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 **Type:** Conference presentation\\ **Type:** Conference presentation\\
 **Presenter:**  Trevor Marshall, PhD\\ **Presenter:**  Trevor Marshall, PhD\\
-**Conference:**  Visiting Professor Lecture Series, organized by the [[http://www.fda.gov/CDER/|Center for Drug Evaluation and Research]], a division of the FDA\\+**Conference:**  Visiting Professor Lecture Series, organized by the [[https://www.fda.gov/CDER/|Center for Drug Evaluation and Research]], a division of the FDA\\
 **Location:**   Bethesda, MD\\ **Location:**   Bethesda, MD\\
 **Date:**  March 7, 2006\\ **Date:**  March 7, 2006\\
-**Related content:** [[http://autoimmunityresearch.org/fda-visiting-professor-7mar06.ram|Video of lecture (.ram file)]]\\ +**Related content:** [[https://autoimmunityresearch.org/fda-visiting-professor-7mar06.ram|Video of lecture (.ram file)]]\\  
 +**See also:** [[https://autoimmunityresearch.org/transcripts/Transcript_2006_Marshall_FDA_Visiting_Prof.pdf|Transcript with slides]] \\
 **Notes:** full citation for this presentation((Marshall TG: Molecular genomics offers new insight into the exact mechanism of action of common drugs - ARBs, Statins, and Corticosteroids. FDA CDER Visiting Professor presentation, FDA Biosciences Library, Accession QH447.M27 2006)) **Notes:** full citation for this presentation((Marshall TG: Molecular genomics offers new insight into the exact mechanism of action of common drugs - ARBs, Statins, and Corticosteroids. FDA CDER Visiting Professor presentation, FDA Biosciences Library, Accession QH447.M27 2006))
  
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 ====Two dimensional representation==== ====Two dimensional representation====
  
-We can also produce two-dimensional molecular representations, which are extremely useful when we are trying to figure out whether we are looking at an agonist or an antagonist – whether the drug is acting to enhance the operation of the receptor, or to block the operation of the receptor. Here is the same ARB in the same CCR2b binding pocket, but now you’ve got the details of atomic interactions. You’ve got each of the residues, each of the amino acids in the receptor, and specific lines showing which of the ligands or which of the ARB atoms are within bonding distance – certainly a Van De Wiel system. And here is the hydrogen bond. The hydrogen bonds are very important because they tend to be quite a lot stronger and they orient the molecules in the receptor. +We can also produce two-dimensional molecular representations, which are extremely useful when we are trying to figure out whether we are looking at an agonist or an antagonist – whether the drug is acting to enhance the operation of the receptor, or to block the operation of the receptor. Here is the same ARB in the same CCR2b binding pocket, but now you’ve got the details of atomic interactions. You’ve got each of the residues, each of the amino acids in the receptor, and specific lines showing which of the ligands or which of the ARB atoms are within bonding distance – certainly a Van Der Waal's distance. And here is the hydrogen bond. The hydrogen bonds are very important because they tend to be quite a lot stronger and they orient the molecules in the receptor. 
  
-But in general, you only go to these representations when you’re going for extreme detail. It’s far too complex otherwise. +But in general, you only go to the 2D-representation when you’re going for extreme detail. It’s far too complex otherwise. 
  
 ====Models of pathogenic genomes==== ====Models of pathogenic genomes====
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 We can also model pathogenic genomes. Here is a protein, protein SAR0276, which is a putative membrane protein within the genome of the methicillin-resistant-Staphylococcus-aureus species MRSA252 – Staphylococcus protein. “Docked” into it (or mated with it or bound to it) is a molecule of the ARB “Olmesartan”. In an upcoming paper, “Molecular Genomics Identify ARBs as a new class of Antibacterial – it’s all a matter of dose,” we show how this ARB can be expected to inhibit the actions of the protein, and thereby disrupt the function of the MRSA252 organism.  We can also model pathogenic genomes. Here is a protein, protein SAR0276, which is a putative membrane protein within the genome of the methicillin-resistant-Staphylococcus-aureus species MRSA252 – Staphylococcus protein. “Docked” into it (or mated with it or bound to it) is a molecule of the ARB “Olmesartan”. In an upcoming paper, “Molecular Genomics Identify ARBs as a new class of Antibacterial – it’s all a matter of dose,” we show how this ARB can be expected to inhibit the actions of the protein, and thereby disrupt the function of the MRSA252 organism. 
  
