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.

One good thing about Trump’s election (maybe two)

Two comments on the election then back to some neuropharmacology and neuropsychiatry which will likely affect many of you (because of some state ballot initiatives).

First: Over the years I’ve thought the mainstream press has become increasingly biased toward the left (not on the editorial page which is fine) but in supposedly objective reporting. Here are just two post election examples

#1 Front page of the New York Times 9 Nov — the first sentence from something they characterize as ‘News Analysis’

““Donald John Trump was elected the 45th president of the United States on Tuesday in a stunning culmination of an explosive, populist and polarizing campaign that took relentless aim at the institutions and long-held ideals of American democracy.”

#2 Front page of the New York Times 10 Nov — more ‘News Analysis’ — Here’s the lead “Populist Fury may Backfire”. Don’t they wish.

I’ll never complain about this sort of thing again (well at least not for four years). Why? Because I’ve been reading the Wall Street Journal, The New York Times, The Nation and the National Review for probably 50 years, and Trump as the antiChrist is the first thing I’ve ever seen all four agree on. This biased coverage simply no longer matters. If it did, Trump would have lost and lost big. This just confirms the marked loss of credibility that the mainstream media has suffered.  People aren’t as dumb as the elites think they are.

Second: Political correctness and attempts to control speech so as not to offend lost big. That’s very good for us all right and left (although the impetus for speech control has switched to the left from the right over the past 56 years) — see https://luysii.wordpress.com/2015/11/22/from-banned-in-boston-to-banned-in-berkeley-in-55-years/

What do the state ballot initiatives have to do with neuropharmacology? Just this. Voters in California, Massachusetts and Nevada approved recreational marijuana initiatives Tuesday night, and several other states passed medical marijuana provisions.

I don’t think this is good. One of the arguments in its favor is that marihuana isn’t as bad as alcohol, which may be true, but if marihuana isn’t all good why add it to the mix? We don’t have a good handle on marihuana use, but it is likely to increase if it’s legal.

Why do I think this is bad (particularly for adolescents)? It is likely that inhibiting synaptic feedback isn’t a good thing for a brain which is pruning a lot of them (which happens in normal adolescence as the thickness of the cerebral cortex shrinks).

There have been many explanations for the decline in College Board Scores over the years. This has led to their normalization (so all our children are above average). If you’re a correlation equals causation fan, plot the decline vs. time of atmospheric lead. It is similar to the board scores decline. Or you can plot 1/adolescent marihuana use vs. time and get a similar curve. The problem, of course, is that we have no accurate figures for use.

Here’s the science — it’s an old post, but little has happened since it was written to change the science behind it

Why marihuana scares me

There’s an editorial in the current Science concerning how very little we know about the effects of marihuana on the developing adolescent brain [ Science vol. 344 p. 557 ’14 ]. We know all sorts of wonderful neuropharmacology and neurophysiology about delta-9 tetrahydrocannabinol (d9-THC) — http://en.wikipedia.org/wiki/Tetrahydrocannabinol The point of the authors (the current head of the Amnerican Psychiatric Association, and the first director of the National (US) Institute of Drug Abuse), is that there are no significant studies of what happens to adolescent humans (as opposed to rodents) taking the stuff.

Marihuana would the first mind-alteraing substance NOT to have serious side effects in a subpopulation of people using the drug — or just about any drug in medical use for that matter.

Any organic chemist looking at the structure of d9-THC (see the link) has to be impressed with what a lipid it is — 21 carbons, only 1 hydroxyl group, and an ether moiety. Everything else is hydrogen. Like most neuroactive drugs produced by plants, it is quite potent. A joint has only 9 milliGrams, and smoking undoubtedly destroys some of it. Consider alcohol, another lipid soluble drug. A 12 ounce beer with 3.2% alcohol content has 12 * 28.3 *.032 10.8 grams of alcohol — molecular mass 62 grams — so the dose is 11/62 moles. To get drunk you need more than one beer. Compare that to a dose of .009/300 moles of d9-THC.

As we’ve found out — d9-THC is so potent because it binds to receptors for it. Unlike ethanol which can be a product of intermediary metabolism, there aren’t enzymes specifically devoted to breaking down d9-THC. In contrast, fatty acid amide hydrolase (FAAH) is devoted to breaking down anandamide, one of the endogenous compounds d9-THC is mimicking.

What really concerns me about this class of drugs, is how long they must hang around. Teaching neuropharmacology in the 70s and 80s was great fun. Every year a new receptor for neurotransmitters seemed to be found. In some cases mind benders bound to them (e.g. LSD and a serotonin receptor). In other cases the endogenous transmitters being mimicked by a plant substance were found (the endogenous opiates and their receptors). Years passed, but the receptor for d9-thc wasn’t found. The reason it wasn’t is exactly why I’m scared of the drug.

