Vancomycin: last line of defence

Vancomycin in the snow

One of the most important discoveries of the 20th Century was antibiotics: chemicals that kill bacteria but not their human hosts. It changed the shape of human society as people began to survive septic cuts, everyday horrific infectious diseases, syphilis and tuberculosis.

The world we live in is almost unimaginably healthy compared to 100 years ago, largely thanks to this discovery. Death in childhood is rare rather than commonplace. Plagues – sweeping epidemics of often bacterial infection – are also far less common, and the coupling of antibiotics and antiseptics is so effective in fending off opportunistic bacteria during invasive surgery that surviving it is no longer the miracle it once was.

With antimicrobial resistance very much our minds, this Christmas our chosen antibiotic is Vancomycin: the last line of defence in fending off bacteria.

Exploiting competition

Vancomycin, like many antibiotics, is sourced (somewhat counter-intuitively) from bacteria. Essentially, humans nick weapons from the perpetual warfare between competing bacteria, and use them to create more precise instruments.

Bacteria are constantly vying with each other for resources to gain the upper hand. Many of them are equipped for sophisticated chemical warfare: releasing hideous poisons to kill other bacteria, or at least supress their growth. Sometimes the chemical combatants include fungi, but the basic modality is always the same: secrete a powerful chemical that directly interferes with the day-to-day business of your competitors (but not your own), and keep pumping it out until you own the field.

By isolating these poisons, we put bacteria’s chemical arsenal to work for our own ends. In the case of Vancomycin, some very clever people isolated the bacteria that manufactures it from a soil sample from Borneo.

Vancomycin makes it almost impossible for certain types of bacteria to build a cell wall. It binds to a commonly used connector (a complex peptide, like a protein polymer) halfway through the wall-building process, and prevents the wall components from sticking together (i.e. crosslinking). Because many bacteria use the same type of connector, the strategy is effective for defeating a broad range of competitors.

Vancomycin is a particularly effective antibiotic due to its site of action. Cell-wall ‘construction’ must happen outside of the cell, so the victim is prevented from resisting by pumping out the chemical poison – the most common form of defence.

What, no ribosome?

Vancomycin looks like a rather complex peptide, but full of oddities, with strange amino acids present. That’s because it’s not made in a ‘normal’ manner, that is, printed out by a ribosome. It is made instead via non-ribosomal synthesis involving a chain of complex proteins – each of which is hundreds to thousands of amino acids long – linking up Vanomycin’s bespoke amino acids in a highly specified manner.

This is bafflingly inefficient and complex. But then, when you’re in an arms race with your soil-bound competitors, no metabolic or energy expense is spared to be top bacteria in your region.

Clever synthesis

Amazingly, human chemists can imitate the sophisticated process of making Vancomycin, and synthesise it directly. That is certainly not true for every antibiotic or chemical – sometimes we still need to get bacteria or fungi to make things for us.

This precious chemical arrives in hospital every day, only to be administered if all other antibiotics fail. It has to be given intravenously. as it can’t be absorbed via the gut. Hundreds of thousands of lives are saved every week thanks to this re-purposed weapon of bacterial chemical warfare.

The Resistance

But dark clouds are gathering on the horizon: there are now more microbes that are resistant even to this last line of defence. Some bacteria are working out how to tweak their cell wall to prevent Vancomycin from messing it up.

Sadly, many life-scientists and chemists have been concentrating their efforts on making drugs for more common, non infectious diseases. But if a bacterium can pull together several modes of resistance into one package, it could easily win the war on a massive scale, sweeping across world and putting humans firmly back to the pre-antibiotic-medicine era – with all its hardship and untimely death.

Forward-thinking public research institutions are aware of this danger, and invest heavily in research to find the next ‘last resort’ antibiotic. Responsible research and policy programmes also pursue ways to improve the stewardship of our current arsenal. For example, the excessive use of antibiotics in agriculture creates a hyper-competitive environment in the soil, wherein many more bacteria evolve stronger and stronger resistance in order to survive.

Vancomycin and other antibiotics have held the bacterial army at bay for a while, but the clock is ticking, and the enemy is at the door.

Aspirin: the first modern drug

Aspirin in the snow

Preparations derived from willow have been a regular feature of the human medicine cabinet for centuries: Ancient Egyptians drank willow ‘tea’ to relieve pain, and the Classic Greek physician Hippocrates wrote about the remedy in 400 BC. But it took a team of German chemists in 1897, working for Bayer, to synthesise a pure compound related to the active substances in willow, acetylsalicylic acid. They packaged it up neatly in pill form, and sold it under its trademarked name, Aspirin, which quickly became a household word.

