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. 2023 Dec 29;15(1):29–35. doi: 10.1021/acsmedchemlett.3c00161

Discovery of Novel PDEδ Autophagic Degraders: A Case Study of Autophagy-Tethering Compound (ATTEC)

Jingying Bao †,‡, Zhenqian Chen †,‡, Yu Li ‡, Long Chen ‡, Wei Wang ‡, Chunquan Sheng ‡,*, Guoqiang Dong ‡,*
PMCID: PMC10788939  PMID: 38229750

Abstract

graphic file with name ml3c00161_0008.jpg

The autophagy-tethering compound (ATTEC) technology has emerged as a promising strategy for targeted protein degradation (TPD). Here, we report the discovery of the first generation of PDEδ autophagic degraders using an ATTEC approach. The most promising compound 12c exhibited potent PDEδ binding affinity and efficiently induced PDEδ degradation in a concentration-dependent manner. Mechanistic studies confirmed that compound 12c reduced the PDEδ protein level through lysosome-mediated autophagy without affecting the PDEδ mRNA expression. Importantly, compound 12c was much more effective in suppressing the growth in KRAS mutant pancreatic cancer cells than the corresponding PDEδ inhibitor. Taken together, this study expands the application scope of the ATTEC approach and highlights the effectiveness of the PDEδ autophagic degradation strategy in antitumor drug discovery.

Keywords: Targeted protein degradation, autophagy-tethering compound, PDEδ, KRAS, antitumor activity

Targeted protein degradation (TPD) is a groundbreaking technology in chemical biology and medicinal chemistry, which has gained considerable interest in recent years.1,2 Developed by Crews’ group, proteolysis targeting chimera (PROTAC) is the most popular method in TPD.3−5 PROTAC is a heterobifunctional molecule that hijacks an endogenous E3 ubiquitin ligase to recruit a protein of interest (POI), inducing ubiquitination of POI and subsequent degradation via the ubiquitin proteasome system (UPS).4,6 Despite the outstanding degradation potency, PROTAC is overly reliant on a limited number of E3 ligases, and genomic alterations in E3 ligases also result in acquired resistance to PROTACs.7−9 These limitations prompted the development of new degradation technologies based on the autophagy-lysosomal pathway (ALP),10 such as lysosome-targeting chimera (LYTAC),11 autophagy-targeting chimera (AUTAC),12 AUTOphagy-TArgeting Chimera (AUTOTAC),13 and autophagy-tethering compound (ATTEC).14 These new technologies possess the potential to expand the scope of accessible degradation targets, ranging from extracellular proteins to cellular organelles.10

Macroautophagy is the most widely studied form of autophagy and is essential for maintaining protein homeostasis.15,16 Macroautophagy is initiated by the formation of phagophores and autophagosomes, which engulf various cargos (e.g. biomolecules, damaged organelles and protein aggregates), and then transfer them into lysosomes for degradation (Figure 1A).16 During the process, the autophagy protein LC3 attaches to the phagophore/autophagosome membrane, participating in all the steps and playing a central role in the recruitment of cargos into the autophagosome.10,16 Recently, Lu’s group proposed a novel degradation approach termed ATTEC that hijacks LC3 to recruit POI, tethering POI directly to the autophagosome for subsequent degradation (Figure 1A). As a proof-of-concept, four bispecific molecules (i.e., GW5074, ispinesib, AN1, and AN2, Figure 1B and Figure S1 in the Supporting Information) were identified as molecular glues that concurrently bound to the mutant Huntington (mHTT) proteins and LC3 proteins.14 These compounds could effectively degrade mHTT proteins and rescue disease-relevant phenotypes. Inspired by the design principle of PROTAC, Lu’s group developed ATTEC degraders for lipid droplets (LDs) by combining LC3 ligands (GW5074 and AN2) with LD-binding probes, leading to the successful elimination of LDs via autophagy.17 Similarly, Ouyang’s group successfully applied the ATTEC approach to degrade BRD4 using GW5074 (1) as the LC3 warhead.18 Previously, our group identified ispinesib (2) as an effective LC3 warhead and designed several potent autophagic degraders of nicotinamide phosphoribosyltransferase (NAMPT).19 Despite the progress made in this field, successful examples of ATTEC degraders are still rather limited. There is an urgent need to expand the application scope of targets and further verify the feasibility of ATTEC.

Figure 1.

