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.

 

Why drug discovery is so hard: reason #20 — competitive endogenous RNAs

The chemist will appreciate le Chatelier’s principle in action in what follows.  We are far from knowing all the players controlling cellular behavior.  So how in the world will we find drugs to change cellular behavior when we don’t know all the things affecting it.  The prveiously unknown cellular player to enter the lists are competitive endogenous RNAs (ceRNAs).  For details see Cell vol. 147 pp. 344 – 357, 382 – 395 ’11.   The background the pure chemist needs for what follows can all be found in the category “Molecular Biology Survival Guide.

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.  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.

9 Feb ’12 — my wife just sent me the following quote “Nature does nothing uselessly”  – Aristotle

For a list of the other 19 reasons and links please see https://luysii.wordpress.com/2011/11/21/a-new-category/

Why drug discovery is so hard reason #19 — ribosomal profiling

Reason #19 why drug discovery is so hard — we are far from knowing all the players in the cell.  (For the first 18 see https://luysii.wordpress.com/2011/11/21/a-new-category/). Here’s a shocker showing how little we know about proteins.  You’d think that, by now, we’d know just about everything about them — how they are made (including splicing variants) from the same gene.  How they are destroyed.  But we don’t.

[ Cell vol. 147 pp. 789 - 802 '11 ] Is an incredible paper, showing that of 5000 protein coding genes in mouse embryonic stem cells, translation of the mRNA begins at 13,454 initiation sites, with 65% of the mRNAs having more than one site where translation begins (start sites), 16% had more than 4 start sites.   All the background a pure chemist needs to understand all this is in the Category “Molecular Biology Survival Guide for Chemists”.

The start sites could be within the coding section of the gene, giving amino truncated products, or upstream (5′ to) the coding section giving proteins with an amino terminal extensions.  A recent paper [ Proc. Natl. Acad. Sci. vol. 109 pp. 197 - 202 '12 ] gives an example of just how important an amino truncated protein can be.  Checkpoint kinase 1 (Chk1) is a crucial regulator of the cell cycle, preventing mitosis from occuring in cells with damaged DNA.  An amino terminally truncated variant (due to alternative splicing, not different initiation) of Chk1 binds Chk1 and represses its activity, letting the cell cycle proceed.  DNA damage results (by a complicated mechanism) in phosphorylation of Chk1, relieving the inhibition by the amino truncated variant, and allowing Chk1 to stop the cell cycle.

The authors also found a class of short RNAs coding for multiple small proteins (they call them sprcRNAs — short polycistronic ribosome associated coding RNAs.)  These short proteins (or peptides if you wish — when a peptide is long enough to be called a protein is a matter of taste) weren’t known.

So now we have a whole bunch of new proteins in the cell, most related to known ones.  Could the drugs we have be affecting the new ones rather than what we’ve thought was their actual target?

The way this was found is almost as interesting as what they found.  It involves a technique called ribosomal profiling.  For background on the ribosome see https://luysii.wordpress.com/2012/01/09/molecular-biology-survival-guide-for-chemists-v-the-ribosome/.

The ribosome is large — a roughly spherical blob 250 – 300 Angstroms in diameter, with the active site of protein synthesis nearly in the center of the molecule.  The messenger RNA within an active ribosome is protected from enzymes which can destroy it (nucleases). So chop up all the RNA in the cell, disassemble the ribosome, then use reverse transcriptase to make a DNA copy of the messenger RNA that’s left, and sequence all of it (using Illumina deep sequencing).

By using inhibitors of either translation initiation (harringtonine) or progression, it is possible to find translation start sites, along with their distribution.  You can also find out just how fast ribosomes are translating mRNA (about 6 amino acids/second in this system).

A new category

On any given week, readers of Derek Lowe’s “In the Pipeline” blog are likely to find posts detailing new layoffs, downsizing, shutting down of whole research groups by big pharma.  Why is this happening?  The short answer is that useful new drugs aren’t being found.  The long answer is that to find a drug, you ought to know what it is that you want the drug to do.  The longer answer is that we don’t understand what’s going on inside cells and organisms well enough to even know what we want a drug to target.

I’ve always been impressed with how little we know about what’s going inside our cells, let alone what’s going on between them, despite unbridled biochemical and molecular biological hubris to the contrary.  Remember nonCoding RNA — it codes all right, just not for protein.  Remember Junk DNA — since we didn’t know what it did, it received the name, despite the fact that over 50% and probably all our DNA is transcribed into RNA.

Over the years, I’ve written lots of posts about our ignorance about these matters.  This is exactly why finding new and better drugs is so hard.  I’ve collected them into a new category “Aargh! Big pharma sheds chemists.  Why?”  They all assume that you know your chemistry (as do all the posts on this blog).  How much molecular and cellular biology readers know is unclear.  However all the background a chemist should need to read this is found in another category “Molecular Biology Survival Guide (for the chemist)” which contains 5 background articles.

Here’s a brief heads up about what each article in the category contains and a link to it.  Note that they don’t appear in an particular logical sequence, just in the order they were written, newest first.  More will be added.

1. https://luysii.wordpress.com/2011/11/20/life-may-not-be-like-a-well-but-control-of-events-in-the-cell-is-like-a-box-spring-mattress/ concerns feedback, and how it obliterates the simple minded notion of control.

2. https://luysii.wordpress.com/2011/10/20/more-troubles-for-the-poor-pharmacologist/ — concerns Miraculin, a molecule which acts as an agonist in some situations and an antagonist in others.  How many of our drugs might act like this.

