Why trying to remove aBeta was plausible

The recent collapse of the latest attempt to remove the main constituent of the Alzheimer plaque, the aBeta peptide (gantenerumab from Roche) is just the latest in a long sad story.

Monoclonal after monoclonal antibody targeting aBeta has failed.  It certainly is time to move on and try new approaches.

The companies pursuing monoclonals were not stupid.  Their approach was (but no longer is) quite reasonable in view of the clinical and experimental evidence implicating the aBeta peptide as causative of Alzheimer’s  Before moving on, here are some of the reasons why.

First (and probably the best) is the mutation that protects against Alzheimer’s disease.  As most of you know, the aBeta peptide (39 to 42 amino acids) is part of a much larger protein the Amyloid Precursor Protein (APP) which contains 639 to 770  amino acids.  This means that enzymes must  cut it out.  Such enzymes (called proteases) are finicky, cutting only between certain amino acids.  In what follows A673T stands for the 673rd position which normally has amino acid Alanine (A) there.  Instead there is amino acid Threonine (T).   The enzyme cleaving at 673  is Beta Secretase 1 (BACE1).

       [ Nature vol. 487 pp. 153 ’12 ] A mutation in APP protects against Alzheimer’s disease.   First the genome sequence APP of 1,795 Icelanders  were studied to look for low frequency variants.  They found a mutation A673T adjacent to the site that is cleaved by beta secretase 1 (BACE1) which doesn’t vary — it’s gamma secretase which cleaves at variable sites leading to Abeta40, Abeta42 formation.  The mutation is at position 2 in Abeta.  The mutation results in a 40% reduction in the formation of amyloidogenic peptides  in vitro (293T cells transfected with variant and normal APP). Amazingly, a different variant at 673 (A673V — V stands for the amino acid Valine) — increases Abeta formation.    Because BACE1 can’t cleave APP containing the A673T mutation, alternative processing of APP at another site the alpha site (which is within aBeta preventing formation of the full 39 – 43 amino acid peptide).

So if you can’t make the full aBeta peptide you don’t get Alzheimer’s (or have less chance of getting it).

Then there are the mutations in the part of APP which code for the aBeta peptide which increase the risk of Alzheimer’s.  They cause the different familial Alzheimer’s disease.   Now that we know the actual structure of the aBeta amyloid fiber, we can understand how they cause Alzheimer’s disease.  This is more strong evidence that the aBeta peptide is involved in the causation of Alzheimer’s disease.

You’ll need some protein chemistry chops to understand the following

Recall that in amyloid fibrils the peptide backbone is flat as a flounder (well in a box 4.8 Angstroms high) with the amino acid side chains confined to this plane.  The backbone winds around in this plane like a snake.  The area in the leftmost loop is particularly crowded with bulky side chains of glutamic acid (single letter E) at position 22 and aspartic acid (single letter D) at position 23 crowding each other.  If that wasn’t enough, at the physiologic pH of 7 both acids are ionized, hence negatively charged.  Putting two negative charges next to each other costs energy and makes the sheet making up the fibril less stable.

The marvelous paper (the source for much of this) Cell vol. 184 pp. 4857 – 4873 ’21 notes that there are 3 types of amyloid — pathological, artificial, and functional, and that the pathological amyloids are the most stable. The most stable amyloids are the pathological ones.  Why this should be so will be the subject of a future post, but accept it as fact for now

In 2007 there were 7 mutations associated with familial Alzheimer’s disease (10 years later there were 11). Here are 5 of them.

Glutamic Acid at 22 to Glycine (Arctic)

Glutamic Acid at 22 to Glutamine (Dutch)

Glutamic Acid at 22 to Lysine (Italian)

Aspartic Acid at 23 to Asparagine (Iowa)

Alanine at 21 to Glycine (Flemish)

All of them lower the energy of the amyloid fiber.