Aspirin is arguably the first over-the-counter, modern drug. It was made via a defined chemical process, rather than isolated from a complex mixture, formulated in a portable, easy-to-ingest pill, and used to alter our elaborate chemical schemes.

Before Aspirin, chemists prescribed some ‘inorganic’ chemical molecules and numerous distillations, infusions and derivations of natural products. But the systematic, mass-produced and mass-marketed Aspirin was a game-changer.

How Aspirin works

Aspirin works mainly by half-mimicking a lipid (a half fat, half polar molecule) that gets converted by an enzyme into a signalling molecule – one of a (rather grandly named) family of prostaglandins. Prostaglandins have a huge range of functions and are found in most of our tissues.

Prostaglandin’s main job is to signal that inflammation is occurring in the body. When there is damage or inflammation, prostaglandin synthase enzymes (cyclooxygenases COX1 and COX2) are activated. They grab a rather ordinary lipid and convert it into a signalling molecule.

Aspirin looks enough like the polar part of that lipid to dive right into this enzyme. Aspirin donates an acetyl group to COX1’s protein, which puts a chemical spanner in its works, permanently stopping this enzyme’s function.

This is the major mechanism of Aspirin, and part of the reason why it blocks the sensation of pain, but the entire story of how it works is still a bit of a mystery.

While it’s permanently putting COX1 out of action, Aspirin is also inhibiting COX2 (reversibly) and preventing platelets from making thromboxane A2. That’s what keeps the platelets in blood less sticky, and why Aspirin is given to patients after heart surgery or stroke.

Indeed, it has been suggested that a regular low dose of Aspirin is just a general good once you are over a certain age.

Yes but why?

This uncertainty about a drug’s modes of action is more common than you might think. Certainly, for many of the earlier drugs, people had known for some time that they had some effect, but without understanding their molecular basis.

The precise mechanisms of action of many commonly prescribed drugs are still ambiguous. Many drugs – Aspirin included – seem to work precisely because they do more than one thing, making it even more complex to work out how these drugs work. In the modern era it is increasingly hard to get drugs without a precise mechanism of action through regulation.  However, over a century of precise use – and over a millennium of experimental use – has given us huge confidence in Aspirin’s utility as a medicine.

Serotonin: happy signals

Serotonin in the snow

Small molecules can be great for sending biochemical signals between cells, or between different parts of a cell, because they can diffuse rapidly in water – notably, the water in your body. Big, multicellular organisms rely on hundreds of such molecules to get on with the business of living.

To be effective as a signaller, a molecule must:

  • Be easy to produce (or convert)
  • Be small enough to diffuse readily
  • Have a unique shape

Furthermore, as one can’t control where such a molecule goes, it should not be of any other use in the body. It would be a disaster, for example, if you were to try out a molecule for sending a specific signal, and it turned out to also be very much at home producing energy.

Serotonin and tryptophan

Serotonin is a good example of an effective signalling molecule. A close chemical relative of the amino acid tryptophan, it is responsible for our overall happiness and mood. Serotonin is certainly not specific to humans – it is a very ancient signalling molecule, used by even the most basic of animals.

Amino acids, like tryptophan, are the building blocks of proteins. Each one is like a tiny Lego brick in one of those complicated Lego kits, with its own, unique ‘side chain’. Many amino acids are manufactured in the body, but some cannot be made by animals (humans can’t make nine of them). Across all animals, a subset of five or so must come from a plant (or bacterium).

Tryptophan, one of only a handful of essential amino acids, is the most chemically distinctive of the lot, with a chunky, two-ring side chain. It can’t be made internally, yet all animals must ingest it to survive. That could explain why serotonin – a derivative of tryptophan – indicates the presence of food in the body.

‘Good food’ and happiness

When food enters the gut of every animal – from the simplest, not-much-more-than-gut-and-tentacles miniature animal to the mighty blue whale – serotonin (made from tryptophan, which can’t be made in the body) is released, signalling ‘good food’ and activating digestion.

But giving the go-ahead for digestion is just one of serotonin’s happy signals – there are many. For example, to no one’s surprise, its signals branched out from ‘food ok’ in all creatures to ‘happiness’ in more sophisticated animals, which have more concentrated neural circuits in the brain for performing more complex actions.

Many neurons have serotonin receptors, and parts of the mammalian brain are assigned to pump out serotonin, which transmits signals between neurons. Without the direct link to digestion, serotonin is now a ‘pure’ signalling molecule in the brain, released by some neurons, taken up by others, and recaptured to return to baseline for future use.

