The peculiar story of PTC124

Several years ago a very, very cool drug was discovered - PTC124. This one was inhibiting NMD (nonsense mediated mRNA decay), and since NMD is implicated in several deseases, PTC124 was of great interest. I have discussed this story here.

Well, it seems that there is a twist in this story. PTC124 was discovered using firefly luciferase (FLuc) as a reporter. And now is seems that it rather than acting on the NMD level, PTC124 interacts with the FLuc itself and modulates its activity!

What's especially interesting, is that work on PTC124 continues, and many more papers are getting published, not necessarily using FLuc... So what was it? Just a fluke?

...this story is somehow similar to that one.

References:

Auld, D. S., Lovell, S., Thorne, N., Lea, W. A., Maloney, D. J., Shen, M., Rai, G., et al. (2010). Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124 Proceedings of the National Academy of Sciences of the United States of America, 107(11), 4878–4883. PIMD 20194791

Auld, D. S., Thorne, N., Maguire, W. F., & Inglese, J. (2009). Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression Proceedings of the National Academy of Sciences of the United States of America, 106(9), 3585–3590. PIMD 19208811

Abort! Abort!

Sometimes things go so wrong that it is just easier to start all over again. Bacteria have these situations too - it's not just us, humans! - and the central dogma of molecular biology (DNA replication, transcription and translation) is no exception.

In essence all the three steps of the central dogma share the very same basic topology: there is a message that gets read, there is a tool that reads it and there is a product. It looks like so:

Say, in the case of translation mRNA (the message) gets read by the ribosome (the tool) and protein (the product) is produced. And when things go wrong, there are three things you can abort: the message, the product and the tool. Let us see how it goes.

Replication

DNA polymerase (the tool) reads the DNA (the message) and produces DNA (the product). And when wrong nucleotide is incorporated, DNA polymerase can excise it and continue making the product using so called  proof-reading mechanism. Complete abortion of the growing DNA strand does not happen, and if mistake is done, it is done and you live with it. Surely, there are ways to fix it later (recombination and so on), but not on the spot, during the replication.

Transcription


RNA polymerases can proof-read too. However, many more things can be done. Special set of transcription factors, called GreA and GreB in bacteria and TFSII in eucaryotes, can activate intrinsic hydrolytic activity of the RNA polymerase and cleave off the growing product. Stalled complex is resolved and now we can try again.

Translation


First, there is a proof-reading mechanism, but rather than cutting off the mis-incorporated letter, GTP is hydrolyzed by GTPase EF-Tu which brings the aminoacyl-tRNA.

Second, if the mistake is done, and wrong amino acid was incorporated after all, bacterial class-1 release factors RF1 and RF2 become prone to peptide-release independent of the stop codon, thus removing the product (the growing protein chain). In mitochondria translational system is bacterial-like, but much more insane, and several (as many as 4 in humans!) class-1 release factors are present, with some of them lacking the ability to recognize the stop codon at all (ICT1, for example), and these resolve stalled ribosomal complexes by cutting off the peptide as well as their bacterial counterparts.

Third, bacterial toxins such RelE and the like are resolving ribosomal complexes by cutting the message (mRNA) rather than the product. Calling them toxins is rather misguiding, they are more of the rescue factors.

And lastly, eukaryotic translational factors Dom34 and Hbs1 (related to termination factors eRF1 and eRF3) are splitting the stalled ribosome into subunits, re-setting the tool.

So it seems the further we move from the DNA, the more dispensable the production complex becomes: in the case of DNA polymerases we have only proof-reading, RNA polymerases can do that and also cleave the message, and translational machinery can do it all: cutting the message (RelE), cutting the product (release factors) and resetting the tool by splitting the ribosome into subunits (Dom34 and Hbs).

