Phenyl Dihydrouracil: An Alternative Cereblon Binder for PROTAC Design

Abstract

Thalidomide and its analogues are frequently used in PROTAC design. However, they are known to be inherently unstable, undergoing hydrolysis even in commonly utilized cell culture media. We recently reported that phenyl glutarimide (PG)-based PROTACs displayed improved chemical stability and, consequently, improved protein degradation efficacy and cellular potency. Our optimization efforts, aiming to further improve the chemical stability and eliminate the racemization-prone chiral center in PG, led us to the development of phenyl dihydrouracil (PD)-based PROTACs. Here we describe the design and synthesis of LCK-directing PD-PROTACs and compare their physicochemical and pharmacological properties to those of the corresponding IMiD and PG analogues.

Proteolysis-targeting chimeras (PROTACs) have received considerable attention in recent years as a promising novel paradigm in drug discovery.¹ PROTACs are heterobifunctional molecules rationally designed to bring the desired target protein in close proximity to an E3 ubiquitin ligase, and consequently induce its ubiquitination and proteasomal degradation. A typical PROTAC consists of three distinct structural motifs, with one binding to the protein of interest, another binding to an E3 ligase complex, and a linker tethering them together.² The initial proof-of-concept PROTACs reported by Crews and Deshaies in 2001 were designed to recruit NF-κBα (IκBα) to the E3 ligase complex SCF-βTRCP.³ The discovery of the thalidomide cereblon-dependent mechanism of action a decade later enabled a major breakthrough in 2015 when the Crews⁴ and Bradner⁵ laboratories independently reported thalidomide-based PROTACs designed to degrade BRD4, a member of a small family of Bromodomain and Extra Terminal (BET) proteins.⁶ In the following years cereblon recruiting emerged as one of the most utilized PROTAC design strategies, mostly due to the favorable drug-like properties of its ligands, the immunomodulatory imide drugs (IMiDs) such as thalidomide.

However, IMiDs are well-known to be inherently unstable,⁷ readily undergoing hydrolysis even under relatively mild conditions, such as incubation in commonly utilized cell culture media.⁸ We recently reported that phenyl glutarimide (PG)-based PROTACs retained cereblon affinity while displaying improved chemical stability and, consequently, improved protein degradation efficacy and cellular potency.⁸⁻¹⁰ We also found PG-based PROTACs displaying improved selectivity profile with respect to the recruitment of classical off-target neosubstrates such as GSPT1.^9,11 However, while PG- PROTACs proved more stable than the corresponding IMiD-based PROTACs, they are still liable to hydrolyze in cell culture media. Moreover, the C-3 carbon of the glutarimide ring in PG was found to readily racemize in cell culture media, with up to 24% inversion over 24 h.⁸ The configurational instability was observed even in DMSO stock solutions stored at room temperature. Consequently, in our PROTAC programs we use racemic PG as the warhead, which is synthetically wasteful and pharmacologically inefficient approach since the CRBN affinity rests only in the R-enantiomer.⁸

In the present study, we describe our efforts to address these issues. We postulated that replacing the glutarimide C-3 carbon in the phenyl glutarimide moiety with a nitrogen atom would not only address the racemization but would also result in further improved chemical stability. Following our earlier finding that pre-T cell receptor and LCK signaling are a common therapeutic vulnerability in T-cell acute lymphoblastic leukemia (T-ALL),¹² we recently reported the development of highly potent and selective LCK PROTACs.¹⁰ The PROTACs were designed using dasatinib as an LCK ligand and the PG as a cereblon-directing moiety. Our lead PG-PROTAC 1 (SJ11646) showed potent degradation of LCK, and a superior cytotoxicity in LCK-activated T-ALL cell lines and primary leukemia samples compared with dasatinib itself.¹⁰ Here, we replaced the PG warhead in PROTAC 1 with the corresponding phenyl dihydrouracil (PD)-based moiety, to obtain PD-PROTAC 2. The impact of this modification on CRBN binding, chemical stability, LCK degradation, cytotoxicity in T-ALL cell line, ternary complex formation, and in vitro ADME properties was then evaluated. PD-based PROTACs have been recently disclosed in patent literature, although their general properties such as chemical stability, physicochemical profile, cytotoxicity, and overall comparison with corresponding PG and classical IMiD derivatives were not discussed.^13,14

