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Role of Nitric Oxide and Cyclic GMP in Cell Signaling and Drug Development

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Role of Nitric Oxide and Cyclic GMP in Cell Signaling and Drug Development
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2017
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Nitric oxide and cyclic GMP are common and important messengers in cell regulation with more than 150,000 research publications in numerous areas of biology. They participate in blood flow, blood pressure, platelet aggregation and blood clotting, cardiac, gastrointestinal, renal, neuronal and immunologic functions, inflammation, atherosclerosis, wound healing, and stem cell and cancer proliferation to name a few of their effects. The pathways for their formation and biochemical/biological effects have permitted an understanding of the mechanism of action and/or discovery of many important drugs. Defining their biochemical pathways and biological effects have permitted drugs to be developed to treat hypertension, heart attacks, blood clotting, pulmonary hypertension, endothelial dysfunction, bacterial toxin diarrhea, erectile dysfunction, infections, wound healing and cancer. These possible therapeutic applications have further stimulated the research interests in cell signaling pathways. Murad F Nobel Lecture. Bioscience Reports 19, 133-154, 1999. Murad F Shattuck Lecture. New England Journal Med. 355, 2003-2011, 2006.
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a year. it's always a treat. to come to the lindau meetings to meet your nobel laureate colleagues and interact with a lot of talent you know people it's a lot of fun. i'm a clinical pharmacologist i'll tell you what that means surely. but i was planning to go to medical school.
and as i finish college they learned about a new program that was just beginning in cleveland.
the chairman of the pharmacology arbiter of sutherland. who is an indie decided.
that future scientist and professor should be trained both in medicine in basic science and he was instrumental in creating the indy ph d. program in cleveland the first one to happen in the united states and i decided to join the program.
so i traded medicine pharmacology biochemistry of my research has been an interpreter ology cardiology i've tried some training and physiology probably train to lot of my family got a little concerned about how many years i trade but by becoming a clinical pharmacologist it printed me.
to chase problems in the multiple tissues and organs and i enjoyed that because it was very rewarding to learn these new disciplines in languages to communicate both in medicine in basic science.
sutherland enrol in nineteen fifty seven. discovered the first interest sell you a second messenger cyclic and pig. they were interested in the mechanism of that an effort and glucagon it deuce glycogen degradation to produce school costs and i joined the nineteen fifty eight and and for the next twelve years as a student in a fellow.
worked with cycle game peter show that numerous hormones in many different issues but enhanced cycling and p. per production to produce a variety of physiological biological effects. in nineteen seventy one year old son got the nobel prize for that discovered a and i'll show you shortly there are other messengers in most of them have received a nobel prizes. but in the late nineteenth sixty's another cyclically tide was discovered by some chemists cyclic want to see lot of us it. and as a student one of my projects was the search for compounds that might inhibit cyclic a p production in activity and and one cyclic a g.p. came along i thought perhaps it would antagonize the effects of cyclic and peace so i switched my interest as i was finishing line fellowship of aging going to. the faculty at university of virginia more and more my interest turned to cycle of g.d.p. and that led a few years later to discover later dockside not review some of that history for you. i'm going to try to avoid a lot about chemistry. but i want to present the concepts of cell communication and how it often works. and how this information can lead to the discovery and development one of many drugs and if we look back. perhaps forty or fifty percent of all the pharmaceuticals on the market relate to sell singling messengers either mimicking the effects are blocking their effects on these by a chemical pathway it's so it's been a gold buying for doug drug development discovered a the first slide summer. this is this concept of cell communication. these are three populations of cells that will talk to each other.
