Development of High-Efficiency Mitochondrial Protease Targeting Chimeras (MtPTACs) for Targeted Degradation of POLRMT and Beyond

Abstract

Despite the broad success of targeted protein degradation (TPD), achieving efficient degradation within mitochondria remains a challenge. Herein, we report a highly active degrader targeting human mitochondrial RNA polymerase (POLRMT) based on the Mitochondrial Protease Targeting Chimeras (MtPTACs) strategy. The lead compound, MTP-B1, is a highly potent MtPTAC to achieve essentially complete target degradation (D _max > 98%) within 24 h at sub-micromolar concentrations. Mechanistically, MTP-B1 circumvents the compensatory transcriptional upregulation induced by POLRMT inhibitors and sustains downstream signaling suppression in washout assays. Remarkably, under the dosing regimens tested, MTP-B1 exhibited greater in vivo antitumor efficacy than the co-administration of a POLRMT inhibitor and a ClpP activator, highlighting the therapeutic potential of targeted protein degradation in mitochondria. These findings, further supported by the extension to a second target, dihydroorotate dehydrogenase (DHODH), provide high-efficiency MtPTACs as a powerful and generalizable modality for mitochondrial medicine.

1. Introduction

Targeted protein degradation (TPD) has emerged as a transformative paradigm in drug discovery, offering a distinct, event-driven mechanism to ablate disease-relevant proteins previously considered “undruggable”. − Among various TPD modalities, heterobifunctional degraders such as proteolysis-targeting chimeras (PROTACs), which exploit the ubiquitin–proteasome system (UPS), have demonstrated remarkable success in both chemical biology and clinical development. − Complementary lysosome-dependent strategies, including lysosome targeting chimeras (LYTACs), autophagosome-tethering compounds (ATTECs), and autophagy-targeted chimeras (AUTACs), have further expanded the degradable proteome to encompass extracellular, membrane-associated, and autophagy-accessible substrates (Figure ). ,

1.

Comparison between classical TPD strategies and mitochondrial protein degraders MtPTACs.

Despite these advances, precise and selective degradation of proteins residing within membrane-enclosed organellesparticularly mitochondriaremains a largely unmet challenge. Mitochondria are physically segregated from the cytosolic UPS and lysosomal pathways, , the mitochondria rely on an autonomous, proteasome-independent quality control system, rendering canonical TPD strategies ineffective. ,

To overcome this barrier, the concept of mitochondrial protease-targeting chimeras (MtPTACs) was recently proposed, leveraging the endogenous mitochondrial protease ClpP (Figure ). , Despite its conceptual appeal, however, the practical implementation of MtPTACs remains highly challenging. Current MtPTACs are often hampered by limited degradation efficiency and unsatisfactory pharmacological properties, which paradoxically result in degraders that exhibit substantially lower therapeutic potency than their corresponding small-molecule inhibitors. As a consequence, the superior efficacy theoretically promised by mitochondrial protein degradation has yet to be convincingly demonstrated. Therefore, the development of high-efficiency MtPTACs capable of inducing rapid and near-complete target degradation, while delivering superior therapeutic outcomes, remains an urgent and unmet need.

Human mitochondrial RNA polymerase (POLRMT) serves as a good tool protein target to validate this technology. , Accumulating evidence indicates that POLRMT is overexpressed at both the mRNA and protein levels in multiple cancer types, and genetic depletion of POLRMT suppresses key oncogenic processes, including tumor cell proliferation, migration, invasion, and angiogenesis. − In previous studies, our group has identified multiple potent POLRMT inhibitors that exhibit robust antitumor activity across a range of cancer models, both in vitro and in vivo. − However, classical small-molecule inhibitors of POLRMT often trigger a compensatory transcriptional upregulation mechanism, where the cell attempts to restore mitochondrial function by increasing target protein levels or activity, potentially limiting long-term therapeutic efficacy. We hypothesized that converting these inhibitors into degraders could abrogate this feedback loop, leading to deeper and more sustained suppression of mitochondrial function.

