Discovery of an orally bioavailable, CNS active pan-mutant RET kinase heterobifunctional degrader

Abstract

Point mutations and chromosomal fusions of the Rearranged During Transfection (RET) transmembrane receptor tyrosine kinase cause constitutive substrate-free activation, driving numerous human cancers. RET-selective kinase inhibitors (selpercatinib, pralsetinib) are in current clinical use for RET-driven tumors. However, the emergence of resistance mutations, such as those at the solvent-front G810 residue, results in reduced efficacy. We sought to exploit the event-driven pharmacology of targeted protein degradation to achieve pan-mutant activity against RET-driven cancers with a single selective RET degrader, while utilizing non-phthalimide cereblon (CRBN) ligands to discover orally bioavailable heterobifunctional degraders. Here we describe the medicinal chemistry efforts that led to compound 20, an orally bioavailable, brain-penetrant, pan-mutant and pan-fusion RET heterobifunctional degrader.

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Introduction

Precision oncology relies upon the identification and pharmacological manipulation of oncogenic driver proteins. Mutated R̲e̲arranged During T̲ransfection (RET) receptor tyrosine kinase was identified as an oncogene in thyroid^1,2 and non-small cell lung cancers (NSCLC).^1,2 RET possesses minimal function in healthy adult tissues, being involved in renal development,^2,3 spermatogonial stem cell maintenance,⁴ neural crest cell proliferation, differentiation, and survival,⁵ and the development and maintenance of neurons of the central and peripheral nervous systems.^1,2,6 Substrate binding to RET results in homodimerization of the extracellular domain, activating the intracellular kinase domain and initiating PI3K-AKT and RAS-MAPK signaling.^2,7–10

Oncogenic RET mutations come in two general classes. Chromosomal rearrangements, found in a variety of cancers including NSCLC and papillary thyroid cancer (PTC),^11,12 lead to novel protein fusions² where the extracellular substrate binding domain is replaced,^1,2,12,13 causing constitutive dimerization^1,12,14 and thus activation of the RET kinase domain.¹⁵ Oncogenic point mutations² common in familial thyroid cancers lineages such as medullary thyroid cancer (MTC) and multiple endocrine neoplasia (MEN),^11,12 cause constitutive activation¹⁵ either via transmembrane or extracellular domain cysteine mutations, causing aberrant disulfide binding and dimerization,^1,12,15 or via activating point mutations¹² in the kinase domain.

Since all oncogenic RET proteins retain the kinase domain,^1,2,12–14 small molecule inhibition of RET kinase was pursued as a therapeutic modality. Multikinase inhibitors, such as vandetanib (Fig. 1, compound 1)^2,16 and cabozantinib (compound 2),^2,17 provide limited clinical efficacy due to dose-limiting toxicities^2,5,12 and the evolution of the RET gatekeeper resistance mutation V804L/M.^12,18–20 Selective RET inhibitors selpercatinib (compound 3)²¹ and pralsetinib (compound 4)^22–24 were developed to improve clinical outcomes and overcome V804L/M resistance, and demonstrate robust clinical effects^25–27 on RET driven cancers, with selpercatinib eliciting a 44% overall response rate and a 24.5 month median duration of response.²⁵ However, clinical resistance to RET inhibitors^28,29 arises from bypass mutations^14,30 (NTRK,^31,32 MET,^32,33 ALK^32,34 Ras-MAPK^29,34) or secondary RET mutations reducing the binding of RET inhibitors,^12,35 such as solvent front mutations to G810,^12,36,37 necessitating the development of next generation RET-selective therapies^38–41 [TPX-0046 (compound 5),^42,43 vepafestinib (compound 6),^44,45 LOX-260 (ref. 45)] (Fig. 1).

Fig. 1 Representative multikinase and selective RET inhibitors. a) VEGFR2-targeted multikinase inhibitors which entered clinical use for RET-driven cancers. b) Current RET-selective inhibitors in clinical use. c) Next-generation RET selective inhibitors with activity against RET mutations known to confer resistance to selpercatinib and pralsetinib.

As an alternative to enzymatic inhibition, targeted protein degradation (TPD) offers several mechanistic differences with the potential to maintain pharmacological efficacy in the face of emerging on-target mutations. First, a degrader's effect may be dominated by cooperative protein–protein interactions between the kinase and the E3 ligase during ubiquitination rather than ligand binding.⁴⁶ Therefore, reduced target affinity may not reduce pharmacodynamic effect.⁴⁷ Second, degraders act catalytically,^48,49 and rapid degradation kinetics could compensate for a loss in binding affinity to the target protein.⁴⁶ Third, degrader pharmacology is driven by proteasomal degradation downstream of the transient formation of the target-protein and E3 ligase ternary complex, rather than sustained target occupancy.^50–52 With this pharmacology, a degrader with reduced target binding affinity can elicit a comparable pharmacodynamic effect to an inhibitor with greater target binding affinity. These combined characteristics may allow degraders to prove more resilient to resistance via inhibitor induced target protein mutations. As such, there is growing interest in using TPD to address RET-driven cancers.^53–60

