The Discovery of Protheolysis Targeting Chimeras (PROTACs), Evolution, Design and Perspectives in Modulating Inflammatory Diseases

Abstract

Currently, most drugs have their mode of action based on occupation-oriented pharmacology (proteins modulation through temporary inhibition by association and dissociation). Alternative modes of actions are welcome to exploit current known targets (in a more selective way, for example) as well as those known as “undruggable”. PROTAC (protheolysis targeting chimera) technology provides a compelling new approach that is based on an event-driven mode of action, exploring simultaneous binding to a protein and an E3 ligase, leading to targeted protein degradation, modulating the proteins levels. After all progress over the past two decades and the recent level of interest, clearly targeted protein degradation mode of action could become a therapeutic modality in the future. Furthermore, several PROTACs are under clinical trials, even already progressing to phase 3 (i.e., ARV-471). In this review we addressed the PROTACs discovery process, mode of action, evolution, current stage, their design, applications to modulating inflammatory conditions and the future directions of this promising drug discovery modality, including problems that still need to be overcome.

Keywords:
PROTAC; event-driven mode of action; undruggable targets; hybrid compounds; E3 ligase

1. Introduction

In the last 20 years, the drug discovery process has seen a groundbreaking paradigm shift with the discovery of targeted protein degradation (TPD), a therapeutical strategy of inducing the depletion and/or reduction of a disease-causing protein via hijacking the endogenous protein degradation machineries.¹,²,³ During this period, the focus has changed from complete attention to the classical targets of traditional therapies (e.g., enzymes, ion channels, protein kinases, G protein-coupled receptors (GPCRs)), to also include those “unreachable” of high biological function interests, such as targets considered “undruggable”.⁴

These “undruggable” targets are molecular entities that have proven challenging to modulate with traditional small-molecule therapies but that remained of great therapeutic interest.¹,⁵ Generally, they include proteins without enzymatic function, such as scaffolding proteins and transcription factors,⁶,⁷ and play crucial roles in various diseases, such as cancer, neurodegenerative disorders, and inflammatory and infectious diseases. Estimative counts that nearly 400 human “undruggable” proteins are disease-related.⁸,⁹ As characteristics, these targets have broad active sites and functional interfaces that are flat and lacking defined pockets, which are challenging for small molecules to bind to.⁸

The traditional and well-succeeded design of occupancy-driven small molecule compounds, for single or multitarget compounds, has been used during the last several decades in drug discovery.¹⁰,¹¹ This strategy is focused on modulating target proteins’ function by occupying their active, orthosteric or allosteric sites. However, occupancy-driven drug discovery, despite having beautiful exceptions, is not always feasible and efficient for all drug targets, several times needing well-defined binding sites and preferentially high affinity.¹ From this point, the TPD approach emerged as innovative and a key therapeutic modality to exploit current known targets (in a more selective way or overcoming potential resistances, for example) as well as those called “undruggables” by means of an event-driven new mode of action (MOA).⁵,¹²,¹³

Probably the main class of small molecules acting as TPD are proteolysis-targeting chimeras (PROTACs), first described in 2001 by Crews and co-workers.¹⁴ Since many protein levels are regulated by ubiquitin-dependent proteasome system (UPS), they designed these compounds as TPD aiming at hijacking the cellular machinery to degrade a protein of interest (POI), as an effective way to modulate the protein abundance of normal and/or diseased cells. Indeed, the PROTACs were designed to be hetero-bifunctional compounds able to bring together a POI and the UPS degradation machinery, which do not usually form such a complex.

In this review, we will briefly address the PROTACs discovery process, MOA, evolution, current stage, their design, applications to modulating inflammatory conditions and the future directions of this promising drug discovery modality, including problems that still need to be overcome.

1.1. The UPS system behind the discovery of PROTACs

The proteostasis process, which is indeed protein homeostasis, denotes the intricate and interrelated cellular processes employed to control the concentration, conformation, and subcellular localization of proteins.¹⁵ This complex system encompasses a wide array of pathways governing protein synthesis, folding, transport, and disposal.¹⁶ In eukaryotic cells, damaged proteins or organelles can be effectively eliminated through proteasomes,¹⁷ which manage the degradation of short-lived proteins and soluble misfolded proteins through the UPS,¹⁸ a major pathway that degrades intracellular proteins.

In this pathway (Figure 1), proteins are targeted for degradation by the proteasome in a three-step process involving ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin protein ligases (E3), which coordinate the transfer of ubiquitin (Ub) molecules to the target protein (substrate) with high specificity through formation of isopeptide linkages between the C-terminal carboxyl group of a Ub moiety and an ε-NH₂ group on a lysine on the protein substrate.¹⁹ Ub is a small eukaryotic regulatory molecule (76-amino acid), which is responsible for marking proteins (through polyubiquitination) for degradation via the above described proteasome system. After the linkage of first Ub to the protein, the next Ub molecules are always linked to one of the seven lysine residues (K6, K11, K27, K29, K33, K48, K63) or the N-terminal methionine of the previous Ub molecule.²⁰ Poly-ubiquitinated proteins in K48 (one of the most abundant) are often targeted to proteasome for degradation (Figure 1). Poly-ubiquitinated proteins in K48 (one of the most abundant) are often targeted to proteasome for degradation. The human genome is estimated to encode more than 600 E3 ligase,²¹,²² each one with high selectivity for different subsets of proteins.

Figure 1
Schematic representation of ubiquitin-proteasome system (UPS) process of protein degradation. The ubiquitin molecule (Ub) is linked to ubiquitin-activating enzymes (E1) (EC:6.2.1.45) through an ATP-dependent process that is subsequently transferred to ubiquitin-conjugating enzymes (E2) (EC:2.3.2.23) through interaction with E2. In a parallel process, ubiquitin protein ligases (E3) (EC:2.3.2.26) recognize and binds to the target protein (depicted in cartoon form). After, the complexed E3 catalyzes the transfer of the ubiquitin molecule from E2 to the target protein. Thus, a sequential process results in the polyubiquitination of the substrate, targeting it for the subsequent proteasome degradation.

The utilization of this protein degradation system for therapeutic applications drew inspiration from researches conducted on viruses and plants. More than 20 different viruses can hijack the human UPS system to promote their own survival and replication.²³ For example, human papillomavirus types 16 (HPV-16) and 18 (HPV-18) possess the E6 protein that recruits the human E3 ligase to ubiquitylate p53, resulting in its degradation.²⁴ Due to the fast degradation of ubiquitinated proteins within cells and nature inspiration, Craig M. Crews and Raymond J. Deshaies¹⁴ raised the idea that manipulating the UPS pathway could serve as an effective method for regulating the protein levels in both healthy and diseased cells. This approach led to the discovery of PROTACs.

1.2. Discovery and mode of action of PROTACs

Despite the occupancy-driven pharmacology MOA has been successful for many biological targets, this MOA can be challenging for modulating all kinds of targets. Specifically, it can be less effective for proteins lacking enzymatic activity, such as scaffolding proteins or those that rely on protein-protein interactions (PPIs).²⁵ Drugs that work by occupying a target site are most effective when the target site remains occupied. Those with low potencies often require high drug doses that can lead to unwanted side effects due to off-target binding at higher drug concentrations.²⁶ Additionally, resistance to inhibition by occupancy-driven therapies can be developed in many diseases, including cancer and bacterial infections.²⁷,²⁸ As a result, many researchers have been exploring new drug classes that use alternatives MOA.

Under this perspective, in the early 2000s, PROTACs were developed as a novel class of small molecules with a new MOA, being designed to harness the cellular machinery responsible for protein degradation. The first PROTAC (named Protac-1), synthesized in 2001,¹⁴ was the in vitro proof of concept for the biological application of this class of compounds, and confirmed the expectations for its MOA. Protac-1 was specifically designed to target methionyl aminopeptidase 2 (MetAp-2), identified as the potential target of the potent angiogenesis inhibitors ovalicin and fumagillin. Protac-1 comprised two different components attached by a linker: ovalicin (ligand for the POI) and a 10-amino acid phosphopeptide DRHDSGLDSM derived from nuclear factor-κB inhibitor-α (NF-ΚBIΑ, also known as IΚBΑ), which is recognized by the E3 ligase β-transducin repeat-containing E3 ubiquitin-protein ligase (β-TRCP) (Figure 2a). Protac-1 acted as a molecular bridge, promoting the interaction between MetAp-2 and β-TRCP, allowing the ligase to ubiquitylate METAP2 and inducing its degradation.¹⁴

Figure 2
(a) Structure of first described PROTAC (Protac-1); (b) PROTACs hetero-bifunctional nature including POI ligand, a linker, and a ligand for E3 ligase.

As for Protac-1, the development of PROTAC technology was based on its hetero-bifunctional nature, which includes a ligand for the POI, a ligand for recruiting ubiquitin E3 ligase, and a linker (Figure 2b).²,²⁹ This unique heterobifunctional character allows simultaneously binding to both the POI and a ubiquitin E3 ligase, forming a ternary complex that activates the UPS for the degradation of the POI (Figure 2).²,²⁹,³⁰

The event-driven nature of PROTACs allows them to act catalytically (considered sub-stoichiometric and an innovative pharmacodynamic modality), triggering the degradation of multiple molecules of the POI with just one PROTAC molecule (Figure 3). This distinctive ability to induce protein degradation and the event-driven MOA distinguish PROTACs as having unique therapeutic potential when compared to classical occupancy-driven therapeutics (Figure 3).³¹,³²

Figure 3
Illustration comparing occupancy- and event-driven MOA. In occupancy-driven MOA the small molecule modulates the POI by means of an association-dissociation occupancy of active site (non-catalytic). In event-driven MOA (catalytic), protein function is modulated by degradation, with PROTAC initiating a degradation cascade involving POI ubiquitination followed by its 26S proteasomal degradation.

