Autophagy, ubiquitous in eukaryotic cells (from yeast to mammals), is an antioxidant response mechanism in cells under oxidative stress and a lysosomal degradation process relatively conserved throughout evolution [1]. It counteracts adverse effects caused by oxidative stress, playing a significant role in cell protection. Previous studies have shown that apoptosis can be inhibited by inducing autophagy. In contrast, inhibition of autophagy may promote apoptosis and inhibit tumor growth [2], [3]. Under intracellular and extracellular stress conditions, the autophagy rate is increased to maintain the survival and proliferation of tumor cells, thereby facilitating tumor growth and tumor cell invasion, metastasis and diffusion [4]. In addition, autophagy attenuates cell damage by eliminating potential toxic substances and increasing cell adaptability [5]. Therefore, autophagy is essential for maintaining cell homeostasis, playing a crucial role in the pathophysiological processes of anti-aging, cell differentiation and development, immunity and elimination of microorganisms and tumors [6], [7].

Autophagy is generally classified into three major types (Fig. 1): macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) [8], [9]. Macroautophagy (referred to hereafter as autophagy) is characterized by the formation of autophagosomes (Fig. 1A). Microautophagy is initiated by lysosomal invagination, and no independent double-membrane autophagosome is formed (Fig. 1B). In contrast to the other two types of autophagy, CMA is not associated with vesicles (Fig. 1C). CMA shows certain selectivity, and the chaperone HSPA8 is often used to specifically degrade target proteins with a unique recognition pentapeptide motif (KFERQ-like) [10]. The lysosome-associated membrane protein 2A (LAMP2A) receptor identifies an exposed KFERQ sequence and binds it, guiding the motif-carrying protein as cargo to lysosomes [11].

Similar to most cellular events, the whole process of autophagy is regulated by autophagy-related genes (ATGs) and autophagy-related proteins. To date, approximately 41 ATG proteins in yeast cells have been discovered, and most of these proteins are evolutionarily conserved [12], [13]. The homologs of many of the genes encoding these proteins have been described in higher eukaryotes; these genes include Beclin1 (BECN1), Vacuolar Protein-Sorting 34 (Vps34), ultraviolet irradiation resistance-associated gene (UVRAG), Vacuole membrane protein 1 (VMP1), and Human tumor protein p53 inducible nucleoprotein 2 (TP53INP2) [14].

The function and dysfunction of these ATGs and proteins are closely related to the occurrence and development of various human diseases, including neurodegenerative diseases, cardiomyopathy, infectious diseases, Type 2 diabetes, fatty liver disease, and cancer [15], [16]. Moreover, the many related regulatory proteins in the autophagy pathway provide a way to positively and negatively affect autophagy. At present, the development of autophagy inhibitors used in cancer therapy mainly focuses on lysosomal inhibitors, such as chloroquine (CQ) and HCQ, and ATG proteins that are essential for autophagy, including Unc51-like kinase 1 (ULK1), ATG4b and Vps34 [17]. ULK1 is a serine/threonine protein kinase that is essential for autophagy initiation [18]. MRT67307 and MRT68921 effectively inhibit ULK1 and block autophagy in cells in vitro [19]. SBI-0206965 also selectively inhibits ULK1/2 (ULK1/2 IC50 = 108/711 nM). FMK-9a, a covalent ATG4b inhibitor with an IC50 of 80 nM, shows excellent inhibition efficiency on ATG4b in vitro [20]. However, an important unresolved problem encountered in the development of potent autophagy inhibitors is the inability to decide whether to block autophagy by interfering with early steps on the autophagy pathway to prevent autophagosome formation, by blocking the maturation stage, or by blocking the lysosomal degradation stage.

Among the key molecules involved in the initiation of autophagy, Vps34 is the only Class III lipid kinase responsible for the generation of phosphatidylinositol 3-phosphate (PI3P), which mediates the initiation of autophagosomal biological genes [21]. Therefore, small molecule inhibitors targeting Vps34 have the potential to regulate the autophagic processes. Notably, Vps34 plays an important role in heart and liver function, and complete Vps34 inhibition may lead to liver hypertrophy, hepatic steatosis, and cardiac hypertrophy in mammals [22], [23]. Therefore, it is important to identify new small-molecule Vps34 antagonists that offer new opportunities for drug discovery and provide new perspectives for understanding the molecular mechanisms underlying autophagy without triggering the cardiac and liver side effects described above.

