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Felicites Rapon
Recently, chemically engineered molecules have been produced to incite the degradation of a given protein. These molecules are called PROTACs [1], which stands for PROteolysis-TArgeting Chimeras.
How does the degradation of a protein work?
The degradation of a protein, also called proteolysis, is achieved by the mechanism of ubiquitination. This mechanism adds a Ubiquitin protein tag onto a protein to be degraded. Actually, ubiquitin markers signal to the cell that the protein degradation can take place. After a chain reaction of ubiquitination, the cell will direct the poly-ubiquitinated protein towards the proteasome. The protein will then be taken over by the proteasome, a specific cellular structure composed of multiple enzymes that will degrade the protein. Once degraded, the protein will be in the form of multiple peptides (small sequences of amino acids joined together by bonds) [2].
Therefore, to be effective, PROTACs must specifically target both a protein to be degraded and the enzyme complex involved in ubiquitination. They have two functions: they are bifunctional.
How can PROTACs be related to Cancer therapy?
By enabling the degradation of a specific protein, PROTACs have proved to be interesting drug candidates against cancer. They present a new therapeutic approach that overcomes the limitations of small-molecule inhibitors (SMI). Actually, just like antibiotic resistance, drug resistance exists for SMI and cancer cells. Cancer cells will alter the proteins targeted by the inhibitors through mutations and/or upregulation in order to disable their interaction with the inhibitor. In fact, with these SMIs, the inhibitory effect on the protein depends on a reversible interaction, which means that this interaction between the protein and its inhibitor is quite sensitive to changes of structure, especially if the protein undergoes mutations.[3,4]
However, with PROTACs therapy, which results in complete degradation of the targeted protein, cancer cells will need to produce this protein again, making PROTACs molecules more robust and potent than SMIs.
To date, ARV-110 and ARV-471 [5] are the first clinical-stage PROTACs designed for the treatment of prostate (2nd most common cancer in men [6]) and breast (most common cancer in women worldwide [6]) cancer, respectively.
But, how can such molecules be designed?
As introduced above, PROTACs are bifunctional molecules. It means they will bind to 2 different elements thanks to 2 structures attached by a linker (Figure 1). The first structure will target the protein of interest (POI) to be degraded. The second structure, at the opposite side of the PROTAC, will target the E3 ubiquitin ligase. Indeed, for the latter, the molecule will bind to a subunit (Substrate Receptor) of the E3 ligase complex called CRL for Cullin-RING ligase. This complex is composed of the E2 enzyme, which carries an Ub protein. Once the PROTAC is attached to the POI and the E3 ligase complex, the Ub protein bound to the E2 enzyme will be transferred to the POI, the POI will be ubiquitinated and by a chain reaction will end up being polyubiquitinated (a chain of Ub proteins).
Figure 1: Degradation of a protein of interest (POI) via a PROTAC molecule
Image created with BioRender.com
Thanks to the illustration (Figure 1), we can easily see that instead of only blocking the molecule’s activity like inhibitors do, PROTACs considerably reduce the ratio of POI by promoting its degradation: this is called proteolysis.
However, we can also see that it can be detrimental to activate this proteolysis continuously as the protein and thus its activity are definitely lost in all the cells where the PROTAC was active. In a potential cancer treatment, targeting the degradation of a protein overexpressed in cancer cells and normally expressed in normal cells could be harmful, as its degradation would function in both cells, resulting in the death of cancerous and non-cancerous cells.
One way to control this proteolysis is to activate it via a light-dependent signal. This would allow proteolysis to be controlled in space and time to avoid off-target effects that occur with many drugs currently on the market. Off-target effects of a drug molecule are defined as effects that occur due to the binding of the drug to an area other than the expected and targeted one, thus causing some unwanted effects in the body.
An American team [7] has therefore designed a PHOTAC (PHOtochemically TArgeting Chimeras) to meet this need for space-time degradation. A PHOTAC is no longer bifunctional but trifunctional because it includes a photoswitchable linker. A photoswitchable linker means that it will react chemically differently when irradiated with electromagnetic radiation such as visible light or UV light. The researchers found a molecule that, upon irradiation, changes its conformation and allows or prevents the binding of PHOTACs with the POI and/or with the E3 ligase complex: azobenzene.
A conformational change means that the molecule will keep the same amount of atoms, but their spatial arrangement will change due to the rotation of these atoms after irradiation. We call these molecules isomers.
What is the azobenzene molecule?
Azobenzene is a molecule that combines 2 phenyl rings with a double bond N=N (azo group with 2 nitrogen atoms). This azo group is at the origin of the cis- and trans- conformation. Indeed, this double bond defines a plane (Figure 2A) so a cis- (or Z-) isomer has its 2 phenyl rings on the same side while a trans- (or E-) isomer has its 2 phenyl rings on both sides of the azo group (Figure 2B).
