From an autophagic initiator to multi-functional drug target

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UNC-51-like kinase 1: From an autophagic initiator to multi-functional drug target Lan Zhang, Liang Ouyang, Yongzhi Guo, Jin Zhang, and Bo Liu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01684 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Journal of Medicinal Chemistry

UNC-51-like kinase 1: From an autophagic initiator to multi-functional drug target Lan Zhang#, Liang Ouyang#, Yongzhi Guo, Jin Zhang, Bo Liu* State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu 610041, China

ABSTRACT UNC-51-like kinase 1 (ULK1), known as an ortholog of the yeast Atg1, is the serine-threonine kinase and the autophagic initiator in mammals. Accumulating evidence has recently revealed the kinase domain structure of ULK1 and its post-translational modifications, as well as further elucidated its regulatory autophagic pathways and associations with diverse human diseases. Interestingly, a series of small molecules have been recently reported to target ULK1 or ULK1-modulating autophagy, which may provide a clue on exploiting them as novel candidate drugs. Taken together, this review discusses how ULK1 acts as an autophagic initiator for modulation of its intricate mechanisms, as well as how ULK1 becomes a multi-functional target for potential therapeutic applications. Key words: UNC-51-like kinase 1 (ULK1); Autophagy; Autophagic pathway; Cancer; Neurodegenerative disease; Small molecule

INTRODUCTION Autophagy, a highly conserved process for lysosomal degradation, can digest some long-lived proteins or damaging organelles. The autophagic process can be regulated by about thirty-five autophagy-related (ATG) genes.1 Among them, UNC-51-like kinase 1 (ULK1), also known as the ortholog of Atg1, which was firstly cloned in yeast, has the same autophagy-initiating function in mammalians.2 In the year of 2016, Yoshinori Ohsumi won the Nobel Prize for his contribution to autophagy research, including his discovery of Atg1 in yeast.

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Of note, the ULK complex is required to initiate the autophagic process. It is composed of ULK1, as well as other three members mATG13, focal adhesion kinase family interacting protein of 200KDa (FIP200) and ATG101 to initiate autophagosome formation. More recently, accumulating studies have demonstrated that ULK1 has some types of post-translational modifications, such as phosphorylation and ubiquitylation.3,4 The above-mentioned modifications of ULK1 may regulate its multiple autophagic signaling pathways, which may be closely involved in diverse diseases including cancer, neurodegenerative disease, infectious diseases, diabetes and cardiovascular diseases.5 In addition, some recent reports have also demonstrated that a number of small-molecule compounds can modulate autophagy by directly or indirectly targeting ULK1 to benefit the therapeutic strategies.6,7 In this review, we focus on discussing how ULK1, as an autophagic initiator regulates the ULK complex and its relevant autophagic pathways, as well as how ULK1 will be gradually regarded as a potential novel target for future drug discovery.

ULK1 STRUCTURE AND FUNCTION Composition of ULK1 and its complex ULK1, a mammalian homolog of Atg1 with an overall similarity of 29%, is a cytoplasmic kinase whose open reading frame (ORF) is composed of 1,050 (Homo sapiens) or 1,051 (Mus musculus) amino acids with molecular weight of 112.6 or 113 kDa.2,8 ULK1 possess an N-terminal kinase domain (KD) (residue 16-278) and a C-terminal

domain

(CTD)

(residue

833-1,050)

containing

two

tandem

microtubule-interacting and transport (MIT) domains. The KD and CTD are speculated to be highly conserved whether in yeast or mammals.8 In addition, the region connecting the KD and MIT for approximately 500 amino acids is less conserved and is named proline/serine-rich (PS) region for its abundance with Pro and Ser (Figure 1).8 The crystal structure of ULK1 KD has recently been determined to have a typical fold, which is similar to the KD of most extensively characterized protein kinase A (PKA), conserved among protein kinase superfamily. The KD consists of one helical C-lobe and 2


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the other N-lobe as the two globular folds, which is composed of a α-helix named αC and a five stranded β-sheet. Moreover, adenosine triphosphate (ATP) is usually utilized by protein kinases by binding to the cleft which is formed between the two lobes, and also covered by a lid which is named the P-loop or Gly-rich loop.7 Of note, the ULK complex is formed by ULK1 with mATG13, FIP200 and ATG101, which may interact with each other to convey its downstream signaling pathways (Figure 1). In 2008, FIP200 was firstly identified to interact with ULK1 in mammalian cells, which is essential for autophagosome formation.9 Subsequently, mATG13 was found to participate in the ULK complex formation, associated with ULK1 and FIP200 in 2009.10 In addition, its binding sites on ULK1 is mapped into C-terminal regions that contain residues 829-1,051. More recently, the binding site on ULK1-mATG13 has been further established by a short motif at the C terminus of mATG13, which is composed of the last 3 amino acids Thr478, Leu479 and Gln480 (human isoform 2).11 mATG13 and FIP200 may together regulate ULK1 kinase activity under normal conditions. Either of them can exert its function normally in a compensatory mechanism, and the maximal activity of ULK1 can be reached by cooperation of mATG13 and FIP200. In 2009, ATG101, as a mATG13 binding protein that functions together with ULK1, mATG13 and FIP200, was firstly identified in mammalian cells.12 Although the intricate mechanism of ATG101 remains unclear, it is one of the essential components of the ULK complex in mammals. Interestingly, there is not any homolog or functional equivalent of ATG101 in yeast.13

