Aflatoxin: a cancer-causing glue

In our previous blog post, we announced our Aflatoxin Challenge – a new series of Foldit puzzles designed to tackle a common poison known as aflatoxin. This week, Baker Lab scientist ianh offers a more detailed picture of the chemistry behind aflatoxin's harmful effects.

Most people on Earth consume aflatoxins every day. Aflatoxins are compounds produced by certain fungi that can grow in or on almost all grains and groundnuts. Aflatoxins are known hepatocarcinogens, meaning they cause liver cancer in high doses. Liver cancer is the third leading cause of cancer death globally, with 83% of cases occurring in East Asia and sub-Saharan Africa where aflatoxin exposure is highest.

What makes aflatoxin so toxic?

Surprisingly, aflatoxin itself isn’t toxic. Once ingested, our body uses its normal metabolic processes to try to break it down. It turns out a metabolic product of aflatoxin – not aflatoxin itself – is harmful, and the chemistry behind its toxicity is both frightening and familiar.

One of the metabolic enzymes that acts on aflatoxin is CYP3A4, a vitally import liver enzyme tasked with breaking down different complex molecules. Its normal targets are molecules produced by our own bodies, like the sex hormones testosterone and estrogen, but CYP3A4 can also safely chew up some of the complex chemicals we put in our body, such as caffeine (found in coffee and tea) and lidocaine (a local anesthetic commonly used by dentists).

When CYP3A4 metabolizes a chemical it changes that molecule’s structure in some way. When it acts on aflatoxin, it adds a chemical feature – chemists call this new feature an epoxide group.

Extreme chemistry

Epoxide groups are highly reactive, meaning they are unstable on their own and like to bond with other nearby chemicals. This extreme reactivity can be harnessed for many practical applications, including adhesion, electrical insulation, and industrial manufacturing. Some of the strongest glues ever made – epoxy glues – are based on epoxide chemistry.

Once CYP3A4 converts aflatoxin into epoxy-aflatoxin, the compound doesn’t wait around for long. It quickly reacts with other chemicals in our cells, especially amines.

One of the best places to find amines in our cells in our DNA. Each of the four letters of DNA – A, T, G, and C – is an amine. Epoxy-aflatoxin reacts especially strongly with guanosine (G), forming a nearly-unbreakable bond. This permanently damages DNA.

Cancer-causing mutations

DNA damage is problematic, not only because it interferes with the natural processes of the damaged cell, but because this damage can be passed on to descendants when the damaged cell replicates. In this way, a single error in a single cell may be amplified over time to affect a large population of cells.

The liver is especially prone to this cascade because it (1) expresses high levels of the CYP3A4 protein that metabolizes aflatoxin, and (2) because liver cells reproduce at faster rates than cells of other organs, allowing DNA mutations to accumulate and propagate much more quickly.

Human DNA includes a gene that encodes for the tumor-suppressing protein p53. The normal role of the p53 protein is to police DNA damage in the cell. It recognizes DNA damage and can initiate DNA repair processes or, in extreme cases, induce cell death. Without functional p53 protein, DNA damage runs rampant in a cell. Dysfunctional p53 is strongly associated with many forms of cancer in humans.

It seems that one particular guanosine in the p53 gene is especially susceptible to aflatoxin damage. Damage to this guanosine is propagated to descendant cells as a G -> T mutation in the p53 gene; the G -> T mutation in the p53 gene results in a ARG -> SER mutation in the p53 protein. The p53 protein falls victim to the very type of DNA damage it is meant to avert! This mutated version of p53 is exceedingly common in liver cancer patients.


In summary: aflatoxin causes DNA damage in the liver, resulting in a population of cells with defective p53 and prompting tumor growth.

Help us design a protein to break down this toxic compound by playing the latest Foldit puzzle, 1445: Aflatoxin Challenge: Round 2 with Insertions!

( Posted by  ianh 81 2490  |  Tue, 10/31/2017 - 17:26  |  0 comments )
5

Destroying a Silent Killer in the Food System

We are launching an uncommon collaboration between UC Davis, University of Washington, Northeastern University, Mars Incorporated, Thermo Fisher Scientific, and the global public, to engineer a protein tailor-made for the degradation of aflatoxins.

Imagine a naturally occurring poison – known to cause liver cancer – which 4.5 billion people around the world are chronically exposed to1.

You don’t have to imagine. The poison exists in the form of aflatoxins, which are compounds produced by certain fungi that can grow in or on almost all grains and groundnuts1. Aflatoxins are known hepatocarcinogens, meaning they cause liver cancer, and are considered a Class 1 carcinogen by the International Agency for Research on Cancer, part of the UN World Health Organization. Liver cancer is the third leading cause of cancer death globally, with 83% of cases occurring in East Asia and sub-Saharan Africa2-3.

