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Archive for the ‘Glycobiology’ Category


With new data in hand, our first preprint on SARS-cov-2 receptor binding domain (RBD) interacting with heparin now has a sibling, which demonstrates that heparin inhibits the infection of Vero cells by SARS-cov-2

Some of the key points of the team’s new work are:

  1. Inhibition of viral infectivity in a Vero cell model by heparin, which is a better inhibitor for SARS-cov-2 than SARS-cov.
  2. Analysis of the interactions of a more extended library of model heparins with the SARS-cov-2 receptor binding domain. As with many other heparin-binding proteins, these data show that while sulfation is critical for RBD binding, the amount of sulfate is not, but instead it is the spatial arrangement of sulfate groups that is most important.

Together the data point to heparin being a potentially useful therapeutic to reduce infectivity.

From a bioinformatics standpoint, we would expect heparin to inhibit virus infection, since the ACE2 binding site on the protein overlaps the heparin binding site. Moreover, heparin was established to inhibit SARS-cov infectivity and the two viruses have good sequence homology in their RBDs, including the ACE2 binding site and the putative overlapping heparin binding site.

However, to determine if this is actually the case takes time. It is nearly a month since the the team’s initial preprint (now updated with low molecular weight heparins), which was the first demonstration of an interaction between the SARS-cov-2 receptor binding domain and heparin. In the ensuing month, the usual lab gremlins have been working overtime to thwart us. In particular we should cite the Scale up Protein Production gremlin, who is a really awkward so and so. Then there is the Devil’s very own side kick, the Surface Gremlin, who has the uncanny ability to mess up the most careful of preparations of sensing surfaces. Anyway, we know them well and wrestled them into submission, though it did take a couple of all nighters to finally send them packing to some other lab (rest assured, they will not stay with you forever, they know where we work and have 24/7 access to our building, they will be back here and leave you alone for a while).

We have a large library of sugars and sugar-related structures to investigate, courtesy of friends and colleagues in the glycoworld. Some relate to potential therapeutics, others to mechanism. So our work is far from finished on this front.

It has been a lot of fun, with the team’s work varying in intensity from the equivalent of lazing in the sun in front of the trench, to long stints in the lab, including a couple of 22 h ones.

It has also been profoundly rewarding. There are some strange features of the interaction of the SARS-cov-2 receptor binding domain with heparin, not the least of which is the need for SDS to regenerate heparin surfaces with bound SARS-cov-2 receptor binding domain, rather than the more usual 2 M NaCl. This is not unprecedented, as many moons ago one of John Gallagher’s team saw something similar with the interaction of thrombospondin with heparin – I think in that case we had to use 1 M urea to regenerate surfaces.

Cellular heparan sulfate is integral to the infectivity of many pathogens and exogenous heparins have been found to be effective inhibitors. In the case of respiratory viruses this extends beyond the coronaviruses to include influenza viruses. HIV and herpes have long been known to be inhibited by heparins, while more recently this has been shown for Zika and other flaviviruses. On the parasite front, Plasmodium falciparun rosetting is inhibited by heparin and a literature search using your favourite pathogen and ‘heparin OR heparan sulfate’ will pull out many more examples.

Somewhat surprisingly, particularly since heparins lacking anticoagulant activity show good inhibition of pathogens, none of this has hit the clinic yet. There are a number of reasons behind this. Heparin and its derivatives are not single chemical entities, which makes companies nervous in relation to regulatory approval. Moreover, this is not blockbuster territory, partly because once one heparin drug is in use, it will not be so difficult to make another that is not covered by the patent. This relates in part to the fact that heparins are not single chemical entities and the myriad routes to making derivatives. There are, for example, a number of different proprietary low molecular weight heparins, though these are not clinically equivalent. Then there is the market. Many of those in need of such therapeutics are poor and a ‘blockbuster’ drug with a hefty price tag is not going to have much of a market.

On the plus side, it is unlikely that a heparan sulfate-dependent pathogen will be able to evolve resistance to a heparin therapeutic – heparan sulfate is too fundamental to metazoan biology for it to be bypassed. So perhaps covid19 will mark the point at which heparins start to be tested clinically as therapeutics in infectious diseases.

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E-mail I sent out to the research group today.

Dear All,

From now on we really do need to reduce lab work and enforce strictly social distancing, something we stated last week.

The first transmissions from Scousers who picked up the virus on match night (bars, clubs, hotels) from Athletico fans will occur this coming week and next week; we will then get F2, F3 (F number related to contact: primary = F1, a contact’s contact = F2 etc.) transmission.

Given the current lack of social distancing in shops and of course since pubs, etc. all open, we can expect a robust increases in numbers. Since numbers are not counted, only acute hospital admissions, any numbers need to be x10 or x20 to get a true view of actual cases. In addition, though the young do not die, it appears that a few have long-term respiratory issues. We also have the first evidence of re-infection from a 70 year old man on the Diamond Princess. Granted patient old, but this is consistent with this family of viruses not causing a substantial and long-lived adaptive immune response. It also suggests there is a possibility that the world has changed for the foreseeable future and will not return to 2019 for years.