Herein, we report the development of a series of highly potent MtPTACs that induce ClpP-dependent degradation of POLRMT. Through systematic optimization, we identified the lead compound MTP-B1, which represents a substantial leap over previous strategies. MTP-B1 is a highly potent MtPTAC to achieve essentially complete degradation (D _max > 98%) of an endogenous mitochondrial protein within 24 h at sub-micromolar concentrations. Mechanistically, MTP-B1 promotes formation of a POLRMT–ClpP ternary complex, effectively bypassing the compensatory transcriptional upregulation observed with POLRMT inhibitors and sustaining downstream mitochondrial suppression in washout experiments. Importantly, we provide the first in vivo evidence that targeted degradation of POLRMT elicits significantly superior antitumor efficacy compared to the combinatorial regimen of a POLRMT inhibitor and a ClpP activator. Furthermore, we extended this strategy to dihydroorotate dehydrogenase (DHODH), a classical and clinically relevant mitochondrial drug target, achieving similarly efficient degradation. Collectively, these findings establish high-efficiency MtPTACs as a powerful and generalizable modality for selective manipulation of mitochondrial proteins and open new avenues for therapeutic intervention in mitochondrial-driven diseases.

2. Results and Discussion

2.1. Rational Design of MtPTACs for Degrading POLRMT

Similar to PROTACs, MtPTACs are designed as heterobifunctional molecules composed of three functional elements: a high-affinity ligand that binds the POI, a ClpP-activating moiety that selectively engages the mitochondrial protease, and an appropriate linker connecting these two components (Figure A). To construct POLRMT-targeting MtPTACs, P1, a potent POLRMT inhibitor developed in our laboratory, was selected as the POLRMT-binding module. Differential scanning fluorimetry (DSF) assay demonstrated robust binding of P1 to POLRMT, as evidenced by pronounced ligand-induced thermal stabilization (Figure S1). This robust target engagement establishes P1 as a suitable POLRMT-binding module for MtPTAC construction. Structural analysis of the P1–POLRMT complex revealed that the piperazine moiety is solvent-exposed, rendering it a favorable site for linker installation (Figure B). In parallel, the Graves group reported a series of small molecules that directly bind to and activate the mitochondrial protease ClpP, among which TR79 displayed potent ClpP agonistic activity (EC₅₀ = 414.49 nM) (Figures B and S2). Notably, the extended alkyl side chain of TR79 offers a synthetically accessible conjugation handle without substantially compromising its ClpP-activating activity. Building on this scaffold, TR79 was subsequently adopted as a recruiting moiety in targeted protein degradation strategies, supporting its utility in mitochondrial protein degradation system design. Given that linker architecture critically influences MtPTAC performance by modulating physicochemical properties and enabling productive target–protease interactions, we systematically optimized linker length, flexibility, and rigidity. To this end, hydrophilic polyethylene glycol chains, flexible aliphatic carbon chains, and more rigid motifs, including cyclic linkers were explored to evaluate their impact on POLRMT degradation efficiency.

2.

Design of novel POLRMT-targeting MtPTACs based upon POLRMT inhibitor compound P1 and ClpP agonist TR79. (A) Schematic representation of the MtPTAC platform, illustrating bifunctional recruitment of POLRMT and ClpP within mitochondria. (B) Structural design and modular composition of POLRMT-targeting MtPTACs based on P1 and TR79.

Guided by the strategy outlined above, P1 and TR79 were conjugated through diverse linker architectures to generate 22 POLRMT-targeting MtPTACs (Figure A,B). To evaluate the degradation efficiency of POLRMT-targeting MtPTACs, MIA PaCa-2 cells, a pancreatic cancer cell line with high endogenous POLRMT expression, were treated with the indicated compounds at concentrations of 0.5, 1, and 5 μM. After 24 h incubation, POLRMT protein levels were analyzed by Western blotting (Figure C). Treatment with MtPTACs resulted in POLRMT degradation to varying extents, highlighting the critical influence of linker composition and architecture on MtPTAC-mediated degradation efficiency. In the A series (MTP-A1–MTP-A10), all compounds in this series effectively induced POLRMT degradation at a concentration of 5 μM. Notably, compound MTP-A2, featuring a two-carbon linker, exhibited the most potent activity, achieving more than 80% POLRMT degradation even at 0.5 μM. Compound MTP-A7, featuring a nine-carbon linker, also induced pronounced POLRMT degradation at 1 and 5 μM. In the B series, the amide linkage proximal to the POLRMT-binding moiety was removed to evaluate whether increased linker flexibility would enhance degradation efficiency. Notably, compound MTP-B1, featuring a two-carbon linker, retained potent POLRMT degradation activity. In addition, compound MTP-B6, bearing an eight-carbon aliphatic linker, also exhibited appreciable POLRMT degradation, further underscoring the importance of linker length and composition. In the C series, rigid linkers motifs were introduced to assess whether increased linker rigidity could further enhance POLRMT degradation activity. Although these compounds were still capable of inducing POLRMT degradation at 5 μM, their overall degradation efficiency was reduced compared with analogues bearing flexible linkers. Collectively, compounds MTP-A2 and MTP-B1, both bearing a two-carbon linker, displayed the most potent POLRMT degradation, while compounds MTP-A7 and MTP-B6 also demonstrated appreciable degradation efficacy. Accordingly, these four compounds were selected for subsequent studies.

