Targeted protein degradation is the cell’s own cleaning system of unwanted proteins. Recently scientists are focusing on induced protein degradation for drug discovery. This unique degradation system can destroy targeted proteins where drug can’t bind and this is how they offer an excellent advantage.

New technologies like PROTACS is getting numerous attentions in pharmaceutical industry as this technique has various advantages. These advantages of (PROTACS) are as follows¹

· It can reduce systemic drug exposure.

· It can target those proteins which are not targeted by common drugs.

· Large and unwieldy, protacs defy conventional wisdom on what a drug should be. But they also raise the possibility of tackling some of the most indomitable diseases around.

Regulation of cellular functions is critical for cell survival and proliferation. The ubiquitin proteasome system (UPS) is a system with in our body that plays a impactful role in protein homeostasis¹. Recently, small molecules have been used to selectively induce the degradation of a variety of interesting target proteins. This technique is called proteolysis-targeting chimeras (PROTACs)².

PROTAC is a new strategic system which involves in the degradation of proteins within the cell and they utilize ubiquitin-protease system for it³. Ubiquitin‐protease system is a natural system present in our body for cleaning harmful proteins from the cell⁴. PROTAC strategically utilize this system.

PROCESS OF UBIQUITINATION:

Ubiquitination is the process of attachment of ubiquitin molecule to the target protein molecule. And this is done by the iso peptide bond formation. This bond is developed between the carboxyl group of the ubiquitin molecule and the lysin residue of the target protein molecule. The process of ubiquitination involves three different enzymes at three different steps. Ubiquitin is attached by three enzymes sequentially ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3)^5-8. In step1 the enzyme known as ubiquitin activating enzyme is used. It harvests the energy which is released from the ATP hydrolysis. In this step the C domain of the ubiquitin molecule is activated by ATP and a thio-ester bond is formed (Figure1). After this step E2-Ub molecule is formed. And in the final step, E3 fixes with the E2-Ub intermediate^9-10. And the E3s exert a very special role within this cascade as they bring both reaction partners in close spatial proximity. And E3 simultaneously enhance the rate of ubiquitin transfer in a catalytic way^11-13.

Fig.1: Diagram of Ubiquitination procedure

Few study suggests that Rpn11 can be coordinated with ubiquitin receptors for early substrate targeting. N-terminal which is ubiquitin-like (UBL) is present in Ubiquitin-receptor. The UBL domains can be characterized by 19S proteasome caps¹⁴.

Proteasomes:

Proteasomes are basically protein complexes which is responsible to destroy unwanted proteins by proteolysis. Proteolysis is a chemical process that breaks peptide bonds. Enzymes involved in such reactions are called proteases. Two principle proteolytic systems present in the eukaryotic cells are lysosomes and proteosomes.

Proteasomes are cylindrical multi subunit proteases. These are generally found in both prokaryotes and eukaryotes. In eukaryotes these are present in both nucleus and cytosol. Eukaryotic proteasomes are mainly of two types. One is 20S proteasome and another is 26S proteasome. And these are arranged as four rings of heptamers stacked upon one another^15-17.

Fig. 2: 20S proteasome and 26S proteasome

Targeted Proteasomal Degradation:

Advances in genetics helped the researchers to identify and validate highly interesting drug targets. For many years’ drug development process is extensively focused on small molecules like enzymatic inhibitors, antibodies etc^18-22.

Small-molecule-induced proteolysis generally offers an alternative strategy. Proteolysis uses the drug-like properties of small molecules^23-5.

This approach offers the potential to reach beyond the limitations of traditional drug discovery and target the ‘‘undruggable’’ proteome, as binding to the protein of interest (POI) does not need to be connected to inhibition of the POI since it induces its removal from the system^25-27.

PROTACs:

Small molecule PROTACs (PROteolysis-Targeting Chimeras) with even smaller E3 binding ligands were discovered²⁸. The first ever, discovered in 2008, consisted of a non-steroidal androgen receptor ligand which was a selective androgen receptor (SARM) modulator, an MDM2 (Mouse Double Minute 2) receptor based ligand called nutlin and a PEG based linker. This SARM-nutlin based PROTAC successfully targeted ubiquitination of the androgen receptor. Subsequently many other PROTACs have been developed. Till 2015, more than 30 PROTACs have come into play, many among which exhibit nanomolar and in-vivo functional effects²⁸.

Small molecule PROTACs catalytically indulge in targeted protein degradation, therefore, making the degradation process sub-stoichiometric²⁹.

The GFP-HaloTag7-targeting PROTAC made use of a small molecule to join a VHL (von Hippel-Lindau) E3 ligase. It had a DC₅₀value of 19 nM, making >90% degradation of GFPHaloTag7 possible. In some cell lines, DC₅₀ values of <2 nM were reported for VHL-based small-molecule PROTACs involving BET (Bromodomain and Extra-Terminal) protein and RIPK2 (Receptor Interacting Serine/Threonine Kinase 2). Striking observation was that the half-maximal values for inhibiting target proteins (IC₅₀values) could be greater than that of PROTACs for degrading target proteins (DC₅₀ values).

In induced protein degradation using PROTACs, remarkably, the depletion occurred within 1-2 hours, and the target protein reductions were sustainable.

Treatment of HEK 293 (Human Embryonic Kidney 293) cells with HaloPROTAC3 resulted in 50% of GFP-HaloTag7 degradation between 4 and 8 h, and at 24 h, the level of GFP-HaloTag7 was controlled at as minimal as about 10%. The remaining depletion after the removal of the PROTAC occurred over 24 or up to 48 h and was sustained.

