Looking for a cure for the common cold? Well, the preliminary testing in mice and human cell cultures is in and it looks like Dr. Rider, along with Scott Wick, Christina Zook, Tara Boettcher, Jennifer Pancoast, and Benjamin Zusman, have done just that; and then some. (Update: Dr. Rider informed me that Jennifer and Ben have had to leave the team)
The therapeutic Dr. Rider has come up with, named DRACO, has stopped infections by every virus it has been challenged with including rhinovirus (the common cold) and H1N1 influenza (the flu). The World Health Organization reports that between three and five million severe flu infections and 250,000 to 500,000 deaths occur each year worldwide, and Dr. Rider and his team may well have given us the weapon we need to stop it and many other viral infections.
The image to the right is a page out of the patent application Dr. Rider filed for DRACO (#7566694) and it lists the different viral, and even some bacterial, infections that DRACO is expected to stop and HIV is among them. Sadly though, the one grant the team has from the National Institute of Health is only sufficient to support one full-time researcher. Two team members have already had to leave but the remaining four are doing what they can. Each one contributes 25% of their time to the project; barely enough to keep it going, let alone build on the incredible results thus far and get this landmark therapeutic into our hands.
The business of killing the viruses that cause human disease, unlike the bacteria, has not met with a great deal of success to date. If you have a bacterial infection you take an antibiotic and in most cases it will be destroyed. Granted, there is the unfortunate side effect of killing our symbiotic flora; the bacteria in and on our bodies that outnumber our own cells and help us in several ways including digestion. And overuse of antibiotics may lead to the rise of the so called super bugs that will be resistant to all of those antibiotics (See it? Kill all those not resistant and if any are resistant, they will have no competition for food and will thus flourish where they would have otherwise been kept in check by the competition. There is more to it, but that’s one crux.). But, they have saved countless lives and turned bacterial infections, by and large, into mere inconveniences. We should note, though, that they are currently used in bulk to pre-treat livestock and increase their size; a practice that may end up being the source of the super-bug that causes great harm to humanity (MacDonald & McBride, 2009).
Destroying viruses indiscriminatingly, on the other hand, provides a favorable outcome. Though this author’s opinion is that they played a critical role in early evolution, I’ve not read of any benefits bestowed on humans by viruses like those gained from the bacteria that live in and upon us. Though I haven’t seen it even hinted at in any research, one remotely possible negative side effect could involve our symbiotic flora. Most viruses are bacterial phages (kill bacteria) so I had to at least consider the possibility that viruses, say in our mucus membranes, may perhaps maintain bacterial population levels. Our immune system is setup such that molecules like proteins that are commonly found in our bodies (called self-antigens) in the absence of an infection are not targeted so it would be feasible. However, I contacted a leading researcher in viral populations and he confirmed that no research to date suggests this viral population control is occurring.
Where there have been studies on viruses in bacterial communities, a milliliter of seawater was shown to contain millions of bacteria, but, also about 10 million “virus like particles” keeping the bacterial population in check (Breitbart & Rohwer, 2005). In any event, DRACO in its current form would be administered most often as a cure for an active infection and it destroyed by our cells in a few days (Rider, Zook, Boettcher, Wick, Pancoast, & Zusman, 2011), so if there was any uptick in bacterial populations it would probably be short lived.
Why the lack of excitement and funding?
Given not only the potential of DRACO demonstrated by the science I will present, but its effectiveness already demonstrated by Dr. Rider and his team, why aren’t there flags flying and a Manhattan Project-level effort to bring it to your Doctor’s office? Conspiracy theories aside, there may be more mundane reasons behind the lack of excitement. As far as the U.S. government is concerned, it only funds basic research, but what of the silence among the people?
Many may know nothing of DRACO, but the reason for that may be that we have become reluctant to express excitement and spread the word about advances in medical science because many don’t translate well outside the lab or, even when they do unforeseen dangers crop up after everyone gets in line. Take gene therapy for example. As it was emerging in the public mind and the notion of an end to all that ails us spread quickly around the globe; first a wet blanket, then tragedy struck. Trial after trial failed, and even when cures seemed at hand, a systemic immunological response to the virus-based transports that delivered the curative genes occurred and Jesse Gelsinger, a trial participant, died (Thomas, Erhardt, & Kay, 2003).