with evolution we went from single cells to multicellular organisms and cells had to learn how to talk to each other to survive. in this cartoon there are three different populations of cells. i'm going to call cell type one and iran will sell it could be a central neuron in your brain or could be a peripheral neuron one years i'm going to call cell type to it into a single cell whining all of your blood vessels it's only one cell think it was thought to be unhurt. we know that's not true there are many interesting messagers the come from these into fuel cells that regularly smooth muscle regulate formed elements platelets white cells and center. and i'm going to call cell type three a smooth muscle cell in the wall of that blood vessel. they're all going to communicate. if cell type one isn't your own will sell the messenger that it produces releases we call a neurotransmitter. if it's intended can sell we call the messenger hormone. for some macrophages look for site we call them growth factor cytokines are key workers. and these messenger molecules that come out of the cells into the synaptic space or bloodstream. can be very simple amino acids taking the lead but the peptides they can you be big hormone such as the natural pounds from to tear. if they're small molecules they're usually bound to a carrier protect them from degradation pronger halfway. the small the larger molecules are usually protected quite well. and what they will do his home throughout the body two adjacent tissues are distance either way to go find their target and they identify their target by the presence of a protein often the central goal membrane protein embedded in that number. and these first messengers buy into this protein we call it receptor in the pine by a three dimensional confirmation of fit like a key to lock so only the right message or talks to the right receptor and only the right receptor identifies right messenger. now sometimes as the receptor inside of the cell as with thyroid hormone and some of the steroid but most often their the membrane. in this first messenger. doesn't necessarily have to go inside the cell to do its business by binding to its receptor it induces a biochemical cascade that often enhances the formation in accumulation of an interest cellular second messenger. the first such second message he was like looking at it. then came calcium thinking psychology and could then came the cost and size across to clients then came days the simplest row then came the trackside so they're now a half a dozen or so intercell your messengers in hundreds of these first messengers. and many of the messengers rock resulted in trucks that many of them resulted in nobel prizes. nitric oxide is a very unique messenger because it's a free radical with that i'm sure electronics outer shell so it's very reactive it's also a guess it's also look at cycle so i can go wherever it once you go into the mitochondria go to the nucleus become out of the self go back in. and it can come out and attached to another carrier go to a distance was also a hormone it's the first message your it functions as an interest cellular messenger and a lot of could a pair of conceptions local acting material as well as a hormone no other molecules can do that. when it comes closer perhaps across the clintons. these messages can function inside of the cell which they're made but with nitric oxide come out and regulate the bio chemistry biology the jason cells are distant cells and when it goes over to sell type three the smooth muscle cell in the wall of that blood vessel because it's relaxing. and by dilating the blood vessels a relaxing with smooth muscle you enhance blood flow oxygen delivery nutrient delivery lower blood pressure.
i think tank trick oxide is a very old molecule i think his life but she and the messengers had to be simple initially it could be complicated.
and the happened to occur in organisms that were probably in the deep trips to the ocean you're all of this magnitude that was very toxic with transition that all in all sorts of gases and to survive they had to detoxify these materials. and what you create the biochemistry to detoxify when i turn it around and start using it to send information to each other and i think that's how it happened i can't prove that but i think that's make sense to be biologically about chemicals. we know today the nitric oxide it is released into the atmosphere whenever you combust fossil fuels any substance with nitrogen one as bird makes a family of nitrogen oxides you know into ago no two in two or three its cetera were called. fox's you know x. apostrophe us they released into the atmosphere in his you heard from dr leanest discussion yesterday they can interact with the ozone layer which is the ultraviolet filter to prevent global warming so that the bleats goes on and participates in global warming that's one of the effects of fossil fuels. we also know this how some very important cardiovascular drugs work from our research in the seventy's nitroglycerin like your press said. our programs that are converted to electric got excited about it and i'll tell you more about that charlotte. and five. so nitric oxide was viewed as a toxic pollutants for many years. and when we came along and said it's also interesting messenger in the body does all these a marvelous things nobody believed this for ten or fifteen years it was very difficult to convince the world that this was going to be an important messenger took a while i was disappointing. but also prevented us to do all the important work during the period of time we did an awful lot of work because we were very excited about our findings. but if you heard i think yesterday for dr i read her show you've got to believe in your work and got to persist do something had been done before because that makes it more fun thing. on the right hand side of the slide are some of the biological effects of nitric oxide. it's by no means a complete list. we discovered the first biological effects in nineteen seventy six seventy seven today there are a hundred and fifty thousand research publications and the introduction of its become one of the most popular areas of medical research there about eighty or ninety thousand publications and cycle could be a. similar number with cycle of g.d.p. so in these areas have become quite popular in their popular because they have led to the discovery and development of a lot of important molecules for medical diseases. this is a partial list in that time doesn't permit me to go through everything it does not try to give you an overview of flavor in the next twenty or thirty minutes. this is how cyclic nucleotides are produced a as a student i worked a cyclic a.p. i found a lot of hormones regulating cycle k.p. production i switched to