Category Archives: Chemistry (relatively pure)

Forgotten but not gone — take II

The RNA world from whence we sprang strikes again, this time giving us a glimpse into its own internal dynamic.  18 months ago I wrote the following post — which will give you the background to follow the latest (found at the end after the (***)

Life is said to have originated in the RNA world.  We all know about the big 3 important RNAs for the cell, mRNA, ribosomal RNA and transfer RNA.  But just like the water, sewer, power and subway systems under Manhattan, there is another world down there in the cell which doesn’t much get talked about.  These areRNAs, whose primary (and possibly only) function is to interact with other RNAs.

Start with microRNAs (of which we have at least 1,500 as of 12/12).  Their function is to bind to messenger RNA (mRNA) and inhibit translation of the mRNA into protein.  The effects aren’t huge, but they are a more subtle control of protein expression, than the degree of transcription of the gene.

Then there are ceRNAs (competitive endogenous RNAs) which have a large number of binding sites for microRNAs — humans have a variety of them all with horrible acronyms — HULC, PTCSC3 etc. etc. They act as sponges for microRNAs keeping them bound and quiet.

Then there are circular RNAs.  They’d been missed until recently, because typical RNA sequencing methods isolate only RNAs with characteristic tails, and a circular RNA doesn’t have any.  One such is called CiRS7/CDR1) which contain 70 binding sites for one particular microRNA (miR-7).  They are unlike to be trivial.  They are derived from 15% of actively transcribed genes.  They ‘can be’ 10 times as numerous as linear RNAs (like mRNA and everything else) — probably because they are hard to degrade < Science vol. 340 pp. 440 – 441 ’17 >. So some of them are certainly RNA sponges — but all of them?

The latest, and most interesting class are the nonCoding RNAs found in viruses. Some of them function to attack cellular microRNAs and help the virus survive. Herpesvirus saimiri a gamma-herpes virus establishes latency in the T lymphocytes of New World primates, by expressing 7 small nuclear uracil-rich nonCoding RNAs (called HSURs).  They associate with some microRNAs, and rather than blocking their function act as chaperones < Nature vol. 550 pp. 275 – 279 ’17 >.  They HSURs also bind to some mRNAs inhibiting their function — they do this by helping miR-16 bind to their targets — so they are chaperones.  So viral Sm-class RNAs may function as microRNA adaptors.

Do you think for one minute, that the cell isn’t doing something like this.

I have a tendency to think of RNAs as always binding to other RNAs by classic Watson Crick base pairing — this is wrong as a look at any transfer RNA structure will show. https://en.wikipedia.org/wiki/Transfer_RNA.  Far more complicated structures may be involved, but we’ve barely started to look.

Then there are the pseudogenes, which may also have a function, which is to be transcribed and sop up microRNAs and other things — I’ve already written about this — https://luysii.wordpress.com/2010/07/14/junk-dna-that-isnt-and-why-chemistry-isnt-enough/.  Breast cancer cells think one (PTEN1) is important enough to stop it from being transcribed, even though it can’t be translated into protein.

*****

[ Proc. Natl. Acad. Sci. vol. 116 pp. 7455 – 7464 ’19 ] The work reports a fascinating example of that early world in which the function of one denizen (a circular RNA called cPWWP2A) binds to another denizen of that world (microRNA 579 aka miR-579) acting as a sponge sopping up so it can’t bind to the mRNAs for angiopoetitin1, occludin and SIRT1.

So what you say?  Well it may lead to a way to treat diabetic retinopathy. How did they find cPWWP2A?  They used the Shanghai BIotechnology Company Mouse Circular RNA microArray which measures circular RNAs.  They found that 400 or so that were upregulated in diabetic retinopathy and another 400 or so that were downregulated.  cPWWP2A was on of the 3 top upregulated circular RNAs in diabetic retinopathy.  cPWWP2A comes from (what else?) PWWP2A, a gene coding for a protein which specifically binds the histone protein H2A.Z.

Overexpression of cPWW2PA or inhibition of miR-579 improves retinal vascular dysfunction in experimental diabetes.

So here is all this stuff going on way down there in the RNA world, first interacting with other players in this world and eventually reaching up to the level we thought we knew about and controlling gene expression.  It’s sort of like DOS (Disc Operating System) still being important in Windows.

How much more stuff like this is to be discovered controlling gene expression in us is anyone’s guess

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Apologies to Hamlet

Apologies to Shakespeare and Hamlet.  Serotonin does “more things in heaven and Earth, Horatio, than are dreamt of in your philosophy.”  How about chemically modifying histones?We all know about serotonin and depression (or at least we think we know).  Block serotonin reuptake by the releasing neuron and bingo you’ve  cured depression (sometimes).  Do not ask the lecturer which of the 15 known serotonin receptors in the brain the increased serotonin actually binds to and what effects the increased levels produce after binding (and which are important for the alleviation of depression).The two body organs producing the most serotonin are the brain and the gut.  Chemical modification of proteins by serotonin has been known for 10 years.  The enzyme responsible is transglutaminase2, it takes the NH2 group of serotonin and replaces the NH2 of glutamine with it — forming an isopeptide bond.

