No, this post is not about how your blood pressure rises when surprised by a snake. Instead, this is a narration of the discovery of one of the most successful medications against hypertension, courtesy of the biology of certain snakes coupled with human curiosity and ingenuity.
Fair warning: Biochemical and pharmacological geeking out ahead (no equations or structures, though)! 😄
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Heart disease. Very few medical conditions are as traumatic and fearsome as sudden-onset heart disease. Traditionally, the heart is synonymous with life itself, and in fact, for quite a long time, the very end of human life was synonymous with the termination of heart function alongside the cessation of breathing.[1]
Not surprisingly, from the perspective of the medical sciences, cardiac physiology and pharmacology are some of the most active areas of research. For such a vital organ group, the cardiovascular system structure is deceptively simple. As a first approximation, the cardiovascular system in vertebrates essentially consists of a muscular pump, the heart,[2] connected to an intricate network of blood vessels. But functionally, it is so much more. The circulatory system is exquisitely controlled at multiple levels, including heart rate, the diameter of the blood vessels, and the composition and physical properties of the blood itself, among many other factors. All of these control mechanisms are indispensable to sustain life. When these mechanisms are disrupted, many pathologies can emerge, both chronic and of sudden onset. It is a fact that cardiovascular diseases include some of the major causes of mortality worldwide, making it a significant public health issue. In the United States alone, hundreds of thousands of people suffer from heart attacks per year. As far as chronic conditions are concerned, hypertension takes the prize, with close to one billion sufferers worldwide. It is important to point out that uncontrolled hypertension can lead to various sudden-onset cardiovascular conditions like heart attacks and stroke. Interestingly, pathological variations in blood pressure also seem to play a role in neurological disorders such as Parkinson’s and Alzheimer’s diseases and others.[3] Thus, it is no wonder that cardiovascular diseases and their treatment are two of the most intensively studied areas of clinical medicine.
The main topic of this post is a family of medications whose invention was directly related to the study of the venom of certain snakes. The prototype of these medications is Capoten® (Captopril). This medication was inspired by the venom of a pit viper (Bothrops jararaca), and it was approved in 1981 to treat high blood pressure. Its biochemical target is the angiotensin-converting enzyme (ACE).
This is a fascinating story, but before that, let’s talk a bit about snakes.
Snakes. Not many animals induce in us the wide range of emotions that snakes do. People hate them, fear them, and oftentimes even love them. However, the truth is that not many people are indifferent to snakes, particularly venomous ones. These organisms have an indisputable place in our legends and traditions, and humans have been familiar with their healing properties for millennia. A strong indication of this fact is the universally recognized symbol of the modern medical sciences, the caduceus, a staff with a snake (only one) around it. This symbol dates from the ancient Greeks’ times and recognizes the immensely important role of certain snakes and their venoms in the practice of clinical medicine.
Many species of snakes are venomous, and as all venomous organisms do, these snakes use their venom as an integral strategy for prey capture, as a predator deterrent, or both. As such, these venoms interfere with many different physiological processes in the target organism, particularly those that tend to limit movement when disrupted.
The systems affected by the venom frequently include the nervous and circulatory systems, but the venom’s effect are not limited to that. In yet another example of evolutionary convergence, most snake venoms display the presence of hyaluronidase which is used as a spreading factor for the venom, a property shared by other animals.
Initially, the impetus for the study of venomous snakes was to try and find antidotes to their venoms to be used as therapeutic agents against snakebite. In fact, even today, snakebites are a worrisome public health problem. This problem is especially acute in parts of Africa, Asia, Latin America, and Oceania. The current annual estimates of the incidence of snakebites indicate that there are up to 2.5 million snakebites, of which about 5 % (close to 125,000) result in fatalities.
Since ancient times, some types of snake venoms were used as therapeutic agents by themselves, not just to find antivenoms. However, it was not until the early 1900s that concerted efforts to study venoms to obtain new medications were organized. Still, it was only in the 1960s that the true endeavors towards actual rational drug design were beginning to be worked on based on venom-derived compounds. The concept of rational drug design is usually associated with modern bioinformatics and genomic techniques, but in reality, this drug discovery model began in the 1970s. This research started with the invention of the first medication derived from venom, the antihypertensive Captopril.
Hypertension. Hypertension is one of the most common cardiovascular pathologies. Close two one billion people worldwide are affected by it; this is close to 1/7th of the world population! To function properly, the body needs to maintain adequate levels of blood pressure (BP). Very low blood pressure is as dangerous as hypertension.