Indeed, while both inhibitors and degraders share the common objective of diminishing functional proteins, their mechanisms differ: inhibition governs protein function, while degradation manages protein abundance. The main advantage of PROTACs MOA, like in Protac-1, is the depletion of the entire target and thereby disrupts both enzymatic activity and nonenzymatic functions. This comprehensive approach enables PROTACs to tackle potential resistance encountered in current therapeutic treatments.³³,³⁴,³⁵,³⁶ Moreover, PROTACs are also less affected by increases in target expression and mutations in the target protein, as their catalytic action requires only low doses.³⁷,³⁸

1.3. PROTACs mechanistic highlights

The main point of PROTAC-induced degradation MOA resides in the ternary complex (POI-PROTAC-E3) formation, enabling POI polyubiquitination and subsequent proteasomal degradation (Figure 3). Established mathematical models applied to this ternary complex formation³⁹,⁴⁰ predict a bell-shaped dependency on PROTAC concentration (Figure 4a).⁴¹ In the classical occupancy-driven MOA, the amount of effect increases depending on how many of the targets the compound can bind to, ultimately generating a response. As receptor occupancy approaches 100%, the observed effect is diminished (S-shape of concentration-response). For PROTACs, the increase in concentration leads to augmentation of the ternary complex, activating the protein degradation. Differently from the occupancy-driven MOA, exceeding the ideal dose can generate a phenomenon called “hook effect” occurs, diminishing degradation activity due to the formation of an unproductive binary complex (Figure 4a).⁴² Furthermore, interactions between the POI and E3 may stabilize or destabilize ternary complex formation (Figure 4b). A cooperative factor (α = K_{d binary}/K_{d ternary}) provides a measure of susceptibility to ternary complex formation and can be positive or negative. Positive cooperativity (α > 1) occurs when stabilizing PPIs between the POI and E3 promote the ternary complex formation, in which the hook effect can be effectively minimized, which ultimately leads to an amplified production of ternary complex. On the other hand, negative values (α < 1) mean that destabilizing factors are abrogating the ternary complex formation.⁴³

Figure 4
(a) Increasing concentration of PROTAC leading to formation of ternary complex (ideal concentration), and the binary complex and “hook effect” (when concentration is too high). PROTAC compound; POI ligand (yellow), E3 ligase (blue); (b) positive and negative cooperativity working on stabilizing or destabilizing, respectively, PPIs interactions. Favorable interactions (blue wave line), unfavorable interactions like charge repulsion and/or steric clashes (red curves).

The first experimental demonstration of the ternary complex was only described in 2017 by Ciulli and co-workers⁴⁴ laboratory from a complex involving MZ1 (PROTAC) and both the bromodomain of Brd4^BD2 (POI) and VHL (Von Hippel-Lindau disease tumor-suppressor protein, E3 ligase). The crystal structure revealed valuable insights into the interactions among BRD4, VHL, and the PROTAC linker. Through assessments using diverse biophysical methods, the study demonstrated positive cooperativity. This cooperative effect was found to enhance the potency and selectivity of PROTAC MZ1, leading to the induced degradation of specific members within the BRD family (Figure 5).⁴⁴

Figure 5
(a) Crystal structure of the complex PROTAC MZ1 (POI ligand– linker–E3 ligase ligand), Brd4^BD2 and VHL (PDB 5T35); (b) electrostatic potential map showing the charged zipper contacts between Brd4^BD2 residues D381 and E383 with E3 VHL residue R108; and E438 from Brd4^BD2 with E3 VHL residue R69. Blue dashed lines indicate hydrogen bonds. The image and the electrostatic potential were generated in UCSF Chimera alfa version 1.17.⁴⁵ Red surfaces represent negative electrostatic potentials and blue surfaces represent positive electrostatic potentials.

Many times, one can hear that the good affinity of a given PROTAC for its POI is not the most important point for a good degradation. Indeed, because of occupancy-driven MOA, Medicinal Chemists are used to thinking that good affinities can be converted into good activities. However, the event-driven nature of PROTACs is completely associated with the ternary complex formation. As described above, its formation can occur with positive cooperativity due to favorable/stabilizing PPIs,⁴⁴ which can ultimately lead to better activities and potencies. Indeed, Wurz et al.⁴⁶ showed that degradation potency and initial rates of degradation correlate well with the ternary complex binding affinity and cooperativity, not depending only on binary affinities (i.e., POI ligand - POI or E3 ligase ligand - E3 ligase). For example, an ibrutinib PROTAC derivative (1) exhibiting a low binary binding affinity (K_d = 11 µM) towards the Bruton’s tyrosine kinase (BTK) demonstrated potent induction of its degradation, with a half-maximal degradation concentration (DC₅₀) of 1.1 nM and a maximum degradation efficacy (Dmax) of 87% (Figure 6). Many efforts are still needed to understand the ternary complex and cooperativity effects. It is important to bear in mind that due to the catalytic MOA, positive cooperativity is not a perfect rule for good PROTACs, as demonstrated in some examples in which cooperativity was not a main factor for efficient degradation.⁴⁷

Figure 6
Ibrutinib and PROTAC 1 structures. Comparison between binary (half-maximal inhibitory concentration, IC₅₀) and ternary (half-maximal degradation concentration, DC₅₀) potencies of PROTAC 1.

2. Evolution of PROTACs until the Present

Since the discovery and first description of PROTACs by Crews group,¹⁴ the technology has hugely expanded from molecules studied in cell lysates and cell culture to studies in animals and animal disease models, finally reaching clinical trials in humans. PROTACs have also spread from an exclusive academic universe at the beginning to the pharmaceutical and biotechnological industries (Figure 7).

Figure 7
Milestones of the PROTACs’ discovery and development and publications about PROTACs since the first relate in 2001.

Regarding publications in PROTACs and based on the Scopus database, we analyzed the period from 2001 to the end of 2023. Curiously, the entry “PROTAC” was not the most precise since some articles described this acronym for fields other than the proteolysis target chimeras. Thus, our analysis was made with the entries “PROTAC” or “PROTACs” and “proteolysis” or “E3”.

After the discovery of Protac-1 in 2001, a clear gap with minor publications was observed until 2015, when an inflection point started, with an impressive increase from 2017. This first period was marked by peptidic PROTACs (e.g., 2) targeting the degradation of the androgen and estrogen nuclear receptors (AR and ER, respectively), expanding the target scope.⁴⁸ A proof of concept that PROTACs could modulate protein degradation intracellularly was achieved when microinjections of these AR and ER targeting PROTACs demonstrated that they could function in an intact cell. However, these PROTACs lacked good cell permeability (Figure 7).

The subsequent development of PROTACs involved incorporating a peptide from hypoxia-inducible factor 1 subunit-α (HIF1α), a fragment able to recruit the VHL E3 ligase in intact cells, eliminating the need for microinjection.⁴⁹ A shorter peptide fragment of HIF-1α was later incorporated into a PROTAC targeting aryl hydrocarbon receptor nuclear trans20240160 (ARNT).⁵⁰ Despite this PROTAC’s “first generation” (indeed considered “bioPROTACs” today) confirmed promising applications inducing specific degradation of desired targets, the peptide nature of these compounds led to poor cell permeability that summed to the low micromolecular activities, hampered their use as therapeutic products (Figure 7).⁷

The undesirable cell penetration parameters led to efforts to overcome this problem and, in 2008, Crew and co-workers⁵¹ developed the first small-molecule PROTAC targeting AR (4), increasing dramatically the targets reported⁵² to be degraded by PROTACs, consisting of nonsteroidal AR ligand (SARM), a ligand targeting E3 ligase from murine double minute 2 (MDM2-p53 PPI inhibitor, nutlin), and a polyethylene glycol (PEG)-based linker.⁵¹ These compounds were more readily taken up by cells than previous peptide-based PROTACs and more likely to be developed into drugs (Figure 7).²⁹

During the next years, the discovery of multiple other E3 ligases, including cereblon (CRBN) (5),⁵³,⁵⁴ cell inhibitor of apoptosis protein (cIAP) (6),⁵⁴,⁵⁵ and later the VHL (7),⁵⁶,⁵⁷ led to a significant advancement of the PROTAC technology. Special attention and development have been made to VHL and CRBN ligands. While many peptidomimetics of VHL ligand class, with high affinity for the homonym E3 ligase, were developed,⁵⁸,⁵⁹,⁶⁰ as well as further structure-activity relationship (SAR) performed to improve physical-chemical properties maintaining similar affinities towards VHL E3 ligase, the immunomodulatory drugs (IMiDs), including thalidomide, pomalidomide, and lenalidomide, were found to target the E3 CRBN at the molecular level (Figure 7).⁶¹,⁶²,⁶³,⁶⁴,⁶⁵,⁶⁶

Despite promising applications for PROTACs have been demonstrated until the beginning of 2010s,¹,⁷ no in vivo activity evidence was reported until 2013, when PhosphoPROTACs were described as the first in vivo proof of concept of PROTACs, being able of inhibiting tumor growth in murine models.⁶⁷ Moreover, PhosphoPROTACs have distinguished between receptor tyrosine kinase (RTK) signaling pathways by incorporating different peptide sequencers as POI recruiting moiety (Figure 7).

The following years experienced an increase on PROTACs interest and development. In 2014, the first peptide based antiviral PROTACs against HBV was developed;⁶⁸ and in 2015, CRBN and VHL E3 recruiting ligands were used to develop CRBN- and VHL-based PROTACs such as the Halo-PROTACs,⁶⁹ and PROTACs targeting the bromodomain and extraterminal (BRD/BET) family of epigenetic proteins.⁷⁰ In 2016, a new PROTAC class was discovered by Astex Pharmaceuticals,⁷¹ the CLIPTACs (in cell clickformed proteolysis targeting chimeras). These PROTACs could be formed intracellularly by biocompatible reactions such as an inverse electron demand Diels-Alder reaction after treating cells, sequentially, with cell permeable compounds.

