Small Molecules Targeting Translation
In the present day, the vast majority of pharmaceutical therapeutics target specific proteins or enzymatic reactions. However, due to conformational challenges, in addition to other issues, a wide number of such proteins remain “undruggable” with current technologies. One solution put forward to address these issues has been to target these proteins before they undergo translation, thus essentially knocking down their production. While research in the area has often been beset by serious side-effects, the recent approval of Onpattro, a RNAi molecule considered a first-in-class drug by the FDA, has provided impetus for other pharmaceutical manufacturers to plough funding into the area.
Below, we give our take on the four most relevant papers on the topic of small molecules targeting translation.
Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain
PLoS Biology, 2017; 15(3), p.e2001882.
Lintner, N.G., McClure, K.F., Petersen, D., Londregan, A.T., Piotrowski, D.W., Wei, L., Xiao, J., Bolt, M., Loria, P.M., Maguire, B. and Geoghegan, K.F
Cardiovascular disease (CVD) is the leading cause of death in Western countries, with high cholesterol a significant causal factor. Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma LDL cholesterol levels by preventing the recycling of the LDL-receptor. Humans with loss-of-function mutations in PCSK9 have a significantly reduced LDL cholesterol profile and a subsequently reduced risk of CVD, with no obvious adverse health effects. Recently, screening analysis identified a molecule which can reduce PCSK9 levels, through inhibition of its translation, although the exact mechanism remained elusive. Here, authors investigated PF-06446846, a more optimised small molecule, in order to investigate these mechanisms and potential impacts both in vitro and in vivo.
Key Findings
- PF-06446846 was demonstrated to specifically reduce the levels of PCSK9 translation, while having very little impact on the translation of other molecules.
- Mutations in the mRNA sequence that resulted in common or synonymous codon output did not influence the effectiveness of PF-06446846, indicating the mRNA sequence is not the target of this drug. Two frameshift mutations, resulting in near similar mRNA sequence but significantly altered amino acid sequence resulted in a loss of inhibition of PCSK9 translation.
- In analysing the safety and efficacy effects in vivo, at the highest dose tested (50mg/kg), it was found that PF-06446846 successfully reduced levels of circulating PCSK9 and blood LDL cholesterol, with no impact on HDL cholesterol. There was a slight reduction in food consumption, although with no impact on body weight. Slight reductions in red blood cell mass were observed, although these reductions were significantly increased for white blood cells. It is possible these changes may be related to the observed reduction in LDL cholesterol.
- PF-06446846 was found to not cause widespread ribosome stalling, but did lead to an increase in stalling early on the PSCK9 transcript.
Implications
PF-06446846 directly and specifically inhibits translation of PCSK9 by acting on the translating human ribosome. The mechanism of action of this small molecule reveals the potential of therapeutically modulating the human ribosome with small molecules as a means to target previously “undruggable” proteins.
Translation control of the immune checkpoint in cancer and its therapeutic targeting
Nature Medicine, 2019. 25(2), pp.301–311
Xu, Y., Poggio, M., Jin, H.Y., Shi, Z., Forester, C.M., Wang, Y., Stumpf, C.R., Xue, L., Devericks, E., So, L. and Nguyen, H.G.
Immune checkpoint therapy is an emerging therapy in the treatment of cancer. The immune system naturally targets out-of-control tumour growth, while also maintaining self-tolerance of health cells through immune checkpoints. However, many tumours take advantage of these immune checkpoints, overexpressing them and thus inhibiting cytotoxic tumour cell death. While transcriptional control of these immune checkpoint molecules has been reported in the literature, the landscape of the post-transcriptional and translational aspects of this process remains largely uncharacterised. Here, researchers delve into these features, utilising ribosome profiling in an in vivo mouse model of hepatocellular carcinoma (HCC).
Key Findings
- A mouse model overexpressing MYC and KRAS led to drastically increased metastasis compared to either gene overexpressed alone. These tumours also demonstrated increased infiltration of immune cells, such as neutrophils and macrophages, which has been associated with increased angiogenesis and metastasis. Oddly, the infiltration of cytotoxic CD8+ T-cells, associated with anti-tumour immunity, also increased.
- RNA-Seq transcriptome analysis of the MYC/KRAS tumour murine model strongly correlated with human HCC, suggesting this is a good model for future study. Additionally, gene expressions profiles were specifically altered at the translational level for the MYC/KRAS model, compared to KRAS alone.
- Notably, one of the genes distinctly upregulated at the translational level in this MYC/KRAS model, compared with KRAS alone, was PD-L1, known to be involved in tumour evasion of the immune system.
- In the KRAS model, ribosomes tended to accumulate at the start codons of upstream ORFs, while in the MYC/KRAS model they accumulated at the start codon of the main ORF. Mutations of the uORF start codons resulted in increased downstream translation Interestingly, the MYC/KRAS model has significantly increased eIF2α phosphorylation, a key component of the eIF2 ternary complex, which is known to assist in bypassing of uORFs.
- Mice injected with a mutant KRAS gene, in addition to a mutation leading to translational overexpression of PD-L1, developed a tumour phenotype similar to that seen with the MYC/KRAS model, suggesting that this phenotype is dependent on PD-L1-mediated immune evasion.
