Rational design of bifunctional protein degrader drugs - Ubiquitin (Ub) tags regulate multiple properties and functions of proteins in cells. Proteasomal degradation of
target proteins is a well-established means whereby the Ub proteasome system (UPS) controls protein content.
Enzymes called Ub E3 ligases conjugate Ub to target proteins by forming an isopeptide bond between the ε-
amino group of the target protein lysine and the carboxylate of the C-terminal glycine of Ub. Conjugation of
multiple Ubs forms poly-Ub chains at any of its seven lysines (K), and K6, K11, K27, K29, K33, K48, and K63
Ub chains having various roles are present in all tissues. Recently, several groups designed small molecules
that bind to an E3 ligase at one end and a target protein at the other, physically facilitating ubiquitylation of the
target protein, which is then degraded. This hijacking of a ligase to ubiquitylate a desired protein has launched
a new class of drug called PROTACs (PROteolysis TArgeting Chimeras). PROTAC-based approaches for
therapeutics offer several advantages: 1) selective, catalytic degradation of the target; 2) conversion of weak
binders into selective PROTAC drugs; 3) degradation of overexpressed or mutant targets; and 4) maximal
degradation from limited target engagement. To date, cereblon and VHL ligase binders have been most
commonly used as vehicles to ubiquitylate target proteins such as nuclear receptors, kinases, transcription
factors, and neuronal proteins tau and α-synuclein. A major problem has hindered development of new PROTAC
drugs, however. Chemical optimization of PROTAC molecules depends on rapid evaluation of synthesized
compounds to guide the synthetic strategy for producing drug candidate molecules. Assays currently available
are labor intensive and do not provide results to the medicinal chemists fast enough – often, a week is required.
In Phase I, a facile in vitro method employing Ub ligases cereblon and HDM2 was developed to screen for
potential PROTAC drugs; PROTAC-mediated ubiquitylation of selected proteins was recapitulated in vitro in a
way that mimics observed PROTAC-dependent ubiquitination and degradation of these proteins in vivo,
achieving the aims of Phase I. In phase II, the utility of this method will be expanded to include representative
members of all Ub ligase families (cullin families, RING finger ligases, Hect family ligases, and SUMO ligase),
increasing the biochemical and chemical space for PROTAC drug discovery. To scale up PROTAC screens, a
microtiter plate-based, high throughput method will be established to monitor in vitro PROTAC drug discovery,
and biochemical and Ub mass spec proteomics will be employed to demonstrate that target protein lysines
ubiquitylated in vitro are correlated with in vivo PROTAC mediated degradation of target proteins.
Commercialization of the microtiter plate based PROTAC system will have a major impact on academic research
as well as PROTAC drug discovery.