Post-translational modifications of ULK1 ULK1 is well-known as a ubiquitously expressed protein kinase which can localize to the autophagosomal membranes in mammalian cells.14 ULK1 is able to be directly activated by several post-translational modifications of crucial stress sensors, such as AMP-activated protein kinase (AMPK) and mammalian target of rapamycin complex 1 (mTORC1). mTORC1, a negative regulator of autophagy can phosphorylate ULK1 at Ser637 (Mus musculus)/Ser638 (Homo sapiens) and Ser757 (Mus musculus)/Ser758 (Homo sapiens) or directly phosphorylate mATG13 at Ser258 to prevent ULK1 activation 3



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as well as to disrupt the interaction between ULK1 and AMPK.15-17 AMPK can relieve the mTORC1-mediated inhibition of ULK1 by inhibiting Raptor, a member of mTORC1, by phosphorylation at Ser722 and Ser792. It can also directly activate ULK1 by phosphorylation at Ser317, Ser467, Thr574, Ser555, Ser637 (Mus musculus)/Ser638 (Homo sapiens) and Ser777 to stimulate autophagy.16,18 In addition, protein phosphatase magnesium-dependent delta isoform (PPM1D) can interact and dephosphorylate ULK1 at Ser637 to trigger genotoxic stress-induced autophagy, which is dependent on p53 activation.19 Moreover, ULK1 can be phosphorylated by type I interferon receptor (IFNR) at Ser757, which is an phosphorylation site of mTORC1 to inhibit ULK1. ULK1 can be activated after the engagement of type I IFNR, and then its activated form is depend on some intermediate kinases; thereby phosphorylating p38 MAPK.20 More interestingly, ULK1 autophosphorylation at Thr180 promotes its ubiquitylation and degradation by kelch-like protein 20 (KLHL20), which governs the degradation of the Beclin1 complex in prolonged starvation (Figure 2A, Table S1).21 In addition to these above-mentioned phosphorylation modifications, ULK1 has other types of post-transcriptional modifications, such as ubiquitylation and acetylation. Autophagy and Beclin1 regulator 1 (AMBRA1), can interact with the E3-ligase TRAF6 to support the ubiquitylation of ULK1 by Lys63-linked chains.4 Additionally, ubiquitylation of ULK1, regulated by phosphorylation on its carboxy terminus, can be induced by neural precursor cell-expressed developmentally downregulated gene 4-like (NEDD4L) at Lys925 and Lys933, which is key to the protection of excessive autophagy.22 And, ULK1 phosphorylates at three different sites (Ser929-Ser930-Thr931) on the same target region of ULK1 for NEDD4L, which is a preparative process for its ubiquitylation and degradation.23 Importantly, tat-interaction protein, 60ku (TIP60), can acetylate ULK1, and its activation can be modulated by glycogen synthase kinase 3β (GSK3β) via phosphorylation at Ser86. Instead of single regulation mode, this process directly links two diversified

modification

patterns,

phosphorylation

and

acetylation,

via

a

GSK3β-TIP60-ULK1 pathway (Figure 2A, Table S1).24 Together, these findings focus on elucidating the partial structure (kinase domain) of ULK1 and the function of the ULK 4


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complex (ULK1-mATG13-FIP200-ATG101), which plays the key role in the autophagy initiation and its regulatory autophagic pathways. Moreover, further evidence also reveals different types of post-translational modifications of ULK1, including phosphorylation, ubiquitylation and acetylation. These connections between the post-translational modifications of ULK1 and its regulatory autophagic pathways are discussed in details in the following section.

AUTOPHAGIC PATHWAYS REGULATED BY ULK1 Upstream autophagic pathways regulated by ULK1 Of note, AMPK is inactive under normal conditions, and mTORC1 can be associated with the ULK complex by interacting between ULK1 and Raptor directly. When nutrient supply is limited, ULK1 also phosphorylates Raptor at Ser855, Ser859, Ser792 and Ser863, thereby inhibiting mTORC1 signaling by hindrance of substrate binding to Raptor.25 When ATP/AMP ratio decreases, AMPK is activated and then it directly inhibits mTORC1.16 AMPK and mTORC1 can interact and thus leading to mTORC1 phosphorylation. It can also result in disassembling of mTORC1 and ULK1, thus activating the ULK complex in conducting autophagosome formation.4 AMPK can also interact with the ULK complex directly in a mTORC1-defcient manner. Interestingly, AMPK can also be negatively regulated by ULK1-mediated phosphorylation thereby constituting a negative regulatory feedback loop.26 More recently, it has been reported that ULK1 can block phosphorylation of AMPK alpha-subunit at Thr172 but phosphorylates Ser108 in the beta1-subunit (Figure 2A, Table S1).27

