Discovery of an Aldo-Keto reductase 1C3 (AKR1C3) degrader

The study addresses therapeutic resistance mechanisms in advanced prostate cancer mediated by AKR1C3 and the androgen receptor splice variant ARv7. AKR1C3, a NAD(P)(H)-dependent oxidoreductase, is overexpressed in multiple cancers and contributes to androgen biosynthesis, tumor proliferation, and chemotherapy resistance. It stabilizes AR full-length and splice variants, particularly ARv7, thereby promoting resistance to androgen receptor signaling inhibitors (ARSIs) such as enzalutamide, darolutamide, and apalutamide. Ubiquitination and degradation of AR-FL and ARv7 increase when AKR1C3 levels are low, but are suppressed when AKR1C3 is overexpressed. The authors hypothesized that targeted degradation of AKR1C3 would destabilize ARv7 and counter ARSI resistance. Leveraging the PROTAC modality, which can catalytically remove target proteins via the ubiquitin-proteasome system and potentially target “undruggable” proteins, the study aimed to design and evaluate the first AKR1C3 degrader that also induces ARv7 degradation.

AKR1C3 has been implicated across hormone-dependent cancers and hematologic malignancies in regulating differentiation, proliferation, apoptosis, and drug resistance. Therapeutic strategies have variably sought selective AKR1C3 inhibition (e.g., in castration-resistant prostate cancer to suppress androgen synthesis) or pan-AKR1C family inhibition to combat chemotherapy resistance in leukemias. Prior reports indicate AKR1C3 stabilizes AR-FL and AR splice variants, particularly ARv7, driving resistance to ARSIs. Small-molecule AKR1C3 inhibitors (e.g., indomethacin, BMT4-158) can degrade the target at high concentrations. The authors’ previous medicinal chemistry delivered potent and highly selective AKR1C3 inhibitors (compounds 1–3), including inhibitor 3 (AKR1C3 IC50 = 43 nM; >2300-fold selectivity over AKR1C2). PROTACs have demonstrated advantages over inhibition including catalytic target removal and improved pharmacodynamics; an AR degrader PROTAC has entered clinical trials, but no degraders targeting AR splice variants have advanced similarly. These data motivated the design of an AKR1C3-targeting PROTAC to concurrently degrade AKR1C3 and ARv7.

Design and docking: The AKR1C3-binding warhead was derived from the authors’ selective inhibitor series. Molecular docking (SeeSAR 12.1) into the AKR1C3 active site (PDB 3UG8) guided linker attachment and length to ensure the PROTAC’s E3 ligase ligand remained solvent-exposed while maintaining key hydrogen bonds (amide carbonyl to Tyr55 and His117) for AKR1C3 inhibition. A triazole-PEG2 linker was selected, and cereblon engagement was achieved via lenalidomide. Synthesis: The AKR1C3 warhead (propargyl ether, intermediate 14) was synthesized via a previously optimized route using a tert-butyldiphenylsilyl-protected phenol, with protection/deprotection and cross-coupling steps to assemble the biphenyl core, followed by O-propargylation. The lenalidomide-PEG2-azide linker (18) was generated from an azide-PEG precursor via tert-butyl bromoacetate installation, deprotection to the acid, conversion to the acyl chloride, and coupling to lenalidomide. The final PROTAC 5 was obtained via Huisgen cycloaddition (“click” chemistry) between warhead 14 and linker 18, followed by saponification to the acid. Enzyme assays: Recombinant AKR1C1–AKR1C4 were prepared. Dehydrogenase activity was measured by NADH formation at 340 nm using S-tetralol as substrate. IC50 values for inhibitors (warhead 4, PROTAC 5, comparator 3) against AKR1C3 and AKR1C2 were determined under conditions where substrate concentrations equaled Km, with inhibitor titrations in quadruplicate. Cell culture: 22Rv1 and LNCaP cells were cultured in RPMI 1640 with 10% FBS; where indicated, charcoal-stripped serum (CSS) media was used to elevate AKR1C3 expression. LNCaP1C3 cells stably overexpressing AKR1C3 were utilized. Compounds included PROTAC 5, warhead 4, inhibitor 3, lenalidomide (cereblon ligand), and enzalutamide (ENZ). Protein degradation assays: Western blots measured AKR1C3, AKR1C1/2, and ARv7 over time. For small molecules (3, 4) and warhead (4), degradation was assessed at 43 nM (IC50 of 3) or 1 µM at 0–72 h. For PROTAC 5, time-course at 10 nM from 0–72 h and low-dose (1 nM) assessments were performed. DC50 values at 72 h were derived from concentration–response data. Proteasome dependency was tested by MG132 (3 µM, 2 h) pretreatment prior to 5 exposure (10 nM, 4 h). Cereblon engagement/competition was probed with lenalidomide pretreatment (3 µM, 2 h) followed by 5 (10 nM, 72 h). Cell viability: MTT/MTS assays measured viability after 72 h exposure across concentrations (0.001–10 µM) in 22Rv1 (normal and CSS media), LNCaP (AKR1C3-null), and LNCaP1C3 (AKR1C3-overexpressing) cells, alone and with ENZ co-treatment. DMSO vehicle controls and multiple replicates supported statistical analysis.

