Amyloid research enters a new era thanks to breakthrough technologies

Parkinson’s, Alzheimer’s, Diabetes mellitus type 2, Huntington’s, Fatal familial insomnia, prion’s, among many others, are diseases with a common contributor: Amyloids. These are aggregates of proteins that become folded into the wrong shape, allowing many copies of that protein to stick together and form large fibrils. These fibrils disrupt the healthy physiological function of nearby tissues and organs.

Over the last 150 years, technical developments have repeatedly created new horizons in amyloid research, including biophysical and chemical analyses, which have retrieved lots of information related to the mechanistic aspects of fibril formation and possible links with pathogenesis. But in the last couple of years, technologies such as Ion Mobility coupled to Electrospray Ionization Mass Spectrometry (ESI-IM-MS) and Microscale Thermophoresis (MST) have appeared to revolutionize amyloids research, particularly in the characterization of the aggregation process and the screening for amyloid ligands and binding partners.

2015: For the first time, a study challenged the scientific principle about Alzheimer protein Amyloid beta

A comprehensive knowledge of the number of molecules and shape that a protein has when it begins to aggregate is crucial for the design of drugs capable of breaking them up or preventing their formation and the corresponding disease.

In Alzheimer’s disease, the protein that aggregates is called Amyloid beta (Aβ). Two of the most common variants of Aβ are Aβ40 and Aβ42, with the latter being the variant most closely associated with Alzheimer's disease. The literature reports that while Aβ40 self-aggregates to sequentially form dimers, trimers and tetramers, Aβ42 self-aggregates to form pentamers and hexamers.

Among the paradigms in the Alzheimer’s disease field, pentamers and hexamers of Aβ42 have been established as the building blocks for Aβ aggregation. These findings have been cited more than 1000 times and consequently numerous studies have been based on this premise. However, a new study has shown that Aβ40 and Aβ42, actually goes through exactly the same aggregation states. The question then is, what accounts for these differences?  And the answer lies on the technologies that have been used so far and artifacts given by previously used ones.

Results published to date are biased by the technique most widely used to study Aβ aggregates: SDS-PAGE. Most groups rely on this technique because it is characterized by the need for a small amount of sample and therefore is used for more straightforward studies. But the aforementioned study demonstrates that pentamers and hexamers form artifactually from dimers and trimers in the presence of SDS. Thus, SDS-PAGE provided flawed information on the order and distribution of Aβ42 oligomers.

Using a new approach though, based on mass spectrometry, namely ESI-IM-MS, it was shownthat the oligomer distribution for Aβ40 and Aβ42 was the same, comprisingmainly dimers and trimers.

ESI-IM-MS not only has the potential for the characterization of oligomers and to identify chemical modifications, but also to provide dynamic and structural information on the oligomers present in the sample. Thanks to these features, yet another paradigm in the field was challenged, which states that higher order Aβ oligomers adopt a β-sheet structure. This study also demonstrated that Aβ dimers and trimers are globular and lack defined secondary structure, indicating that major structural rearrangements occur during fibril formation. It should be noted that up to now, drug design has been based on the premise of interfering with the beta-sheet structure, so the authors believe that this strategy should be reconsidered and recommend caution when using SDS-PAGE to study Aβ oligomers.

These huge changes on the way scientist used to look at Aβ amyloids have been made thanks to new technologies as well as critical thinking. They highlight the importance of defining the structures and properties of the species involved in Aβ aggregation; it is only through this level of molecular detail that it will be possible to interfere with Aβ aggregation in a rational manner. Specifically, by identifying Aβ40 and Aβ42 dimers and trimers as the earliest oligomers formed during Aβ aggregation, they now constitute the earliest forms to be considered in the design of therapeutic strategies targeting Aβ oligomerization.

2015: Protein-ligand binding affinity measurements can finally be analyzed on supramolecular aggregates such as amyloid fibrils

In Parkinson’s disease, intracellular deposits of the intrinsically disordered protein α-synuclein, form a major characteristic of the pathology. To date, no cure for this disease exists, a consequence at least in part, of the lack of fundamental understanding of the mechanism of aggregation and its associated toxicity, as well as the incomplete characterization of the interactions between aggregates of α-synuclein and other compounds, including small molecules and proteins. But why studying these interactions? Well, they are important for both diagnostic (e.g. for positron emission tomography) and therapeutic purposes (e.g. for targeted aggregation inhibitors). In this context, there is an urgent need for experimental techniques that can be used for high throughput screening to identify such compounds.

Protein aggregates are situated though, at the boundary between soluble and insoluble structures and thus are challenging to study, because classical biophysical techniques, such as scattering, spectroscopic and calorimetric methods, are not well adapted for their study. Standard techniques, such as isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) can provide important information, but suffer from a number of limitations, including high levels of sample consumption (ITC), potential surface artifacts (SPR) and high sensitivity to solution conditions (both ITC and SPR).