Interestingly, the serotonylation of histones is quite specific.  Only glutamine #5 on histone H3 is modified this way.  For the reaction to occur lysine #4 on histone H3 must be trimethylated (H3K4Me3) — now you can begin to see the combinatorial possibilities of the various histone modifications known.  Over 130 post-ranslational modifications of histones were known by 2013 [ Cell vol. 155 p. 42 ’13 ].

The H3K4Me3Q5Ser is enriched in euchromatin and correlates with permissive gene expression.  Changing glutamine #5 to something else so it can’t be serotonylated changes the transcription pattern, and deficits in cellular differentiation.  You can read more about it in Nature vol. 567 pp. 464 – 465, 535 – 539 ’19 ]

Another research idea yours for the taking

How many of our 20,000 or so protein coding genes are essential for human existence?  There is a way to find out with no human experimentation whatsoever.  Even better, probably all the data is out there.  Looking at it the right way, finding and collating it is where you come in.  Be warned, it would be a lot of work.

Previous work [ Science vol. 350 pp. 1028 – 1029, 1092 – 1096, 1096 – 1101 ’15 ] came up with the idea that only 2,000 or so of our protein coding genes were truly essential.  The authors cleverly looked at a ‘near haploid’ chronic myelogenous leukemia cell line (KBM7).  Then because only one copy of a gene was present, they systematically knocked out gene after gene using CRISPR and looked at viability.

Similar work in yeast stated that only 1,000 of its 6,000 protein coding genes were essential.

But this is single cell stuff.  What about living breathing people?

Where is this data?  How should it be interrogated?  See if you can figure it out before reading further.

Probably more has been done since Science vol. 337 pp. 64 – 69 ’12 sequenced just the portion of our genome coding for proteins (the exomes) in 1,351 Europeans and 1,088 Africans.  Each individual had 35 premature termination codons, meaning that the gene likely didn’t produce a functional protein.  The average person also had 13,595 single nucleotide polymorphisms (from the standard genome), and probably some of them a less than functional protein.

Do you see how you could use this sort of thing to find out which genes are essential to our existence?

People sequence exomes because it’s easy and because the exome accounts for only 2% of our genome.

My guess is that probably a million exomes have been sequenced thus far, if not more.

So all you have to do is look at all million exome sequences and all 20,000 protein coding genes, and see —

In one of the Sherlock Holmes stories the following dialog appears

Gregory (Scotland Yard): “Is there any other point to which you would wish to draw my attention?”
Holmes: “To the curious incident of the dog in the night-time.”
Gregory: “The dog did nothing in the night-time.”
Holmes: “That was the curious incident.”

The curious incident would be a gene which never (or rarely) had a premature termination codon in the 1,000,000 or so exomes.  That would imply that it was essential for the existence of a living breathing human being.

Cute !  Well I’m a retired neurologist with no academic affiliation — take the idea and run with it.

Addendum 31 Mar ’19 – I received the following comment from Bryan

You may be interested in reading this pre-print on the topic:
Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes https://www.biorxiv.org/content/10.1101/531210v2

To which I replied
    • Bryan– thanks for the link. It was a good enough idea that the people at the Broad Institute had thought of it and carried it out. As people in grad school used to say when they got scooped on a paper — at least we were thinking well.

      It was hard to tell from reading the preprint whether there were genes with no pLoF (predicted loss of function) proving them essential. They do say that the 678 genes essential for human cell viability (characterized by CRISPR screening were ‘depleted’ for pLoF.

 

Good to see Charlie’s still at it

Good to see Charlie Perrin is still pumping out papers, and interesting ones to boot.  I knew him in grad school.  He’s got to be over 80.

This one —J. Am. Chem. Soc. 141, 4103 (2019) –is about something that any undergraduate organic chemist can understand (if not the techniques he used) — keto/enol tautomerism, in which the hydrogen bounces between two oxygens, so that, given N molecules in solution, N/2  have the hydrogen bound to one oxygen and N/2 have it bound to the other.

No so in what Charlie found — a compound where the hydrogen is smack dab in the middle.  Some fancy NMR techniques were used to show this.

Hydrogen bonds are extremely subtle (which is why we don’t understand water as well as we might).  Due to the small mass of the proton it isn’t appropriate to treat the proton in hydrogen bonded systems as a classical particle.  When quantum mechanics enters, aspects such as zero point motion, quantum delocalization and tunneling come into play.  These are called quantum nuclear effects (aka Ubbelohde effects).

Why Organic Chemistry should always be taken (and passed) by pre-meds — take II

An old friend’s mother died of a ruptured intracranial aneurysm and he asked me what his risk was.  So I looked up my old notes on the medical literature that I took when I was in practice (copied below).  They show once again why someone who can’t pass organic chemistry doesn’t belong in medicine.  They are far too out of date to be of clinical use, and hopefully more work has been done since I retired in 2000.

But look at the notes.  All are in reputable journals and have been refereed.  But they conflict.  You have to evaluate this data to give decent advice, just as you have to weigh conflicting steric effects, electronegativity, bond strength, electrostatic effects in solving an organic chemistry problem.  Memorization of the various effects is necessary, but you have to keep them in your head and weigh them.   A perfect memory alone just won’t do.

Here are my notes, followed by the first post on this point (which was almost 10 years ago). You don’t have to go to medical school to see how conflicting they are.