ULK1 and Beclin1 Beclin1, known as a mammalian ortholog of yeast Atg6, is a downstream physiological target of ULK1. It can be associated with vacuolar protein sorting 34 (VPS34) and VPS15 to form a complex, which serve as a binding partner of some proteins that are capable of regulating autophagy.28 In Beclin1-VPS34 complex I composed of Beclin1, VPS34, VPS15 and ATG14L, ATG14L can recruit ULK1 to Beclin1, promoting Beclin1 5



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phosphorylation. While in Beclin1-VPS34 complex II composed of Beclin1, VPS34, VPS15 and UV radiation resistance-associated gene protein (UVRAG), UVRAG binds a minority of the total Beclin1 to represent a fraction of the Beclin1-associated VPS34 activity.29 Under the conditions of mTORC1 inhibition or amino acid starvation, activation of ULK1 can phosphorylate Beclin1 on Ser14 (Mus musculus)/Ser15 (Homo sapiens), increasing the activity of the Beclin1-containing complex to meet the requirement for autophagic induction, which is also required and conserved in C. elegans.28 Moreover, SESN2 can interact with ULK1 and thus promoting ULK1-dependent phosphorylation of Beclin1 at Ser14, which is essential to bind with Parkin before mitochondrial translocation.30 Additionally, ULK1 can also phosphorylate ATG14 at Ser29 in a mTOR-dependent manner, thus resulting in an increasing ATG14-VPS34 lipid kinase activity to modulate autophagy.31 Interestingly, Beclin1 can also be phosphorylated at Ser30 by ULK1 to activate ATG14-VPS34 complex, which is completely independent of ULK1-mediated phosphorylation of Beclin1 and ATG14.32 Besides, AMBRA1 can be bound to dynein complex by binding with dynein light chain 1 (DLC1) in an inactive state. When autophagy induction does occur, AMBRA1 can be released from the dynein complex by the ULK1-mediated phosphorylation and translocate to the Endoplasmic reticulum (ER), together with Beclin1-VPS34 for the autophagosome formation (Figure 2B, Table S1).33

Other autophagic pathways modulated by ULK1 ULK1 phosphorylates ATG4B on Ser316 to disrupt ATG4B activity by affecting the ATG4B-LC3 complex formation.34 Folliculin (FLCN) complex can be regulated by ULK1, whose phosphorylation at Ser406, Ser537 and Ser542 are induced by the overexpression of ULK1. FLCN interacts with GABA(A) receptor-associated protein (GABARAP) which specifically promotes ULK kinase activation dependent on the ULK1 LC3-interacting region (LIR) motif. FLCN-GABARAP association can be modulated by either folliculin-interacting protein (FNIP)-1 or FNIP2, and thus being further modulated by ULK1.35 Autophagy-dependent stimulator of interferon genes (STING) is subsequently 6


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phosphorylated at Ser366 by ULK1 after its activation of transcription factors interferon regulatory factors 3 (IRF3) and nuclear factor kappa B (NF-κB), as well as its delivery of TANK-binding kinase 1 (TBK1) to endosomal/lysosomal compartments.36 ULK1 activation occurs following disassociation from its repressor AMPK, and it is elicited by cyclic dinucleotides (CDNs) generated by the cGAMP synthase.

36

The degradation of

Bcl-12/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) that can be induced by hypoxia inducible factor 1 (HIF-1) under hypoxia, needs ULK1 activation by AMPK directly.37 ULK1 can interact with FUN14 domain containing 1 (FUNDC1), phosphorylating it at Ser17, which may enhance the binding of FUNDC1 with LC3. The mutant of FUNDC1 lacking for binding ability with ULK1 can block the translocation of ULK1 to mitochondria and thus inhibiting mitophagy.38 Additionally, ULK1-mediated phosphorylation of Ser339 in the co-chaperone cell division cycle protein 37 (Cdc37), which is a ULK1 substrate, compromises the recruitment of client kinases to Cdc37 and heat shock protein 90 (Hsp90).39 Moreover, DENN domain-containing protein 3 (DENND3), as a guanine nucleotide exchange factor (GEF) for Rab12, is phosphorylated by ULK1 at Ser554 and Ser572 thereby activating Rab12 to facilitate autophagosome trafficking and induce autophagy (Figure 2B, Table S1).40 As mentioned above, these ULK1-regulated autophagic pathways, mainly involved in AMPK, mTORC1, and Beclin1 play their crucial roles in the autophagy process. Moreover, these pathways may further connect with each other to construct the autophagic signaling network, which has many links to many human diseases, such as cancer, neurodegenerative disease, infectious diseases in the following section.

ULK1 AND HUMAN DISEASES Of note, ULK1 can regulate its downregulated autophagic pathways that are closely involved in diverse human diseases, such as cancer, neurodegenerative disease, infectious diseases, diabetes and cardiovascular diseases. Accumulating evidence has recently revealed that ULK1 may be regarded as a novel potential drug target in the treatment of several diseases. Thus, in this section, we summarize the relationships 7



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between ULK1 and different diseases, which may help exploiting the complicated autophagic mechanisms of ULK1 in human diseases.