Figure 1

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Design and mechanism of PDEδ autophagic degraders. (A) Schematic illustration of the POI degradation and design of PDEδ ATTECs. The figure was generated using Biorender (https://biorender.com). (B) Chemical structures of LC3 ligands. (C) Chemical structures of representative PDEδ inhibitors.

KRAS is a common mutated oncogene in pancreatic cancer, with mutations occurring in approximately 90% of patients.20 The oncogenic function of KRAS proteins depends on their localization and enrichment in the cell membranes, and PDEδ plays a key role in the transfer of KRAS from the cytosol to the cell membrane. Therefore, KRAS-PDEδ protein–protein interaction (PPI) has become an emerging target in pancreatic cancer.21,22 In recent years, a number of small molecule KRAS-PDEδ inhibitors have been reported (Figure 1C), such as compound 3 and deltazinone (4).22−24 Our group also identified several potent PDEδ inhibitors with low nanomolar affinity.25−27 Although these inhibitors effectively blocked the KRAS-PDEδ PPI, they were limited by poor antitumor activity due to the rapid release of inhibitors from PDEδ induced by endogenous Arl2.23 Thus, it is critical to develop novel strategies that target PDEδ more effectively.

Previously, using the PROTAC approach, we have designed and synthesized the first PDEδ degrader with improved antitumor activity over the corresponding inhibitor, demonstrating the distinct advantages of PDEδ degradation over inhibition.28 Herein, the ATTEC strategy was applied to construct novel PDEδ autophagic degraders by coupling PDEδ inhibitors and LC3 ligands with suitable linkers (Figure 1A). Among them, PDEδ ATTEC 12c was particularly effective in inducing PDEδ degradation via autophagy and showed enhanced antiproliferative potency against KRAS mutant pancreatic cancer cells. This study further validated ATTEC as an effective TPD technology and provided a promising lead compound targeting the KRAS-PDEδ interaction.

Typically, the ATTEC degrader is a bifunctional molecule composed of three components: a ligand of the POI, a flexible linker, and an LC3 warhead (Figure 1A). The PDEδ ATTEC functions by inducing proximity between PDEδ and LC3 through the formation of a ternary complex, leading to the recruitment and engulfment of PDEδ into an autophagosome and ultimately promoting lysosome-mediated degradation. First, we selected compounds 1 and 2 as LC3 warheads because of their successful application in BRD4 and NAMPT ATTEC degraders. The key step in designing a bifunctional molecule is to identify a suitable site to attach the linker without disrupting the critical interactions with LC3. Previously, we have constructed an LC3 fluorescent probe based on compound 2 and verified that the primary amine is an appropriate linkage site.19 Here, a novel LC3 fluorescent probe 5 was designed by connecting the FITC group at the phenolic hydroxyl group of compound 1 (Figure 2A, Scheme S1 in the Supporting Information). To confirm the distribution of compound 5, a colocalization experiment was performed in HeLa cells. After treatment with the probe for 2 h, the cells were stained with lysosome probes (Red) for another 1.5 h. As shown in Figure 2B, green and red fluorescence together overlapped in the lysosomes, indicating that compound 5 could localize and fuse into the lysosomes. Therefore, the hydroxyl group of compound 1 was used as a favorite site for attaching the linkers.

Figure 2.

Figure 2

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Colocalization experiment of a compound 1-based LC3 fluorescent probe. (A) Design of fluorescent probe for LC3 by attaching the FITC group to compound 1. (B) Fluorescence images of HeLa cells treated by compound 5 (green) and a lysosome probe (red). Scale bar: 10 μm.

Compounds 3 and 4 were identified as highly potent and selective PDEδ inhibitors with binding affinities in the low nanomolar range.23,24 Importantly, these compounds demonstrated high specificity without any nonspecific interactions with other proteins, thus making them ideal candidates for designing ATTECs. The cocrystal structure of compound 3 in complex with PDEδ (Figure 3A) revealed that this small molecule occupied the prenyl binding pocket of PDEδ and formed five hydrogen bonds with Cys56, Arg61, Gln78, Met118, and Tyr149, respectively. The terminal carboxyl group pointed toward the solvent region of PDEδ (Figure 3A), providing a tethering site for introducing the linker and LC3 ligand. Furthermore, SAR studies confirmed that labeling of compound 3 at the carboxylic acid with a long length linker was tolerated without affecting its binding affinity with PDEδ.23 The predicted binding model of compound 4 with PDEδ indicated that the terminal benzyl group was exposed to the solvent (Figure 3B), making it a suitable site for linker attachment. To facilitate the synthesis of ATTECs, the 2-phenylpropyl scaffold in compound 4 was removed and the remaining amide group was served as a handle for linker installation. As a result, a series of PDEδ ATTECs were designed by connecting different LC3 ligands and PDEδ inhibitors with various lengths of alkane and ethylene glycol (PEG) linkers (Figure 3C).