Probably, 2019 was a landmark year in which PROTACs shifted from laboratory proof of concepts to a translational exploration when compounds ARV-110 (NCT03888612) and ARV-471 (NCT04072952) entered in clinical trial phase 1 targeting the androgen receptor (AR) and estrogen receptor (ER), respectively (Figure 7). The interest for PROTACs as pharmaceutical products also had a considerable increase after 2019 when the number of patents with the term “PROTAC” was analyzed in Scifinder database. For instance, the number of patents since the first Crews patent in 2002 until 2019 was 57. Only in 2020, 48 patents were registered while this number grew to 176 registers last year. Worth of note, antitumoral and anti-inflammatory compounds correspond to the major applications described inside the patents, with anticancer responding to 61%, though.

The increasing interest of pharmaceutical companies accelerated the translation process of PROTACs from the basic research to the clinical trials and as reflected in the increase in patents number. Furthermore, the clinical significance of the PROTAC approach became evident through the initiation of many clinical trials with PROTAC-based molecules until today (data shown in section 2.1.1).

2.1. Present

The total number of 470 publications in PROTACs last year and 1467 in a 5-years period (2019-2023) shows how this field has grown since its first description in 2001 (Figure 7). The importance of PROTACs is measured not only by the number of publications, but also the high-impact journals in which we can find these publications: Journal of Medicinal Chemistry (149), European Journal of Medicinal Chemistry (110), Cell Chemical Biology (46), ACS Medicinal Chemistry Letters (36), and Journal of the American Chemical Society (36).

Regarding targets reached, in 2019, a review from Sun et al.³⁷ described that around 40 proteins could be degraded by PROTACs at that time. The same group updated this number for about 130 degradable targets in 2021,³⁸ showing an impressive increase of 90 targets in two years. Currently, consulting the PROTAC-DB (an electronic repository of structural and experimental data about PROTACs with POI and chemical structures of POI ligands, along with biological activities and physicochemical properties),⁷² the number of these targeted proteins has increased to 323 (an increase of 70% in degradable targets in only 2 years),⁷² indicating that the era of protein degradation has indeed arrived.

Maybe one of the main appeals to PROTACs is the possibility of reaching “undruggable” targets. Indeed, some compounds were developed as degraders of proteins lacking a catalytic site or a small-molecule binding site like aberrant Tau, present in frontotemporal dementia.⁷³,⁷⁴,⁷⁵ The most frequently mutated gene family in cancers,⁷⁶,⁷⁷,⁷⁸,⁷⁹ rat sarcoma virus (RAS) (Kirsten (KRAS), neuroblastoma (NRAS) and Harvey (HRAS)), was at one time termed as “undruggable” and was a model for the development of many PROTACs. Today, modulating RAS using PROTACs is still an open avenue for discovering RAS therapies and understanding its basic cell biology.⁸⁰ Nevertheless, the use of PROTAC for degrading these proteins, particularly those engaged in PPIs, remains somehow restricted due to challenges in identifying small-molecule binders for these proteins.⁸¹ New techniques are welcome to identify new chemical entities that bind to these “undruggable” POI, like the recently described site-specific and fragment-based covalent ligand screening using liquid chromatography-tandem mass spectrometry (LC-MS/MS).⁸¹

In contrast, PROTACs are widely used for targeting kinases (n = 195 kinases), today representing around 60% of all described PROTACs’ targeted proteins. At first sight, it appears to be countersense since kinases are already an explored target by the classical occupancy-driven pharmacology MOA. The main point here is that many kinases possess well-established and potent inhibitors or high affinity ligands. These compounds can be readily modified to incorporate linkers, keeping adequate binding affinity. Furthermore, kinases exhibit deep binding pockets, facilitating the binding of PROTACs, and prompting interaction between the kinases and the E3 ligases, leading to ubiquitination and eventual degradation.

Additionally, PROTACs may solve one of the main problems of adenosine 5’-triphosphate (ATP)-competitive kinase inhibitors, the lack of selectivity due to the high degree of homology of ATP binding sites between kinases.³⁸ When nonselective inhibitors are used as POI ligands for designing new PROTACs, both the protein ligands and the degraders can bind to the corresponding kinases. However, in the case of PROTACs this recognition induces specific protein-protein interactions between the POI and E3 ligase, forming a ternary complex that can be very specific. This two-step recognition mechanism contributes to the selective degradation of targets.³⁸ This curious characteristic is because functional residues in proteins tend to be highly conserved over evolutionary time. Jack et al.⁸² described an analysis with 524 distinct enzymes, demonstrating a highly evolutionary conservation of amino acid residues near the catalytic site, while in contrast, sites in protein-protein interfaces are only weakly conserved. The lack of selectivity for ATP-competitive kinase inhibitors resides in the interaction of these compounds with the highly conservative sites of the enzymes, while in PROTACs, the ternary complex conduct the E3 ligase to a lesser conservative region of enzymes, i.e., the protein-protein interfaces, possibly giving completely different modes of interactions.

An interesting example was described by Bondeson et al.⁸³ From a high-throughput competitive binding assay, they discovered that foretinib at 10 µM could bind to more than 130 different kinases (Figure 8). Taking foretinib as model for the POI ligand, they designed and synthesized two PROTACs (8 and 9) using different E3 ligase ligands (CRBN (8) and VHL (9)). PROTACs 8 and 9, at the same concentration, significantly changed the binding profile, in which 8 retained binding to 52 kinases while 9 bonded 62 kinases and were able to only degrade less than 10 kinases. Additionally, they demonstrated that PROTAC 8 effectively degraded the therapeutically relevant protein p38a/MAPK14, despite weak binding affinity (Figure 8).⁸³

Figure 8
Use of non-selective inhibitor foretinib as POI ligand for designing new selective PROTACs.

Common observed drug resistance via mutations (close to the inhibitor binding pockets), gain of scaffolding function or overexpression of drug targets in long term treatments with traditional kinase inhibitors are explanations to the current option for new PROTACs. The event-driven MOA of PROTACs, resulting in catalytic removal of POI, can evade drug resistance from long-term selection pressure by degrading target proteins.⁸⁴ Furthermore, studies have revealed that PROTACs can induce the degradation of many mutated kinases like BTK. This highlights the potential of TPD for treating diseases, e.g., chronic lymphocytic leukemia (CLL), resulting from kinase resistance to ibrutinib due to mutations.⁸⁵,⁸⁶,⁸⁷ While the ibrutinib MOA is to covalently bind to cysteine in the ATP binding pocket of BTK, exactly where the mutation occurs (C481S), PROTAC derivatives were not designed to covalently bind to BTK, inducing the degradation of both wildtype and mutant (C418S) forms.³⁴,⁴⁶,⁴⁶,⁸⁵,⁸⁸

2.1.1. Clinical proof-of-concept

All the attractive characteristics of PROTACs described herein had their clinical validation when ARV-110 (AR) and ARV-471 (ER) entered in clinical trials in 2019. The oral efficacy and safety of ARV-110 have been proven in managing metastatic castrated prostate cancer (mCRPC),⁸⁹,⁹⁰,⁹¹ while ARV-471 has demonstrated promising results in treating breast cancer.⁹² Since them, an increasing number of protein targets have emerged to develop clinical degraders and 21 new PROTACs entering in clinical trials as single chemical entities or as pharmaceutical combination with other drugs, and nowadays, 20 clinical trials are still running. These PROTACs degraders are based on different E3 ligands, but mainly on CRBN-based ligands.

While the first two PROTACs modulated androgen (AR) and estrogen (ER) receptors, many other targets raised such as B-cell lymphoma-extra large (BCL-X_L), bromodomain-containing protein 9 (BRD9), signal transducer and activator of transcription 3 (STAT3) (Table 1 and disclosed structures in Figure 9). Currently, almost all clinical trials are focused on research for anticancer PROTACs. However, recently (in 2021) the first clinical degrader of interleukin-1 receptor associated kinase 4 (IRAK-4) for autoimmune inflammation-diseases entered in clinical trials, the KYMERA’s degrader KT-474.⁹³ Worth of note, KT-474 is already in phase 2 clinical trials and is showing that degraders for inflammatory conditions might be an interesting and promising field.

