Category Archives: Aargh ! Big pharma sheds chemists. Why?

Further (physical) chemical elegance

If the chemical name phosphatidyl serine (PS) draws a blank, read the verbatim copy of a previous post under the *** to find out why it is so important to our existence. It is an ‘eat me’ signal when there is lots of it around, telling professional scavenger cells to engulf the cell showing lots of PS on its surface.

Life, as usual, is more complicated. There are a variety of proteins exposed on cell surfaces which bind to phosphoserine. Not only that, but exposing just a little PS on the surface of a cell can trigger a protective immune response. Immune cells binding to just a little PS on the surface of another cell proliferate rather than eat the cell expressing the PS. This brings us to Proc. Natl. Acad. Sci. vol. 111 pp 5526 – 5531 ’14 that explains how a given PS receptor (called TIM4) acts differently depending how much PS is present.

Some PS receptors such as Annexin V have essentially an all or none response to PS, if they bind at all, they trigger a response in the cell carrying them. Not so for TIM4 which only reacts if there is a lot of PS around, leaving cells which express less PS alone. This allows these cells to function in the protective immune response.

So how does TIM4 do this? See if you can think of a mechanism before reading the rest.

In addition to the PS binding pocket TIM4 has 4 peripheral basic residues in separate places. The basic residues are positively charged at physiologic pH and bind to the negative phosphate group of phosphatidyl serine. The paper doesn’t explain how these basic residues don’t bind to the other phospholipids of the cell surface (such as phosphatidyl choline or sphingomyelin). In any even TIM4 will be triggered only if these groups also bind PS, leaving cells which show relatively little PS alone. Clever no?

For the cognoscenti, the Hill coefficient of TIM4 is 2 while that of Annexin V is 8 (describing more than explaining the all or none character of Annexin V binding).

****
Flippase. Eat me signals. Dragging their tails behind them. Have cellular biologists and structural biochemists gone over to the dark side? It’s all quite innocuous as the old nursery rhyme will show

Little Bo Peep has lost her sheep
and doesn’t know where to find them
Leave them alone, and they’ll come home
wagging their tails behind them.

First, some cellular biochemistry. The lipid bilayer encasing all our cells is made of two leaflets, inner and outer. The composition of the two is different (unlike the soap bubble). On the inside we find phosphatidylethanolamine (PE), phosphatidylserine (PS). The outer leaflet contains phosphatidylcholine (PC) and sphingomyelin (SM) and almost no PE or PS. This is clearly a low entropy situation compared to having all 4 randomly dispersed between the 2 leaflets.

What is the possible use of this (notice how teleology invariably creeps into cellular biology)? Chemistry is powerless to explain such things. Much as I love chemistry, such truths must be faced.

It takes energy to maintain this peculiar distribution. The enzyme moving PE and PS back inside the cell is the flippase. It requires energy in the form of ATP to operate. When a cell is dying ATP drops, and entropy takes its course moving PE and PS to the cell surface. Specialized cells (macrophages) exist to scoop up the dying or dead cells, without causing inflammation. They recognize PE and PS by a variety of receptors and munch up cells exposing them on the surface. So PE and PS are eat me signals which appear when there isn’t enough ATP around for flippase to use to haul PE and PS back inside. Clever no?

No for some juicy chemistry (assuming that you consider transport of a molecule across a lipid bilayer actual chemistry — no covalent bonds to the transferred molecule are formed or removed, although they are to the transporter). Well it certainly is physical chemistry isn’t it?

Here are the structures of PE, PS, PC, SM http://www.google.com/search?q=phosphatidylserine&client=safari&rls=en&tbm=isch&tbo=u&source=univ&sa=X&ei=bDRLU5yfHOPLsQSOnoG4BA&ved=0CPABEIke&biw=1540&bih=887#facrc=_&imgdii=_&imgrc=qrLByG2vmhWdwM%253A%3BwAtgsTPwCxeZXM%3Bhttp%253A%252F%252Fscience.csumb.edu%252F~hkibak%252F241_web%252Fimg%252Fpng%252FCommon_Phospholipids.png%3Bhttp%253A%252F%252Fscience.csumb.edu%252F~hkibak%252F241_web%252Fcoursework_pages%252F2012_02_2.html%3B1297%3B934.

There are a few things to notice. Like just about every lipid found in our membranes, they are amphipathic — they have a very lipid soluble part (look at the long hydrocarbon changes hanging below them) and a very water soluble part — the head groups containing the phosphate.