In the dark (initial state), the trans PHOTAC (composed of the trans form of azobenzene) is the most stable. According to its absorption spectrum, there is an absorption peak for a wavelength around 390 nm. Thus, upon irradiation at 390 nm, the photons associated with the radiation will excite the trans form and change its conformation to the cis form.
Figure 3 : (UV-Visible) Absorption spectra of PHOTAC-I-3 following irradiation. Adapted from Reynders, Martin et al. [2]
This change in conformation is due to the energy carried by these photons. This cis form will no longer show an absorption peak around 390 nm but around another wavelength (Figure 3).
To return to the trans form, we can either irradiate the cis form at the same wavelength as its absorption peak (>450 nm), or do a thermal relaxation, i.e., use heat to return to the initial state.
Thus, PHOTACs are based on the principle of photoisomerization (photoswitch), and their confirmation change will be decisive for the degradation of a protein (Figure 4).
Figure 4 : PHOTAC mechanism following irradiation. Image created with BioRender.com
How were the PHOTACs designed?
In the paper, the authors designed 2 types of PHOTACs. Both use the E3 ligase complex called CRL-CRBN (the PHOTAC will bind specifically the CRBN protein of the E3 ligase complex) while the targeted POI is different. PHOTAC-I targets BRD2-4 which are proteins of the BET (bromodomain and extra terminal) family, they play a role in cell growth. PHOTAC-II targets FKBP12, another type of protein (Figure 5).
Figure 5 : PHOTAC-I and PHOTAC-II design. Image created with BioRender.com
Here, only the process for obtaining a PHOTAC-I will be described (Figure 5).
The authors started by taking the high-affinity inhibitor (JQ1) of the BET protein (BRD2-4), so binding is already very specific and strong. From this JQ1, the molecule called dBET was obtained, which is a PROTAC targeting some BET proteins. So dBET is composed of a CRBN substrate (E3 ligase complex substrate), a linker and JQ1. From dBET, they formed different forms of PHOTAC-I such as PHOTAC-I-3, one of the most efficient.
The synthesis of a compound means the production in a chemical way of such a compound.
PHOTACs synthesis starts with lenalidomide (CRBN substrate), then the azo group is added by a diazotization phenomenon, and by a second reaction the azobenzene structure is obtained. After obtaining the azobenzene, they have to add the linker and then the inhibitor JQ1 (to target the POI) through a peptide coupling reaction (condensation of a carboxyl group (-COOH) and an amine group (-NH2) to form a peptide bond: a covalent bond).
One step of the synthesis allowed them to easily obtain a PHOTAC library with different linker sizes. This is the coupling step with the HATU molecule and a diaminoalkane (a molecule with 2 amine groups (-NH2), in green on Figure 6).
Figure 6 : Synthesis of PHOTAC-I-3 starting from lenalidomide. Adapted from Reynders, Martin et al. [2]
After the synthesis, the properties of azobenzene and PHOTAC were analyzed. For example, the fatigue resistance of the photoswitch linker which consists in measuring to what extent after a certain number of irradiation cycles (trans to cis and then cis to trans) the PHOTAC will be degraded and will lose its activity. Here, we can see that there is no apparent degradation over 5 hours (Figure 7C).
They also measured at different wavelengths the ratio between cis and trans isomers, called the photostationary state (PSS). As explained earlier, after irradiation at 390 nm, the cis form will be dominant, then after increasing the wavelength of the irradiation, the amount of trans form will increase as we return to the initial state (Figure 7E).
Figure 7 :
Fraction of trans-PHOTAC-I-3 in the PSS (C).
Reversible switching and photochemical stability of PHOTAC-I-3 (E). From Reynders, Martin et al. [2]
What are the results in RS4;11 cells (a cancer cell type used in the paper)?
RS4;11 cells are acute lymphoblastic leukemia cells, a cancer affecting the lymphocytes-a type of white blood cells.
PHOTAC-Is targets the degradation of BRD2-4 proteins, this degradation can be measured with the cell viability rate. Indeed, BRD2-4 proteins are linked to cell proliferation and differentiation processes as it plays a crucial role in the regulation of gene transcription. Consequently, if only PROTACs had been designed, they would also have affected the proliferation of normal cells.
The researchers showed that the EC50 (half maximal effective concentration) is the concentration of the molecule for which there is half of the effect (here cell viability). The lower the EC50, the lower the concentration of this molecule is required: efficacy is achieved more easily. (Figure 8A). This EC50 is lower for PHOTAC upon 390 nm irradiation than in the dark by a factor of 7.
Finally, the authors analyzed the expression level of the proteins using a biochemical technique called Western blot. With this technique, the proteins are separated according to their molecular weight and are visualized as a band whose intensity represents the protein expression level. It can be seen (Figure 8B) that upon irradiation, between 3 µm and 100 nm, there is a loss or a small amount of BRD2-4 proteins which shows that degradation has occurred.