This takes nothing away from neither the historic and often thankless work of our medical researchers, nor even the great potential in gene therapy. So many of our great researchers work hard throughout their lives knowing the chances of a breakthrough are slim and the pay scale is disproportionately low given what they contribute to our lives; including saving them. The simple fact is that their work is as challenging as it comes and yet they soldier on often driven only by the hope of alleviating human suffering.
Now, that’s not to say that the basic elements of life are complicated; they are not. The essence of life’s mechanisms is that we contain molecules which can bind to other molecules, and when they do, they tend to change shape or break apart. When they do that, they often gain the ability to bind to other molecules causing them to do the same and finally to interact with our DNA and change the protein makeup of a cell or even cause it to destroy itself. At the conceptual level, it’s actually quite simple. However, these molecular interactions have been developed and refined over a few billion years and that has led to the evolution of the complexity inherent in human physiology. And working at this level often requires knowledge of subatomic forces and even quantum level interactions so though the expression science is rigorous; errors do occur and negative elements in our human nature occasionally surface.
Why DRACO is Different
So why am I hoping to get everyone excited about DRACO? Well, for starters, each DRACO molecule is built from elements that our cells already use to defend against viral infection. And while it is not uncommon today to use bits and pieces of our proteins in drug development, these pieces are special and, when combined, they tie together two pathways involved in viral immunity to overcome the survival mechanisms viruses have evolved to possess. It will require a little Molecular Biology to make this stuff more clear, and I will provide it in what I hope is a quickly digestible presentation in the next section.
Another reason DRACO is different is that it binds to viral double-stranded RNA molecules regardless of the RNA sequence encoded. That means that it will indiscriminately bind to viral double stranded RNA regardless of what virus produced it and what mutations that virus may have experienced (Nanduri, Carpick, Yang, Williams, & Qin, 1998). So, unlike flu vaccines which rely on predicted viral migration for the coming season, it is not necessary to build a new DRACO for every different virus type.
Finally, there is the protein from which DRACO takes its ability to bind viral double-stranded RNAs (dsRNA). I will give more detail below but for now what is critical is that the part of that protein that actually makes contact and binds to the viral dsRNA is the same in more than 20 other proteins which also bind dsRNAs (Nanduri, Carpick, Yang, Williams, & Qin, 1998). This is very important because it means that the “sequence is conserved”. In other words, the amino acid sequence in this part of the protein is found in many other proteins that also bind to dsRNAs. What that translates to is that it works so well at binding dsRNAs that evolutionary refinement has produced the best sequence for the job and these proteins all share it. Lather, rinse, repeat… works every time.
After the following brief interlude to give what I hope is a very clear explanation of molecular biology at a conceptual level, I will give the detail on DRACO.
A Little Molecular Biology
Believe it or not molecular biology at a conceptual level is simple. The basic tenant is that a gene encodes a protein that is “transcribed” (written) onto RNA (messenger RNA or “mRNA”), and that mRNA is “translated” into a protein by a cellular component called a Ribosome. Now there are a few variations on the theme, but understanding this will give you an understanding of the significance of DNA and the generation of the proteins that do the bulk of the work in a cell. To complete the picture, in different tissues and in the presence or absence of different substances a particular gene’s transcription can be turned on or off; thus the diversity of cell types, their functions and changes within throughout life.
Another fundamentally important element describes the work of proteins in detecting and responding to chemical changes within and without the cell. Past the essentials like keeping the cell alive, changes often involve the arrival of proteins and other molecules released by the endocrine system (thyroid gland, pituitary gland, etc.) and neighboring cells; molecular signals. The number of different molecular signals in the biology of life is vast and when combined in thought with the intricacies of the signal propagation and response, we see that the cell contains the most complex communication system we have ever encountered. (and about 10,000 of them can fit on the head of a pin… the head of a pin! I’m looking at you Intel!)
Like the Operating System and programs on your computer have different jobs but are both written using computer languages, the cells of our body have programs and an operating system. The language is chemistry and the interactions between proteins, lipids (fats), and DNA are like the programs and OS; these interactions are known in the parlance as molecular, or cellular, signaling. These signaling programs are used in virtually every aspect of cellular life including survival, reproduction, and communicating with neighboring cells.
Our cells are wonderful members of their communities. They are constantly checking their surroundings (the Extracellular Fluid, or ECF), responding to what is found there, and letting their neighbors know if anything needs to be done based on what they find. It’s one for all and all for one as each cell and cell type does what they do best for themselves, and the community. (John Nash would be proud) The way this Community Watch program is carried out is through cellular signaling involving receptors, migration, conformational change (a molecule changing shape) or cleavage (a part of the protein gets cut off), and usually transcription. The action continues past transcription as the new proteins created interact with others inducing changes and maybe initiating other signal pathways.