-In this particular case, the way we came across the protein and realized that it was actually a GPCR family was by using standard genomic search techniques. Beside the molecule was CCR2b, which is the molecule I showed you a little while ago, the one that’s prevalent on myocytes and forces the myocytes to migrate to areas of trauma. This particular protein is in the MRSA genome, and its function? We don’t know. But if it’s ever expressed by the organism, and if the ARB is present in the blood stream of the individual, the ARB will go after that particular protein and bind into it quite firmly.+In this particular case, the way we came across the protein and realized that it was actually a GPCR family was by using standard genomic search techniques, by homology with CCR2b, which is the molecule I showed you a little while ago, the one that’s prevalent on monocytes and causes the monocytes to migrate to areas of trauma. This particular protein is in the MRSA genome, and its function? We don’t know. But if it’s ever expressed by the organism, and if the ARB is present in the blood stream of the individual, the ARB will go after that particular protein and bind into it quite firmly.
  
 Why do I have this here? Well, it turns out this whole search that led to the presentation here today started off with some papers back in the early nineties, where biochemists that were working on the development of ARBs found that unless they applied a bactericide to kill any bacteria in the tissue samples, the radiolabelled ARB was taken up by the bacterial organism and not by the tissue that was under test. At the time I had no idea what was going on or why this would be the case, and the biochemists didn’t care about it. They just made sure they killed the bacteria in any tissue samples before they did their testing. Why do I have this here? Well, it turns out this whole search that led to the presentation here today started off with some papers back in the early nineties, where biochemists that were working on the development of ARBs found that unless they applied a bactericide to kill any bacteria in the tissue samples, the radiolabelled ARB was taken up by the bacterial organism and not by the tissue that was under test. At the time I had no idea what was going on or why this would be the case, and the biochemists didn’t care about it. They just made sure they killed the bacteria in any tissue samples before they did their testing.
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 ====Do ARBs only affect AG2R1?==== ====Do ARBs only affect AG2R1?====
  
-Now, what we’ve done is a very large computer search, using computer service, running Linux and some software which automatically docks ligands or drugs into proteins. We’ve taken some hypothetical proteins where we don’t have an x-rayed structure, so we can’t be precisely sure that these are the correct shape (but we think they are), and then we’ve taken known proteins VDR and PPARg which have actually been photographed with x-ray technique – in order to find out exactly where the atoms are. And we’ve matched up some drugs with the receptors. As you can see from this table, most of the ARBs and statins have some affinity for angiotensin2 receptor which is a GPCR (that’s a receptor like we were looking at earlier, a membrane receptor), CCR2b (which is a putative model again), and VDR and PPARg (which are both nuclear receptors).+Now, what we’ve done is a very large computer search, using computer servers, running Linux and some software which automatically docks ligands or drugs into proteins. We’ve taken known proteins and some hypothetical proteins as well where we don’t have an x-rayed structure, so we can’t be precisely sure that these are the correct shape (but we think they are), and then we’ve taken known proteins VDR and PPARg which have actually been photographed with x-ray technique – in order to find out exactly where the atoms are. And we’ve matched up some drugs with the receptors. As you can see from this table, most of the ARBs and statins have some affinity for angiotensin2 receptor which is a GPCR (that’s a receptor like we were looking at earlier, a membrane receptor), CCR2b (which is a putative model again), and VDR and PPARg (which are both nuclear receptors).
  