How were the various receptors for mind benders found? You throw a radioactively labelled drug (say morphine) at a brain homogenate, and purify what it is binding to. That’s how the opiate receptors etc. etc. were found. Why did it take so long to find the cannabinoid receptors? Because they bind strongly to all the fats in the brain being so incredibly lipid soluble. So the vast majority of stuff bound wasn’t protein at all, but fat. The brain has the highest percentage of fat of any organ in the body — 60%, unless you considered dispersed fatty tissue an organ (which it actually is from an endocrine point of view).

This has to mean that the stuff hangs around for a long time, without any specific enzymes to clear it.

It’s obvious to all that cognitive capacity changes from childhood to adult life. All sorts of studies with large numbers of people have done serial MRIs children and adolescents as the develop and age. Here are a few references to get you started [ Neuron vol. 72 pp. 873 – 884, 11, Proc. Natl. Acad. Sci. vol. 107 pp. 16988 – 16993 ’10, vol. 111 pp. 6774 -= 6779 ’14 ]. If you don’t know the answer, think about the change thickness of the cerebral cortex from age 9 to 20. Surprisingly, it get thinner, not thicker. The effect happens later in the association areas thought to be important in higher cognitive function, than the primary motor or sensory areas. Paradoxical isn’t it? Based on animal work this is thought to be due pruning of synapses.

So throw a long-lasting retrograde neurotransmitter mimic like d9-THC at the dynamically changing adolescent brain and hope for the best. That’s what the cited editorialists are concerned about. We simply don’t know and we should.

Addendum 11 Nov ’16:  From an emerita nonscientific professor friend of my wife. “Much of the chemistry/pharmacology etc. is way beyond me, but I did get the drift of the conversation about marihuana and am glad to now have even a simplified concept of what it does to the brain. Having spent the last 20 years working with undergraduate and graduate students, I’ve seen first hand the decline in cognitive ability.” 

Scary ! ! ! !

Having been in Cambridge when Leary was just getting started in the early 60’s, I must say that the idea of tune in turn on and drop out never appealed to me. Most of the heavy marihuana users I’ve known (and treated for other things) were happy, but rather vague and frankly rather dull.

Unfortunately as a neurologist, I had to evaluate physician colleagues who got in trouble with drugs (mostly with alcohol). One very intelligent polydrug user MD, put it to me this way — “The problem is that you like reality, and I don’t”.

Should pregnant women smoke pot?

Well, maybe this is why college board scores have declined so much in recent decades that they’ve been normed upwards. Given sequential MRI studies on brain changes throughout adolescence (with more to come), we know that it is a time of synapse elimination. (this will be the subject of another post). We also know that endocannabinoids, the stuff in the brain that marihuana is mimicking, are retrograde messengers there, setting synaptic tone for information transmission between neurons.

But there’s something far scarier in a paper that just came out [ Proc. Natl. Acad. Sci. vol. 112 pp. 3415 – 3420 ’15 ]. Hedgehog is a protein so named because its absence in fruitflies (Drosophila) causes excessive bristles to form, making them look like hedgehogs. This gives you a clue that Hedgehog signaling is crucial in embryonic development. A huge amount is known about it with more being discovered all the time — for far more details than I can provide see http://en.wikipedia.org/wiki/Hedgehog_signaling_pathway.

Unsurprisingly, embryonic development of the brain involves hedgehog, e,g, [ Neuron vol. 39 pp. 937 – 950 ’03 ] Hedgehog (Shh) signaling is essential for the establishment of the ventral pattern along the whole neuraxis (including the telencephalon). It plays a mitogenic role in the expansion of granule cell precursors during CNS development. This work shows that absence of Shh decreases the number of neural progenitors in the postnatal subventricular zone and hippocampus. Similarly conditional inactivation of smoothened results in the formation of fewer neurospheres from progenitors in the subventricular zone. Stimulation of the hedgehog pathway in the mature brain results in elevated proliferation in telencephalic progenitors. It’s a lot of unfamiliar jargon, but you get the idea.

Of interest is the fact that the protein is extensively covalently modified by lipids (cholesterol at the carboxy terminal end and palmitic acid at the amino terminal end. These allow hedgehog to bind to its receptor (smoothened). It stands to reason that other lipids might block this interaction. The PNAS work shows this is exactly the case (in Drosophila at least). One or more lipids present in Drosophila lipoprotein particles are needed in vivo to keep Hedgehog signaling turned off in wing discs (when hedgehog ligand isn’t around). The lipids destabilize Smoothtened. This work identifies endocannabinoids as the inhibitory lipids from extracts of human very low density lipoprotein (VLDL).

It certainly is a valid reason for women not to smoke pot while pregnant. The other problem with the endocannabinoids and exocannabinoids (e.g. delta 9 tetrahydrocannabinol), is that they are so lipid soluble they stick around for a long time — see https://luysii.wordpress.com/2014/05/13/why-marihuana-scares-me/

It is amusing to see regulatory agencies wrestling with ‘medical marihuana’ when it never would have gotten through the FDA given the few solid studies we have in man.