Beyond liver cancer, aflatoxins are associated with worsened health outcomes in the developing world. These fungal toxins have been shown to negatively impact the immune system, rendering vaccines less effective, and to reduce vitamin absorption and recovery rates from malnutrition4.

Aflatoxin in Our Food System

So how does this potent carcinogen get into – and stay in – our food? In developed countries, laws limit the amount of aflatoxins allowed in food for humans, livestock, and pets. These laws are backed by expensive monitoring and advanced food safety technologies.

In the developing world, aflatoxin limits are set but often unenforced. Under-regulated food manufacturing, subsistence agriculture, small-scale farming, and lower healthcare budgets all contribute to the aflatoxin contamination problem. Over the past 50 years, when food and feed has been sampled in developing countries, the majority has been found to contain aflatoxins well above the legal limits5. A recent study in India found that 100% of collected samples of grain flour were contaminated, with an average aflatoxin concentration three times higher than the limit allowed in the United States6-7.

Aflatoxins affect more than just the plants we eat. When fed to cattle, they transform into a more potent toxin which accumulates in milk. A 2016 study in urban low-income areas in Nairobi found that 100% of the milk tested had detectable levels of aflatoxin, with 63% of the samples above the legal limit8.

This global health issue is also a trade and economic problem. Before scientists understood how harmful aflatoxins can be, Africa accounted for more than three quarters of the global peanut export market. Its current share is about 4%, partly due to its inability to meet aflatoxin standards outside of Africa. This costs Africa $1 billion per year in lost peanut revenue alone. Other African crops are similarly affected.

Climate change may make the world’s aflatoxins problem worse. The fungi which produce aflatoxins thrive in hot and humid conditions. Right now many parts of the world are not well suited for these deadly fungi, but rising temperatures could allow for more growth in more areas, increasing the threat of exposure. In just the last five years, cases of aflatoxin contamination have been increasing in Europe9.

Mitigation of Aflatoxin

How can we tackle aflatoxin? Removing aflatoxins from our food is extremely difficult. To date, there appears to be no ‘best practice’ for minimizing exposure other than strong enforcement of legal limits and systemic use of safe agricultural practices commonly adhered to in developed countries.

Several approaches for managing and degrading aflatoxins are currently in practice, but none are widely considered effective. At present, microbial, enzymatic, and bio-control approaches are preferred. Each has its own complications, ranging from expensive multi-day treatments to low efficacy in certain environments. The underlying issue is that there is no cost-effective and efficient system to remove aflatoxins from our food system5,10.

Our Challenge For You

Through Foldit (run from University of Washington and Northeastern University), anyone in the world can help to optimize an enzyme that we hypothesize could be capable of degrading a susceptible lactone ring in aflatoxin. Chemical degradation of this lactone ring has been demonstrated to decrease aflatoxin toxicity by more than 20-fold11. However, the enzyme in its current state is unable to perform this reaction. We are asking Foldit players to restructure the active site of this enzyme.

The goal is to enable the enzyme to effectively interact with the partially degraded Aflatoxin B1 molecule that we have modeled inside. Once we develop a highly effective enzyme capable of degrading the toxin, enzymes could readily be added to feed and food to remove toxins in real-time, complementing the current techniques for aflatoxin management and control12.

The Siegel Lab at the UC Davis, through the support of Mars, will select the top player designs to experimentally characterize. Thermo Fisher Scientific will donate gene synthesis services, encoding player designs through its proprietary DNA synthesis platforms. Using these genes, researchers in the Siegel Lab will produce the player-designed proteins and determine whether they are capable of degrading Aflatoxin B1, the type of aflatoxin that is most potently carcinogenic.

Over the next few months, we’ll be releasing a series of puzzles in which Foldit players can manipulate the enzyme model with escalating degrees of control (starting with simple site directed mutagenesis and leading to full redesign with insertion/deletion of protein segments). We will continue to provide feedback on previous rounds of player designs in order to further explore this enzyme’s ability to degrade aflatoxin. The first puzzle of the series, 1440: Aflatoxin Challenge: Round 1, is available now!

By participating in these Aflatoxin Challenge puzzles, the players agree that all player designs will be available permanently in the public domain, and the players will not seek intellectual property protection over the designs created as part of the challenge.