Results and discussion

Since the RET kinase domain (KD) is common to all oncogenic RET mutants,¹³ and the KD expresses multiple surface lysines, we developed a model system using a HEK293T cell line expressing RET-KD with a C-terminal HiBiT fusion tag to screen for RET heterobifunctional degraders. Initial assessment of RET-KD degradation was performed using our internal Kinodestroyer™ library (a similar approach was used to explore the degradable kinome).⁶¹ Multiple Kinodestroyer™ series effectively degraded the RET KD model system through cereblon (CRBN) and the proteasomal machinery (Fig. S1†). Focusing on the compound series bearing the promiscuous kinase binder CTx-0294885,⁶² we identified that a solvent front exit vector adjacent to the hinge binding motif with shorter linkers generally resulted in efficient degradation (exemplified by compound 7).

The phthalimide CRBN ligand of compound 7 brings with it two limitations. First, phthalimide-based CRBN ligands often enable the off-target degradation of CRBN neosubstrates, such as IKZF1; and second, the high polar surface area and multiple hydrogen bond acceptors of the phthalimide ligand increase the challenge of discovering orally bioavailable heterobifunctional degraders. To address these limitations, an internal library of CRBN ligands with reduced neosubstrate degradation and polar surface area was explored.^63–67 Gratifyingly, the phthalimide CRBN ligand could be replaced with a di-anilino CRBN ligand, without adversely affecting RET degradation, while eliminating the degradation of CRBN neosubstrates (Fig. 2, compound 8).

Fig. 2 Initial progression from non-selective degraders to RET selective pyrazolopyridine degraders showed that the structure–activity relationship (SAR) observed in the linker and CRBN binding region translated between targeting ligand series.

In parallel to the exploration of the CRBN ligand, the degradation selectivity of compound 7 was investigated. As previously observed with kinase degraders,^61,68–70 heterobifunctional degraders based on the promiscuous kinase ligand CTx-0294885 are more selective than the parent inhibitor, degrading 19 kinases in a global proteomic experiment (GPE) (Fig. S2a†) rather than the 235 kinases CTx-0294885 binds.⁶² To further increase degradation selectivity, a RET-selective pyrazolo-pyridine inhibitor^71,72 was selected for further targeting ligand and degrader development to further reduce binding to the human kinome. Incorporating the pyrazolo-pyridine targeting ligand with the linker and CRBN binder from the most active library hit provided compound 9, which efficiently degrades both RET wild-type (WT) KD and the resistance mutant RET G810R KD, and reduced off-target kinase degradation from 19 to 2 kinases in GPE (Fig. S2b†).

To ensure our RET-KD model system accurately predicts cellular effects, we correlated RET KD HiBiT degradation with full length mutant or fusion RET degradation. LC-2/ad cells bearing a CCDC6-RET fusion protein and TT cells with a RET C634W mutant were treated with compound 9, Western blot analysis of RET protein levels correlated with the degradation of HiBiT tagged RET KD. In all 3 systems, compound 9 achieved rapid, deep degradation of RET resulting in a significant reduction in TT and LC-2/ad cell viability (Fig. 3, GI₅₀ = 10 nM, E_max = 10%). To ensure that the observed RET degradation was CRBN mediated, compound 10 was synthesized, bearing an N–Me glutarimide, which prevents interactions with CRBN. Compound 10 failed to degrade RET (Table 1, HiBiT RET KD E_max 80%), but still effectively inhibited RET (ADP-Glo = 12 nM) and inhibited LC-2/ad cell growth (Fig. S3†), indicating that RET degradation was CRBN dependent. Therefore, compound 9 was explored further.

Fig. 3 RET selective degrader, compound 9, demonstrated potent and efficient RET KD, full length RET, and RET fusion degradation. A) The HiBiT RET-KD model system shows comparable degradation to full-length RET C634W and CCDC6-RET fusion protein as determined by Western blot. B and C) Compound 9 demonstrated superior cell growth inhibition effects to pralsetinib and selpercatinib in B) TT cells bearing mutant oncogenic RET C634W and C) LC-2/AD cells bearing oncogenic CCDC6-RET fusion protein.

Table 1 Exploration of the degradation and selectivity effects of targeting ligand modification^a

Compound	R =	X =	RET KD^b DC50/Emax (nM/%)	RET G810R KD^b DC50/Emax (nM/%)	VEGFR DC50/Emax (nM/%)	pVEGFR2 IC50/Emax (nM/%)	RET-WT^c IC50 (nM)
a DC50 represents the concentration needed to reduce HiBiT tagged RET-KD or VEGFR2 to 50% of the vehicle control maximum. IP represents the inflection point of the curve when 50% response is not achieved. Emax represents the minimum amount of HiBiT tagged RET-KD or VEGFR remaining after compound treatment, or the minimum amount of pVEGFR detected by Homogeneous Time-Resolved Fluorescence (HTRF). IC50 represents the concentration needed to inhibit 50% of the autophosphorylation of VEGFR, or to inhibit 50% of the generation of ADP by RET kinase as determined by ADP-Glo. b KD as an abbreviation for kinase domain. c RET IC50 determined by ADP-Glo.
9		H	2/15%	46/27%	145/25%	445/24%	5
10		Me	IP 1532/80%	>10 mM/90%	>10 mM/84%	—	12
11		H	2/9%	50/37%	—/84%	726/29%	—
12		H	1/17%	74/42%	—/65%	1071/25%	15
13		H	2/14%	330/42%	—/60%	>2000/47%	11
14		H	71/35%	—/71%	N/A	—	961
15		H	5/23%	103/37%	—/65%	>10 mM/61%	44

