A review on cullin neddylation and strategies to identify its inhibitors for cancer therapy

Abstract

The cullin-RING E3 ligases (CRLs) are the biggest components of the E3 ubiquitin ligase protein family, and they represent an essential role in various diseases that occur because of abnormal activation, particularly in tumors development. Regulation of CRLs needs neddylation, a post-translational modification involving an enzymatic cascade that transfers small, ubiquitin-like NEDD8 protein to CRLs. Many previous studies have confirmed neddylation as an enticing target for anticancer drug discoveries, and few recent studies have also found a significant increase in advancement in protein neddylation, including preclinical and clinical target validation to discover the neddylation inhibitor compound. In the present review, we first presented briefly the essence of CRLs' neddylation and its control, systematic analysis of CRLs, followed by the description of a few recorded chemical inhibitors of CRLs neddylation enzymes with recent examples of preclinical and clinical targets. We have also listed various structure-based pointing of protein–protein dealings in the CRLs' neddylation reaction, and last, the methods available to discover new inhibitors of neddylation are elaborated. This review will offer a concentrated, up-to-date, and detailed description of the discovery of neddylation inhibitors.

Introduction

The protein concentration inside the cells must be maintained up to a certain level to preserve the homeostatic conditions, like regulation of gene expression and cell cycle progression (Yu et al. 2020). As the central dogma is a very slow process, therefore protein degeneration leads to a fast and non-reversible condition that diminishes various important regulatory proteins within the cells (Kordbacheh and Kasko 2021). Many of these proteins are the key regulators of various transcription factors. Thus, in response to external stimuli, the rapid turnover of these proteins is needed (Ross et al. 2021). In eukaryotes, the major procedure involved in the degradation of proteins is the ubiquitin protostomes system (UPS) which is involved in ATP-dependent sequential reactions comprising three significant stages of peptide modification along with large chains of poly-ubiquitin molecules that target proteins for further processing via 26S proteasome (Paccosi and Proietti-De-Santis 2021). The reaction is called ubiquitylation which depends on three main enzymes including the E1 activating enzymes, E2 conjugating enzymes, and the E3 ligases for successive transmission of the ubiquitin molecule towards the substrate proteins (Zhao et al. 2014). The importance of a protein combined through a solo chain of ubiquitin or polyubiquitin can vary, relating to various biological activities such as endocytosis, cell signaling, immune response, DNA replication, as well as DNA damage and repair events. Therefore, the dysregulation of UPS can cause several disorders, including various forms of cancers, neurodegenerative disorders, and several metabolic syndromes (Zhou et al. 2021). The UPS control destruction of various proteins, including key mediators of approximately 12 essential signaling cascades besides fundamental regulators of transcription and cell cycle progression (Zou and Zhang 2021). The E3 ligases also named E3 ubiquitin ligase (E3UL) are those multi-protein complex molecules among UPS, whose specificity relay on mostly post-translational modifications via many factors including the ubiquitin-like molecule called neural-precursor-cell-expressed developmentally down-regulated gene 8 (NEDD8). Recently, the NEDD8 conjugation pathway known as neddylation has been proved as a target for the development of new anticancer drugs (Wang et al. 2011). The best-known function of NEDD8 is the activation of E3UL, especially the cullin-RING Ligases (CRLs) (Baek et al. 2020). According to some previous studies, some suggest that CRLs substrates have tumor suppressor characters due to their involvement in DNA damage, cell cycle progression, and cellular stress response (Liu et al. 2009). The neddylation stimulates CRLs for ubiquitylation and acts on certain tumorigenic-suppressive substrates (Zhao et al. 2014). Pointing neddylation in the cancer cells leads to the inactivation of CRLs due to which accumulation of tumor suppresser substrates is prohibited. Thus, a powerful chemotherapeutic strategy can be established by targeting the neddylation pathway (Li et al. 2021a, b). It has been discovered for a long time that the inhibitors of the proteasome can induce apoptosis in human cancer cells. Several studies have also authenticated that UPS is a capable antitumor target, ultimately conducting approval of a landmark proteasome inhibitor bortezomib (Velcade), by the US Food and Drug Administration (FDA), for the handling of relapsed mantle cell lymphoma (MCL) and multiple myeloma (MM) tumors (Yu et al. 2020). In 2009, the finding of a NEDD8 activating enzyme (NAE) blocker MLN4924 (Pevonedistat) and accompanying clinical testing set a breakthrough which proved the neddylation as a dynamic pathway for anticancer drug discovery (Schulman and Harper 2009). The continuous efforts have been carried out thereafter to identify new drugs as neddylation inhibitors from which some drugs have shown very capable anticancer properties (Li et al. 2021a, b). In this article, we describe the structural design of NEDD8 protein, neddylation pathway, the systematic analysis of CRLs, and targeting the neddylation pathway for cancer therapy. Then we discuss current progress in controlling the enzyme-mediated sequence of neddylation, surveyed by various published chemical neddylation blockers. The last portion of the paper is covered by opportunities for aiming the protein–protein interactions (PPIs), some available approaches used for the detection of novel drug candidates for targeting the neddylation. Note that, unless mentioned, "neddylation" in this article applies to "cullin neddylation."