Here’s why

Glutamic Acid at 22 to Glycine (Arctic) — glycine is the smallest amino acid (side chain hydrogen) so this relieves crowding.  It also removes a negatively charged amino acid next to the aspartic acid.  Both lower the energy

Glutamic Acid at 22 to Glutamine (Dutch) — really no change in crowding, but it removes a negative charge next to the negatively charged Aspartic acid

Glutamic Acid at 22 to Lysine (Italian)– no change in crowding, but the lysine is positively charged at physiologic pH, so we have a positive charge next to the negatively charged Aspartic acid, lowering the energy

Aspartic Acid at 23 to Asparagine (Iowa) –really no change in crowding, but it removes a negative charge next to the negatively charged Glutamic acid next door

Alanine at 21 to Glycine (Flemish) — no change in charge, but a reduction in crowding as alanine has a methyl group and glycine a hydrogen.

As a chemist, I find this immensely satisfying.  The structure explains why the mutations in the 42 amino acid aBeta peptide are where they are, and the chemistry explains why the mutations are what they are.

It’s time to look elsewhere.  The best this class of drug (monoclonal antibodies against aBeta) offers is lecanemab which slows the rate of decline by a measly 27%.   This is very small beer

While big pharma was far from stupid to intensively (and expensively) to give the monoclonals the old college try in the past (for the reasons cited above), they would be incredibly stupid to continue this line of attack.

4 diseases explained at one blow said the protein chemist — part 2 — TDP43

A brilliant paper [ Science vol. 377 eabn5582 pp. 1 –> 20 ’22 ] explains how changing a single amino acid (proline) to another  can cause 4 different diseases, depending on the particular protein it is found in (and which proline of many is changed).

There is so much in this paper that it will take several posts to go over it all.  The chemistry in the paper is particularly fine.  So it’s back to Biochemistry 101 and the alpha helix and the beta sheet.

A lot of the paper concerns TDP43, a protein familiar to neurologists because it is involved in FTD-ALS (FrontoTemporal Dementia — Amyotrophic Lateral Sclerosis) and ALS itself.

I actually saw a case early in training.  I had been taught that ALS patients remained cognitively intact until the end (certainly true in my experience — think of Stephen Hawking), so here was this ALS case who was mildly demented.  My education, deficient at that time, so I’d never heard of FTD-ALS, had me writing in the chart “we’re missing something here”.  These were calmer times in the medical malpractice world.

TDP43 is a protein with a lot of different parts in its 414 amino acids.  There are two regions which bind to RNA (Rna Recognition Motifs { RRMs } ), and a glycine rich low complexity domain at the carboxy terminal end.

TDP43 proteins are found in the neuronal inclusions of ALS (interestingly, these weren’t recognized when I was in training).  The low complexity domain of TDP43 aggregates and form fibers.  Some 50 different mutations have been found here in patients.

Just this year the cryoEM structure of TDP43 aggregates from two patients with FTD-ALS were described [ Nature vol. 601 pp. 29 – 30, 139 – 143 ’22 ].  It appears to be a typical amyloid structure with all 79 amino acids (from # 282 Glycine to #360  Glutamine) in a single plane.  Here’s a link to the actual paper — https://www.nature.com/articles/s41586-021-04199-3.  It is likely behind a paywall, but if you can get it, look at figure 2 p. 140, which has the structure.  Who would have ever thought that a protein could flatten out this much.

Both structures were from TDP-43 with none of the 24 mutations known to cause FTD-ALS.

But that’s far from the end of the story.  The same area of TDP43 can also form liquid droplets (perhaps the precursor of the fibers).  But that’s where the brilliant chemistry of [ Science vol. 377 eabn5582 pp. 1 –> 20 ’22 ] comes in.

That’s for next time.  After that, I should be finished with Needham and will have time to write about 6 or so of the interesting papers I’ve run across in the past 6 months.

Amyloid Structure at Last ! 3 The Alzheimer mutations

I am republishing this post from last October, because the excellent paper I’m going to write about has similar thinking.

Although the chemistry explaining why these mutations are associated with Alzheimer’s disease is exquisite and why they point to ‘the’ cause of Alzheimer’s disease — the amyloid fibril, billions have been spent in attempts to remove amyloid fibrils with no useful therapeutic result (and some harm)

Here’s the old post

The structure of the amyloid fibril formed by the aBeta42 peptide exactly shows why certain mutations are associated with hereditary Alzheimer’s disease.   Here is a picture

https://www.alzforum.org/news/research-news/danger-s-bends-new-structure-av42-fibrils-comes-view

Scroll down to the picture above “Bonds that Tie”