Table 1
Status and characteristics of PROTACs currently under clinical trial evaluations

Year	Comp	PROTAC	ROA	Target	Main indications	E3	CP	NCT number	Status
2019	Arvinas	ARV-110	oral	AR	metastatic castration resistant prostate cancer	CRBN	I/II, Ib	NCT03888612	A, NR
							Ib	NCT05177042	A, NR
2019	Arvinas / Pfizer	ARV-471	oral	ER	ER+/HER2- locally advanced or metastatic breast cancer	CRBN	I	NCT05538312	C
								NCT05930925
								NCT05652660
								NCT05673889
								NCT06005688
								NCT05463952	A, NR
								NCT05501769
								NCT05732428
							I/II	NCT05548127	R
								NCT04072952
								NCT06206837	NYR
							II	NCT05549505	A, NR
								NCT06125522	R
								NCT05573555
								NCT01042379
							III	NCT05909397	R
								NCT05654623
2020	Bristol Myers Squibb	CC-94676	oral	AR	metastatic castration-resistant prostate cancer	CRBN	I	NCT03554993	C
								NCT04958291
2021	BeiGene	BGB-16673		BTK	B-cell malignancies	CRBN	I	NCT05294731	R
								NCT05006716
2021	Nurix Therap.	NX-2127	oral	BTK	relapsed/refractory B-cell malignancies	CRBN	I	NCT04830137	A, NR
2021	Nurix Therap.	NX-5948	oral	BTK	relapsed/refractory B-cell malignancies	CRBN	I	NCT05131022	R
2021	Haisco	HSK29116		BTK	relapsed/refractory B-cell malignancies	CRBN	I	NCT04861779	R
2021	Lynk	LNK-01002		Ras GTP-ase	myelofibrosis (PV-MF, ET-MF) or acute myeloid leukemia	CRBN	I	NCT04896112	W
2021	Accutar Biotech	AC682	oral	ER	locally advanced or metastatic ER+ breast cancer	CRBN	I	NCT05489679	T
							I	NCT05080842	A, NR
2021	Arvinas	ARV-766	oral	AR	metastatic castration-resistant prostate cancer	undisclosed	I/II	NCT05067140	R
2021	Dialectic Therap.	DT2216	i.v.	BCL-XL	solid tumor, hematologic malignancy	VHL	I	NCT04886622	C
2021	Foghorn Therap.	FHD-609	i.v.	BRD9	advanced synovial sarcoma	undisclosed	I	NCT04965753	A, NR
2021	Kymera / Sanofi	KT-474	oral	IRAK-4	autoimmune diseases, atopic dermatitis (AD) or hidradenitis suppurativa (HS)	CRBN-based	I	NCT04772885	C
							II	NCT06058156,	R
								NCT06028230
2022	Hinova	HP518		AR	metastatic castration-resistant prostate cancer	CRBN	I	NCT05252364	C
							I	NCT06155084	NYR
2022	Kintor	GT-20029		AR	prostate cancer	CRBN	I	NCT05428449	C
2022	Arvinas	ARV-110		AR	ARV-110 in combination with abiraterone	CRBN	I	NCT05177042	A, NR
							I/II	NCT03888612	A, NR
2022	Kymera	KT-413	i.v.	IRAK-4	relapsed or refractory B-cell NHL	CRBN	I	NCT05233033	S
2022	Kymera	KT-333	ND	STAT3	lymphoma, large granular lymphocytic leukemia, solid tumors	undisclosed	I	NCT05225584	R
2022	C4 Therap.	CFT8634	oral	BRD9	synovial sarcoma	CRBN	I/II	NCT05355753	T
2022	C4 Therap.	CFT8919	oral	EGFR-L858R	non-small-cell lung cancer	CRBN	I	FDA permission, not yet started	–
2022	Accutar Biotech	AC176	oral	AR	prostate cancer	undisclosed	I	NCT05241613	A, NR
2022	Cullgen	CG 001419	oral	TRK	cancer and other indications	CRBN	I/II	FDA permission, not yet started	–
2022	Astellas Pharma	ASP3082	i.v.	KRAS G12D	solid tumor	undisclosed	I	NCT05382559	R

• PROTAC: protheolysis targeting chimera; ROA: route of administration; CP: clinical phase; NCT numbers: National Clinical Trial (NCT) identifier number; AR: androgen receptor; CRBN: cereblon; A: active; NR: not recruiting; ER: estrogen receptor; C: completed; R: recruiting; NYR: not yet recruiting; BTK: Bruton’s tyrosine kinase; PV-MF: polycythemia vera myelofibrosis; ET-MF: essential thrombocythemia myelofibrosis; W: withdrawal; T: terminated; i.v.: intravenous; VHL: Von-Hippel-Lindau; BRD9: bromodomain-containing protein 9; IRAK-4: interleukin-1 receptor associated kinase 4; NHL: non-Hodgkin lymphoma; S: suspended; STAT3: signal transducer and activator of transcription 3; EGFR-L858R: L858R mutant form of the epidermal growth factor receptor; FDA: Food and Drug Administration; TRK: tropomyosin receptor kinase; KRAS G12D: Kirsten rat sarcoma virus gene mutation G12D.

Figure 9
All disclosed structures of PROTACs under clinical evaluations.

2.1.2. Current drawbacks of PROTACs and possible avenues to improvements

Despite many advantages discussed above in this review, PROTACs still have space for improvement in some areas that can be considered disadvantageous,⁹⁴ such as:

(i) PROTACs have some drawbacks in terms of potential toxicities, stemming from either on-target or off-target protein degradation. PROTACs may lack selectivity, leading to the degradation of other proteins (off-target effect) or causing unselective degradation of the POIs in undesired tissues (on-target effect). This challenge can be partially addressed using other administration ways, like topical PROTACs that have proven to mitigate systemic exposure and associated side effects. A recent illustration is compound GT20029 (structure not yet disclosed), targeting AR and currently undergoing phase I clinical trials in China.⁹⁵ However, in cases where systemic administration is necessary, targeted delivery systems can potentially overcome issues related to poor selectivity. In contrast to conventional small molecules, PROTACs typically present greater complexities in terms of drug metabolism and pharmacokinetics (DMPK) and need more comprehensive safety evaluations.⁹⁶ The metabolites of PROTACs, particularly those formed through linker cleavage, have the potential to competitively bind to the POI or the E3 ligase. This competitive binding may “antagonize” the degradation of the POI, consequently diminishing the efficacy of the original PROTAC. Hence, there is a need to develop innovative approaches for characterizing pharmacokinetics and metabolite profiling.⁹⁶

(ii) PROTACs are molecules with properties laying outside Lipinsk’s rule of five that can negatively affect their pharmaceutical effects.⁹⁷,⁹⁸,⁹⁹ As an illustration, PROTACs possess a higher molecular weight (MW) in comparison to conventional small-molecule inhibitors, potentially posing a pharmacokinetic challenge to their cellular permeability.¹⁰⁰ However, upon oral administration, PROTACs have demonstrated the ability to induce degradation of the POIs in any accessible cells as proved in clinical trials. Recent studies¹⁰¹,¹⁰² reveal an intriguing aspect of PROTACs, indicating that their properties can somehow contribute to their cell-permeability. An example from a PEG-linker PROTAC demonstrated varying conformations in different environments. In aqueous solvents like extra- and intra-cellular compartments, the PROTAC assumed elongated and polar conformations. Conversely, in apolar solvents such as chloroform, simulating the cell membrane interior, the PROTAC adopted conformations with a smaller polar surface area. It suggests that the flexibility of PROTACs could facilitate a kind of chameleonic behavior, allowing the PROTAC surface to adapt to the solvent, ensuring good solubility in both polar and apolar compartments and, consequently, enabling cellular permeability.¹⁰¹,¹⁰²

(iii) A current obstacle in advancing proteolysis targeting chimeras (PROTACs) lies in the empirical nature of structure-activity relationships (SARs) associated with linker length as well as the flexibility, that is a crucial parameter to be considered during design and development. More than only influencing PK properties,¹⁰³,¹⁰⁴ length and flexibility “play important roles in inducing the ternary complex formation. In this context, employing macrocyclization or conformational restriction strategies to rigidify the linkers, and so fixing POI and E3 ligands in the bioactive conformation, may promote the formation of ternary complexes and improve the degradation profile of the PROTAC.⁹⁶

(iv) At elevated concentrations, PROTACs exhibit “hook effects”, wherein the competitive formation of a target POI-PROTAC or E3 ligase-PROTAC binary complex occurs, leading to diminished efficacy. This phenomenon has been observed in various studies⁹⁴,⁹⁷,¹⁰⁵ and can lead to problems regarding differences in metabolic capabilities between populations.

(v) Despite previous theories, it is already possible to identify PROTACs’ resistance emerging through genomic alterations in the core components of E3 ligase complexes.¹⁰⁶ This is an additional challenge for PROTAC development arising from the potential development of resistance in cells treated with degraders containing CRBN and VHL E3 ligase recruiters.¹⁰⁶ Thus far, a limited number of E3 ligases (e.g., CRBN, VHL, MDM2, IAP) are used for PROTAC development (discussed in section 3.2). CRBN and VHL E3 ligases are generally considered to be ubiquitously expressed in humans, showing limited selectivity of PROTACs in cancer cells over normal cells. There are some exceptions when tumor enrichment of an E3 ligase coincides with the dependence of the tumor on expression of that ligase. An analysis with CERES scores in DepMap of E3 ligases across multiple tumor cell lines indicated these correlations.¹⁰⁷ This discovery has enabled the recognition of E3 ligases and other genes associated with the UPS, with some better-characterized than others (e.g., WD repeat-containing protein 82 (WDR82) and cell division cycle 20 (CDC20)), that demonstrate significant tumor essentiality across various cancer cell types.¹⁰⁸ Ligases exhibiting these profiles offer the advantage of reducing the likelihood of tumor cells developing resistance to PROTACs through ligase-based mechanisms.