      [ New England J. Med. vol. 341 pp. 1344 – 1350 ’99 ] 626 first degree relatives of 160 patients with subarachnoid hemorrhage were screened for aneurysm by MRI angiography.  Aneurysms were found in 25/626 (not much higher than the literature would imply in any of us — they use the figure of 2.3%) — total number of aneurysms were 33.  18/25 had surgery and 11/18 had a decrease in function (disabling in 1).    They estimate the increase in life expectancy due to the surgery at 2.5 years.   They don’t think the morbidity of surgery is worth it.  The study is from the Netherlands.
      These results can’t be extrapolated to cases were there is more than one member affected by aneurysm (they may have a higher yield of aneurysms, and the risk of rupture may be different).   The screening led to 5 angiographies in patients who didn’t turn out to have an aneurysm — thus exposing a normal person to risk.
      [ Brit. Med. J. vol. 320 pp. 141 – 145 ’00 ] A study of 6175 patients with aneurysmal subarachnoid hemorrhage and 14781 first degree relatives (of whom 11640 were children followed for 108933 patient years showed 19 subarachnoid hemorrhages during followup.   This is an increased risk 3 times that of the general population — however, this translates to an absolute risk of under 1/500 per year.
      [ J. Neurosurg. vol. 66 pp 522 – 528 ’87 ]  A review of the literature on familial aneurysms shows that familial aneurysms tend to rupture at a smaller size and when the patient is younger.   There is a similar incidence of multiple aneurysms and predominance of females over males with multiple aneurysms in the familial cases.  Anterior communicating artery aneurysms are slightly less frequent.  In sibling pairs, the aneurysms occur at the same or at mirror sites and rupture within the same decade twice as frequently as randomly selected nonfamilial aneurysm patient pairs.
      [ Stroke vol. 27 pp. 630 – 632 ’96 ] Familial subarachnoid hemorrhage is said to account for 6 – 9% of all such cases.  The outcome is said to be worse in familial than sporadic subarachnoid hemorrhage.
      [ Stroke vol. 25 pp. 2028 – 2037 ’94 ]  Since the initial report in ’54, there have been 238 families with 560 affected members reported in the literature through ’93. Only 3% of these families had 5 or more affected.   Siblings of an affected male proband are more likely to be affected than siblings of an affected female.  After review of 73 families, the authors conclude that no single pattern of inheritance can account for all families (unsurprise ! ).
        [ Neurosurg. vol. 12 pp. 214 – 216 ’83 ] A family with 4 members with intracranial aneurysms is reported.  Two of these were in an unusual location, the distal anterior cerebral artery.
        The 5th case report of identical twins with multiple aneurysms is given [ Acta. Neurochir. vol. 95 pp 121 – 125 ’88 ]
        [ Neurosurg. vol. 20 pp 226 – 239 ’87 ]  A prospective study of 579 consecutive patients with subarachnoid hemorrhage was done.  1/250 siblings had an aneurysm, but an aneurysm occurred in another family member in 1/14.
      [ Stroke vol. 22 pp. 1426 – 1430 ’91 ]  3 families (among 175 patients with spontaneous dissections of the cervical arteries seen at the Mayo Clinic between 1970 and 1989) were found with intracranial aneurysms.  No patient had both conditions.  Both Ehlers Danlos type IV (ED – IV ) and Marfan’s syndrome can have aneurysm and cervical artery dissection as components.
       [ Stroke vol. 25 pp. 2028 – 2037 ’94 ]  Intracranial aneurysms have been associated with the following hereditary disorders.  However, only polycystic kidney disease, Ehlers Danlos, Marfan’s neurofibromatosis and pseudoxanthoma elasticum are at increased risk of aneurysm.  The others may be fortuitous.  Among the others a alpha-glucosidase deficiency, alpha-antitrypsin deficiency, alkaptonuria, Fabry’s disease, hereditary hemorrhagic telangiectasia, Noonan’s syndrome, tuberous sclerosis, and multiple endocrine neoplasia type I syndrome.
      [ Brit. Med. J. vol. 311 pp. 288 – 289 ’95 ] A study of the first degree (1290) and second degree (3038) relatives of 163 patients with subarachnoid hemorrhage from the Netherlands showed that 10/1290 first degree and 4/3038 second degree relatives had had a subarachnoid hemorrhage.  This is a 6 fold higher risk for first degree relatives than the population at large (however, fewer than 1% of first degree relatives had had a subarachnoid hemorrhage).   3 other studies (which the authors criticize) hadn’t found this.  [ Stroke vol. 27 pp. 7 – 9 ’96 ] A further study of this group showed that hypertension was 2.3 times as common in first degree relatives, stroke was 1.8 times as common and coronary heart disease was 1.9 times as common in first degree relatives (as compared to second degree relatives).  Thus the increased risk of subarachnoid hemorrhage in first degree relatives may reflect an increase in known risk factors for subarachnoid hemorrhage rather than a ‘new’ defect in the arterial walls.
       [ Arch. Neurol. vol. 52 pp. 202 – 204 ’95 ] A much higher incidence of subarachnoid hemorrhage in first degree relatives of the 149 cases of subarachnoid hemorrhage in Seattle over 2 years is reported.  An astounding 11.4% of cases had a first degree relative with a history of subarachnoid hemorrhage (vs. 6.4% of controls through random digit dialing).    When I take family histories (which I do for every patient I see), I don’t get anything nearly this high (I think, but I’ll have to look).   Another study estimated that the percentage of first degree relatives should be 5.5% [ Stroke vol. 23 pp. 1024 – 1030 ’92 ].
     [ Neurol. vol. 53 pp. 982 – 988 ’99 ] Another study on aneurysm risk of first degree relatives of patients who suffered a subarachnoid hemorrhage from an intracranial aneurysm.  There were 193 index patients and 626 first degree relatives studied 78% of those eligible).    Aneurysms were found in 25/626 — a 4% incidence.  The group with aneurysm didn’t have a high number of atherosclerotic risk factors.    This only twice the 2.3% prevalence of unruptured aneurysms in the general population.    The rate of subarachnoid hemorrhage in first degree of aneurysmal bleeders is 3- 7 fold that of the general population.   Given the only twofold increased prevalence of aneurysm found in this study, this may mean that there may be two types of aneurysms which run in families — the bleeding kind and the nonbleeding kind.
     [ Stroke vol. 27 pp. 1050 – 1054 ’96 ] In a study of 30 patients with ruptured aneurysm from 14 families in which another member had an aneurysm 24/30 were women.
        [ Lancet vol. 349 pp. 380 – 384 ’97 ] A study from Finland screened first degree relatives over the age of 30 of index cases of subarachnoid hemorrhage with magnetic resonance angiography (MRA)  There were 698 available of whome 438 were screened with magnetic resonance angiography.  38/438 had aneurysms (families with polycystic kidney disease, Marfan’s, Ehlers Danlos IV were excluded).
        [ Can. J. Neurol. Sci. vol. 24 pp. 326 – 331 ’97 ] The Saguenay Lac Saint Jean area of Quebec contains  ~ 300,000 people (all inbred).  The incidence of familial aneurysm is very high (related to the total aneurysm burden) and 144/502 individuals with ruptured intracranial aneurysm had another affected family member (first to third degree relative).   However, they think this is due to accidental aggregation as the families are large (average number of siblings is 7 ! ).
        [ Neurol. vol. 51 pp. 1125 – 1130 ’98 ] A study of 125 relatives of patients in 23 families in which 2  more individuals had aneurysmal subarachnoid hemorrhage.  116 had no history of aneurysm themselves and 7/116 had an asymptomatic ruptured aneurysm.  9 had a history of aneurysm and 3/9 had new asymptomatic intracranial aneurysms.   MRA was used to study the 116 and CT angiography was used to study the 9.
Here is the first post on the subject, written almost 10 years ago