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Despite improved selectivity relative to the initial hit compound 7, compound 9 both inhibited and degraded VEGFR-2 (compound 9, pVEGFR2 inhibition IC₅₀ 445 nM, VEGFR DC₅₀/E_max 145 nM/25%), an off target of vandetanib and cabozantinib resulting in dose limiting toxicity. Thus, we explored whether targeting ligand modification could modulate VEGFR-2 activity. Exploration of the targeting ligand revealed that modifications of the P-loop binding region of the RET targeting ligand could modulate degradation selectivity. Removing the terminal aromatic ring reduced VEGFR-2 degradation without reducing RET degradation (compound 11, RET DC₅₀/E_max 2 nM/9%, VEGFR DC₅₀/E_max >10 μM/84%), though further reduction in the P-loop binding region reduced maximal RET G810R KD degradation (compounds 12–14, RET E_max 42–71%). Elaboration of the N-acetyl piperazine found in compound 11 to a 4-isopropylamide, 4-ethyl piperidine provided the best overall selectivity against VEGFR-2 degradation and inhibition, despite a reduction in RET degradation E_max (compound 15, RET DC₅₀/E_max 5 nM/23%, VEGFR DC₅₀/E_max >10 μM/65%, pVEGFR IC₅₀ >10 μM). Unfortunately, targeting ligand modifications did not enhance RET G810R degradation (Table 1).

With a targeting ligand in hand, improvements in the catalytic efficiency and overall selectivity of RET G810R degradation were sought via linker modification. Initial efforts focused on modification of the spacer between the targeting ligand and linker. Replacing the 1,4-phenyl ring with an N-substituted pyrazole retained VEGFR degradation selectivity, while improving both RET WT and G810R degradation 5-fold vs. compound 15 (Table 2, compound 16 RET DC₅₀/E_max 2 nM/13%, RET G810R DC₅₀/E_max 24 nM/24%, VEGFR DC₅₀/E_max >10 μM/51%). We therefore assessed the PK properties of compound 16 in mice, and observed moderate clearance and poor oral bioavailability (CL 31 mL min⁻¹ kg⁻¹, % F <1).

Table 2 Linker exploration and the effects on RET degradation efficiency and mouse PO PK^a

Compound	RET KD^b DC50/Emax (nM/%)	RET KD^b (G810R) DC50/Emax (nM/%)	RET-WT^c IC50 (nM)	VEGFR DC50/Emax (nM/%)	pVEGFR2 IC50/Emax (nM/%)	Cl (mL min^−1 kg^−1)	PO DN AUCInf (ng h mL^−1)	F
a DC50 represents the concentration needed to reduce HiBiT tagged RET-KD or VEGFR2 to 50% of the vehicle control maximum. IP represents the inflection point of the curve when 50% response is not achieved. Emax represents the minimum amount of HiBiT tagged RET-KD or VEGFR remaining after compound treatment, or the minimum amount of pVEGFR detected by HTRF. IC50 represents the concentration needed to inhibit 50% of the autophosphorylation of VEGFR, or to inhibit 50% of the generation of ADP by RET kinase as determined by ADP-Glo. F% represents the oral bioavailability as determined by comparison of the dose normalized PO administration AUC0-last and the dose normalized IV administration AUC0-last. b KD as an abbreviation for kinase domain. c RET IC50 determined by ADP-Glo.
15	5/23%	103/37%	43.6	—/65%	>10 mM/61%	—	—	—
16	1.9/13%	24/24%	9	>10 mM/51%	1162/30%	31	8.09	0.88%
17	1.8/13%	19/21%	3.5	>10 mM/92%	671/17%	16	181	18%
18	1.4/11%	19/24%	—	—	660/18%	15	601	53%
19	1.6/8%	19/21%	1.7	—	>2670/43%	12	325	23%
20	1.7/8%	21/22%	4.25	—	5141/42%	18	319	39%

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To improve clearance and oral bioavailability of RET-selective heterobifunctional degraders, we continued to explore linker modifications, with an eye towards making the physicochemical properties more drug-like by removing hydrogen bond donors and acceptors, rigidifying, and/or contracting the linker. Introducing a quaternary 4-piperidinol retained RET WT and G810R degradation activity, while significantly improving oral bioavailability and clearance (compound 17, RET DC₅₀/E_max 2 nM/13%, RET G810R DC₅₀/E_max 19 nM/21%, CL 16 mL min⁻¹ kg⁻¹, % F 18%). This unexpected result was investigated by molecular dynamics simulations, revealing that, compared to compound 16, compound 17 more frequently occupied conformations with a lower solvent accessible surface area (SASA) (Fig. 4). Presumably the collapsed conformation decreased the effective ePSA, thereby improving membrane permeability.^73–76 Given the improved in vivo profile and modified targeting ligand present in compound 17, selectivity was once again assessed by proteomic studies (Fig. S4†). Gratifyingly, compound 17 further reduced the number of off targets observed compared to compound 9 from 7 to 2, AURKA and PARP14.