Everyone needs to put a barrier at home, in the entrance, so nothing that touches the outside world gets in without a wash or disinfectant on the outside (this applies to shopping deliveries, since you have no idea what is on the packaging… Soapy water does the trick, since as we know detergents destroy membranes. I note the canny Scousers cleared out all washing up liquid and soap from my local Asda yesterday!

We all continue to monitor our temperatures morning and evening, as we have done for the last week.

Children should not go to nursery or school – I have pulled mine from school and I know a number of parents have done the same. This is because they have the least symptoms (the younger you re the lesser your symptoms), but of course are very good at infecting others. If this means you have to be at home, then so be it.

At work and in the lab we will do social distancing as follows (any ideas welcome):

Passing people in the corridor, walk sideways, so with your back to them.

At the bench: with full PPE we should be fine. All training has to be done at a distance of at least 1.5 m. Ditto troubleshooting. This will be interesting ! It will slow things down, since a learner may set something up, then move away for the expert to come in and check. But it is safe.

There needs to be a full set of gloves where we put on lab coats, so we have PPE before we enter the lab area. Currently we put on lab coat and then go to our bench where we have gloves – we should glove up at the entrance.

We enter the lab area by the door where out lab coats are. No more wandering along the corridor and by Lab C from Lab D. If a queue at lab entrance, make space, turn to the wall.

Desks: DO NOT SIT WITHIN 2 M OF SOMEONE, so if there is someone at a neighbouring desk, you cannot sit at your desk.  Stick to the 2 m rule. In any event, we should not be using the common office, except to set up distance operation of IT equipment

It is obvious that we will be locked down in a week or so, as the infection and death numbers rise, with only  essential work (maintenance, e.g, living material that cannot be frozen/stored and virus work) allowed.

Outside of work, avoid travel that involves other people (bus, train, etc.), since there is no social distancing and no masks yet).

I will be in on Monday to do some plasma cleaning, where Hao and I will work together at a comfortable distance! Later in the week we will be working on the virus protein – Hao and I will figure how we can operate in the CD/SPR lab with  someone on both instruments and other people involved coming and out. It we had masks it would be much easier.

As you are all on the research side you may not be aware that a of this Monday the University has very wisely cancelled all face-to-face teaching; all teaching is online. We can use this opportunity to tidy up a bit (no project students, which will increase space available, and so make social distancing easier.

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Thursday last week (Feb 27) Mark was up from Keele and popped his head around my office door – not a surprise, as he is often here to do circular dichroism on various heparin-binding proteins – to announce that Marcelo had managed to make some SARS-CoV-2 S1 receptor binding domain. Mark had asked Hao,  my postdoc, to do some SPR measurements to see if it bound heparin.

Later in the day I went over to the SPR/CD lab to find Courtney, Mark’s PhD student and Mark beavering away on the CD. A quick discussion. Hao had finished some work on our first grade A heparin functionalised SPR surface, so we set about injecting the SARS-CoV-2 surface protein (Spike) S1 Receptor Binding Domain – a one shot experiment, as amounts of protein were limited, so we injected 1 mL at 500 µL/min (I like high flow rates as mixing is way better, though still far from perfect).

Bingo.

Still the control to do, since though confident in the surface’s resistance to non-specific binding, we still needed to be sure. So we collected the effluent fraction with the Receptor Binding Domain and then passed that through the pump (we are still sorting out fluidics etc., instrument only arrived a month ago) onto the reference channel (streptavidin, no heparin). Small drop due to mismatch in refractive index of the receptor binding domain and the running buffer, and then nothing. Back to running buffer and we go to baseline, no binding.

We agreed we should write up the experiments and put them out as a preprint. The work was not done in a vacuum. SARS-1 receptor binding domain binds heparin and its interaction with cellular heparan sulfate looks to be important in viral adhesion and cell entry (as for many viruses) and the corresponding region of the SARS-2 protein is identical. As we note in the preprint, this may provide a route to first line therapy, while we await a vaccine and other therapeutics.

The experiments depended on many other factors, which are worth considering, when we think about how research and innovation actually occur.