Why Organic Chemistry should always be taken (and passed) by pre-meds

Back when I was posting on “The Skeptical Chymist”, the editor (Stuart Cantrill), told me that noises were being made about dropping organic chemistry from the pre-med curriculum and asked me to comment. I didn’t because the idea seemed so ridiculous. There is no possibility of really understanding anything about cellular biology, drug action, molecular biology etc. etc. without a firm grounding in organic chemistry. You simply must have some idea what vitamins, proteins, DNA and RNA and the drugs you’ll be using look like and how they chemically interact — which is what organic chemistry gives you the background for. Not that you can stop there — but all medical schools teach biochemistry — which starts at organic chemistry and takes off from there. Organic certainly helped me follow molecular biology as it exploded starting in the 60s.

Cynics might say that docs don’t synthesize things or crystallize the drugs they use. Knowing what’s going on under the hood is just esthetic filigree. Just tell them what ‘best practice’ is, and let them follow it like robots. Who cares if they know the underlying science. People drive cars without really understanding what a carburator or a manifold does (myself included).

It wasn’t until I got about 400 pages into the magnificent textbook of Organic Chemistry by Clayden, Greeves, Warren and Wothers (only 1100 action packed pages to go !) that the real answer became apparent. The stuff is impossible to memorize. Only assimilating principles and applying them to novel situations will get you through — exactly like the practice of medicine.

Let us suppose you have an eidetic memory, and know the best treatment for every condition. You wouldn’t have to know any science at all, would you?

What’s wrong with this picture? First of all, there isn’t a best treatment known for every condition. Second, every doc will see conditions and problems that simply aren’t in the books. When I first started out, I was amazed at how much of this there was. I asked an excellent internist who’d been in practice for 30 years if he’d seen it all. He thought he saw something completely new each week. Third, conditions occur in combinations, and many patients (and nearly all the elderly) have many more than one problem. The conditions and treatments interact in a highly nonlinear fashion. The treatment for one problem might make another much worse (see below).

Here is a concrete example using a familiar person (Sonia Sotomayor) and a disorder which should be known to all (the new Swine Flu which swept America and the world this spring). Let’s say that you’re that lucky soul with the perfect memory who knows all the best treatments (well those that exist anyway) and as such you’ve been given the responsibility of taking care of her.

It is public knowledge (e.g. Wikipedia) that Justice Sotomayor has had diabetes since age 8, requiring insulin since that time. Pictures show, that like many diabetics, she is overweight — depending on how tall she is I’d guess by 25 – 45 pounds. Influenza is usually a disease of the fall and winter, and the new Swine Flu is now down in South America, but it’s likely to sweep back up here this fall. We know it’s extremely infectious, but so far fortunately rather benign. There is no guarantee that it will stay that way. Suppose that while down in S. A. it mutated and has become more virulent (a possibility that the CDC takes extremely seriously).