This brings us to [ Proc. Natl. Acad. Sci. vol. 111 pp. E1334 - E1343 '14 ] Which describes ATP8A2 (aka the flippase). Interestingly, the protein, with at least 10 alpha helices spanning the membrane, and 3 cytoplasmic domains closely resembles the classic sodium pump beloved of neurophysioloogists everywhere, which pumps sodium ions out of neurons and pumps potassium ions inside, producing the equally beloved membrane potential of neurons.

Look at those structures again. While there are charges on PE, PS (on the phosphate group), these molecules are far larger than the sodium or the potassium ion (easily by a factor of 10). This has long been recognized and is called the ‘giant substrate problem’.

The paper solved the structure of ATP8A2 and used molecular dynamics stimulations to try to understand how it works. What they found is that transmembrane alpha helices 1, 2, 4 and 6 (out of 10) form a water filled cavity, which dissolves the negatively charged phosphate of the head group. What happens to those long hydrocarbon tails? The are left outside the helices in the lipid core of the membrane. It is the charged head groups that are dragged through by the flippase, with the tails wagging along behind them, just like little Bo Peep.

There’s a lot more great chemistry in the paper, particularly how Isoleucine #364 directs the sequential formation and annihilation of the water filled cavities between alpha helices 1, 2, 4 and 6, and how a particular aspartic acid is phosphorylated (by ATP, explaining why the enzyme no longer works in energetically dying cells) changing conformation of all 10 transmembrane helices, so that only one half of the channel is open at a time (either to the inside or the outside).

Go read and enjoy. It’s sad that people who don’t know organic chemistry are cut off from appreciating such elegance. There is more to esthetics than esthetics.

Why drug discovery is so hard: Reason #25 — What if your drug target is really a pointer to the real target?

Any drug safely producing weight loss would be a big (or small) pharma blockbuster. Those finding it should get on the boat to Sweden. Finding a target to attack is the problem. Here’s one way to look. Take lots of fat people, lots of thin people and see what in their genomes differentiates them (assuming anything does). Actually what was done was to look at type II diabetics (non-insulin dependent) the vast majority overweight and controls. The first study involved the genomes of nearly 5,000 diabetics and controls. How did they interrogate the genomes? At the time of the work it was impossible to completely sequence this many genomes.

It’s time to speak of SNPs (single nucleotide polymorphisms). Our genome has 3.2 gigaBases of DNA. With sequencing being what it is, each position has a standard nucleotide at each position (one of A, T, G, or C). If 5% of the population have one of the other 3 at this position you have a SNP. Already 10 years ago, some 7 MILLION SNPs had been found and mapped to the human genome.

The first study found some SNPs associated with obesity in the diabetics. This tells where to look for the gene. A second study with nearly 9,000 diabetics and controls, replicated the first.

Then the monster study, with 39,000 people [ Science vol. 316 pp. 889 - 894 '07 ] found FTO (FaT mass and Obesity associated gene) on chromosome #16. The 16% of Caucasian adults with two copies of the variant SNP in FTO were 1.67 times more likely to be obese. An intense flurry of work showed that the gene coded for an oxidase, using iron and 2 oxo-glutaric acid (alphaKG for you old timers). The enzyme removes methyl groups from the amino group at position #6 of adenine and the 3 position of thymine. Before this time, no one really paid much attention to them. Subsequently we’ve found 6 methyl adenine in a mere 7,676 mRNAs. Just what it does when it’s there, and why the cell wants to remove it is currently being worked out.

Clearly FTO is a great target for an obesity drug. Of course they knocked the gene out in the mouse. The animals were normal at birth, but at 6 weeks weighed 30 – 40% less than normal mice. FTO as a drug target looked even better after this.

It was somewhat surprising that the SNP was in an intron in the gene. This meant that even in the obese the protein product of the FTO gene was the same as in the skinny. Presumably this could mean more FTO, less FTO or a different splice variant. If some of this molecular biology is above your pay grade, the background you need is in 5 posts starting with https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/.

It was somewhat surprising that FTO levels were the same in people with and without the fat SNP. That left splice variants as a possibility.

The denouement came this week [ Nature vol. 507 pp. 309 - 310, 371 - 375 '14 ]. The intron containing the SNP in FTO produces obesity by controlling another gene called IRX3 which is a mere 500,000 nucleotides away. The intron of FTO binds to the promoter of IRX3 turning the gene on resulting in more IRX3. Mice lacking a functional copy of IRX3 have a 25 – 30% lower body mass. As any C programmer would say, FTO is the pointer not the data.