Moreover, there is nothing wrong with elevated blood pressure when the situation merits it, as in the well-known fight-or-flight response, where the body automatically prepares for emergencies. The fight-or-flight response includes complex mechanisms through which, among other things, the body allocates its resources, one of them being blood circulation, to handle the emergency better. For example, in this situation, the digestive and reproductive systems are transiently shut down while you flee or fight (there is something very wrong with you if you think about food or reproduction when being chased by a bear; just saying).
Physiologically, the fight or flight response is only meant to be active for short periods. Once the emergency is over, everything goes (or should go) back to normal. Blood pressure should rise when one is working out, for example, or while under exertion for any other circumstance. Again, once physical activity decreases, blood pressure should decrease in turn and eventually go back to resting levels.
The problem arises when blood pressure remains abnormally elevated for more extended periods and out of sync with periods of physical activity. Under these chronic conditions, high BP becomes a risk factor for various pathologies, such as cardiovascular and cerebrovascular events and kidney disease, among other ailments. By the way, when chronic hypertension is of unknown origin, it is called essential hypertension. The current understanding of essential hypertension indicates that several genetic and environmental factors can contribute to its origin. For example, essential hypertension tends to run in families, strongly suggesting a genetic link, hinting at biochemical abnormalities in the patient. On the other hand, some of the environmental factors known to contribute to the pathogenesis of essential hypertension include diet, stress, lifestyle, and other diseases like diabetes, for example.
Physiologically, there are many ways to modulate blood pressure. Some of these ways include the production of endogenous substances by the body, which upon binding to their appropriate receptors, induce a specific physiological change that affects blood pressure in some way. A subset of pharmacology explores the study of such endogenous substances, termed autopharmacology, a term coined in the 1930s by Sir Henry Dale, one of the founding fathers of modern pharmacology. He defined the term as the study of the “…pharmacological effects of natural substances in tissue that are released under a variety of pathological conditions…”.
These endogenous substances are collectively named autacoids. Autacoids are cell-produced compounds that act like hormones but at a much more local level. In general, autacoids have a brief duration of action and induce their physiological effects near the site where they are synthesized.
Autacoids include many types of familiar molecules like the immunomodulatory compound histamine (the small compound responsible for many allergic reactions; this is where the name antihistamine comes from). Other well-known autacoids include neurotransmitters, hormones, and related molecules. The story of the very first venom-derived approved drug relates to one of these autacoids, the small peptide bradykinin, as we will see next.
Captopril. The narrative of any scientific discovery is inevitably tinted by the human point of view. Nonetheless, the history of Captopril is well-documented. In addition to its importance to the medical sciences, this story is also important to illustrate the initial efforts to develop rational drug design practices. As in any modern interdisciplinary effort, many people were involved in the genesis of Captopril as an antihypertensive agent, from the generation of the concept to the actual approval and marketing of the drug. This account is, by necessity, an abbreviated one.
This story’s central (non-human) protagonist is a snake, specifically the Brazilian pit viper, Bothrops jararaca.
Pit vipers comprise a group of some 150 species that include some very familiar species, like rattlesnakes. All pit vipers share a common characteristic: the presence of so-called pit organs located between the eyes and the nostrils of the snake. These organs endow the animal with the ability to detect light at infrared wavelengths; in other words, they can see heat. This ability is a handy feature as these snakes usually prey on small mammals like mice. The vipers’ heat-sensing ability complements their senses of sight and smell.
As the name implies, the Brazilian pit viper hails from South America. They are generally found in the rainforest, but it is not uncommon to find them in other habitats. These vipers are not particularly large snakes; their average size is about 60 cm (close to 23 inches) long. In general, the females are larger than the males. Since their habitat significantly overlaps with human settlements, this snake represents a significant danger to people in such areas.[4] Close to 90 % of snakebite cases in South America are attributed to this particular species. Of course, the dangerous nature of this snake resides not in its size but its venom. Partly because of their relative size and partly because of the specific composition of the venom, female Brazilian pit vipers are more lethal than males. At any rate, one of the main symptoms of their bite in people is an extreme and, therefore very dangerous, decrease in blood pressure. This particular effect of the venom was the origin of the idea to study it, with the purpose of finding substances capable of lowering blood pressure.