References:

Borukhov S, Sagitov V, & Goldfarb A (1993). Transcript cleavage factors from E. coli. Cell, 72 (3), 459-66 PMID: 8431948

Toulmé F, Mosrin-Huaman C, Sparkowski J, Das A, Leng M, & Rahmouni AR (2000). GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming. The EMBO journal, 19 (24), 6853-9 PMID: 11118220

Pedersen K, Zavialov AV, Pavlov MY, Elf J, Gerdes K, & Ehrenberg M (2003). The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site. Cell, 112 (1), 131-40 PMID: 12526800

Orlova M, Newlands J, Das A, Goldfarb A, & Borukhov S (1995). Intrinsic transcript cleavage activity of RNA polymerase. Proceedings of the National Academy of Sciences of the United States of America, 92 (10), 4596-600 PMID: 7538676

Kassavetis GA, & Geiduschek EP (1993). RNA polymerase marching backward. Science (New York, N.Y.), 259 (5097), 944-5 PMID: 7679800

Richter R, Rorbach J, Pajak A, Smith PM, Wessels HJ, Huynen MA, Smeitink JA, Lightowlers RN, & Chrzanowska-Lightowlers ZM (2010). A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome. The EMBO journal, 29 (6), 1116-25 PMID: 20186120

Shoemaker CJ, Eyler DE, & Green R (2010). Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay. Science (New York, N.Y.), 330 (6002), 369-72 PMID: 20947765

Atkinson GC, Baldauf SL, & Hauryliuk V (2008). Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components. BMC evolutionary biology, 8 PMID: 18947425

Antonicka H, Ostergaard E, Sasarman F, Weraarpachai W, Wibrand F, Pedersen AM, Rodenburg RJ, van der Knaap MS, Smeitink JA, Chrzanowska-Lightowlers ZM, & Shoubridge EA (2010). Mutations in C12orf65 in patients with encephalomyopathy and a mitochondrial translation defect. American journal of human genetics, 87 (1), 115-22 PMID: 20598281

Zaher HS, & Green R (2009). Quality control by the ribosome following peptide bond formation. Nature, 457 (7226), 161-6 PMID: 19092806

specific inhibition of nonsense-mediated mRNA decay by small molecule

The last step of protein synthesis is called translation termination. During this step the stop codon is recognized by the protein factor called "release factor" and finished protein is cleaved off the tRNA. Mutations which cause premature termination (nonsense mutations) lead to shortened protein which is usually defective, and in order to avoid accumulation of these proteins such mRNA are recognized by nonsense mediated mRNA decay (NMD) machinery and degraded.

This is usually a good thing, but imagine that the gene which has this nonsense mutation is very important for survival of the cell, and imagine that the truncated version is still somewhat functional. In this case it would be beneficial to produce it even if it is somewhat compromised: something is better than nothing.

Therefore in some cases NMD is actually a cause of a desease, such as Duchenne muscular dystrophy and cystic fibrosis - over-zealous NMD removes all the damaged mRNAs and no protein product is produced. In this case suppression of NMD is needed.

Treatment with antibiotic gentamicin wich causes error-prone translation is a solution, but unfortunately gentamicin is not specific for NMD and causes errors on all the steps of translation. Specific inhibition of NMD was sought after for many years, and finally a smal molecule which inhibits it was discovered.

The molecule is called PTC124, and we know that it does the trick (inhibits NMD) and works well against the DMD. It was discovered in 2007, and by now we still don't know to what it binds (ribosome? NMD factors?) and what is the mechanism. The reason for that is that it is extremely complicated to construct an in vitro system for studying PTC124 (you will need purified translational and NMD components, and that is a lot of components). So for now we have no idea how it works, but it does!

Update: ...or may be it doesn't!

References:

Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P, Paushkin S, Patel M, Trotta CR, Hwang S, Wilde RG, Karp G, Takasugi J, Chen G, Jones S, Ren H, Moon YC, Corson D, Turpoff AA, Campbell JA, Conn MM, Khan A, Almstead NG, Hedrick J, Mollin A, Risher N, Weetall M, Yeh S, Branstrom AA, Colacino JM, Babiak J, Ju WD, Hirawat S, Northcutt VJ, Miller LL, Spatrick P, He F, Kawana M, Feng H, Jacobson A, Peltz SW, & Sweeney HL (2007). PTC124 targets genetic disorders caused by nonsense mutations. Nature, 447 (7140), 87-91 PMID: 17450125

Manuvakhova M, Keeling K, & Bedwell DM (2000). Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system. RNA (New York, N.Y.), 6 (7), 1044-55 PMID: 10917599