Fig. 4 SASA analysis demonstrated that compound 17 adopted conformations with reduced solvent accessible surface area at lower energy levels than compound 16.

In this campaign, linker contraction also proved beneficial to both catalytic efficiency and PK properties. Shorter linkers with fewer rotatable bonds maintained low clearance in mice and, when combined with a 4-piperidinol fragment, improved oral bioavailability (compound 18, RET DC₅₀/E_max 1.4 nM/11%, RET G810R DC₅₀/E_max 19 nM/24%, CL 15 mL min⁻¹ kg⁻¹, % F 53%). Surprisingly, continuing to reduce linker length, such as in compound 19, decreased VEGFR-2 inhibition while maintaining RET degradation, although the oral bioavailability decreased (compound 19, RET DC₅₀/E_max 1.6 nM/8%, RET G810R DC₅₀/E_max 19 nM/21%, pVEGFR-2 IC₅₀ >2670 μM, CL 12 mL min⁻¹ kg⁻¹, % F 23%). Introducing a more metabolically stable pyrazolo-pyrazine targeting ligand to compound 19 provided the best balance of degradation activity, selectivity, and PK properties (compound 20, RET DC₅₀/E_max 1.7 nM/8%, RET G810R DC₅₀/E_max 21 nM/22%, pVEGFR-2 IC₅₀ = 5141 μM, CL 18 mL min⁻¹ kg⁻¹, % F 39%), and was therefore selected for in vivo investigation.

Since compound 20 demonstrated efficient degradation of both WT and G810R RET, the degradation effects on other mutants of interest were assessed in HiBiT cell lines bearing G810S/C RET KD. Compound 20 efficiently degraded RET G810S/C, with a DC₅₀ of 3 and 12 nM and E_max of 25% and 31%, respectively (Table S1†). Next the cellular viability effects of compound 20 were profiled in a panel of Ba/F3 cell lines transfected with full length KIF5B-RET fusions bearing kinase domain resistance mutants of interest, specifically clinically observed gatekeeper (V804L),^12,18–20 solvent front (G810R),^12,36,37 hinge (Y806N)¹⁸ or roof (L730I)¹⁸ mutants (Fig. 5). Compared to current and investigational next generation RET selective inhibitors, compound 20 provided broad mutant coverage, efficiently killing cells bearing all tested KIF5B-RET fusions, while the tested RET-selective inhibitors all demonstrated reduced efficacy against at least one mutant form, supporting the theory that degraders require less binding affinity than inhibitors and may be able to address target protein mutations causing resistance to inhibitors.⁴⁷ Importantly, this cell killing effect was not general to all cells, as compound 20 only shows detrimental effects on the proliferation of HepG2 cells at 10 μM (Fig. S6†). Notably, these experiments were conducted in Ba/F3 cell lines, a murine cell line containing mouse CRBN. Despite the residue differences in the thalidomide binding domain between mouse and human CRBN,⁷⁷ the degradation rate of compound 17 in Ba/F3 cells transfected with human RET was similar to the rate in human cell lines (Fig. S5†).

Fig. 5 Growth inhibition effects of RET-selective inhibitors and degraders in Ba/F3 cells transfected with RET mutants known to cause resistance to RET-selective inhibitors. Compound 20 demonstrated more cross-mutant growth effects than known RET-selective inhibitors. A) Growth inhibition effects in cells bearing solvent front mutations. TPX-0046 and compound 20 demonstrated robust growth inhibition. B) Growth inhibition effects in cells bearing gatekeeper mutations. Selpercatinib, pralsetinib, and compound 20 demonstrated robust growth inhibition. C) Growth inhibition effects in cells bearing hinge binding region mutations. Compound 20 demonstrated superior growth inhibition effects. D) Growth inhibition effects in cells bearing rooftop mutations. TPX-0046 and compound 20 demonstrate growth inhibition effects.

The use of glutarimide-based cereblon ligands can raise the potential for undesired off-target degradation effects due to the resurfacing of cereblon enabling the interaction with known neosubstrates such as GSPT1, SALL4, and IKZF1. Given the known potential for undesired effects due to the degradation of CRBN neosubstrates, such as teratogenicity for SALL-4 degraders, we profiled compound 20 in a panel of cell lines bearing HiBiT-tagged neosubstrates. While compound 20 showed selectivity against GSPT1 and IKZF1, unfortunately degradation of SALL4 was observed (6.1 μM, 36% E_max, Table 3).