What if she gets the new Swine flu next month? At this point there is no ‘best treatment’ known. Diabetics don’t do well with infections — they get more of them, and have more complications when they do. Her diabetes is certainly going to get worse. What if some think the ‘best treatment’ is corticosteroids (which is often used for severe lung infections) — which will really raise hell with her diabetes? Should you give it? Recall that corticosteroid use during the Asian SARS epidemic (another serious lung infection) seemed to hurt rather than help (Journal of Infection, Volume 51, Issue 2, Pages 98-102). There is no data to help you here and you and your patient don’t have the luxury of waiting for it. Don’t forget that her father died at 42 of heart disease. That could be relevant to what you do. Suppose, like many overweight diabetics she has high blood pressure and elevated lipids as well. How will that affect her management?

Your perfect eidetic memory of medicine will not be enough to help you with her management — you are going to have to think, and think hard and apply every principle of medicine you know to a new and unfamiliar situation with very little data to help you.

Sounds like Organic Chemistry doesn’t it? Anyone without the particular type of mind that is able absorb and apply multiple and (often) conflicting principles doesn’t belong in medicine. A hardnosed mathematician I audited a course from a few years ago, said that people would come up to him saying that if they couldn’t pass Calculus, they wouldn’t get into medical school. He felt that if they couldn’t, he didn’t want them in medical school (I’m not sure he told them this — probably he did). The same thing holds in spades for Organic Chemistry.

Stock tip — update

The FDA approved esketamine (Spravato) last week (see copy of original post at the end).  I had recommended buying Johnson and Johnson if the FDA approved it.  I think it’s a good long term buy, but there is no rush for the following reason — Esketamine is not a drug you can get a prescription for and take on you own. Because of the psychiatric side effects it must be administered in a SPRAVATO REMS.

Risk Evaluation and Mitigation Strategy (REMS): SPRAVATO™ is available only through a restricted program called the SPRAVATO™ REMS because of the risks of serious adverse outcomes from sedation, dissociation, and abuse and misuse.

Important requirements of the SPRAVATO™ REMS include the following:

  • Healthcare settings must be certified in the program and ensure that SPRAVATO™ is:
    • Only dispensed in healthcare settings and administered to patients who are enrolled in the program.
    • Administered by patients under the direct observation of a healthcare provider and that patients are monitored by a healthcare provider for at least 2 hours after administration of SPRAVATO™.
  • Pharmacies must be certified in the REMS and must only dispense SPRAVATO™ to healthcare settings that are certified in the program.

So you can’t go to some shady practitioner who’ll say you have treatment resistant depression and get some (e.g. the pill pushers for opiates, ‘medical’ marihuana  etc. etc.)

So there aren’t going to be hordes of users right away, although the stuff I’ve read implies that there will be eventually.

If you have a subscription to Cell have a look at vol. 101 pp. 774 – 778 ’19 by the guys at Yale who did some of the original work.  If not content yourself with this.

They are refreshingly honest.

Was the Discovery of Ketamine’s Antidepressant Serendipitous?Of course. However, its discovery emerged from the testing of a novel mechanistic hypothesis related to the pathophysiology of depression.”

Basically the authors rejected the regnant theory of depression, namely that the cause was to be found in monoamine neurotransmission (e.g. by dopamine, norepinephrine, serotonin).  There was some evidence that the cerebral cortex was involved in depression (not just the monamine nuclei of the brainstem), so they looked at the two major neurotransmitters in brain (glutamic acid, and GABA), and chose to see what would happen if they blocked one of the many receptors for glutamic acid, the NMDA receptor.  They chose ketamine to do this.
Here’s what they found,  A single dose of ketamine produced antidepressant effects that began within hours peaked in 24 – 72 hours and dissipated within 2 weeks (if ketamine wasn’t repeated).  This was in 50 – 75% people with treatment resistant depression.  Remarkable 1/3 of treated patients went into remission.    There simply has never been anything like this, which is why I thought the drug would be a blockbuster.
There is a lot of speculation about just which effect of esketamine is crucial (increase in glutamic acid release with AMPAR stimulation, brain derived neurotrophic factor (BDNF) release, TrkB receptor stimulation, mTORC1 activation, local protein synthesis, restoration of functional connectivity in functional MRI.   In animals one sees a rapid proliferation of dendritic spines.
As promised – here’s a copy of the first post

Stock tip

The past performance of stock recommendations is no guarantee that it will continue — which is fortunate as my first tip (ONTX) was a disaster.  I knew it was a 10 to one shot but with a 100 to 1 payoff.  People play the lottery with worse odds.  Anyway ONTX had a rationale — for the gory details see — https://luysii.wordpress.com/2016/06/01/in-a-gambling-mood/

For those brave souls who followed this recommendation (including yours truly) here’s another.

On 4 March 2019 if the FDA approves esketamine for depression, buy Johnson and Johnson.  Why?  Some people think that no drug for depression works that well, as big Pharma in the past only was reporting positive studies.  The following is from Nature 21 February 2019.

Depression drug A form of the hallucinogenic party drug ketamine has cleared one of the final hurdles towards clinical use as an antidepressant. During a 12 February meeting at the US Food and Drug Administration (FDA) in Silver Spring, Maryland,an independent advisory panel voted 14 to 2 in favour of recommending a compound known as esketamine for use in treating depression.