I don’t know if big or small pharma was at work finding inhibitors or enhancers of FTO function, but this paper should have brought them to a screeching halt. The FTO/IRX3 story just shows how many pitfalls there are to finding new drugs, and why the search has shown relatively little success recently. We are trying to alter the function of an incredibly complex system, whose workings we only dimly understand.

Why drug discovery is so hard: Reason #24 — Is the 3′ untranslated region of every mRNA a ceRNA?

We all know what proteins do. They act as enzymes, structural elements of cells, membrane proteins where drugs bind etc. etc. The background the pure chemist needs for what follows can all be found in the category “Molecular Biology Survival Guide.

We also know that that the messenger RNA for any given protein contains a lot more information than that needed to code for the amino acids making up the protein. Forget the introns that are spliced out from the initial transcript. When the mature messenger RNA for a given protein leaves the nucleus for the cytoplasm where the ribosome translates it into protein at either end it contains nucleotides which the ribosome effectively ignores. These are called the untranslated regions (UTRs). The UTRs at the 3′ end of human mRNAs range in length between 60 and 4,000 nucleotides (average 800). It costs energy to store the information for the UTR in DNA, more energy to synthesize the nucleotides which make it up, even more to patch them together to form the UTR, more to package it and move it out of the nucleus etc. etc.

Why bother? Because the 3′ UTR of the mRNA contains a lot of information which tells the cell how much protein to make, how long the mRNA should hang around in the cell (among many other things). A Greek philosopher got here first — “Nature does nothing uselessly” – Aristotle

Those familiar with competitive endogenous RNA (ceRNA) can skip what follows up to the ****

Recall that microRNAs are short (20 something) polynucleotides which bind to the 3′ untranslated region (3′ UTR) of mRNA, and either (1) inhibit its translation into protein (2) cause its degradation. In each case, less of the corresponding protein is made. The microRNA and the appropriate sequence in the 3′ UTR of the mRNA form an RNA-RNA double helix (G on one strand binding to C on the other, etc.). Visualizing such helices is duck soup for a chemist.

Molecular biology is full of such semantic cherry bombs as nonCoding DNA (which meant DNA which didn’t cord for protein), a subset of Junk DNA. Another is the pseudogene — these are genes that look like they should code for protein, except that they don’t because of lack of an initiation codon or a premature termination codon. Except for these differences, they have the nucleotide sequence to code for a known protein. It is estimated that the human genome contains as many pseudogenes (20,000) as it contains true protein coding genes [ Genome Res. vol. 12 pp. 272 - 280 '02 ]. We now know that well over half the genome is transcribed into mRNA, including the pseudogenes.

PTEN (you don’t want to know what it stands for) is a 403 amino acid protein which is one of the most commonly mutated proteins in human cancer. Our genome also contains a pseudogene for it (called PTENP). Interestingly deletion of PTENP (not PTEN) is found in some cancers. However PTENP deletion is associated with decreased amounts of the PTEN protein itself, something you don’t want as PTEN is a tumor suppressor. How PTEN accomplishes this appears to be fairly well known, but is irrelevant here.

Why should loss of PTENP decrease PTEN itself? The reason is because the mRNA made from PTENP, even though it has a premature termination codon, and can’t be made into protein, is just as long, so it also contains the 3′UTR of PTEN. This means PTENP is sopping up microRNAs which would otherwise decrease the level of PTEN. Think of PTENP mRNA as a sponge.

Subtle isn’t it? But there’s far more. At least PTENP mRNA closely resembles the PTEN mRNA. However other mRNAs coding for completely different proteins, also have binding sites in their 3′UTR for the microRNA which binds to the 3UTR of PTEN, resulting in its destruction. So transcription of a completely different gene (the example of ZEB2 is given) can control the abundance of another protein. Essentially its mRNA is acting as a sponge, sopping up the killer microRNA.

It gets worse. Most microRNAs have binding sites on the mRNAs of many different proteins, and PTEN itself has a 3′UTR which binds to 10 different microRNAs.

So here is a completely unexpected mechanism of control of protein levels in the cell. The general term for this is competitive endogenous RNA (ceRNA). Two years ago the number of human microRNAs was thought to be around 1,000 (release 2.0 of miRBase in June ’13 gives the number at 2,555 — this is unlikely to be complete). Unlike protein coding genes, it’s far from obvious how to find them by looking at the sequence of our genome, so there may be quite a few more.

