What is docosahexenoic acid and why should you care?

Why should drug chemists care about docosahexenoic acid — it’s a fairly trivial organic structure as these things go – a 22 carbon straight chain carboxylic acid with 6 double bonds — https://en.wikipedia.org/wiki/Docosahexaenoic_acid. However the structure is decidedly non-random (see later)

Docosahexenoic acid turns out to be crucial for the function of the blood brain barrier (BBB), something that makes it very difficult to get drugs into the brain. Years of work have shown that the only drugs able to get through the BBB are small lipid soluble molecules of mass under 400 kiloDaltons with fewer than 9 hydrogen bonds. Certainly not a large group of drugs. The more we know about the BBB, the more likely we’ll be able to figure out something to circumvent it.

The BBB was known to exist more than 100 years ago. Ehrlich found that dyes injected into the circulation were rapidly taken up by all organs except the brain. His student E. Goldmann found that dye injected into the CSF stained the brain but not other organs.

The barrier has at least two components — (1) a very tight seal between the cells lining brain blood vessels (e.g. the endothelium) — see the end of the post — (2)very low transfer across the endothelial cell from the vessel lumen. The latter is called transcytosis and involves formation of small vesicles at the lumenal surface of the endothelium, migration across the endothelial cell with release of vesicle content on the other side.

In general there are two mechanisms of transcytosis — clathrin coated pits, and caveolae. Brain endothelium shows very low rates of transcytosis. There aren’t any coated pits (no explanation I can find) and the rate of caveolar transcytosis is very low.

Dococsahexaenoic acid is the reason for the low rate of caveolar transcytosis. Here is why.

[ Nature vol. 509 pp. 432 – 433, 503 – 506, 507 – 511 ’14 Neuron vol. 82 pp. 728 – 730 ’14 ] An orphan transporter, MFSD2a (Major Facilitator Superfamily Domain containing 2a) is selectively expressed in the BBB endothelium. It is REQUIRED for formation and maintenance of BBB integrity. Animals lacking MFSD2a show uninhibited bulk transcytosis across the endothelium. The animals show no obvious defects in the junctions between the endothelial cells. Pericytes (cells in the brain layer after the endothelium) are important in keeping the levels of MFSD2a at normal levels as animals lacking them show the same defects in the BBB as those lacking MFSD2a. Even though knockouts don’t have much of a BBB, they have normal patterning of vascular networks.

MFSD2a is the major transporter of docohexaenoic acid (DHA), an omega3 fatty acid (more later). DHA isn’t made in the brain and must be transported into it. Knockouts have reduced levels of DHA in the brain accompanied by neuronal loss in the hippocampus and cerebellum and microcephaly. Human cases due to mutation are now known (11/15). Transport of DHA and fatty acids into the brain across the BBB occurs only in the form of esters with lysophosphatidylcholines (LPCs) but not as free fatty acids in a sodium dependent manner. The phospho-zwitterionic headgroup of of LPC is essential for transport. MFSD2a ‘prefers’ long chain fatty acids (oleic, palmitic), failing to transport fatty acids with chain lengths under 14.

So MFSD2a inhibits transcytosis at the same time it promotes fatty acid transport into the brain. Major Facilitator Superfamily (MFS) proteins use the electrochemical potential of the cell to transport substrates. The best known MFSs are the glucose transporters (GLUT1 – 4).

So the blood brain barrier is due in part to the lipid transport activity of MFSD2a which gives BBB endothelium a different lipid composition (with lots of docosahexenoic acid) ) than others, inhibiting caveolar transport. Increased DHA levels are associated with membrane cholesterol depletion, as well as displacement of caveolin1 (the major protein involved in this form of transcytosis) from caveolae.

It is likely that MFSD2A acts as a lipid flippase, transporting phospholipids, including DHA containing species from the outer to the inner plasma membrane leaflet (where caveolin1 binds).

What is so hot about docosahexenoic acid — 22 carbons all in a row, a carboxyl group and 6 double bonds. We’re not talking fused ring systems, alkaloids, bizarre functional groups etc. etc.