1. Williams, J. H.; Phillips, T. D.; Jolly, P. E.; Stiles, J. K.; Jolly, C. M.; Aggarwal, D., Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am J Clin Nutr 2004, 80 (5), 1106-22.
2. Schatzmayr, G.; Streit, E., Global occurrence of mycotoxins in the food and feed chain: facts and figures. World Mycotoxin Journal 2013, 6 (3), 213-222.
3. Ajani, J.; Chakravarthy, D. V. S.; Tanuja, P.; Pasha, K. V., Aflatoxins - A Review. Indian Journal of Advances in Chemical Science 2014, 3, 49-60.
4. Liu, Y.; Wu, F., Global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment. Environ Health Perspect 2010, 118 (6), 818-24.
5. Williams, J. H.; Phillips, T. D.; Jolly, P. E.; Stiles, J. K.; Jolly, C. M.; Aggarwal, D., Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am J Clin Nutr 2004, 80 (5), 1106-22.
6. Kumar, P.; Mahato, D. K.; Kamle, M.; Mohanta, T. K.; Kang, S. G., Aflatoxins: A Global Concern for Food Safety, Human Health and Their Management. Front Microbiol 2016, 7, 2170.
7. Ramesh, J.; Sarathchandra, G.; Sureshkumar, V., Survey of market samples of food grains and grain flour for Aflatoxin B1 contamination. Int. J. Curr. Microbiol. Appl. Sci 2013, 2 (5), 184-188.
8. USDA, Mycotoxin Handbook. 2015.
9. Kiarie, G.; Dominguez-Salas, P.; Kang’ethe, S.; Grace, D.; Lindahl, J., Aflatoxin exposure among young children in urban low-income areas of Nairobi and association with child growth. African Journal of Food, Agriculture, Nutrition and Development 2016, 16 (3), 10967-10990.
10. Herrera, M.; Anadón, R.; Iqbal, S. Z.; Bailly, J. D.; Ariño, A., Climate Change and Food Safety. In Food Safety: Basic Concepts, Recent Issues, and Future Challenges, Selamat, J.; Iqbal, S. Z., Eds. Springer International Publishing: Cham, 2016; pp 149-160.
11. Ehrlich, K. C.; Moore, G. G.; Mellon, J. E.; Bhatnagar, D., Challenges facing the biological control strategy for eliminating aflatoxin contamination. World Mycotoxin Journal 2015, 8 (2), 225-233.
12. Lee, L. S.; Dunn, J. J.; DeLucca, A. J.; Ciegler, A., Role of lactone ring of aflatoxin B1 in toxicity and mutagenicity. Experientia 1981, 37 (1), 16-17.
13. Olempska-Beer, Z. S.; Merker, R. I.; Ditto, M. D.; DiNovi, M. J., Food-processing enzymes from recombinant microorganisms—a review. Regulatory Toxicology and Pharmacology 2006, 45 (2), 144-158.

( Posted by 81 2490  |  Fri, 10/13/2017 - 20:57  |  6 comments )
8

New Classroom Puzzle Series

As many of you know, playing Foldit can be a very effective way to learn about protein structure and folding, as well as other topics in biochemistry. Many teachers have taken advantage of this facet in the past, asking students to play Foldit as part of the class. However, we have not typically designed puzzles in Foldit for the express purpose of biochemistry education.

This winter, Professor Scott Horowitz will be teaching a biochemistry course at the University of Denver, with an emphasized use of Foldit to illustrate specific biochemical topics. Students will read primary literature in biochemistry, and will have access to custom Foldit puzzles that are designed to complement the assigned papers. Ideally, the puzzles will give the students an intuitive feel for the biochemical systems they are learning about, and enable them to better understand the papers they are reading. (And yes, the students will be graded, in part, based on their Foldit score.)

But first, we’re asking for the help of you, the Foldit community! Before assigning these puzzles to students, we’d like to get some feedback from our regular Foldit players—veterans and newcomers alike. Over the next couple of months, we’ll be posting a series of “Classroom” puzzles, with the course reading when possible, and we want to hear your thoughts and opinions about how to make them more useful for undergraduate students.

Check out the first puzzle of the series, 1434: Classroom Puzzle: Phosphates in Biology! This puzzle focuses on the structure of RNA, and the role of phosphorus in biology. The puzzle description links to two related articles: the first is a landmark 1987 paper by Frank Westheimer discussing the unique role of biological phosphates; the second is a controversial 2010 paper by Wolfe-Simon et al. suggesting arsenic as a viable substitute for phosphorus. Please leave comments and suggestions on the puzzle page!