For a good analogy, you can think of it this way; when a parent’s aural receptors, their ears, pick up a sound wave of a particular frequency, say that of a thud, many chemicals, adrenaline for example, begin to be produced in their brain which produce many changes in the brain’s state, including a heightened awareness to sound waves from those receptors (ears). Also, their brain starts to formulate a plan to check and see which child has produced that thud and make sure they are safe… before they punish her or him for playing ball in the house. And this was all started by sound waves of a particular frequency reaching those ears. (ok, ok, a range of frequencies…)
Very often you will find these receptors embedded in the membranes of our cells. You can think of them kind of like ears, only these receptors are very specifically tuned and they are proteins not ears. If our ears were setup like these receptors, we might have two for every frequency in the range of human hearing. Cellular membrane receptors though, rather than sound waves, are usually “specific for” (tuned to) a particular sequence of amino acids in another protein. The amino acid sequence within a protein makes the atoms of the molecule take a specific shape and the amino acid sequence in the receptor’s “binding area” also creates a certain shape. And when some sequence segment in a protein creates a shape that is complementary to (aligns with chemically) the shape on some area of a receptor, like a key that fits a lock, the protein will bind to the receptor.
The protein that binds to a given receptor is known as that receptor’s “ligand” and when that binding starts a chain reaction of subsequent bindings and conformational changes in other proteins, it is the initiator of a molecular signal. That signal usually ends with new proteins being created and those new proteins drive the changes the cell displays in response to the signal. Understanding how this chain reaction, or “signal propagation”, takes place is again; simple at the conceptual level. When a receptor binds its ligand it will undergo a conformational change in response to the new atomic forces exerted on the protein by its bound ligand. Often, this exposes parts of the proteins that were previously folded within and therefore not exposed on the “surface” of the protein. (like a heavily chewed straw cupped in your hand, only some parts of the straw contact your skin) These newly exposed regions are usually on the end of the receptor that is within the cell membrane or in the intracellular fluid (ICF) (see Figure 2). This is commonly referred to as the “activation” of the receptor. The newly exposed regions often bind yet another protein that happens to be floating in the ICF near the membrane where the receptor is embedded. Once it binds, it too undergoes a conformational change or cleavage which results in the exposure of a part of that protein that was previously hidden. And this sequence of binding, conformational change or cleavage, and further binding is how the signal propagates.
Figure 1 – Simple Cellular Signal
Now that you have an idea of how a signal pathway plays out in the cytoplasm, we can consider the last two major factors in signaling; translocation and translation. First is “translocation” which is simply the transporting of a molecule from one location within the cell to another by some mechanism. The way it usually happens is that one of those newly exposed regions on an activated protein allows it to attach to a protein whose job it is to transport proteins into the cell’s nucleus.
Once there, the activated protein can bind and, in turn, activate what are known as transcription factors. Again, big name, simple job; they bind to a specific area on our DNA usually near the start of a gene, undergo conformational change or cleavage which causes them attract RNA transcription proteins. These proteins, in turn, transcribe the gene’s sequence by building an mRNA (messenger) whose nucleotide sequence matches that of the gene. This mRNA molecule is then transported out of the nuclease to another group of proteins called a ribosome. The ribosome then “reads” the mRNA and assembles a new protein by building a chain of amino acids in the order specified by the mRNA.
This is the “translation” of the mRNA and it works like this. There are four nucleotides that make up RNA and every group of three in the sequence indicates a particular amino acid. Just like paint-by-number where a number indicates each of the colors to be used in painting, an RNA sequence is kind of like a shorthand for amino acids. Where in a painting 10 might indicate green, in our cells a ribosome reading “UGU” along a RNA sequence will add a cysteine amino acid to the new protein chain. (U is Uracil, G is Guanine)
The transcription and translation of new proteins is the most common end to cellular signals and while this pathway I’ve provided here is a simplification, it is a simplification in number only. There are usually many proteins and transcription factors involved not only propagating the signal but also regulating it as there are proteins that can inhibit the signal and others than can enhance it. However, what you now have a handle on is the essence of cellular signaling and you can feel proud because no human being up until recently knew how it worked.