ULK1 in cancer Downregulation of ULK1 has recently been found in several types of cancer tissues. Under this circumstance, the activation of ULK1-regulated autophagic cell death would be a promising therapeutic strategy. For instance, the reduced expression of ULK1 is found to be associated with tumor progression, together with reducing autophagy, suggesting that ULK1 is a new prognostic factor in breast cancer.41 Recently, ULK1 has been found to be downregulated in breast cancer based upon the cancer genome atlas (TCGA) and tissue microarray (TMA) analyses, which is particularly remarkable in triple-negative breast cancer (TNBC).6, 42 On the other hand, upregulation of ULK1 was also discovered in other types of cancer tissues; therefore, the inhibition of ULK1-regulated cytoprotective autophagy would be another promising therapeutic strategy. For example, AMPK (p-Thr172)-ULK1 (p-Ser555) pathway is shown to be related with the bromodomain and extra-terminal domain (BET) inhibitor JQ1-induced autophagy in leukemia stem cells (LSCs) resistant to the BET inhibitors, which reveals that pro-survival autophagy is a potential mechanism in resistance of acute myeloid leukemia (AML) and LSCs to JQ1.43 ULK1, combined with LC3B, would improve prognosis assessment of the patients, which were demonstrated to be an important prognostic factor for hepatocellular carcinoma (HCC).44 High expression of ULK1 may be served as a useful marker for short biochemical progression (BCP)-free survival and overall survival in patients with metastatic prostate cancer (PCa) after androgen deprivation therapy (ADT).45 ULK1 activity is also found to be required for type I IFN-induced anti-proliferative responses and anti-neoplastic effects on myeloproliferative neoplasms.20 According to the Janus role of autophagy (autophagic cell death or cytoprotective autophagy) in cancer, activation or inhibition of ULK1-modulated autophagy should be used in specific tumor types, respectively (Figure 3).

8


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ULK1 in neurodegenerative diseases Currently, ULK1 has been demonstrated to be a potential new target in neurodegenerative diseases, such as Huntington's disease (HD) and Parkinson’s disease (PD). ULK1 is reported to inhibit p70S6K under the condition of starvation, and further elucidating ULK1 and its target microRNAs miR-595 and miR-4487 as potential new biomarkers in PD.46 The phosphorylation of ATG14 induced by ULK1 is at Ser29 in an mTORC1-dependent manner, which regulates ATG14-Vps34 lipid kinase activity for regulation of autophagy in HD. ULK1-dependent phosphorylation of Beclin1 and ATG14, as well as ATG14-associated Vps34 activity are compromised in the Gln175 knock-in HD mouse models. The phosphorylation of ATG14 is downregulated during proteasomal inhibition-dependent general proteotoxic stress, and this reduction is partly regulated by p62-mediated segregation of ULK1 to undissolving cellular fraction.31 In addition, ULK1 influences neuronal cell viability, and enhances p70S6K phosphorylation at Thr389, leading to the reduction of MN9D viability under MPP+ (1-methyl-4-phenylpyridium iodide) treatment.47 ULK1-related autophagy can be regulated by a chromosome 9 open reading frame 72 (C9ORF72)/Smith-Magenis syndrome chromosomal region candidate gene 8 (SMCR8)-containing complex, which is identified in both amyotrophic lateral sclerosis (ALS)

and frontotemporal dementia (FTD) (Figure 3).48,49 As mentioned above,

activation of ULK1-regulated cytoprotective autophagy would be a promising therapeutic strategy in neurodegenerative diseases.

ULK1 in infectious diseases Hitherto, several studies have demonstrated that ULK1 can modulate its regulatory autophagic pathways in some infectious diseases. For instance, ULK1 is a determinant in parasite killing process which is induced by genetic or pharmacologic inhibition of FAK and epidermal growth factor receptor (EGFR).50 During prion infection, activation of the AMPK-ULK1 signaling pathway is beneficial for induction of autophagy which may be involved in the clearance of damage factors in prion-infected brain tissues.51 Phosphorylation of ULK1 mediated by human immunity-related GTPase M (IRGM) is 9



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triggered by hepatitis C virus (HCV).52 Annexin A2 (AnxA2) can regulate autophagy which contributes to host immunity against pseudomonas aeruginosa via AKT1-mTOR-ULK1 axis.53 Human immunodeficiency virus type-1 (HIV) release can be decreased by histone deacetylase (HDAC) inhibitors via the ULK1-involved mechanism.54 The ULK1-dependent mitophagy is involved in protection against immunopathology triggered by influenza A virus.55 In addition, ULK1 is required in the formation of Brucella-containing vacuole (BCV) conversion into the autophagic features (aBCV) (Figure 3).56 According to the aforementioned studies, ULK1-modulated autophagy plays a cytoprotective role in such infectious diseases; however, targeting ULK1-regulated

autophagy could be a new

approach for future potential therapeutics.