Physiology and biochemistry of NEDD8

The NEDD8 is translated as 81 amino acid protein, which is almost 60% identical and 80% homologous to the ubiquitin molecule with 9 kDa molecular mass (Santonico 2019). It was first isolated from the mouse brain and then further research was carried out. Research suggests that the protein's stability may be improved in certain cases by covalent modification using the NEDD8 enzyme (Watson et al. 2011). Ribosomal proteins, whose stability may be improved by neddylation and sequestration in nuclear aggregates, have already been described. Hence, it's possible to enhance the stability of several proteins using NEDD8 post-translational modifications. Oved et al. were the first to establish that NEDD8 conjugation may cause the degradation of a protein when they proved active epidermal growth factor receptor (EGFR) was neddylated and subsequently destroyed, most likely in lysosomes (Enchev et al. 2015). Several other NEDD8-substrates have been discovered to be degrading in recent years. NEDD8 substrates are complicated because the cell may employ the enzyme in the position of ubiquitin, this process is termed as "atypical neddylation," which complicates our understanding of the enzyme's substrates even more. This may arise when NEDD8 levels inside the cell are elevated, or even when ubiquitin levels within the cell are depleted (Soucy et al. 2010). Many additional proteins that increase stability because of covalent alteration with NEDD8 have also been discovered in the literature. Hypoxia-induced factors (HIF-1 and HIF-2), which are substrates for NEDD8 modification, were discovered, and the levels of HIF-2 increased because of neddylation (Zheng and Tao 2020). Another research found that, after hypoxia induction, neddylation helped to stabilize HIF-1 levels in tumor cells, suggesting that it may assist tumors to survive in hypoxic settings more effectively. The transforming growth factor type-II (TGF-type-II) receptor neddylation facilitated the transportation of the receptors to early endosomes rather than caveolin-containing compartments, at which receptor would indeed be ubiquitinated and degraded (Li et al. 2021a, b). As a whole, these results suggest that post-translational modification of a peptide with NEDD8 may help to maintain the substrate, perhaps by shielding the targeted protein from ubiquitination. Structurally, the mature NEDD8 seems to have glycine at its carboxyl-terminal end that is chemically bonded to a lysine residue in the target protein (Soucy et al. 2010). It also possesses a globular ubiquitin-fold domain (UFD) in its structure. According to previous research, it has been revealed that regardless of their resemblances, the NEDD8 and ubiquitin molecules have some areas in their structure holding few charges and polarity (Fig. 1a, b). Those charged areas are regarded as a basic key factor of these proteins (Enchev et al. 2015). The NEDD8 tail is solution flexible. Upon interaction with neddylation and de-neddylation enzymes, it adopts different extended structures and, similar to ubiquitin, ends with a Gly–Gly arrangement that is linked with covalent targets. To mediate various PPIs, two surface-exposed hydrophobic patches, Ile44 and Ile36, are essential, and both are preserved in NEDD8 structures (Fig. 1c). The attachment of NEDD8 to its target protein comprises a series of reactions involving E1, E2, and E3 enzymes, respectively. These enzymes help NEDD8 conjugate with the target protein molecule (Watson et al. 2011).

Fig. 1.

The structural representation of NEDD8 protein. a, b NEDD8-specific charged surface patches structural representations. Acidic blotches on two views of the NEDD8 rotated 180° around the y-axis are shown in red and the basic surfaces blue. The interactions between NEDD8 and ubiquitin could be caused on these surfaces. c Structural description of the hydrophobic patches of NEDD8, which attribute to the interaction of most recognized interfaces with binding partners. The side chains that help Ile36 Patch are displayed in pink, Ile44's blue residue, and β1–β2 is shown in green. The Ala72 is also shown, which handles NEDD8 and ubiquitin discrimination by the respective E1 enzymes

The enzymatic cascade of neddylation

The neddylation cascade begins with NEDD8 maturation. The reaction of mature NEDD8 synthesis is catalyzed by two enzymes ubiquitin C-terminal hydrolase isozyme 3 (UCHL3), and NEDD8-specific protease 1 (NEDP1) also called human de-neddylase 1 (DEN1) or SUMO-1 (Santonico 2019). The C-terminal 5-amino acid residues of NEDD8 are removed by these enzymes to convert the precursor form into the mature form. Once the C-terminus of NEDD8 is exposed by this processing step, it may be used to activate NEDD8 and all of its downstream activities. In contrast to other UBL precursors, such as SUMO or ubiquitin, DEN1 is extremely selective for NEDD8 precursors and therefore does not process any of the other UBL precursors (Schulman and Harper 2009). The NEDD8 after becoming matured is activated by NAE, a heterodimer that comprises amyloid-β precursor protein-binding protein 1 (APPBP1) and ubiquitin-activating enzyme 3 (UBA3) (Enchev et al. 2015). The activation of NEDD8 via NAE requires ATP and forms the NEDD8-AMP molecule before transferring NEDD8 to cysteine of NAE through the thioester bond. The NAE-NEDD8 complex then transfers NEDD8 to the E2 enzyme, which has two forms which are UBE2M and UBE2F, respectively (Cockram et al. 2021). An interaction between NEDD8-charged NAE and its homologous E2s that promotes the production of NEDD8-E2 thioesters is responsible for the unique specificity of the NEDD8 pathway. A combination of the ionic interactions and conformational flexibility of this specific hydrophobic interaction allows NAE to recognize its two distinct E2s and thus integrates further specificity concerning CRLs modification, as UBC12 and UBE2F have been found to neddylate different CRLs (Soucy et al. 2010). The last step includes the interaction of the E2 enzyme with the E3 enzyme to attach NEDD8 with the lysine residue of the substrate (CRLs) protein molecule (Baek et al. 2020). The shift of NEDD8 from UBC12 or UBE2F towards the relevant CRLs may need a ligase-like activity, including the five defectives in cullin neddylation 1 protein (DCN1) and similar proteins being hypothesized to operate as facilitators of particular cullin neddylation (Zou and Zhang 2021). The E3 ligases are further categorized according to domain existing within core protein into various kinds which include the homologous to E6-AP carboxyl terminus (HECT), Really Interesting New Gene RING-box proteins 1 (RBX1), and RBX2 [also known as regulators of cullin 1 (ROC1) and ROC2/SAG, respectively], casitas B-lineage lymphoma (c-CBL), murine double minute 2 (MDM2), inhibitors of apoptosis (IAPs), tripartite motif-containing 40 (TRIM40), SCFFBXO11, ring finger protein 111 (RNF111) and the RNA polymerase-II transcription factor B unit 3 (TFB3) class including the TFIIH/NER complex subunits, respectively (Wang et al. 2011). To increase the amount of ubiquitin transferred to the substrate protein, the RING-box proteins serve as a scaffold for binding the E2 ubiquitin complex to the substrate protein directly. Before ubiquitin is transferred to its target substrate, HECT ligases form a catalytic thioester bond with the HECT domain's C-terminal lobe, as opposed to RING-type neddylation ligases (Stuber et al. 2021).