A recent study demonstrated for the first time that Microscale Thermophoresis (MST) is a valuable method for screening for ligands and partners of even such highly challenging samples as supramolecular protein aggregates, such as amyloid fibrils. Due to their polymeric nature, these types of samples are substantially more difficult to handle than most soluble monomeric proteins and this study presents a comprehensive protocol for the use of MST for amyloid-binding assays.

Binding affinities were obtained for a nanobody that binds to monomeric α-synuclein in excellent agreement with a previously determined value obtained from ITC measurements. Its binding affinity to the oligomers was also obtained, which had not previously been reported, partly due to the challenge of obtaining sufficient quantities of pure oligomers. This issue was been able to overcome by exploiting the low sample requirements of MST.

The binding of the small molecule epigallocatechin gallate (EGCG), one of the main constituents of green tea, to α-synuclein aggregates was also investigated by MST. This molecule has been reported to bind to various species on the aggregation pathway of α-synuclein and even to remodel mature amyloid fibrils and other protein aggregates, but its affinity for oligomers had not been previously measured. With this technique it was possible to obtain the binding constants of EGCG to α-synuclein amyloid fibrils and oligomers as well.

The possibility of characterizing novel types of ligands that bind to protein aggregates, both small and large molecules, at high throughput and using minute sample quantities is a highly valuable addition to the experimental toolbox available for the development of diagnostic and therapeutic strategies against protein missfolding diseases. In particular, it should be emphasized that oligomeric structures, which have been highlighted as the most toxic species on the aggregation pathway, often occur only at low concentration and with a short lifetimes and hence experimental methods that are rapid and require only small amounts of sample are vital for their study.

The anti-life discovery that have saved more lives in history

During world war II, the battle against disease-causing micoorganisms saw an innovation that has saved more sick and injured people than any single invention in history: Penicillin, also known as the magic bullet.

From ancient times, doctors and patients have dreamed of a drug that would make people well after getting an infection. In the 1920s, Alexander Fleming serendipitously discovered that the mold penicillium produced a “juice” that would kill other organisms: the anti-life discovery. Fleming published his work, but was never able to purify Penicillin nor produce it in mass quantities and his work was left behind. But after a decade, everything changed….. and history was made.

An accidental discovery: more than a contaminated plate

"When I woke up just after dawn on September 28, 1928, I certainly didn't plan to revolutionize all medicine by discovering the world's first antibiotic, or bacteria killer, but I suppose that was exactly what I did" Fleming said.

By 1927, Fleming had been investigating the properties of staphylococci. He was already well-known from his earlier work, and had developed a reputation as a brilliant researcher, but his laboratory was often untidy. On September 1928, Fleming returned to his lab having spent August on holiday with his family. Before leaving, he had stacked all his cultures of staphylococci on a bench in a corner of his lab. Once Fleming got into his lab, he picked up his plates and noticed that one culture was contaminated with a fungus, and that the colonies of staphylococci immediately surrounding the fungus had been destroyed, whereas other staphylococci colonies farther away were normal, “That’s funny”, he said. Fleming showed the contaminated culture to his former assistant Merlin Price, who reminded him, "That's how you discovered Lysozyme." Fleming grew the mould in a pure culture and found that it produced a substance that killed a number of disease-causing bacteria. He identified the mould as being from the Penicillium genus, and, after some months of calling it "mould juice", named the substance it released Penicillin on  March 7, 1929.

Beauty on the making

Fleming had neither the lab resources nor the chemistry background to take the next giant steps of isolating the active ingredient of the penicillium mold juice, purifying it and how to use it.

That task fell to Dr. Howard Florey, an Australian professor of pathology at Oxford University. It’s been said that he was a master at extracting research grants from tight-fisted bureaucrats and an absolute wizard at administering a large laboratory filled with talented but quirky scientists. He had long been interested in the ways that bacteria and mold naturally kill each other. His work on Penicillin began in 1938 (10 years after Fleming’s discovery!!), thanks to one of his brightest employees, the biochemist Dr. Ernst Chain, a german jewish refugee from Nazy Germany.

In 1938, they decided to study some natural antibacterial compounds, Chain (his interest piqued by Fleming’s 1929 article), suggested penicillin and Florey went along with him. That was the very beginning.