If you need some refreshing on the general structure of amyloid, have a look at the first post in the series — https://luysii.wordpress.com/2021/10/11/amyloid-structure-at-last/

Recall that in amyloid fibrils the peptide backbone is flat as a flounder (well in a box 4.8 Angstroms high) with the amino acid side chains confined to this plane.  The backbone winds around in this plane like a snake.  The area in the leftmost loop is particularly crowded with bulky side chains of glutamic acid (single letter E) at position 22 and aspartic acid (single letter D) at position 23 crowding each other.  If that wasn’t enough, at the physiologic pH of 7 both acids are ionized, hence negatively charged.  Putting two negative charges next to each other costs energy and makes the sheet making up the fibril less stable.

The marvelous paper (the source for much of this) Cell vol. 184 pp. 4857 – 4873 ’21 notes that there are 3 types of amyloid — pathological, artificial, and functional, and that the pathological amyloids are the most stable. The most stable amyloids are the pathological ones.  Why this should be so will be the subject of a future post, but accept it as fact for now

In 2007 there were 7 mutations associated with familial Alzheimer’s disease (10 years later there were 11). Here are 5 of them.

Glutamic Acid at 22 to Glycine (Arctic)

Glutamic Acid at 22 to Glutamine (Dutch)

Glutamic Acid at 22 to Lysine (Italian)

Aspartic Acid at 23 to Asparagine (Iowa)

Alanine at 21 to Glycine (Flemish)

All of them lower the energy of the amyloid fiber.

Here’s why

Glutamic Acid at 22 to Glycine (Arctic) — glycine is the smallest amino acid (side chain hydrogen) so this relieves crowding.  It also removes a negatively charged amino acid next to the aspartic acid.  Both lower the energy

Glutamic Acid at 22 to Glutamine (Dutch) — really no change in crowding, but it removes a negative charge next to the negatively charged Aspartic acid

Glutamic Acid at 22 to Lysine (Italian)– no change in crowding, but the lysine is positively charged at physiologic pH, so we have a positive charge next to the negatively charged Aspartic acid, lowering the energy

Aspartic Acid at 23 to Asparagine (Iowa) –really no change in crowding, but it removes a negative charge next to the negatively charged Glutamic acid next door

Alanine at 21 to Glycine (Flemish) — no change in charge, but a reduction in crowding as alanine has a methyl group and glycine a hydrogen.

As a chemist, I find this immensely satisfying.  The structure explains why the mutations in the 42 amino acid aBeta peptide are where they are, and the chemistry explains why the mutations are what they are.

Abeta peptide, Amyloid structure, Arctic familial Alzheimer mutation, Dutch familial Alzheimer mutation, Familial Alzheimer’s disease, Flemish familial Alzheimer mutation, Iowa familial Alzheimer mutation, Italian familial Alzheimer mutation. No

Amyloid Structure at Last ! 3 The Alzheimer mutations

The structure of the amyloid fibril formed by the aBeta42 peptide exactly shows why certain mutations are associated with hereditary Alzheimer’s disease.   Here is a picture

https://www.alzforum.org/news/research-news/danger-s-bends-new-structure-av42-fibrils-comes-view

Scroll down to the picture above “Bonds that Tie”

If you need some refreshing on the general structure of amyloid, have a look at the first post in the series — https://luysii.wordpress.com/2021/10/11/amyloid-structure-at-last/

Recall that in amyloid fibrils the peptide backbone is flat as a flounder (well in a box 4.8 Angstroms high) with the amino acid side chains confined to this plane.  The backbone winds around in this plane like a snake.  The area in the leftmost loop is particularly crowded with bulky side chains of glutamic acid (single letter E) at position 22 and aspartic acid (single letter D) at position 23 crowding each other.  If that wasn’t enough, at the physiologic pH of 7 both acids are ionized, hence negatively charged.  Putting two negative charges next to each other costs energy and makes the sheet making up the fibril less stable.

The marvelous paper (the source for much of this) Cell vol. 184 pp. 4857 – 4873 ’21 notes that there are 3 types of amyloid — pathological, artificial, and functional, and that the pathological amyloids are the most stable. The most stable amyloids are the pathological ones.  Why this should be so will be the subject of a future post, but accept it as fact for now

In 2007 there were 7 mutations associated with familial Alzheimer’s disease (10 years later there were 11). Here are 5 of them.