Regulatory factors of neddylation

The regulation of neddylation is achieved via different proteases by removing NEDD8 from target proteins like COP9 signalosome (CSN) and NEDP1, which also takes part in the maturation of NEDD8 as well in de-neddylation reaction. Other de-neddylation enzymes include ataxin, USP21, and UCH-L1. Among all these, only NEDP1 and CSN are NEDD8-specific enzymes. While the rest are thought to be involved in regulating de-ubiquitination also (Li et al. 2021a, b). Normally, during the neddylation of CRLs, the lysine fragment of the C-terminus triggers a conformational modification in its structure, that interrupts the cullin associated NEDD8 dissociative protein 1 (CAND1) and leads its allosteric connection along with stimulating the CRLs to facilitate the transmission of ubiquitin from activated E2 to the target protein (Stuber et al. 2021). After the reaction, deneddylase protein fixes with the neddylated CRLs, which results in the de-neddylation of CRLs via separation of the CRLs matrix and the discharge of E2 and target protein molecule until the next round of neddylation (Soucy et al. 2010). The conservation of CRLs requires CAND1-mediated competitive neddylation and allows it to be integrated with various protein receptors to facilitate the neddylation of many other different substrates and ensure cell survival (Newton et al. 2008). The mechanism of neddylation and de-neddylation of CRLs, CAND1 assembly, and substate ubiquitination is described in Fig. 2. Several additional proteins have been identified as having the ability to control neddylation in various ways. The DCN1 discovered from yeast is one of them which regulate neddylation (Enchev et al. 2015). The human genomic codes intended for DCN1-like proteins are labeled as DCN1-DCNL5. Possibly the DCNLs are the most important factors which regulate neddylation in human cells (Wang et al. 2011). The DCN1 fixes the N-terminus of UBE2M and CRLs and acts as basic support for E3 ligase to guide NEDD8 towards the lysine residue of CRLs for initiating the neddylation (Baek et al. 2021). All DCNLs strongly interact with the CAND1, and promote the release of old substratum adapters, and associate them with core CRLs complexes (Keuss et al. 2016). It is believed that DCNLs are specifically for the CRLs family in vivo. For example, in humans, the DCN1 regulates the neddylation of Cul1 and Cul3 but not the Cul4 (Liu et al. 2009). The DCNL1 also localizes along with UBCL2 inside the nucleus of cells to perform its function. Moreover, both DCNL1 and DCNL2 are mono-ubiquitylated in a reaction requiring the ubiquitin associated domain (UBA domain) which directs their nuclear export (Wang et al. 2011). Likewise, the excess interpretation of DCNL1 causes a marked increase in neddylated protein levels in human cells (Enchev et al. 2015). Another regulator of neddylation is Glycyl-tRNA synthetase (GlyRS) an essential enzyme for protein formation. The GlyRS strongly interacts with NEDD8, APPBP1, and UBE2M to enhance the neddylation (Mo et al. 2016). Another protein that regulates neddylation is Tfb3 which helps in the conjugation of NEDD8 with UBE2M (Santonico 2019). Some studies have revealed a piece of surprising information that the infections caused by bacteria can also lead to inhibition of neddylation in eukaryotic cells. Recently research has recognized that the NEDD8 also acts as a target of cycle inhibiting factor (Cif) which is produced by enterohemorrhagic and enteropathogenic bacteria and prevents eukaryotic cell propagation by delaying the cell cycle in human cells (Enchev et al. 2015). The NEDD8 along with Cif localizes within the nucleus of the host cell and leads accumulation of NEDD8 conjugated CRLs. The co-immunoprecipitation exposed that the Cif interrelated with CRLs through binding with the neddylated forms of CRLs (Watson et al. 2011).

Fig. 2.

The representation of neddylation and de-neddylation pathways and their enzymatic cascade. a CRLs neddylation, b CRLs assembly and CAND1 displacement, c SUBSTRATE ubiquitination, d releasing of substrate and CSN attachment, e de-neddylation of CRLs, f CAND1 attachment

The systematic analysis of CRLs and neddylation

It is estimated that 20% of cellular proteins may be targeted and destroyed by the UPS after activation by the neddylation of CRLs. The CRLs are versatile E3UL that hold a large part of the mediated protein turnover in eukaryotic cells (Li et al. 2021a, b). As they monitor a wide range of cellular activities, it would not be surprising that their neddylation serves essential roles in both physiological and pathological conditions including cancer cell progression in humans. The family of mammalian CRLs includes eight participants from Cul1 to Cul7 and PARC, which are categorized by a specific domain of CRLs homology (Baek et al. 2020). The CRLs attack countless substrates and consequently affect a broad variety of cellular mechanisms (Fig. 3) such as DNA replication, cell cycle, gene transcription, apoptosis and oxidative stress, cellular growth and development, cell signaling, transcription monitoring, epigenetic integrity, and cancer metastasis (Baek et al. 2021). They act as a molecular scaffold binding to an adaptor protein and a substrate receptor protein at its N-terminus, along with a RING protein (RBX1 or RBX2) at its C-terminus 2005 (Soucy et al. 2010). They have seemed to be broadly represented in both the cytoplasm and nucleus, but no convincing evidence suggests that their activity is regulated by cellular localization (Keuss et al. 2016). The initial crystallographic structures of CRLs showed a quite enlarged configuration where they serve as compact scaffold proteins (Hassanpour and Dehghani 2017). Most of the short-lived proteins are substrates of CRLs, especially for Cul1, which includes regulators for the cell cycle, epigenetic regulatory molecules, peptides for translation, and autophagy regulation (Bradford and Jin 2021). While the HIF1α is a key regulator of Cul2 and plays a major role in monitoring the reactions to hypoxia (Wang et al. 2021a, b). The Cul2 also forms a complex with VHL protein that causes mutations that contribute to the development of VHL disease, characterized by multiple tumorigeneses in many organs. VHL was accidentally identified as the first neddylation suppressor to expand the NEDD8 substratum variety beyond the CRLs (Enchev et al. 2015). However, Cul3 has a major substrate known as NRF2, an antioxidant transcription factor concerned with free radicals and oxidative stress (Wang et al. 2011). As per the literature, the substrates of Cul4 play a crucial role in the DNA replication and response against DNA damage, Cul4A is found in an area often amplified with breast cancer and the mice resistant to UV-induced skin tumors were deficient in Cul4A (Liu et al. 2009). Whereas, the Cul5 substrates are frequently associated with autophagy and virus-induced responses (Li et al. 2021a, b). Kaposi’s sarcoma-related herpesvirus (KSHV) showed a similar mechanism for the degradation of VHL and p53 molecules in collaboration with Cul5 (Okumura et al. 2016). The Cul7 and its substrates are yet hardly researched, with quite limited substrates outlined until now (Baek et al. 2020). Few studies revealed that Cul7 genetic mutations are being associated with several inherited diseases. There have been no currently offered structures that either reveal the three-dimensional structure of Cul7 and PARC (Skaar et al. 2014). The PARC reacts with tumor suppressor protein p53 and also induces p53 dependent cell death. A recent experiment on inhibition of neddylation was conducted and the data shows that because of CRLs ubiquitination around 20% of all intracellular proteins were patent for proteasomal decay (Sun et al. 2020a, b).