 There are some that don’t dock, and there are some that dock with quite high affinity. Losartan, for example, would not normally inhibit the function of the VDR at the concentrations that the drug is normally administered. But other drugs such as Telmisartan clearly significantly affect the operation of both VDR and PPARg at normal concentrations. This graph is in nanomoles. So with a 25 milligram daily dose of Olmesartin, for example, it will create a bloodstream concentration that will affect up to around 10 nanomolar affinity; and certainly at the 0.04 and 0.3 nanomolar, there will be very great interaction.  There are some that don’t dock, and there are some that dock with quite high affinity. Losartan, for example, would not normally inhibit the function of the VDR at the concentrations that the drug is normally administered. But other drugs such as Telmisartan clearly significantly affect the operation of both VDR and PPARg at normal concentrations. This graph is in nanomoles. So with a 25 milligram daily dose of Olmesartin, for example, it will create a bloodstream concentration that will affect up to around 10 nanomolar affinity; and certainly at the 0.04 and 0.3 nanomolar, there will be very great interaction. 
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 I’ve highlighted the two residues that are mutated, here. This residue is mutated in Bos Taurus, and this residue is mutated in Cavia porcellus. You can see I’ve got a Candesartan, an ARB, bound into the binding pocket, and there’s actually an oxygen on the Candesartan that is very tightly bound to the Isoleucine 193 of Bos Taurus. I’ve highlighted the two residues that are mutated, here. This residue is mutated in Bos Taurus, and this residue is mutated in Cavia porcellus. You can see I’ve got a Candesartan, an ARB, bound into the binding pocket, and there’s actually an oxygen on the Candesartan that is very tightly bound to the Isoleucine 193 of Bos Taurus.
  
-====Bos taurus & Cavia porcellus====+====AT2R1 mutation - Bos taurus & Cavia porcellus====
  
 One of the things that we found in our study was that we couldn’t (as accurately as we expected) match up the expectations for the binding affinity of Candesartan that were listed in the NDA with the binding affinity that we were simulating in our receptor. Then when we had a look and realized that the NDA had been done with Bos taurus, a protein from the animal genome, we suddenly realized that there is a huge difference in affinity at this point; and that tended to make us feel a little bit more comfortable with our model of the angiotensin receptor. One of the things that we found in our study was that we couldn’t (as accurately as we expected) match up the expectations for the binding affinity of Candesartan that were listed in the NDA with the binding affinity that we were simulating in our receptor. Then when we had a look and realized that the NDA had been done with Bos taurus, a protein from the animal genome, we suddenly realized that there is a huge difference in affinity at this point; and that tended to make us feel a little bit more comfortable with our model of the angiotensin receptor.
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 But the biggest surprise we had was the extremely high affinity which the ARBs had for VDR and PPAR-gamma, which are nuclear receptors. These are not on the membrane of the phagocyte, but in the nucleus of the phagocyte. And they are key to the operation of the immune system. So while it was reasonable that these highly flexible, polar ligands (the ARBs and statins) might well have a good affinity for other GPCRs (membrane proteins) other than angiotensin2 type 1 receptor, we never expected them to have such a high affinity for the nuclear receptors. But the biggest surprise we had was the extremely high affinity which the ARBs had for VDR and PPAR-gamma, which are nuclear receptors. These are not on the membrane of the phagocyte, but in the nucleus of the phagocyte. And they are key to the operation of the immune system. So while it was reasonable that these highly flexible, polar ligands (the ARBs and statins) might well have a good affinity for other GPCRs (membrane proteins) other than angiotensin2 type 1 receptor, we never expected them to have such a high affinity for the nuclear receptors.
  
-VDR and PPAR-gamma are located in the nucleus of cells, and are some of the molecules which cooperate, using a complex interplay of dimerization (dimerization is where proteins bind to each other to form multiple complexes called dimers) with activation by a variety of ligands which transcribe genes from the host DNA into messenger-RNA. In turn this RNA will be translated by the ribosomes into long protein strands, and they are then folded into the final shape - for example SPCA, SPCR shape. There are lots of enzymes, lots of folds. They’re folded by, obviously electrostatic forces, but also by enzymes, and there is some feeling that other nuclear receptors are involved in some protein folding as well.+VDR and PPAR-gamma are located in the nucleus of cells, and are some of the molecules which cooperate, using a complex interplay of dimerization (dimerization is where proteins bind to each other to form multiple complexes called dimers) with activation by a variety of ligands which transcribe genes from the host DNA into messenger-RNA. In turn this RNA will be translated by the ribosomes into long protein strands, and they are then folded into the final shape - for example that GPCR shape - with lots of folds. They’re folded by, obviouslyelectrostatic forces, but also by enzymes, and there is some feeling that other nuclear receptors are involved in some protein folding as well.
  
