Design, Synthesis and Biological Evaluation of α-Synuclein Proteolysis-Targeting Chimeras.

α-Synuclein aggregation under pathological conditions is one of the causes of related neurodegenerative diseases. PROTACs (proteolysis targeting chimeras) are bifunctional small molecules that induce a post-translational erasure of proteins via the ubiquitination of target proteins by E3 ubiquitin ligase and subsequent proteasomal degradation. However, few research studies have been conducted for targeted protein degradation of α-synuclein aggregates. In this article, we have designed and synthesized a series of small-molecule degraders 1-9 based on a known α-synuclein aggregation inhibitor sery384. In silico docking studies of sery384 with α-synuclein aggregates were accomplished to ensure that the compounds bound to α-synuclein aggregates specifically. The protein level of α-synuclein aggregates was determined to evaluate the degradation efficiency of PROTAC molecules on α-synuclein aggregates in vitro. The results show that compound 5 had the most significant degradation effect, with DC50 of 5.049 µM, and could induce the degradation of α-synuclein aggregates in a time- and dose-dependent manner in vitro. Furthermore, compound 5 could inhibit the elevation of the ROS level caused by overexpression and aggregation of α-synuclein and protect H293T cells from α-synuclein toxicity. Conclusively, our results provide a new class of small-molecule degraders and an experimental basis for the treatment of α-synuclein related neurodegenerative diseases.

Chemodivergent Staudinger Reactions of Secondary Phosphine Oxides and Application to the Total Synthesis of LL-D05139ß Potassium Salt.

Unprecedented Staudinger reaction modes of secondary phosphine oxides (SPO) and organic azides are herein disclosed. By the application of various additives, selective nitrogen atom exclusion from the azide group has been achieved. Chlorotrimethylsilane mediates a stereoretentive Staudinger reaction with a 2-N exclusion which provides a valuable method for the synthesis of phosphinic amides and can be considered complementary to the stereoinvertive Atherton-Todd reaction. Alternatively, a 1-N exclusion pathway is promoted by acetic acid to provide the corresponding diazo compound. The effectiveness of this protocol has been further demonstrated by the total synthesis of the diazo-containing natural product LL-D05139ß, which was prepared as a potassium salt for the first time in 6 steps and 26.5 % overall yield.

Xyloketal B Reverses Nutritional Hepatic Steatosis, Steatohepatitis, and Liver Fibrosis through Activation of the PPARα/PGC1α Signaling Pathway.

Nonalcoholic fatty liver disease (NAFLD) represents a class of disorders including hepatic steatosis, steatohepatitis, and liver fibrosis. Previous research suggested that xyloketal B (Xyl-B), a marine-derived natural product, could attenuate the NAFLD-related lipid accumulation. Herein, we investigated the protective mechanism of Xyl-B in a high-fat diet (HFD) mice fatty liver model by combining a quantitative proteomic approach with experimental methods. The results showed that the administration of Xyl-B (20 and 40 mg·kg-1·day-1, ip) ameliorated the hepatic steatosis in HFD mice. Proteomic profiling together with bioinformatics analysis highlighted the upregulation of a cluster of peroxisome proliferator-activated receptor-α (PPARα) downstream enzymes mainly related to fatty acid oxidation (FAO) as key changes after the treatment. These changes were subsequently confirmed by bioassays. Moreover, further results showed that the expression levels of PPARα and PPARγ coactivator-1α (PGC1α) were increased after the treatment. The related mode-of-action was confirmed by PPARα inhibition. Furthermore, we evaluated the PPARα-mediated anti-inflammatory and antifibrosis effect of Xyl-B in methionine-choline-deficient (MCD) mice hepatitis and liver fibrosis models. According to the results, the histological features were improved, and the levels of inflammatory factors, adhesion molecules, as well as fibrosis markers were decreased after the treatment. Collectively, these results indicated that Xyl-B ameliorated different phases of NAFLD through activation of the PPARα/PGC1α signaling pathway. Our findings revealed the possible metabolism-regulating mechanism of Xyl-B, broadened the application of xyloketal family compounds, and may provide a new strategy to curb the development of NAFLD.