psychology it turns out the pathways are very very similar in fact the adelaide cyclists as the make cycle k.p.. in the quiet only speculation is that makes likely to be have a lot of homology they probably originated from the same and sister jean anything there probably was a gene protein a one point that made either cycle nucleotide if you can you take what amino acid in the catalytic obeyed the quiet cyclists can make cyclic a and had two and cyclists can make. psychology. and they convert nucleotides either a.t.p. the cyclic eight mpg or g.t. peed in psychology and the entire process the alpha phosphate cycle eyes and back in the two position of rivals and three position of rivals for the cycle nucleotide. it turns out that there are seven i so forms of what cyclists seven different gene product. the sol utilise a form as a header a timer with an alphabet a sudden it and it's the receptor for nitric oxide. nitric oxide bias to heene press that a group heene pops offer the pocket and activates the inside of several hundred fold to make psychology of the incredible amplification and that's often the way signaling pathways work a single molecule can affect and. designed with a high turnover number to make lots of other messengers so this application all the way along with step a kindness phosphorylation lots of proteins they regulate lots of other activities. there's also redundancy because all of these messengers talk to each other. that makes it a lot more complicated but also more interesting lot more fun. the cycle nucleotides are regulated their levels of tissues by the rate of formation as well as the rate of degradation. and their destroyed by a family the science called cycle nucleotide fast but i guess traces that cleaved the fos what i justified to make the corresponding my office it. there are eleven cyclically tide us what history says. some hydra life cycle k.p. so i realize cycle g.p. some had lived above. the one that's familiar to all of you in the audience is the type five fast that x. rays which selectively hide realize the cycle of g.d.p. and the reason you know about it is because that's the target for by anger that's how it works by aggregate him a set inside permitting the nerves in the blood vessels of the penis. it's really centric outside make more cyclists g.p. or visibility taishan should dial up the blood vessels they fill with blood that's the mechanism and physiology of the people actually. it also does the same thing another blood vessels.
and this area has become it interesting field for drug discovery obviously. now many of the messengers cyclic a.p. cycle g.d.p. kalsu bicyclists real active a protein kinase as there are several hundred protein kinase some of them transfer the game of phosphate eighty p. to syrian are three main residence of proteins.
some transfer the phosphate from a.t.p. to tie receiving residues they're called tire still kindness and the syrian three any kind he says are regulated by to sell your messengers cycle came p cycle g.d.p. calcium days with israel. the terrace the kind he says are regulated by growth factors inside a cat's they don't have messenger molecules. these kindnesses phosphor late a variety of proteins. the proteins can be insides think any structural proteins and when they're phosphor limit or d fuss for lead their activities can change they can be activated are inhabited and their structure and function can change as well so this basically is the mechanism for single transmission and and transaction. communication simple problem is you have all these ice of farms in a variety of compartments and you don't know who's talking to who sometimes that's what makes it difficult because what you like to have our drugs that are selective in specific the influence one pathway and one physiological effect. biological effect another to avoid side effect in taxes.
i'm going to skip to some but chemistry. ago. in the process of studying want only cyclists and discovery were soluble and particularly so for a wee accidentally found a side hydroxyl me and so the nitrate activating the site utilise a form of the answer.
big surprise. we often include a side of her buffers in the refrigerator cold room to inhibit bacterial growth that's how we stumbled onto it. my goal is to figure out how four months regulated psychology and the formation that when you know marginalize tissues the hormone effects are lost you and coupled the signalling pathways. but we had now three small molecules inside hydroxyl been sort of night try they would increase likely g.n.p. in tacticians in slices and cultures and activate the insein and sell three extract and i thought if we understood how they activated the enzyme perhaps we can reconstitute a hormone. which was my goal i wanted to fight hormone signaling pathways so i can interrupt or accelerate the pathway is possible therapeutic agents for other diseases as we did with cyclic aid. well it became quite a mystery story because we put these on all sorts of tissues and cells and one of the tissues i worked with that the time because i knew cycle k.p. relaxed smooth muscle i suspected the cycle. g m p would contract with us. and we develop the trickiest with muscle bottle and we put a side so the nitrate drexel mean and the smooth muscle preparations the increase likely g.o.p. is they did in other tissues. but the tissue didn't contract that relaxed big surprise again. i then remembered as a resident working in the intensive care units taking care of patients with heart attacks. and what we do with patients with heart attacks is give them visit islanders nitroglycerin matter press side to lower blood pressure so the partisan have to work so hard and can heal faster we call it decreased after a lot of work lot on the heart so we put i could listen like your press said. but on the smooth muscles take us relaxation is the were thought to and from work of others the big surprise once they to activity quite was likely to increase like what you pay so now we had a family of compounds that were vases violators smooth muscle access that all activated quite only side. plus the psyche of life of work. and we coined the term nature of the as a child actors and the question was there so different structure there has to be a comment intermediate what is it and it took us about three or four years to figure that out and it turned out to be detrick access and we first generated by trackside early december nineteenth seventy six and. if humid it activated all of our site look well as cyclists preparation so that was the eureka ball but we realised my goodness a free radical a gasket activated site had never been shown before and i thought what interesting chemistry this is going to be can be complicated because it's a free radical the gas but isn't it could be.