Table 3 In vitro and in vivo profile of compound 20

Specific measure		Compound 20
a DC50 represents the concentration needed to reduce HiBiT tagged GSPT1, SALL4, or IKZF1 to 50% of the vehicle control maximum. Emax represents the minimum amount of HiBiT tagged GSPT1, SALL4, or IKZF1 remaining after compound treatment. b IC50 represents the concentration needed to inhibit 50% of the binding of a CRBN ligand modified with a NanoBRET 590 dye to NanoLuciferase tagged CRBN in a HEK293T cell line. c LD50 represents the concentration at which the cell density has been reduced by 50% from baseline. Emax represents the cell growth observed at the maximum concentration tested as a percentage of the growth of the cells in the negative control. d hERG IC50 determined by automated patch clamp method (SyncroPatch 384PE). e CYP inhibition and TDI were performed in human liver microsomes using the non-dilution approach. f Plasma protein and brain homogenate binding determined by ultracentrifugation. g Solubility determined by shake-flask method at 37 °C. h formulation: 40% PEG 400, 60% of a 10% solution of 10% HP-β-CD in water. i K p and Kp,uu calculated based on individual animals, and the individual ratios were then averaged to determine the Kp and Kp,uu. Outlier animals were excluded from averages. j Average of two animals. k Average of three animals. l T 1/2 could not be determined as plasma levels did not return to baseline by 24 h.
In vitro selectivity
Neosubstrate selectivity
GSPT1 HiBiT (24 h)	DC50 (μM)/Emax (%)^a	>10/100
SALL4 HiBiT (6 h)	DC50 (μM)/Emax (%)^a	6.1/36
IKZF1 HiBiT (6 h)	DC50 (μM)/Emax (%)^a	>10/54
CRBN binding: NanoBRET	IC50 (nM)^b	10
ADMET
HepG2 viability (72 h)	LD50 (μM)/Emax (%)^c	6.4/−99
hERG inhibition	IC50 (μM)^d	1.54
CYP inhibition (TDI)
1A2	IC50 (μM)^e (ratio)	>10 (1)
2B6		>10 (1)
2C19		>10 (1)
2C8		>10 (1)
2C9		>10 (1)
2D6		>10 (1)
3A4-M		2.6 (3.9)
3A4-T		>10 (1)
Hepatocyte Cl int,scaled,WS
Mouse	mL min^−1 kg^−1	38
Rat		17
Dog		18
Cyno		21
Human		13
Plasma protein binding
Mouse	% unbound^f	1.9
Rat		1.1
Dog		2.4
Cyno		2.5
Human		5.7
Brain homogenate binding
Mouse	% unbound^f	0.14
Rat		0.24
Solubility^g
SGF	μM	1024.45
FaSSIF		3.19
FeSSIF		55.47
In vivo PK
IV administration (1 mg kg ^−1 )		Mouse	Rat
CL	mL min^−1 kg^−1	18.4	25.5
T 1/2	h	5.3	8.7
V d,ss	L kg^−1	7.8	18.2
AUClast	h ng mL^−1	814	572
k p
2 h^j		0.12
8 h^k		0.26
24 h^k		—
K p,uu
2 h^k		0.009
8 h^k		0.020
24 h^k		—
PO administration (10 mg kg ^−1 )
C max	Ng mL^−1	312	35.4
T 1/2	h	ND^l	ND^l
AUClast	h ng mL^−1	3192	475
% F		39	8
k p
4 h^j		0.12
8 h^k		0.22
24 h^k		—
K p,uu
4 h^j		0.009
8 h^k		0.016
24 h^k		—

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To understand the potential to develop compound 20, the in vitro ADME characteristics were profiled (Table 3). Compound 20 showed moderate stability to hepatic clearance across species, which, when combined with the relatively high plasma protein binding across species, indicated the potential for low in vivo clearance. However, compound 20 possessed undesirable off target effects against Cyp3A4 (IC₅₀ = 2.57 μM, TDI ratio = 3.9) and hERG (IC₅₀ = 1.54 μM). Additionally, compound 20 was poorly soluble in FaSSIF and FeSSIF (3.19 and 55.47 μM, respectively) presenting a potential formulation challenge.

Despite the low solubility, compound 20 was dosed to fasted male CD1 mice and fasted male Sprague Dawley rats by IV and PO administration. After IV administration (1 mg kg⁻¹), compound 20 showed scalable PK, with moderate CL (Table 3: mouse: 18.4 mL min⁻¹ kg⁻¹; rat: 25.5 mL min⁻¹ kg⁻¹), a high volume of distribution (Table 3: mouse: 7.8 L kg⁻¹; rat 18.2 L kg⁻¹), and moderate exposure (Table 3: mouse: 814 h ng mL⁻¹; rat: 572 h ng mL⁻¹). After oral administration (10 mg kg⁻¹) in mice, compound 20 showed good oral bioavailability (Table 3: F = 39%), whereas in SD rats, the oral bioavailability was low (Table 3: F = 8%). As in vivo efficacy and PD experiments leveraged mice as the model species, we examined the CNS exposure of compound 20 in male CD1 mice after IV administration at 2, 8, and 24 hours and after oral administration at 4, 8, and 24 hours. Compound 20 crossed the BBB after both IV and oral administration. Examining the brain exposure after PO administration (10 mg kg⁻¹) at and shortly after the T_max, compound 20 crossed the blood brain barrier (Table 3: K_p 0.12–0.22 from 4–8 h), but with high brain homogenate binding (Table 3: 0.14% unbound) resulting in reduced free exposure (Table 3: K_p,uu 0.009–0.020 from 4–8 hours). At 24 hours, the compound was fully cleared from the brain.

