out of the 12 authors of the original paper (one could not be contacted) published a formal retraction of the conclusions of the paper:
We wish to make it clear that in this paper no causal link was established between MMR vaccine and autism as the data were insufficient. However, the possibility of such a link was raised and consequent events have had major implications for public health. In view of this, we consider now is the appropriate time that we should together formally retract the interpretation placed upon these findings in the paper, according to precedent.
Then in February of 2010, the Lancet finally formally retracted the whole paper:
..., it has become clear that several elements of the 1998 paper by Wakefield et al. are incorrect, contrary to the findings of an earlier investigation. In particular, the claims in the original paper that children were “consecutively referred” and that investigations were “approved” by the local ethics committee have been proven to be false. Therefore we fully retract this paper from the published record.
This was in response to the results of an inquiry by the British General Medical Council, convened in July 2007. In a decision released almost three years later, the General Medical Council fitness to practice panel found Wakefield guilty of multiple instances of serious professional misconduct. The Panel states that he was dishonest and misleading, that he breached his duties, that he repeatedly breached fundamental principles of research medicine, that he failed to ensure that the factual information contained in the paper was true and accurate, that he was intentionally dishonest, irresponsible and misleading, and that his actions were contrary to the clinical interest of the patient, and an abuse of his position of trust as a medical practitioner. The Panel concluded that his conduct brought the medical profession into disrepute. Finally, on May 24th, 2010, he was struck off the UK medical register, revoking his license to practice medicine in the UK.

Every aspect of Dr Wakefield's claim has been disproven and discredited, numerous times over.  Yet the meme persists, possibly because it provides a simple explanation to a complex problem, provides and answer that so many parents desperately search for, and gives something to blame.  But the truth is: there is no link between vaccines and autism - there never was.  So vaccinate your kids!