(vi) New E3 ligase scaffolds must be developed. CRBN-based structures (the most used scaffold) are thalidomide like derivatives that are known as possible degradation inducer of Structural Design of PROTACs. From this optics, PROTACs must be as selective as possible to prevent the degradation of proteins caused by the CRBN ligands themselves.⁶⁴,¹⁰⁹

(vii) As explained before, the number of targets that can be degraded by PROTACs has greatly expanded. However, most of them are “druggable” targets while some others are still challenge to tackle via small-molecule degraders.¹ For instance, modulation of targets lacking well defined binding sites and hydrophobic pockets have received growing attention with the use of new potential strategies as oligonucleotide-based PROTACs.¹,⁹⁶,¹¹⁰,¹¹¹

3. Design of PROTACs

From a structural perspective, the success of designing PROTACs for targeting different proteins, as quickly discussed above, depends on integrating three distinct chemical moieties: a ligand for POI, an E3 ligase binder, and a linker connecting these two components.¹²,³⁷,¹⁰⁵ In a Medicinal Chemistry point of view, the design strategy of PROTACs is not different from the so called “Molecular Hybridization”, which is a strategy that aims to combine two or more molecules (or parts of them) in a new, single chemical entity.¹¹²,¹¹³ Identifying the proper combination of these three elements is the first and pivotal step, needing a thorough examination of the structural features of both the E3 ligase and the POI. The formation of a ternary complex is completely dependent on the spatial orientation and alignment of both elements (POI and E3 ligase ligands), which plays a pivotal role in conducting the ubiquitin-protein knockdown process. As so, the design is specially influenced by the attachment position used to link the E3 ligase and POI ligands, a parameter that can be anticipated based, for example, on the binding mode of the POI and its ligand from a co-crystallized structure or by using computational tools. Additionally, the length and flexibility of the linker connecting the two moieties, i.e., POI and E3 ligase, can markedly impact potency and selectivity.⁵⁶,⁸³,¹⁰⁵,¹¹⁴,¹¹⁵

3.1. The POI ligand requirements

The structural requirements for the POI ligand choice/exploration always depends on which target is supposed to be used. Regarding these ligands, a diverse range of warheads has been documented,¹¹⁶ encompassing non-covalent, irreversible, and reversible covalent ligands, as well as allosteric variants.¹¹⁷ Good consultation sources of POI ligands are the PROTAC-DB⁶⁴,¹⁰⁹ database and PROTACpedia.⁷²,¹¹⁸ Both serve as valuable resources for PROTAC development by gathering the chemical structures, biological activities, and physicochemical properties of POI ligands.

As described within topic “2.1. Present”, most of the current proteins targeted by PROTACs are “druggable” targets. For these POI, structural information can many times be found, such as crystal structures with bounded ligands or their SAR, guiding the design of new degraders. Based on these data, it is feasible to identify optimal linker attachment points in solvent-exposed regions that do not interact with the POI. Two examples of degraders starting from co-crystallized ligands are described in Figure 10. The potent IRAK-4 (enzyme involved in inflammatory processes) degrader 10 was designed based on the crystal structure of PF-06650833 (a phase II IRAK-4 inhibitor) inside the IRAK-4 kinase domain, showing solvent exposed vector at 4-position of isoquinoline scaffold. This position was selected to the linker attachment and led to the PROTAC 10 with a DC₅₀ of 151 nM (Figure 10a).¹¹⁹ The second example describes a similar approach for the discovery of the GPX4 degrader, the GDC-11. From the covalent inhibitor ML 162, crystallized with GPX4, three sites that were observed turned to the solvent and able to be modified. To the original anisole moiety, an amide linker and a pomalidomide based E3 ligase ligand were attached giving the PROTAC GDC-11, which presented a moderate GPX4 degradation efficacy of 33% at 10 µM (Figure 10b).¹²⁰

Figure 10
(a) Design of PROTAC 10 based on the crystal structure (PDB 5UIU) of phase II IRAK-4 inhibitor PF-06650833; (b) discovery of the GPX4 degrader GDC-11 from the co-crystallized covalent inhibitor ML 162 (PDEB 6HKQ). The image and the hydrophobic surfaces were generated in UCSF Chimera alfa version 1.17.⁴⁵ Red surfaces represent hydrophobic regions and blue surfaces represent hydrophilic surfaces.

Nevertheless, the absence of a well-defined ligand capable of engaging the intended target limits early-stage drug discovery initiatives, particularly those focused on novel biological targets characterized by poorly understood pharmacology. These “undruggable targets” need more technologies helping the discovery of new compounds useful as POI ligands. However, it is important to remember that the affinity of the ligand to the POI is not the main focus on PROTACs, since their mechanism of action is based on an event-driven sub-stoichiometric catalytic MOA (Figure 3).⁵,¹²,¹³ Thus, techniques like fragment based drug discovery (FBDD), which is frequently used to discover new ligands (usually with low affinities) can be a valuable starting point for the discovery of new POI ligands moieties.¹²¹ Indeed, many successful examples of PROTACs used the FBDD at the beginning of their discovery process including CST905 (BRAF^V600E-PROTAC), SIAIS100 (BCR-ABL-PROTAC) and 11 (CDK9-PROTAC) (Figure 11).¹²²,¹²³,¹²⁴

Figure 11
Discovered PROTACs in which the FBDD process was applied at the discovery of the POI ligand moiety (yellow).

Maybe the most successful example of FBDD in the drug discovery process is the BCL-X_L-PROTAC DT2216, that is currently in phase 1 clinical trials (Table 1 and Figures 9 12).¹²⁵ This PROTAC was developed from a nuclear magnetic resonance (NMR)-based screening that furnished two active fragments (12 (300 μM) and 13 (4300 μM)) binding in adjacent sites. Both fragments were linked by the sulfonamide group, leading to compound 14 (half-maximal inhibitory concentration (IC₅₀) = 93 nM) (Figure 12). A lead optimization process was carried out with further modifications in this structure, specifically outside the active pocket, aiming at improving hydrophobic interaction and the π-π stacking with BCL-X_L resulted in compound ABT-737 (15) (IC₅₀ = 83 pM). Due to its low solubility and high lipophilicity, which resulted in poor pharmacokinetic properties, optimization of compound 15 continued by replacing the hydrophobic groups with hydrophilic ones. Furthermore, the crystal structure of compound 15 (PDB 2YXJ) indicated that the tertiary amine was turned to an open space, and to this group, the linker and VHL E3 ligase were attached, giving the compound DT2216 (Figure 12).

Figure 12
FBDD applied to the discovery of the BCL-X_L-PROTAC DT2216 (phase 1 clinical trials) from the two fragment hits (12 and 13) identified by NMR (PDB 1YSG). The image and the hydrophobic surfaces were generated in UCSF Chimera alfa version 1.17.⁴⁵ Red surfaces represent hydrophobic regions and blue surfaces represent hydrophilic surfaces.

3.2. The linkers as fundamental moieties

Linkers play a crucial role in the activities of PROTACs. The adaptability of the linkers significantly influences the overall degradation efficiency of a specific PROTAC. Parameters such as the distance between the POI and the UPS, and the presentation and accessibility of reactive lysine residues on the POI to the E2 are vital factors, ultimately determined by the characteristics of the linker unit. Nonetheless, the connection between the spatial arrangement of lysine residues on the POI surface, the structure, and interconnection of poly-Ub chains, and the effectiveness of degradation remains inadequately comprehended.¹¹⁴,¹²⁶ Guidelines for formulating potent PROTAC linker designs, from scratch, are regrettably absent and, to date, studies on the SAR of linkers are predominantly empirical, and the design of linkers still poses a bottleneck.

The appropriate combination of length, hydrophilicity, and rigidity in ligand-connecting linkers has been demonstrated to influence various properties of PROTACs such as pharmacodynamic (PD), and pharmacokinetic (PK) characteristics such as cellular permeability, metabolic stability, solubility, and, of course, biological activity.¹¹⁴,¹²⁷,¹²⁸ This combination forms the basis for the successful design of effective PROTACs. Optimization of linker composition is crucial for each ligand pair (POI and E3 ligase), especially concerning the length and conjugation sites on each ligand.⁴⁶,¹¹⁴ A commonly employed strategy involves creating a library of PROTACs incorporating linear unsaturated aliphatic linkers of variable lengths, typically ranging from a few atoms to 29 atoms. This process continues until a suitable spatial orientation is identified, proving productive for ternary complex formation between the target POI and the E3 ubiquitin ligase.¹,¹⁰³,¹¹⁴ The most potent PROTAC linkers are selected through systematic and extensive iterations, incorporating different chemical motifs.

Typically, longer aliphatic linkers, including polyethylene glycol and other glycol chains, have produced substantial contributions to protein-ligand interactions within the ternary complex.¹²⁹,¹³⁰,¹³¹,¹³² They have played a role in stabilizing the orientation of the complex through cooperative binding. However, there are cases where the energy gained in the ternary complex from new PPIs is counteracted by the entropic cost associated with reduced PROTAC flexibility.¹⁰³ Recently, there has been a shift from flexible, linear alkyl- and PEG linkers to more rigid structures, including alkyne, and cyclic scaffolds like piperazine, piperidine, and triazole (Figure 13). This sentence is particularly true if we have a look to the disclosed structures from compounds in clinical trials in Figure 9, in which nine of eleven PROTACs have more rigid linkers.

Figure 13
The most common linker moieties used in PROTAC design according to chain flexibility.

One prevalent approach to combine POI and an E3 ligase binders involves the utilization of click chemistry, such as the copper-catalyzed Huisgen 1,3-dipolar cycloaddition reaction described by Sharpless and co-workers,¹³³ Meldal and co-workers,¹³⁴ and Bertozzi and co-workers,¹³⁵ which incorporates an azide and an alkyne moiety given an 1,2,3-triazol. An extensive and insightful review on this technique applied to PROTACs was provided by Pasieka et al.,¹³⁶ covering a wide range of aspects related to the topic. Beyond the broad applicability and excellent compatibility of this linking strategy, the resultant triazole ring may offer a metabolic advantage compared to linear linkers being more susceptible to oxidative metabolism in vivo, as highlighted by Xia et al.¹³⁷ Furthermore, improving linker rigidification can potentially reduce entropy loss within the system.