In vivo pharmacology

NSCLC cancers with RET fusion driver mutations are well-established model systems for assessing the efficacy of RET targeted therapies. Further, RET fusions often redistribute to the cytoplasm,^1,5 situating RET in the same cellular compartment as the proteasomal machinery, while RET mutant proteins tend to remain associated with the cell membrane. Therefore, a RET degrader may offer maximal effect in the context of RET-fusion proteins, and PDX models of a RET-driven NSCLC bearing the common KIF5B-RET fusion (CTG-0838)^{23,71,78–80} and a colorectal cancer model bearing the common CCDC6-RET fusion (CR2518)⁷⁹ were selected as in vivo proof of concept systems to assess the anti-tumor efficacy of compound 20.

For both PDX models, patient-derived cells were implanted subcutaneously into BALB/c mice and treated with either the standard of care RET-selective inhibitor selpercatinib (50 mg kg⁻¹, PO, BID),⁸¹ or compound 20 (5 mg kg⁻¹, IV, QD or 30 mg kg⁻¹, PO, QD). Both IV and oral administration of compound 20 produced tumor regression comparable to selpercatinib in both models (Fig. 6B and F), characterized by rapid, sustained degradation of KIF5B-RET as assessed by Western blot (Fig. 6H). This analysis was only performed on the CTG-0838 tumors, as the end of study CR2518 tumors were too small to analyze. In a separate, PD focused study, a single administration of compound 20 at the same doses led to a >50% reduction in KIF5B-RET after 5 hours (Fig. S7†). To demonstrate that our model was working as intended, RET pathway inhibition was monitored using pSHC as a RET pathway biomarker.²³ The level of pSHC in tumor samples was assessed by Western blot and showed robust reduction in pSHC levels at 5 hours, which was retained at 24 hours, indicating potent and sustain inhibition of RET signalling by compound 20.

Fig. 6 RET selective degrader, compound 20, displayed moderate oral bioavailability that translated into effective in vivo activity. A) PK profile of compound 20 showed moderate clearance and moderate bioavailability driving reasonable exposure. B) When dosed to mice implanted with CR2518 colorectal cancer patient-derived cells with a driving CCDC6-RET fusion mutation, IV dosed compound 20 provided comparable efficacy to the clinical RET selective inhibitor selpercatinib, and (C) was well tolerated. D) In intracranial CR2518 tumor bearing mice, compound 20 crossed the BBB, showing high intracranial tumor concentration and (E) caused CCDC6-RET degradation and reduced pSHC levels as detected by Western blot. F) Compound 20 dosed orally to mice implanted with CTG-0838 NSCLC patient-derived cells with a driver KIF5B-RET mutation provided comparable efficacy to the clinical RET selective inhibitor selpercatinib at a lower dose, (G) was well tolerated, and (H) resulted in significant reduction of both KIF5B and pSHC levels intratumorally 5 h after dosing.

Up to 25% of NSCLC bearing driver RET fusions result in brain metastases.^82–84 To address these metastases, RET targeted therapies must be able to cross the brain blood barrier (BBB) in sufficient concentration to drive comparable effects as seen in peripheral tumors. Based on the PK results demonstrating that compound 20 crossed the BBB in CD-1 mice, mice bearing an intracranially implanted orthotopic CCDC6-RET colorectal cancer PDX organoid were given a single dose oral dose of 30 mg kg⁻¹ of compound 20, and plasma and tissue levels of compound 20, as well as CCDC6-RET and pSHC protein levels, were monitored over 24 hours. In PK studies, compound 20 crossed the BBB with a K_p,uu of 0.009–0.016, while in PK/PD studies, compound 20 accumulated in the intracranial tumor, causing rapid and robust reduction of CCDC6-RET and pSHC levels, comparable to the effects observed in peripheral tumor models (Fig. 6D and E). The CNS activity of compound 20 was comparable to the activity seen with selpercatinib in the same model (Fig. S8†).

Chemistry

The synthesis of these RET degraders was designed such that the P-loop binding region and the CRBN binder could both be varied independently, allowing maximal flexibility in the synthesis of RET heterobifunctional degraders. To this end, the route design initiated with the parallel synthesis of the targeting ligand (Scheme 1) and the CRBN ligand (Scheme 2).

Scheme 1 Targeting ligand synthesis.

Scheme 2 CRBN ligand synthesis.