Viruses are small “agents” that can infect all type of organisms from humans to bacteria and can only replicate inside an infected cell. They consist of two or three elements; their genetic material (DNA or RNA), a protein coat called a capsid which protects this material, and some contain a lipid “envelope” around the capsid. They enter the cell in various ways involving interactions between the capsid, or envelope, and the cell membrane. These interactions are usually based on a viral protein with several Arginines strung together. Interestingly, it is this “Arginine rich” sequence that has been replicated and added to many drugs, including DRACO, to allow them to pass through the cell membrane.
There are several reasons why viral infections are difficult to stop. For starters, they are not living entities and therefore do not contain proteins critical to life that are necessarily ubiquitous in an evolutionarily constrained form (that means: too much mutation in the gene and the protein function is lost and the organism dies so they remain largely the same throughout in most species). Though not one single genetic element has been found that is shared between all viruses (Rohwer & Edwards, 2002), as we will see, there are those shared within groups of viruses called families. There are common genes within viral taxonomies (Breitbart & Rohwer, 2005), but a gene that is common does not necessarily imply that everyone has exact same sequence of nucleotides. Therefore, the protein produced by the gene in one virus may have a different amino acid sequence than it does in another. The result of this may be that a treatment or vaccine that successfully targets one virus, may not be successful against another.
Another reason combatting viral infections is very difficult is because of the immense diversity and numbers in which they exist in nature. Take about 53 gallons of seawater and you will find about 5,000 different types of viruses and there are estimated to be 1031 viruses on the Earth. Viruses are classified by characteristics such as their genomic medium, DNA or RNA, and structural diversity. Even just classifying them is a monumental job as there is more structural diversity among viruses than there is in all the plants or animals on the planet. (Breitbart & Rohwer, 2005)
However, while the diversity within a small environment, like 53 gallons of sea water, is high, the diversity on a global scale is relatively low. This means that in a small environment there are a large number of different viruses to be found, however, within the next 53 gallons you will find mostly the same virus types (Breitbart & Rohwer, 2005).
Given this immense diversity, it is easy to see why the “targeted” approach so promising in cancer therapies actually may not be feasible for defeating viral infections. Flu shots, for example, target epitopes (short amino acid sequences within a given protein) in two proteins found on the surface of viruses. The proteins are hemagglutinin (H) and neuraminidase (N) thus the names we hear like H1N1. There are different hemagglutinin proteins found on different viral strains, thus the H1, H2, H3, etc. The same holds for neuraminidase (N1, N2…) and each year the World Health Organization analyzes data to determine which strains are moving in force. The results of the analysis determine which flu strains are targeted by flu shots.
Natural Viral Defense
Two major molecular signal pathways are stimulated in our cells in response to viral infection. The first is considered part of our immune system and is known as the Interferon Response, named as such because the proteins produced were observed to interfere with viral replication. The second pathway induces apoptosis (programmed cell death); an everyday occurrence for hundreds of millions of our cells that we will discuss below.
To understand the magnitude of the mobilization resulting from the detection of a virus within one of our cells, consider that the transcription of several hundred genes is initiated by the Interferon Response. This means that hundreds of new proteins are created within the cell and their impact is definitive. Signals are sent out to other cells in the area and if the cell was proceeding through mitosis (replication), the process is halted. Not only is viral RNA transcription halted, but all RNA transcription is halted. Not only is viral RNA destroyed by a protein named RNase L, but are those produced by the host cell. And finally, the second pathway, apoptosis is initiated and the cell kills itself. All this action is undertaken in an attempt to halt further infection though the fate of the infected cell is always death. (Randall & Stephen, 2008)
With the depth and severity of the cell’s response to viral infection it’s reasonable to wonder why we get sick and even die from viral infections (note, death occurring during viral infections is actually often due to a secondary bacterial infection). However, viruses have been evolving longer than any cells, let alone human cells, have existed on Earth (Koonin, Senkevich, & Dolja, 2006) and, today, they primarily infect bacteria which are orders of magnitude more numerous than we are. So, it comes as no surprise that they have evolved methods to avoid detection, destruction, and to overcome attempts to block their replication. Often, these measures only allow the virus to delay the onset of the Interferon Response, but if the delay is long enough, a substantial infection will result and we become ill.