The first two articles introducing the utilization of cu-catalyzed azide-alkyne cycloaddition (CuAAC) to PROTAC synthesis were concurrently published in a special issue of the Journal of Medicinal Chemistry. The research conducted by Jung and co-workers¹³⁸ focused on the synthesis of triazole-base (Sirt), a family of histone deacetylases implicated in the pathogenesis of various diseases, including inflammation.¹³⁸,¹³⁹ A Sirt2-selective and potent compound (SirReal) was chosen as the ligand for the POI, while the well-established CRBN ligand thalidomide was selected as the E3 ligase binder. For the first time the use of a triazole linker led to novel degraders and induced the degradation process of Sirt by facilitating the formation of a ternary complex (Figure 14).¹³⁸ In the second work, Wurz et al.¹⁴⁰ developed PROTACs targeting BRD4 utilizing the well-known JQ-1 moiety,¹⁴¹ which binds to BRD4, along with E3 ligase binders from both the VHL and CRBN classes, connected by PEG linkers. Proximity and protein degradation assays confirmed the capability of the triazole-based PROTACs 16-18 to form the ternary complex, thus inducing proteasome-mediated degradation (Figure 14).¹⁴⁰

Figure 14
Synthesis of first-in-class 1,2,3-triazole-based PROTACs 16 as Sirt2 degraders and 17 and 18, as BRD4 degraders.

Another interesting feature involved with linkers is the possible photochemical modulation of PROTACs activity, enabling spatiotemporal control of PROTAC-mediated protein degradation, which has potential to avoid side effects.¹⁴² Two possible photochemically controllable PROTACs (PHOTACs) are known: (i) photocaged, that is a modified PROTAC designed by caging the PROTAC, thus leading to an inactive degrader, in which the light irradiation can remove the substituent leading to an active PROTAC able to conduct the protein degradation.¹⁴³ This approach, despite applied to linkers, can be also used in POI and E3 ligase binders; and (ii) photoswitchable PROTACs, that is applied specially to linkers. This photochemical modulation is an alternative approach to locally activate PROTACs by means of a photoisomerization.

Pfaff et al.¹⁴⁴ designed the photoswitchable PROTAC 19 by integrating a bistable ortho-tetrafluoroazobenzenes (ortho-F₄-azobenzenes) linker between the POI ligand and the E3 ligase ligand. So far, azobenzenes are the most common class of photoswitches used for the photo-control of biomolecules.¹⁴⁵ The lead PROTAC structure chosen was ARV-771, where the linker’s length between the POI ligand and the E3 ligase ligand is approximately 11 Å. As illustrated in Figure 15, substituting the PEG-based linker in ARV-771 with ortho-F₄-azobenzene resulted in an anisomeric photo PROTAC pair.¹⁴⁴ The trans-PROTAC 19 (active form) maintained an optimal distance of 11 Å between both ligands, while cis-PROTAC 19 (inactive form) presented a shorter distance of only 8 Å. Photoswitch occurred upon exposure to 530 nm irradiation (visible light), in which trans-PROTAC 19 could be converted into cis-PROTAC 19. Conversely, under 415 nm irradiation, cis-PROTAC 19 could be transformed into trans-P ROTAC 19. Intriguingly, trans- P ROTAC 19 induced the degradation of BRD2 but not BRD4 in Ramos cells after 18 h, while no apparent degradation was observed with cis-PROTAC 19. In contrast to photocaged PROTACs, photoswitchable PROTACs provide a reversible on/off switch for targeted protein degradation.¹⁴⁴

Figure 15
Photoswitchable BET PROTAC design: substitution of the PEG linker in ARV-771 by an ortho-F₄-azobenzene generating anisomeric photo-PROTAC 19 pair of isomers with considerable changing in the distance between POI ligand and E3 ligase binder moieties.

3.3. The E3 ligase universe in expansion

The selection of the E3 ligase ligand plays a crucial role in determining the ultimate success of PROTACs, since they are the “responsible moiety” to recruit the degrader machinery.

The human genome contains two members of the E1 enzyme family, approximately 40 E2, and over 600 E3 ubiquitin ligases.¹⁴⁶ While our comprehension of substrate recognition and the regulation of ubiquitination remains incomplete, the genome’s choice of approximately 600 E3 ligases demonstrates the capacity to ubiquitinate a significantly larger number of protein substrates in a controlled manner, exhibiting considerable specificity.¹⁴⁷ Playing a vital role in protein ubiquitination, E3 ligases contribute to substrate selection and influence the efficiency of the ubiquitin cascade.²² The success of a protein knockdown depends directly on the selection of the best E3 ligase. Different degradation profiles can be observed on a specific target based on the recruited E3 ligase.¹⁴⁸

A beautiful example of this specificity was demonstrated by Ciulli and co-workers,¹⁴⁹ when they reported the development of a leucine-rich repeat kinase 2 (LRRK2) (implicated in Parkinson’s disease and inflammatory processes) PROTAC XL01126,¹⁵⁰ as an alternative LRRK2-targeting strategy. Initial design and screenings of PROTACs based on ligands for the E3 ligases VHL, CRBN, and cIAP identified the best degraders, always containing a thioether-conjugated VHL ligand, while other E3 ligase ligands presented none or insipient activity (Figure 16). A structural optimization in a second step led to the discovery of XL01126 as a fast and potent degrader of LRRK2 in multiple cell lines, with DC₅₀ varying within 15-72 nM, high cell permeability, positively cooperative ternary complex with VHL and LRRK2 (α = 5.7) (Figure 16). It also presented interesting PK properties being orally bioavailable (F = 15%) and penetrating to the blood-brain barrier (BBB) after either oral or parenteral dosing in mice.¹⁴⁹

Figure 16
Development of the LRRK2 PROTAC XL01126 by screening different ligands for the E3 ligases: von VHL, CRBN, and cIAP.

Each E3 ligase has its own specificities and, despite the vast number of known E3 ligases, until now not many of them have been successfully used as PROTACs’ targets, as exemplified until here, in this review: e.g., VHL, MDM2, cIAP, and CRBN. Thus, a substantial universe of E3 ligases is still unexplored, holding promise for targeted protein degradation. Consequently, E3 ubiquitin ligases are garnering attention as appealing drug targets, given their implication and dysregulation in various diseases.⁵³,⁹⁶ A significant amount of the initial efforts has focused on pairing VHL or CRBN with various target proteins to optimize resource utilization and accelerate advancement of PROTACs and indeed, these are the most common E3 ligase ligands found in “PROTACs’ world”.

We proceeded with an analysis on PROTAC-DB⁷² for mapping the relevance of each E3 ligase ligand to the PROTACs’ development until today. CRBN (65%, n = 3530) is so far the most used ligand, followed by VHL (29%, n = 1578), the IAP family (XIAP, cIAP, and IAPs; 4%, n = 190) and MDM2 (1%, n = 56) (Figure 17). Main representants of these E3 ligase ligand families are illustrated in Figure 18. The most used E3 ligases explored (CRBN and VHL) are considered to have low tissue-specific expression and already presented reports of punctual mutations in cancer models. This demonstrates the importance of developing new ligands for E3 ligase and some are already ongoing, like DCAF11, DCAF15, DCAF16, FEM1B, RNF114, RNF4,¹⁵¹,¹⁵²,¹⁵³,¹⁵⁴,¹⁵⁵ that are depicted as “others” in Figure 19, still representing an incipient 1% of total examples, within 34 PROTACs. Despite a few examples, the main structures of these new E3 ligase ligands are disclosed in Figure 19. The analysis of PROTACs undergoing clinical trials was impacted by five compounds (22%) for which the structures have not yet been disclosed. Within the disclosed structures, again CRBN is for sure the most applied (74%, n = 17), with VHL presenting only 1 compound (4%) (Figure 17).

Figure 17
Representative analysis of E3 ligase ligand families described on PROTAC-DB⁷² (accessed on 02-19-2024) and under current clinical trials.

Figure 18
Main representants of the most used E3 ligase ligands families according to the correspondent E3 ligase. The black wave line represents the point of attachment to the linker.

Figure 19
Representants of the “other” used E3 ligase ligands families according to the correspondent E3 ligase.

Nowadays there is increasing interest in academia and industry for identifying E3 ligases with unique expression profiles to enable tissue- and cell-type-specific target degradation.¹ Recent discoveries¹⁵⁶ suggested that E3 ligases with tissue-selective expression profiles may present unique therapeutic opportunities, even though their mechanisms have not yet been fully elucidated. For example, specific E3 ligases from the central nervous system (CNS) have emerged, including ring finger protein 182 (RNF182) and tripartite motif-containing protein 9 (TRIM9).¹⁵⁷,¹⁵⁸ These ligases are particularly noteworthy for addressing targets associated with neuronal diseases, possibly allowing CNS-specific therapeutic targeting, and avoiding systemic off-target and toxicity effects, despite no PROTAC has been developed until now for these ligases. The same idea can be used to treatments focusing on degrading specific proteins in cells with improvement of a determined E3 ligase, such as the F-box protein 44 (FBXO44) that is enriched in some tissues but not specific to any.¹⁵⁹,¹⁶⁰

4. PROTACs Developed for Inflammation Processes

4.1. PROTACs developed for histone deacetylases (HDACs)

After their introduction in cancer therapy, histone deacetylases (HDACs) inhibitors (HDACi) gained attention for application in other diseases as well. HDACs play crucial roles in inflammatory diseases such as asthma, rheumatoid arthritis (RA), and chronic obstructive pulmonary disease (COPD).¹⁶¹,¹⁶²,¹⁶³,¹⁶⁴ Understanding and intervening in the functions of specific isoforms that contribute to inflammation provide opportunities for the development of novel therapeutics. In RA, specifically, HDAC3i significantly suppressed most interleukin (IL)-1β-inducible genes targeted by pan-HDACi. The same result was reproduced by silencing HDAC3 expression. These results identify HDAC3 as a potential therapeutic target in the treatment of inflammatory and autoimmune diseases and indicated that the degradation of HDAC3 would be an interesting approach for this purpose.¹⁶²,¹⁶³,¹⁶⁵

Cao et al.¹⁶⁵ described the development of PROTACs targeting HDAC3 for degradation, containing pomalidomide (21) as CRBN-based E3 ligase recruiter, linked to a class of I HDAC inhibitors (22) with the ortho-aminoanilide subunit (Figure 20a). The nucleus ortho-aminoanilide was chosen because derivatives of this class exhibited specific slow-tight binding to the active site of class I HDACs 1, 2, and 3, a feature that could be advantageous in the development of PROTACs.¹⁶⁶,¹⁶⁷ Moreover, two notable inhibitors from this class, entinostat and CI994, are currently undergoing clinical trials.