Figure 8 : Viability of RS4;11 cells after treatment with PHOTAC-I-3 for 72 hours in the dark or following irradiation (A). Immunoblot analysis after treatment of RS4;11 cells with PHOTAC-I-3 for 4 hours at different concentrations (E). Adapted from Reynders, Martin et al. [2]
To conclude :
The article shows that azobenzene has many advantages allowing it to be used as a photoswitch for PHOTACs.
Indeed, azobenzene is a photochromic molecule (hence its photoswitch property). This means that it can change color upon irradiation (light absorption). Thus, a colorimetric assay can be performed to measure the amount of trans isomers compared to cis isomers. This principle called photochromism is at the origin of tinted sunglasses.
This paper also shows that it is quite easy to design such PHOTACs targeting different CRLs but also different POIs and this from an existing library of PROTACs. Thus, we could treat many diseases ranging from cancers to neurodegenerative diseases.
However, further analyses need to be performed, especially regarding the mechanisms behind the conformational change and the change in affinity for the POI or the substrate receptor, for example. Further experiments are required to better understand the size and position of the linker and its role in the photoswitch properties. For example, for PHOTAC-I-9 which has a different position of the azobenzene within the linker, there is no difference in degradation under irradiation (Figure 9).
Figure 9 : PHOTAC-I-3 and PHOTAC-I-9 chemical structure. Adapted from Reynders, Martin et al. [2]
These analyses could be performed with molecular docking, a field of structural biology that predicts and models from mathematical calculations what the orientation of one molecule will be with respect to another.
Concretely?
PHOTACs should be considered as prodrugs which, after light-triggered isomerization of azobenzene, will be functional and thus play their role as drugs.[8]
However, some modifications have to be done in order to be used as oncological phototherapy treatment.
Actually, the exposure of UV (300-400 nm) causes DNA and tissues damage yet UV light stimulation is used here in order to induce the azobenzene change of conformation. To bypass this problem, instead of using the azobenzene, we could use azobenzene analogues which change conformation under red or Near-Infrared (NIR)-light stimulation (780-2500nm) [9]. Thus, with a NIR-light laser, we could not only avoid tissue toxicity due to UV irradiation but also be able to target the deeper layers of solid tumors since the higher the wavelength, the deeper the penetration tissue.
Moreover, the sensitivity of azobenzene to light but also hypoxia (it means the lack of oxygen) and enzymes makes this molecule and therefore the PHOTAC eligible for a specific drug-delivery system such as hypoxia-resposive nanoparticules (1-100 nm) [9,10]. Those nanoparticules encapsulate the PHOTAC and by a hypoxia-induced stimulation due to the hypoxic tumoral microenvironement will deliver specifically the PHOTAC to cancer cells.
Edited by Rachel Cherney
References :
[1] Sakamoto, K M et al. “Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation.” Proceedings of the National Academy of Sciences of the United States of America vol. 98,15 (2001): 8554-9. doi:10.1073/pnas.141230798
[2] Kaiser G, 2022, Polypeptides and Proteins, Microbiology, Biology LibreTexts, accessed 13 June 2023, <https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_(Kaiser)/Unit_7%3A_Microbial_Genetics_and_Microbial_Metabolism/19%3A_Review_of_Molecular_Genetics/19.1%3A_Polypeptides_and_Proteins>
[3] Burke, Matthew R et al. “Overcoming Cancer Drug Resistance Utilizing PROTAC Technology.” Frontiers in cell and developmental biology vol. 10 872729. 25 Apr. 2022, doi:10.3389/fcell.2022.872729
[4] An, Sainan, and Liwu Fu. “Small-molecule PROTACs: An emerging and promising approach for the development of targeted therapy drugs.” EBioMedicine vol. 36 (2018): 553-562. doi:10.1016/j.ebiom.2018.09.005
[5] Pfizer 2023, Arvinas and pfizer announce, accessed 13 June 2023, <https://www.pfizer.com/news/press-release/press-release-detail/arvinas-and-pfizer-announce-upcoming-vepdegestrant-arv-471>
[6] World Cancer Research Fund International, 2020, Prostate cancer statistics, Breast cancer statistics, accessed 13 June 2023, <https://www.wcrf.org/cancer-trends/>
[7] Reynders, Martin et al. “PHOTACs enable optical control of protein degradation.” Science advances vol. 6,8 eaay5064. 21 Feb. 2020, doi:10.1126/sciadv.aay5064
[8] Zhu, Jundong et al. “Triggered azobenzene-based prodrugs and drug delivery systems.” Journal of controlled release : official journal of the Controlled Release Society vol. 345 (2022): 475-493. doi:10.1016/j.jconrel.2022.03.041
[9] Jerca, Florica Adriana et al. “Advances and opportunities in the exciting world of azobenzenes.” Nature reviews. Chemistry vol. 6,1 (2022): 51-69. doi:10.1038/s41570-021-00334-w
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