As shown in Table 1 and Figure S1, PD-PROTAC 2 retained the CRBN affinity (52 ± 19 nM) measured in our fluorescent polarization assay using a Cy5-conjugated lenalidomide as a fluorescent probe, although the affinity was around 35-fold lower when compared to PG analogue 1 (1.4 ± 0.2 nM). To monitor PROTAC-induced ternary complex formation between LCK and CRBN proteins, we employed a proximity-based AlphaLISA assay. In this assay, the PD-PROTAC yielded the characteristic bell-shaped profile over a range of concentrations. Luminescence emission increased at lower concentrations of PROTAC as the ratio of ternary complex was increasing and decreased at higher concentrations due to saturation of CRBN and LCK binding sites, exhibiting the so-called “hook effect”. As expected, dasatinib itself showed no signal in this assay. Interestingly, despite the lower CRBN affinity, PD-PROTAC 2 still displayed a considerably higher AlphaLISA signal amplitude than PG–PROTAC 1, suggesting a greater extent of ternary complex stabilization (Figure 1A).

Table 1. LCK PROTACs: Structures, Cereblon Affinity, LCK Protein Degradation Potency, and KOPT-K1 Cell Cytotoxicity.

^aCell viability assessed by Cell-Titer-Glo after 72 h of incubation. All values are the mean of three independent experiments. Error represents SD.

^bDegradation determined by HiBiT-LCK at 4 h. All values are the mean of three independent experiments. Error represents SD.

^cCRBN-DDB1 binding determined by fluorescent polarization assay. Error represents SEM (n = 3).

^dData previously reported in Hu et al.¹⁰

Figure 1.

LCK PROTACs evaluation: (A) compound-induced ternary complex formation of His-CRBN-DDB1 and GST-LCK in AlphaLISA assay; (B) LCK protein levels in LCK-HiBiT-tagged KOPT-K1 cells after 4 h of incubation with increasing concentrations of PROTACs; (C) KOPT-K1 cell viability after 72 h of incubation; (D) PROTAC stability in KOPT-K1 cell culture media; (E) KOPT-K1 cell viability after 24 h of incubation; (F) immunoblot analysis for LCK protein after the treatment of KOPT-K1 cells over 4 h with increasing concentrations of PD–PROTACs 2 and 5. Degradation values were calculated using quantified band intensities from immunoblots (Figure S2), and DC₅₀ values calculated based on the average of three independent experiments.

To measure PROTAC effects on the LCK protein levels in T-ALL cells, we employed the HiBiT technology.¹⁵ LCK-HiBiT-tagged KOPT-K1 cells were generated by tagging the endogenous LCK protein using CRISPR-Cas12a technology, and PROTACs were evaluated in a time- and dose-responsive manner over 4 h. The extent of LCK degradation induced by PD-PROTAC 2 in this assay was found to be similar to PG-PROTAC 1 and thal-PROTAC 3, with DC₅₀ values of 15 nM and 6 nM and 13 nM, respectively (Table 1 and Figure 1B).

Interestingly, whereas a modification of the linker handle in PG-PROTAC 4 resulted in a very little change in the overall profile in respect to the lead PG analogue 1, it did bring significant improvement in the PD-PROTAC series. Indeed, PD-PROTAC 5 proved to be the most effective LCK degrader to date, with a DC₅₀ of 0.8 nM in the LCK HiBiT assays, which is an order of magnitude greater potency in the LCK-HiBit assay than the corresponding PG-analogue 4 (Table 1). PD-PROTAC 5 also produced one of the highest amplitudes in the LCK-CRBN AlphaLISA assay (Figure 1A), suggesting a high ternary complex stabilization and rationalizing its superior LCK degradation potency despite lower CRBN binding affinity.

We next evaluated the effect of PROTACs on KOPT-K1 cell viability. We have previously shown that this human T-ALL cell line is highly sensitive to LCK inhibition and degradation.¹⁰ Cells were treated with compounds over 72 h, and their viability was assessed using the CellTiter-Glo assay kit (Promega). The LC₅₀ values were determined by the proprietary software Robust Investigation of Screening Experiments (RISE), developed in house on the Pipeline Pilot platform (Biovia, v. 17.2.0). Consistent with the AlphaLISA and HiBit data, PD-PROTAC 2 displayed a very high potency in this assay, too, with LC₅₀ of 5 pM (Table 1). Notably, in comparison to PD analogue 2, the corresponding thalidomide-based PROTAC 3 showed a 2000-fold lower cytotoxicity in the KOPT-K1 cells, despite having an almost identical LCK protein degradation potency (Table 1, Figure 1C). This data disconnect can be rationalized by a poor stability of the thalidomide PROTAC in the KOPT-K1 cell culture media (t_1/2 = 13 h), when compared to PG and PD analogues, both having half-life >48 h in this media (Figure 1D).