Half the answer is that the double bonds are NOT randomly arranged. The 6 occur all in a row (but with methylene groups between them). This tells the chemist that they are not conjugated, hence the chain is probably not straight. Think how unlikely the arrangement is considering the way 6 double bonds and 9 methylenes COULD be arranged in a chain (2^15). Answer 5 ways depending on where the arrangement starts relative to the end of the chain.

The other half is that all the double bonds are cis, making it very unlikely that the 21 carbon chain can straighten out and cross the membrane. Lots of DHA means a very disordered membrane, which may be impossible to caveolin1 (and clathrin) to bind to.

So even though it’s years and years since I left organic chemistry, it permits the enjoying of the biochemical esthetics of the blood brain barrier.

The tight junctions between endothelial cells are primarily responsible for barrier function. These tight junctions are found only in the capillaries and postcapillary venules of the brain. Endothelial cells of the brain have few pinocytotic vesicles and fenestriae. [ Neuron vol. 71 p. 408 ’11 ] The brain vasculature has the thinnest endothelial cells, with the tightest junction and a higher degree of pericyte coverage coverage (‘up to’ 30%). [ Neuron vol. 78 pp. 214 – 232 ’13 ] The tight junctions are made from occludin, claudins and junctional adhesion molecules, and are closer to the lumen than the adherens junctions (which also link endothelial cells to each other) made by the cadherins (E, P and N). (ibid p. 219) TLR2/6 specific stimuli.

The powers that be at the New York Times have decided that Hillary is toast

Kremlinologists used to carefully look at who was in what position in the stands reviewing the annual May Day parade to understand behind the scenes power struggles and who was currently on top. Here in the States, we are more fortunate. All we need to do is read the letters to the editor on the editorial page of the New York Times.

Just like “Home on the Range”, never is heard a discouraging word (or a letter which disagrees with the editorial stances of the Times).

So today (15 May 2017) 4 letters appeared concerning a column written 7 May by Frank Bruni titled “Hillary Clinton’s Absolution” — all highly critical of Hillary and Bill.

A few quotes:

“The Clintons have been running the Democratic party like a personal fief for 25 years”.

“But to ruminate about how Hillary would have won if only the Russians, WikiLeaks and James Comey didnot do her in — well, I have had enough of this.”

“Frank Bruni” seems obsessed enough about Hillary Clinton’s feelings to write an opera.”

“But it’s also her (Hillary’s) supporters in the DemocraticParty leadership who need to seek redemption for abandoning the working class voters who once formed the core constituency of their party.”

Do you think the publication of these 4 letters was unintentional? By the purest of coincidences publication occurred the very day Hillary announced the formation of a new PAC to oppose Trump — http://fortune.com/2017/05/15/hillary-clinton-launches-political-action-committee-dedicated-to-liberal-causes/

The same day a full page article title “How Democracy Collapsed” concerning Venezuela. The word elites appears 3 times, the word socialism not at all.

A Touching Mother’s Day Story

A Touching Mother’s Day Story

Yes, a touching mother’s day story for you all. It was 50 years ago (yes half a century ago ! ! ), and I was an intern at a big city hospital on rotation in their emergency room. The ER entrance was half a block from an intersection with a bar on each corner. On a Saturday night, we knew better than to try to get some sleep before 2AM or until we’d put in 2 chest tubes (to drain blood from the lungs, which had been shot or stabbed). The bartenders were an intelligent lot — they had to be quick thinking to defuse situations, and we came to know them by name. So it was 3AM 50 years ago and Tyrone was trudging past on his way home, and I was just outside the ER getting some cool night air, things having quieted down.

“Happy Mother’s day, Tyrone” sayeth I

“Thanks Doc, but every day is Mother’s day with me”

“Why, Tyrone?”

“Because every day I get called a mother— “

Life at 250 Atmospheres pressure 1.8 tons/square inch

Tube worms (actually a form of mollusc) live on the depths of the ocean floor where there is almost no light, and very little oxygen. Just as plants use light energy to remove electrons from water to form oxygen and fix carbon, passing the stolen electrons back to oxygen taxing it though intermediary metabolism, symbiotic bacteria living in the worms remove electrons from hydrogen sulfide (H2S) formed by the hydrothermal vents on the seafloor. . How did the tube worms get this far down? By riding decaying wood down there. [Proc. Natl. Acad. Sci. vol. 114 pp. E3652 – E3658 ’17 ] This is the wooden-steps hypothesis [Distel DL, et al. (2000) Nature 403:725–726] which states that the large chemosynthetic mussels (ship worms) found at deep-sea hydrothermal vents descend from much smaller species associated with sunken wood and other organic deposits, and that the endosymbionts of these progenitors made use of hydrogen sulfide from biogenic sources (e.g., decaying wood) rather than from vent fluids.