So most microRNAs bind the 3′UTR of more than one protein (the average number is unclear at this point), and most proteins have binding sites for microRNAs in their 3′UTR (again the average number is unclear). What a mess. What subtlety. What an opportunity for the regulation of cellular function. Who is going to be smart enough to figure out a drug which will change this in a way that we want. Absence of evidence of a regulatory mechanism is not evidence of its absence. A little humility is in order.

*****

If this wasn’t a scary enough, consider the following cautionary tale — Nature vol. 505 pp. 212 – 217 ’14. HMGA2 is a protein we thought we understood for the most part. It is found in the nucleus, where it binds to DNA. While it doesn’t transcribe DNA into RNA, it does bind to DNA helping to form a protein complex which binds to DNA which effectively helps promote transcription of certain genes.

Well that’s what the protein does. However the mRNA for the protein uses its 3′ untranslated region (3′UTR) to sop up microRNAs of the let-7 family. The mRNA for HMGA2 is highly overexpressed in human cancer (notably the very common adenocarcinoma of the lung). You can mutate the mRNA for HMGA2 so it doesn’t produce the protein, just by putting a stop codon in it near the 5′ end. Throw the altered mRNA into a tissue culture of an lung adenocarcinoma cell line, and the cell become more proliferative and grows independently of being anchored to the tissue culture plate (e.g. anchorage independence, a biologic marker for cancer).

So what? It means that it is possible that every mRNA for every protein we make is acting as a ceRN A. The authors conclude the paper with ” Such dual-function ceRNA and protein activities necessitate a deeper exploration of the coding genome in biological systems.”

I’ll say. We’re just beginning to scratch the surface. The control mechanisms within the cell continue to amaze (me) by their elegance and subtlety. I doubt highly that we know them all. Yet more reasons that drug discovery is hard — we are mucking about with a system whose workings we only dimly understand.

Ligand binding is an inherent property of proteins — another reason for drug side effects. Reason #23 — Why drug discovery is so hard

Proteins bind ligands with exquisite specificity. Is this due to natural selection, or is the binding of small molecules an inherent property of proteins? If you consider an alpha helix as a rod 11 Angstroms wide with 3.5 Angstroms of height for every turn, you’ll see that it’s impossible to pack such items into a spherical structure without creating 3 dimensional spaces of some sort. Even when you line seven them up parallel to each other there is space between them. In fact such a structure is one of the favorite targets of the medicinal chemist (the 7 transmembrane helix G protein coupled receptor), with a space in the center of the bundle for ligand binding.

A paper in the current (4 June ’13) issue of PNAS (vol. 110 pp. 9344 – 9349) looks at the question in an unusual way. Certainly spaces exist in naturally occurring proteins (e.g. proteins which have been shaped by natural selection). They found that the spaces in them (which they call pockets) fall into about 400 groups.

Then they looked at a library of proteins designed with no other goal in mind, than the formation of a structure which was 1. stable and 2. compact. They found the same 400 pockets. So the spaces are what the late Stephen Jay Gould called a spandrel, something which exists as an accidental byproduct due to the existence of something else.

In the discussion of the paper the authors state “we conclude that ligand-binding promiscuity is likely an inherent feature resulting from the geometric and physical–chemical properties of proteins.”

What does this mean for the medicinal chemist? No matter how selective the drug (ligand) is for the protein its designed to hit, the 20,000 or so proteins making us up are likely to have other places for it to bind. This makes the design of drugs without side effects nearly impossible.

Why Drug Discovery Is So Hard – Reason #22b — Drugs aren’t always doing the things we think they are

One of the things the AIDS virus does to make ‘curing’ AIDS so difficult is hiding. It integrates a DNA copy of its RNA genome into the genome of immune cells (and God knows what else) where it just sits quietly. Activation of the immune cell to fight infection often leads to emergence and production of more virus. One promising mode of therapy is preventing the DNA copy from entering our genome in the first place. The AIDS virus (aka HIV1) produces a protein called Integrase which does that. This has led to the development of integrase inhibitors.