This hunch was correct, and scientists found several examples of blood pressure-lowering substances. One of such substances found in the venom was bradykinin, discovered and isolated from the venom of B. jararaca in the 1940s by Drs. Wilson T. Beraldo and Mauricio Rocha e Silva, both from Brazil. Even though bradykinin was originally discovered in the Brazilian pit viper, it is also an autacoid produced in the human body. Bradykinin is formally classified as an inflammatory mediator, and its primary function is to lower blood pressure. Bradykinin lowers blood pressure mainly by dilating blood vessels due partly to its stimulation of the release of a potent vasodilator, nitric oxide, an endogenous molecule that is also the same substance released when cardiac patients take a nitroglycerin pill.
In time, research showed that bradykinin was a small molecule, specifically a short peptide (only nine amino acids long), and therefore amenable to chemical synthesis. However, upon testing synthetic bradykinin, it was immediately apparent that something was missing from the overall picture. The researchers found that “natural” bradykinin isolated from snake venom proved to be more potent and long-lasting than the synthetic version. Moreover, when synthetic bradykinin was tested in the presence of snake venom, it displayed more potent effects than when tested by itself. These results hinted at a biochemical puzzle worth investigating, and the task fell on another associate of Dr. Rocha e Silva, a postdoctoral researcher named Dr. Sergio Ferreira.
Dr. Ferreira joined the research group of Dr. Rocha e Silva in the 1960s, virtually right out of medical school. His first scientific love was neurophysiology, but at the time, there were no opportunities in this research area in his native Brazil. Therefore he ended up in the laboratory of Dr. Rocha e Silva, a twist of fate that proved very fortunate for Dr. Ferreira.[5] Based on careful research on the specific venom components of B. jararaca, it became evident that there were additional substances that enhanced the hypotensive activity of bradykinin. Dr. Ferreira and colleagues determined that this substance was yet another small peptide itself, with an undetermined sequence at the time.
In 1965, Dr. Ferreira published the discovery of the first such substance. Eventually, several similar peptides were discovered (but not fully characterized at the time) and collectively called the bradykinin potentiating factor (BPF). These peptides differed slightly in their length, and interestingly, they were found not only in the Brazilian pit viper venom but also in the venom of several related species. These small peptides enhanced the activity of bradykinin by preventing its deactivation by an as yet unknown enzyme. BFP turned out to be essentially bradykinin’s bodyguard!
The plot thickened when Dr. Ferreira started working with Dr. John Vane of England’s Royal College of Surgeons. Dr. Vane had a longstanding interest in the biology of hypertension, among other research interests. In fact, Dr. Vane shared the 1982 Nobel Prize of Physiology or Medicine in part for his studies on the analgesic and anti-inflammatory mechanisms of aspirin, a line of research done in collaboration with Dr. Ferreira.
The collaboration between these two scientists was quite fruitful, as Dr. Ferreira and Dr. Vane published no less than 25 joint papers between 1967 and 1997.[6] At the suggestion of Dr. Vane, Dr. Ferreira tested his bradykinin-potentiating factor against an enzyme that displayed a mechanism of action very much related to the genesis of hypertension. This enzyme was called angiotensin-converting enzyme (ACE), and Dr. Ferreira’s experiment paid off, as we will see soon.
Before continuing with this story, we need to take a brief detour to explain the relationship between ACE and hypertension. It is not as direct as it would seem. Several enzymes and peptide precursors are involved in this process.
Very briefly, when normal blood pressure drops, this change is sensed by specific cells in the kidneys, which take notice and release an enzyme called renin into the bloodstream. This enzyme by itself does not affect blood pressure. The renin substrate is a 400-plus amino acid long protein called angiotensinogen, produced by the liver. Renin then cuts a portion of angiotensinogen containing the first ten amino acids. This ten amino acid peptide is called angiotensin I, which by itself only has a modest effect on blood pressure.
A second enzyme, angiotensin-converting enzyme (ACE, see above) cleaves the last two amino acids of angiotensin I, generating an eight amino acid peptide, angiotensin II. ACE is produced and activated in the lungs. Now, angiotensin II is a rather active chemical species, which increases blood pressure through various mechanisms: it narrows the diameter of the blood vessels (a phenomenon called vasoconstriction), it signals the activation of vasopressin, an enzyme that causes the body to retain water, and induces the release of adrenaline and noradrenaline, two substances that further increase vasoconstriction. Additionally, angiotensin II stimulates the release and activation of another enzyme, aldosterone, which modulates water retention. The combination of vasoconstriction and increased water volume are the two factors that directly influence an increase in blood pressure.