Highly efficient synthesis of benzodioxins with a 2-site quaternary carbon structure by secondary amine-catalyzed dual Michael cascade reactions.

Salicylic acids and substituted ynones were employed as substrates to afford a class of valuable 4H-benzo[d][1,3]dioxin-4-ones with a 2-site quaternary carbon structure in up to 92% yield by secondary amine-catalyzed dual Michael cascade reactions under mild reaction conditions. The α,ß-unsaturated ketone as the key intermediate in the cascade process was successfully separated and characterized. As a result, a new reaction route for ynone species is demonstrated, which is totally different from the existing allenamine activation model.

Xyloketal B exerts antihypertensive effect in renovascular hypertensive rats via the NO-sGC-cGMP pathway and calcium signaling.

Xyloketal B (Xyl-B) is a novel marine compound isolated from mangrove fungus Xylaria sp. (No 2508). We previously showed that Xyl-B promoted endothelial NO release and protected against atherosclerosis through the Akt/eNOS pathway. Vascular NO production regulates vasoconstriction in central and peripheral arteries and plays an important role in blood pressure control. In this study, we examined whether Xyl-B exerted an antihypertensive effect in a hypertensive rat model, and further explored the possible mechanisms underlying its antihypertensive action. Administration of Xyl-B (20 mg·kg-1·d-1, ip, for 12 weeks) significantly decreased the systolic and diastolic blood pressure in a two-kidney, two-clip (2K2C) renovascular hypertensive rats. In endothelium-intact and endothelium-denuded thoracic aortic rings, pretreatment with Xyl-B (20 µmol/L) significantly suppressed phenylephrine (Phe)-induced contractions, suggesting that its vasorelaxant effect was attributed to both endothelial-dependent and endothelial-independent mechanisms. We used SNP, methylene blue (MB, guanylate cyclase inhibitor) and indomethacin (IMC, cyclooxygenase inhibitor) to examine which endothelial pathway was involved, and found that MB, but not IMC, reversed the inhibitory effects of Xyl-B on Phe-induced vasocontraction. Moreover, Xyl-B increased the endothelial NO bioactivity and smooth muscle cGMP level, revealing that the NO-sGC-cGMP pathway, rather than PGI2, mediated the anti-hypertensive effect of Xyl-B. We further showed that Xyl-B significantly attenuated KCl-induced Ca2+ entry in smooth muscle cells in vitro, which was supposed to be mediated by voltage-dependent Ca2+ channels (VDCCs), and reduced ryanodine-induced aortic contractions, which may be associated with store-operated Ca2+ entry (SOCE). Taken together, these findings demonstrate that Xyl-B exerts significant antihypertensive effects not only through the endothelial NO-sGC-cGMP pathway but also through smooth muscle calcium signaling, including VDCCs and SOCE.

Studies on the Design and Synthesis of Marine Peptide Analogues and Their Ability to Promote Proliferation in HUVECs and Zebrafish.