Several inhibitors targeting Vps34 have been identified in recent years; however, a review that comprehensively summarize these inhibitors and discuss their structure–activity relationships is lacking. Thus, to promote the fruitful development of small-molecule autophagy inhibitors targeting Vps34, we comprehensively review the molecular mechanisms of autophagy and the structure–activity relationship of the known Vps34 inhibitors, and provide some potential therapeutic perspectives.

Autophagy, which degrades damaged or excess cellular components into basic biomolecules that are then recycled in the cytoplasm, is often considered a survival-promoting mechanism that protects cells under stressful conditions [24]. Macroautophagy is the main autophagy pathway. The formation of a typical autophagosome is evolutionarily conserved, and autophagy usually includes four critical steps: initiation, elongation, maturation and degradation.

Under the effect of autophagy-inducing signals (starvation, injury or metabolic stress), a large number of double-membrane compartments from the endoplasmic reticulum, mitochondria and other organelles are generated in the cytoplasm, and then, the double-membrane compartments extend on two sides to form autophagic vesicles [25]. ULK1 forms the ULK1 initiation complex with ATG13, ATG101 and FIP200, driving the formation of an autophagosome (Fig. 2A). Studies have demonstrated that multiple mechanisms are involved in the regulation of autophagy initiation, among which mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the autophagy of cells through the activation of ULK1 [26], [27]. Under the condition of adequate nutrition, mTORC1 phosphorylates ULK1 and ATG13 in the ULK1 complex, thereby inhibiting the elongation of the autophagy membrane. [26], [28], [29] When cells are starved or treated with mTOR inhibitors, the ULK1 complex dissociates from mTORC1, resulting in the dephosphorylation of ULK1- and ATG13-specific residues, which catalyzes the activation of the ULK1 protein and promotes the phosphorylation of other residues in ATG13 and FIP200, contributing to the initiation of autophagy [30], [31]. Therefore, mTORC1 may be associated with the activation of ULK1 catalytic activity. In addition, 5′-AMP-activated protein kinase (AMPK) is an important kinase involved in cellular energy sensing and signaling regulation during autophagy [32], [33].

Activation of Class III phosphoinositide 3-kinase (PI3K-III) promotes the formation of PI3P on the lipid bilayer membrane and then initiates autophagy [34]. The formation of Vps34 Complex 1(PIK3C3-C1) is also critical during the initial stage of nucleation/autophagosome formation (Fig. 2A) [35]. PIK3C3-C1 is composed of Beclin1, Vps15, Vps34, ATG14L and Ambra1, among which Beclin1 is the mammalian homolog of yeast ATG6/Vps30 [36], [37], [38], [39], [40]. PIK3C3-C1 is responsible for producing PI3P by activating the PI3K-III protein, thereby recruiting effectors such as WD repeat domain phosphoinositide-interacting protein (WIPI), which interacts with phosphatidylinositol, and double FYVE-containing protein 1 (DFCP1), which is located in the isolation membrane. Because it contains two FYVE domains that can combine PI3P; thus, DFCP1 is often used as a marker protein of early autophagosomes [41], [42].

As a key regulator of autophagy and endocytosis sorting, the formation of Vps34 Complex I involves multiple autophagy-related proteins. Initially, Vps34 is activated by binding to Vps15, and the pair then binds Beclin1 to form the Vps34-Vps15-Beclin1 complex. Then, ULK1 is phosphorylated to form Ambra1, which interacts with the Vps34-Vps15-Beclin1 complex on microtubules to form PIK3C3-C1. PIK3C3-C1 is released from microtubules and transported to the endoplasmic reticulum, the main site of autophagosome formation [43], [44]. During the nucleation of autophagosomes, PIK3C3-C1 phosphorylates phosphatidylinositol (PI) to generate PI3P, which is crucial for autophagy vesicle membrane extension and ATG protein recruitment of autophagy vesicles [34], [45]. Moreover, the Beclin1/PI3K-III complex interacts with DFCP1, ATG2 and WIPI and recruits other ATG proteins involved in membrane elongation [46]. When the Beclin1/PI3K-III complex combines with other regulatory proteins, it can selectively participate in different stages of autophagy. For example, when binding to ATG14L, the Beclin1/PI3K-III complex participates in the formation of autophagy vesicles; when it combines with UVRAG, autophagic vesicles can undergo maturation and transportation [47], [48].