3. https://luysii.wordpress.com/2011/07/17/weve-found-the-mutation-causing-your-disease-not-so-fast-says-this-paper/ — Very disturbing work — showing that mutations thought to cause epilepsy don’t always do so (depending on genetic background).  By implication this is true for other diseases as well, and makes it unlikely that we will ever uncover ‘the’ cause of many diseases.

4. https://luysii.wordpress.com/2011/03/02/we-dont-know-all-the-players-which-is-why-finding-good-drugs-is-so-hard/ — This is the real reason drug discovery is so hard.  This work describes some newly found players which determine how much protein actually gets made by a given cell.

5. https://luysii.wordpress.com/2011/02/02/medicinal-chemists-do-you-know-where-your-drug-is-and-what-it-is-doing/  This describes  a paper which shows that amitryptyline, a drug we thought we understood, has a radically different action, and in cystic fibrosis of all things.  Hint:  it involves ceramide a newly discovered second messenger inside the cell.

6. https://luysii.wordpress.com/2010/12/29/tidings-of-great-joy-for-synthetic-organic-chemists-anyway/ — Shows had all sorts of small (under 1000 Dalton) molecules of intermediary metabolism bind to cellular proteins (not necessarily on their active sites) and affect their function.  Checking this out gives us a whole new group of druggable targets — again assuming we know everything the protein is doing.

7. https://luysii.wordpress.com/2010/11/14/protein-mutation-the-view-from-the-bedside-and-the-lab/ — discusses two different views of proteins — that of the clinician, for whom even a single mutation causes disease, and that of the population geneticist who shows that in a given population each  proteins contain tons of them which have no significant effects at all.

8. https://luysii.wordpress.com/2010/09/08/positive-allosteric-modifiers-exciting-and-humbling/ — discusses allosteric modifiers of protein function, and how hard it will be to find them, since our understanding of all the conformations a protein can assume and how to shift from one to the other is so sketchy.

9. https://luysii.wordpress.com/2010/08/12/2-new-kinds-of-genes-who-knew-we-didnt/  One is about genes coding for multiple very small peptides — which essentially have been ignored, since one way of looking for protein coding genes, it to search DNA for sequences lacking stop codons.

10. https://luysii.wordpress.com/2010/07/29/tolstoy-rides-again-autism-spectrum-disorder/ — Concerns this nebulous disease and the fact that most kids with it have a genetic defect peculiar to that family.  Given that, how do you find a single drug to treat it (assuming treatment is needed — something contentious at this point).

11. https://luysii.wordpress.com/2010/07/14/junk-dna-that-isnt-and-why-chemistry-isnt-enough/ How a pseudogene for a tumor suppressor determines the actual level of the tumor suppressor.  The first inkling of what turns out to be a year later a very widespread phenomenon (ceRNA –aka competitive endogenous RNA)  – a future post will discuss how this introduces a whole new layer of control in the cell.

12. https://luysii.wordpress.com/2010/07/01/why-linearity-is-not-enough/  Essentially #1 all over again — I plagiarized myself.

13. https://luysii.wordpress.com/2010/06/16/bad-news-on-the-cancer-front/  A mere 50,000 single nucleotide changes between normal tissue and one highly sequenced adenocarcinoma of the lung.  Doubtless, not all are causative, and doubtless that different tumors will have different mutations, but it’s hard to see a generalized drug for cancer.

14. https://luysii.wordpress.com/2010/02/17/organic-chemistry-under-assault-ii/  Why even knowing 6 genetic causes of a well understood disease (Parkinsonism — due to a deficiency of dopamine) doesn’t really give you a clue as to a way to treat it.

15. https://luysii.wordpress.com/2010/02/14/organic-chemistry-under-assault/ A mystery — figuring out how a well known toxin does what it does

16. https://luysii.wordpress.com/2009/11/09/some-humility-is-in-order/  Despite having studied hemoglobin and sickle hemoglobin out the gazoo for 60 years, we still don’t have a small molecule inhibitor of hemoglobin sickling, showing how paltry our understanding of protein dynamics actually is.

17. https://luysii.wordpress.com/2009/10/20/vegetarians-are-wimps-science-now-tells-us-why/  A fun post showing how even something ‘natural’ has unexpected effects inside our bodies.

18. https://luysii.wordpress.com/2009/09/25/are-biochemists-looking-under-the-lamppost/ — Explores the possibility that side chains of proteins can chemically interact with each other to form new and exciting compounds.  The example is green fluorescent proteins, but see a recent comment, about 5 home grown cofactors that proteins make for themselves.  Iss more of this going on in our cells.  We won’t know until we look.

19.  Added 12 Jan ’12 – https://luysii.wordpress.com/2012/01/12/why-drug-discovery-is-so-hard-reason-19-ribosomal-profiling/.  More than half of 5000 proteins studied in a mouse embryonic stem cell have multiple variants due to synthesis initiation at multiple sites on the mRNA.  This is distinct from splice variants (which also occur).

2o. Added 29 Jan ’12 – http://luysii.wordpress.com/2012/01/29/why-drug-discovery-is-so-hard-reason-20-competitive-endogenous-rnas/.  How mRNAs for one protein act as sponges, sopping up microRNAs which might otherwise be decreasing the levels of another protein.

21. Added 7 Mar ’12  – https://luysii.wordpress.com/2012/03/07/why-drug-discovery-is-so-hard-reason-21-rna-sequences-wont-help-you-determine-function/.  RNAs with almost no sequence similarity can have very similar functions.  Using human lincRNAs to cure developmental defects in Zebrafish

22. Added 18 Mar ’12 – https://luysii.wordpress.com/2012/03/18/why-drug-discovery-is-so-hard-reason-22-drugs-arent-doing-what-we-think-they-are/.  Hallucinogens don’t excite your brain, they put parts of it to sleep.  Who knew?