Other than target protein degradation, their downstream signaling cascades also met with rapid and sustainable inhibition.

For example, p-ALK (Phospho-Anaplastic lymphoma kinase) and p-STAT3 (Phospho-Signal transducer and activator of transcription 3), the ALK (Anaplastic lymphoma kinase) downstream markers were reported to be significantly inhibited after treating with AL-depleting PROTACs for 2 h. Thereafter, ALK-depleting PROTACs were washed out, followed by inhibition of ALK downstream signaling which lasted for 10h.

PROTACs exhibited high specificity and selectivity for their target proteins. Several studies involving proteomes suggest that the high selectivity of these PROTACs can be extended beyond the intrinsic binding specificity of the corresponding target binding ligands.

Interestingly, JQ1, the thienotriazolodiazepine BET inhibitor lacked selectivity towards BRD2 (Bromodomain-containing protein 2), BRD3 (Bromodomain-containing protein 3), and BRD4 (Bromodomain-containing protein 4). However, development of JQ1-based PROTACs MZ1 and AT1 resulted in selective depletion of BRD4.

A. Choice of PROTAC Warheads and Linkers:

The effects of different variables on PROTAC efficacy and target protein were explored selectively, using two E3 ligands, three targeting ligands, and four different linkers involving a diversity of chemical space²⁵. This study brought about several intriguing conclusions.

First, choosing the protein targeting ligand largely influences PROTAC selectivity and degradation activity.

Second, the cell permeability might be more influenced by the PROTAC than target-protein degradation.

Third, the recruited E3 ligase can also significantly influence the ability of the PROTAC to degrade different substrates.

B. Hook Effect:

The Hook Effect, a frequent observation in studies involving protein degradation, accounts for a reduction in the degradation of the target protein at relatively higher concentrations of the PROTACs^30-32. This effect is mostly a characteristic of three component systems. Reportedly, at higher concentrations of PROTACs (higher than their DC50 values), auto inhibition of the formation of E3: PROTAC: target ternary complex occurs. This occurs due to increased concentrations of PROTAC: E3 and PROTAC: target binary complexes.

C. Limitations and Possibilities:

Owing to the large size of small molecule PROTACs, their oral bioavailability is being affected. Currently their use is limited to intraperitoneal, subcutaneous and intravenous use³³.

Although small molecule PROTACs proved extremely successful in enhancing target selectivity, some off-targets were also noticed, which may serve as the target of the target ligands. Small molecule PROTACs can therefore, sometimes fail at degrading their potential targets.

Since 2015, most reported small molecule PROTACs aimed at target proteins by involving VHL or CRBN E3 ligases. By searching for ligands that bind to other E3 ligases and developing the E3 ligases, development of PROTACs can be sped up.

HYDROPHOBIC TAGGING:

Incorporation of a ligand into a hydrophobic tag for a POI (Protein of Interest) could be done to mimic a partially unfolded state³⁴. In this way, the same cellular control that degrades terminally unfolded proteins also recognizes a hydrophobically tagged protein.

● Her3 degradation:

Her3 shares sequential similarity with epidermal growth factor receptor, but differs from the ErbB family of receptor tyrosine kinases in that its active site is devoid of key catalytic residues. Therefore, it is a pseudokinase and is difficult to be pharmacologically targeted.

First, Her3 ligand was recognized by ATP competitive compounds in an assay involving fluorescence energy transfer²⁰. Addition of an acrylate improved the potency and generated a highly selective covalent ligand to Her3.

Subsequently, an adamantyl moiety was coupled to generate a powerful Her3 degrader that abolished almost all Her3 dependent signaling pathways.

● Androgen Receptor Degradation:

Androgen Receptor (AR) has been a well-defined oncology target. Although, early stages of prostate cancer were successfully treated by aromatase inhibitors and AR antagonists, development of resistance rendered them unsuccessful.

The first selective androgen receptor down regulator (SARD) was developed by adding alkyl fluoryl chain of fulvestrant to dihydroandrosterone.

Parallelly, adamantyl moiety was added to the AR agonist RU59063 to generate a pure antagonist, possessing the ability to degrade AR³⁵.

Functions of the ubiquitin-proteasome system:

As it coordinated the degradation of short-lived proteins, the ubiquitin-proteasome system regulates many important cellular processes. The synchronized proteolysis of cyclins and cyclin-dependent kinase inhibitors is very important for cell cycle progression^23-24. Disruption of this process sometimes leads to cell cycle arrest²⁴. The proteasome has the ability to control gene expression by degrading transcription factors like NF-κB, p53, c-Jun, c-Myc, c-Fos, HIFlα²⁵. Perhaps the best characterized of these is NF-κB, the collective name for inducible dimeric transcription factors. Activated NF-κB is present in the cell nucleus. NF-κB is a significant regulator of cell proliferation, apoptosis, immune and inflammatory responses, and it controls the gene expression.

CONCLUSION:

The targeted protein degradation is a very complex and relatively less understood method. Many of the gene expression is controlled by this degradation method. That’s why this process is having great impact to cause cancer and other diseases. Future medicines and treatment strategies are truly going to be depended on this process of protein degradation.

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Received on 06.01.2020 Modified on 25.08.2020

Research J. Pharm. and Tech. 2021; 14(9):5047-5050.

DOI: 10.52711/0974-360X.2021.00880