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Artikel in Cell:

Small molecule targeting of transcription-replication conflict for selective chemotherapy

The Independent:

Scientists develop pill that destroys solid cancer tumours in early research

Pharma boy Derek Lowe in Science:

A New Mode of Cancer Treatment

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2 Aug 2023
By Derek Lowe

Let’s talk about a really interesting new paper in Cell Chemical Biology, which is also getting some pickup in the wider press. It’s about a molecule designated AOH1996, which seems to have a unique mode of action in tumor cells, one that might make it more more selective for those as compared to normal ones.

The key target here is a protein called PCNA (from its old name of “proliferating cell nuclear antigen”). If you look at that link, you’ll see its very appealing structure in the cell - three PCNA proteins come together and encircle a DNA strand, and the outside of this ring has binding spots for a lot of other proteins involved in replication (such as DNA polymerase epsilon), in DNA repair (ditto), in things like making epigenetic signaling marks on DNA-histone complexes, and in chromatin remodeling in general. That makes it sound like a pretty important protein, and so it is - PCNA is absolutely essential in replication, and it was first noticed as something that got expressed heavily in cell nuclei during DNA synthesis as a runup to cell division.

And this brings up a fundamental conflict in the life of a cell. DNA is constantly being transcribed (read off into RNA for the later translation to proteins as well as other uses), and there are huge numbers of proteins and processes involved in that. Replication for cell division is a whole other process, one that also involves a huge number of proteins descending onto the DNA (every single bit of it, as the entire genome is copied for the daughter cell to come). You can imagine that these two mighty networks of DNA-associated machinery might bang into each other from time to time, and so they do - this review, entitled “Transcription as a Threat to Genomic Integrity”, will take you through a lot of this. From a strict cell-replication standpoint, it would be better if the DNA was locked up in a vault where nothing could mess with it until it came time to make a copy, but that’s impossible: day-to-day (and minute-to-minute) cell biology requires constant messing around with that same DNA. Winding and unwinding around histone proteins, huge transcriptional complexes landing on particular sequences and starting the synthesis of mRNA molecules, transcription factors sitting around on their preferred sequences and blocking/promoting such readouts - the activity around a cell’s DNA makes beehives look pretty laid-back.

Cancer cells, many of whom are constantly replicating, are under particular stress in this regard, and a lot of chemotherapy drugs are specifically interfering with replication and DNA repair pathways. The discovery of a “cancer-associated PCNA” (caPCNA) isoform made that a particularly interesting target. Back about ten years ago there were several reports of small molecules and cell-penetrating peptides as PCNA ligands, and these continue to show up in the literature (although as far as I know, none of them have progressed further). But a peptide in this area has made it through Phase I in the clinic. PCNA continues to be targeted whenever some new mode of action comes along that might make it actionable.

The current molecule is a traditional direct small molecule binder that is selective for caPCNA over the regular type, which is a very attractive advantage to explore. The team behind it has been working on it for several years now to validate that mechanism, and the new paper linked first above is their report of going all the way into animal models. AOH1996 is a very unremarkable-looking molecule - to be honest, it looks like the sort of stuff that you used to see in old combinatorial chemistry libraries in the late 90s and early 2000s, a couple of aryl-rich groups strung together with amide bonds. It’s certainly not going to be the most soluble stuff in the world, but they seem to have been able to formulate it. But I’m definitely not going to make fun of any chemical structure that works!

Mechanistically, it binds to a particular region that’s different in caPCNA (as shown by some very nice structural biology results), and this binding stabilizes the protein’s interaction with the largest subunit (RPB1) of RNA polymerase II - in fact, it stabilizes it so thoroughly that the RPB1 protein gets targeted for degradation by the cellular housekeeping machinery! Binding of AOH1996 also weakens its association with actively-transcribed chromatin regions, and this causes accumulation of double-stranded DNA breaks DSBs), which is just the kind of thing you want to mess up a cancer cell’s replication process (and just the kind of thing you don’t want happening to other cell types). This is an example of deliberately amplifying the transcription/replication conflict - things get so messed up that the transcriptional complex has to be cleared.

The new paper shows preclinical toxicity testing in two species (mice and dogs), which is what you need to get to human trials. It seems to pass those very well, with no signs of trouble at 6x the effective dose in either species. And if you were throwing DSBs all over the place in normal tissues, believe me, you’d see tox. It is clean in an Ames test, for example. As for efficacy, in cell assays the concentration needed for 50% growth inhibition across 70 different cancer cell lines averaged around 300nM, while it showed no toxic effects on various non-cancer lines up to 10 micromolar (at least a 30x window). The affected cells show cell-cycle arrest, replication stress, apoptosis, and so on. And application of AOH1996 along with other known chemotherapy agents made the cells much more sensitive to those, presumably because they couldn’t deal with those on top of the problems that AOH1996 was already causing.

It also shows growth arrest in xenograft tumors in mouse models, with a no-effect dose at least six times its effective dose, and combination therapy with a topoisomerase inhibitor showed even more significant effects. The compound has entered a Phase I trial in humans on the basis of the above data, and I very much look forward to seeing it advance to Phase II, where it will doubtless be used in combination with several existing therapies. I hope that human cancers will prove vulnerable to this new mode of attack in the clinic, and that they are not able to mutate around it with new forms of caPCNA too quickly, either. The comparison with the peptide agent mentioned above will be especially interesting, too. There’s only one way to find out - good luck to everyone involved!