3.

Design of POLRMT-targeting MtPTACs and preliminary degradation activity screening. (A, B) Chemical structures of the designed POLRMT-targeting MtPTACs. (C) Evaluation of POLRMT degradation activity of POLRMT-targeting MtPTACs.

2.2. MTP-B1 was Identified for Further Activity Evaluation

Based on the initial POLRMT degradation activity screening, the four most active compounds were selected for quantitative evaluation of their degradation potency. Dose–response assays revealed that compounds MTP-A2 and MTP-A7 exhibited DC₅₀ values of 407.4 nM and 596.6 nM, with maximal degradation (D _max) of 98.62% and 95.33%, respectively (Figure A–C). Compounds MTP-B1 and MTP-B6 showed DC₅₀ values of 187.2 nM and 577.2 nM, achieving D _max of 98.18% and 96.82%, respectively (Figure D–F). Collectively, these results demonstrate dose-dependent POLRMT degradation for all four compounds, with compound MTP-B1 exhibiting the highest potency. Moreover, MTP-B1 also demonstrated pronounced degradation potency in pancreatic cancer AsPC-1 cells, with a DC₅₀ value of 416.9 nM (Figure S3). Based on its superior activity, compound MTP-B1 was advanced for subsequent studies. A time-dependent degradation assay was then performed by treating cells with 1 μM MTP-B1 and monitoring POLRMT protein levels at the indicated time points (Figure G). The results showed that MTP-B1 induced more than 50% POLRMT degradation within 16 h and near-complete degradation was achieved by 24 h, demonstrating that MTP-B1 rapidly and efficiently promotes POLRMT degradation. Notably, upon MTP-B1 treatment, an additional lower-molecular-weight POLRMT species emerged concomitantly with POLRMT degradation. Consistent with this observation, a similar lower-molecular-weight POLRMT species was previously reported and demonstrated to represent a degradation intermediate generated during ClpP-mediated proteolysis of POLRMT. In our study, the intensity of the lower-molecular-weight POLRMT species progressively decreased over time and became barely detectable at higher compound concentrations or following prolonged compound exposure (Figure D,G). To assess the durability of POLRMT degradation induced by MTP-B1, a washout recovery assay was conducted. MIA PaCa-2 cells were treated with 1 μM MTP-B1 for 24 h to induce POLRMT degradation, followed by compound removal and longitudinal monitoring of POLRMT protein levels (Figure H–I). Notably, no appreciable recovery of POLRMT was detected within 24 h after washout, and only partial restoration was observed at 48 h. These results indicate that MTP-B1 induces a sustained and long-lasting degradation of POLRMT that persists well beyond compound withdrawal. This persistence may reflect a combination of target protein turnover kinetics and longer-lived downstream regulatory effects following initial degradation. To further validate the degradation activity of MTP-B1, immunofluorescence analysis was performed to assess POLRMT protein levels. Consistent with the biochemical results, MTP-B1 treatment resulted in a dose-dependent reduction in POLRMT fluorescence intensity, providing confirmation of its POLRMT-degrading activity (Figure J,K).

4.

Evaluation of the degradation activity of optimized compounds targeting POLRMT. (A–C) Dose-dependent degradation of POLRMT protein induced by compounds MTP-A2 and MTP-A7 in MIA PaCa-2 cells. Cells were treated with increasing concentrations of MTP-A2 or MTP-A7, followed by immunoblot analysis to assess POLRMT protein levels. (D–F) Dose-dependent POLRMT degradation induced by compounds MTP-B1 and MTP-B6 in MIA PaCa-2 cells, as determined by Western blot analysis following treatment with increasing compound concentrations. (G) Time-dependent degradation of POLRMT protein in MIA PaCa-2 cells upon treatment with compound MTP-B1. (H–I) Reversibility of MTP-B1-induced POLRMT degradation assessed by compound washout experiments in MIA PaCa-2 cells. (J–K) Immunofluorescence analysis validating compound MTP-B1-induced degradation of POLRMT protein. MIA PaCa-2 cells were treated with compound MTP-B1, followed by immunofluorescence staining to visualize POLRMT expression. Representative images and quantitative analysis confirm a marked reduction in POLRMT signal upon MTP-B1 treatment, consistent with the degradation observed in immunoblot assays. The above-mentioned data are representative of three independent experiments (n = 3). Statistical significance was assessed using an unpaired two-tailed Student’s t-test for comparisons between two groups and one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparisons test for comparisons of multiple treatment groups with a single control group. Significance levels are indicated as follows: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