Synthesis of the targeting ligand started with pyrazolopyridine 21a or pyrazolopyrazine 21b, which was then coupled with the exit moiety by a Suzuki reaction. Once the exit moiety was in place, the P-loop attachment point was deprotected to a phenol, and subsequently activated as the triflate. This triflate was coupled to the pyridine of the extended P-loop binding region, through another Suzuki reaction. To this pyridine was added the relevant amine by an S_NAr reaction, followed by capping with the desired moiety to fill the P-loop pocket.

In parallel to the synthesis of the targeting ligand, CRBN ligand synthesis began by coupling the linker to the aniline of the CRBN ligand. When the full fragment was not commercially available, this was done via either a Suzuki reaction of tert-butyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate with 4-bromo-3-fluoro-nitrobenzene, or via an S_NAr reaction of the relevant piperidine with 3,4-difluoronitrobenzene. Then, the nitro group was reduced to the amine under Pd/C or Fe conditions, and the aniline was added to racemic bromo-glutarimide to result in relevant CRBN binder.

Once the targeting ligand and CRBN ligand were in hand, the two convergent fragments were coupled together either by an amide coupling using HATU (9–17), or with a reductive amination reaction (19 and 20), to provide the heterobifunctional degrader in good overall yields as racemates at the glutarimide stereocenter. Uniquely, compound 18 was synthesized by building the linker and CRBN ligand onto targeting ligand fragment 26g to provide the racemate 18 in good overall yield (Scheme 3).

Scheme 3 Degrader synthesis.

Discussion and conclusion

In this study, we designed a series of RET-selective degraders to overcome a broad range of RET mutants that drive resistance to small molecule inhibitors. Initial screening of a library of unselective kinase inhibitor-derived degraders revealed that multiple different scaffolds induced RET degradation, with the solvent front as a common exit vector. Initial SAR exploration focused on CRBN ligands, specifically non-phthalimide based ligands, which predispose heterobifunctional degraders to a physicochemical property space that may enable oral bioavailability, and are known to avoid the degradation of CRBN neosubstrates.^63–67 This effort led to the discovery of heterobifunctional degraders employing a di-anilino CRBN ligand, which maintained RET degradation without degrading CRBN neosubstrates like IKZF1, unlike the phthalimide CRBN ligand derived initial hits.

Utilizing this early SAR information, we explored heterobifunctional degraders based around the RET-selective pyrazolo-pyridine targeting ligand. Early degraders, such as compound 9, showed robust activity against WT RET, but a significant reduction in activity against the solvent front mutant G810R, and undesired off-target degradation of VEGFR. Exploration of the pyrazolo-pyridine targeting ligand revealed that P-loop substitutions increased selectivity for RET, however, these modifications were insufficient to improve degradation of the mutant of interest G810R.

Investigation of the linker SAR revealed that the functional group identity at the solvent front enabled efficient G810R mutant RET degradation, as observed in compound 16. Modifications to the linker of this compound resulted in the development of orally bioavailable and CNS-penetrant RET degraders. Compound 20 inhibited the growth of cells bearing clinically observed RET resistance mutants, reflecting the pan-mutant profile desired. When administered by oral gavage to mice bearing RET-fusion tumors, compound 20 demonstrated robust efficacy and CNS penetrance, with sufficient exposure to elicit intracranial RET degradation comparable to peripheral RET degradation.

These findings supported our hypothesis that exploiting the mechanistic differences between degradation and inhibition might provide a means to overcome target protein mutations driving resistance to inhibitors. Compound 20 demonstrated that pan-mutant activity could be achieved with a degrader, and that such a degrader could achieve oral bioavailability and CNS penetrance sufficient to achieve activity against peripheral and intracranial disease. Future efforts include improving the ADME profile and overall PK to support the development of potential clinical RET degraders.

Experimental

Chemical synthesis

See ESI† for synthetic methods.

Cell culture

HEK293T-RET WT HiBiT cells were generated from HEK293T cells obtained from American Type Culture Collection (ATCC, Manassas, VA; catalogue number CRL-3216) were transduced with pCDH-EF1 mammalian expression vectors generated in-house to ectopically express wild-type or G810R RET kinase domain (AA 658-1114) fused with a C-terminal HiBiT tag. Expression of the fusion protein was driven by the EF1 promoter, and transduced cells were selected with puromycin. All HEK293T-HiBiT cell lines were grown in DMEM media supplemented with 10% fetal bovine serum.

HEK293 cells expressing KDR/VEGFR2 were obtained from SibTech (Brookfield, CT, USA; catalog # SBT021.293, clone 4D7), and were modified in-house by endogenously tagging KDR/VEGFR2 with a C-terminal HiBiT tag via CRISPR knock in.