Fig. 3.

The CRLs and their associations with different cellular pathways, including DNA replication, signal transduction, cell cycle, oncogene regulation, gene transcription, apoptosis, and oxidative stress. The proteins taking part in various processes are also mentioned

Neddylation and cancer

Cancer is the world’s second-largest cause of death (Ferlay et al. 2021). Therefore, a major health issue for all human communities (Enchev et al. 2015). Sadly, it is a kind of disease that causes alterations in the molecular mechanisms of cells, therefore it represents a major difficulty for its specific diagnosis, followed by treatment effectiveness (Soucy et al. 2010). Experts have studied different phases of cancer and showed that certain gene mutations are involved in the development of various forms of cancers by contributing to the emergence of unusual cells (Hassanpour and Dehghani 2017). The study on the role of neddylation in liver cancer cell lines suggested neddylation was frequently hyper elevated in cancer cells and was associated with poor prognosis. Recently, in 2021, the neddylation in lung cancer cells was studied which revealed that within the cells of both adenocarcinoma of the lungs and squamous cell carcinomas, the whole neddylation mechanism, such as NEDD8 enzymatic conjugation and global-protein neddylation was over-activated (Zhou et al. 2019). The survival of patients with a high expression of neddylation was much worse than in lung adenocarcinoma patients with decreased neddylation expression (Yu et al. 2020). Previously research conducted on the Chinese hamster cell line showed that the cell line contains a temperature-sensitive mutation in the NEDD8 conjugation path. The cells at non-permissive temperature pass over consecutive S-phase deprived of prevailing the mitosis and accumulate DNA molecule via multiple cell divisions (Handy et al. 2011). This alteration could be liberated through transfection by the means of APPBP1 vector expression, which is a crucial part of NAE heterodimer (Schabla et al. 2019). Interestingly, another study suggested that the replication feature of the chromatin licensing and DNA replication factor-1 (Cdt-1) can be controlled via Cul4A and Cul1 mediated neddylation for the suitable replication of DNA during each cell cycle stage (Handy et al. 2011). Besides this, they also suggested that any overexpression or knockdown of Cdt-1 or endogenous suppressor of Cdt-1 leads to genomic uncertainty and replication in ts41 cell lines (Chen et al. 2020). Besides CRLs some other proteins were also testified being associated with NEDD8 (Zhao et al. 2014), like mouse double minute 2 (Mdm2) (Kim et al. 2021a, b), tumor suppressor p53 (Wen and Wang 2021), cancer suppressor protein like Von Hippel–Lindau (VHL) (Yang et al. 2020), ribosomal protein (L11) (Li et al. 2020), EGFR (He et al. 2021), liver kinase B1 (LKB1) and protein kinase B also known as Akt kinases (Chen et al. 2020). Likewise, it has been proved that the overexpression of F-box proteins like S-phase kinase-associated protein-2 (Skp2) is directly associated with cancer development and the suppression of Skp2 leads to tumor regression by its associated CRLs (Cai et al. 2020). Scientists have revealed that by incorporating many F-box molecules, the CRLs destroy around 20% of all regulatory proteins of cells related to the progression of cancer (Handy et al. 2011). Besides that, regulating p27 is also evidenced in various forms of cancers (Russo et al. 2020), and decreased levels of p27 are associated with reduced consequences of prostate cancer (Chen et al. 2020), non-small cell lung cancer (Liu et al. 2020), colorectal carcinoma (Das et al. 2020), and MCL (Lucendo et al. 2020). The LKB1 and Akt kinases are very much crucial for the metabolism of the liver (Sun et al. 2020a, b). According to existing data, almost 40% of lung tumor cell lines have shown Cul1 overexpression (Kim et al. 2021b). Major components of the CRLs family, their substrates, cellular functions, and association with cancer are summarized in Table 1. LKB1 and Akt are novel potential targets of the neddylation. Alterations in neddylation can enhance the LKB1 and Akt steadiness, therefore it is very much significant to induce metabolic interruptions via neddylation (Zhou et al. 2021). The Hu antigen R (HuR) is an important RNA-binding protein that has a crucial role in cell proliferation, survival, and differentiation. It also maintains the structure of mRNA (Qin et al. 2020). Existing data and plenty of research have given the assumption that expression of HuR is usually improved in several cancer cells that means HuR is linked to tumorigeneses (Elcheva and Spiegelman 2021). Another research suggested that HuR is linked with Mdm2 and is stabilized via neddylation in Mdm2 dependent method, shielding HuR from decay (Papatheofani et al. 2021). Cancer cell lines have larger ratios of neddylated protein substrates than normal fibroblasts, and abnormal NEDD8 conjugation has been shown in cancer cells (Park et al. 2020). HSC4 oral carcinoma cells transfected with Ubc12 that inhibits neddylation were shown to be anti-proliferative, showing that cancer cells have abnormally high neddylation levels, which contributes to the enhanced cell proliferation (Soucy et al. 2010).

Table 1.