2.3. Mechanistic Investigation of ClpP-Dependent POLRMT Degradation Induced by MTP-B1

To elucidate the mechanism underlying MTP-B1-induced POLRMT degradation, a ligand competition assay was first performed to determine whether target engagement is required for its activity. Cells were co-treated with 1 μM MTP-B1 in the presence of either the POLRMT inhibitor P1 or the ClpP ligand TR79, followed by analysis of POLRMT protein levels. Treatment with MTP-B1 alone resulted in robust POLRMT degradation, whereas co-treatment with either P1 or TR79 markedly attenuated this effect (Figure A), indicating that MTP-B1-induced degradation depends on simultaneous engagement of both POLRMT and ClpP. We next examined whether canonical protein degradation pathways contribute to MTP-B1-induced POLRMT degradation. Co-treatment with the NEDD8-activating enzyme inhibitor MLN4924, or the classic autophagy inhibitor Chloroquine (CQ) failed to restore POLRMT protein levels (Figure B,C), indicating that POLRMT degradation is not mediated by either the CRL-dependent ubiquitination or the autophagy–lysosome pathway. In contrast, co-treatment with the proteasome inhibitor Bortezomib (BTZM) completely abrogated POLRMT degradation (Figure B,C). Moreover, we further examined the potential involvement of the ubiquitin–proteasome system using MG132, another mechanistically distinct proteasome inhibitor that lacks inhibitory activity against ClpP. MG132 had no effect on MTP-B1-induced POLRMT degradation. (Figure S4). Given prior evidence that BTZM can inhibit ClpP proteolytic activity, the observed attenuation of MTP-B1-induced POLRMT degradation may be attributable to inhibition of ClpP activity. To determine whether MTP-B1 accelerates POLRMT turnover, a cycloheximide (CHX) chase assay was performed. Cells were treated with CHX to block new protein synthesis in the presence or absence of MTP-B1, and POLRMT protein levels were monitored over time (Figure D–F). The results showed that MTP-B1 significantly accelerated the decline of POLRMT compared with control, demonstrating that MTP-B1 reduces intracellular POLRMT levels by accelerating its protein degradation rather than inhibiting its synthesis. Additionally, by introducing a sterically bulky tert-butyl group onto the phenyl ring of the TR79 ligand to disrupt its binding to ClpP, we synthesized the corresponding inactive control compound, N1 (Figures G andS5). The ClpP-activating activity of N1 was subsequently evaluated at the enzymatic level. In contrast to MTP-B1, which robustly activated ClpP proteolytic activity, N1 exhibited no detectable ClpP activation (Figure H). In parallel, the POLRMT-degrading activity of N1 was evaluated in MIA PaCa-2 and AsPC-1 cells. Consistent with its lack of ClpP activation, N1 failed to induce any appreciable degradation of POLRMT in either cell line (Figure I–L). To further validate the requirement of ClpP in MTP-B1-induced POLRMT degradation, ClpP was silenced using siRNA. Knockdown of ClpP markedly attenuated the degradation of POLRMT induced by MTP-B1, establishing the ClpP dependence of this process (Figure M). We further assessed mitochondrial localization of MTP-B1 and detected pronounced accumulation within isolated mitochondria, confirming its efficient mitochondrial enrichment (Figure S6). To determine whether MTP-B1 affects POLRMT expression at the transcriptional level, quantitative PCR (qPCR) was performed to measure POLRMT mRNA levels. The results showed that treatment with MTP-B1 had no significant effect on POLRMT mRNA abundance, whereas the POLRMT inhibitor P1 induced a marked upregulation of POLRMT mRNA expression (Figure N). Furthermore, we performed quantitative proteomic profiling to assess changes in intracellular protein abundance following MTP-B1 treatment. The results showed that MTP-B1 treatment significantly reduced POLRMT protein abundance, accompanied by a decrease OXPHOS-associated protein NDUFAF2 (Figure S7A). In contrast, parallel proteomic analysis of TR79 revealed no significant reduction in POLRMT abundance, indicating that the observed POLRMT depletion was specifically induced by MTP-B1 rather than resulting from a nonspecific effect of TR79 treatment (Figure S7B). Collectively, these results demonstrate that MTP-B1 promotes POLRMT degradation through a binding-dependent, ClpP-mediated proteolytic mechanism that is independent of ubiquitination and proteasomal pathways and does not affect POLRMT transcription.

5.