Figure 20
New PROTACs based on ortho-aminoanilide compounds. (a) Design and evaluation of PROTACs 23a and 23b using CRBN E3 ligase ligand; (b) design and evaluation of PROTAC 23a and 23b using VHL E3 ligase ligand.

The authors¹⁶⁵ synthesized a series of PROTACs varying the linker length between pomalidomide and the HDAC inhibitor subunit. PROTAC 23a was capable of degrading HDAC3 at a concentration of 10 μM in murine macrophage cell line RAW 264.7 macrophages, while HDAC1 and HDAC2 were also degraded at higher concentrations though. Curiously, it was observed that the efficacy of PROTAC-mediated degradation was somehow cell selective depending on the type of cell line used, as no degradation of HDAC3 was observed in A549 cells. The presence of the para-fluoro ortho-aminoanilide group in 3b provided increased selectivity for HDAC3. Despite compound 23a has been more potent as HDACs inhibitor, compound 23b exhibited higher potency and enhanced selectivity for HDAC3 degradation compared to 23a, with a DC₅₀ value of 0.32 μM in RAW 264.7 macrophages.¹⁶⁵

A novel PROTAC (25) that links the HDAC inhibitor ligand (24) to VHL E3 ligase recruiter, instead of pomalidomide (21), was able to induce the degradation of HDAC3 in THP-1 and HeLa cells, with a DC₅₀ value of 0.6 nM and a Dmax of approximately 90% in THP-1 cells (Figure 20b). While degradation of HDAC8 was observed at much higher concentrations, HDACs 1, 2, 4, and 6 remained unchanged. PROTAC 25 caused a decrease in HDAC3 levels at low micromolar concentrations in primary human macrophages. Additionally, 25 was able to reduce the secretion of pro-inflammatory cytokines such as TNF-α and IL-6, in levels notably superior to that observed for the HDAC3 inhibitor prototype 24 in the same assay. Macrophage polarization, that is crucial in the inflammatory process, was also evaluated. In this context, compound 25 was able to prevent the polarization of M0 to M1-like macrophages in response to stimulation in a cell culture of primary human macrophages. Overall, the results obtained are good evidences suggesting that PROTACs targeting HDAC3, such as PROTAC 25, have interesting anti-inflammatory potential.¹⁶³

Histone deacetylase 6 (HDAC6) emerged as a promising therapeutic target for treating various diseases.¹⁶⁸,¹⁶⁹ A recent investigation¹⁶⁴ demonstrated the significance of HDAC6 in the activation of the nucleotide oligomerization domainlike receptors family pyrin domain containing 3 (NLRP3) inflammasome, indicating that modulating HDAC6 activity holds potential for the treatment of numerous inflammatory disorders. In 2021, the PROTAC 27 was described demonstrating low toxicity and the ability to efficiently and selectively degrade HDAC6 in different cell lines, including activated THP-1. The PROTAC 27 was designed using the E3 ligase ligand pomalidomide (21) (CRBN-based), and the potent HDAC6 inhibitor (26) (IC₅₀ = 128.6 µM), a compound designed by combining structures from the natural product indirubin and the pharmacophore for HDACi: hydroxamic acid group (ZBG)-linker-cap (Figure 21a).¹⁷⁰,¹⁷¹ The authors demonstrated, for the first time, that using PROTACs to selectively degrade HDAC6 is a good and efficient strategy to block NLRP3 inflammasome activation in lipopolysaccharide (LPS)-induced mice model. Moreover, PROTAC 25 was able to produce an anti-inflammatory activity in vivo attenuating NLRP3 inflammasome activation in LPS-induced mice, suggesting that in vivo NLRP3 inflammasome activation depends on HDAC6 (Figure 21a).¹⁷²

Figure 21
Structures and activities of HDAC6 PROTACs. (a) Design and evaluation of PROTAC 25 using CRBN E3 ligase ligand; (b) design and evaluation of CRBN E3 ligase derivatives PROTAC 27 and PROTAC 28 (with methylated CRBN E3 ligase ligand as negative control).

Following the idea of NLRP3 modulation by HDAC6, two new PROTACs (27 and 28) were described recently¹⁷³ with the aim of investigating the effect of HDAC6 deficiency on NLRP3-mediated IL-1β release. The design of PROTAC 27 was based on the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the CRBN-based E3 ligase recruiter, thalidomide (Figure 21b). PROTAC 28 was designed as a negative (non-degrading) control, being able to bind to HDAC6 but lacking the ability to induce degradation. The strategy of CRBN-moiety methylation is quite common to negative controls because the free N–H in glutarimide ring is essential for recruiting the E3 ligase. PROTAC 27 significantly reduced HDAC6 levels in THP-1 macrophages at low concentrations of 0.1 µM and with maximum degradation at 10 µM, without affecting cell viability at same concentrations (Figure 21a). On the other hand, PROTAC 28, as expected, could not degrade HDAC6. The biological evaluations indicated that both PROTACs (27 and 28) significantly reduced IL-1β release in a concentration-dependent manner, suggesting that HDAC6 degradation is not necessary to inhibit NLRP3 inflammasome-mediated IL-1β release.¹⁷³

The next example is about sirtuin 2 (Sirt2) degraders. Sirt2 is a nicotinamide adenine dinucleotide (NAD⁺)-dependent histone deacetylases, classified as HDAC class III¹⁷⁴,¹⁷⁵ In 2018, Schiedel et al.¹³⁸ described the development of PROTAC 16 combining, as POI ligand, an interesting and selective inhibitor (29) for Sirt2, which is implicated with the anti-inflammatory response,¹⁷⁶ with thalidomide, as E3 ligase ligand. The attachment position (meta or para) came from observation of crystal structure of 29 with Sirt2 (PDB4RMI) (Figure 22b). In this work, for the first time, the construction of the PROTAC linker used the Cu^I-catalyzed azide-alkyne cycloaddition to joint an azide derivative of thalidomide to the Sirt inhibitor 29. PROTAC 16 was able to chemically induce the Sirt2 degradation potently and selectively, resulting in hyperacetylation of the microtubule network, accompanied by an increase in process elongation (Figure 22b).¹³⁸

Figure 22
(a) Selective ligand of Sirt2 chosen as POI ligand and its binding mode to the enzyme evidencing the benzyl ring turned to the solvent. Positions meta and para as attachment points; (b) PROTAC 16 structure with the triazole link and CRBN E3 ligase recruiter. The image and the hydrophobic surfaces were generated in UCSF Chimera alfa version 1.17.⁴⁵ Red surfaces represent hydrophobic regions and blue surfaces represent hydrophilic surfaces.

4.2. PROTACs developed for miscellaneous inflammation-related targets

The IRAK-4 is a member of serine-threonine kinase family, playing a significant role in the regulation of interleukin-1 receptors (IL-1R) and Toll-like receptors (TLRs) related signaling pathways.¹⁷⁷ Recognition of foreign pathogens and inflammatory signals by these receptors promotes activation that leads to the production of pro-inflammatory cytokines via the NF-κB pathway. The IRAK-4 mediated inflammation and related signaling pathways contribute to inflammation in autoimmune diseases and cancers. Therefore, targeting IRAK-4 to develop single-target, multi-target inhibitors and PROTAC degraders is an important direction for the treatment of inflammation and related diseases.¹¹⁹,¹⁷⁸,¹⁷⁹

GlaxoSmithKline researchers developed a PROTAC with potential to inhibit multiple pro-inflammatory cytokines in peripheral blood mononuclear cells (PBMCs).¹¹⁹ From the analysis of the IRAK-4 co-crystallized with an inhibitor, the researchers verified that the position 4 of the isoquinoline ring was exposed to the solvent, being an optimal position for modifications. PROTAC design started by choosing the ideal E3 ligase recruiter. Firstly, compounds containing the IRAK-4 ligand subunit, flexible linkers and VHL (PROTAC 29), CRBN (PROTAC 30) and IAP (PROTAC 31) as E3 ligase ligands were synthesized (Figure 23).

Figure 23
Discovery process of IRAK-4 PROTAC 10 from the initial screening of different E3 ligase ligands, followed by optimization of IRAK-4 ligand moiety and linker optimization.

The authors identified that the majority of synthesized compounds could bind to IRAK-4 (IC₅₀ ranging from 0.022 to 21 nM), but only the VHL-PROTACs (PROTAC 29 ) were effective to degrade IRAK-4 (50% degradation at 3 μM). Once found the best match structure (IRAK-4 ligand-linker-E3 ligase ligand), they tried to optimize the IRAK-4 ligand. The authors hypothesized that a more potent IRAK-4 ligand could result in a more effective IRAK-4 degradation, reason why they optimized the ligand using PF-06650833 as a prototype, altering the lactam ring. This modification resulted in a PROTAC 32 with DC₅₀ = 259 nM in PBMC. After, aiming to improve the activity of PROTAC 32, efforts were directed to linker optimization focusing on modulating polarity and flexibility of the chain. The best combined result was obtained by changing the aliphatic chain by a spirocyclic pyrimidine linker, resulting in the PROTAC 10 with DC₅₀ = 151 nM in PBMC (Figure 23). To confirm that the real degradation mechanism was proteasome dependent, assays were performed in the presence of epoxomycin (a proteasome inhibitor), observing a non-existent change regarding the IRAK-4 levels in the tests with epoxomycin. Next, they sought to investigate the IRAK-4 potential kinase independent role in TLR mediated signaling. The PROTAC was monitored for cytokine inhibition upon TLR7/8 stimulation in PBMCs and were capable of completely blocking IL-6 secretion as well as a wider panel of cytokines.