Back in the day, I had a suite of IAsys optical biosensors – brilliant instruments, with a vibrostirrer so we had proper mixing and never had issues with mass transport. The company bit the dust sadly, so these became legacy and then were scrapped. As a back up a colleague had a Biacore, but that packed up too. Personally I never liked these, as they are too ‘closed’ and I much prefer instruments that are open, without any ‘black box’ or inaccessible elements. Last year a new colleague, Roy Goodacre, hosted a seminar by Jean-François Masson from Montreal on SPR. So I went along and was taken by the instrument Jean-François had developed – open architecture in terms of fluidics, small, portable (he developed it to measure explosive residues in ground water used by communities near Canadian Army ranges). We had a chat after the seminar, and I contacted the company he has set up to commercialise the instrument. Some months later, when I had the time, I went around the Biochemistry Department with my hat and we put together the cash and bought a P4SPR, delivered end January 2020. I then set out plumbing in fluidics and making biotinylated oleyl ethelene glycol self assembled monolayers on the gold surface, with help from Richard Nichols in our Chemistry department who has a plasma cleaner.

So a group of people happy to work together with no discussion regarding ownership etc. and willing to stump up a few £ to buy a modest, yet powerful piece of kit. This really boils down to trust in your colleagues, be they PhD students or Professors and it is a real pleasure to work at ground level in such an environment. It is also an environment where you can innovate, something that still excites me.

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I have written the following to my Brexit Party Representatives in the European Parliament. At the heart of parliamentary democracy is the idea that our representatives do indeed represent the interests of their constituents, regardless of Party politics. Of course interests have to be balanced but when these are win-win, there can be no reason for not representing a particular interest. As the matter is not a personal one, then I have also put it on my blog, since there is no reason for secrecy.

I will, of course, post any further correspondence, unless it is confidential for some reason.

“Dear Ms Fox (& cc’ed to Mr Nielsen and Mr Bull)

I am writing to you as one of your constituents regarding an issue which affects me personally and the region. As a University Professor I have over the years been awarded research funds from various Framework Programmes. Most recently, I am part of the €4M FET-OPEN programme “ArrestAD”. This aims to test a new paradigm for Alzheimer’s Disease screening and lay the foundation for a new class of drugs that would arrest the disease.

FET-OPEN projects are very much blue skies and in our case we appear to have hit the jackpot. The trials of the diagnostic in Paris and Warsaw  are quite spectacular and our own work has shown that the targets which ArrestAD has proposed are eminently druggable.

ArrestAD is a 4 year programme. As in any blue skies research, towards the end of the penultimate year a decision is made by the research team whether we should apply for a new, larger project, under one of the translational programmes available under H2020, or to can the idea, in the event it isn’t going to deliver.

Since ArrestAD is delivering its promise, the team will be going forward and applying for a translational programme. This will involve further clinical centres and greater industry participation, since we need more patients and, for drug development, far greater resource.

Alzheimer’s being what it is, ArrestAD obviously impacts widely and not just on myself: there are substantial social and economic ramifications for our region, the UK, and beyond.

The problem we face is that with a so-called No Deal Brexit, the UK loses access to funding from H2020 and the future framework programme. There is no  legislation in the UK Parliament that would guarantee funding as a 3rdcountry. The upshot is that Brexit will prevent my continued contribution to this likely life-changing research programme. Importantly, it will prevent the UK from reaping economic benefit (clinical trials, pharmaceutical industry).

As my representative in the European Parliament, it is imperative that you work to find a solution, which ensures that the drug development arm of this project remains based in the UK, and that the UK is able to participate fully in the wider clinical trials of the diagnostic. I think you would agree that given the impact of this dreadful disease, this is in everyone’s interests.”

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Friday Pat Eyers pushed our two papers on new screens we have developed for sulfotransferases up onto Biorxiv. More about the history of this work later. For now the briefest of summaries.

The heparan sulfate 2-O sulfotransferase paper is here

and the tyrosine sulfotransferase paper is here.

The key messages are:

(1) Mimetics of PAPS, the universal sulfate donor, that inhibit sulfotransferases are present in kinase inhibitor libraries.

(2) We demonstrate selectivity, in that some compounds inhibitor one sulfotransferase better than they do the other.

(3) PAPS mimetics look like providing a rich vein of sulfotransferase inhibitors of varying selectivity, rather like ATP mimetics have done for kinases.

(4) We have two very effective high throughput screens, which means no sulfotransferase is now beyond our reach.

Sulfation has been frustrating due to the lack of chemical tools to selectively inhibit a particular sulfotransferase. With these two papers we can foresee such tools in the not too distant future and with these, we can unpick the role of sulfation in biology, from development, through homeostasis to disease.

Exciting times!

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Congratulations to Aiseta!

On Monday 4 December Aiseta Baradji successfully defended her thesis. A long journey and a hard one as ever with its ups and downs, surprises and a certain amount of head scratching over data that push us in new directions. In the end a great thesis that will be consulted in the labs of her supervisors for a long time. Now onto the next phase.

 

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Two postdoc positions are available in my lab.

Both are part of the larger, European Commission-funded FET-Open programme, ArrestAD, which has recently been funded.

Position 1 aims to characterise heparin-binding proteins in Alzhiemer’s disease.

Position 2 aims to develop inhibitors to Golgi sulfotransferases. (more…)

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