The substrate proteins of CRLs and their linkages with different forms of cancer

Substrates	Functions	CRLs	Connections with cancer	References
Cyclin E	Regulation of cell cycle	Cul1	Irregular expression of cyclin E is related to tumor production and breast cancer progression	Jang et al. (2020)
mTOR	Cell growth and proliferation	Cul1	Dysregulated in various human tumors	Shah and Chen (2020)
Cdt-1	DNA replication and licensing factors	Cul1 and Cul4	Overexpression is associated with genomic instability, dysregulated in various human tumors	Che et al. (2020)
NRF2	Stress response transcription factor	Cul3	Associated with chemoresistance. Down-regulated in various human tumors	Kobayashi et al. (2004)
c-Jun	AP1 transcription factor	Cul1	It is identified as a powerful oncoprotein	Hryniewicz-Jankowska et al. (2021)
β-catenin	Transcription factor	Cul1	Up-regulation is associated with colon and prostate cancer	Ramachandran and Ciulli (2021)
BimEL	Pro-apoptotic tumor suppressor protein	Cul1	The level decreased in transformed cells because of over degradation	Bi et al. (2020)
c-Myc	Cell proliferation regulator	Cul1	It is identified as an influential oncoprotein	Sweeney et al. (2020)
Emi1	APC inhibitor	Cul1	Overexpression is associated with ovarian cancer	Schrock et al. (2020)
P27	Cyclin-dependent kinase inhibitor	Cul1 and Cul4	Overexpression is associated with several cancers and is linked with poor prognosis	Sharma and Nag (2014)

Targeting neddylation for cancer therapy

The selection of molecular mechanisms as targets for antitumor drug development requires careful assessment of a number of parameters. pharmacologically manipulable pathways must be essential to cancer cell viability, be vulnerable to alteration or deregulation, and not be lethal to normal cells when disrupted (Wang et al. 2011). The NEDD8 conjugation route is an interesting avenue for the discovery of new drugs given these prerequisites for rational drug design. The importance of protein neddylation in various genetic systems is crucial (Rambacher et al. 2021). The NEDD8 pathway's relevance in cancer has been reported in several studies as described in the above section. The use of crystallography to study the NAE in complex with its substrates has provided crucial insights into critical protein–protein interactions as well as the catalytic mechanism of the E1 enzyme, which is responsible for the initial neddylation process, amongst other discoveries (Qiu et al. 2020). As a result, tiny compounds that interfere with the activity of NAE including its binding partners have been developed (Soucy et al. 2010). A neddylation inhibitor may have a superior toxicity/efficacy profile when related to a proteasome inhibitor since proteasome inhibition impacts the processing of all polyubiquitinated proteins, while a neddylation inhibitor would only affect proteins that have been polyubiquitinated by CRLs. Moreover, some anti-proliferative proteins like p27, IkBa are attached with polyubiquitin, which results in their degradation (Gai et al. 2021). As a result, a NAE inhibitor must increase the amounts of anti-proliferative molecules in the body, hence inhibiting cell growth (Yu et al. 2020). A proteasome inhibitor would indeed affect these proteins, but also all numerous different regulatory proteins (peptides that are often pro- or antiproliferative and/or had little to do with cell proliferation) and house-keeping proteins (proteins that have to be turned over due to general wear and tear) as well as other proteins that are damaged by the proteasome (Wu et al. 2020). The fact that it impacts the metabolism of essential anti-proliferative proteins would lead some to believe that neddylation inhibitors would have a more selective anti-tumor effect than a proteasome inhibitor (Chang et al. 2021). However, rather than inhibiting the proteasome, which has an impact on a broad spectrum of proteins, inhibiting substrate-specific ubiquitin E3 ligases might provide a possible therapeutic opportunity for cancer treatment with a more tailored approach (Jevtić et al. 2021). Moreover, proteins that are involved in degradation seem to be an excellent way of treating cancer (Hong et al. 2020). As the best-highlighted feature of neddylation alteration is to trigger CRLs, which guide several anti-apoptotic polymers for the proteasomal degradation in tumor cells. Therefore, the neddylation is validated as an attractive target for cancer (Gatti et al. 2020). Certainly, the procedure of neddylation is unusually stimulated because of overexpression of NEDD8, and many enzymes involved in neddylation including APPBP1, UBE2M, DCN1, and RBX1 in several forms of cancers like glioblastoma, lung cancer, colon cancer, liver cancer, and squamous cell carcinoma (Park et al. 2020). Besides this, it has been proved that elevated levels of all these enzymes are related to the poor prognosis of the patient (Chang et al. 2021). These findings together provide a clear sign that neddylation alteration is a capable target for anticancer (Handy et al. 2011). NAE is an ATP-dependent molecule that triggers the neddylation, and in the structure of NAE, there are three main important domains. A catalytic cysteine transthiolation domain to create an E1-NEDD8 thioester bond. Another domain is for acyl adenylate construction and a UFD domain for interaction with the E2 (Park et al. 2020). Based on the structure of enzymes involved in neddylation, many approaches are developed to create the inhibitors of NEDD8 activation (Santonico 2019). In some studies, the activation of NAE was inhibited via ATP-competitive inhibitors to prohibit the binding of ATP with NAE, which is very crucial for the beginning of neddylation cascades (Jevtić et al. 2021). Some experiments were based on imitation of the NEDD8-AMP complexes by creating a NEDD8-composite adduct like the NEDD8 adenylate, but then blocking efficient intra-enzyme reactions. An example of this is the MLN4924 compound (Enchev et al. 2015). The MLN4924’s success in a few preliminary studies supports and emphasizes the importance of designing other neddylation pathway inhibitors that may prove novel anticancer agents (Zhong et al. 2012). Until now, various inhibitors of neddylation have been identified via using different cell lines and different sorts of accumulated CRLs substrates are being studied (Table 2). The chemical structure of some identified inhibitors of NAE is shown in Fig. 4.

Table 2.

The reported inhibitors of neddylation

Compounds	Cell lines	Targets	Accumulated CRLs substrates	Clinical trials	References
MLN4924	HCT-116	NAE	c-Jun, P27, NRF1 α	Phase I and Phase II	Soucy et al. (2009)
Compound-1	HCT-116	Pan-E1	NA	NA	Chen et al. (2011)
TAS4464	CCRF-CEM	NAE	NRF2, P27, CDT1	Phase I	Yoshimura et al. (2019)
ABP1	NA	Pan-E1	NA	NA	An and Statsyuk (2013)
LZE	MCF-7, Bcl-7402	NAE	NA	NA	Zhang et al. (2014)
M22	A549	NAE	P53, P27, CDT1	NA	Lu et al. (2016)

NA not available

Fig. 4.

