Recently, the Manfred Jung group of the University of Freiburg in Germany published a summary report on the application of PROTAC in epigenetics, focusing on the latest progress of PROTAC on epigenetics targets. PROTAC is a heterobifunctional molecule that can use the natural ubiquitin-proteasome pathway to selectively degrade target proteins. This emerging technology has been applied to a variety of selective degradation targets, including (nuclear) receptors, kinases, and epigenetic proteins. Many PROTACs have been designed in the field of epigenetics.

Epigenetics is a rapidly developing field of research. In the past few decades, three major epigenetic mechanisms (such as DNA methylation, histone modification, and non-coding RNA) have been intensively studied, and many proteins have been identified as epigenetic modifiers. Gene regulation, cell signaling and metabolic pathways and pathogenic processes play an important role. At present, a variety of inhibitors have been reported for epigenetic targets and some have been approved for cancer treatment, but long-term use will cause drug resistance problems, and these problems are expected to be solved by PROTAC technology.

Introduction to PROTAC

The mechanism of action of PROTAC

PROTAC can use the natural ubiquitin proteasome pathway to induce the degradation of target proteins. Its structure can be divided into three different parts, namely, ligands targeting the target protein (POI), ligands targeting E3 ubiquitin ligase, and ligation Linker with two ligands. This dual function allows PROTAC to simultaneously bind the target protein and E3 ligase and form a ternary complex (POI:PROTAC:E3), causing polyubiquitination of the target protein and degradation by the proteasome.

Compared with traditional occupancy-based small molecule inhibitors, PROTAC has many advantages, for example, long drug efficacy, high target selectivity, high phenotypic inhibition rate, catalytic, can completely inhibit multi-domain proteins, and because PROTAC does not limit to the active site, it can target any accessible protein area, so that the degradation of "non-drugable" proteins can be achieved.

History of PROTAC

The term PROTAC started in 2001, when Sakamoto et al. reported a peptide-based degradation agent for methionine aminopeptidase-2, which was poor due to high molecular weight and structural instability. Until the discovery of E3 ligase ligands with improved pharmacokinetic characteristics, the first all-small molecule PROTAC targeting androgen receptors was designed. In the following years, high-efficiency degradants for a variety of targets were reported, including kinases, (nuclear) receptors, and epigenetic targets. New PROTAC technologies were also born, such as dTAG system, HaloPROTAC, phosphoPROTAC, PHOTAC and lysosomal targeting chimera (LYTAC).

Recently, great efforts have been made in the PROTAC design, the formation of ternary complexes and the structure of Linker, these factors are the main driving factors for the successful degradation of proteins. The formation of the ternary complex can be characterized by the synergy coefficient α, which is defined as the ratio of the dissociation constant of the binary complex (PROTAC: POI or PROTAC: E3) and the ternary complex (POI: PROTAC: E3). Recently, it has been found that α> 1 has a positive correlation with high-efficiency degradation ability and low Hook effect. Therefore, it is desirable to characterize the formation of complexes through the synergy coefficient α, but there are also some examples showing that synergy is not essential for effective protein degradation. Moreover, the formation of ternary complexes does not necessarily lead to protein degradation.

In 2018, Nowak et al. reported the crystal structure of several different PROTACs forming a ternary complex. From the crystal structure of the complex, it can be seen that the interface between the two proteins has only a weak interaction. The study found that the linker length of PROTAC and the binding position with the complex affect the binding conformation of the BRD4 and CRBN complex. After further evaluation of different Linkers, it was found that Linkers with certain lengths and structures are beneficial to the formation and stability of the ternary complex, but the Linker design is still elusive.

Therefore, when designing PROTAC, in addition to considering the formation of ternary complexes and the composition of Linker, PROTAC stability, cell permeability, target abundance, and target re-synthesis rate should also be considered.

PROTAC design

To date, the calculation methods used for PROTAC structural design and prediction of the ternary complex structure include: (a) docking PROTAC target protein and E3 ligase respectively to estimate the minimum length of Linker; (b) molecular dynamics studies Or MELD exchange simulation to predict the structure of the ternary complex formed; (c) protein-protein docking, which provides a possible model for the E3 ligase/POI complex; (d) conformation sampling method, which can provide a reasonable binding conformation. Drummond and Williams used the MOE modeling software package to perform the most detailed computer simulation of the PROTAC-mediated ternary complex. These methods have been verified to be feasible, but the structural prediction of the PROTAC/CRBN/BRD4 complex is far from satisfactory, which may be due to the weak interaction between the proteins in the complex.

To be continued in Part II…