ULK1 in diabetes and cardiovascular diseases Interestingly, ULK1-modulated autophagy is also reported in diabetes and cardiovascular diseases. For instance, ULK1-mediated autophagy cascade is involved in activation of the NLR pyrin domain containing 3 (NLRP3)-apoptotic speck protein containing a caspase recruitment domain (ASC), related to type 2 diabetes (T2D).57 Impaired AMPK/ULK1 signaling can suppress autophagic activation in proximal tubules, which underlies T2DM-induced worsening of renal ischemia/reperfusion (I/R) injury.58 In cardiovascular diseases, reduced activation of ULK1 is associated with the therapeutic effect of food restriction, an autophagic inducer on post-infarction cardiovascular dysfunction.59 The ULK1 inactivation induced by increased mTOR partly can mediate protective autophagy in high glucose-induced cardiomyocyte injury (Figure 3).60 As mentioned above, the roles of ULK1 seem to be complicated in both diabetes and cardiovascular diseases; therefore, the strategy targeting ULK1-modulated autophagy should be further explored. In summary, these findings would shed new light on exploiting the intricate mechanisms of autophagy in human diseases. More importantly, it may provide a basic for discovery of more relevant small-molecule compounds targeting ULK1 for further potential therapies. 10


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TARGETING ULK1 IN AUTOPHAGY FOR POTENTIAL THERAPIES Hitherto, a number of small molecules have been identified to regulate ULK1/ULK1-mediated

autophagic

pathways,

which

is

of

great

benefit

to

autophagy-related therapies. Depending on the differentiations of expression level and specific regulations of ULK1 in diseases, such compounds can be further divided into two types, inhibitors or activators of ULK1, which can down-regulate or up-regulate ULK1 to exert its distinctive function for potential therapeutic implications.

ULK1 inhibitors Recently, compound 6 (1), known as a ULK1 inhibitor, has been screened out using a standard 32P-ATP radioactive assay with MBP as the substrate, with a high affinity (IC50 = 8 nM).7 Unfortunately, the non-specificity of compound 6 toward ULK1 in autophagy restricted its usage in follow-up study. To further improve the selectivity for ULK1, a novel inhibitor scaffold of ULK1 was explored. Compound 3 (2) was synthesized as a less potent inhibitor of ULK1 (IC50 = 120 nM), although it showed good selectivity throughout the kinome.61 In addition, SBI-0206965 (3), as an FAK inhibitor, was also demonstrated to be highly selective ULK1 (IC50 = 108 nM) and ULK2 (IC50 = 711 nM) inhibitor. SBI-0206965 can suppress ULK1-mediated phosphorylation of VPS34 to regulate autophagy. Notably, SBI-0206965 can synergize with the mTOR inhibitors to kill tumor cells, as a rationale for their combined use in clinic.62 Using above-mentioned biochemical assay, another research group found MRT67307 (4), a TBK1 inhibitor, potently inhibited both ULK1 (IC50 = 45 nM) and ULK2 (IC50 = 38 nM). MRT68921 (5), as an analog of MRT67307, processed tremendously increased affinity for ULK1 (IC50 = 2.9 nM) and ULK2 (IC50 = 1.1 nM). However, MRT67307 and MRT68921 also showed non-specificity toward ULK1. In addition, MRT68921 can inhibit autophagosome formation in ULK1/2 double-knockout MEF cells co-incubated with recombinant wild-type ULK1, indicating that MRT68921 inhibits autophagy in an ULK1-dependent manner.63 More recently, indazole-derived ULK1 inhibitors were identified by performing an in silico HTS campaign 11



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and structural modifications. Compound 3g (6) showed potent affinity against ULK1 (IC50 = 45 nM), which is highly equivalent to MRT67307. Under the in vitro drug metabolism assessment, compound 3g exhibited excellent stability in microsomes of human, rat, and mouse, as well as negligible CYP inhibition activity.64 Besides those targeted ULK1 inhibitors, WP1130 (7) was found to inhibit deubiquitinases, leading to the increase of ULK1 ubiquitination, the transfer of ULK1 to aggresomes, as well as the inhibition of ULK1 activity, which can inhibit the ULK complex and autophagy.65

ULK1 activators Recently, a small-molecule compound LYN-1604 (8), has been reported as the first ULK1 activator designed based upon the crystal structure of ULK1 KD. LYN-1604 possessed a high affinity for ULK1 with an EC50 of 18.94 nM, which is determined by ADP-Glo kinase assay. Based on site-directed mutagenesis and in vitro ULK1 kinase assay, Lys50 together with Leu53 and Tyr89, was identified to be crucial for the binding of ULK1. LYN-1604 also exhibited good anti-proliferative activity against breast cancer cells, especially in MDA-MB-231 cells (IC50 = 1.66 µM). It was able to induce cancer cell death, associated with autophagy by the ULK complex and showed a good therapeutic effect on TNBC in vivo, suggesting the importance of the combination of multidisciplinary in discovering novel potential ULK1 modulators in the future.42 Further, other small-molecule compounds have also been reported to modulate the ULK1-mediated autophagic signaling pathways. For instance, Temozolomide (9) can induce autophagy via the ATM AMPK ULK1

axis

for

the

treatment

of

O6 methylguanine

DNA

methyltransferase negative gliomas.66 Tetrandrine (10) decreases human oral cancer SAS cells viability via induction of autophagic cell death by augmentation of p-ULK1 and p-mTOR levels.67 Baicalein (11) is reported to induce autophagic cell death by the activation of AMPK-ULK1 pathway, as well as by the inhibition of mTORC1 in breast carcinoma MDA-MB-231 cells.68 As mentioned above, these small-molecule compounds that directly or indirectly target ULK1 or ULK1-modulating autophagy are summarized herein (Table 1). 12