Mechanistic validation of POLRMT degradation induced by POLRMT-MtPTAC MTP-B1. (A) Ligand competition assay evaluating the effect of competing ligands on MTP-B1-induced POLRMT degradation at 1 μM MTP-B1. (B, C) Investigation of the degradation pathway mediated by MTP-B1. (D–F) CHX chase assays were performed by co-treating cells with CHX (50 μM) and MTP-B1 (1 μM) to evaluate the effect of MTP-B1 on POLRMT protein stability. (G) A caging group was introduced into the ClpP ligand moiety to abolish ClpP activation, thereby generating the inactive negative control compound N1. (H) The ClpP agonistic activity of the negative control compound was evaluated, and the results showed that N1 exhibited no detectable ClpP activation. (I–L) Degradation activity of the negative control compound N1 toward POLRMT in MIA PaCa-2 and AsPC-1 cells. (M) Assessment of MTP-B1-induced POLRMT degradation in MIA PaCa-2 cells following ClpP knockdown by siRNA. (N) The effects of the MTP-B1 or P1 on POLRMT mRNA expression levels were evaluated using qPCR. The above-mentioned data are representative of three independent experiments (n = 3). Statistical significance was assessed using an unpaired two-tailed Student’s t-test for comparisons between two groups and one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparisons test for comparisons of multiple treatment groups with a single control group. Significance levels are indicated as follows: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

2.4. MTP-B1-Induced Formation of the POLRMT–MTP-B1-ClpP Ternary Complex

Given the ClpP dependence of MTP-B1-induced POLRMT degradation, we sought to determine whether POLRMT and ClpP are brought into proximity through formation of a ternary complex. First, we examined whether MTP-B1 promotes spatial proximity between POLRMT and ClpP by immunofluorescence colocalization analysis. Following 500 nM MTP-B1 treatment for 12 h, partial overlap in the subcellular distributions of POLRMT and ClpP was observed, consistent with enhanced proximity of the two proteins (Figures A and S8). To substantiate the interaction between POLRMT and ClpP, co-immunoprecipitation (Co-IP) assays were performed. Notably, ClpP was efficiently co-precipitated in the presence of MTP-B1 (Figure B). To further corroborate the formation of the ternary complex, we performed differential scanning fluorimetry (DSF) using purified recombinant proteins. The DSF analysis revealed that MTP-B1 binds efficiently to both POLRMT and ClpP (Figure S9A,B). Furthermore, a His-tagged POLRMT pull-down assay demonstrated that MTP-B1 enhances the interaction between POLRMT and ClpP in a concentration-dependent manner (Figure S9C). Moreover, a structural model of the ternary complex was constructed and assessed using molecular dynamics (MD) simulations (Figure C). The ternary complex remained highly stable throughout a 100 ns MD simulation (Figure D), with MTP-B1 maintaining a stable binding conformation and an RMSD of less than 1 Å, indicating minimal conformational fluctuation (Figure E). In addition, MTP-B1 established persistent interactions with key amino acid residues of both POLRMT and ClpP, consistent with its proposed role in mediating ternary complex formation (Figure F).

6.

MTP-B1-Induced Formation of the POLRMT–MTP-B1–ClpP Ternary Complex. (A) Immunofluorescence colocalization analysis of POLRMT and ClpP. (B) Co-immunoprecipitation (co-IP) assay demonstrating MTP-B1-induced interaction between POLRMT and ClpP. (C) Structural model of the POLRMT–MTP-B1–ClpP ternary complex. (D) Root-mean-square deviation (RMSD) of the protein backbone during the molecular dynamics (MD) simulation. (E) RMSD of MTP-B1throughout the MD simulation. (F) Analysis of key amino acid residues involved in the interactions between MTP-B1 and POLRMT–ClpP following MD simulation.

