PROteolysis TArgeting Chimeras (PROTACs)
Much of drug discovery deals with non-covalent, reversible interactions with the target protein, more recently there are also a class of small molecule therapeutic agents that bind covalently to the target protein these don't require the maintenance of high drug levels to maintain target occupancy. In contrast the PROteolysis Targeting Chimera (PRO- TAC) technology provides an alternative to module biological function by specially using the ubiquitin proteasome system to induce degradation of the target protein DOI.
PROTACs are bifunctional molecules that bind to the target protein and an E3 ligase, the simultaneous PROTAC binding of two proteins brings the target protein in close enough proximity for polyubiquitination by the E2 enzyme associated to the E3 ligase, which flags the target protein for degradation through the proteasome.
The PROTAC is composed of three components.
- A head-group that targets the protein of interest
- A crosslinker
- A second ligand at the opposite end that binds an E3 ligase
Ubiquitin-Proteasome Pathway (UPP)
PROTAC ligand binds to both the protein target and an E3 ubiquitin ligase to form a ternary complex, followed by transfer of ubiquitin from the E2 to the protein substrate. The ubiquitin is attached to a lysine on the target protein, subsequent ubiquitins are then added to a lysine residue of the first added ubiquitin. The ternary complex dissociates and PROTAC is recycled. Polyubiquitinated protein undergoes degradation via 26S proteasomes.
Since the efficacy depends on protein resynthesis rate, pharmacodynamic half-life is often much longer than the PK half-life similar to small molecules with very slow off-rates or covalent inhibitors. It should be noted that this is an intracellular mechanism and would probably unsuitable for some targets e.g. plasma targets.
Ligands targeting the E3 ligases
The ubiquitin system is a major coordinator of cellular physiology through regulation of both protein degradation and signalling pathways. A E3 ubiquitin ligase is a protein that recruits an E2 ubiquitin-conjugating enzyme that has been loaded with ubiquitin, recognises a protein substrate, and assists or directly catalyses the transfer of ubiquitin from the E2 to the protein substrate. The ubiquitin is attached to a lysine on the target protein, subsequent ubiquitins are then added to a lysine residue of the first added ubiquitin. Humans have an estimated 500-1000 E3 ligases but as of yet, only a few have been validated or exploited for PROTAC development, specifically VHL, CRBN, MDM2, and cIAP1.
Early PROTACs used the HIF-1a peptide fragment bearing a cell-penetrating peptide sequence to recruit the Von Hippel-Lindau disease tumor-suppressor protein (VHL) E3 ligase. Later less peptide-like ligands were used still targeting the VHL E3 ligase, however these still suffered from poor cell penetration . The mouse double minute 2 homologue (MDM2) E3 was recruited by using a known MDM2-p53 PPI inhibitor, nutlin, as the E3 ligand with improved cell penetration. The E3 cereblon (CRBN) was identified as the molecular target of the immunomodulatory drugs, thalidomide, pomalidomide and lenalidomid.
Examples of Ligands for Target Proteins
Generally higher affinity ligands work better in Protacs but any ligand with Ki ~1uM or better has a good chance of working. Some work has used natural ligands/peptides but most of the recent work has focussed on the use of known small molecule inhibitors.
Many of the targeted proteins are oncology targets but other indications are also being investigated.
Protein Kinases (Akt, BCR‐Abl, c‐Abl, BTK, anaplastic lymphoma kinase [ALK], CDK9, EGFR, HER2, c-Met, TBK1, CK2, ERK1/2, FLT3, Fak, RIPK2, DAPK1, and PSD‐95)
Nuclear Receptors (ER, AR, and RAR)
Proteins in transcriptional regulation (BRD4, BDR9, Sirt2, HDAC6, TRIM24, IKZH1/3, Pirin, and Smad3)
Regulatory proteins (CRABP‐I/II, TACC3, AHR, FKBP12, ERRα, MCL1 and X‐protein)
Potential neurodegeneration targets (Huntingtin, Tau, a‐synuclein, and PSD‐95)
Metabolic enzymes (MetAP‐2 and DHODH)
DNA repair (PARP1)
In order to identify the optimal vector for ligand attachment it may be necessary to explore a number of alternatives. In the work looking at designing PROTACS for MCL1, a critical pro-survival factor in cancers such as multiple myeloma where MCL1 levels directly correlate to disease progression. a variety of different attachment points on the MCL1 inhibitor A-1210477 were explored DOI.
The linker length and pole composition of PROTACs affect characteristics such as cell permeability, and solubility DOI these linkers contain a mixture of hydrophobic and hydrophilic functionality to balance the hydrophobicity/hydrophilicity of the resulting hybrid compounds.
Kinetics and Stoichiometry
One of the most attractive features of the approach is that a PROTAC molecule acts in a catalytic manner, it only needs to bind a molecule of target once to tag it for degradation, and then is released and set free to bind another molecule of target and carry on, as in a catalytic cycle DOI. Since the efficacy depends on protein resynthesis rate, pharmacodynamic half-life is often much longer than the PK half-life. The importance of the catalytic mechanism was underlined in a recent publication DOI, that compared both covalent binding and reversible binding PROTACs derived from the covalent BTK inhibitor ibrutinib. They demonstrated that a covalent binding PROTAC inhibited BTK degradation despite evidence of target engagement, while BTK degradation was observed with a reversible binding PROTAC.
Whilst the physicochemical properties of these molecules are not ideal for oral administration, ARV-110 An oral androgen receptor PROTAC degrader for prostate cancer has been reported to be active after oral administration Poster.
The first PROTAC is due to enter clinical trials in early 2019.
The Phase 1 study will investigate the safety and tolerability of ARV-110 in patients with mCRPC who have progressed on at least two standard of care treatment regimens and includes exploratory measures of efficacy.
I did not find the structure of ARV-110 but looking at their patents it is likely to be related to the molecule shown below.
Kymera have also described preclinical data on a targeted protein degrader of IRAK4 claiming pharmacokinetic and pharmacodynamic properties within tumours support advancement toward clinical development in MYD88-driven lymphomas in 2020.
Protac-Induced Protein Degradation in Drug Discovery: Breaking the Rules or Just Making New Ones? DOI
PROteolysis TArgeting Chimeras (PROTACs) — Past, present and future DOI
PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer DOI.
First targeted protein degrader hits the clinic DOI
Last Updated 13 March 2019