the difference and interesting so we chased it as i said for ten or fifteen years nobody would believe it. this is what i truly is a violent will do to a blood vessel if you preqin track the blood vessel new york and africa and then after the contract in a stable couple minutes later you had an eye to a visa dilated you increase likely g.d.p. and ten or twenty seconds.
this is followed by relaxation. and after a couple minutes cycle g.p. returns to bazell levels but relaxation pursue this is that mean psychology is not related to relaxation know that's not true. messengers hampshire half last cycle k p g m p nitric oxide don't last very long but activation of the protein kinase in the phosphorylation of proteins last much longer longer half lives and that's what's going on here. i so we realized. that we have figured out how the activities of violators word enough for that was what there was one of the reasons i was invited going to stop. in eighteen forty seven italian chemist made nitroglycerin what campus often do is pick their finger sticking the powdered tasted i'm not sure why they do that but they do they got faster headaches like by graham.
some of the physicians in europe realized nitroglycerin besides being an explosive was also a visa dialer and start using a friendship actors and it was huge for one hundred years to treat engine not knowing how it worked in banking seventy seven seventy eight we figured it was approached road to make my tracks.
after no male bell made his fortune. by making nitroglycerin making it less explosive by combining it with the dye to major centers to make dynamite and as you know the history and some of the young people should go to the internet look up the nobel foundation internet to the electronically seemed like an awful lot about all the laureates but also the history of alfred nobel. interesting story. now it's and pharmacologist. you also learn that when you have an exaggerated material it does something of interest. you should ask yourself is it mimicking in into action as pathway. and i propose a nineteen seventy eight. that hormones must be acting perhaps by converting some and dodging the substance into nitric oxide which also was a messenger that was heresy. i guess a free radicals a messenger your and your crazy. we couldn't prove it because the concentration required was fairly low and the technology did not permit us to ask anything to back satirists oxidation products at the animal or concentrations so i took a few years to develop some new technology to do that we succeeded and by the late eighty's people begin to leave us for. for because we had asked these not a measure it and because of another scientist robert forget. rather french got as a professor at suny downstate in brooklyn and astrobiologist to i happen to know who is interested in contraction in relaxation of blood vessels.
we knew that a number of drugs would cause relaxation of blood vessels and lower blood pressure and animals asked to call the brain to kind of number of others.
his to be but when you put them on this as an organ best the field because relaxation. and he discovered that reason for that was because you were destroying the integrity of the of the feeling. these india feel depended visa dollars require the integrity of the infield. and without the brazilian they did nothing and a contract instead of relax any percent of that work at a seminar in virginia while i was still there. in nineteen eighty. and it turned out that much of his story was similar to our story with my teammates dollars and after seven i took it off to my office i said bob there are three ways to relax with muscle you increase cyclic a.p. or cycle g.d.p. or decrease calcium and that's the way the substances working let's go. figure this out we agreed to collaborate he called the material he came out of into fuel cells e.d.f. and with little dr lexcen factor it had a short half life. turned out we did that collaborate we moved to stanford he went back to new york his wife develop breast cancer he was distracted for a while. e.d.f. is nitric oxide works of cyclists you pick. to make a long story short now this is a blood vessel cartoon show you how these pathways were. on the left is the industrial lining of all your blood vessels on the right is the smooth muscle in the wall of that blood vessel and in red or three categories of compounds that work by increasing cycle g.p. production. in the top biddle the night to have a style leaders are production precursors to make the trip back said some require in science to do it some do it spontaneously in the blood based on ph and oxygen tent. the interactive a societal ice a form of what it's like place to make cycle g.d.p. that activates the cycle g.p. dependent protein kinase we found that fast forward to riding present proteins and smooth muscle we did that with p thirty two incorporation by prelate pulling the eighty people you can display those proteins. and gels and then try to determine what they are with the antibodies are taking the spots and and sequencing one aspect it turns out that many proteins get fast forward some of them lose their phosphate and two proteins lost their phosphate and we identify them as massive like it. so what happens when you increase in no make psychology cycle g. lower side have sought calcium it influences fos when i was tied in the interest sell your calcium stores and their mobilization and the entry of calcium from the outside and mycenae lynching. kinases a calcium cabbage will depend on and side and lower cost of its activity drops in my stuff like chain loses its fossett these are acting filaments last film it's when miles and fast for later the slide together lachey of contraction you take up the phosphate the slight a party relaxation that's the mechanism.