 But these are at the very heart of the genome. All of the proteins produced by the cell come from this DNA transcription process. But these are at the very heart of the genome. All of the proteins produced by the cell come from this DNA transcription process.
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 VDR is the first one we’ll look at. The correct operation of the VDR is key to both the endocrine and the immune system. Some functions of the VDR include: It decreases parathyroid hormone transcription. High levels of VDR generally correlate with low levels of PTH; it decreases the transcription of PTH. VDR is the first one we’ll look at. The correct operation of the VDR is key to both the endocrine and the immune system. Some functions of the VDR include: It decreases parathyroid hormone transcription. High levels of VDR generally correlate with low levels of PTH; it decreases the transcription of PTH.
  
-It regulates the Toll-like receptor 2 and Toll-like receptor 4 expression; and these are receptors which are on phagocytes, and they are part of the innate immune response. In fact, they are key to the innate immune response. VDR regulates them and consequently regulates the  response of the body to bacteria.+It regulates the Toll-like receptor 2 and Toll-like receptor 4 expression; and these are receptors which are on phagocytes, and they are part of the innate immune response. In fact, they are key to the innate immune response. VDR regulates them and consequently regulates the response of the body to bacteria.
  
 It transcribes CAMP (cathelicidin antimicrobial peptide) and that is an endogenous antibiotic that the body makes, which attacks lipopolysaccharides on Gram-negative bacteria. There are a number of endogenous antibiotics, that’s one of them. We know for certain that that is transcribed by the VDR. It transcribes CAMP (cathelicidin antimicrobial peptide) and that is an endogenous antibiotic that the body makes, which attacks lipopolysaccharides on Gram-negative bacteria. There are a number of endogenous antibiotics, that’s one of them. We know for certain that that is transcribed by the VDR.
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 It interacts with cofactors SRC1 and SRC3, SRC steroid receptor cofactors, which are inhibited by P65, which is half of the nuclear factor kappaB. Again, immune system. And we know it’s associated also with the granulocyte-macrophage stimulating factor, another key immune system function.  It interacts with cofactors SRC1 and SRC3, SRC steroid receptor cofactors, which are inhibited by P65, which is half of the nuclear factor kappaB. Again, immune system. And we know it’s associated also with the granulocyte-macrophage stimulating factor, another key immune system function. 
  
-It regulates TGF-beta signaling, and DRIP coactivators (DRIP is D-Receptor Interactive Proteins), all of which regulate cell differentiation and apostosis. There’s a url of a search engine which will specifically search for citations on the VDR, if any of you are interested in looking further. http://www.ihop-net.org.+It regulates TGF-beta signaling, and DRIP coactivators (DRIP is D-Receptor Interactive Proteins), all of which regulate cell differentiation and apostosis. There’s a url of a search engine which will specifically search for citations on the VDR, if any of you are interested in looking further. https://www.ihop-net.org.
  
 There is so much activity in molecular genomics at the moment, looking at the VDR. We published a paper not too long ago, and recently I wanted to look it up on PubMed. So – I’m lazy, and rather than type in the full name and the author, I just typed in the VDR characters and let the VDR carry through. I thought, well, it’ll be somewhere on the first page. No way! There have been forty papers published on VDR since ours in mid-January. That’s a rate of about one a day. And half of those are on the immune system, and the importance of this receptor to the immune system. There is so much activity in molecular genomics at the moment, looking at the VDR. We published a paper not too long ago, and recently I wanted to look it up on PubMed. So – I’m lazy, and rather than type in the full name and the author, I just typed in the VDR characters and let the VDR carry through. I thought, well, it’ll be somewhere on the first page. No way! There have been forty papers published on VDR since ours in mid-January. That’s a rate of about one a day. And half of those are on the immune system, and the importance of this receptor to the immune system.
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 ====PPAR-gamma==== ====PPAR-gamma====
  
-PPAR is another receptor. There are two forms (Peroxysome Proliferator Activation Receptor is the whole acronym) but PPAR affects the generation of lipids, and it also transcribes key immune system genes. And here’s a point which I thought was rather good from one of the papers that I reviewed. PPAR-gamma was “originally discovered as a pivotal regulator of adipocyte differentiation,” but it’s “intimately involved in the regulation of expression of a myriad of genes, that regulate energy metabolism, cell differentiation, apoptosis and inflammation.”+PPAR is another receptor. There are two forms (Peroxysome Proliferator Activation Receptor is the whole acronym) but PPAR affects the generation of lipids, and it also transcribes key immune system genes. And here’s a quote which I thought was rather good from one of the papers that I reviewed. PPAR-gamma was “originally discovered as a pivotal regulator of adipocyte differentiation,” but it’s “intimately involved in the regulation of expression of a myriad of genes, that regulate energy metabolism, cell differentiation, apoptosis and inflammation.”
  