There are five main ways known that viruses can avoid the Interferon Response but we are only going to mention one here; global inhibition of host cell gene expression. This means that the virus has the ability to stop the cell from making any new proteins. (Randall & Stephen, 2008) Even just this one sounds pretty serious, and it is, since most molecular signaling pathways in our cells end with the transcription of the new proteins. However it is important to remember that when talking about cell biology concentrations are important. In other words; how much of this molecule or another is there in the cytosol (intracellular fluid)? So, let’s say some virus has the ability to block the Interferon signal pathway. However, it needs to produce enough of the protein that can produce the interference before the pathway is completely shut down. This interference is usually the result of the targeting of one protein in the pathway, so, if the cell can produce that protein faster than the virus produces its inhibitor only a delay in the Interferon Response will result. The cell will eventually win out and apoptosis will occur, hopefully before viral replication proceeded far enough to infect many of its neighbors.
Protein Modularity: The Chimera
One of the most interesting properties of proteins is the apparent modularity of their structure with regard to function. That is there are discrete functional units, called domains or moieties, within proteins that carry out specific actions such as binding to a site on another protein or pulling the entire protein through a cell’s membrane. Amazingly, these units can be “inserted” into a different protein resulting in the addition of that function to the protein usually without the impacting the function of its existing domains. Even when the insertion results in adverse effects either on the function of the domain inserted or those indigenous to the protein a process called “Directed Evolution” can be used to ameliorate those effects.
The protein into which the domain is added is usually referred to as a Chimera. Chimeras are typically produced by splicing the DNA sequences of interest from the source genes together to create a new sequence. The new sequence is then inserted into the DNA of a bacteria, usually E Coli., and the bacteria then creates the protein through the normal transcription/translations mechanism. When a sufficient number of mitotic cycles have produces a sufficient number of bacteria containing the new protein, a large amount of the chimeric protein will be produced. At this point the bacteria are lysed (cut) so the cytosol spills out into the solution containing the bacteria. That solution is then purified and refined until it contains only the new protein.
Mechanisms of Cellular Uptake
The trick with any therapeutic which, like DRACO, needs to operate within a cell is to transport it across the membrane. Although the cell membrane is very effective at keeping the cytosol (fluid inside the cell) isolated from the extracellular fluid (ECF, fluid outside the cell), the separation is not absolute; molecules can enter the cell from the ECF in several ways. One way, called endocytosis, is actively carried out by the cell itself and mediated by a receptor specific to the molecule within the membrane. When the molecule binds to the receptor the cell’s membrane engulfs it by extending its membrane around it and forming a vesicle as it pinches off inside the cell. Another method is for the molecule to pass directly through the membrane by virtue of chemical interactions between the molecule and those found on the surface of the cell. This is the method employed by viruses and DRACO alike and I will present details below.
Apoptosis, or programmed cell death, is a normal process by which cells self-terminate. This often occurs in response to signals from the cell’s environment or from the detection of mistakes in the DNA replication process during mitosis. Apoptosis causes the death of 50-70 billion cells per day in the average adult (TOCRIS Biosciences). Like most processes in cells, it involves the propagation of a signal carried out by interactions between proteins. As the signal progresses, the cell’s DNA is degraded, the protein skeleton of the cell is dismantled, and finally the cell itself is degraded and consumed by the macrophages of the Immune System. More on this later…
DRACO: Natural Extracts
DRACO, or Double-Stranded RNA Activated Caspase Oligomerizer, is a chimeric protein containing three critical domains. The first provides transport across the cell membrane into the cytosol and even into the cell nucleus. The next is a domain that detects viral double stranded RNA (dsRNA) and binds DRACO to it. The third domain binds to Caspase 9. The beauty in DRACO is in the simplicity and inherent safety of the mechanism by which its effector function, apoptosis, is induced in a cell found to contain viral derived dsRNA. The binding of DRACO to dsRNA and Caspase 9 in itself does nothing. And this is a good thing because dsRNA is also produced by our cells, however they are short in length compared to those of viral origin. In order for DRACO to induce the apoptotic signal, many DRACO molecules must bind to the same dsRNA, thus the strand must be long. When enough DRACOs bind to the same dsRNA, the bound Caspases will automatically activate each other and induce the molecular signal that induces apoptosis.
To understand another reason why DRACO is so effective and safe, one must consider words T.S. Elliot once wrote “Immature poets imitate; mature poets steal”. When it comes to the domains that make up DRACO, Dr. Rider is a mature poet. Instead of taking a synthetic approach and attempting to recreate the cellular components or their function in response to viral infection, he took the domains directly from the human genes that encode the proteins involved in that response to create a chimera.