Not long after Chain, Florey recruited Norman Heatley, an English recently graduated PhD in biochemistry from Cambridge University. He was assigned to come up with a method to produce and extract enough penicillin for Chain to study. And how clever this man was!! Heatley's initial contribution (the first in a remarkable series) was the "cylinder plate," or "penicillinder," to determine how powerful this unknown substance was. The assay plates contained short lengths of glass tubing embedded into bacteria-laden agar, and each tube was filled with a different penicillin solution. The diameter of the growth-free circle of agar around each tube was measured from a glass scale illuminated from underneath. The assay gave rise to the ‘Oxford Unit’ of Penicillin. But this incredibly smart assay created by Heatley had another important capability: it indicated the critical time when a culture should be harvested at the peak of antibacterial activity. Furthermore, this man discovered that the Penicillium growing time could be reduced by reusing the fungus and producing up to twelve crops of the penicillin fluid underneath a single fungal mat.

Chain, along with chemist Edward Penley Abraham on the other hand, worked out a successful technique for purifying and concentrating penicillin. The keys seemed to lie in controlling the pH of the “juice,” reducing the sample’s temperature and evaporating the product over and over (essentially freeze-drying it).  In the first step of penicillin’s purification set up by Chain, the cooled and slightly acidified culture was mixed with ether, which took up the penicillin and left impurities behind. But penicillin was amazingly unstable, and it was this instability, which had defeated earlier scientists including Fleming, precluded conventional separation techniques.

During a meeting in March 1940, Heatley, by nature modest and quiet, listened as Chain and Florey hotly debated why penicillin vanished. Then, half-apologetically, he put forward what he later called a ‘laughably simple’ idea, although heretofore it had crossed no one’s mind. If penicillin could be extracted from a neutral buffer of water into ether, why shouldn’t it be possible to transfer it out of the ether into water made alkaline by passing the mould broth back and forth between acid and alkaline to purify it, like extracting an egg yoke by slipping it between two broken shells? Heatley nailed it and this “back-extraction” method worked brilliantly. The resulting watery solutions of penicillin were freeze-dried into a stable brown powder that was remarkably powerful in a dilution of one to a million. Yet-as the Oxford team later learned-the powder contained only 1% pure penicillin. Nevertheless, this method produced enough penicillin in 7 weeks for Florey to test the effects of the antibiotic in animals.

Soon after, Chain ran down to a laboratory that maintained test animals and requested that two mice be injected with a sample of the extracted penicillin. Though the injection represented a far higher dosage than that administered in Fleming’s similar experiment, the mice survived apparently unharmed; the more-concentrated penicillin had passed its first toxicity test. Florey then directed that the antibacterial properties of penicillin in mice be tested—the crucial step that Fleming had not taken. 

On May 25, 1940, in an experiment monitored by Heatley, eight mice were infected with Streptococcus. An hour later, four of them were given penicillin. Heately wrote in his lab notebook: "After supper with some friends, I returned to the lab and met the professor to give a final dose of penicillin to two of the mice. The controls were looking very sick, but the treated animals seemed very well. I stayed at the lab until 3:45 a.m. by which time all four control animals were dead. It really looks as if P. [penicillin] may be of practical importance”.

In the morning, he was able to tell Florey that the four treated with penicillin were still alive. Brilliant science!

On August 24, 1940, Florey and Chain reported their findings in theLancet: the article electrified research groups around the world that were seeking cures for bacterial disease. It was at that point that Florey realized that he had enough promising information to test the drug on people. But the problem remained: how to produce enough pure penicillin to treat people?

Going big thanks to Norman Heately

In spite of efforts to increase the yield from the mold cultures, it would take 2,000 liters of mold culture fluid to obtain enough pure penicillin to treat a single case of sepsis in a person. Again, Heatley solved the problem. He was a supreme improviser, known for using his skills to cobble together functional efficient laboratory set-ups from whatever he could lay his hands on. First, he automated his back-extraction process with available equipment. This apparatus consisted of milk churns and soft drink bottles connected with yards of glass and rubber tubing, and included a warning bell to signal when a bottle was full or empty. It stood six feet (1.8 m) tall upon a stand made from an old bookcase discarded by the Bodleian Library. Six columns of extracted penicillin from uniform droplets of acidified culture flowed in a counter current direction to the top of a column where it was collected while the spent watery liquid was discharged below.

But Heatley needed to grow more mold juice to keep pace with his extraction machine that processed 12 liters of medium an hour. Because it was wartime, there were great scarcities of everything. With the outbreak of war, resources were limited, and thus the mould had to be grown in whatever they could lay their hands on such as pie dishes, trays, biscuit tins, gasoline cans, sterilized bottles and even old-fashioned bed pans borrowed from the Radcliffe Infirmary, can you imagine that?!