In our previous studies, tripeptide 1 was found to induce angiogenesis in zebrafish embryos and in HUVECs. Based on the lead compound 1, seven new marine tripeptide analogues 2⁻8 have been designed and synthesized in this paper to evaluate the effects on promoting cellular proliferation in human endothelial cells (HUVECs) and zebrafish. Among them, compounds 5⁻7 possessed more remarkable increasing proliferation effects than other compounds, and the EC50 values of these and the leading compound 1 were 1.0 ± 0.002 µM, 1.0 ± 0.0005 µM, 0.88 ± 0.0972 µM, and 1.31 ± 0.0926 µM, respectively. Furthermore, 5⁻7 could enhance migrations (58.5%, 80.66% and 60.71% increment after culturing 48 h, respectively) and invasions (49.08%, 47.24% and 56.24% increase, respectively) in HUVECs compared with the vehicle control. The results revealed that the tripeptide including l-Tyrosine or d-Proline fragments instead of l-Alanine of leading compound 1 would contribute to HUVECs' proliferation. Taking the place of the original (l-Lys-l-Ala) segment of leading compound 1, a new fragment (l-Arg-d-Val) expressed higher performance in bioactivity in HUVECs. In addition, compound 7 could promote angiogenesis in zebrafish assay and it was more interesting that it also could repair damaged blood vessels in PTK787-induced zebrafish at a low concentration. The above data indicate that these peptides have potential implications for further evaluation in cytothesis studies.

Xyloketal B alleviates cerebral infarction and neurologic deficits in a mouse stroke model by suppressing the ROS/TLR4/NF-κB inflammatory signaling pathway.

Xyloketal B (Xyl-B) is a novel marine compound isolated from mangrove fungus Xylaria sp. We previously demonstrated that pretreatment with Xyl-B exerted neuroprotective effects and attenuated hypoxic-ischemic brain injury in neonatal mice. In the present study we investigated the neuroprotective effects of pre- and post-treatment with Xyl-B in adult mice using a transient middle cerebral artery occlusion (tMCAO) model, and explored the underlying mechanisms. Adult male C57 mice were subjected to tMCAO surgery. For the pre-treatment, Xyl-B was given via multiple injections (12.5, 25, and 50 mg·kg-1·d-1, ip) 48 h, 24 h and 30 min before ischemia. For the post-treatment, a single dose of Xyl-B (50 mg/kg, ip) was injected at 0, 1 or 2 h after the onset of ischemia. The regional cerebral perfusion was monitored using a laser-Doppler flowmeter. TTC staining was performed to determine the brain infarction volume. We found that both pre-treatment with Xyl-B (50 mg/kg) and post-treatment with Xyl-B (50 mg/kg) significantly reduced the infarct volume, but had no significant hemodynamic effects. Treatment with Xyl-B also significantly alleviated the neurological deficits in tMCAO mice. Furthermore, treatment with Xyl-B significantly attenuated ROS overproduction in brain tissues; increased the MnSOD protein levels, suppressed TLR4, NF-κB and iNOS protein levels; and downregulated the mRNA levels of proinflammatory cytokines, including IL-1ß, TNF-α, IL-6 and IFN-γ. Moreover, Xyl-B also protected blood-brain barrier integrity in tMCAO mice. In conclusion, Xyl-B administered within 2 h after the onset of stroke effectively protects against focal cerebral ischemia; the underlying mechanism may be related to suppressing the ROS/TLR4/NF-κB inflammatory signaling pathway.

Xyloketal B Attenuates Fatty Acid-Induced Lipid Accumulation via the SREBP-1c Pathway in NAFLD Models.

The goal of this study was to examine the effects of xyloketal B on nonalcoholic fatty liver disease (NAFLD) and to explore the molecular mechanisms underlying its effects in both in vivo and in vitro models. We discovered an association between xyloketal B and the sterol regulatory element-binding protein-1c (SREBP-1c) signaling pathway, which is related to lipid metabolism. Mice were dosed with xyloketal B (5, 10 and 20 mg/kg/d) and atorvastatin (15 mg/kg/d) via intraperitoneal injection once daily for 40 days after being fed a high fat diet plus 10% high fructose liquid (HFD+HFL) for 8 weeks. Xyloketal B significantly improved HFD+HFL-induced hepatic histological lesions and attenuated lipid and glucose accumulation in the blood as well as lipid accumulation in the liver. Xyloketal B increased the expression of CPT1A, and decreased the expression of SREBP-1c and its downstream targeting enzymes such as ACC1, ACL, and FAS. Xyloketal B also significantly reduced lipid accumulation in HepG2 cells treated with free fatty acids (FFAs). These data suggested that xyloketal B has lipid-lowering effects via the SREBP-1c pathway that regulate lipid metabolism. Thus, targeting SREBP-1c activation with xyloketal B may be a promising novel approach for NAFLD treatment.