That point of recapture is the site of action of some antidepressant drugs – though no one is entirely sure how precisely the blocking of serotonin uptake – which increases the amount of signalling serotonin between neurons – really changes mood. But it definitely makes us happier.

ATP: go, go, go!

Phosphates in the snow

If there is one molecule that could be called the ‘currency’ of life, it is ATP: adenosine triphosphate. It belongs to a large class of molecules of life, which are all high in energy and have constituent parts that would – given enough time – much prefer to separate.

ATP has three parts:

1. adenosine, a ‘molecular handle’ comprising a complex double ring,

2. ribose, a five-carbon sugar, and

3. the ‘business end’, with three phosphates in a row. These phosphates would prefer to be whizzing about in the lovely, polar water rather than stuck rigidly in a row. Each has a fearsomely negative charge, so they jostle against one another.

When hydrolysis does cause a phosphate to fall off, ATP is reduced to ADP (adenosine diphosphate), and when the next one goes it becomes AMP (adenosine monophosphate).

However, ATP doesn’t just burn itself away. Left to their own devices, ATP’s phosphates hydrolyse rather slowly, so their stored chemical frustration can be released by proteins as required.

Highly traded currency

ATP is constructed mainly by harnessing proton gradients (see water, the first molecule of Christmas). It is a practical form of energy that fuels virtually every other process in the cell – like transporting molecules in and out of the cell, bringing proteins into contact with each other, moving vesicles about, and making new chemicals and proteins.

There are many similar, high-energy, kinetically stable molecules in life aside from ATP. Notable examples include triphosphates that contain other nucleotide bases: guanine (GTP), and cytosine (CTP). Some things use GTP as an energy source, but ATP is the most widely traded currency for energy.

How proteins plug in

The “A” in ATP is a distinctive shape that nestles comfortably into proteins. Nearly all of the proteins that do use ATP for energy have a special ‘pocket’ just for adenine.

When the adenine nestles into this protein pocket on one side of the protein, that pulls the pent-up phosphates into position on another spot – they are then lined up perfectly for the last one in line to be removed, and energy to be released.

The removal of that last phosphate depends on the protein. The energy can only be released if the protein is exactly the right shape to cut that bond. The cutting of the bond will often move aspects of protein.

Sometimes (as in the case of myosin, a muscle protein) that energy is used for physical movement overall. Sometimes, it’s used to join things together and make a new molecule – like when an amino acid is joined to a tRNA so it can get ready to make new protein.

Sometimes the phosphate is transferred to another molecule – a protein or another small molecule – but more often it is simply released. Once the job is done, the resulting two-phosphate ADP falls out of the pocket, the free phosphate floats off, surrounded by solvating waters, and the cycle starts again.

These cycles are happening billions of times every second in every living thing. Our living is a constant hum of ATP changing to ADP and P, and then being recreated elsewhere.

Origins

In theory, the A – adenosine – could be anything relatively stable with a distinctive, recognisable shape. But it is not by chance that this is exactly the same molecule as part of RNA, best known for its mediation between DNA instruction and protein manufacture.

This goes to the likely origin of life, where everything was RNA: the genetic code, the catalysis engines and the ability to capture energy. Remnants of that RNA world are everywhere in the main, massive enzymes of life, such as the Ribosome – and in every protein which binds ATP. Everywhere, the currency of energy is still this ancient molecule.

ATP in EMBL-EBI data resources

Find out more about ATP on the EMBL-EBI website

 

Water: the life of the party

Water molecule

Water is such an everyday substance – you use water to wash, drink, make food; it is commonplace in our weather and rivers, and surrounds us in oceans. Its ubiquity is not only important for our environment: it is absolutely critical for life.

Water is simple and strong: two tiny, positive hydrogen atoms bonded to one big, fat, negative oxygen atom. This imbalanced charge sets up a strong dipole: oxygen hugs its electrons close, and the two hydrogens are pushed to be far more positive. This dipole makes water almost semi-structured, as the strongly positive side of one water molecule reaches for the strongly negative side of the next partner, and so on.

This endless dance is behind water’s entire approach to life.

Smooth operator

Water can operate as a liquid across a far wider range of temperatures than most molecules of its size. It happily insinuates itself between other polar molecules, allowing them to dissolve.

Water accepts other molecules, from behemoth proteins to miniscule chemicals, lubricating the party so the right molecules can meet and separate with minimum fuss.

But while molecules of all sizes are at home with water, the relationships differ wildly. Small, polar molecules interact with water almost as peers. But larger molecules often drape the water around themselves, kind of like a microscopic cape, or shell.