Figure 3.

Figure 3

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Design of PDEδ ATTECs. (A) Crystal structure of inhibitor 3 in complex with PDEδ (PDB ID: 5ML4(23)). (B) Predicted binding model of compound 4 with PDEδ. The figures were generated using Pymol (http://www.pymol.org/). (C) Design strategy for PDEδ-targeting ATTECs.

The procedures for the synthesis of target compounds are depicted in Schemes 1 and 2. The key intermediates 7 and 8 were prepared according to literature methods.18,23 Amide coupling of compound 7 with different linkers 6a–d afforded compounds 10a–d. After removal of the Boc group by trifluoroacetic acid (TFA), the intermediate amines were coupled with compound 8, followed by removal of the N-Boc from the piperidine scaffold to afford target compounds 11a–d. The key intermediate 9 was synthesized via five steps according to our reported protocols.28 The Boc group of compounds 10a–c was removed, followed by condensation with key intermediate 9 to obtain target compounds 12a–c. Similar reactions in Scheme 1 including deprotection of the Boc group and amide coupling were performed in Scheme 2 to yield target compounds 15a–c and 16a–c.

Scheme 1. Synthetic Route of Target Compounds 11a–d and 12a–c.

Scheme 1

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Reagents and conditions (a) EDCI, HOBT, DIPEA, DMF, rt., 2 h, yield 28–36%; (b) (i) TFA, DCM, rt, 0.5 h; (ii) compound 8, EDCI, HOBT, DIPEA, DMF, rt, 2 h; (iii) TFA, DCM, rt., 0.5 h, yield 18–33%; (c) (i) TFA, DCM, rt, 0.5 h; (ii) compound 9, EDCI, HOBT, DIPEA, DMF, rt, 2 h, yield 16–21%.

Scheme 2. Synthetic Route of Target Compounds 15a–c and 16a–c.

Scheme 2

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Reagents and conditions (a) EDCI, HOBT, DIPEA, DMF, rt, 2 h, yield 44–50%; (b) (i) TFA, DCM, rt, 0.5 h.; (ii) compound 8, EDCI, HOBT, DIPEA, DMF, rt, 2 h; (iii) TFA, DCM, rt, 0.5 h, yield 34–51%; (c) (i) TFA, DCM, rt, 0.5 h; (ii) compound 9, EDCI, HOBT, DIPEA, DMF, rt, 2 h, yield 39–41%.

Initially, the binding affinities of target compounds with PDEδ were evaluated by fluorescence polarization (FP) method described previously.28 Compound 4 served as a positive control. The results revealed that all the target compounds demonstrated nanomolar affinities (Table 1), with the KD values ranging from 51 to 153 nM. Then we assessed their effects on reducing the PDEδ levels in KRAS mutant MiaPaCa-2 cells (human pancreatic cancer) using the Western blotting assays. As shown in Figure 4A and Table 1, except for compound 11a, all of the compounds were able to induce PDEδ degradation at a concentration of 20 μM after 24 h treatment. The degradation potency decreased with the extension of linker length. LC3 warhead play a crucial role in PDEδ degradation. PDEδ ATTECs with compound 1 as the LC3 warhead (compounds 11b–c and 12a–c) showed the best activity in reducing the PDEδ protein level in MiaPaCa-2 cells, with maximum degradation rates in the range of 46–85%. On the other hand, ATTEC degraders with compound 4 as the PDEδ binder (compounds 12a–c and 16a–c) showed better protein degradation activities than corresponding compound 3-based analogous. Among these compounds, compound 12a and 12c significantly induced PDEδ degradation in a concentration-dependent manner (Figure 4B, Figure S2 in the Supporting Information), with DC50 (concentration causing 50% PDEδ degradation) values of 18.0 and 1.7 μM, respectively. Moreover, time-course experiments were performed to evaluate the kinetics of PDEδ degradation in MiaPaCa-2 cells. As shown in Figure 4C, compounds 12a and 12c degraded PDEδ in a time-dependent manner, with degradation starting early at approximately 4 h.