However, he found that amog of all 'fancy' objects, the best one for culturing the mold was the old fashioned enamel bedpan with a side arm through which the culture could be inoculated and harvested!!! Inspired by the bedpan, Heatley designed a square-sided ceramic vessel that could be quickly and inexpensively made by a slipcast process (a technique for the mass-production of pottery and ceramics)

Each utensil held a liter of medium and could be stacked horizontally in the incubator, vertically in the autoclave, and in neat rows lining the walls of the Dunn School operating theater. Heatley fetched the first 174 (of 500) bespoke bedpans from a nearby pottery, in a "bull-nosed Morris two-seater…. groaning under the weight," and incubated them with the mold on Christmas Day 1940.

Six penicillin girls” hired by Florey were in charge of handling the culture pots. Within a month, the now famous "bedpans" produced enough penicillin to justify the beginning of Florey's clinical trials.

When Chain urged that a patent be sought on penicillin, as was usual in German research institutes, Florey refused to enter into such a commercial agreement on a discovery he presumed would benefit all mankind—a decision that long rankled Chain.

Penicillin is tested for the first time in a person

In early January 1941 Florey was ready to test penicillin on humans. The first English patient to whom the drug was administered was a young woman whose cancer was beyond treatment and who had agreed to test penicillin’s toxicity. She showed an alarming reaction—trembling and sharply rising fever. However, Abraham was able to show that impurities in the drug, not the drug itself, had caused the adverse reaction. In February a policeman became the first patient with an infection to be treated with penicillin. He nicked his face working in his rose garden. The scratch, infected with streptococci and staphylococci, spread to his eyes and scalp. Although Alexander was treated with doses of sulfa drugs, the infection worsened and resulted in smoldering abscesses in the eye, lungs and shoulder. Florey and Chain heard about the horrible case at high table one evening and, immediately, asked the Radcliffe physicians if they could try their ”purified” penicillin. No one knew the dosages and the length of treatment required to eliminate various bacterial infections; these parameters were being worked out by just such trials—primitive by today’s standards. After five days of injections , the policeman began to recover, but the penicillin supply had almost run out, and even retrieving penicillin from the man’s own urine (a commonly used procedure in the early clinical trials) failed to save him and died. Florey vowed that from then on he would always have enough penicillin to complete a treatment.

Using Heatley’s homemade contraptions, six patients treated during spring of 1941 at Oxford’s Radcliffe Infirmary used a total of 2 million units of the drug, with only one fatality (the policeman). Remarkably, the Oxford team's entire research effort to this point took just 18 months and was based on a mere 4 million units of Heatley's handmade penicillin, an amount that today represents a daily dose for a single person.

Florey planned an even larger trial, but the problem once again was to obtain enough penicillin. To increase penicillin supplies, Florey approached various British pharmaceutical firms, but only ICI considered itself in a position to accept the challenge (though many later joined the effort). In July 1941, with the help of the Rockefeller Foundation, Florey and Heatley carried the secret to the United States to persuade scientists and companies to undertake the production work that had been so crippled by shortages in the United Kingdom. The entry of the United States into the war in December 1941 altered the course of history in regard to penicillin, and by the end of 1943 its production was the second-highest priority of the U.S. War Department. New climates and traditions of research then clearly emerged — for instance, the British Medical Research Council believed that patenting medicines was unethical. They rejected Chain's urgent requests that the work be protected — a refusal that bore more than a hint of anti-Semitism. American companies patented their production techniques, and Chain's prophecy that he would have to pay royalties to use his own invention proved correct, although whether the Oxford scientists could have patented their preliminary work remains debatable.

Some chemists were confident that they would soon be able to synthesize penicillin from a few organic chemicals. This attitude resulted in a major effort conducted on both sides of the Atlantic to understand the structure of the penicillin molecule as the prerequisite for its eventual synthesis. A structure was proposed by Chain and Abraham and later confirmed by new instrumental techniques for analyzing the structure of organic molecules, including X-ray crystallography, which was practiced by Dorothy Hodgkin, a near neighbor of the Oxford chemists. Unfortunately, Hodgkin’s work on the molecule culminated too late in the war to be used in devising a synthesis for penicillin. Even after 1957, when such a synthesis was created, fermentation continued to underlie the commercial production of penicillin and related antibiotics. But the structural knowledge gained in the war years proved invaluable in developing penicillin-like antibiotics after the war that could be administered more conveniently, were more effective, and had fewer side effects….. and the rest, as they say, is history.

With World War II over and the Nobel Prizes distributed to Fleming, Florey, and Chain for their work on penicillin. Heatley, the genius was left behind. In 1990 he was awarded an honorary Doctorate of Medicine from Oxford. What makes the honor particularly remarkable is that Heatley is the only person to receive that award in the university's 800- year history.

Have you ever taken penicillin? Next time you or your kids do, remember of all the effort and bedpans behind.