This dynamic, fluid medium, with its random motion and friendly attitude towards other polar molecules, is the medium in which life works.

More than a medium

But water is far more than a medium for life.

It accommodates additional protons (hydrogens without their electron) easily, for example making positive H3O+ ions. This is so commonplace that is has its own vocabulary: pH, influenced by acids and alkalis. These extra protons can be donated by other molecules, or on temporary loan from neighbouring water molecules. They often play walk-on roles in enzyme catalysis.

This flow of protons is behind life’s most intensive energy-capture processes. Whether it’s capturing energy from light or releasing energy from complex chemicals, all life exploits proton gradients.

The mysterious movement of protons

Both bacterial cells and the organelles of more complex organisms have membranes that are impermeable to proton movement. By way of specialised proteins inside the cell, they use light or chemical energy to achieve a net movement of protons from one side of the membrane to another.

Protons present mainly in the form of H3O+ on each side. But exactly how one side loses a proton and the other side gains one is only partially understood – it may well involve complex, quantum chemistry. The process ultimately leads to a higher concentration of H+ ‘inside’ the cell or organelle.

At some point, ‘gatekeeper’ proteins release the protons out of the cell, often in equally mysterious ways, and harnessing the energy produced to perform actions. Intriguingly, in bacteria these gatekeepers include the flagella complex: a massive ‘tail’ that acts as a whip-like propeller to move the bacteria along. Other, less exotic but still fearsomely complex schemes make ATP, the energy currency of the cell.

The net movement and release of protons – stored as types of H+ ions on either side of a membrane – is the ultimate coupling that produces the vast majority of energy powering life on this planet. In both bacteria and organelles, proton pressure powers the creation of ATP (another ‘molecule of Christmas’).

Water in EMBL-EBI’s chemical biology resources

Find more information about water in EMBL-EBI data services.

Information and Biology

This is an idle muse on information and biology as I wait for my SFO to Burbank plane (also an experiment in “fast blogging”).

Biology is truly an information science – what are biological systems? They are way more than the atoms that make them up; they are far more than just the molecules that make them up; ultimately they are remarkable systems which can harness the inevitable flow of energy towards heat to their own persistence and, in many animal’s case, information capture and decision making.

And to study biology we absolutely need information science. Our theories are useful (eg, evolution) and true but not predictive in the same way as (say) gravity or the QCD in physics. To understand biology there is a huge amount of measurement, ie, data gathering, but to make sense of that data, we need computers to basically enhance our own capabilities in owning that data. There are practical aspects – humans are not good at the sort of immense book keeping one needs to trawl through datasets – but there are more profound aspects; human pattern recognition is rather cranky, often visualisation centric and is over-eager to find a pattern; computers (when programmed right) are far more level headed.

I remember distinctly the first time I realised this; in a small, cold room in Cold Spring Harbor, aged 19, in 1993, I had written a frameshift tolerant profile-HMM to DNA sequence program – this would later become “Pairwise and Searchwise”. The first profile I ran was my favourite (and still favourite!) protein domain, the RRM. And as the results came back I was both amazed at the number of hits, but rapidly started to worry my program was just identifying garbage – many of the hits did not have the canonical motif to RRMs of [F/Y]x[F/Y]xxF in amino acid code.

Thankfully I didn’t entirely trust my own opinion; I looked carefully at the resulting hits and it dawned on me that profile HMM had taken the data and found something deeper. That in fact the motif was xUxVxF, with U being I/L/V (sometimes F), and V being mainly V. The previous two FxF motifs was in fact a subset of this family, interleaved with the U position between the two Fs. This was in fact the central beta sheet, with an even pattern of hydrophobic residues. This lead to my second, large paper a year later (I was a precocious undergraduate student).

What I learnt was that I could trust the computer – or rather trust it at least as much as trusting my own opinion; the computer had “crunched the numbers” on the input profile HMM and found a different pattern to me; by iterating between the computer and myself “we” had made a discovery. Of course, I had programmed the computer, so the discovery somehow did lead back to me, but there was simply no way I could have done this unaided.

