HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins

Abstract

Small molecule-induced protein degradation is an attractive strategy for the development of chemical probes. One method for inducing targeted protein degradation involves the use of PROTACs, heterobifunctional molecules that can recruit specific E3 ligases to a desired protein of interest. PROTACs have been successfully used to degrade numerous proteins in cells, but the peptidic E3 ligase ligands used in previous PROTACs have hindered their development into more mature chemical probes or therapeutics. We report the design of a novel class of PROTACs that incorporate small molecule VHL ligands to successfully degrade HaloTag7 fusion proteins. These HaloPROTACs will inspire the development of future PROTACs with more drug-like properties. Additionally, these HaloPROTACs are useful chemical genetic tools, due to their ability to chemically knock down widely used HaloTag7 fusion proteins in a general fashion.

Figure 1

Schematic depiction of a bifunctional HaloPROTAC containing chloroalkane (which binds HaloTag7 fusion proteins) and a hydroxyproline derivative which binds VHL. These two motifs would serve to bring the HaloTag7 fusion protein into proximity with the E3 ligase, VHL, leading to the degradation of the HaloTag7 fusion protein by the proteasome.

Figure 2

The average fluorescence per cell compared to vehicle control was measured by flow cytometry after 24 hour treatment with the indicated compounds and concentrations. All compounds were tested in triplicate unless otherwise noted. A) HaloPROTACs containing Degradation Inducing Moiety A lead to nearly 70% degradation of GFP-HaloTag7, when sufficiently long linkers are used. B) HaloPROTAC3 (tested in quintuplicate), which contains Degradation Inducing Moiety B, leads to 90% degradation of GFP-HaloTag7 at 625 nM. GFP-HaloTag7 degradation was measured by flow cytometry after 24 hour treatment with HaloPROTAC and normalized to 24 hour treatment with DMSO. Error bars depict the Standard Error of the Mean (SEM).

Figure 3

A) A study of linker length with Degradation Inducing Moiety B shows that three ethylene glycol units are optimal for the degradation of GFP-HaloTag7. B) Structures of HaloPROTACs that have weaker affinity for VHL. C) Reducing the affinity for VHL attenuates their ability to induce degradation of GFP-HaloTag7, although the effect is not necessarily linear. GFP-HaloTag7 degradation was measured by flow cytometry after 24 hour treatment with HaloPROTAC and normalized to 24 hour treatment with DMSO. All compounds were tested in triplicate except for HaloPROTAC3 (quintuplicate) and HaloPROTAC9 and HaloPROTAC10 (quadruplicate). Error bars depict the SEM.

Figure 4

A) The enantiomers of HaloPROTACs (containing D-amino acid residues) which do not bind VHL do not induce degradation of GFP-HaloTag7, supporting the necessity of VHL binding for activity. B) Pre-treatment with excess ent-HaloPROTAC3 (1 hour) prevents degradation of GFP-HaloTag7 by HaloPROTAC3 after 24 hours. C) Pre-treatment with epoxomicin (4 hours) prevents degradation of GFP-HaloTag7 by HaloPROTAC3 after 20 hours. D)Treatment with VL285 attenuates the ability of HaloPROTAC3 to induce the degradation of GFP-HaloTag7. E) Structure of VL285. All error bars depict SEM. All compounds were tested in triplicate.

Figure 5

A) Comparison of HaloPROTAC3 (quintuplicate) to Hyt36 (triplicate) shows that HaloPROTAC3 is significantly more potent and efficacious. B) HaloPROTAC3 leads to 50% degradation of GFP-HaloTag7 within 4 to 8 hours. C) Significant recovery from 24 hour treatment with HaloPROTAC3 is observed after a 24 hour washout. All data was repeated in triplicate. Error bars depict the SEM.

Figure 6

Fluorescent microscopy shows drastic loss of fluorescence upon 24 hour treatment with HaloPROTAC3 but not the inactive ent-HaloPROTAC3. Comparison of fluorescence images to phase contrast view is shown in Supplemental Figure 2.

Figure 7

Immunoblotting confirms that nearly complete degradation of A) GFP-HaloTag7 is observed after 24 hour treatment with 500 nM HaloPROTAC3, with significant degradation at 50 nM HaloPROTAC3. HaloPROTAC3 can lead to degradation of other HaloTag7 fusion proteins such as B) HaloTag7-ERK1 and HaloTag7-MEK1. As expected, endogenous ERK and MEK are not degraded.

Scheme 1

Synthesis of HaloPROTACs containing Degradation Inducing Moiety A and Degradation Inducing Moiety B. Full synthetic details are found in the Supporting Information.