At 2500 meters down the water pressure is 3750 pounds per square inch. One can only imagine the changes required in the amino acid sequences of their proteins required so they aren’t denatured or aggregated by such pressure.

The idea that life on planetary moons with subsurface oceans (Ganymede, Europa, Titan, Enceladus) could exist is no longer as fantastic as it initially seemed.

If it be found the implications for our conception of our place in the natural world are enormous.

Why wasn’t this mentioned in Genesis or any known creation myth? Assume for the moment that there actually is a creator who made itself known to our ancestors. If it tried to give Abraham, Gautama Budda, Mohammed et. etc. knowledge of these things, it wouldn’t have been believed. Planets? Planets with moons? Please. A few miracles here and there would be all that would be needed.

Impeach Earl Warren ! ! !

Most of the readership is too young to remember billboards all over the South in the 50’s and 60’s crying “Impeach Earl Warren ! ! ! ” He was the chief justice of the Supreme Court which in 1954 rendered the decision that legally enforced racial segregation was unconstitutional.

Now we have a letter in today’s New York Times arguing for a recall presidential election, and an article in the 8 May New Yorker titled “EndGames What would it take to cut short Trump’s Presidency.” Don’t they wish. Hilarious. Santayana must be loving it. “Those who cannot remember the past are condemned to repeat it”

Remember entropy?

Organic chemists have a far better intuitive feel for entropy than most chemists. Condensations such as the Diels Alder reaction decrease it, as does ring closure. However, when you get to small ligands binding proteins, everything seems to be about enthalpy. Although binding energy is always talked about, mentally it appears to be enthalpy (H) rather than Gibbs free energy (F).

A recent fascinating editorial and paper [ Proc. Natl. Acad. Sci. vol. 114 pp. 4278 – 4280, 4424 – 4429 ’17 ]shows how the evolution has used entropy to determine when a protein (CzrA) binds to DNA and when it doesn’t. As usual, advances in technology permit us to see this (e.g. multidimensional heteronuclear nuclear magnetic resonance). This allows us to determine the motion of side chains (methyl groups), backbones etc. etc. When CzrA binds to DNA methyl side chains on the protein move more, increasing entropy (deltaS) and as well all know the Gibbs free energy of reaction (deltaF) isn’t just enthalpy (deltaH) but deltaH – TdeltaS, so an increase in deltaS pushes deltaF lower meaning the reaction proceeds in that direction.

Binding of Zinc redistributes these side chain motion so that entropy decreases, and the protein moves off DNA. The authors call this dynamics driven allostery. The fascinating thing, is that this may happen without any conformational change of CzrA.

I’m not sure that molecular dynamics simulations are good enough to pick this up. Fortunately newer NMR techniques can measure it. Just another complication for the hapless drug chemist thinking about protein ligand interactions.

The incredible combinatorial complexity of cellular biochemistry

K8, K14, K20, T92, P125, S129, S137, Y176, T195, K276, T305, T308, T312, P313, T315, T326, S378, T450, S473, S477, S479. No, this is not some game of cosmic bingo. They represent amino acid positions in Protein Kinase B (AKT).

In the 1 letter amino acid code K is lysine T, threonine, S serine, P proline, Y tyrosine.

All 21 amino acids are modified (or not) one of them in 3 ways. This gives 4 * 2^20 = 4,194,304 possible post-translational modifications. Will we study all of them? It’s pretty easy to substitute alanine for serine or threonine making an unmodifiable position, or to substitute aspartic acid for threonine or serine making a phosphorylation mimic which is pretty close to phosphoserine or phosphothreonine, creating even more possibilities for study.