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CONCLUSIONS UNC-51-like kinase 1 (ULK1), known as an ortholog of the yeast Atg1, is the serine-threonine kinase and the autophagic initiator in mammals. The ULK complex, which consists of ULK1, mATG13, FIP200 and ATG101, is required to initiate the autophagy process. It is well-known that ULK1 can be regulated by some specific stimulations such as homeostasis changes or pharmacological intervention. Although the original biological function of ULK1 is to initiate autophagy, regulations and modifications (mainly referring to phosphorylation) of ULK1 can turn itself into a powerful

regulator

to

function

in

various

pivotal

signaling

pathways

(e.g.,

AMPK-mTOR-ULK1 axis), and thus participating in treatment of several diseases, such as cancer and neurodegenerative diseases. Accumulating evidence has recently indicated the feasibility of targeting ULK1 by some specific small molecules such as LYN-1604, a ULK1 activator that induces ULK1-modulating autophagy-associated cell death in TNBC, suggesting its therapeutic potential by targeting ULK1. With rapid progress of ULK1 function and application, targeting ULK1 or ULK1-modulated autophagy would be a promising therapeutic strategy in the near future. However, some questions have remained unsolved in the coming several years, especially the resolution of full-length crystal structure of ULK1 and even the ULK complex in human. If they do so, more precise targeted ULK1 activators or inhibitors would be discovered and further utilized in the treatment of human diseases. In addition to some canonical ULK1-mediated autophagic pathways, a better understanding of other noncanonical pathways of ULK1 would also provide a possible avenue for further targeted therapies. Currently, a new hope of utilizing ULK1 for targeted therapies may lie in discovering some candidate small-molecule drugs for modulating ULK1-regulated autophagic pathways and even the whole autophagic network. Taken together, elucidation of ULK1, known as the autophagic initiator, would shed light on exploring this kinase as an omnipotent drug target for future therapeutic applications.

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AUTHOR INFORMATION Corresponding Authors *B.L. E-mail: [email protected]. Phone: (+86)28-85164063. Author Contributions #

These authors contributed equally.

Notes The authors declare no competing ﬁnancial interest. Biographies Lan Zhang received his Ph.D. degree in medicinal chemistry from Shenyang Pharmaceutical University in 2015. Then, he joined the faculty of State Key Lab of Biotherapy, West China Hospital, Sichuan University as an assistant research fellow. His current research is focused on the discovery of small-molecule drugs targeting ULK1. Liang Ouyang received his Ph.D. degree in medicinal chemistry from West China School of Pharmacy, Sichuan University in 2010. He began his academic career in 2012 at the State Key Lab of Biotherapy, West China Hospital, Sichuan University as an associate professor. His research interests include identification of novel drug targets in cell death, and structure-based discovery of small-molecule and peptide drugs. Yongzhi Guo is a Ph.D. candidate student at the State Key Lab of Biotherapy, Sichuan University. And now he is studying the autophagic mechanisms of triple negative breast cancer under the guidance of Dr. Bo Liu. Jin Zhang received her Ph.D. degree in medicinal chemistry from Shenyang Pharmaceutical University in 2017. Then, she joined the faculty of State Key Lab of Biotherapy, West China Hospital, Sichuan University as an assistant research fellow. Her current research is focused on the discovery of small-molecule drugs targeting autophagy-related genes. Bo Liu received his Ph.D. degree in Bioinformatics (Drug design) from School of Life Sciences, Sichuan University in 2010. In 2012, he joined the faculty of State Key Lab of Biotherapy, West China Hospital, Sichuan University as an associate professor. His research interests include identification of novel autophagic targets and structure-based 14


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design and discovery of small-molecule candidate drugs in human diseases.

ACKNOWLEDGEMENTS We are grateful to Prof. Shengyong Yang and Prof. Canhua Huang (Sichuan University) for their critical reviews on this manuscript. This work was supported in part by grants from National Key R&D Program of China (Grant No. 2017YFC0909301 and Grant No. 2017YFC0909302) and National Natural Science Foundation of China (Grant No. 81473091, Grant No. 81673290, Grant No. 81673455 and Grant No. 81602953).