[ Proc. Natl. Acad. Sci. vol. 110 pp. 8327 - 8328, 8690 - 8695 '13 ] THe HIV1 integrase is targeted to sites in chromatin by the host protein LEDGF (Lens Epithelium Derived Growth Factor, aka p75). This work shows that the integrase inhibitors blocking the interaction of LEDGF/p75 (a translational coactivator) with the integrase cause something else — they cause AIDS viruses under construction within the cell. to assemble into a noninfectious structure. This happens long after integration and expression of viral RNA and protein. It is they thought that the integrase inhibitors inappropriately stabilize integrase dimers in the viral assembly process.

Who knew? They weren’t designed to do that.

For two more examples along these lines please see

https://luysii.wordpress.com/2012/03/18/why-drug-discovery-is-so-hard-reason-22-drugs-arent-doing-what-we-think-they-are/

http://luysii.wordpress.com/2011/02/02/medicinal-chemists-do-you-know-where-your-drug-is-and-what-it-is-doing/

Why even great drugs have serious side effects in some patients

Finding good drugs is hard enough, but even great ones are often laid low by unexpected side effects.  This has to do with the tremendous genetic variation in people, about which, more later.  But first a true story from the past.

Neurologists treat epilepsy.  There was a period of 17 years when I was in practice when not a single new  drug against epilepsy (anticonvulsant) was introduced in the USA.  Each new drug would seem to be the answer for a small group of patients that nothing had helped before.

Felbamate (Felbatol) was one such anticonvulsant.  It helped people that nothing else touched. In the year after introduction some 150,000 people were taking it.   I had several very happy patients using Felbatol in the 90s.   1 year later the bomb dropped.  Ten cases of total bone marrow failure (aplastic anemia) had developed in patients taking the drug, a lethal complication.  Every neurologist (and probably every physician) got an urgent letter from the FDA.

Normally, unless there is an allergic reaction, anticonvulsants are never stopped suddenly.  They are tapered over a week or two.  Why?  Basically all anticonvulsants are sedating.  People adapt to this, and it’s like driving a car with one foot on the brake.  Remove the brake and the car shoots forward.  So neurologists all over the country brought patients into the hospital as the drug was immediately stopped.  We were quite worried that the previously uncontrolled seizures would flare.

I had one such patient.  Her family was quite worried about the possible side effects of suddenly stopping Felbamate.  I managed to control myself (hopefully) as I told them there was no side effect worse than death.  As risky as it is, there are still about 12,000 people taking the drug (after being carefully told about the risks) according to Wikipedia.  That’s how good a drug it is.

Why wasn’t this terrible complication picked up in the phase I, II, III studies of Felbamate — 10 cases in 150,000 people is 1/15,000, and no drug study for epilepsy was that large back then.  The incidence of epilepsy in adults is probably around 1%, meaning that some 1,500,000 people would have to be screened to find those 15,000.  So effectively there is no way to find such a rare complication before the drug was released.

A paper last month in Science (vol. 337 pp. 100 – 104 ’12) showed why this sort of thing is almost certain to happen again and again.

DNA sequencing is getting faster and cheaper all the time, so large numbers of people can have parts of their genomes sequenced.  A recent post https://luysii.wordpress.com/2012/07/31/how-badly-are-thy-genomes-oh-humanity/ discussed a paper that  sequenced roughly three quarters of the genes coding for proteins in some 2,439 people — e.g. 15,585 protein coding genes.

The Science paper was more circumspect.  They sequenced ‘only’ 202 genes coding for proteins in 14,002 people.  These genes were chosen quite carefully out of the 20,000 or so protein coding genes we have.  The 202 genes were known drug targets — say the neurotransmitter uptake proteins targeted by SSRIs and tricyclic antidepressants, the dopamine receptors targeted by antipsychotics.  So were the 14,002 people chosen to have their genes sequenced.  There were two ‘normal’ populations samples with 1,322 and 2,059 people each, and 12 populations chosen from people with particular diseases.  Most of these were European (12,514/14,002).

The findings essentially explain why we’ll always have rare side effects.  The total amount of DNA sequenced in each individual was 864,000 positions.  They found ‘rare’ variants (e.g. found in less than 1/200 people) quite commonly.  In fact in the group as a whole such rare variants occurred once every 21 positions in the Europeans.  The variants are the single nucleotide variants (SNVs).  Here’s a recap of just what a SNV is (for more detail see the link given above).  90% of the rare variants had never been seen before, even in these 202 proteins of great biologic and medical interest.