the in the field dependent visit dialers only work if the in the field was intact.
because they have the receptor years when asked of calling just mean better credit center spoon muscle doesn't have receptions for the slogans. and when they interact with the into still sell calcium enters the cell activates an enzyme called nitric oxide synthesis the converse the amino acid a large mean that i forgot side which is easier if. that goes over to the smooth muscle compartment it works just like it might have is a child. that tells us when you have patients with heart attacks. you don't want to use in the field will depend of his of dollars to lower their blood pressure to decrease after work because they have in the field is function and i'll tell you more about that surely you want to use direct acting like to his dollars dollar blood pressure. and that's what i learned as a house offices so it all began to come together in your medical training search are often overlaps with the third category of his dilution the age of patents and. when kalani was studying his talent you the cell. he knows the cardiac teacher had great deals. and when you see granules in tissue which usually indicative of stored peptide hormones. the bold and canada isolated those great meal sequence the protein inspect i'd call that feature on a critic factor it turns out there are three peptides in that family can impede be a p.c.p. and we discovered they all work by activating the particularly i so forms of one hundred cyclists so they too were to say. like what you pay but through a different receptor. that gives us ways to elevate cycle of g.d.p. and cells by going through a sizeable pathway are but who the particularly as a pathway through the thoughts with estrogen is where the protein kinase as little surprise that where he will deliver.
and there are three i sue forms of nitric oxide something this.
three different gene products most issues have the sense i've taken her argentine like backside centrally the other about fifty or sixty percent from august's not just to is an interesting one because normally the transcript of protein is not present in tissues and. yes there's been an inflammatory and solved and pro inflammatory cytokines aisle one if you're on camera enough alpha activate enough capital be which regulates transcription of duster. so if you see the last two transcript your protein is indicative of an inflammatory can solve it becomes an inflammatory biomarker. the report. they all catalyze this reaction. they actually ice the quantity no nitrogen of argentine the hydroxyl argentine that's further oxidized the form city nitric oxide. the enzymes have a very complicated array of coal factors they are heene containing proteins they utilize calcium camacho one. they utilize if i'm in f a d. the substrates be taught besides a large enough to hide oxygen and in any b.p.h.. now in the utilize tetra hydride battery if you oxidized those co-factor which means i'm no longer makes nitric oxide that's very important instead it makes you peroxide and nine which will scavenge nitric oxide which is the wisest thing in the world you want to do because when you know. by its it was super oxide makes proxy nitrate which is incredibly toxic will save more about that show.
so have had little bit but almost a year. this is the pathway that we've worked out many tissues.
in by understanding the elements in components in the pathway the biological and chemical sequence each step become somewhat your target for you to go discovered chemicals as potential drug leads to develop products. the cap the pathways are always a little more complicated. patients with hypertension diabetes atherosclerosis tobacco use and perhaps obesity have blood vessels that do not make enough nitric oxide and they don't make it for several reasons and i won't go into the details but by knowing about chemistry and the problems in these patients we can figure out better ways to treat. if their diseases and we don't win nutritional supplements as well as drugs it's a very important disease that most of you in the audience have it when i was at stanford i was able to and work with corner is from young motorcycle students killed themselves on the motorcycle at seventeen eighteen nineteen their corners where happen.
the robot can they look normal this logically but they were making enough to trick us so it's there.
i. these are some of the diseases in opportunities in clinical medicine where this story can be utilized to start treating patients and it's a diverse list.
neuroscience. stem cells cancer gene transcription hormone production the list goes on and on that i'm that work with all these by worked with many of them it's been quite exciting this is why they're hundred fifty thousand papers out there and you can't keep up with the story morris too complicated thank you very much a.
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