ABBREVIATIONS USED ULK1, UNC-51-like kinase 1; Atg, autophagy-related gene; FIP200, focal adhesion kinase family interacting protein of 200KDa; ORF, open reading frame; CTD, C-terminal domain; KD, kinase domain; MIT domain, microtubule-interacting and transport domain; PS region, proline/serine-rich region; PKA, protein kinase A; ATP, adenosine triphosphate; mTORC1, mammalian target of rapamycin complex 1; AMPK, AMP-activated protein kinase; PPM1D, protein phosphatase magnesium-dependent delta isoform; IFNR, interferon receptor; KLHL20, kelch-like protein 20; AMBRA1, autophagy and beclin1 regulator 1; NEDLL4, neural

precursor

cell-expressed

developmentally

down-regulated

4-like;

TIP60,

at-interaction protein, 60ku; GSK3β, glycogen synthase kinase 3β; VPS34, vacuolar protein sorting 34; UVRAG, UV radiation resistance-associated gene protein; DLC1, dynein light chain 1; FLCN, folliculin; GABARAP, GABA(A) receptor-associated protein; LIR, LC3-interacting region; FNIP, folliculin-interacting protein; STING, stimulator of interferon genes; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factors 3; NF-κB, nuclear factor kappa B; CDNs, cyclic dinucleotides; BNIP3, Bcl-12/adenovirus E1B 19 kDa interacting protein 3; HIF-1, hypoxia inducible factor 1; FUNDC1, FUN14 domain containing 1; Cdc37, cell division cycle protein 37; Hsp90, heat shock protein 90; DENND3, DENN domain-containing protein 3; GEF, guanine nucleotide exchange factor; TCGA, the cancer genome atlas; TMA, tissue microarray; TNBC, triple-negative breast

15



Page 16 of 33

cancer; BET, bromodomain and extra-terminal domain; LSCs, leukemia stem cells; AML, acute myeloid leukemia; HCC, hepatocellular carcinoma; BCP, biochemical progression; PCa, prostate cancer; ADT, androgen deprivation therapy; PD, Parkinson’s disease; HD, Huntington's disease; C9ORF72, chromosome 9 open reading frame 72; SMCR8, Smith-Magenis syndrome chromosomal region candidate gene 8; FTD, frontotemporal dementia; ALS, amyotrophic lateral sclerosis; EGFR, epidermal growth factor receptor; IRGM, immunity-related GTPase M; HCV, hepatitis C virus; AnxA2, Annexin A2; HIV, human

immunodeficiency

virus

type-1;

HDAC,

histone

deacetylase;

BCV,

Brucella-containing vacuole; NLRP3, NLR pyrin domain containing 3; ASC, apoptotic speck protein containing a caspase recruitment domain; T2D, type 2 diabetes; I/R, ischemia/reperfusion.

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Small molecule inhibition of the autophagy kinase ULK1 and identification of ULK1 substrates. Mol. Cell 2015, 59, 285-297. (63) Petherick, K. J.; Conway, O. J.; Mpamhanga, C.; Osborne, S. A.; Kamal, A.; Saxty, B.; Ganley, I. G. Pharmacological inhibition of ULK1 kinase blocks mammalian target of rapamycin (mTOR)-dependent autophagy. J. Biol. Chem. 2015, 290, 11376-11383. (64) Wood, S. D.; Grant, W.; Adrados, I.; Choi, J. Y.; Alburger, J. M.; Duckett, D. R.; Roush, W. R. In silico HTS and structure based optimization of indazole-derived ULK1 inhibitors. ACS Med. Chem. Lett. 2017, 8, 1258-1263. (65) Drießen, S.; Berleth, N .; Friesen, O.; Löffler, A. S.; Böhler, P.; Hieke, N.; Stork, B. Deubiquitinase inhibition by WP1130 leads to ULK1 aggregation and blockade of autophagy. Autophagy 2015, 11, 1458-1470. (66) Zou, Y. H.; Wang, Q.; Li, B. L.; Xie, B.; Wang, W. M. Temozolomide induces autophagy via ATM AMPK ULK1 pathways in glioma. Mol. Med. Rep. 2014, 10, 411-416. (67) Huang, A. C.; Lien, J. C.; Lin, M. W.; Yang, J. S.; Wu, P. P.; Chang, S. J.; Lai, T. Y. Tetrandrine induces cell death in SAS human oral cancer cells through caspase activation-dependent apoptosis and LC3-I and LC3-II activation-dependent autophagy. Int. J. Oncol. 2013, 43, 485-494. (68) Aryal, P.; Kim, K.; Park, P. H.; Ham, S.; Cho, J.; Song, K. Baicalein induces autophagic cell death through AMPK/ULK1 activation and downregulation of mTORC1 complex components in human cancer cells. FEBS. J. 2014, 281, 4644-4658.