2.5. MTP-B1 Effectively Suppresses the Expression of Genes Involved in Oxidative Phosphorylation

To further characterize the transcriptional consequences of MTP-B1, transcriptomic analysis was performed. The results revealed that MTP-B1 treatment led to widespread transcriptional reprogramming, with 2,554 genes significantly downregulated and 1,362 genes upregulated (|Log₂ FC| > 1) (Figure A). Pathway enrichment analysis revealed that these differentially expressed genes were significantly enriched in metabolic pathways, prominently including OXPHOS (Figure B). Gene set enrichment analysis (GSEA) further demonstrated a coordinated downregulation of OXPHOS pathways genes (Figure C). Heatmap analysis of OXPHOS-associated genes revealed a global suppression of pathway components upon MTP-B1 treatment (Figure D), with significant downregulation observed for multiple genes involved in mitochondrial respiration, including COX1, COX2, COX3, ATP6, ATP8, ND3, ND4, ND4L, CYTC, NDUFC2, NDUFV3, and UQCRC2 (Figure E). These transcriptomic findings were further validated by RT–qPCR analysis. Consistent with the RNA-seq data, the expression of multiple POLRMT-dependent mitochondrial genes , was also markedly and significantly downregulated (Figure F). ND1, a key mitochondrially encoded gene widely used as a marker of mitochondrial transcriptional activity, was employed to assess the dose- and time-dependent effects of MTP-B1. The MTP-B1 reduced ND1 expression in a clear dose- and time-dependent manner (Figure G,H). Notably, following compound withdrawal, MTP-B1 exhibited a more sustained inhibitory effect on mitochondrial transcription than the POLRMT inhibitor P1, with significant ND1 suppression still observed at 72 h (Figure I).

7.

MTP-B1 Suppresses mitochondrial oxidative phosphorylation. (A) Global transcriptomic analysis identifying significantly differentially expressed genes upon MTP-B1 treatment. (B) Pathway enrichment analysis of differentially expressed genes, highlighting pronounced enrichment in OXPHOS-related pathways. (C) Gene set enrichment analysis (GSEA) demonstrating significant negative enrichment of the OXPHOS gene set. (D) Heatmap visualization of OXPHOS-associated genes showing coordinated transcriptional repression. (E) OXPHOS-related genes significantly downregulated in the transcriptomic dataset. (F) Quantitative RT–PCR validation of representative downregulated OXPHOS genes identified by transcriptomic analysis. (G) Dose-dependent suppression of the mitochondrial transcription–related gene ND1 following MTP-B1 treatment. (H) Time-dependent effects of MTP-B1 and P1 on ND1 expression. (I) Recovery of ND1 expression following compound washout, demonstrating the prolonged and durable transcriptional repression induced by MTP-B1 compared with P1. The above-mentioned data are representative of three independent experiments (n = 3). Statistical significance was assessed using an unpaired two-tailed Student’s t-test for comparisons between two groups and one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparisons test for comparisons of multiple treatment groups with a single control group. Significance levels are indicated as follows: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

2.6. Evaluation of Antitumor Activity of MTP-B1 In Vitro

The in vitro antiproliferative activity of MTP-B1 was evaluated in pancreatic cancer cell lines MIA PaCa-2 and AsPC-1 using the sulforhodamine B (SRB) assay. MTP-B1 exhibited potent antiproliferative activity against MIA PaCa-2 cells (IC₅₀ = 5.08 nM), markedly outperforming the POLRMT inhibitor P1 (IC₅₀ = 358.32 nM) (Figure A). Consistently, enhanced potency was also observed in AsPC-1 cells (IC₅₀ = 57.76 nM for MTP-B1 vs 635.51 nM for P1) (Figure B). To align the exposure duration of the degradation assay with that of the proliferation assay, we determined POLRMT degradation after 7 days of MTP-B1 treatment. Under these time-matched conditions, MTP-B1 exhibited an apparent POLRMT degradation DC₅₀ of 8.81 nM (Figure S10A). To assess the antiproliferative activity of MTP-B1 was associated with POLRMT degradation, we further evaluated N1, a structurally related control compound lacking POLRMT-degrading activity. N1 showed markedly weaker antiproliferative activity against MIA PaCa-2 cells, with an IC₅₀ of 1.71 μM, corresponding to an approximately 337-fold reduction in potency relative to MTP-B1 (Figure S10B). To further evaluate the dependence of MTP-B1 activity on POLRMT abundance, we generated POLRMT-overexpressing MIA PaCa-2 cells (Figure S10C). POLRMT overexpression partially attenuated the antiproliferative activity of MTP-B1, increasing the IC₅₀ from 6.32 nM in OE-NC cells to 94.15 nM in POLRMT-overexpressing cells (Figure S10D). In addition, colony formation demonstrated that MTP-B1 effectively suppressed clonogenic growth of MIA PaCa-2 cells (Figure C). To further elucidate the antiproliferative mechanism of MTP-B1, flow cytometric analyses were performed. MTP-B1 treatment did not significantly affect cell cycle progression or mitochondrial membrane potential (Figures D andS11). In contrast, MTP-B1 induced a dose-dependent increase in intracellular reactive oxygen species (ROS), accompanied by a pronounced elevation in late-stage and total apoptosis (Figure E–G). Collectively, these findings demonstrate that MTP-B1 exerts potent antiproliferative effects in pancreatic cancer cells, which is associated with disruption of cellular redox homeostasis and induction of apoptosis.

8.

