Periodic Reporting for period 1 - HHAT (Hhat inhibition as a novel approach for selective cancer treatment)
Période du rapport: 2021-05-22 au 2023-05-21
The Hedgehog (Hh) pathway is a conserved cell-cell signalling pathway that is normally involved in the development of tissue, but is associated with cancer during maturity. There is strong evidence that Hh signalling plays an important role in pancreatic ductal adenocarcinoma (PDAC), a very deadly (5-year survival rate is 5%) form of cancer, as well as being involved in over 25% of cancer deaths. however, the Hh pathway is highly complex and includes several poorly understood branches. Nevertheless, completely removing Hh signalling has been shown to effectively inhibit PDAC growth. Approved Hh inhibitors Vismodegib and Sonidegib exist, but are not effective against PDAC because they do not completely inhibit Hh signalling. We propose developing alternative Hh targeting drugs that are capable of completely inhibiting Hh signalling, thus providing the tool compounds to start developing a drug against this deadly disease, and cancer more generally. Our approach is based on targeting the protein Hedgehog acyltransferase (HHAT), a critical protein in the Hh pathway. The overall objective of the project is to generate compounds that have the right potency and physicochemical properties to target HHAT in cells and animal models. This objective has been achieved, as we have generated a molecule that has high potency against HHAT, good stability, as well as being well tolerated in mice.
An overview of the work performed within the project:
- Throughout the project, over a hundred novel compounds have been synthesized and tested for their HHAT inhibitory activity.
- The cryo-EM structure of HHAT was solved. This was followed with mechanistic studies, mutational studies and molecular dynamics modelling to describe a catalytic cycle for HHAT functioning.
- Cry-EM strtuctures of eight compounds bound to HHAT were solved at high resolution. This has enabled structure based drug design.
- A high throughput cellular HHAT inhibition assay was developed. The assay uses a Gli-GFP reporter cell line to measure Hh signalling in an automated fashion on the IncuCyte platform. Cellular activity was measured for over thirty compounds.
- Metabolic stability studies were performed in mouse, rat and human microsomes, as well as compound permeability and plasma binding studies. Our top compound was also tested in vivo in mice.
- Results were disseminated through poster presentations at the 2021 FASEB protein lipidation research conference and the 2022 EMBL chemical biology conference.
- structural studies were published in peer reviewed journal Molecular Cell (Coupland et al.,2021, 81, 5025–5038).
- Throughout the project, over a hundred novel compounds have been synthesized and tested for their HHAT inhibitory activity.
- The cryo-EM structure of HHAT was solved. This was followed with mechanistic studies, mutational studies and molecular dynamics modelling to describe a catalytic cycle for HHAT functioning.
- Cry-EM strtuctures of eight compounds bound to HHAT were solved at high resolution. This has enabled structure based drug design.
- A high throughput cellular HHAT inhibition assay was developed. The assay uses a Gli-GFP reporter cell line to measure Hh signalling in an automated fashion on the IncuCyte platform. Cellular activity was measured for over thirty compounds.
- Metabolic stability studies were performed in mouse, rat and human microsomes, as well as compound permeability and plasma binding studies. Our top compound was also tested in vivo in mice.
- Results were disseminated through poster presentations at the 2021 FASEB protein lipidation research conference and the 2022 EMBL chemical biology conference.
- structural studies were published in peer reviewed journal Molecular Cell (Coupland et al.,2021, 81, 5025–5038).
The project has expanded the available toolbox for studying Hedgehog signalling in biology and cancer. First, we have solved the high resolution cryo-EM structure of HHAT bound to several inhibitors. This has improved our mechanistic understanding of the enzyme class, while also enabling us to perform structure based drug design. Furthermore, the project has generated over a hundred new compounds, many of which are vastly more potent than the current state of the art in the literature. Some of these compounds have been validated for in vivo use with unprecedented potency. We expect the impact of this to be that it will allow the investigation of different routes to target the Hedgehog signalling pathway. The highly complex nature of this pathway, with various cell-cell interactions and branching points, makes this particularly useful, as modulating the pathway at different points can have vastly different outcomes. This is relevant because targeting Hedgehog signalling in the clinic has been highly unpredictable and has led to paradoxical results. Our tools will allow us to shed light on those paradoxes. Doing so would have a major impact on how Hedgehog sensitive cancers are analyzed an treated, which in turn could lead to increased survival rates, reduced caretaking costs, improved quality of patient life and more generally a reduction in societal impact for Hedgehog sensitive cancers.