**** Recall that each nucleotide is one of four possibilities (A, T, G, C), and that each 3 nucleotides therefore has 4^3 = 64 possibilities.  61/64 combinations code for amino acids which, since we have only 20 gives a certain redundancy of the famed genetic code.   The other 3 combinations code for no amino acid (usually) and tell the machinery making proteins to stop.  Although crucial to our existence, these are called nonsense codons.

The genetic code is therefore 3fold degenerate (on average).  However, some amino acids are coded for by just 1 combination of 3 nucleotides while others are coded by as many as 6.  So some single nucleotide variants (SNVs) leave the amino acid coded for the same (these are the synonymous SNVs), while others change the amino acid (nonSynonymous SNVs), and possibly protein function.  *****

Certainly, not all of these variants will cause trouble, and our genomes are incredibly fault tolerant, as most of us carry very impaired genes for at least 35 of the proteins (e.g. they are truncated, so not a full protein is made).  Some almost certainly will cause unexpected reactions or side effects from a given drug.  There are so many SNVs out there.

Have Tibetans illuminated a path to the dark matter (of the genome)?

I speak not of the Dalai Lama’s path to enlightenment (despite the title).  Tall people tend to have tall kids. Eye color and hair color is also hereditary to some extent.  Pitched battles have been fought over just how much of intelligence (assuming one can measure it) is heritable.  Now that genome sequencing is approaching a price of $1,000/genome, people have started to look at variants in the genome to help them find the genetic contribution to various diseases, in the hopes of understanding andtreating them better.

Frankly, it’s been pretty much of a bust.  Height is something which is 80% heritable, yet the 20 leading candidate variants picked up by genome wide association studies (GWAS) account for 3% of the variance [ Nature vol. 461 pp. 458 - 459 '09 ].  This has happened again and again particularly with diseases.  A candidate gene (or region of the genome), say for schizophrenia, or autism,  is described in one study, only to be shot down by the next.   This is likely due to the fact that many different genetic defects can be associated with schizophrenia — there are a lot of ways the brain cannot work well.  For details — see http://luysii.wordpress.com/2010/04/25/tolstoy-was-right-about-hereditary-diseases-imagine-that/. or see http://luysii.wordpress.com/2010/07/29/tolstoy-rides-again-autism-spectrum-disorder/.

Typically, even when an  association of a disease with a genetic variant is found, the variant only increases the risk of the disorder by 2% or less.  The bad thing is that when you lump them all of the variants you’ve discovered together (for something like height) and add up the risk, you never account for over 50% of the heredity.  It isn’t for want of looking as by 2010 some 600 human GWAS studies had been published  [ Neuron vol. 68 p. 182 '10 ].  Yet lots of the studies have shown various disease to have a degree of heritability (particularly schizophrenia).  The fact that we’ve been unable to find the DNA variants causing the heritability was totally unexpected.  Like the dark matter in galaxies, which we know is there by the way the stars spin around the galactic center, this missing heritability has been called the  dark matter of the genome.

Which brings us to Proc. Natl. Acad. Sci. vol. 109 pp. 7391 – 7396 ’12.  It concerns an awful disease causing blindness in kids called Leber’s hereditary optic neuropathy.  The ’cause’ has been found. It is a change of 1 base from thymine to cytosine in the gene for a protein (NADH dehydrogenase subunit 1) causing a change at amino acid #30 from tyrosine to histidine.  The mutation is found in mitochondrial DNA not nuclear DNA, making it easier to find (it occurs at position 3394 of the 16,569 nucleotide mitochondrial DNA).

Mitochondria in animal cells, and chloroplasts in plant cells, are remnants of bacteria which moved inside cells as we know them today (rest in peace Lynn Margulis).

Some 25% of Tibetans have the 3394 T–>C mutations, but they see just fine.  It appears to be an adaptation to altitude, because the same mutation is found in nonTibetans on the Indian subcontinent living about 1500 meters (about as high as Denver).  However, if you have the same genetic change living below this altitude you get Lebers.

This is a spectacular demonstration of the influence of environment on heredity.  Granted that the altitude you live at is a fairly impressive environmental change, but it’s at least possible that more subtle changes (temperature, humidity, air conditions etc. etc.) might also influence disease susceptibility to the same genetic variant.  This certainly is one possible explanation for the failure of GWAS to turn up much.  The authors make no mention of this in their paper, so these ideas may actually be (drumroll please) original.