During extension, the autophagosome membrane continuously absorbs the components to be degraded in the cytoplasm and these components reside in the inner part of the membrane, and then, the forming spherical structure closes and is called an autophagosome. Two unique ubiquitin-like conjugation systems play important roles in this process (Fig. 2B): the ATG5-ATG12-ATG16 ubiquitin-like conjugation system and the microtubule-associated protein 1 light chain 3 (LC3) ubiquitin-like conjugation system [49], [50], [51]. The two conjugation systems work together to promote the extension of the autophagosome membrane [52].

When autophagosome formation is completed, LC3-II attached to PE on the outer membrane is cleaved by Atg4 and released back into the cytoplasm [50]. Autophagosomes fuse with lysosomes through the action of the microtubule skeleton induced by of Endosomal Sorting Complex Required for Transport (ESCRT) and Rab GTPases (Rabs), which configure autolysosomes [53]. The fusion between autophagosomes and lysosomes also requires the coparticipation of lysosome membrane protein LAMP-1 and the small GTP-binding protein Rab7 [54], [55], [56]. Other lysosome-associated proteins involved in the maturation process include LAMP2 and UVRAG [47], [56].

After the formation of autolysosomes, a series of acid hydrolases degrade of the isolated cytoplasmic substances in autolysosomes (Fig. 1A) [57]. The small molecules generated by degradation, especially amino acids, are transported back into the cytoplasm, where they are recycled through for protein synthesis, thereby maintaining basic cell functions under starvation conditions.

Vps34 is a member of the PIK kinase family, which is categorized into Class I, Class II and Class III PIK kinases, including p110α, p110β, p110δ, p110γ, PI3K-C2α, PI3K-C2β, PI3K-C2γ and Vps34 [58]. The core catalytic structure of Vps34 (PDB ID: 2X6H), consisting of a helical solenoid domain that forms an interface with a bilobal catalytic domain, includes the C2 domain, a helical domain and a PI3K catalytic domain (Fig. 3A) [58], [59]. The C-terminal helix is critical for Vps34 to maintain catalytic activity and is closely related to the substrate PI binding loop and catalytic domain. Furthermore, the C-terminal helix masks on the catalytic site in the enzyme in closed conformation, inhibiting lipid kinase activity in the absence of substrate (Fig. 3B). The deletion of residues or point mutations in the C-terminal helix significantly impairs lipid kinase activity in the presence of substrate but increases basic ATPase activity in the absence of substrate [59].

Vps34 forms two complexs, PIK3C3-C1 which is composed of Vps34-Vps15-Beclin1-Atg14L (Fig. 3C) and PIK3C3-C2 which is composed of Vps34-Vps15- Beclin1-Vps38 [60]. The variation in a single subunit between the two complexes renders them engaged in distinct cellular processes. PIK3C3-C1 is involved in the initiation of autophagy, while complex II is involved in endocytic pathways [61]. The discussion of complex II will be limited in this paper as the primary focus is on elucidating the role of Vps34 complex I in autophagy.

The kinase domain of Vps34, like other protein/lipid kinases, contains a P-loop, a hinge, and an activation loop (Fig. 3A). However, compared to that of other PI3Ks, the conformation of the ATP-binding site in Vps34 is uniquely narrow, which may be critical to the lack of effective Vps34 inhibitors [62], [63], [64]. Therefore, with the guidance of the Vps34 protein structure, a new generation of Vps34 inhibitors with high potency and selectivity as chemical tools and therapeutic agents will likely be identified and used to investigate the specific relationships between autophagy and tumorigenesis.

Vps34 is essential for autophagy initiation, and the Beclin-1/PI3K-III complex formed by Vps34 and Beclin1 is the key complex for autophagy initiation. In addition to participating in autophagy initiation, Vps34 engages in other cellular processes, including cell proliferation, apoptosis, endocytosis, intracellular vesicular transport, and LC3-associated phagocytosis (LAP) [65], [66], [67], [68].

Vps34 PI3K activity is coordinated via its associated protein chaperone, which plays a central role in autophagy. In addition to being involved in vesicle transport, Vps34 is involved in nutrient sensing and protein synthesis via the action of the mTOR pathway and in signal transduction downstream of G-protein-coupled receptors [69], [70]. However, studies have revealed that Vps34 promotes the occurrence and development of tumors by recruiting PKCδ to activate p62 through phosphorylation [71]. In MCF-7 cells, the transcription of p62 protein was regulated by Vps34. P62 is a substrate protein in the autophagy process, and its main function is to remove ubiquitinated proteins and misfolded proteins [72]. P62 itself is also degraded by the autophagy pathway.