Most of the serines, threonines, tyrosines listed are phosphorylated, but two of the threonines are Nacetyl glucosylated. The two prolines are hydroxylated in the ring. The lysines can be methylated, acetylated, ubiquitinated, sumoylated. I did take the trouble to count the number of serines in the complete amino acid sequence and there are 24, of which only 6 are phosphorylated — so the phosphorylation pattern is likely to be specific and selected for. Too lazy do the same for lysine, threonine, tyrosine and proline. Here’s a link to the full sequence if you want to do it — http://www.uniprot.org/uniprot/P31749

The phosphorylations at each serine/threonine/tyrosine are carried out by not more than one of the following 8 kinases (CK2, IKKepsilon, ACK1,TBK1, PDK1, GSK3alpha, mTORC2 and CDK2)

AKT contains some 481 amino acids, divided (by humans for the purposes of comprehension) into 4 regions Pleckstrin Homology (#1 – #108), linker (#108 – #152) catalytic –e.g. kinase (#152 – #409),regulatory (#409 – #481).

This is from an excellent review of the functions of AKT in Cell vol. 169 pp. 381 – 3405 ’17. It only takes up the first two pages of the review before the functionality of AKT is even discussed.

This raises the larger issue of the possibility of human minds comprehending cellular biochemistry.

This is just one protein, although a very important one. Do you think we’ll ever be able to conduct enough experiments, to figure out what each modification (along or in combination) does to the many functions of AKT (and there are many)?

Now design a drug to affect one of the actions of AKT (particularly since AKT is the cellular homolog of a viral oncogene). Quite a homework assignment.

Is Martin Burke the anti-Christ for synthetic organic chemistry?

Will a machine put synthetic organic chemists out of business. Is its proponent and inventor Martin Burke the anti-Christ? 2 years ago he thought that he’d need 5,000 building blocks to make 282,487 natural products. Now he’s down to 1,400, 20 years and 1 Billion dollars [ Science vol. 356 pp. 231 – 232 ’17 ].

Back in the day we studied the zillions of terpene natural products built from various machinations of just the isopentyl group. Does he really need another 1,399?

The synthesis is a modification of the Suzuki synthesis in which R-B(OH)2 and R’ – X are coupled by palladium to form R -R’. It uses MIDA (HOOC CH2 NCH3 CH2 COOH — N-MethylIminodiAcetic acid) which wraps itself around the boron and shuts down further synthesis.

In 2008 Burke found that MIDA boronates stick to silica when methanol and ether are both present, and then drop off when tetrahydrofuran (THF) is present. This allows catch and release. For purification they can run the compounds through a silica containing vial.

In 2015 some 200 building blocks with the halogen and MIDA capped boronic acid were availablle commercially.

Burke hooked up with a computer scientist to look at the structures of the 282,487 and break them down into fragments needing only carbon carbon bond formation — a fascinating problem in graph theory.

Derek did a post on this a few years ago. Hopefully he’ll do another.

Because they aren’t there

George Mallory tried 3 times to be the first to climb Everest dying on his last attempt. When asked why he was so obsessed, he achieved immortality by saying “because it’s there”. Chemists have spent 60 years trying to synthesize carbon nanobelts “because they aren’t there”.

Well a group of Japanese chemists finally did it [ Science vol. 356 pp. 172 -175 ’17 ] It’s not quite the ultimate belt because the 6 membered rings are staggered as they are in phenacenes –https://en.wikipedia.org/wiki/Phenacene. There are 6 three ringed phenacenes in the structure, and the diameter of the ring is 8.324 Angstroms. There is no question that they got the compound as they crystallized it and have bond lengths for all.

If you look at the paper, this is a zig zag structure rather than a linear poly anthracene. The bond lengths show that every other ring has symmetric bond lengths midway between sp2 and sp3 (e.g. it’s aromatic), while the other ring clearly is not.

It be interesting to measure the chemical shifts of the C-H bonds over the center of the ring — if they could make a paraCyclophane-type molecule bridging the diameter by a (CH2)n moiety.

As long as we’re on the subject what about putting a twist in the ring and making a mobius belt. Mobius molecules are known — http://www.scs.illinois.edu/denmark/wp-content/uploads/gp/2008/Collins-1.pdf — is a very nice review — with a lot of pictures.

The authors think that their work has potential applications — “our synthesis of carbon nanobelt 1 could ultimately lead to the programmable synthesis of single- chirality, uniform-diameter CNTs (30–32) and open a field of nanobelt science and technology”. I think they were just having fun as chemists are wont to do.