Table 1. Binding Affinity and Degradation Activities of PDEδ ATTECsa.

cpds PDEδ (KD, nM) PDEδ degradation (%, 20 μM) cpds PDEδ (KD, nM) PDEδ degradation (%, 20 μM)
11a 57 ± 10 0% 15a 51 ± 19 24%
11b 153 ± 65 46% 15b 71 ± 0.9 32%
11c 52 ± 12 59% 15c 73 ± 3.3 35%
11d 74 ± 19 66% 16a 74 ± 6.1 33%
12a 116 ± 56 51% 16b 94 ± 13 13%
12b 82 ± 38 60% 16c 82 ± 2.0 42%
12c 56 ± 4.3 85% 4 9.3 ± 0.5  
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a

Values represent mean ± SD of at least three independent experiments.

Figure 4.

Figure 4

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Activity of PDEδ autophagic degraders in MiaPaCa-2 cells. (A) Degradation activities of PDEδ ATTECs at 20 μM. (B) Compounds 12a and 12c degraded the PDEδ in a dose-dependent manner. (C) Compounds 12a and 12c degraded the PDEδ in a time-dependent manner.

Given the potent PDEδ binding affinity and degradation efficiency, the antiproliferative activities of PDEδ autophagic degraders 12a and 12c were assessed in two different KRAS mutant human pancreatic cancer cell lines (MiaPaCa-2 and Capan-1 cells) by CCK8 assays. The PDEδ inhibitor 4 was used as a positive control. As illustrated in Table 2, compounds 12a and 12c showed significantly enhanced antiproliferative activities in the suppression of cell proliferation in MiaPaCa-2 and Capan-1 cells. Consistent with protein degradation activity, compound 12c exhibited the best antiproliferative activity (Capan-1 IC50 = 0.8 μM; MiaPaCa-2 IC50 = 1.4 μM), which was significantly more potent than the corresponding PDEδ inhibitor 4. The superior inhibitory activity of PDEδ ATTECs in cell growth compared with inhibitor counterparts highlighted the significant advantages of PDEδ degradation over inhibition. Due to the good degradation efficiency and antiproliferative activity, ATTEC 12c was selected for further functional characterization.

Table 2. Antitumor Activities of Compounds 12a and 12c In Vitroa.

  IC50 (μM)
cpds MiaPaCa-2 Capan-1
12a 25 ± 15 3.5 ± 0.7
12c 1.4 ± 0.4 0.8 ± 0.1
4 >100 >100
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a

Values represent mean ± SD of at least three independent experiments.

The recent research suggests that compound 1 may not interact with LC3.29 To further verify the direct interaction between compound 12c and LC3 protein, MicroScale Thermophoresis (MST) method was used to assess the binding affinity of compound 12c toward LC3. The determined KD value of 187 nM demonstrated the potent and effective binding capability of compound 12c with LC3 (Figure S3 in the Supporting Information). In order to investigate whether compound 12c caused the PDEδ degradation via lysosome-mediated autophagy, the autophagy inhibitor Bafilomycin A1 (Baf-A1)16 was added to evaluate its effect on reversing the PDEδ protein level in MiaPaCa-2 cells. As expected, the total protein level of PDEδ was reversed upon the addition of the autophagy inhibitor (Figure 5A). The mRNA expression levels of PDEδ have a direct effect on its protein content. To further confirm whether the decrease in PDEδ protein level was due to the downregulation of mRNA expression induced by compound 12c, quantitative real-time PCR (qRT-PCR) analysis was performed to measure the PDEδ mRNA expression levels in MiaPaCa-2 cells. As shown in Figure 5B, compound 12c did not cause any significant changes in the PDEδ mRNA level. Furthermore, we used PDEδ inhibitor 1 and LC3 ligand 4 to verify whether the degradation effect of 12c can be reversed. As expected, compound 1 could obviously prevent the degradation of the PDEδ protein induced by compound 12c. Meanwhile, LC3 ligand 4 also showed competitive inhibition of the ability of compound 12c to degrade PDEδ (Figure 5C). Recent studies have indicated that the compound 1-based degrader functions as an PROTAC that degrades target proteins through the ubiquitin proteasome pathway.30 To further validate the degradation mechanism of compound 12c, proteasome inhibitor MG-132 and neddylation inhibitor MLN4924 were employed. Interestingly, pretreatment with MG-132 or MLN4924 could not block the PDEδ degradation induced by compound 12c (Figure S4 in the Supporting Information). In addition, when LC3B was knocked down using siRNA in MiaPaCa-2 cells (Figure S5 in the Supporting Information), the degradation activity of compound 12c was significantly decreased, almost abolishing the degradation of PDEδ. These results demonstrated that compound 12c was a bona fide autophagic degrader that significantly induced PDEδ degradation through lysosome-mediated autophagy without interfering with mRNA synthesis.