Vps34 not only plays a key role in autophagy but can also regulate cancer cell growth by activating the MEK-ERK pathway [73]. Additionally, autophagy has been closely related to the regulation of T-cell activation and differentiation [74], [75]. When cellular metabolism is impaired, T cells fail to differentiate in Vps34-deficient T cells [76]. By using VPS34-IN1 (a selective inhibitor of Vps34), Vps34 inhibition exerted antileukemic activity and induced apoptosis in acute myeloid leukemia (AML) cells, but the viability of normal CD34 + hematopoietic cells was not affected, highlighting the potential targeting of Vps34 as a therapeutic treatment in AML [77].

In recent years, immune checkpoint blocking (ICBs) has been used to great success in the treatment of cancer. By combining immune checkpoint therapy with autophagy inhibitors, the prosurvival effect of autophagy on tumors may be eliminated [78]. Bassam Janji's group found that by reducing Vps34 protein levels in tumor cells or inhibiting Vps34 kinase activity, cold tumors were converted to hot inflamed tumors, thereby enhancing the efficacy of immune checkpoint antibodies against PD-1 and PD-L1 and increasing the efficacy of immunotherapy [79]. Knockdown of the Vps34 protein by utilizing short hairpin RNA (shRNA) in a variety of mouse tumor cells, such as melanoma tumor cells and colorectal tumor cells, effectively inhibited tumor growth and prolonged mouse survival. At the same time, the overall infiltration of immune cells into tumor tissues was significantly increased in a tumor immunity model mice with reduced Vps34 expression, and the infiltration of NK cells and CD4+ and CD8+ T cells, which are closely related to tumor immunity, was also increased [80]. Therefore, reducing the Vps34 protein level in tumor cells or inhibiting the activity of Vps34 kinase may increase the number of immune cells and enhance the tumor-killing effect of cytotoxic cells.

However, knocking down Vps34 protein levels or inhibiting Vps34 kinase activity with Vps34 inhibitors did not inhibit tumor growth in immunodeficient NOD-SCID-IL2rg(−/−) (NSG) mice [79]. In other words, the antitumor function of Vps34 must depend on the immune system. Similar results were obtained in melanoma cell lines, colorectal cancer cell lines, and renal cancer cell lines. These studies indicated that the combination of Vps34 inhibitors and immune checkpoint antibodies showed the real possibility of new targets being developed for the next generation of tumor immunotherapy drugs and the possibility that comprehensive treatment methods will be available for immune checkpoint therapy and prognosis, suggesting new strategies for further increasing the efficacy of immunotherapy.

The regulation of Vps34 activity involves the posttranslational modifications of acetylation, modification, transcription, and ubiquitination and miRNAs [81]. Under autophagy-promoting conditions, the Vps34 complex is activated by AMPK. Activated AMPK directly regulates Vps34 kinase function by phosphorylating Thr163 and Ser165 residues in the C2 domain of Vps34 (Fig. 3A), and AMPK phosphorylates the transcription factor FOXO3 at Ser555, Ser558 and Ser626 to regulate Vps34 transcription [82], [83]. Cofactors, such as Atg14L, UVRAG, Bif-1 and Ambra1, interact with Beclin1, which is a protein upstream of Vps34, to regulate Vps34 and promote the formation of the Beclin1-Vps34-Vps15 core complex, thereby promoting autophagy [14]. The formation of the Beclin1-Vps34-ATG14L complex is also promoted through SUMOylation of Vps34, which is mediated by small ubiquitin-related modifier 1 (SUMO1) [67], [84].

The acetyltransferase p300 specifically mediates the acetylation of Lys771 and Lys29, reducing the affinity of Vps34 for substrate PI and impeding the formation of the Vps34-Beclin1 core complex [85], [86]. During mitotic arrest caused by DNA-damaging agents, CDK1 negatively regulates the interaction between Vps34 and Beclin1 by phosphorylating Vps34 at Thr159. Vps34 is inhibited by K48-induced ubiquitination [87]. Under conditions of autophagy induction, the E3 ubiquitin ligase NEDD4/NEDD4-1 inhibits the K48-linked ubiquitination of Vps34 by recruiting USP13, thereby stabilizing the Vps34 protein [88]. Furthermore, miR-340-5p binds directly and inhibits the expression of Vps34 in esophageal squamous cell carcinoma (ESCC) [89].