( Posted by  bkoep 81 1151  |  Wed, 09/27/2017 - 21:41  |  1 comment )
0

Introducing Small Molecule Design Puzzles

Hi everyone,

Today we are officially launching the Foldit small molecule drug discovery game mode.

To kick off the new game mode, we are releasing a new puzzle on acquired immunodeficiency syndrome (AIDS). It has been over three decades since the CDC reported on the discovery of AIDS and its cause, the human immunodeficiency virus (HIV). Currently, 17 million people globally are being treated for HIV with antiretroviral therapy (WHO Global AIDS Update 2016). A total of 2.1 million people were infected with HIV in 2015 and 1.1 million people died from AIDS, with the majority of patients being from in Africa.


(Source: UNAIDS Global AIDS Update 2016)

Current treatment therapies focus on blocking the enzymes used by HIV to reproduce. One such enzyme is HIV protease, an enzyme which the virus uses to cleave polypeptide precursors. Without a functioning HIV protease, the virus cannot make the proteins needed to produce new virus particles. Several small molecule drugs have been created to block HIV protease, such as saquinavir and amprenavir. These compounds inhibit HIV protease by binding in the enzyme active site, a large cleft running through the middle of the protein, which prevent the enzyme's substrates from binding to the enzyme. HIV protease is a good test case for small molecule design, as there's a wealth of information about what sorts of compounds bind to the enzyme.

For this puzzle, we are interested in what small molecule inhibitors you can design for HIV protease 1. As a starting point, we're giving you the base fragment for saquinavir in the binding pocket. What we'd like you to do is use the new small molecule design tools to alter the chemical structure of the inhibitor ligand to get a better binding (better scoring) ligand.

We recommend that you use the selection interface (Menu->Selection Interface) for this process, as it gives you the most control, but you can also access some of the drug design tools through the "Modes" entry of the original interface. In particular, the Ligand Design tool will be your main tool for altering the identity of the ligand.

In the selection interface, the Ligand Design tool will pop up after clicking on the ligand. (In the original interface, click Ligand Design from the modes menu before clicking on the ligand.) You can then click one or more individual atoms to change the atom's element, or to add and delete bonds, atoms and groups. Keep in mind the advanced GUI (Main/Menu->General Options->Show advanced GUI) enables additional options under View that may help in working with small molecules. (I like the "Cartoon Ligand" view, myself, as it allows you to select ligand hydrogen atoms.)

Explore the tools and different designs you can make for the small molecule! Remember, you can use the Upload for Scientists button for designs that you want us to look at, even if they are not the best-scoring solutions!

-rmoretti & freeradical


P.S. Didn't follow the development of the drug design mode? Catch up with these previous blog posts:

* Foldit drug design introduction
* Foldit Drug Design Part Two
* Foldit Drug Design Blog: Interface Update
* Drug design puzzles coming your way!
* Let's Get Ready for Drug Design Puzzles!
* Devprev Drug Design Update
* Drug Design Update: Tool Talk
* Drug Design Update: Merk Molecular Force Field
* Big Update: Experimental Client (Drug Design)

(Note that some features discussed are for older versions and have changed since.)

( Posted by  rmoretti 78 882  |  Tue, 09/19/2017 - 23:11  |  0 comments )
2

A Visit from a Veteran Foldit Player

A couple of weeks ago, the Foldit team had the pleasure of meeting with veteran Foldit player, Timo van der Laan! Timo, who was visiting Seattle from his home in the Netherlands, met with us to discuss volunteering his programming expertise. Timo's background in automating development processes and structuring documentation will be a huge asset as he works with our team on improving Foldit.

The team discussed numerous potential projects and afterward, bkoep took Timo on a tour of the lab where proteins designed by Foldit players are tested. They examined samples of plasmid DNA, each of which encodes the amino acid sequence of a Foldit player designs, which are inserted into E. coli cells. Then they visited the E. coli incubators where the bacteria are grown, with shaking platforms that keep the growth medium turbulent and well-aerated. Timo was introduced to some of the instruments are used to purify proteins, like a column for size exclusion chromatograph (SEC); as well as other instruments that are used to characterize proteins, like the circular dichroism (CD) spectrometer. Lastly, they viewed some of the protein crystallization trays that have been set up for the numerous projects of the Baker Lab.


bkoep and Timo
bkoep and Timo van der Laan on a tour of the Baker Lab

We thoroughly enjoyed meeting Timo after all these years and are looking forward to working with him on whatever project ends up getting nailed down!

You can read more about these instruments and the design testing process in our previous blog post.

( Posted by  smortier 81 2490  |  Wed, 06/21/2017 - 20:17  |  2 comments )
8
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