The Potential of Proteolytic Chimeras as Pharmacological Tools and Therapeutic Agents

Abstract

The induction of protein degradation in a highly selective and efficient way by means of druggable molecules is known as targeted protein degradation (TPD). TPD emerged in the literature as a revolutionary idea: a heterobifunctional chimera with the capacity of creating an interaction between a protein of interest (POI) and a E3 ubiquitin ligase will induce a process of events in the POI, including ubiquitination, targeting to the proteasome, proteolysis and functional silencing, acting as a sort of degradative knockdown. With this programmed protein degradation, toxic and disease-causing proteins could be depleted from cells with potentially effective low drug doses. The proof-of-principle validation of this hypothesis in many studies has made the TPD strategy become a new attractive paradigm for the development of therapies for the treatment of multiple unmet diseases. Indeed, since the initial protacs (Proteolysis targeting chimeras) were posited in the 2000s, the TPD field has expanded extraordinarily, developing innovative chemistry and exploiting multiple degradation approaches. In this article, we review the breakthroughs and recent novel concepts in this highly active discipline.

Keywords: autophagy; chimera; lysosome; protac; proteasome; targeted protein degradation; ubiquitin.

Figure 1

Schematic representation of the main routes of TPD in the cell. Degradation pathways targeting cargos to the proteasome and to the lysosome are shown. Curved arrows indicate the action of degrader molecules, including distinct types of protacs, molecular glues, lytacs, autacs and attecs, as they are described in the literature. Protacs and molecular glues utilize the E3 ligases shown (blue ovals), directing to the 26S and 30S proteasomes the ubiquitinated targets, which are recruited by means of the Rpn1, Rpn10 and Rpn13 proteasome receptors (dark green ovals). Ubiquitin-independent protacs direct targets to the 20S protesome. Lytacs utilize the M6PR (green) receptor to internalize external proteins to pre-lysosomal compartments, and finally deliver them to the lysosome. Autacs require E3 ligases, such as Parkin, Smurf1 and Lrsam1 (blue ovals), to target ubiquitinated cargo to autophagy receptors (OPTN, NDP53, p62, NBR1 and TAX1BP1) (green ovals), which interact with LC3 to induce cargo engulfment and autophagosome formation. On the other hand, attecs directly link protein targets with LC3, promoting engulfment and autophagosome formation. Autophagosomes are eventually integrated to the lysosome, which will hydrolyze the incoming materials. For abbreviations, see main text.

Figure 2

Protacs and E3 ligases. Schematic representation of the E3 ligases involved in TPD and the protac designed, as they appear in the text. RING ligases contain monomeric (e.g., PARKIN, UBR1), dimeric (e.g., cIAP, MDM2) [15,19] and multimeric forms. The Cullin RING multimeric ligase (CRL) superfamily [20] contains up to seven families, including Skp1-Cullin(Cul1)-Fbox (SCF), the DDB1-Cul4 and the elongin B, C-Cul2/5-SOCSbox protein (ECS). Thus, CRL1^TIR1, CRL2^VHL, CRL4^DDB1 and CRL4^CRBN, mentioned in the text, belong to these families [21,22,23,24]. (A) Structure of Protac-1, the first TPD developed. (B) Small-molecule protacs to target the breakpoint cluster region—Abelson tyrosine kinase (BCR-ABL). (C) Protac for a non-steroidal androgen receptor ligand (SARM) and the MDM2 ligand nutlin. (D) An example of SNIPERs to recruit the homodimeric E3 cellular cIAP1 for the degradation of retinoic acid-binding proteins (CRABP-I and II). (E) The structure of thalidomide and its role as degrader. (F) Protac d9A-2 for the degradation of SLC9A1 from leukemic cells. E3 ligase ligands are shown in red and ligands for the POIs are shown in blue. Black circles show the regions of interaction induced by the degrader molecule. Curved arrows represent the ubiquitination process by the transfer of ubiquitin moieties (red ovals).

Figure 3

Conceptual representation of the different proteolytic chimeras described in Section 3.

Figure 4

(A) Photac targeting the BET family of epigenetic readers BRD2-4. The photoswitchable azobenzene unit is marked in red. (B) Canonical protac ARV-771 and its photocontrolled version, showing the trans-cis isomerization between active and inactive states. (C) Photocaged variants of protacs developed as Brd4 degraders. In all cases, a DMNB group (in blue) is used as a phototrigger.

Figure 5

(A) Top: illustration of the concept of reversible covalent inhibition and application to the design of a protac against the ERRα (protac 1). (B) Examples of non-covalent (NC-1), irreversible covalent (IR-2) and reversible covalent (RC-3) protacs against BTK. (C) Examples of covalent protacs designed as BTK degraders. The Michael acceptor moiety, used as a warhead, is marked in red. Examples of HaloProtacs by functionalization of a VHL ligand with a series of ω-chlorohexyl-PEG linkers (in green).

Figure 6

Design of cliptacs by the in-cell bioorthogonal click reaction of two smaller clickable partners. The moieties suitable for the click reaction are marked in blue and red, while the covalent warhead present in the ERK1/2 ligand is shown in green.

Figure 7

(A) Structures of pyrazinoic acid (POA) and selective estrogen receptor down-regulators (SERDs). (B) Examples of adamantyl and Boc3Arg as hydrophobic tags. Hydrophobic tags are shown in blue.

Figure 8

Variants of TPD directed to lysosomal degradation. (A) Lysosome-targeting chimeras (lytacs). Serine-O-mannose-6-phosphonate, M6Pn (left), represented as a building block to form the poly-(M6Pn) ligand attached to the POI ligand. In this example, a lytac antibody is shown (right). (B) Autophagy targeting chimeras (autacs). Autac 4, with the configuration 2-Phenylindole-3-glyoxyamide-PEG-p-fluorobenzyl guanine, designed to trigger mitophagy. (C) Autophagosome tethering compounds (Attecs). 10O5, 8F20, AN1 and AN2 act as LC3-interacting warheads for the autophagosome tethering strategy.