We also evaluated KOPT-K1 cell viability following a shorter incubation time (24 h), which produced curves that enabled more accurate LC₅₀ calculations for the most potent PROTACs (Figure 1E). At this time point, PG-PROTACs were still the most potent, while there was a very little difference between PD- and thalidomide-based PROTACs (Table 1). We noted the apparent disconnect between the HiBiT and CTG data, which could be attributed to the difference in incubation time (4 h HiBiT, and 24 h CTG), assay readout, or a multitude of other factors that are yet to be fully understood. Interestingly, while following the longer incubation PG- and PD-PROTACs displayed significantly higher cytotoxicity, the thal-PROTAC produced practically identical LC₅₀ values at the two time points (8 nM at 24 h, and 10 nM and 72 h). This finding is consistent with the thal-PROTAC complete hydrolysis by the earlier time point, rendering the longer incubation ineffective.

Immunoblotting analysis was performed to confirm the degree of LCK protein degradation observed in the HiBiT assay. KOPT-K1 cells were treated with increasing concentrations of 2 or 5 over four hours and then assayed for endogenous LCK levels by immunoblotting. Consistent with the HiBiT assay data, under these conditions PD–PROTAC 5 achieved a complete degradation of the LCK protein (D_max > 98.4 ± 0.6%) and an order of magnitude lower DC₅₀ value (0.23 ± 0.13 nM), when compared to 2 that displayed DC₅₀ of 8 ± 6 nM with D_max of 66 ± 7% (Figure 1F and Figure S2).

We then profiled our PROTACs against classical IMiD neosubstrates (GSPT1, IKZF1, CK1α), since the cytotoxicity of CRBN-directing degraders may also arise from off-target degradation induced by a molecular glue mechanism.^16,17 Importantly, none of the tested PROTACs, including thalidomide-based PROTAC 3, affected levels of HiBiT-tagged GSPT1 protein in HEK293T cells¹¹ at concentrations up to 10 μM (Figure S3A). Immunoblotting confirmed that PD-PROTACs 2 and 5 in KOPT-K1 cells did not affect the abundance of GSPT1, nor IKZF1 and CK1α proteins at concentrations up to 100 nM (Figure S3B and C). In addition, neither of the two PD warheads featuring in PROTACs 2 and 5, piperazin-PD and PD-oxo-acetic acid, showed activity in the KOPT-K1 CTG assay, further confirming that a molecular glue-related mechanism did not contribute to the observed cell phenotype (Figure S3D).

We also profiled all PROTACs in Caco-2 permeability and mouse microsomal stability assays and found them all exhibiting typical PROTAC profiles with low cell permeability and high metabolic clearance (Table S1). The PROTACs high mouse microsomal clearance may not be surprising, considering that the parent inhibitor, dasatinib, also showed poor stability in this assay with a half-life of 0.82 h.

In summary, to address the intrinsic instability and racemization issues associated with IMiDs and PG-based ligands, as well as to further expand the options available for CRBN-directing PROTACs, we developed PD as an alternative warhead. Here we demonstrated that PD derivatives retained CRBN affinity and displayed greatly improved chemical stability compared to classical IMiD- as well as PG-based PROTACs. Consequently, PD–PROTAC 2 showed much higher in vitro antiproliferative efficacy compared to its IMiD analogue 3, whereas PD-PROTAC 5 (SJ43489) is the most potent LCK degrader to date. Collectively, these data support the utility of PD as an alternative cereblon warhead for PROTAC design.

Glossary

Abbreviations

ALL

acute lymphoblastic leukemia

CRBN

cereblon

GSPT1

G1 to S phase transition 1

IMiDs

immunomodulatory imide drugs

LCK

lymphocyte-specific protein tyrosine kinase

LCMS

liquid chromatography mass spectrometry

PD

phenyl dihydrouracil

PG

phenyl glutarimide

PROTAC

proteolysis targeting chimeras

FP

fluorescent polarization

DDB1

damaged DNA binding protein 1

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.2c00436.

• Supplementary table, biological assays data, as well as full chemical synthetic methods and ¹H and ¹³C NMR of target compounds (PDF)

Author Contributions

^† J.A.J. and S.Y. contributed equally to this work.

The authors declare the following competing financial interest(s): Z.R. receives consulting fees from Revolution Medicines, Orum Therapeutics, Nyrada, and VZDJ Discovery. J.J.Y. receives research grants from Takeda Pharmaceutical and Astra Zeneca.