Evaluation antitumor activity of MTP-B1 in vitro. (A, B) Antiproliferative effects of MTP-B1 in pancreatic cancer cell lines MIA PaCa-2 and AsPC-1. (C) Suppression of clonogenic growth in MIA PaCa-2 cells by MTP-B1. (D) Effect of MTP-B1 on mitochondrial membrane potential in MIA PaCa-2 cells. (E) Dose-dependent induction of intracellular ROS by MTP-B1 in MIA PaCa-2 cells. (F, G) Apoptotic profiling of MIA PaCa-2 cells following MTP-B1 treatment, showing increased late-stage apoptosis. The above-mentioned data are representative of three independent experiments (n = 3). Statistical significance was assessed using an unpaired two-tailed Student’s t-test for comparisons between two groups and one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparisons test for comparisons of multiple treatment groups with a single control group. Significance levels are indicated as follows: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

2.7. In Vivo Anticancer Efficacy of MTP-B1 in the MIA PaCa-2 Xenograft Model in Mice

Encouraged by the potent in vitro POLRMT degradation and antiproliferative activity, we evaluated the pharmacokinetic profile of MTP-B1 following intravenous administration in mice. MTP-B1 exhibited substantial systemic exposure at 50 mg/kg, with a C _max of 61,966.67 ng/mL, an AUC_0–∞ of 26,100.84 ng·h/mL, and a terminal half-life of 3.47 h. Both C _max and AUC increased more than dose proportionally from 2 to 50 mg/kg, indicating enhanced systemic exposure at the higher dose (Table S1). Based on these findings, the in vivo antitumor efficacy of MTP-B1 was evaluated in a MIA PaCa-2 xenograft model. Tumor-bearing mice were treated intravenously every other day with vehicle, P1 (50 mg/kg) + TR79 (50 mg/kg), P1 (100 mg/kg), or MTP-B1 (25, 50, or 100 mg/kg) for 25 days (Figure A). MTP-B1 treatment resulted in a dose-dependent suppression of tumor growth in vivo. Particularly, administration of 25 mg/kg MTP-B1 achieved a comparable antitumor effect to 100 mg/kg of the POLRMT inhibitor P1. At dose of 50 mg/kg, MTP-B1 produced tumor growth inhibition similar to that observed with the combination of 50 mg/kg P1 and 50 mg/kg TR79. Notably, MTP-B1 at its highest tested dose (100 mg/kg) achieved a tumor growth inhibition (TGI) of 70.1%, markedly outperforming the co-administered POLRMT inhibitor and ClpP activator (Figure B–D). These results demonstrate that, under the specific dosing regimens tested, MTP-B1 exhibited superior in vivo antitumor efficacy compared with P1 alone or the combination of P1 and TR79. No significant body weight loss was observed during the treatment period, and histopathological examination of major organs revealed no apparent treatment-related abnormalities, indicating good in vivo tolerability (Figure E,F). Mechanistically, Western blot analysis of tumor tissues exhibited POLRMT degradation upon MTP-B1 treatment, which was further confirmed by immunofluorescence analysis (Figure G,H). Consistently, immunohistochemical analyses revealed a pronounced reduction in the proliferation marker Ki67 and a significant increase in TUNEL-positive apoptotic cells in MTP-B1-treated tumors, indicating effective suppression of tumor proliferation and induction of apoptosis in vivo (Figure I).

9.

In vivo anticancer efficacy of MTP-B1 in the MIA PaCa-2 xenograft model in mice. (A) Schematic illustration of the in vivo study design and dosing regimen. (B) Representative images of excised tumors from each treatment group at the study end point. (C) Tumor weights of excised xenografts from different treatment groups. (D) Tumor growth curves showing changes in tumor volume during the dosing period. (E) Body weight changes of mice during treatment, indicating overall tolerability. (F) Representative hematoxylin and eosin (H&E)–stained sections of major organs collected at the study end point, showing no overt histopathological abnormalities upon MTP-B1 treatment. (G) Analysis of POLRMT protein levels in excised tumors from different treatment groups. (H) Immunofluorescence staining of tumor sections assessing POLRMT expression. (I) Immunohistochemical staining for Ki67 and TUNEL assay of tumor sections, assessing tumor cell proliferation and apoptosis, respectively. The above-mentioned data are representative of three independent experiments (n = 3). Statistical significance was assessed using an unpaired two-tailed Student’s t-test for comparisons between two groups and one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparisons test for comparisons of multiple treatment groups with a single control group. Significance levels are indicated as follows: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