Synthesis of a Class of Chiral-Bridged Phosphoramidite Ligands and Their Applications in the First Iridium-Catalyzed Asymmetric Addition of Arylboronic Acids to Isatins.

A series of novel chiral phosphoramidite ligands based on chiral-bridged biphenyl backbones have been prepared conveniently and characterized. The ligands complexed with [IrCodCl]2 provided the first iridium catalyst system for the asymmetric addition of arylboronic acids to N-protected isatins with high efficiency. When performed in THF/H2O at 80 °C with 2 equiv of the arylboronic acids, the transformations acquired good to excellent results (up to 98% yield and 95% ee).

Design and synthesis of novel xyloketal derivatives and their protective activities against H2O2-induced HUVEC injury.

In this work, we designed and synthesized a series of amide derivatives (1-13), benzoxazine derivatives (16-28) and amino derivatives (29-30) from xyloketal B. All 28 new derivatives and seven known compounds (14, 15, 31-35) were evaluated for their protection against H2O2-induced HUVEC injury. 23 and 24 exhibited more potential protective activities than other derivatives; and the EC50 values of them and the leading compound 31 (xyloketal B) were 5.10, 3.59 and 15.97 µM, respectively. Meanwhile, a comparative molecular similarity indices analysis (CoMSIA) was constructed to explain the structural activity relationship of these xyloketal derivatives. This 3D QSAR model from CoMSIA suggested that the derived model exhibited good predictive ability in the external test-set validation. Derivative 24 fit well with the COMSIA map, therefore it possessed the highest activity of all compounds. Compounds 23, 24 and 31 (xyloketal B) were further to examine in the JC-1 mitochondrial membrane potential (MMP) assay of HUVECs using flow cytometry (FCM). The result indicated that 23 and 24 significantly inhibited H2O2-induced decrease of the cell mitochondrial membrane potential (ΔΨm) at 25 µM. Collectively, the protective effects of xyloketals on H2O2-induced endothelial cells may be generated from oxidation action by restraining ROS and reducing the MMP.

Xyloketal B attenuates atherosclerotic plaque formation and endothelial dysfunction in apolipoprotein e deficient mice.

Our previous studies demonstrated that xyloketal B, a novel marine compound with a unique chemical structure, has strong antioxidant actions and can protect against endothelial injury in different cell types cultured in vitro and model organisms in vivo. The oxidative endothelial dysfunction and decrease in nitric oxide (NO) bioavailability are critical for the development of atherosclerotic lesion. We thus examined whether xyloketal B had an influence on the atherosclerotic plaque area in apolipoprotein E-deficient (apoE-/-) mice fed a high-fat diet and investigated the underlying mechanisms. We found in our present study that the administration of xyloketal B dose-dependently decreased the atherosclerotic plaque area both in the aortic sinus and throughout the aorta in apoE-/- mice fed a high-fat diet. In addition, xyloketal B markedly reduced the levels of vascular oxidative stress, as well as improving the impaired endothelium integrity and NO-dependent aortic vasorelaxation in atherosclerotic mice. Moreover, xyloketal B significantly changed the phosphorylation levels of endothelial nitric oxide synthase (eNOS) and Akt without altering the expression of total eNOS and Akt in cultured human umbilical vein endothelial cells (HUVECs). Here, it increased eNOS phosphorylation at the positive regulatory site of Ser-1177, while inhibiting phosphorylation at the negative regulatory site of Thr-495. Taken together, these findings indicate that xyloketal B has dramatic anti-atherosclerotic effects in vivo, which is partly due to its antioxidant features and/or improvement of endothelial function.