-Due to the links with fat-cell development, insulin and glucose metabolism, drugs which affect PPAR-gamma are likely to profoundly modulate the lipid metabolism. PPAR-gamma also modulates the immune system, particularly vascular inflammation.+Due to the links with fat-cell development, insulin and glucose metabolism, drugs which affect PPAR-gamma are likely to profoundly modulate the lipid metabolism. PPAR-gamma also modulates the immune system, especially vascular inflammation.
    
 ====PPAR-alpha==== ====PPAR-alpha====
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 But what these nuclear receptors are responsible for is transcription of DNA genes to strands of mRNA, which are then translated (in the ribosomes) into proteins. But what these nuclear receptors are responsible for is transcription of DNA genes to strands of mRNA, which are then translated (in the ribosomes) into proteins.
  
-Now, if you want some basic genomics tutorials, there is a simplified set of flash animations at this particular url which I have found to be particularly simple to understand, and yet quite accurate. http://www.johnkyrk.com/ +Now, if you want some basic genomics tutorials, there is a simplified set of flash animations at this particular url which I have found to be particularly simple to understand, and yet quite accurate. https://www.johnkyrk.com/ 
  
 So now we will look at some simplified 3D animations of these transcription molecules, just enough to give an overview of what the nuclear receptors do, and how the Corticosteroids, ARBs and Statins affect gene transcription. Corticosteroids now, because as we went further and further into this study, we widened out the scope of interest as to what we were looking for.  So now we will look at some simplified 3D animations of these transcription molecules, just enough to give an overview of what the nuclear receptors do, and how the Corticosteroids, ARBs and Statins affect gene transcription. Corticosteroids now, because as we went further and further into this study, we widened out the scope of interest as to what we were looking for. 
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 ====GCR Homidimer “zinc fingers”==== ====GCR Homidimer “zinc fingers”====
  
-What I have there is a close-up of the skeleton of the GCR Homodimer. What that means is there are two GCR ‘zinc finger’ regions here, two proteins, and they are coupled together through this ‘zinc finger’ complex, and they’re sitting on top of DNA. You can see the double strand of DNA here, and these helices – well actually underneath the zinc fingers it’s hard to see; but that helix, and this helix, are responsible for causing the bonds in the centre of the DNA (which are all hydrogen bonds; there are not any molecules here in the centre of the DNA strand) to break apart, by forces from the molecules here, and that causes the particular gene to be transcribed. A very simplified explanation but it’ll do.+What I have there is a close-up of the skeleton of the GCR Homodimer. What that means is there are two GCR ‘zinc finger’ regions here, two proteins, and they are coupled together through this ‘zinc finger’ complex, and they’re sitting on top of DNA. You can see the double stranded DNA here, and these helices – well actually underneath the zinc fingers it’s hard to see; but that helix, and this helix, are responsible for causing the bonds in the centre of the DNA (which are all hydrogen bonds; there are not any molecules here in the centre of the DNA strand) to break apart, by forces from the molecules here, and that causes the particular gene to be transcribed. A very simplified explanation but it’ll do.
  