- eFT508, a drug inhibiting MNK1/2, led to decreased eIF2α phosphorylation and concomitant reductions in PD-L1 translation specifically. This was also associated with a decrease in liver cancer progression and metastasis.
Implications
The identification of this MYC/KRAS model as having strong correlations with human HCC is an important finding, and should be considered in future research into this disease. The finding that PD-L1 is linked to MYC at the level of translation has important implications for the area of cancer immunity and surveillance. Importantly, uncovering the effectiveness of a drug currently undergoing testing in clinical trials raises hope for future treatment of this disease.
The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly
eLife, 2015, 4, p.e05033
Sidrauski, C., McGeachy, A.M., Ingolia, N.T. and Walter, P.
eIF2α is a eukaryotic translation initiation factor. Importantly, it is a target of a number of kinases as part of the integrated stress response (ISR), in order to coordinate and regulate gene expression in response to potentially hazardous conditions. Phosphorylation of this factor under these conditions typically downregulates global gene expression, while upregulating specific genes important to cell survival. However, the ISR has also been implicated in a number of processes, ranging from insulin production to memory consolidation. Here, the authors investigated the impact of ISRIB, a small molecule reducing the effect of eIF2α phosphorylation downstream of the impact of eIF2α-associated kinases.
Key Findings
- Among the genes that were translationally upregulated upon inducing endoplasmic reticulum (ER) stress, the most pertinent were ATF4, ATF5, CHOP, and GADD34. ATF4 and ATF5 are both known targets of the ISR, while CHOP is a pro-apoptotic transcription factor. GADD34 itself is a regulatory subunit of the eIF2α phosphatase.
- ISRIB treatment largely reversed these changes in ER stressed cells, but had little impact on translation in unstressed cells. Treatment with this drug also significantly reduced the formation of stress granules, commonly associated with ER stress. Interestingly, it did not reduce stress granule formation induced in the absence of eIF2α phosphorylation, indicating that it likely acts exclusively through this mechanism.
- Upon treatment of cells with pre-formed stress granules with ISRIB, stress granules promptly dissolve and fade. However, like the above, this activity is limited exclusively to stress granules associated with eIF2α. Additionally, it had no impact on another type of ribonucleoprotein assembly, namely P-bodies.
Implications
This study is an important landmark in eIF2α research, as ISRIB is the first known drug to ablate the impact of eIF2α phosphorylation, downstream of eIF2α kinase activity. As such, it opens up a new avenue for targeting this molecule, especially so in the areas of insulin production and memory and learning.
Cellular response to small molecules that selectively stall protein synthesis by the ribosome
PLoS genetics, 2019, 15(3), p.e1008057–3087
Liaud, N., Horlbeck, M.A., Gilbert, L.A., Gjoni, K., Weissman, J.S. and Cate, J.H
Today, the majority of drugs utilised in treating disease act through inhibiting protein or enzymatic activity. However, a class of so-called “undruggable” proteins represent a challenge to this methodology. One potential solution lies within the area of translatomics, with the aim to repress translation of a specific transcript, or set of transcripts. One problem with this idea is the wide array of off-target effects that come with most translational inhibitors. PCSK9 inhibitors (which reduce blood cholesterol levels) are one class of drugs that are able to selectively inhibit specific transcripts, with comparatively few off-target effects. This paper aimed to investigate, utilising CRISPRi screens and ribosome profiling, which genes modify the impact of this translational inhibitor, in order to gain insights into its mechanism of action and evaluate targets for potential combination therapy. The authors also investigate two variants of this inhibitor (PF8503 and PF846).
Key Findings
- It was found that PF8503 affected the translation of 46 mRNA transcripts, compared to 24 for PF846. It was also more cytotoxic. Due to this factor being favourable in growth-based screens, PF8503 was used going forward.
- It was found that mRNA synthesis and export pathways, as well as apoptosis, DNA repair, and cell cycle pathways were protective against cytotoxicity induced by PF8503. On the other hand, pathways related to the mitochondria, translation, and ribosome biogenesis sensitised cells to this toxicity. Notably, a number of genes involved in ribosome quality control, such as those involved in rescue from ribosome stalling (e.g. ASCC2/ASCC3) also greatly sensitised cells to PF8503.
- The ability of PF8503 to inhibit PCSK9 translation was not affected by ASCC2 or ASCC3 knockdown. Similarly, global translation was not affected by these knockdowns.
- When the same methodology was used to identify which genes impacted on both harringtonine treatment (a general translational initiation inhibitor) and PF8503 treatment, it was found that there was little crossover. However, some pathways, including ribosome biogenesis, the spliceosome, and RNA transport, were shared. Interestingly, aminoacyl-tRNA biosynthesis only impacted cells in the PF8503 group.
Implications
This paper illuminated some of the important molecular players in the cellular response to selective ribosome stalling, in this instance stalling induced by PCSK9 inhibitors. Such research may prove important in cardiovascular health specifically, given the potential role PCSK9 inhibitors may play in lowering blood cholesterol. It is also an important paper in the area of therapeutic drugs targeting translational repression.
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