This motif of extending the human mental processes to tackle unfeasible-for-a-human tasks is now routine in molecular biology – DNA/ genome assembly was painstakingly done by hand in the 1980s / early 1990s (I remember one colleague  sliding pieces of cut out sequence past each other) – you’d be bonkers to do this “by hand” today (it’s an amusing thought!); decoding single cell experiment is unfeasible outside of a computer; modelling Xray diffraction patterns or fitting/rotating EM to make structural models have always needed the extension of the human mind by computers. Given the huge numbers of possibilities being “crunched” statistical methods have to go hand in hand with these computational schemes – it is strong statistics which allows us to “trust” the output

That said, computer programs have their limitations; mainly to do with things outside of the assumptions needed to make the method and statistics work; computers are still surprisingly bad at reconciling contradictory data. The number of oddities, exceptions and weirdness in biological systems plague systematically rolling out computational models. For a long time I thought that gene prediction should be a entirely solved  problem by computer based methods building off other evidence (transcripts; proteins). After close to a decade of watching this first hand in my Ensembl years I’ve realised that not only is there a series of “standard model doesn’t work here” (eg: Ig locus, or protocadherins) but there is a substantial and annoying amount of complex evidence reconciliation which, if you want every gene structure to be as good as possible you have to put a human in the loop. My old collaborator and co-founder of Ensembl, Michele Clamp would rage against people “hand knitting” genes – it was never going to scale – and yet our best laid plans, algorithms and large compute had a frustrating set of cases where you just had to declare X the answer and be done with it. You needed both computers and humans, working together, and of course the computers had to have humans programming and running them (in the early days we used to describe this as “babysitting” the pipeline; watching it crunch data and then it would occasionally fall over, sometimes due to infrastructure failure, sometimes due to weird data; these days the infrastructure is far, far better but the data issues are perennial in biology).

There is something interesting in the air though with these new Neural Networks – CNNs, RNNs, adversarial training. They “just” need data, even unlabelled data, and they very clearly empirically work; image recognition, nanopore base calling, chromatin states are all happy users of these. And it is frustrating that without a formal model inside these beasts, it is hard to know what precisely they are picking up. That is both unsatisfying but also has hidden dangers about generalisation – can we trust these things when they are let loose on all the complex data outside of the training sets.

People are making progress here. The rather wonderful process of reversing these neural networks can provide the “archtype” of what they are looking at (check out https://www.popsci.com/these-are-what-google-artificial-intelligences-dreams-look); with multiple composite layers one can start to have at least some sense of what composite parts are being used. And I am sure smart CS/stats people are going to continue to break this down. But ultimately I am less worried than other people about these beasts; again, humans have to find the datasets and define the objective functions of success; humans have to work out how to shape and present data in the first place, and then how to use the output. Humans still have the responsibility, end to end, of the process, just as I did in my RRM discovery, and ultimately this is just another step of extending the human abilities via computers.

 

 

Is Science right, and how do we know it?

Reflections on reproducibility, digital communication and open science

Is science sound? There has been a sustained discussion about this over the past five years – ever-present in the background, and punctuated by intense public debates, both in the scientific press and more broadly. There is a host of concerns – from reproducibility of science to incentive structures – all focused ultimately on how we know what is true and what is not. The answer is not always straightforward.

Continue reading “Is Science right, and how do we know it?”

The big reveal: Beta galactosidase and cryo-EM

My final Structure of Christmas may look like an unremarkable enzyme, but it heralded the arrival of a game-changing method in structural biology.

My ninth (and final) Structure of Christmas is beta-galactosidase: a pretty run-of-the-mill enzyme that turns compound sugars into monosaccharides. When you put a special dye on it, it turns the dye blue (whee!). It’s a mainstay of molecular biology and millions of students have used it in countless experiments, both fascinating and mundane. It doesn’t have much of a ‘wow’ factor – it’s a solid member of a respectable family of sugar-cleaving enzymes.

What is so special about it is the way its structure was determined.

Continue reading “The big reveal: Beta galactosidase and cryo-EM”

Tropomyosin and actin: Move!

My penultimate structure of Christmas is actually two molecular partners, which work together to make muscle move.

Most of my Christmas structures have been separable units – some large, some small – that float around in cells or cell membranes. But to move physically, organisms need to have more at their disposal than some things floating in solution. For most life forms, movement is managed by proteins working together. A perfect example of this is the beautiful partnership between actin and tropomyosin.

Continue reading “Tropomyosin and actin: Move!”

Antibodies: Defend!

Once a parasite makes it past our outer defences, it encounters some seriously sophisticated weaponry. One of these is the ever-shifting antibody, my seventh structure of Christmas.

Every large organism – you included – is just a feast laid out for any parasite (bacteria, virus or beastie) clever enough to break in and access its carefully amassed energy. Throughout the Billion-Years’ Evolutionary War between hosts and parasites, the host has always been on the defensive, endlessly innovating to fend off invaders.

Continue reading “Antibodies: Defend!”