Glutamic Acid at 22 to Glycine (Arctic)

Glutamic Acid at 22 to Glutamine (Dutch)

Glutamic Acid at 22 to Lysine (Italian)

Aspartic Acid at 23 to Asparagine (Iowa)

Alanine at 21 to Glycine (Flemish)

All of them lower the energy of the amyloid fiber.

Here’s why

Glutamic Acid at 22 to Glycine (Arctic) — glycine is the smallest amino acid (side chain hydrogen) so this relieves crowding.  It also removes a negatively charged amino acid next to the aspartic acid.  Both lower the energy

Glutamic Acid at 22 to Glutamine (Dutch) — really no change in crowding, but it removes a negative charge next to the negatively charged Aspartic acid

Glutamic Acid at 22 to Lysine (Italian)– no change in crowding, but the lysine is positively charged at physiologic pH, so we have a positive charge next to the negatively charged Aspartic acid, lowering the energy

Aspartic Acid at 23 to Asparagine (Iowa) –really no change in crowding, but it removes a negative charge next to the negatively charged Glutamic acid next door

Alanine at 21 to Glycine (Flemish) — no change in charge, but a reduction in crowding as alanine has a methyl group and glycine a hydrogen.

As a chemist, I find this immensely satisfying.  The structure explains why the mutations in the 42 amino acid aBeta peptide are where they are, and the chemistry explains why the mutations are what they are.

 

 

 

 

 

 

Amyloid Structure at Last ! – 2 Birefringence

This was the state of the art 19 years ago in a PNAS paper (vol. 99 pp. 16742 – 16747 ’02).  “Amyloid fibrils are filamentous structures with typical diameters of 10 nanoMeters and lengths up to several microns.  No high resolution molecular structure of an amyloid fibril has yet been determined experimentally because amyloid fibrils are noncrystalline solid materials and are therefore incompatible with Xray crystallography and liquid state NMR.”

Well solid state NMR and cryo electron microscopy have changed all that and we now have structures for many amyloids at near atomic resolution.  It’s probably behind a pay wall but look at Cell vol. 184 pp. 4857 – 4873 ’21 if you have a chance.  I’ve spent the last week or so with it, and a series of posts on various aspects of the paper will be forthcoming.  The paper contains far too much to cram into a single post.

So lacking an Xray machine to do diffraction, what did we have 57 years ago when I started getting seriously interested in neurology?  To find amyloid we threw a dye called Congo Red on a slide, found that it bound amyloid and became birefringent when it did so.

Although the Cell paper doesn’t even mention Congo Red, the structure of amyloid they give explains why this worked.

What is birefringence anyway?  It means that light moving through a material travels at different speeds in different directions.  The refractive index of a material is the relative speed of light through that material versus the speed of light in a vacuum.   Stand in a shallow pool.  Your legs look funny because light travels slower in water than in air (which is nearly a vacuum).

Look at the structure of Congo Red — https://en.wikipedia.org/wiki/Congo_red.  It’s a long thin planar molecule, containing 6 aromatic rings, kept planar with each other by pi electron delocalization.

The previous post contained a more detailed description of amyloid — but suffice it to say that instead of wandering around in 3 dimensional space, the protein backbone in amyloid is confined to a single plane 4.8 Angstroms thick — here’s a link — https://luysii.wordpress.com/2021/10/11/amyloid-structure-at-last/

Plane after plane stacks on top of each other in amyloid.  So a micron (which is 10,000 Angstroms) can contain over 5,000 such planes, and an amyloid fibril can be several microns long.

It isn’t hard to imagine the Congo Red molecule slipping between the sheets, making it’s orientation fixed.  Sounds almost pornographic doesn’t it? This orients the molecule and clearly light moving perpendicular to the long axis of Congo Red will move at a different speed than light going parallel to the long axis of Congo Red, hence its birefringence when the dye binds amyloid.

Well B-DNA (the form we all know and love as the double helix) has its aromatic bases stacked on top of each other every 3.4 Angstroms.  So why isn’t it birefringent with Congo Red?  It has a persistence length of 150 basePairs or about .05 microns, which means that the average orientation is averaged out, unlike the amyloid in a senile plaque

There is tons more to come.  The Cell paper is full of fascinating stuff.