Identification of marine neuroactive molecules in behaviour-based screens in the larval zebrafish.

High-throughput behavior-based screen in zebrafish is a powerful approach for the discovery of novel neuroactive small molecules for treatment of nervous system diseases such as epilepsy. To identify neuroactive small molecules, we first screened 36 compounds (1-36) derived from marine natural products xyloketals and marine isoprenyl phenyl ether obtained from the mangrove fungus. Compound 1 demonstrated the most potent inhibition on the locomotor activity in larval zebrafish. Compounds 37-42 were further synthesized and their potential anti-epilepsy action was then examined in a PTZ-induced epilepsy model in zebrafish. Compound 1 and compounds 39, 40 and 41 could significantly attenuate PTZ-induced locomotor hyperactivity and elevation of c-fos mRNA in larval zebrafish. Compound 40 showed the most potent inhibitory action against PTZ-induced hyperactivity. The structure-activity analysis showed that the OH group at 12-position played a critical role and the substituents at the 13-position were well tolerated in the inhibitory activity of xyloketal derivatives. Thus, these derivatives may provide some novel drug candidates for the treatment of epilepsy.

Discovery of Effective Dual PROTAC Degraders for Neurodegenerative Disease-Associated Aggregates.

The aggregation of specific proteins is a histopathological hallmark in various neurodegenerative diseases (NDs), among which Alpha-synuclein (α-Syn) and tau have received increased attention. The targeted protein degradation (TPD) strategy has been studied in the treatment of NDs, but multitarget bifunctional molecules have been ignored. Herein, a series of effective dual PROTAC degraders were developed, which could degrade α-Syn aggregates and total tau simultaneously. The degradation effects were evaluated in vitro, and the results showed that T3 could significantly knockdown α-Syn aggregates and total tau in the degradation efficiency with DC50 of 1.57 ± 0.55 and 4.09 ± 0.90 µM, respectively. Further mechanistic exploration showed that the degradation effect was mediated by the ubiquitin-proteasome system (UPS). Additionally, the therapeutic efficacy of T3 was confirmed in an MPTP-induced PD mouse model. Our results suggest that these dual PROTACs may provide a potential therapeutic strategy for NDs.

Discovery of the First Potent DYRK2 Proteolysis Targeting Chimera Degraders.

Dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) has been identified as a promising oncogenic driver of several types of cancer and is considered to be a critical cancer therapeutic target. Several inhibitors of DYRK2 have been reported, but no degraders have been found yet. In this work, we designed and synthesized the first series of proteolysis-targeting chimeras (PROTACs) using curcumin and its analogs as warheads to target and degrade DYRK2. The results of degradation assays showed that the compound CP134 could effectively downregulate the intracellular DYRK2 level (DC50 = 1.607 µM). Further mechanism of action experiments revealed that CP134 induced DYRK2 degradation through the ubiquitin-proteasome system. Altogether, CP134 disclosed in this study is the first potent DYRK2 degrader, which could serve as a valuable chemical tool for further evaluation of its therapeutic potential, and our results broaden the substrate spectrum of PROTAC-based degraders for further therapeutic applications.

Targeting EGFR degradation by autophagosome degraders.

Several generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been developed for the treatment of non-small cell lung cancer (NSCLC) in clinic. However, emerging drug resistance mediated by new EGFR mutations or activations by pass, leads to malignant progression of NSCLC. Proteolysis targeting chimeras (PROTACs) have been utilized to overcome the drug resistance acquired by mutant EGFR, newly potent and selective degraders are still need to be developed for clinical applications. Herein, we developed autophagosome-tethering compounds (ATTECs) in which EGFR can be anchored to microtubule-associated protein-1 light chain-3B (LC3B) on the autophagosome with the assistance of the LC3 ligand GW5074. A series of EGFR-ATTECs have been designed and synthesized. Biological evaluations showed that these compounds could degrade EGFR and exhibited moderate inhibitory effects on certain NSCLC cell lines. The ATTEC 12c potently induced the degradation of EGFR with a DC50 value of 0.98 µM and a Dmax value of 81% in HCC827 cells. Mechanistic exploration revealed that the lysosomal pathway was mainly involved in this degradation. Compound 12c also exhibited promising inhibitory activity, as well as degradation efficiency in vivo. Our study highlights that EGFR-ATTECs could be developed as a new expandable EGFR degradation tool and also reveals a novel potential therapeutic strategy to prevent drug resistance acquired EGFR mutations.