The transport domain of the DRACO protein is a short sequence of Arginines (an amino acid) which form a structure that is drawn to the outer surface of the cell membrane and then bind to molecules contained within. The binding is strong enough to pull the DRACO molecule into the membrane and through both layers, yet weak enough to break as it passes through the inner layer; releasing into the cytosol (Wender, Galliher, Goun, Jones, & Pillow, 2008). To determine the effectiveness of the transport domain, the team employed florescent tags with which they can track the location of the DRACO molecules. These tests showed that the poly-arginine transport, along with two other configurations, were successful in taking DRACO not only into the cytosol, but into the nucleus as well. And this success was encountered not only in cultured human cells but also in mice.
Detection and Binding
To enable DRACO to detect and bind dsRNAs, Dr. Rider selected a domain from one of our proteins that does that very job. This protein is known by the recommended name (ready for it?) “Interferon-Induced, double-stranded RNA-activated protein kinase” or (thankfully) the alternative short name “PKR” and it, as its name implies, binds to dsRNA. PKR is “constitutively expressed” in our cells, which means that it is always present. However, normally PKR is in an inactive state meaning that something needs to happen to it before it will perform its function and that something is its binding with viral derived dsRNA. Essentially PKR is a Pattern Recognition Receptor (PRR) and recognizes dsRNAs via their structure rather than any particular sequence of nucleotides making it the logical choice for DRACO. This is because its ability to bind will not be effected by mutations in the genetic sequences of any given virus. PKR plays a critical role in our cellular defense against viruses and it has been shown to play this role in a range of viruses including HIV and HSV (Herpes Simplex Virus). (Sadler & Williams, 2008)
Once activated, PKR has been shown important in many aspects of the defense against viral infection. This includes halting mRNA translation and initiating the ubiquitination (targeting for destruction) of a protein called Cell Division Cycle 2 (Cdc2) thus arresting cellular mitosis (Sadler & Williams, 2008). But all Dr. Rider was interested in from PKR was the domains that allow it to bind to viral dsRNAs. So, why did I tell you about the important functions of PKR if all we care about here is the binding domains? Because, a protein that plays several critical roles in cell physiology will have evolved highly efficient and effective domains; and that makes the dsRNA Binding Motifs (RBMs) of PKR a great choice for a DRACO.
Now, given the fact that we all know that viral infections can not only be fatal, but chronic (persisting for long periods), doesn’t that suggest that PKR is flawed in its ability to contribute to a successful mitigation of a viral infection and therefore confers on DRACO equal deficiencies? Well, no, because the methods viruses have evolved to defeat the response of our include targets other than PKR, and even those that do target PKR generally inhibit downstream interactions and events resulting from PKR activation (Randall & Stephen, 2008).
There are three commonly described signal pathways that lead to apoptosis named; the intrinsic, the extrinsic, and the Perforin/Granzyme (intrinsic; “from the inside”, extrinsic; “from the outside”, and Perforin… well, never mind, your T Cells know what they are doing). The extrinsic starts with the binding of a cell surface receptor, but we will focus on the intrinsic since it is relevant to our discussion.
The intrinsic pathway starts with the mitochondria and is sometimes described as more than one pathway given that more than one stimuli can initiate the signal pathway. Examples of the stimuli include radiation, toxins, hypoxia (low oxygen), hyperthermia, viral infections, and free radicals. In any case, the mitochondria will effectively lyse (rupture of the membrane), spilling its previously sequestered contents into the cytoplasm. Important among them is Cytochrome C which binds and activates another protein named Apaf-1 which then binds a protein known as Caspase-9 inducing in it a conformational change. This change allows Caspase-9 molecules to bind together and when they are in close proximity they activate each other. This “cross-activation”, as it’s known, starts a signaling cascade and commits the cell to apoptosis. (Elmore, 2007)
Join to overcome
So we have two critical elements of our cell’s viral defense; detection/interferon response and apoptosis and they seem pretty potent, so why do people get sick and even die from viral infections? (Note, though that people usually die from secondary bacterial infections not the viral infection itself) As I eluded to above, the reason is that both the Interferon Response signal pathway and the apoptotic pathway can be blocked by the various counter-measures that viruses have evolved. So what is so great about DRACO? One might say that I haven’t really made that case; only highlight probable causes of its failure. After all, if DRACO combines key domains from the very cellular proteins whose pathways can be blocked, how can DRACO be effective in the face of these viral counter-measures?
