Targeted protein knockdown using small molecule degraders

Abstract

Small molecule probes of biological systems have traditionally been designed to bind to and inhibit the active sites of their protein targets. While this class of pharmacological agents has been broadened by the development of a small number of allosteric and protein-protein interaction (PPI) inhibitors, conventional drug design still excludes 'undruggable' proteins that are neither enzymes nor receptors. Recent years have seen the emergence of new classes of small molecules that can target hitherto undruggable proteins by recruiting the cellular proteostasis machinery to selectively tag them for degradation. These molecules, especially the class known as Proteolysis Targeting Chimera (PROTACs), represent a paradigm shift in chemical genetics, but their most tantalizing potential is as novel therapeutic agents. This review briefly summarizes the preclinical development of small molecule-based protein degraders, and describes the recent improvements in the technology that have positioned PROTACs on the cusp of entering the clinic.

Fig. 1. PROTAC mechanism of action

PROTACs bind a POI (not necessarily at an active site) and enforce proximity with an E3 ligase. The ternary complex thus formed is favored by PPIs between the POI and the E3. Ubiquitin transfer follows, enabling the formation of the polyubiquitin signal on the POI. Degradation occurs via the proteasome, and the unharmed PROTAC is regenerated to carry out successive rounds of ubiquitination.

Fig. 2. Incremental progress in PROTAC development

a) Early peptidic PROTAC (named PROTAC-1) targeted MetAP-2 by recruiting it to the β-TRCP SCF E3 ligase. b) SARM-nutlin PROTAC that used the MDM2 E3 ligase for degradation of AR. c) SNIPER(TACC-3)-1, a PROTAC targeting TACC3 using the cIAP E3 ligase ligand bestatin. d) An IAP based PROTAC (SNIPER(ER)-87) that preferentially recruited XIAP over cIAP to target ERα for degradation.

Fig. 3. VHL- and CRBN-based PROTACs with in vivo activity

a) ARV-771, a JQ-1 based VHL recruiting BET PROTAC, that was shown to be active in CRPC models in vivo. b) BETd-246, a PROTAC that used a novel BET bromodomain binding chemotype conjugated to thalidomide to degrade BRD2/3/4 and was shown to have activity against a patient-derived xenograft (PDX) model of treatment resistant breast cancer.

Fig. 4. Hydrophobic tagging mechanism of action

Adamantyl moiety containing hydrophobic tags bind a POI (not necessarily at an active site), and result in protein degradation through one or both of two possible mechanisms: recruitment of protein quality control chaperones for which the hydrophobic adamantyl group signals protein unfolding, or by bona fide protein unfolding local to the POI ligand binding site due to the bulky nature of the large hydrocarbon moiety. Similar to PROTACs, hydrophobic tags are liberated intact subsequent to protein degradation, and can effect successive rounds of POI destruction.