Based on these facts, the researchers reasoned that any chemical compound that prevents the conversion of angiotensin I into angiotensin II has an excellent chance of being developed into an antihypertensive medication. This in fact was the rationale that led Dr. Vane to suggest testing Dr. Ferreira’s bradykinin potentiating factor on the ACE. Surprisingly, it turned out that ACE was the same enzyme that inactivated bradykinin and was the “natural intended target” of the bradykinin potentiating factor.
At around the same time, in 1968, Dr. Y.S. Bakhle, a colleague of Dr. Vane, demonstrated that an extract of the Brazilian pit viper venom could inhibit ACE isolated from dog lung tissue. This information sparked the collaboration between two scientists at the pharmaceutical company Squibb, Drs. Miguel Ondetti and David Cushman, a chemist and a biochemist, respectively. Coincidentally, at the time, Dr. Vane was a consultant for Squibb, and he was the one who actually suggested working on the angiotensin-converting enzyme. One could say that Dr. Vane was the actual catalyst for the eventual development of Captopril!
Originally, Vane suggested that Ferreira, Ondetti, and Cushman collaborate in the project, but it was not to be. Instead, Ferreira became a scientific competitor of Ondetti and Cushman in the search for the specific peptides that composed the Bothrops jararaca bradykinin potentiating factor, which was also known to be an ACE inhibitor. The explicit hope was to obtain a compound with the potential of being developed into a medication. Dr. Ferreira isolated the first successful substance from the venom, a pentapeptide (5 amino acids long) that potentiated the action of bradykinin, which they called BPP5a. Although effective in its anti-ACE activity and antihypertensive properties, it was not very stable in the sense that enzymes quickly degraded it in the body.
On the other hand, Ondetti and Cushman were able to isolate and fully characterize six longer peptides from the venom, which showed anti-ACE activity. They determined that two specific terminal sequences in these peptides: Tryptophan-Alanine-Proline, and Phenylalanine-Alanine-Proline, proved optimal for the observed activity. In addition, one of the peptides that they found was exceptionally stable, a nine amino acid peptide called teprotide, on account of the presence of four proline amino acids in its sequence.
The presence of this specific amino acid, proline, became essential to this story. The main disadvantage of teprotide, a burden shared by most peptides, is that they are usually tough to develop as oral medications, in significant part because peptides tend to be degraded by digestive enzymes. This means that such compounds need to be administered by injection (almost no one’s favorite method of taking medication). This fact almost caused the end of this specific line of investigation, yet the researchers persevered. Eventually, teprotide (of course, in injectable form) was the first compound clinically tested in hypertensive individuals, which yielded auspicious results.
Based on additional lines of research, Cushman and Ondetti hypothesized that a small molecule composed of two amino acids at most was the way to go in the search for ACE inhibitors amenable to oral administration. They synthesized some sixty-plus variations of such compounds to screen for structure-function relationships; this practice is a well-established pharmacological approach. They realized that a succinic acid derivative of the amino acid proline was a promising compound.
Their insight paid off with the eventual synthesis of Captopril. In addition, their efforts were rewarded by the Albert Lasker-DeBakey Clinical Medical Research Award (The Lasker awards, for short). These are a set of prestigious awards given since 1945. They are colloquially known as “America’s Nobels”, and, in more than one case, the awarding of a Lasker signals the eventual Nobel Prize award.
In the end, this discovery and development of Captopril paved the way for additional compounds based on the Captopril example that have proven useful as antihypertensive agents, all of it due to the keen observations and carefully designed experiments that integrated the specific expertise, ideas, and abilities of a wide variety of researchers.
This story is an example of biomedical science at its best.
PICTURE CREDIT: https://eol.org/pages/51899701
[1] Nowadays, other factors that are used to define the end of life include the absence of brain electrical activity among others.
[2] Which in humans, it beats between 2 and 4 billion times over a healthy lifespan.
[3] Please see Campdelacreu (2014), de Toledo et al. (2010), and Skoog I, Gustafson D (2006).
[4] For more information on the natural history of this species please see: http://animaldiversity.org/accounts/Bothrops_jararaca/
[5]Dr. Ferreira eventually went on to have a distinguished career himself, being involved in the development of captopril, participating in the elucidation of the mechanism of action of aspirin, and helping establish the quite unexpected role of morphine as a peripheral painkiller. Please see Downey (2008).
[6] Roughly one paper every fourteen months or so. Source: http://www.ncbi.nlm.nih.gov/pubmed.
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