The chemical structure of few NAE inhibitors that were discovered by targeting neddylation and NAE-UE2M interactions

The MLN4924 as neddylation inhibitor and its preclinical and clinical progression

The MLN4924 is an inhibitor that targets NAE specifically (Baek et al. 2021). The research focused on MLN4924 has shown an unexpected linkage between neddylation and various other important mechanisms of cells (Gai et al. 2021). It was identified in 2009 via the high throughput screening (HTS) method by the millennium pharmaceutics through repeated enhancements in therapeutic designs (Zhao et al. 2014). It displayed a potent and extremely selective compound that influences blockage of E2-NEDD8 binding in a purified mixture of enzymes by a half-maximal inhibitory concentration (IC50) of 4 nmol/L (Yu et al. 2020). Until now several experiments have shown the effects of MLN4924 on different cancer cell lines and inhibition of mouse xenograft models (Table 3). The NAE starts binding of NEDD8 with MLN4924 to create NEDD8-MLN4924 complexes, which block the neddylation of CRLs and causes their abolishing along with the inhibition of E3UL activity (Li et al. 2021a, b). These all events lead accumulation of CRLs substrates and cause cell cycle arrest, DNA damage, and apoptosis (Wang et al. 2011). By nature, the complex of MLN4924-NEDD8 is analogous to NEDD8 adenylate. The inhibitory effects of MLN4924 are extremely specific to NAE and SUMO activating enzyme (SAE) in enzymatic assays (Du et al. 2021). The first-ever reported study on preclinical demonstration of MLN4924 was conducted by Soucy and colleagues. The experimental data revealed a concept of targeting NEDD8 associated with protein turnover as a novel effective anticancer approach. In their experiment, they showed that the treatment with MLN4924 interrupts the turnover of CRLs mediated proteins and also induced apoptosis in various forms of cancers (Fig. 5). Strangely, their data confirmed that the MLN4924 leads to cell death which was directly associated with dysregulation of the S-phase of DNA formation (Chang et al. 2021). Moreover, the administration of MLN4924 in mice possessing human xenografts showed inhibitory effects on tumor development. The overall results of the experiment suggested that NAE inhibition is a novel tactic with very effective broad applications in tumor treatment (Yu et al. 2020). Besides this, the treatment of MLN4924 in hematologic as well as solid tumors in mouse xenografts led to improve regression of cancer at well-tolerated concentrations and schedules (Aubry et al. 2020). A single subcutaneous (SC) dose of MLN4924 inhibited neddylated CRLs in nude mice and caused DNA damage and apoptosis (Shi et al. 2020). Recently it was revealed that MLN4924 prevents angiogenesis and inflammation, and amplifies the microenvironment of tumor cells to suppress tumorigenesis. This was accomplished because of the downregulation of CRLs neddylation (Gatti et al. 2020). The significant proportion of preclinical research to date using MLN4924 concentrates on its effectiveness in cancers favorably affecting adults (Yu et al. 2020). The Pediatric Preclinical Testing Program (PPTP) has studied its activities in their impressive set of childhood malignancies models. To identify specific pediatric carcinoma that can take the most advantage of NAE inhibition, they tested in vitro and in vivo effectiveness of MLN4924 in tumor modules (Foster et al. 2021). Results demonstrated that the all-experimental models they studied resulted in a significant increase in survival without evidence of recurrence. Further analyses of the particular model in their studies revealed that sub-pieces of glioblastomas, rhabdomyosarcomas, and neuroblastomas were particularly susceptible to MLN4924 treatment (Islam et al. 2021). These findings are a solid justification for further studying potential MLN4924 applications in the treatment of cancers in childhood (Shi et al. 2020). MLN4924 has also been evaluated for advanced solid tumors, lymphomas, and myeloma and advanced AML in early phase clinical studies. Preliminary data from these trials submitted to the American Society of Clinical Oncology (ASCO) in 2009 showed that MLN4924 treatment resulted in the accumulation of substrates associated with target inhibition in the peripheral blood and skin of patients (Scholz et al. 2020). The preclinical synergistic antitumor activity of AML xenografts was shown to be well tolerated combinedly by MLN4924 + Azacitidine with promising clinical activity in patients with untreated AML (Sekeres et al. 2021). In another study, inhibition of NAE positively modulates T-cells polarization in vitro and increased the production of interferon-β. These findings have been also summarized in vivo using immuno-compromised mouse models (Wang et al. 2021a, b). Moreover, the T-cells exposed to MLN4924 recovered NAE activity and maintained their response to the stimulation of the T cell receptor as well as cytotoxicity. This data highlights the possible immune consequences for chronic lymphocytic leukemia (CLL) and lymphoid malignancies for targeting neddylation (Cordo’ et al. 2021).

Table 3.

The outline of effects of MLN4924 on mouse xenografts and different tumor cell lines

Tumor sources	IC50 (µM) of MLN4924	Cell lines	Repression of mouse xenografts	References
Lung cancer	1.03	H460	H522	Soucy et al. (2009)
Lung cancer	0.13	Calu-6	Calu-6	Soucy et al. (2009)
Colorectal cancer	0.1	HCT-116	HCT-116	Milhollen et al. (2010)
Acute myeloid leukemia	0.21	PL-21 HL-60 MOLM-13	HL60	Swords et al. (2018)
Diffuse large B-cell lymphoma (DLBCL)	0.03	U2932	Primary human DLBCL	Milhollen et al. (2010)
DLBCL	0.10	OCI-Ly10	OCI-Ly10	Wang et al. (2011)
DLBCL	0.03	OCI-Ly19	OCI-Ly19	Wang et al. (2011)

Fig. 5.

