New PROTAC Drug Degrades Cancer's Immortality Enzyme TERT

Telomerase, composed of the reverse transcriptase TERT and its RNA template TERC, is reactivated in 80–90% of human cancers, enabling unlimited cell division. Beyond telomere maintenance, TERT also supports cancer survival through non-catalytic roles including enhanced DNA damage repair, transcriptional regulation, mitochondrial ROS suppression, and modulation of apoptotic thresholds. Conventional telomerase inhibitors—including imetelstat, the first FDA-approved agent in this class—primarily block TERT's enzymatic function but may leave these protein-interaction-driven activities intact, limiting their therapeutic ceiling.

To address this gap, the research team developed NU-PRO-1, a covalent proteolysis targeting chimera (PROTAC) designed to eliminate TERT protein entirely rather than just silence its enzymatic activity. The design began with structure-based computational docking using both Tribolium castaneum TERT (tcTERT) and a high-resolution cryo-EM human TERT structure, identifying the primer grip cysteine (C931 in hTERT) as the covalent attachment site. The team synthesized a library of six initial PROTAC candidates linking their previously developed covalent inhibitor NU-1 to the VHL E3-ligase ligand (S,R,S)-AHPC via variable-length PEG linkers, with attachment at either the para or meta positions of NU-1's difluorophenyl tail.

Screening in MCF7 human breast cancer cells revealed that meta-linked PROTACs outperformed para-linked variants, and linker length was critical—the two-unit PEG meta variant (A₂m) achieved 69% TERT degradation at just 0.3 μM. Guided by updated hTERT docking data showing a key hydrogen bond between the para-fluoro group and R631, the team then synthesized NU-PRO-1, which restored the para-fluorine while retaining the meta linker attachment. NU-PRO-1 demonstrated superior degradation potency over A₂m, achieving effective TERT reduction at 0.1–0.4 μM within 8 hours. A full time-course experiment showed degradation beginning at 2 hours, nadir near 10 hours, with TERT levels rebounding toward baseline by 24 hours—indicating transient degradation kinetics consistent with covalent PROTAC behavior.

Mechanism-of-action studies confirmed NU-PRO-1 acts specifically through the ubiquitin-proteasome pathway: pre-treatment with the proteasome inhibitor MG132, the Cullin-RING ligase inhibitor MLN4924, or competitive VHL ligands (AHPC-HCl or VH-298) all blocked TERT degradation. Critically, a non-covalent control PROTAC (A₂m-nc, with the reactive warhead methylene removed) showed markedly reduced activity, validating the importance of covalent TERT engagement for efficient degradation of this low-abundance target.

To probe functional consequences, cells were irradiated with 6 Gy and assessed 24 hours later for persistent DNA double-strand break markers (53BP1 and γH2AX foci). At the TERT-degrading dose (0.3 μM), NU-PRO-1 delayed DNA repair more effectively than NU-1 alone. At a higher, non-degrading dose (1.0 μM)—which inhibits catalytic activity but not protein abundance—NU-PRO-1 behaved similarly to NU-1, suggesting the enhanced radiosensitization is specifically attributable to TERT protein loss rather than catalytic inhibition alone. These findings argue that TERT's non-catalytic functions meaningfully contribute to DNA damage response in cancer cells.