If such environmental influences on the phenotypic expression of genetic changes are common, it might be yet another explanation for why drug discovery is so hard.  Consider CETP (Cholesterol Ester Transfer Protein) and the very expensive failure of drugs inhibiting it. Torcetrapib was associated with increased deaths in a trial of 15,000 people for 18 – 20 months.  Perhaps those dying somehow lived in a different environment.  Perhaps others were actually helped by the drug

The Harvard Chemistry Department Reunion — Part III

Readers of “In The Pipeline” know how grim it is out there for chemists in Big Pharma (not so much in academia, assuming they get in).  I was interested to talk to Harvard chemistry PhDs minted in the past 10 years for their take on this.

First, a caveat.  At my 50th college reunion, our area was well attended by younger graduates (we had a free drinks for all policy).  One of them remarked that we looked pretty good for a bunch of people in their 70′s.  Of course the few hundred or so dead ones weren’t there, and presumably those down on their luck or their finances weren’t there either.  The same probably goes for the PhD’s who decided to attend. (The grad students and post-docs were mostly there for the free eats according to my wife).  So even though it’s likely an unrepresentative sample, I did talk to 15 – 20 relatively recent PhDs.

They all agreed that there was relatively little job security in big pharma.  Did a Harvard degree help?  Most thought not in terms of retention, but a few said that getting a job in big pharma would be next to impossible for a PhD from a program not in the top ten (of which Harvard presumably is a member).

Interestingly, most of the recent PhD’s were in big pharma.  2 of them were patent attorneys.

The grad students agreed that the zeitgeist was that times were bad, and most hoped that things would be better when they finished.  Some said they could take a post-doc to wait things out further.

I did meet a remarkably adventurous individual at the chemistry reunion.  The following day was the big conference for anyone who ever got a Harvard graduate degree (in anything).  His PhD was in economics, and he worked for NASDAQ and with essentially no technical background, he decided to listen in to the chemists.  There were 8 brief presentations by faculty in the afternoon and 7 of them had obvious medical applications.  This impressed the economist, but a fellow grad student of my era (and current department chair) told us that it was nearly impossible to get a grant for anything else.  Showing that it’s an ill wind that blows nobody any good, he said that he picked up an excellent and very experienced NMR jockey for his department from one of Pfizer’s many bloodlettings.  Ordinarily, he’d have little hope of hiring someone of that caliber.

Only one of the 8 presentations had anything to do with synthetic organic chemistry, but it was one that Woodard would have loved, a pseudosymmetric molecule, built from the inside out rather than from the outside in.  Even so, the molecule, a natural product, had medical implications.

Back in the 60s there were plenty of grad students and postdocs from the Indian subcontinent (mainly Sikhs).  This time, just one. The Asian contingent back then (all postdocs) was largely Japanese.  This time mostly Chinese, including 2 grad students from Beijing.

Finally:

Libraries have certainly changed. The library in the Harvard Chemistry Building is a beautiful wood paneled affair with comfortable chairs and big elegant wooden tables. All the returnees on our tour of the department wanted to see it. The graduate student leading our group noted that she almost never goes there, getting what she wants from her computer.

The library was unchanged, except for the fact that there was no one in it about 11AM. The librarian came out of her den anxious to talk to a few living breathing humans, and wouldn’t let us go. Solitary confinement is hell.

Addendum 30 April 11 — On getting to the handouts from the affair, there is an interview with John Lechleiter CEO of Eli Lilly, who started out as a bench chemist after receiving hisPhD in ’80 from guess where in Chemistry and Chemical Biology.  Interesting.

Why drug discovery is so hard: Reason #22 — Drugs aren’t doing what we think they are

50 or so years ago, Cambridge apocrypha had it that Timothy Leary, put LSD into the punch at a party to observe its effects on social behavior (an early double blind experiment).  A student, having imbibed, decided he was God and could walk across Massachusetts avenue with impunity, losing his life in the process, his death being hushed up by Harvard.  It could have been an urban myth, but it was widely prevalent, showing that even the highly intelligent aren’t immune to this sort of thing.

So we all knew (and know) that LSD and other hallucinogens causes a degree of excitement.  We then assume that excitement is synonymous with increased brain activity, correct?  Wrong says [ Proc. Natl. Acad. Sci. vol. 109 pp. 1820 - 1821, 2138 - 2143 '12 ] !