The suggested mechanism for MLN4924's inhibitory action on tumor growth has been described. MLN4924 inhibits CRLs by blocking cullin neddylation, causing a buildup of CRLs substrates in the presence of MLN4924. The cancer cells' development is inhibited because of a variety of key biological responses, such as apoptosis, oxidative stress, and G2 cycle arrest, which are all responsible for the growth suppression of cancer cells

The Compound-I as neddylation inhibitor

In 2011, an experiment on the kinetic affinity of MLN4924, Compound-I, and purified ubiquitin was performed and results showed that Compound-I played a major role in the maintenance of UPS and can modulate various biological pathways. It inhibited the exchange activities of UAE-dependent ATP-PPIs by blocking UAE thioester bonds (Chen et al. 2011). Another research suggested that Compound-I inhibited the activity of NAE in the cell-based assay and in vitro assays within micromolar ranges (Hyer et al. 2018). The cytotoxicity of cancer cells was examined using MTT assay, the results showed that Compound-I was moderator toxic against cancer cells at 10 mM IC50. Their experimental data also suggested that Compound-I acts as a non-covalent competitive suppressor of NAE and caused an accumulation of CRLs substrates by inhibition of NAE (Wang et al. 2011).

The TAS4464 compound as neddylation inhibitor

TAS4464 was recently discovered in 2019 by Taiho Pharmaceutical as a small molecule inhibitor by HTS and structure-based virtual screening (SBD) techniques (Ochiiwa et al. 2021). According to the findings of the experiment, TAS4464 treatment inhibited the neddylation of CRLs and induced accumulation of various substrates, including p27, Cdt-1 in human cancer cell lines. To date, TAS4464 is a very potent inhibitor of NAE showing antitumor activities in both solid and hematological tumors with prolonging target inhibition (Yoshimura et al. 2019). Another study revealed that TAS4464 selectively blocked NAE during in vitro assay parallel to the UBE2M-NEDD8 thioester via an IC50 of 0.995 nmol/L, which is almost ten times higher than MLN4924 (10.5 nmol/L) (Muraoka et al. 2019). An experiment based on MM treated with TAS4464 suggested that TAS4464 treatment is a promising target to treat MM via both individuals and combination therapy (Ochiiwa et al. 2021).

The LZ3 compound as neddylation inhibitor

Zhang et al. (2014) employed a novel approach merging covalent docking by virtual screening technology (VS) and identified three covalent NAE inhibitors, of which LZ3 was the strongest compound discovered via in vitro testing. The covalent binding mechanism for LZ3 was then supported by a cell-based assay which showed that LZ3 reacts with NAE with a similar pattern as MLN4924 reacts (Zhang et al. 2014). The MTT assay displayed growth inhibition in various tested cancer cell lines with 12–29 μM IC50. The western blot results confirmed the inhibitory effects of LZ3 on neddylation via suppression of binding of Ubcl2-NEDD8 in MCF-7 cell lines (Yu et al. 2020).

The adenosyl sulfamate ABP1 compound as a neddylation inhibitor

Based on the structure of Compound-I another compound was created and named ABP1 that forms a covalent adduct resembling Compound-I and MLN4924 (Barghout and Schimmer 2021). The ABP1 serves as an activity-based probe for in vitro neddylation assay and detects NAE activity inside intact cells (Misra et al. 2017). Experimental data also revealed that ABP1 displayed a decrease in the levels of SUMO, ubiquitin, and NEDD8 conjugation in cell-based assays. The same research group further designed another compound, ABP A3, based on ABP1 discovery. The ABP A3 suppresses NEDD8 conjugation in A549 cells (An and Statsyuk 2013).

The M22 compound as a neddylation inhibitor

In 2016, a SBVS experiment was conducted in a compound library comprising almost 50,000 compounds to identify the inhibitors of NAE. The computational docking and grading supported by molecular evaluation and target validation lead to the emergence of 1-benzyl-N-(2,4-dichlorophenethyl) piperidine 4-amine (M22) as a consistent NAE inhibitor compound. The M22 showed inhibition of NAE in various cancer cell lines and suppression of tumor in AGS xenografts in nude mice, besides reducing acute toxicity in the zebrafish model (Ni et al. 2020). Further research showed that the small molecule M22 is a non-covalent NAE inhibitor that exhibits significant antitumor properties in both cell-based assays and in vitro techniques (Hyer et al. 2018). Its activities were also compared to another compound: candesartan cilexetil (CDC) (Ni et al. 2020). Based on all the above findings it has been proved that targeting NAE is a very important tool for anticancer drug identification and in the future, further efforts should be done to identify inhibitors against neddylation via targeting other enzymes of neddylation for improved selectivity with estimated lesser toxicity.

The latest techniques used to detect the neddylation inhibitory compounds

The process of discovery of neddylation inhibitor is a quite hectic and multistep procedure comprising many systematic practices to discover and characterize various compounds leading towards the establishment of hits and then legalize them broadly through biological trials to achieve the rank of a viable therapeutic drug (Zhong et al. 2014). Detection of MLN4924 compound and subsequent preclinical and clinical findings provide a piece of firm evidence that via targeting the neddylation pathway the new promising inhibitors can be discovered for anticancer therapeutic implications (Skaar et al. 2014). The target-based screening of natural and synthetic compounds is considered an important tool for the development of anticancer drugs (Dadashpour and Emami 2018). As neddylation is a process having dynamic enzymatic cascades and constantly changing conformations with diverse interaction surfaces hence, targeting PPIs involving neddylation via structural-based drug strategy is always a challenge for researchers (Zhang et al. 2014). During the past 10 years, different neddylation inhibitors were reported and now several alternative approaches have been developed for the identification of neddylation inhibitors. Some of the latest popular techniques are described in the following sections of the article.