What’s so hot about esketamine?  First its mechanism of action is completely different than the SSRIs, Monoamine oxidase inhibitors, or tricyclic antidepressants.

As you likely know, antidepressants usually take a few weeks to work at least in endogenous depression.  My clinical experience as a neurologist is slightly different, as I only used it for patients with disease I couldn’t help (end stage MS etc. etc.) where the only normal response to the situation was depression.  They often helped patients within a week.

I was staggered when I read the following paper back in the day.  But there was no followup essentially.

archives of general psychiatry volume 63 pp. 856 – 864 2006
The paper is not from St. Fraudulosa Hospital in Plok Tic, but from the Mood Disorders Research Unit at the National Institute of Mental Health.
Here are the basics from the paper

Patients  Eighteen subjects with DSM-IV major depression (treatment resistant).

Interventions  After a 2-week drug-free period, subjects were given an intravenous infusion of either ketamine hydrochloride (0.5 mg/kg) or placebo on 2 test days, a week apart. Subjects were rated at baseline and at 40, 80, 110, and 230 minutes and 1, 2, 3, and 7 days postinfusion.

Main Outcome Measure  Changes in scores on the primary efficacy measure, the 21-item Hamilton Depression Rating Scale.

Results  Subjects receiving ketamine showed significant improvement in depression compared with subjects receiving placebo within 110 minutes after injection, which remained significant throughout the following week. The effect size for the drug difference was very large (d = 1.46 [95% confidence interval, 0.91-2.01]) after 24 hours and moderate to large (d = 0.68 [95% confidence interval, 0.13-1.23]) after 1 week. Of the 17 subjects treated with ketamine, 71% met response and 29% met remission criteria the day following ketamine infusion. Thirty-five percent of subjects maintained response for at least 1 week.

Read this again: showed significant improvement in depression compared with subjects receiving placebo within 110 minutes after injection, which remained significant throughout the following week.

This is absolutely unheard of.  Yet the paper essentially disappeared.

What is esketamine?  It’s related to ketamine (a veterinary anesthetic and drug of abuse) in exactly the same way that a glove for your left hand is related to a right handed glove.  The two drugs are optical isomers of each other.

What’s so important about the mirror image?  It means that esketamine may well act rather differently than ketamine (the fact that ketamine worked is against this).  The classic example is thalidomide, one optical isomer of which causes horrible malformations (phocomelia) while the other is a sedative used in the treatment of multiple myeloma and leprosy.

If toxic side effects can be avoided, the market is enormous.  It is estimated that 25% of women and 10% of men will have a major depression at some point in their lives.

Initially, Esketamine ( SPRAVATOTM)  will likely be limited to treatment resistant depression.  But depressed people will find a way to get it and  their docs will find a way to give it.  Who wants to wait three weeks.  Just think of the extremely sketchy ‘medical indications’ for marihuana.

Does gamma-secretase have sex with its substrates?

This is a family blog (for the most part), so discretion is advised in reading further.   Billions have been spent trying to inhibit gamma-secretase.  Over 150 different mutations have been associated with familial Alzheimer’s disease.  The more we know about the way it works, the better.

A recent very impressive paper from China did just that [ Science vol. 363 pp. 690- 691, 701 eaaw0930 pp. 1 –> 8 ’19 ].

Gamma secretase is actually a combination of 4 proteins (presenilin1, nicastrin, APH1 (anterior pharynx defect) and PEN-2 (presenilin enhancer 2). It is embedded in membranes and has at least 19 transmembrane segments.  It cleaves a variety of proteins spanning membranes (e.g it hydrolyzes a peptide bond — which is just an amide).  The big deal is that cleavage occurs in the hydrophobic interior of the membrane rather than in the cytoplasm where there is plenty of water around.

Gamma secretase cleaves at least 20 different proteins this way, not just the amyloid precursor protein, one of whose cleavage products is the Abeta peptide making up a large component of the senile plaque of Alzheimer’s disease.

To get near gamma secretase, another enzyme must first cleave APP in another place so one extramembrane fragment is short.  Why so the rest of the protein can fit under a loop between two transmembrane helices of nicastrin.  This is elegance itself, so the gamma secretase doesn’t go around chopping up the myriad of extracellular proteins we have.

The 19 or so transmembrane helices of the 4 gamma secretase proteins form a horseshoe, into which migrates the transmembrane segment of the protein to be cleaved (once it can fit under the nicastrin loop).

So why is discretion advised before reading further?  Because the actual mechanism of cleavage involves intimate coupling of the proteins.    One of the transmembrane helices of presenilin1 unfolds to form two beta strands, and the transmembrane helix of the target protein unfolds to form one beta strand, the two strands pair up forming a beta sheet, and then the aspartic acid at the active site of gamma secretase cleaves the target (deflowers it if you will).  Is this sexual or what?

All in all another tribute to ingenuity (and possibly the prurience) of the blind watchmaker. What an elegant mechanism.

Have a look at the pictures in the Science article, but I think it is under a paywall.

Stock tip

The past performance of stock recommendations is no guarantee that it will continue — which is fortunate as my first tip (ONTX) was a disaster.  I knew it was a 10 to one shot but with a 100 to 1 payoff.  People play the lottery with worse odds.  Anyway ONTX had a rationale — for the gory details see — https://luysii.wordpress.com/2016/06/01/in-a-gambling-mood/

For those brave souls who followed this recommendation (including yours truly) here’s another.