 Now here is that same complex, but with a different perspective. You can see there, the DNA at the bottom. In this case, every single atom in the complex has been labeled. We can select a different type of display here – we’ll just select the normal ribbon configuration. You can see the DNA strands, and here is the receptor we saw on the previous slide. I just wanted to show you the back side of the DNA. You can see particularly the gaps across the centre of the nucleic acids. Those are hydrogen bonds that fill those gaps. Those are broken by the nuclear receptors as they cause the gene transcription into RNA.  Now here is that same complex, but with a different perspective. You can see there, the DNA at the bottom. In this case, every single atom in the complex has been labeled. We can select a different type of display here – we’ll just select the normal ribbon configuration. You can see the DNA strands, and here is the receptor we saw on the previous slide. I just wanted to show you the back side of the DNA. You can see particularly the gaps across the centre of the nucleic acids. Those are hydrogen bonds that fill those gaps. Those are broken by the nuclear receptors as they cause the gene transcription into RNA. 
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 ====Drug displacement of activator from receptor==== ====Drug displacement of activator from receptor====
  
-What we have here is per cent saturation on the left hand axis, zero to 100 per cent, and drug concentration across the bottom. We have the normal curve which indicates the IC50, which is equal to the bound natural ligand + Kd (where Kd is the disassociation function, or -log10(Ki). Ki, that is, for what we had on the other slide. What I’m looking at is the homologous bindings of VDR. In other words, we’re not assuming any saturation of either the drug or the receptor. They’re just homologous bindings to the VDR, with Simvastatin versus 1,25-D (which is in orange), and Telmasartin versus 1,25-D (which is in yellow). And I put a note there that the Prednisolone Kd is very similar to Simvastatin and it will have a similar displacement of the active 1,25-D from the VDR.+What we have here is per cent saturation on the left hand axis, zero to 100 per cent, and drug concentration across the bottom. We have the normal curve which indicates the IC50, which is equal to the bound natural ligand + Kd (where Kd is the disassociation function, or -log10(Ki). Ki, that is, for what we had on the other slide. What I’m looking at is the homologous bindings of VDR. In other words, we’re not assuming any saturation of either the drug or the receptor. They’re just homologous bindings to the VDR, with Simvastatin versus 1,25-D (which is in orange), and Telmisartan versus 1,25-D (which is in yellow). And I put a note there that the Prednisolone Kd is very similar to Simvastatin and it will have a similar displacement of the active 1,25-D from the VDR.
  
 The reason the yellow band is so wide is because the concentration at the lower end of the yellow band is the concentration of 1,25-D in blood, and the concentration at the upper end is the predicted concentration inside the cell. It’s quite a bit higher inside the cells of course, somewhere in the region of one to two nanomolar. That’s been determined in vitro, approximately. The Kd value was derived from modeling. The reason the yellow band is so wide is because the concentration at the lower end of the yellow band is the concentration of 1,25-D in blood, and the concentration at the upper end is the predicted concentration inside the cell. It’s quite a bit higher inside the cells of course, somewhere in the region of one to two nanomolar. That’s been determined in vitro, approximately. The Kd value was derived from modeling.
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 The same with Atorvastatin. It has a strong affinity for PPAR-gamma and alpha, but not too much on the thyroids. The same with Atorvastatin. It has a strong affinity for PPAR-gamma and alpha, but not too much on the thyroids.
  
-The thing that’s really clear from this is that every ARB and every statin is a little bit different in its activity profile. So even though your clinical medicine looks at “a statin” as being “a statin,” and really doesn’t pay very much attention to whether it’s Primastatin or Atorvastatin, Lipitor or so forth, there actually is a huge difference in their profile in terms of what receptors they are affecting in the human body.+The thing that’s really clear from this is that every ARB and every statin is a little bit different in its activity profile. So whereas clinical medicine looks at “a statin” as being “a statin,” and really doesn’t pay very much attention to whether it’s Primastatin or Atorvastatin, Lipitor or so forth, there actually is a huge difference in their profile in terms of what receptors they are affecting in the human body.
  
 But one thing that’s common to them all is that all of the statins affect PPAR-alpha and PPAR-gamma. Some of the statins also affect VDR, notably Simvastatin, and Lovastatin marginally. But one thing that’s common to them all is that all of the statins affect PPAR-alpha and PPAR-gamma. Some of the statins also affect VDR, notably Simvastatin, and Lovastatin marginally.
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 That’s not unreasonable if you think about it. You’ve got a drug that’s targeted at lipids, and it goes after the PPAR receptors. That could be the primary mode of action - we don’t know. But certainly the job of an expert in genomics is to point it out, so that the in vitro work and the clinical work can go away and say, “Oh yes, that’s true, this is a major function of this class of drugs, and the major reason why they act the way they do.” That’s not unreasonable if you think about it. You’ve got a drug that’s targeted at lipids, and it goes after the PPAR receptors. That could be the primary mode of action - we don’t know. But certainly the job of an expert in genomics is to point it out, so that the in vitro work and the clinical work can go away and say, “Oh yes, that’s true, this is a major function of this class of drugs, and the major reason why they act the way they do.”
  