Secondary metabolites of a mangrove endophytic fungus Aspergillus terreus (No. GX7-3B) from the South China Sea.

The mangrove endophytic fungus Aspergillus terreus (No. GX7-3B) was cultivated in potato dextrose liquid medium, and one rare thiophene compound (1), together with anhydrojavanicin (2), 8-O-methylbostrycoidin (3), 8-O-methyljavanicin (4), botryosphaerone D (5), 6-ethyl-5-hydroxy-3,7-dimethoxynaphthoquinone (6), 3ß,5α-dihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (7), 3ß,5α,14α-trihydroxy-(22E,24R)-ergosta-7, 22-dien-6-one (8), NGA0187 (9) and beauvericin (10), were isolated. Their structures were elucidated by analysis of spectroscopic data. This is the first report of a natural origin for compound 6. Moreover, compounds 3, 4, 5, 7, 8 and 10 were obtained from marine microorganism for the first time. In the bioactive assays in vitro, compounds 2, 3, 9 and 10 displayed remarkable inhibiting actions against α-acetylcholinesterase (AChE) with IC50 values 2.01, 6.71, 1.89, and 3.09 µM, respectively. Furthermore, in the cytotoxicity assays, compounds 7 and 10 exhibited strong or moderate cytotoxic activities against MCF-7, A549, Hela and KB cell lines with IC50 values 4.98 and 2.02 (MCF-7), 1.95 and 0.82 (A549), 0.68 and 1.14 (Hela), and 1.50 and 1.10 µM (KB), respectively; compound 8 had weak inhibitory activities against these tumor cell lines; compounds 1, 2, 3, 4, 5, 6 and 9 exhibited no inhibitory activities against them.

Synthesis and neuroprotective action of xyloketal derivatives in Parkinson's disease models.

Parkinson's disease (PD) is the second most common neurodegenerative disease affecting people over age 55. Oxidative stress actively participates in the dopaminergic (DA) neuron degeneration of PD. Xyloketals are a series of natural compounds from marine mangrove fungus strain No. 2508 that have been reported to protect against neurotoxicity through their antioxidant properties. However, their protection versus 1-methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity is only modest, and appropriate structural modifications are necessary to discover better candidates for treating PD. In this work, we designed and synthesized 39 novel xyloketal derivatives (1-39) in addition to the previously reported compound, xyloketal B. The neuroprotective activities of all 40 compounds were evaluated in vivo via respiratory burst assays and longevity-extending assays. During the zebrafish respiratory burst assay, compounds 1, 9, 23, 24, 36 and 39 strongly attenuated reactive oxygen species (ROS) generation at 50 µM. In the Caenorhabditis elegans longevity-extending assay, compounds 1, 8, 15, 16 and 36 significantly extended the survival rates (p < 0.005 vs. dimethyl sulfoxide (DMSO)). A total of 15 compounds were tested for the treatment of Parkinson's disease using the MPP+-induced C. elegans model, and compounds 1 and 8 exhibited the highest activities (p < 0.005 vs. MPP+). In the MPP+-induced C57BL/6 mouse PD model, 40 mg/kg of 1 and 8 protected against MPP+-induced dopaminergic neurodegeneration and increased the number of DA neurons from 53% for the MPP+ group to 78% and 74%, respectively (p < 0.001 vs. MPP+ group). Thus, these derivatives are novel candidates for the treatment of PD.