Ba/F3 cell lines bearing mutant RET proteins were generated from Ba/F3 cells obtained from Leibniz Institute DSMZ (DSMZ No. ACC 300, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture GmbH, Braunschweig, Germany). Ba/F3 cells ectopically expressing the KIF5B-RET (V804L) fusion protein were created by lentiviral transduction of the parental BA/F3 cell line with lentivirus produced using a pCDH-EF1 mammalian expression vector plasmid containing the KIF5B-RET(V804L) construct with a C-terminal HiBiT tag and IRES Puro (Kif5b_RET(V804L)-HiBiT (CrownBio) IL3-independent). Transduced cells were selected with puromycin. Expression of the fusion protein is driven by the EF1 promoter.

Ba/F3 cells ectopically expressing the KIF5B-RET(G810R) fusion protein were created by lentiviral transduction of the parental BA/F3 cell line with lentivirus produced using a pCDH-EF1 mammalian expression vector plasmid containing the KIF5B-RET(G810R) construct with a C-terminal HiBiT tag and IRES Puro (Kif5b_RET(G810R)-HiBiT (CrownBio) IL3-independent). Transduced cells were selected with puromycin. Expression of the fusion protein is driven by the EF1 promoter. Ba/F3 cell lines ectopically expressing the following RET fusions and/or mutations were engineered by the same method: KIF5B-RET L730I, and KIF5B-RET Y806N. All Ba/F3 cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS).

LC-2/AD cells were obtained from Sigma Aldrich (catalogue number 94072247) and grown in a 1 : 1 mixture of RPMI 1640 media and HAMS F12 media supplemented with 10% fetal bovine serum (FBS).

TT cells were obtained from ATCC (catalogue number CRL-1803) and grown in RPMI 1640 media supplemented with 10% fetal bovine serum and 10% sodium pyruvate.

KELLY cells were obtained from DSMZ (DSMZ No. ACC 300, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture GmbH, Braunschweig, Germany) and were grown in RPMI 1640 media supplemented with 10% FBS and 2 mM sodium pyruvate.

NCIH929 cells were obtained from ATCC (Manassas, VA, Catalogue Number CRL-9068) and were grown in RPMI 1640 media supplemented with 10% FBS and 0.5 mM 2-mercaptoethanol.

HepG2 cells were obtained from ATCC (Manassas, VA, Catalogue Number HB-8065) and were grown in MEM media supplemented with 10% FBS.

Animal studies

All procedures related to animal handling, care, and treatment were conducted in compliance with all applicable regulations and guidelines of, and approved by, the relevant Institutional Animal Care and Use Committee (IACUC), specifically the IACUC of CrownBio for studies utilizing the xenograft CR2518 model in mice, the IACUC of Champions Oncology for studies utilizing the xenograft CTG-0838 model in mice, and the IACUC of Syngene International Limited for PK studies.

Biological methods

See ESI† for detailed in vitro and in vivo methods.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

The authors declare the following competing financial interest(s): the authors declare competing financial interests as C4 Therapeutics, Inc. stockholders and employees.

Acknowledgements

The authors thank the chemistry team at Syngene International Limited who assisted with the synthesis of RET degraders: Aditya Kumar (Project Leader), Prakash Kumar, Rashmi Jayarama, Bhushanarao Dogga, Krishna Tulishetti, Nagarjuna Pothaladevi, Jagadeesh Muthannagari, Suresh Tummala, Sandhya Devaraj, Ramakrishna Betrala, and Kiran Hirbhagat. The authors also thank the chemistry team at WuXi International Inc who assisted with the synthesis of RET degraders: Chuang Yang, Hudantai Liu, Wenjian Lian, Tao Lv, and Jiabin Hu.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Experimental details of pharmacology and synthetic chemistry. See DOI: https://doi.org/10.1039/d5md00337g

‡ Current Address: Aleksia Therapeutics, Waltham, MA, USA.

§ Current Address: Acrivon Therapeutics, Boston, MA, USA.

¶ Current Address: Photys Therapeutics, Waltham, MA, USA.

|| Current Address: Thermo Fischer Scientific, USA.

** Current Address: MD Anderson Cancer Center, Houston, TX, USA.

†† Current Address: Superluminal Medicines Inc, Boston, MA, USA.

‡‡ Current Address: Tower Semiconductor, Migdal Haemek, Israel.

§§ Current Address: Expansion Therapeutics, Boston, MA, USA.

¶¶ Current Address: Treeline Therapeutics, Boston, MA, USA.

|||| Current Address: Spanish National Cancer Center, Madrid, Spain.

*** Current Address: Flagship Pioneering FL94, Cambridge, MA, USA.

††† Current Address: Larkspur Biosciences, Watertown, MA, USA.

‡‡‡ Current Address: Day One Biopharmaceuticals, Suffolk County, MA, USA.

§§§ Current Address: Otsuka Pharmaceuticals Companies, USA.

¶¶¶ Current Address: Remix Therapeutics, Watertown, MA, USA.

|||||| Current Address: Psivant Therapeutics, Boston, MA, USA.

**** Current Address: Nexo Therapeutics, Littleton, CO, USA.

†††† Current Address: PostEra, Cambridge, MA, USA.

‡‡‡‡ Current Address: Deciphera Pharmaceuticals, Waltham, MA, USA.

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