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is a transcription factor responsible for regulating multiple cellular responses, including proliferation, survival and differentiation. CREB is induced by a variety of growth factors and inflammatory signals and subsequently mediates the transcription of genes containing a cAMP-responsive element. Several immune-related genes possess this cAMP-responsive element, including IL-2, IL-6, IL-10, and TNF-α. In addition, phosphorylated CREB has been proposed to directly inhibit NF-κB activation by blocking the binding of CREB binding protein to the NF-κB complex, thereby limiting pro-inflammatory responses. EP300 (E1A-binding protein P300) is defined as an acetyltransferase that can acetylate histone and has been broadly studied in several chronic diseases, including cancer and inflammation.¹⁸⁰,¹⁸¹,¹⁸²

Following the same strategy of the GlaxoSmithKline group cited above, Cheng-Sánchez et al.¹⁸³ used a crystallographic structure of CREB-binding protein (CBP)/EP300 with a co-crystallized ligand (CPI-1612) as a starting point for the development of PROTACs. Analysis of this X-ray structure (PDB 6V8N) showed that the methyl group on the pyrazole moiety pointed outside the pocket, thus revealing an optimal attachment position to add the linker and E3 ligase ligands. Firstly, they evaluated the most used E3 ligase recruiters (VHL, IAP and CRBN) and aliphatic/PEG short linkers (n = 2 and 3). In the first moment, the PROTACs did not degrade CBP/EP300 and the authors hypothesized that maybe the sizes of the linkers were not ideal. New PROTACs were synthesized with larger linkers (n = 4-7). The authors assessed if the compounds were able to form a ternary complex between CRBN and the CBP catalytic core using fluorescent-based technology for detecting proteinprotein interactions (FluoPPI). Using FluoPPI technology, it was possible to detect that compounds with larger linkers were able of forming a ternary complex between CBP/EP300 and CRBN. Moreover, western blotting in LP1 cells demonstrated that compounds with longer linkers were able to degrade CBP/EP300, suggesting that the absence of ternary complex formation was responsible for the inactivity of PROTACs with shorter linkers. The results showed that CRBN-recruiting was better than VHL/IAP and increased linker length led to active CBP/EP300 degraders and the main representant of the series as the PROTAC dCE-1 (Figure 24).

Figure 24
Discovery of PROTAC dCE-1. (a) Selective and potent ligand of CBP, CPI-1612, was chosen as POI ligand and its binding mode to the enzyme (PDB 6V8N) evidenced the methylpyrazole ring turned to the solvent; (b) PROTAC dCE-1 structure with the linker and CRBN E3 ligase recruiter. The image and the hydrophobic surfaces were generated in UCSF Chimera alfa version 1.17.⁴⁵ Red surfaces represent hydrophobic regions and blue surfaces represent hydrophilic surfaces.

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) sits downstream of the pattern recognition of the nucleotide-binding oligomerization domain (NOD) receptors NOD1 and NOD2. Stimulated NOD1 and NOD2 interact with RIPK2 and lead to the activation of NF-κB and mitogen-activated protein kinases (MAPK), followed by the production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-12/23. Defects in NOD/RIPK2 signaling pathway are associated with numerous inflammatory diseases, including asthma, sarcoidosis, inflammatory bowel disease and multiple sclerosis.¹⁸⁴,¹⁸⁵,¹⁸⁶

Initially, Mares et al.¹⁸⁶ planned the new PROTACs through the combination of a RIPK2 ligand and E3 ligase recruiters (VHL, CRBN and IAP) by a PEG linker. All synthesized PROTACs were effective in degrading RIPK2, so the authors focused their efforts on IAP-recruiting E3 ligase. PROTAC 33 proved to be a potent degrader of RIPK2 in THP-1 cells, also inhibiting TNF-α release in a human whole blood assay (Figure 25). Despite the good results, the physicochemical and pharmacokinetic properties were not adequate, so researchers have focused efforts on optimizing these factors. Using medicinal chemistry optimization strategies, the researchers modified RIPK2 binder, IAP ligase binder and linker, obtaining PROTAC 34 (Figure 25). Even with the physical-chemical and pharmacokinetic improvements and the capacity to degrade RIPK2, their cellular degradation potency was modest, impacting the dose required to deliver high levels of in vivo degradation of RIPK2. One more time, the authors attempted to optimize the linker and IAP binder regions through structural modifications. The additional methylene group in PROTAC 35 increased the RIPK2 inhibitory potency in more than 10-fold, which translated into significantly improved cellular degradation potency in human PBMCs, besides completely inhibiting the release of TNFα, IL1β, IL-6, and IL-10 (Figure 25).

Figure 25
Design and optimization of PROTACs for RIPK2 degradation.

NF-ΚB represents a family of inducible transcription factors, regulating a large array of genes involved in different processes of the inflammatory responses. In macrophages, excessive or chronic activation of NF-ΚB regulates the expression of genes for inflammatory responses and mediates the release of pro-inflammatory factors and chemokines, thus contributing to the pathogenesis of many inflammatory diseases. Inhibition of NF-ΚB activation in macrophages is a promising strategy to prevent or treat these conditions. BTK is a nonreceptor cytoplasmic tyrosine kinase in the Tec family of protein tyrosine kinases. Aberrant activation of B-cells is demonstrated to play a central role in the pathogenesis of B-cell malignancies, autoimmune diseases and inflammation.¹⁸⁷,¹⁸⁸,¹⁸⁹

Using computational molecular modeling tools, Buhimschi et al.¹⁹⁰ developed new PROTACs based on ibrutinib. Docking the crystal structures of BTK and cereblon ternary complex showed that the eight-atoms linker is nearing the minimal length needed to bridge the two binding sites. Afterwards, the researchers synthesized MT-802, a PROTAC that degrades BTK with a DC₅₀ of 9.1 nM in Namalwa cell line (Figure 26).

Figure 26
Design of conformational constrained linkers in the development of new BTK PROTACs based on ibrutinib.

Based on studies by Buhimschi et al.,¹⁹⁰ Huang et al.¹⁹¹ evaluated the influence of a new linkers on PROTAC based ibrutinib-CRBN. Initially, ligands containing a piperazine ring attached to PEGs of different lengths were synthesized, but neither of the compounds with a semi-rigid linker proved to be better than the flexible MT-802 prototype. The semirigid piperidine PROTACs reduced BTK levels in Ramos cells in the range of 28-61% at 100 nM, while MT-802 reduced 75% at 100 nM. PROTACs with an even more conformationally constrained linkers were synthesized. These new PROTACs containing two cycles linkers reduced BTK levels in the range of 31-99% at 100 nM, with PROTAC 36 being the most active, containing a piperazine and an azetidine linker. The authors evaluated the time and concentration-dependent degradation effects against BTK in Ramos cells and PROTAC 36 degraded BTK with DC₅₀ = 3.18 nM, while MT-802 showed DC₅₀ = 63.31 nM in the same cell. PROTAC 36 degraded BTK in Mino cells with DC₅₀ = 7.07 nM and MT-802 DC₅₀ = 35.55 nM. PROTAC 36 suppressed IL-6 and IL-1β secretion (protein levels) in LPS-stimulated RAW 264.7 cells as measured by enzyme-linked immunosorbent assay. PROTAC 36 also decreased the IL-1β and IL-6 levels in the ZIP animal model. These results suggest that PROTAC 36 effectively inhibited NF-κB-mediated production of pro-inflammatory chemokines, indicating its therapeutic potential for inflammatory diseases (Figure 26).

5. Conclusions

In this review, we summarize the discovery, development, design, and importance of PROTACs. Since the first report, when Protac-1 was developed, many advances have been done. Today the interest for PROTACs is well spread in both academic and industry fields with high level of investments. Despite having only 23 years since its discovery, PROTACs are hugely impacting the drug discovery process due to their special event-driven mode of action. For sure, during this time many advances have improved the technique as the strong development on new linkers, E3 ligase ligands, PD and PK improvements, etc. However, as discussed, some drawbacks still need to be overcome for the complete success of PROTACs like potential toxicities, high molecular weights, difficulties with PK (like crossing barriers), better understanding of SAR, etc. With all the current success of PROTACs, the event-driven mode of action is moving around being applied to other classes of TPDs like the AUTAC,¹⁹² LYTAC,¹⁹³ “molecular glues”,¹⁹⁴ RIBOTAC,¹⁹⁵ HomoPROTAC,¹⁹⁶ multitargetPROTACs,⁹⁶ etc.

But will PROTACs and derivatives indeed become a new major class of pharmacological treatments or only continuing an excellent pharmacological tool? Maybe in a couple of years we can come back through this text with the answer and see what else happened.

Acknowledgments

First, the authors would like to thank Prof Eliezer Barreiro for all his commitment over the years disseminating knowledge and defending good practices in Medicinal Chemistry in Brazil. Authors would also like to thank the National Council for Scientific and Technological Development (CNPq, 314723/2021-8), and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, No. E-26/210.134/2018 and 210.018/2020) for the financial support. This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES), finance code 001.

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Edited by

Editor handled this article: Carlos Maurício R. de Sant’Anna (guest) Prof Eliezer Barreiro, thanks for all your groundbreaking and enthusiastic contributions to Brazilian Medicinal Chemistry. It is a great honor for us, your scientific son and your grandsons, having you as part of our histories.

Publication Dates

• Publication in this collection
  02 Sept 2024
• Date of issue
  2024

History

• Received
  27 Feb 2024
• Published
  13 Aug 2024