1 research outputs found
p53 transcriptional activity as a tool to uncover novel and diverse druggable targets in cancer
The transcription factor p53 is one of the most studied tumour suppressors with over 90 000
publications in PubMed referring to the protein. It is also the most frequently mutated gene
across all cancer types with around 50% of cancers presenting as mutant p53, and when it is
not mutated, it is frequently inactivated to circumvent its tumour suppressor function.
Therapeutic targeting of both mutant and wild-type p53 has been a key focus ever since its
first discovery as “the guardian of the genome”. For our drug development programme, we
have focused on visualising the induction of p53 transcriptional activity as a readout for a
desirable phenotype. This screen used two stably transfected reporter cell lines, the T22
murine fibroblasts, and the ARN8 human melanoma cell line. Using this forward chemical
genetic approach, we have entered into our drug development programme in a target-blind
manner.
For Paper I we screened 30 000 compounds in both T22 and ARN8 cells and selected those
that were capable of increasing p53 transcriptional activity in the ARN8 tumour cells, but not
in the T22 murine fibroblasts. We selected a compound from the hits that had a drug-like
structure as well as possessing a chiral centre and christened it HZ00. HZ00 was found to
induce p53 protein in a dose-dependent manner, selectively kill tumour cells whilst inducing
a reversible G1 arrest in normal human dermal fibroblasts (HNDFs), and increase p53
synthesis at early timepoints without stabilising the protein or increasing levels of p53
mRNA. HZ00 also synergised with the inhibitor of p53 degradation, nutlin 3, both in vitro
and in vivo in a tumour xenograft model. Following target deconvolution using a knowledgebased
approach we identified DHODH, a key enzyme in the de novo pyrimidine nucleotide
synthesis pathway, as the target of HZ00. At this point we re-screened 30 000 compounds in
ARN8 cells that were previously screened in the T22 cell line for another study. We found
that those that were able to activate p53 in ARN8 cells also largely inhibited DHODH. This
yielded 12 other chemotypes capable of inhibiting DHODH. At this point we tested HZ00
analogues and identified a much more potent compound we named HZ05. HZ05
phenocopied HZ00 and demonstrated enantiomer-selective inhibition of DHODH with (R)-
HZ05 inhibiting DHODH with an IC50 of 11 nM. We obtained a crystal structure of (R)-
HZ05 in complex with DHODH and found that it occupied the same quinone tunnel as the
known inhibitors brequinar and teriflunomide (A77 1726). HZ05 caused a number of tumour
cells to accumulate in S-phase. We found that a slower cycling cell line, U2OS, required pretreatment
with HZ05 to accumulate cells in S-phase prior to treatment with nutlin 3a to
achieve tumour cell kill, as co-treatment resulted in G1 arrest. We therefore theorised that
accumulating cells in S-phase with high levels of p53 predisposed them to cell kill upon
application of a blocker of p53 degradation.
The first sets of compounds found back in 2008 by the Laín laboratory were the tenovins.
Tenovin 1 was the first compound identified from the screen, which used the T22 murine
fibroblasts to establish its ability to activate p53 transcriptional activity in the reporter assay.
Tenovin 1 was, however, not particularly soluble and therefore a more soluble analogue
called tenovin 6 was synthesised. Tenovin 6 elicited many of the same cellular phenotypes as
tenovin 1, and therefore target identification was conducted using tenovin 6. Tenovin 6 was
subsequently identified as an inhibitor of SirT1 and SirT2 in a yeast genetic screen,
biochemical assays and further target validation in mammalian cells. Tenovin 1 and 6
displayed a very similar profile – they both induced p53 transcriptional activity and both
increased acetylation of both p53 and tubulin. This is where the similarity ends, however, as
it was discovered, through extensive structure-activity relationship studies, that the targeting
profiles of both molecules was markedly different.
In Paper II we built upon previous studies that identified tenovin 6 as a compound capable of
inhibiting autophagy. In this paper we conducted structure-activity relationships using
tenovin analogues to understand the mechanism by which tenovins affect autophagy. We
confirmed that tenovins capable of perturbing autophagy do so by inhibition of autophagic
flux, in a similar manner to chloroquine, by raising the pH of lysosomes. We also isolated the
portion of the molecule, a tertiary amine at the end of an aliphatic chain, as the reason for
blockage of autophagic flux. Finally, we found that blockage of autophagic flux by tenovins
is required to eliminate tumour cells in culture and that this blockage of autophagy is capable
of killing mutant B-Raf tumour cells arrested in G1 by vemurafenib treatment.
In Paper III we further explored the targeting profile of the tenovins and tested whether
tenovins were capable of inhibiting DHODH. We found that tenovins 1 and 6 were capable
of inhibiting DHODH at 113 nM and 500 nM respectively. We also conducted a thermal shift
assay and identified tenovins 1, 6 and 39OH as being capable of interacting with DHODH in
vitro. We then obtained a crystal structure of tenovin 6 occupying the same quinone tunnel as
HZ05, brequinar and teriflunomide. Phenotypically, tenovin 1 and 33 had their ability to
induce p53 transcriptional activity ablated upon addition of either uridine or orotate, but not
dihydroorotate, whilst tenovin 6 had its ability to induce p53 transcriptional activity partially
prevented by addition of uridine or orotate. Tenovin 39 and 39OH displayed no difference
upon supplementation. Tenovin 1 and 33 also had their growth inhibitory effect markedly
reduced upon orotate or uridine supplementation, but no other tenovin, including 6, showed
any effect of supplementation. We also discovered another target of the tenovins – the ability
to inhibit nucleoside uptake. We discovered that uridine uptake was blocked by tenovin 6, 33,
39, 39OH and 50. This paper, therefore, highlights the shifting targeting profile of the
tenovins due to small molecular changes and that a phenotypic readout may remain static
even as the targeting profile changes, as well as highlighting both the benefits and cautions of
targeting multiple disparate targets in cells.
Unlike our other projects, Paper IV focused on understanding the structure and function of
DHODH. We studied a purified DHODH lacking the transmembrane domain using native
protein nano-electrospray mass spectrometry (nESI-MS). Firstly, we identified MS
conditions that allowed for the DHODH to spray and isolated a high m/z range that
corresponded to the molecular weight of the enzyme plus the bound FMN cofactor. Ion mass
spectrometry was conducted to differentiate between the holo- and apo- DHODH, with the
holo-DHODH corresponding to a compact formation suggesting that folded DHODH with
FMN present can be preserved in the gas phase. We next incubated lipids that constitute the
human mitochondrial membrane with DHODH and analysed the interaction in the gas phase.
Complexes with both PE and CDL were evident, but complexes with PC were not easily
detected. The next finding was that an intact protein-cofactor complex was required for the
DHODH inhibitor, brequinar, to bind thus confirming that brequinar binding to DHODH is
not random, but requires properly structured DHODH. Finally, MD simulations were
conducted using both full length and truncated protein associated with a model PE bilayer.
These models established that DHODH sits on the surface of the lipid bilayer loosely and is
anchored in place by the transmembrane helix and this anchorage holds DHODH in the
correct orientation to allow insertion of coenzyme-Q10 into the quinone tunnel of DHODH