To understand why the domain combination that is DRACO has been undeterred in actual testing against viruses using these counter measures, first consider that the Interferon Response induces the transcription of hundreds of proteins. These anti-viral proteins can stop viral replication, signal other cells to enter an anti-viral state, and induce apoptosis; but their translation (production) can be blocked. DRACO, however, does not rely on the Interferon Response or those proteins produced as a result thereof. DRACO can induce apoptosis without the need for the Interferon Response and it can enter all cells, so signaling the neighbors of the problem is not required. So, even if the Interferon Response signaling pathway is blocked, DRACO’s ability to function will not be impeded.
Fair enough, one might say, but you wrote above that viral counter measures also target the apoptotic pathway which is DRACO’s effector function (the way it does its job) so that would be enough to render DRACO useless at least against viruses that can do that. However, it turns out that those counter measures most often target the initiation of the signal and DRACO acts near the middle. For example, consider that the majority of viruses block apoptosis in a way analogous to how Dr. Rider built DRACO. While they obviously didn’t use their brilliant minds to search for a potent domain to add to a protein of their own. They did evolve a domain in a protein common to many viruses that is also found in human proteins and, of course, produces the same function. In humans it is found within the so-called “Bcl family” of proteins and, interestingly, some members are a help in apoptotic signaling (pro-apoptotic) and some block it (anti-apoptotic).
The domain is known as the BH domain and whether a given member of the Bcl family is anti- or pro- apoptotic, seems to be determined in large part by the number of these BH domains it contains. For example, in humans the Bcl-2 protein is anti-apoptotic and, no surprise the viral version of the protein, vBcl-2, is also anti-apoptotic. To inhibit apoptosis, Bcl-2, and its viral counterpart (homolog), gather up the pro-apoptotic members of the Bcl family that stick in mitochondrial membranes and inactivate them thus blocking the apoptotic signal pathway from proceeding. (Galluzzi, Brenner, Morselli, Touat, & Kroemer, 2008)
So where does DRACO act in the apoptotic signal pathway? It acts downstream of these actors as it contains the Caspase-9 binding domain from Apaf-1 which we discussed earlier. Once activated, Caspase-9, in turn, activates Caspase-3 which then leads to the activation of proteins already in the cytosol that cut up the chromatin (uncoiled DNA) and destroy the cell.
So all is perfectly well? Well, no, not perfectly. I did manage to find one paper that described a virus containing a protein that inhibits Caspase-3 from carrying out its functions (Wang, Sharp, Koumi, Koentges, & Boshoff, 2002). However, for those rare viruses, Dr. Rider assured me “If we do run into a virus that is resistant to DRACO for whatever reason, we have a large number of tools we can use to solve the problem.” Gotta love him! And isn’t it great that he is at MIT using his brilliant brain to alleviate human suffering rather than in some Wall Street firm flipping bits to get rich?
But there still is the matter of a virus producing a protein that can shut down new protein production in the cell, how can DRACO be effective against viruses that can do that? Well, as we will see below DRACO is effective even against these viruses as evidenced by its defeat of an infection by an Influenza A virus. These viruses produce a protein called NS1 that inhibits processing and export of cellular mRNAs (Randall & Stephen, 2008) which means that they never make it out of the nucleus and therefore are never transcribed. While this can shut down the Interferon Response long enough to allow the virus to replicate and escape to infect other cells, DRACO’s function is unaffected. This is because DRACO’s effector signaling components, the Caspases, are constitutively (always) present in the cytosol so no new protein synthesis is required to propagate the signal and start apoptosis.
Which Viruses and Why…
So out of the large number of viruses that can infect humans, how many will DRACO be able to stop? Virtually all of them. This is because, all viruses produce it as a byproduct of the process of creating their proteins and replicating their genome. Now, as I eluded to above, there are other ways viruses can evade the dsRNA binding domains of the Interferon Response. Some developed measures to hide their dsRNA either in the host cell nucleus or cellular vesicles, and others produce a protein that can bind to it, thus blocking host proteins from doing the same. However, biological systems tend not to be perfect, often far from it. A small amount of viral dsRNA eludes the virus’ attempts to hide it and while it is often insufficient for the Interferon Response, it seems to be sufficient to set DRACO into action.
