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.
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 79 1027 | Wed, 09/27/2017 - 21:41 | 1 comment )
Introducing Small Molecule Design Puzzles
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.
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!
* 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 79 803 | Tue, 09/19/2017 - 23:11 | 0 comments )
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.
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 79 1369 | Wed, 06/21/2017 - 20:17 | 2 comments )
Since our last blog post, we've carried out an x-ray diffraction experiment with one of our protein crystals. We were lucky that the protein crystal yielded high quality diffraction data, and from this data we were able to solve the first-ever crystal structure of a protein designed by Foldit players—a near-exact match to the designed structure! Below we explain a bit more about x-ray diffraction. In a later post, we'll examine the final structure in more detail.
First, the protein crystal is harvested from the drop using a small loop of nylon, about 0.3 mm across. Protein crystals are often very fragile, so looping the crystal requires a steady hand (i.e. optimal coffee dosage). Even in the loop, the crystal is still immersed in an aqueous solution, with the surface tension of the water helping to keep the crystal in the loop. The loop is rapidly submerged in liquid nitrogen, at a temperature of about -200ºC, which quenches most of the thermal motion of molecules in the crystal.
Once frozen, our looped crystal is mounted on a robotic arm that positions the loop in the path of an x-ray beam. During x-ray exposure, the crystal is kept under a steady stream of cold nitrogen gas to limit temperature increases in the crystal. X-rays have a high energy, and a protein crystal can only endure so much exposure to x-rays before it starts to degrade. The protein lattice could disintegrate from the increased thermal motion of individual protein molecules, or else the x-rays could trigger chemical reactions within the protein, distorting its structure.
X-rays are simply a type of electromagnetic radiation with a very short wavelength—in this case about one angstrom. In an x-ray diffraction experiment, it's important that all radiation has exactly the same wavelength and is focused into a very narrow beam. With our crystal mounted in the path of the x-ray beam, an x-ray detector is positioned behind the crystal, and measures incident x-rays after they strike the crystal and are diffracted by electrons of the protein molecules within. Because of the regular arrangement of atoms in the protein crystal, diffracted x-rays undergo constructive interference in particular directions. This occurs when two equivalent "slices" of the crystal are oriented to coincide with the wavelength of the x-rays. Wherever constructive interference occurs, the detector registers an especially intense signal, shown as a dark spot on the image below. Taken together, these spots comprise a diffraction pattern.
Above is an x-ray diffraction pattern from a protein crystal. In the inset at the right, we can see that some spots seem to have duplicates which are slightly offset. This indicates that there are actually two identical crystals in the path of the x-ray, lying in slightly different orientations. Most likely, the crystal cracked in two during freezing. Fortunately, the image-processing software we use is sophisticated enough to correct for this issue.
The spacing and position of spots is governed by the size and shape of the crystal’s unit cell, the repeating unit that makes up the crystal. The intensity of each spot is determined by the distribution of electrons within the unit cell (i.e. the positions of atoms in the protein). Every atom of the unit cell contributes to each spot in the diffraction pattern. If you could change the electron density around just one atom of your crystallized protein, this would alter the intensity of every spot in the diffraction pattern!
Notice that spots farther from the center of the detector tend to be less intense. More distant spots contain higher resolution data about the electron density of the protein. If we adjust the contrast of this image, we can discern spots close to the edge of the detector. This protein diffracts x-rays to a resolution limit of 1.20 Å! In an electron density map derived from these diffraction patterns, we should be able to distinguish the positions of individual atoms.
If the crystal is rotated relative to the x-ray beam, then we would observe another diffraction pattern, as the new orientation produces constructive interference in different directions. We typically measure a new diffraction pattern at rotation intervals of 0.5 degrees, eventually rotating the crystal a total of 180 degrees (sometimes less for highly-symmetric crystals) to collect a complete dataset. This dataset was collected with a state-of-the-art detector that can measure individual photons; collecting a full dataset takes no more than a few minutes. In the early days of protein crystallography, it could take a whole day to collect a complete dataset!
The processing and interpretation of a these x-ray diffraction patterns is a complex, technical procedure, and we won't go into it here. But suffice it to say, this x-ray diffraction data revealed a full, high-resolution crystal structure of this Foldit player-designed protein!
Congratulations to Waya, Galaxie, and Susume who contributed to this solution in Puzzle 1297! All players should check out Puzzle 1384 to explore the refined electron density map from this data, and see if you can fold up the protein sequence into its crystal structure! We'll follow up later with a more detailed comparison of the designed model and the final crystal structure.( Posted by bkoep 79 1027 | Tue, 05/30/2017 - 04:59 | 3 comments )