Xyloketal B exhibits its antioxidant activity through induction of HO-1 in vascular endothelial cells and zebrafish.

We previously reported that a novel marine compound, xyloketal B, has strong antioxidative actions in different models of cardiovascular diseases. Induction of heme oxygenase-1 (HO-1), an important endogenous antioxidant enzyme, has been considered as a potential therapeutic strategy for cardiovascular diseases. We here investigated whether xyloketal B exhibits its antioxidant activity through induction of HO-1. In human umbilical vein endothelial cells (HUVECs), xyloketal B significantly induced HO-1 gene expression and translocation of the nuclear factor-erythroid 2-related factor 2 (Nrf-2) in a concentration- and time-dependent manner. The protection of xyloketal B against angiotensin II-induced apoptosis and reactive oxygen species (ROS) production could be abrogated by the HO-1 specific inhibitor, tin protoporphyrin-IX (SnPP). Consistently, the suppressive effects of xyloketal B on NADPH oxidase activity could be reversed by SnPP in zebrafish embryos. In addition, xyloketal B induced Akt and Erk1/2 phosphorylation in a concentration- and time-dependent manner. Furthermore, PI3K inhibitor LY294002 and Erk1/2 inhibitor U0126 suppressed the induction of HO-1 and translocation of Nrf-2 by xyloketal B, whereas P38 inhibitor SB203580 did not. In conclusion, xyloketal B can induce HO-1 expression via PI3K/Akt/Nrf-2 pathways, and the induction of HO-1 is mainly responsible for the antioxidant and antiapoptotic actions of xyloketal B.

Targeted Degradation of Alpha-Synuclein by Autophagosome-Anchoring Chimera Peptides.

Targeted protein degradation (TPD) confers knockdown of "undruggable" targets such as alpha-synuclein (αSyn), a pathogenic protein in multiple neurodegenerative diseases. Though many of these proteins were mainly degraded through the autophagy-lysosome pathway (ALP), few TPD tools harnessing the ALP were reported. Herein, we developed a strategy termed autophagosome-anchoring chimera (ATACC), in which the protein of interest (POI) can be anchored to microtubule-associated protein-1 light chain-3B (LC3B) on the autophagosome with the assistance of an LC3-interacting region (LIR)-containing bifunctional peptide, and the selective autophagy of the POI is thus facilitated. A series of αSyn-targeting ATACC peptides were designed and synthesized. Biological evaluations demonstrated that these compounds could degrade αSyn specifically and effectively through a "chemical-induced cargo recognition-ALP degradation" mechanism. The neuroprotective effects of ATACC peptide P1 were further validated in vitro and in vivo. Collectively, our results provided a new TPD tool and revealed a potential therapeutic strategy against synucleinopathies.

Discovery of Small-Molecule Degraders for Alpha-Synuclein Aggregates.

Alpha-synuclein (αSyn) species, especially the oligomers and fibers, are associated with multiple neurodegenerative diseases and cannot be directly targeted under the conventional pharmacological paradigm. Proteolysis-targeting chimera technology confers degradation of various "undruggable" targets; however, hardly any small-molecule degrader for αSyn aggregates has been reported yet. Herein, by using the probe molecule sery308 as a warhead, a series of small-molecule degraders for αSyn aggregates were designed and synthesized. Their degradation effects on αSyn aggregates were evaluated on a modified pre-formed fibril-seeding cell model. Compound 2b exhibited the highest degradation efficiency (DC50 = 7.51 ± 0.53 µM) with high selectivity. Mechanistic exploration revealed that both proteasomal and lysosomal pathways were involved in this kind of degradation. Moreover, the therapeutic effects of 2b were tested on SH-SY5Y (human neuroblastoma cell line) cells and Caenorhabditis elegans. Our results provided a new class of small-molecule candidates against synucleinopathies and broadened the substrate spectrum of PROTAC-based degraders.