As evidence, DRACO has been effective against every virus tested and Dr. Rider had this to say “we have successfully demonstrated that DRACO works against all the negative-stranded ssRNA (single stranded) viruses we have tested thus far, including two different influenza strains, two members of the arenavirus family of hemorrhagic fever viruses (Tacaribe and Amapari), and two different Guama bunyavirus strains (related to hantaviruses). Thus we have demonstrated DRACO efficacy against negative-stranded ssRNA viruses, positive-stranded ssRNA viruses, dsRNA viruses, and DNA viruses.”
Now you may be wondering “what does that mean”? That means that despite existing research suggesting that some RNA viruses don’t produce dsRNA, they actually do (Weber, Wagner, Rasmussen, Hartmann, & Paludan, 2006). Why did the experiments fail to show this? Because there are limits to the detection methods we use today and one of those limits is a lower bound to the amount of the material that must be present for it to be detected. DRACO, on the other hand, appears to be far more efficient as it functions with dsRNA levels below threshold of detection.
The proof, they say, is in the pudding and while I’m not sure who “they” are and what kind of pudding they are peddling, for DRACO the proof is in the “tissue survival after infection” pudding and even the early results are silencing the critics (again, I don’t know who they are, but if I did… I would show them Dr. Rider’s paper). Earlier I mentioned some methods viruses employ to evade the Interferon Response, including hiding their dsRNA byproducts in the nucleus, and here I describe the test results that prove DRACO is undeterred.
Let’s start with a perennial favorite; the flu which is caused by anyone of “the influenza family of viruses”. The flu shots offered to us every year contain a quantity of two inactive or partially active Influenza A viruses and one Influenza B virus that the CDC determined will be the members of the family infecting us in the coming season. Now we know DRACO isn’t concerned with variations in surface and structural proteins whose results we use to characterize viruses, but with Influenza, there is a catch. The influenza virus has been shown to hide its dsRNA in the infected cell’s nucleus. It has been reported that no dsRNA “intermediates” (process byproducts) are found in the cytosol of cells infected with an Influenza A virus; the same one used in DRACO testing. The researchers found viral derived dsRNA in the nucleus but as expected but they did not detect it in the cytosol. (Wisskirchen, Ludersdorfer, Müller, Moritz, & Pavlovic, 2011)
However, Dr. Rider and his team has shown that DRACO is not only transported into the cytosol, but also the nucleus. So how do we think DRACO fared when challenged it with Influenza A? A picture is worth a thousand words, so let’s have a look. To the right, you can see in both images in the top row, there are plenty of cells happily bathing. (They are human Hepatocytes; liver cells) In the bottom row which shows those cells after infection with the H1N1 virus used in that study, and things are very different. Cells that were not protected by DRACO are on the left, at least they were; those protected by DRACO are on the right. Cells that were not treated with DRACO died within 3 days of infection, however, the cells on the right, even after 72 days, show no sign of viral CPE. What is viral CPE? A fine question… CPE is “cytopathogenic effect” and it means that something, H1N1 in this case, caused disease in cells (cyto = “cell”, pathos = “feeling, suffering”, and genic = “to produce”).
By the end of the initial tests, Dr. Rider and his team tested DRACO against 15 viruses in human cell cultures and in mice. Eleven of those replicated in the cytoplasm and four replicated in the nucleus, and DRACO stopped them all. These viruses even produced the protein containing dsRNA binding domains which they use to hide their dsRNA from the Interferon Response (LIN, LAN, & ZHANG, 2007) but DRACO kept the cells alive none the less.
How long to transition (when can I get it at my drugstore)?
Wonderful, you might be saying, Dr. Rider and his team should share the Nobel Prize, but when can my doctor start writing prescriptions? Not just yet. Great, you might be saying now, DRACO is just like the others after all; a fantastic success in the lab, but given that I am not, and have no intention of becoming, a mouse; it won’t help me.
You are, of course, justified in feeling that way, but this common scenario need not play out with DRACO. As I mentioned earlier Dr. Rider and his team are committed to improving health and saving lives as evidence by the fact that they are finding ways to continue their work despite a very sad lack of adequate funding. But therein lies the key: funding.
The government, will only fund basic research and no pharmaceutical or biotech has stepped up to fund the work. So, perhaps we can step in, en masse, to help Dr. Rider in his attempt to make history of human suffering from viral infections. If more than 10,000,000 people can contribute to the re-election campaign of a certain President of the United States, just think what we can do with DRACO.
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