• First AKR1C3-targeting PROTAC (compound 5) discovered; dual degradation of AKR1C3 and ARv7 demonstrated.
• Enzyme potency/selectivity: Warhead 4 AKR1C3 IC50 = 62 ± 1 nM; PROTAC 5 AKR1C3 IC50 = 77 ± 2 nM (similar to inhibitor 2; slightly less potent than 3 at 43 nM). Selectivity vs AKR1C1/2 diminished relative to 3: warhead 4 ~146-fold; PROTAC 5 ~116-fold (vs >2300-fold for 3).
• Degradation efficacy (22Rv1): PROTAC 5 degraded AKR1C3 starting at 4 h post-treatment at 10 nM, reaching ~75% maximal reduction by 72 h. ARv7 was concomitantly degraded by 72 h. AKR1C1/C2 also decreased to a lesser extent.
• DC50 at 72 h: AKR1C3 DC50 = 52 nM; ARv7 DC50 = 70 nM; AKR1C1/C2 DC50 = 49 nM.
• Superiority vs inhibitors: At 10 nM, PROTAC 5 significantly degraded AKR1C3 whereas small-molecule inhibitors 3 and 4 did not. Small-molecule inhibitor 3 at 1 µM showed time-dependent target degradation by 72 h, but was less efficient than warhead 4 and markedly inferior to PROTAC 5 at low nanomolar concentrations.
• Mechanism: AKR1C3 degradation by 5 was blocked by proteasome inhibitor MG132 (3 µM, 2 h pretreatment; assessment at 4 h), indicating proteasome dependence. A concentration-dependent ‘hook effect’ was observed above ~1 µM, consistent with ternary complex saturation. Lenalidomide pretreatment (3 µM) induced AKR1C3 degradation and trended to attenuate PROTAC effect, though interpretation is complicated by lenalidomide’s intrinsic activity against AKR1C3 in this context.
• Cell viability: In 22Rv1 (moderate AKR1C3), PROTAC 5 reduced viability significantly at 0.01, 0.1, and 10 µM after 72 h; 1 µM alone was not significant, likely due to the hook effect. Enzalutamide (25 µM) alone had no effect (resistant line), but co-treatment with 5 at 1 µM significantly reduced viability, overcoming ENZ resistance. In 22Rv1 grown in CSS (high AKR1C3), 5 produced a dose-dependent reduction from 0.01 µM with ~55% reduction at 10 µM; co-treatment with 0.1 µM 5 and ENZ significantly reduced viability. LNCaP (AKR1C3-null) showed no effect from 5, while ENZ reduced viability. LNCaP1C3 (AKR1C3-overexpressing) became ENZ-resistant; 5 reduced viability at 1 and 10 µM, confirming on-target action.

The findings validate the hypothesis that targeted degradation of AKR1C3 destabilizes ARv7 and can overcome resistance mechanisms to ARSIs in prostate cancer. PROTAC 5 retains AKR1C3 inhibitory potency while adding catalytic protein removal via cereblon-mediated ubiquitination, leading to robust and time-dependent degradation of AKR1C3 and concomitant ARv7 loss. The proteasome dependency (MG132 sensitivity) and the observed hook effect support a bona fide PROTAC mechanism. Efficacy correlates with AKR1C3 expression: PROTAC 5 reduces viability in AKR1C3-expressing models, potentiates enzalutamide in resistant 22Rv1 cells, and is inactive in AKR1C3-null LNCaP cells, while regaining activity in AKR1C3-overexpressing LNCaP1C3. Although selectivity over AKR1C1/2 is reduced relative to the most selective inhibitor, partial AKR1C1/2 degradation may contribute to overcoming therapy resistance where pan-AKR1C activity is advantageous. Overall, the study establishes AKR1C3 degradation—along with secondary ARv7 loss—as a promising strategy to mitigate ARSI resistance in advanced prostate cancer.

This work reports the first PROTAC degrader of AKR1C3 (compound 5) that also degrades ARv7 and, to a lesser extent, AKR1C1/2. PROTAC 5 outperforms selective small-molecule inhibitors at low nanomolar concentrations, induces proteasome-dependent degradation starting at 4 h with ~75% maximal reduction by 72 h, and restores sensitivity to enzalutamide in resistant prostate cancer cells. These results provide a compelling chemical probe and a potential therapeutic strategy targeting the AKR1C3/ARv7 axis to counter ARSI resistance. Future work should optimize warhead, linker, and E3 ligase ligand combinations to enhance potency, isoform selectivity, and pharmacokinetics, and evaluate in vivo efficacy and safety.

• Reduced isoform selectivity of the warhead and PROTAC versus the most selective inhibitor (compound 3), potentially causing partial pan-AKR1C degradation.
• Evidence of a hook effect at higher concentrations (>1 µM), which may limit activity at certain dose ranges.
• Proteasome inhibitor MG132 was toxic over longer incubations, restricting mechanistic assessment to early time points (4 h).
• Lenalidomide’s intrinsic impact on AKR1C3 complicates competitive binding interpretations in combination experiments.
• Discrepancy between biochemical potency and cellular activity likely due to cell penetration issues of carboxylic acid-containing compounds.