On 4 March 2019 if the FDA approves esketamine for depression, buy Johnson and Johnson.  Why?  Some people think that no drug for depression works that well, as big Pharma in the past only was reporting positive studies.  The following is from Nature 21 February 2019.

Depression drug A form of the hallucinogenic party drug ketamine has cleared one of the final hurdles towards clinical use as an antidepressant. During a 12 February meeting at the US Food and Drug Administration (FDA) in Silver Spring, Maryland,an independent advisory panel voted 14 to 2 in favour of recommending a compound known as esketamine for use in treating depression.

What’s so hot about esketamine?  First its mechanism of action is completely different than the SSRIs, Monoamine oxidase inhibitors, or tricyclic antidepressants.

As you likely know, antidepressants usually take a few weeks to work at least in endogenous depression.  My clinical experience as a neurologist is slightly different, as I only used it for patients with disease I couldn’t help (end stage MS etc. etc.) where the only normal response to the situation was depression.  They often helped patients within a week.

I was staggered when I read the following paper back in the day.  But there was no followup essentially.

archives of general psychiatry volume 63 pp. 856 – 864 2006
The paper is not from St. Fraudulosa Hospital in Plok Tic, but from the Mood Disorders Research Unit at the National Institute of Mental Health.
Here are the basics from the paper

Patients  Eighteen subjects with DSM-IV major depression (treatment resistant).

Interventions  After a 2-week drug-free period, subjects were given an intravenous infusion of either ketamine hydrochloride (0.5 mg/kg) or placebo on 2 test days, a week apart. Subjects were rated at baseline and at 40, 80, 110, and 230 minutes and 1, 2, 3, and 7 days postinfusion.

Main Outcome Measure  Changes in scores on the primary efficacy measure, the 21-item Hamilton Depression Rating Scale.

Results  Subjects receiving ketamine showed significant improvement in depression compared with subjects receiving placebo within 110 minutes after injection, which remained significant throughout the following week. The effect size for the drug difference was very large (d = 1.46 [95% confidence interval, 0.91-2.01]) after 24 hours and moderate to large (d = 0.68 [95% confidence interval, 0.13-1.23]) after 1 week. Of the 17 subjects treated with ketamine, 71% met response and 29% met remission criteria the day following ketamine infusion. Thirty-five percent of subjects maintained response for at least 1 week.

Read this again: showed significant improvement in depression compared with subjects receiving placebo within 110 minutes after injection, which remained significant throughout the following week.

This is absolutely unheard of.  Yet the paper essentially disappeared.

What is esketamine?  It’s related to ketamine (a veterinary anesthetic and drug of abuse) in exactly the same way that a glove for your left hand is related to a right handed glove.  The two drugs are optical isomers of each other.

What’s so important about the mirror image?  It means that esketamine may well act rather differently than ketamine (the fact that ketamine worked is against this).  The classic example is thalidomide, one optical isomer of which causes horrible malformations (phocomelia) while the other is a sedative used in the treatment of multiple myeloma and leprosy.

If toxic side effects can be avoided, the market is enormous.  It is estimated that 25% of women and 10% of men will have a major depression at some point in their lives.

Initially, Esketamine ( SPRAVATOTM)  will likely be limited to treatment resistant depression.  But depressed people will find a way to get it and  their docs will find a way to give it.  Who wants to wait three weeks.  Just think of the extremely sketchy ‘medical indications’ for marihuana.

 

Proline rides again !

Proline is a kinky amino acid.  Kinky in the sense that it is only one of the twenty with a fixed configuration of its alpha carbon because of the ring (which may be why there is more of it in organisms living at high temperature) and kinky in the sense that when present in alpha helices it produces a kink.  The previous post shows how it is used to schlep the body weight’s worth of ATP we make each day out of our mitochondria — https://luysii.wordpress.com/2019/01/30/3939/.

Well here it is in one of the marijuana receptors (CB1).  Binding of delta9 THC in the 7 transmembrane alpha helix bundles of the G Protein Coupled Receptor (GPCR) causes an alteration in the kink allowing transmembrane helix 6 (TM6) to move outward toward the cytoplasm, creating a cavity on the intracellular side, where the G protein trimer can bind.

You can read much more about this in an exquisite paper [ Cell vol. 176 pp. 448 – 458 `19 ] describing the CB1 receptor bound to a synthetic ligand 20 times more potent that delta-9 tetrahydrocannabinol (delta9 THC).  It is a cryoEM study which used 177,000 projections to come up with a 3 Angstrom resolution structure of CB1 bound to MBDB-FUBINACA in complex with its G protein trimer.  They had to use a single chain variable fragment (scFv6) along with a positive allosteric modulator (PAM) called ZCZ-011 to stabilize the complex.

MBDB-FUBINACA is a story in itself.  It is presently the fentanyl of synthetic cannabinoids, which “has been linked to thousands of hospitalizations and numerous fatalities”  [ New England Journal of Medicine vol. 376 pp. 235 – 242 ’17 ].  I’m surprised I’ve never heard of it — have you? But then I’ve been retired from clinical practice for some time. Perhaps the mainstream press, pushing marihuana legalization as it has been, kept it quiet, or more likely there have been no further episodes of mass intoxication from the AMB-FUBINACA (aka the zombie drug) since 2017.