-The same with the ARBs. Some of them have a high affinity for the VDR and PPARs. Candesartin, you see, does not have high affinity for VDR and PPAR, but  have a look over here at the thyroid receptors that Candesartin has a very high affinity too.+The same with the ARBs. Some of them have a high affinity for the VDR and PPARs. Candesartin, you see, does not have high affinity for VDR and PPAR, but have a look over here at the thyroid receptors that Candesartin has a very high affinity to.
  
 **Audience Question:** To what extent do we know about thyroid function? I see the numbers are all over the place. How much is thyroid function affected when these numbers are low, as we know that there are replacements?  **Audience Question:** To what extent do we know about thyroid function? I see the numbers are all over the place. How much is thyroid function affected when these numbers are low, as we know that there are replacements? 
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 **Audience Comment:** Actually, a lot of drugs are. **Audience Comment:** Actually, a lot of drugs are.
  
-**Answer:** Well, yes, that is whole job of its own just to get the word out.+**Answer:** Well, yes, that is the whole job of the scientist just to get the word out.
  
 **Audience Comment:** I would encourage you to do that, because to me, that seems to be the next level of what needs to go on here. **Audience Comment:** I would encourage you to do that, because to me, that seems to be the next level of what needs to go on here.
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 **Audience Comment:** Understood.  **Audience Comment:** Understood. 
  
-**Answer:** There are some issues with the way the modeling is done. Typically, we work on the receptor as being a fixed receptor. But no protein is fixed. It varies, the position varies from front to side, not only Van der Wiel forces but also the hemodynamic forces and other things. When you are dealing with the larger proteins, the modeling of the rings and the deformation of the rings becomes very significant, as it does with the steroids. The steroids are devilishly difficult to deal with because you have got the four ring conformation that you have to deal with.+**Answer:** There are some issues with the way the modeling is done. Typically, we work on the receptor as being a fixed receptor. But no protein is fixed. It varies, the position varies from front to side, not only Van der Waal forces but also the hemodynamic forces and other things. When you are dealing with the larger proteins, the modeling of the rings and the deformation of the rings becomes very significant, as it does with the steroids. The steroids are devilishly difficult to deal with because you have got the four ring conformation that you have to deal with.
  
 **Audience Comment:** But then, looking at the next iteration, we would be looking the various genetic snips, whatever, of the various pathways in TNF metabolism and such. Because that is where we are trying to figure this out. I am a Rheumatolgist. We are trying to figure this out with TNF inhibitors—and all the biologics that have been released. We think they are going to do something and then we just can not seem to find it, for example. **Audience Comment:** But then, looking at the next iteration, we would be looking the various genetic snips, whatever, of the various pathways in TNF metabolism and such. Because that is where we are trying to figure this out. I am a Rheumatolgist. We are trying to figure this out with TNF inhibitors—and all the biologics that have been released. We think they are going to do something and then we just can not seem to find it, for example.
  
-**Answer:** I have got this feeling—it is not a hypothesis, just a feeling—but there is something somewhere that activates all these GCPRs in extreme infection. Everything goes wrong; the eyes, the everything. Do not know what it is. TNF-alpha is always a good place to start looking, also Interferon-gamma, some of those. And yes, we do have that on the books, as it were, but it is an extra level of complexity beyond where we are at now. We just mastered the steroids and we thought that was great. +**Answer:** I have got this feeling—it is not a hypothesis, just a feeling—but there is something somewhere that activates all these GCPRs in extreme infection. Everything goes wrong; the eyes, the everything. Do not know what it is. TNF-alpha is obviously a good place to start looking, also Interferon-gamma, some of those. And yes, we do have that on the books, as it were, but it is an extra level of complexity beyond where we are at now. We just mastered the steroids and we thought that was great. 
  
 **Audience Comment:** Great. Thank you very much.  **Audience Comment:** Great. Thank you very much. 
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