2.8. Application of MtPTACs to the DHODH Target

Building on our successful application of the MtPTAC strategy to induce selective degradation of POLRMT, we next sought to evaluate the generalizability of this approach by extending it to the other mitochondrial target, dihydroorotate dehydrogenase (DHODH). , For DHODH ligand selection, to ensure potent target engagement, we chose P2, a derivative of the clinically advanced DHODH inhibitor BAY 2402234 (Figure A). Based on the binding mode, we identified that the phenyl moiety of P2 is oriented toward a solvent-exposed region (Figure B). Accordingly, a carboxyl group was introduced at this position, and the resulting derivative P3 retained potent DHODH inhibitory activity (DHODH, IC₅₀ = 5.23 nM) (Figure C). Accordingly, using P3 as the DHODH-binding ligand, we conjugated it to TR79 via vary linkers to generate eight DHODH-targeting MtPTACs (MTP-D1–MTP-D8) (Figure D). Among them, MTP-D7 and MTP-D8 displayed robust DHODH degradation activity (Figure E). Moreover, MTP-D7 and MTP-D8 induced DHODH degradation in a concentration-dependent manner, with MTP-D7 exhibiting a DC₅₀ value of 2.09 μM (D _max = 85.32%) and MTP-D8 showing a markedly enhanced potency with a DC₅₀ of 0.76 μM (D _max = 92.15%) (Figure F–H). Time-dependent analyses revealed that MTP-D8 rapidly promotes DHODH degradation, with substantial degradation observed within 16 h (Figure I,J). MTP-D8 showed potent antiproliferative effects in A549 cells, with an IC₅₀ value of 0.11 μM, which was substantially lower than that of the DHODH inhibitor P3 (IC₅₀ = 0.97 μM) (Figure K).

10.

Design and evaluation of DHODH-targeting MtPTACs. (A) Chemical structures of reported DHODH inhibitors. (B) Structure-based binding mode analysis guiding the rational design of DHODH-targeting MtPTACs. PDB ID: 6QU7. (C) Introduction of a carboxyl group preserves DHODH inhibitory activity. (D) Chemical structures of the designed DHODH-targeting MtPTACs. (E) Primary DHODH degradation activity screening of DHODH-targeting MtPTACs. (F) Dose-dependent activity of the optimized compound MTP-D7. (G) Dose-dependent activity of the optimized compound MTP-D8. (H) DC₅₀ values of compounds MTP-D7 and MTP-D8; (I, J) Time-dependent degradation profiles of compound MTP-D8. (K) Antiproliferative activities of MTP-D8 and the DHODH inhibitor P2. The above-mentioned data are representative of three independent experiments (n = 3). Statistical significance was assessed using an unpaired two-tailed Student’s t-test for comparisons between two groups and one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparisons test for comparisons of multiple treatment groups with a single control group. Significance levels are indicated as follows: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3. Conclusion

TPD has shifted the therapeutic paradigm from occupancy-driven inhibition to event-driven elimination of disease-relevant proteins. While classical TPD approaches like PROTACs, LYTACs, AUTACs, and ATTECs primarily exploit the ubiquitin–proteasome system or lysosome-dependent pathways, these are inherently limited for intramitochondrial targets due to the unique double-membrane structure and autonomous protein quality control of mitochondria. To overcome this, mitochondrial protease-targeting chimeras (MtPTACs) have been developed to exploit endogenous mitochondrial proteases, such as ClpP, enabling selective degradation of proteins within the mitochondria. In this work, we report the development of highly efficient MtPTACs that induce ClpP-dependent degradation of mitochondrial protein POLRMT. Lead compound MTP-B1 achieves near-complete degradation (D _max > 98%) of POLRMT within 24 h at sub-micromolar concentration. Compared with conventional POLRMT inhibitors, MTP-B1 overcomes compensatory transcriptional upregulation and enables sustained suppression of mitochondrial transcription. MTP-B1 exhibits robust antitumor activity both in vitro and in vivo. Notably, in vivo administration of MTP-B1 at low doses achieved antitumor efficacy comparable to that of high-dose POLRMT inhibitors. Importantly, we provide the first in vivo evidence that targeted degradation of POLRMT affords markedly superior antitumor efficacy compared with combined treatment using a POLRMT inhibitor and a ClpP activator. Extension of this strategy to DHODH further highlights the generality of MtPTACs as a powerful platform for selective manipulation of mitochondrial proteins and therapeutic exploration in mitochondria-associated diseases.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.6c01255.

• Additional biological data, experimental methods, and details of compound synthesis and characterization (PDF)

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C.C., L.L., and X.L. contributed equally to this work.

The authors declare no competing financial interest.