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

Combinatorial expression of GPCR isoforms affects signalling and drug responses

G-protein-coupled receptors (GPCRs) are membrane proteins that modulate physiology across human tissues in response to extracellular signals. GPCR-mediated signalling can differ because of changes in the sequence1,2 or expression3 of the receptors, leading to signalling bias when comparing diverse physiological systems4. An underexplored source of such bias is the generation of functionally diverse GPCR isoforms with different patterns of expression across different tissues. Here we integrate data from human tissue-level transcriptomes, GPCR sequences and structures, proteomics, single-cell transcriptomics, population-wide genetic association studies and pharmacological experiments. We show how a single GPCR gene can diversify into several isoforms with distinct signalling properties, and how unique isoform combinations expressed in different tissues can generate distinct signalling states. Depending on their structural changes and expression patterns, some of the detected isoforms may influence cellular responses to drugs and represent new targets for developing drugs with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling that considers how combinatorial expression of isoforms in a particular cell type, tissue or organism collectively influences receptor signalling and drug responses

Isolation and characterization of a novel human RGS mutant displaying gain-of-function activity

Regulator of G protein signaling (RGS) proteins play a crucial role in the adaptation of cells to stimulation by G protein-coupled receptors via heterotrimeric G proteins. Alterations in RGS function have been implicated in a wide range of disease states, leading to many researchers focusing on controlling the action of these regulatory proteins. Previous studies have centered on reducing or inhibiting the action of RGS proteins, utilizing inactive mutants or small molecular RGS inhibitors. Here we describe the isolation and characterization of a novel human RGS4 mutant which displays enhanced or gain-of-function (GOF) activity. RGS4(S30c) demonstrates GOF activity both in an in vivo yeast-based signalling pathway and in vitro against the G alpha(o1) subunit contained in an alpha(2A)-adrenoreceptor-G alpha(C3511)(o1) fusion protein. Mutational analysis of serine 30 identified a number of alternative substitutions that result in GOF activity. GOF activity was retained upon transposition of the serine 30-cysteine mutation to the equivalent serine residue in human RGS16. As with previously identified GOF mutants, RGS4(S30C/S30F/S30K) demonstrate increased steady state protein levels, however these mutants also demonstrate enhanced GAP activity through an additional mechanism distinct from the increased protein content. The identification of human RGS mutants with GOF activity may provide novel therapeutic agents for the treatment of signaling-based diseases and the ability to transpose these mutations to other human RGS proteins extends their application to multiple pathways. (C) 2007 Elsevier Inc. All rights reserved

Isolation and characterization of a novel human RGS mutant displaying gain-of-function activity

Regulator of G protein signaling (RGS) proteins play a crucial role in the adaptation of cells to stimulation by G protein-coupled receptors via heterotrimeric G proteins. Alterations in RGS function have been implicated in a wide range of disease states, leading to many researchers focusing on controlling the action of these regulatory proteins. Previous studies have centered on reducing or inhibiting the action of RGS proteins, utilizing inactive mutants or small molecular RGS inhibitors. Here we describe the isolation and characterization of a novel human RGS4 mutant which displays enhanced or gain-of-function (GOF) activity. RGS4(S30c) demonstrates GOF activity both in an in vivo yeast-based signalling pathway and in vitro against the G alpha(o1) subunit contained in an alpha(2A)-adrenoreceptor-G alpha(C3511)(o1) fusion protein. Mutational analysis of serine 30 identified a number of alternative substitutions that result in GOF activity. GOF activity was retained upon transposition of the serine 30-cysteine mutation to the equivalent serine residue in human RGS16. As with previously identified GOF mutants, RGS4(S30C/S30F/S30K) demonstrate increased steady state protein levels, however these mutants also demonstrate enhanced GAP activity through an additional mechanism distinct from the increased protein content. The identification of human RGS mutants with GOF activity may provide novel therapeutic agents for the treatment of signaling-based diseases and the ability to transpose these mutations to other human RGS proteins extends their application to multiple pathways. (C) 2007 Elsevier Inc. All rights reserved

Combinatorial expression of GPCR isoforms affects signalling and drug responses.

G-protein-coupled receptors (GPCRs) are membrane proteins that modulate physiology across human tissues in response to extracellular signals. GPCR-mediated signalling can differ because of changes in the sequence1,2 or expression3 of the receptors, leading to signalling bias when comparing diverse physiological systems4. An underexplored source of such bias is the generation of functionally diverse GPCR isoforms with different patterns of expression across different tissues. Here we integrate data from human tissue-level transcriptomes, GPCR sequences and structures, proteomics, single-cell transcriptomics, population-wide genetic association studies and pharmacological experiments. We show how a single GPCR gene can diversify into several isoforms with distinct signalling properties, and how unique isoform combinations expressed in different tissues can generate distinct signalling states. Depending on their structural changes and expression patterns, some of the detected isoforms may influence cellular responses to drugs and represent new targets for developing drugs with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling that considers how combinatorial expression of isoforms in a particular cell type, tissue or organism collectively influences receptor signalling and drug responses

Combining native and ‘omics’ mass spectrometry to identify endogenous ligands bound to membrane proteins

Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, ‘nativeomics’, unifying ‘omics’ (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function

Pharmacovigilance of herbal medicines

Pharmacovigilance is essential for developing reliable information on the safety of herbal medicines as used in Europe and the US. The existing systems were developed for synthetic medicines and require some modification to address the specific differences of medicinal herbs. Traditional medicine from many different cultures is used in Europe and the US which adds to the complexities and difficulties of even basic questions such as herb naming systems and chemical variability. Allied to this also is the perception that a ‘natural’ or herbal product must be safe simply because it is not synthetic which means that the safety element of monitoring for such medicines can be overlooked because of the tag associated with such products. Cooperation between orthodox physicians and traditional practitioners is needed to bring together the full case details. Independent scientific assistance on toxicological investigation, botanical verification can be invaluable for full evaluation of any case report. Systematic pharmacovigilance is essential to build up reliable information on the safety of herbal medicines for the development of appropriate guidelines for safe effective use