Hallucinogens like LSD and psilocybin bind to lots of neurotransmitter receptors (serotonin alone has at least 14, and this doesn’t count the splice variants).  Still, the best correlation of hallucinogenic activity is with agonist activity at one serotonin subtype, the serotonin 2A receptor (5HT2AR). In man, the psychedelic activity of psilocin is blocked by pretreatment with 5HT2AR antagonists.

There are now noninvasive methods to study brain activity in man.  The most prominent one is called BOLD, and is based on the fact that blood flow increases way past what is needed with increased brain activity.  This was actually noted by Wilder Penfield operating on the brain for epilepsy in the 30s.  When the patient had a seizure on the operating table (they could keep things under control by partially paralyzing the patient with curare) the veins in the area producing the seizure turned red.  Recall that oxygenated blood is red while the deoxygenated blood in veins is darker and somewhat blue.  This implied that more blood was getting to the convulsing area than it could use.

BOLD depends on slight differences in the way oxygenated hemoglobin and deoxygenated hemoglobin interact with the magnetic field used in magnetic resonance imaging (MRI).  The technique has had a rather checkered history, because very small differences must  be measured, and there is lots of manipulation of the raw data (never seen in papers) to be done.  10 years ago functional magnetic imaging (fMRI) was called pseudocolor phrenology.

Another newer technique called arterial spin labeling perfusion also measures blood flow.

Both techniques were used on 15 ‘experienced’ hallucinogen users, who received either placebo or psilocin (the active metabolite  of psyilocybin) IV.  The druggies also rated the intensity of their experiences.

The surprising finding is that decreases in blood flow (implying decreased neuronal activity) occured in areas of the brain ‘implicated’ (e.g. not proven) in psychedelic drug actions.  Even more interesting is that the intensity of the experience  correlated with decrements in blood flow.

This constitutes yet another example of why drug discovery is hard.  Even when we know the observable effects of a given drug, our theories of how the drug does what it does, can be widely off base — in this case bass ackwards.  So if you were screening for an antihallucinogen, the incorrect theory would lead you seriously astray.  This is why big pharma is dropping research on CNS drugs — they haven’t had much success, and the theories to guide them may be flat out wrong.

Why drug discovery is so hard: Reason #21 — RNA sequences won’t help you determine function

We are just beginning to understand all the things RNA does in the cell, despite its importance obvious to all for half a century (think messenger RNA which goes back that far).  This means that RNA is likely to be a target of useful drugs.  Posts #4, #11 and #20 concern some of the more newly discovered effects of RNA in the cell.

While we’re still discovering proteins with no obvious resemblance  in their amino acid sequence to known proteins, most of them do have some resemblance we’ve seen before.  So if we see a kinase-like domain, or a group of 7 rather hydrophobic sequences, we have a leg up on what that protein is actually doing.

A similar attack (comparing sequences to RNAs of known function) should help us figure out what some of the RNAs in the cell not coding for protein are actually doing.  If you see a mistyke in this sentence, you still probably know what I meant (e.g. how that word is meant to function in the sentence).  That’s the hope underlying the technique anyway

Recent work in the zebrafish [ Cell vol. 147 pp. 1537 - 1550 '11 ] shows that this isn’t very likely in the RNA world. For some background on large intervening nonCoding RNAs (lincRNAs — aka lncRNAs) see http://luysii.wordpress.com/2011/03/02/we-dont-know-all-the-players-which-is-why-finding-good-drugs-is-so-hard/.  The zebrafish has become a plaything of embryologists (because it is transparent, and because like most fish (except sharks) it is a vertebrate.

At any rate the work found some 550 distinct lincRNAs in the zebrafish.  But only 29 had detectable sequence similarity with lincRNAs in mammals (which are just as numerous).  Even though chromosomes have been scrambled many times over geologic time, many genes near each other in the zebrafish are near each other in humans as well (the term for this is synteny).  This means one can look at DNA to see where the lincRNA is binding in two organisms, and infer that they’re doing something similar physiologically if they are binding to a syntenic site.

So they did this and found some  lincRNAs with almost no sequence similarity to each other binding to identical syntenic sites in man and zebrafish.  Next they used antisense reagents targeting the small regions of the lincRNAs conserved between us and fish and produced developmental defects (in the fish)  Amazingly, despite very little sequence similarity, human orthologs (determined by synteny) could prevent the embryological defects.

So in this case at least, and probably more generally, we’re not going to be able to look at the sequence of lincRNAs (or the many other types of non messenger RNAs present in the cell) and infer what they are doing.  This will make drug discovery in this area even harder.

 

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