Table of Contents graphic

23



Page 24 of 33

Figure Legends

Figure 1. The structure and activation of ULK1. Domain structure of ULK1 is composed of the respective domains, including kinase domain (KD), proline/serine-rich (PS) region and C-terminal domain (CTD) containing MIT1/2. The upstream kinases AMPK and mTORC1 are shown above ULK1, which may play the crucial roles in regulating ULK1 by phosphorylation (amino acid sites are based upon sequence in mouse). ULK1 can be activated by several stress signals (shown on the right-hand side), such as amino acid/serum starvation, low energy and glucose deprivation. Some small-molecule compounds targeting ULK1 are listed on the left-hand side, including LYN-1604 as a ULK1 activator as well as compound 6, SBI-0206965 and MRT68921 as ULK1 inhibitors.6-12,15-18, 42,62,63

24


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Figure 2. Post-translational modifications of the ULK complex and autophagic pathways regulated by ULK1. (A) ULK1 can form a quaternary complex with mATG13, FIP200 and ATG101 to be the center of the whole ULK1-related regulation network. Upstream signaling such as AMPK and mTORC1 regulate the ULK complex via phosphorylation, ubiquitylation and acetylation in post-transcriptional modification processes. For instance, ULK1 can be activated by AMPK phosphorylation at Ser317, Thr574, Ser467, Ser555, Ser637 and Ser777, or directly inhibited by mTORC1 phosphorylation at Ser637 and Ser757. (B) ULK1 can transmit signals received from upstream to downstream pathways, notably, Beclin1 pathway (composed of Beclin1, VPS34, ATG14L and VPS15) by phosphorylation of ATG14L at Ser29, as well as phosphorylation of Beclin1 at Ser30 and Ser14, 25



Page 26 of 33

respectively.4,15-19,21-28,31-32,34-36,38-40

Figure 3. ULK1-regulated autophagic pathways in diverse human diseases. ULK1 (shown as the crystal structure of ULK1 KD, PDB code: 4WNP7) has been widely reported to be involved in a number of autophagic pathways in diverse diseases, such as cancer

(TNBC,

Pca,

Acute

Myeloid

Leukemia,

Myeloproliferative

Neoplasms),

neurodegenerative diseases (PD, FTD, ALS), infectious diseases (Prion Infection, Pseudomonas Aeruginosa Infection, Parasite, Influenza A Virus, HCV, HIV), diabetes (Type 2 Diabetes, T2DM-I/R) and cardiovascular diseases (Cardiomyocyte Injury).

6,20,

42,43,45-55,57,58,60

26


Page 27 of 33 1 2 3 4 5 6 7 8 9 10 Compound 11 12 13 1 14 15 (compound 6) 16 17 18 19 20 21 2 22 23 (compound 3) 24 25 26 27 28 29 30 3 31 (SBI-0206965) 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Table 1. Small-molecule compounds targeting ULK1/ULK1-mediated autophagy Function

ULK1 inhibitor

ULK1/2 inhibitor

ULK1/2 inhibitor

Chemical Structure

Target/Pathways

Biological Activity

Cell Type

Disease

Reference

ULK1

ULK1 (IC50 = 8 nM)

-

-

7

ULK1/ULK2

ULK1 (IC50 = 120 nM) ULK2 (IC50 = 360 nM)

-

-

61

ULK1/ULK2

ULK1 (IC50 = 108 nM) ULK2 (IC50 = 711 nM)

U87MG cells, Mouse KrasG12D/p53-/NSCLC cells, A549 cells

Lung cancer

63

27


Journal of Medicinal Chemistry 1 2 3 4 5 6 7 4 8 9 (MRT67307) 10 11 12 13 5 14 15 (MRT68921) 16 17 18 19 20 21 6 22 23 (compound 3g) 24 25 26 27 28 7 29 (WP1130) 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 28 of 33

ULK1/ULK2

ULK1 (IC50 = 45 nM) ULK2 (IC50 = 38 nM)

Mouse Embryonic Fibroblast (MEFs)

-

63

ULK1/2 inhibitor

ULK1/ULK2

ULK1 (IC50 = 2.9 nM) ULK2 (IC50 = 1.1 nM)

Mouse Embryonic Fibroblast (MEFs)

-

63

ULK1 inhibitor

ULK1

ULK1 (IC50 = 45 nM)

-

-

64

-

HEK293 cells, HeLa cells, U2OS cells

Cervical cancer and osteosarcoma

65

ULK1/2 inhibitor

Autophagy inhibitor

ULK1, USP9X

28


Page 29 of 33 1 2 3 4 5 6 7 8 9 10 8 11 (LYN-1604) 12 13 14 15 16 17 9 18 19 (Temozolomide) 20 21 22 23 24 25 10 26 (Tetrandrine) 27 28 29 30 31 32 33 34 11 35 (Baicalein) 36 37 38 39 40 41 42 43 44 45 46 47


ULK1 activator

ULK1

ULK1 (EC50 = 18.94 nM) MDA-MB-231 (IC50 = 1.66 µM)

Autophagy inducer

ATM-AMPK-ULK1 pathways

-

U87MG cells, U251 cells

Glioma

66

Autophagy inducer

Caspase-dependent and LC3-I and LC3-II-dependent pathways

-

Human oral cancer SAS cells

Oral cancer

67

ULK1-related pathway activator

AMPK/ULK1 pathway

-

PC-3 cells, MDA-MB-231 cells

Colorectal and breast cancer

68

MDA-MB-231 cells

Triple-negative breast cancer

42

29


Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

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“-“ indicates relevant information is not provided or determined in the reference.

30


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164x69mm (300 x 300 DPI)



473x486mm (300 x 300 DPI)


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559x376mm (200 x 200 DPI)


From an autophagic initiator to multi-functional drug target

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