I’ve never knowingly used marihuana.  Frankly it scares me — for why please see — https://luysii.wordpress.com/2014/05/13/why-marihuana-scares-me/.

There are 4 molecular switches buried in GPCRs [ Current Med. Chem. vol. 19 pp. 1090 – 1109 ’12 ]

1. The ionic lock switch between the D/E R Y sequence at the cytoplasmic end of TM3 and E286 at the cytoplasmic end of TM6 (single letter amino acid code used) –http://130.88.97.239/bioactivity/aacodefrm.html

2. TM3 – TM7 lock switch.  In rhodopsin it is between the protonated Schiff base of lysine and a glutamic acid and it broken on light activation,.=

3. Toggle switch linked with the n P x x Y motif in TM7 (x stands for any amino acid) — much more about this later in the post.

4. Transmission switch — produced by agonist binding, the outward movement of TM6 to to ligand binding creating a hole fo the G protein to bind to the receptor on the cytoplasmic side.

So why did I call the Cell paper exquisite?  Because of the molecular detail it provides about just how MDMB FUBINACA activates CB1.  Here’s the structure of AB-FUBINACA — https://en.wikipedia.org/wiki/AB-FUBINACA.   Both look like drugs designed by a committee.  They both have a para-iodophenyl group, an amide, and a fused indole ring with an extra nitrogen (imidazole ring — I never could keep heterocyclic nomenclature straight).    MDMB has a methyl ester (in place of the amide) and a tertiary butyl group (in place of the isoPropyl group).

I don’t have time to look up how Pfizer came up with it.  The FUBINACAs do not resemble delta9 THC at all — https://en.wikipedia.org/wiki/Tetrahydrocannabinol.

The pictures in the paper show how the hydrophobic aromatic side chains of FIVE phenylalanines and 2 tryptophans create a nice oily space for delta9 THC and MBDB-FUBINACA to bind.

F200 (phenylAlanine 200) and W356 are the toggle twin switch which stabilize the inactive conformation of CB1.  The rotation of F200 to interact with the imidazole of FUBINACA, allows W356 to rotate outward, changing the kink produced the the proline #358  in TM6 allowing the helix to straighten and rotate outward toward the cytoplasm, creating a cavity for the G protein to bind to.

Definitely a tour de force for the blind watchman.

Let’s hear it for the blind watchmaker

The blind watchmaker had a lot of foresight in choosing to use a rather  funky looking amino acid (proline) resembling none of the others.  A lot of kindness was also shown to structural molecular biologists by two of the watchmaker’s henchmen – Burkholderia gladioli and the common daisy.

All appear in a fascinating paper [ Cell vol. 176 pp. 435 – 447  ’19 ] in which the structure and better the mechanism of action of the mitochondrial ADP/ATP translocase, a molecule of some interest since our mitochondria make our body weight of ATP each day and need some way to get it out into the cytoplasm where it is used.

The molecule has quite a job to do, getting the rather large ATP molecule out to the intermembrane space (and thence out to the cytoplasm) without allowing protons to sneak out with it, since it is the proton gradient which is used to power ATP synthase the exquisite machine which makes ATP.   This is quite a trick as no chemical moiety is as small as a proton.

The translocase has two states — one in which it is open to the mitochondrial matrix (called the m-state) and another in which it is open (eventually) to the cytoplasm — called the c-state. In the m-state the cytoplasmic portion is shut, and in the c-state the membrane portion is shut.

The rather wierd looking molecule bongkrekic acid  made by Burkholderia gladioli  https://en.wikipedia.org/wiki/Bongkrek_acid binds to the translocase fixing it in the m-state.  Atractyloside, made by daisies binds to the molecule fixing it in the c-state.  They made life much easier for the structural biologist and cryoEMographers who wrote the paper.

Proline comes in because when placed in an alpha helix, proline’s 5 membered ring structure fixes the alpha carbon so that it is essentially inflexible, meaning that it can’t get into the conformation that the other 19 amino acids can get into when an alpha helix is formed.  Translation — proline is a helix breaker, forming a kink in the helix.

The translocase contains 3 modules of 100 amino acids each of which has 2 alpha helices, one of them containing a proline causing a kink in the helix.  The prolines are in the middle of the helix.  The ATP channel is formed by the 6 helices.

Essentially in the middle of the membrane, the kinked alpha helices form a pivot (fulcrum), so the helices rock back and forth, opening one side while simultaneously shutting the other, permitting ATP to bind near the fulcrum without letting anything else through, when the pivot shifts   — out goes the ATP (without letting protons sneak past).

There is far more beautiful protein chemistry on display.  There is a conserved signature motif Proline x Aspartic acid/Glutamic acid X X Lysine/Arginine at the carboxy terminal end of one of the helices of each other 3 modules — this forms a salt bridge shutting the channel on the matrix side.  Glycine and other small amino acids (alanine) allow close packing of the helices on the cytoplasmic side.

It is unfortunate that the most of humanity doesn’t have the background to appreciate the elegance and beauty of Nature’s solution to the problem.  I say Nature rather than God to be scientifically correct, but it’s elegant chemistry like this that makes it hard for me to accept that it arose by the machinations of a blind watchmaker.