Progress has been slow but not for want of trying

Progress in the sense of therapy for Alzheimer’s disease and Glioblastoma multiforme is essentially nonexistent, and we could use better therapy for Parkinsonism. This doesn’t mean that researchers have given up. Far from it. Three papers all in last week’s issue of PNAS came up with new understanding and possibly new therapeutic approaches for all three.

You’ll need some serious molecular biological and cell physiological chops to get through the following.

l. Glioblastoma multiforme — they aren’t living much longer than they were when I started pracice 45 years ago (about 2 years — although of course there are exceptions).

The human ZBTB family of genes consists of 49 members coding for transcription factors. BCL6 is also known as ZBTB27 and is a master regulator of lymph node germinal responses. To execute its transcriptional activity, BCL6 requires homodimerization and formation of a complex with a variety of cofactors including BCL6 corerpressor (BCoR), nuclear receptor corepressor 1 (NCoR) and Silencing Mediator of Retinoic acid and Thyroid hormone receptor (SMRT). BCL6 inhibitors block the interaction between BCL6 and its friends, selectively killing BCL6 addicted cancer cells.

The present paper [ Proc. Natl. Acad. Sci. vol. 114 pp. 3981 – 3986 ’17 ] shows that BCL6 is required for glioblastoma cell viability. One transcriptional target of BCL6 is AXL, a tyrosine kinase. Depletion of AXL also decreases proliferation of glioblastoma cells in vitro and in vivo (in a mouse model of course).

So here are two new lines of attack on a very bad disease.

2. Alzheimer’s disease — the best we can do is slow it down, certainly not improve mental function and not keep mental function from getting worse. ErbB2 is a member of the Epidermal Growth Factor Receptor (EGFR) family. It is tightly associated with neuritic plaques in Alzheimer’s. Ras GTPase activation mediates EGF induced stimulation of gamma secretase to increase the nuclear function of the amyloid precursor protein (APP) intracellular domain (AICD). ErbB2 suppresses the autophagic destruction of AICD, physically dissociating Beclin1 vrom the VPS34/VPS15 complex independently of its kinase activity.

So the following paper [ Proc. Natl. Acad. Sci. vol. 114 pp. E3129 – E3138 ’17 ] Used a compound downregulating ErbB2 function (CL-387,785) in mouse models of Alzheimer’s (which have notoriously NOT led to useful therapy). Levels of AICD declined along with beta amyloid, and the animals appeared smarter (but how smart can a mouse be?).

3.Parkinson’s disease — here we really thought we had a cure back in 1972 when L-DOPA was first released for use in the USA. Some patients looked so good that it was impossible to tell if they had the disease. Unfortunately, the basic problem (death of dopaminergic neurons) continued despite L-DOPA pills supplying what they no longer could.

Nurr1 is a protein which causes the development of dopamine neurons in the embryo. Expression of Nurr1 continues throughout life. Nurr1 appears to be a constitutively active nuclear hormone receptor. Why? Because the place where ligands (such as thyroid hormone, steroid hormones) bind to the protein is closed. A few mutations in the Nurr1 gene have been associated with familial parkinsonism.

Nurr1 functions by forming a heterodimer with the Retinoid X Receptor alpha (RXRalpha), another nuclear hormone receptor, but one which does have an open binding pocket. A compound called BRF110 was shown by the following paper [ Proc. Natl. Acad. Sci. vol. 114 pp. 3795 – 3797, 3999 – 4004 ’17 ] to bind to the ligand pocked of RXRalpha increasing its activity. The net effect is to enhance expression of dopamine neuron specific genes.

More to the point MPP+ is a toxin pretty selective for dopamine neurons (it kills them). BRF110 helps survival against MPP+ (but only if given before toxin administration). This wouldn’t be so bad because something is causing dopamine neurons to die (perhaps its a toxin), so BRF110 may fight the decline in dopamine neuron numbers, rather than treating the symptoms of dopamine deficiency.

So there you have it 3 possible new approaches to therapy for 3 bad disease all in one weeks issue of PNAS. Not easy reading, perhaps, but this is where therapy is going to come from (hopefully soon).