Figure 5.

Figure 5

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Compound 12c degraded PDEδ via autophagy and induced apoptosis in MiaPaCa-2 cells. (A) MiaPaCa-2 cells were pretreated with Baf-A1, followed by the treatment of compound 12c for another 24 h. (B) The mRNA level of PDEδ induced by compound 12c. “ns” indicates no significant difference. (C) MiaPaCa-2 cells were pretreated with compounds 4 and 1, followed by the treatment of compound 12c for another 24 h. (D) Flow cytometry analysis of the apoptosis induced by compound 12c in MiaPaCa-2 cells.

To explore whether compound 12c affects downstream signaling pathways of KRAS by degrading the PDEδ protein, MiaPaCa-2 cells were stimulated with epidermal growth factor (EGF) for 5 min, followed by treatment with different concentrations of compound 12c for 12 h. The results verified that compound 12c significantly downregulated the expression levels of p-AKT in a concentration-dependent manner (Figure S6 in the Supporting Information). To further investigate the mechanism of action of compound 12c, we conducted flow cytometry assays with Annexin V/PI staining to examine its antitumor effects (Figure 5D). Treatment with compound 12c resulted in a significant increase in the apoptosis of MiaPaCa-2 cells after 24 h. At a concentration of 1 μM, the apoptosis rate induced by compound 12c was 22.23%, which was higher than that of compound 4 at 20 μM (apoptosis rate: 18.78%). The apoptosis rate was increased to 33.25% at 10 μM, indicating that compound 12c induced apoptosis in a dose-dependent manner. These results demonstrated that compound 12c, the most potent PDEδ autophagic degrader, possessed strong antitumor activity by inducing apoptosis in MiaPaCa-2 cells.

In summary, using the ATTEC technology, the first class of PDEδ autophagic degraders composed of different LC3 ligands and PDEδ inhibitors was successfully designed and synthesized. The most promising compound 12c efficiently induced PDEδ degradation through lysosome-mediated autophagy without interfering with PDEδ mRNA synthesis. As compared with the PDEδ inhibitor, compound 12c demonstrated significantly enhanced antiproliferative potency in KRAS mutant pancreatic cancer cells and induced apoptosis in dose-dependent and time-dependent manner. PROTAC is currently the most popular method for TPD. Using this approach, we have designed and identified several PDEδ PROTACs as potent PDEδ degraders.28 Interestingly, compound 12c, developed using the ATTEC approach, exhibited superior degradation activity and antitumor potency compared with the corresponding PDEδ PROTAC in MiaPaCa-2 cells. This highlights the advantages of autophagic degradation over proteasome-mediated degradation for targeted degradation of PDEδ. Taken together, this study further validated the effectiveness of ATTEC in TPD technology, broadened the scope of degradable targets, and provided a promising lead compound targeting the KRAS-PDEδ interaction.

Glossary

Abbreviations

TPD

targeted protein degradation

ATTEC

autophagy-tethering compound

PROTAC

proteolysis targeting chimera

ALP

autophagy-lysosomal pathway

LYTAC

lysosome targeting chimera

AUTAC

autophagy-targeting chimera

POI

protein of interest

qRT-PCR

quantitative real-time PCR

NAMPT

nicotinamide phosphoribosyltransferase

TFA

trifluoroacetic acid

FP

fluorescence polarization

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00161.

  • Experimental section, Figures S1–S6, Scheme S1, chemistry, 1H NMR, 13C NMR, and ESI-MS of representative compounds (PDF)

Author Contributions

§ J.B., Z.C., and Y.L. contributed equally. The manuscript was written through contributions of all authors.

This work was supported by the National Natural Science Foundation of China (grant 82273779 to G. D., 82204211 to W.W., and 82030105 to C.S.), the National Key Research and Development Program of China (grant 2022YFC3401500 to C.S.).

The authors declare no competing financial interest.

Supplementary Material

ml3c00161_si_001.pdf (1.2MB, pdf)

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