The detection of CRLs inhibitors and DCN1–UBE2M interactions through HTS

The HST assay is based on the time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescent resonance energy transfer (FRET) protocols. In these assays, a signal is formed once a donor and an acceptor particle reach closer and result in FRET (Cote et al. 2019). In the advanced form of TR-FRET, the homogenous time-resolved fluorescence (HTRF) is associated with the time-resolved (TR) array of fluorescence (Emami-Nemini et al. 2013). The traditional HTS has been very beneficial for the identification of neddylation inhibitors (Wang et al. 2011). Until now, the most potent inhibitor of neddylation including MLN4924 as mentioned above was discovered through the traditional HTS method (Buckley and Crews 2014). Recently, in 2020, an experiment was conducted based on the identification of CRLs inhibitors via the HTS method. The experiment was based on the screening of 17,000 compounds, around 1700 compounds were approved by FDA, 600 compounds were got from plant resources whereas, some compounds were synthetic drug-like small molecules. The result of the experiment suggested that gossypol a natural compound got from cottonseed is a potent inhibitor of CRLs neddylation. Further biochemical results revealed the gossypol blocks neddylation of Cul1 and Cul5 via direct binding to Cul1-RBX1 and Cul5-SAG complex. They suggested that Cul5-H572 plays a very crucial role in gossypol binding (Yu et al. 2020). Recently, an inhibitor of DCN1 known as piperidinyl urea was discovered via HTS based on TR-FRET assay. A further study developed the HTRF protocol for inhibitors of DCN1-UBE2M. Moreover, another research group used the k48 di-Ub test, which attempts to discover the inhibitors of E3UL (Hammill et al. 2018). The in vitro enzymatic assay was performed via incubation of purified NAE, UBE2M, Cul1-RBX1. A FRET signal was created representing a firm di-Ub bond. Using this experiment contributed to the detection of suramin as a promising CRLs inhibitor (Su et al. 2020).

The discovery of CRLs inhibitors and DCN1-UBE2M interactions via amplified luminescent proximity homogenous assay (AlphaScreen)

The AlphaScreen is an outstanding technique for screening various target compounds. It offers a modest and effective means to evaluate the consequences of biomolecular interactions and actions of compounds and is widely used for biomarker quantification, drug discovery, and cell signaling researches (Houck and Kavlock 2008). Its capability to access the post-translational modifications of proteins offers innovative usages in drug screening research. As some events of cell signaling including sumoylation, phosphorylation, proteolysis, and glycosylation remain difficult to measure, but the AlphaScreen protocol has now challenged these procedures. The AlphaScreen is widely used nowadays to measure the interactions of various proteins involved in neddylation and ubiquitination (Weitzel 2011). In 2013, an experiment was conducted on the cellular effects of NAE inhibitor via the AlphaScreen method. The targets were to access the total NAE activity, neddylated Cul1, and the detection of thioester formation of NAE (Soucy et al. 2010). For in vitro transthiolation assay of NAE-UBE2M, the AlphaScreen was performed for the identification of a small molecule inhibitor. The NAE, biotinylated UBE2M, and Flag-tagged NEDD8 were added in a reaction mixture to establish in vitro reaction of enzymes (Zhao et al. 2014). For signal detection, the donor beads of streptavidin and acceptor beads of anti-flag labels were used. Another researcher used AlphaScreen for the detection of NAE-NEDD8 interactions. The reaction mixture was comprising His-NEDD8, GST-NAE, UBE2M, and mg ATP. The acceptor beads were Ni²⁺ whereas the donor beads were having a glutathione coat. The Rhodium-III compound was discovered as an NAE blocker in the study (Yu et al. 2020).

Detection of neddylation inhibitors via virtual screening technology (VS)

The VS has emerged as a promising method complementing HTS techniques. It was started for drug discovery in 1997 and has experienced rapid growth in pharmaceutical research to date (Romasanta et al. 2021). The VS can be divided into two diverse categories: named ligand-based (LBVS) and structural-based (SBVS) techniques (Rica et al. 2021). Many NAE inhibitors mentioned in the previous sections were identified through VS strategies. The basic principle for appropriate VS is the high-resolution of crystalline assemblies of the targeted protein and interrelated particles. The SBVS becomes an important method for discovering chemical compounds, lead stabilization, and a promising alternative for HTS and LBVS (Laplante and Zhang 2021). An experiment was based on the covalent plotting and investigation of the DCN1-UBE2M interface sites, an UBE2M peptide of 12-residue was first recognized and manufactured as the shortest peptide with a powerful attraction to the DCN1 molecule (Yang et al. 2021). The chronological subtraction of amino acid from this 12-residue peptide leads to the formation of a tetrapeptide molecule, yet the lightest molecule for patent interaction (Soucy et al. 2010). Using this tetrapeptide, widespread medicinal chemistry experiments were completed through continuous assessment of probe selectivity, effectiveness, and lastly leading to the detection of the effective, DCN1 inhibitors named DI-591 and DI-404, respectively (Houck and Kavlock 2008). Several additional examples can be found throughout the further neddylation inhibition studies. The upcoming attempts in drug detection and development will certainly be the convergence of all existing methods, counting HST, VS, along with some other creative techniques, for a stronger accuracy rate. Although the assembly of the high resolution of each enzyme used in these researches was primarily determined, more comprehensive co-crystal assemblies of proteins at various stages of neddylation reaction are highly required. For example, the UBE2M and RBX1 related structures are already present, but RBX2’s co-crystal structure and its obligatory associates are still unsolved. Such as, for the neddylation of Cul5, the RBX2 molecule works only with UBE2F (Horn-Ghetko et al. 2021) followed through the ubiquitylation of various substrates, counting pro-NOXA protein, which is a pro-apoptotic protein (Busche et al. 2021). The underlying mechanisms of the preferential pairing of E3UL with UBE2M and respective CRLs substrates are still unclear. To categorize highly selective inhibitors directing these interactions, future research should be oriented towards resolving structures of these special pairs of NAE, UBE2M, and CRLs.

The verification and confirmation of neddylation inhibitors

When it comes to the competitive realm of drug research, finding a successful "hit" is an extremely unusual occurrence. In the year 2008, the pharmaceutical sector in the United States invested $65 billion in the search for new pharmacological therapies. Only one anticancer compound was authorized by the FDA during the same year (Pillaiyar et al. 2020). The term “Hit to lead” (H2L) also known as lead generation is a phase in initial drug discovery where the small molecule hits from HTS are assessed and undergo inadequate optimization to categorize the capable lead complexes (de Esch et al. 2021). Here we outlined usually applied tactics from the HTS to evaluate and refine the target neddylation inhibitors, which results in the declaration of lead molecules. Various cellular assays have been established for the validation of neddylation inhibitors. The basic techniques to authenticate the target binding of proteins are the cellular thermal shift assay, co-immunoprecipitation assay (Maneiro et al. 2021), and the biotinylated pull-down assay respectively (Laplante and Zhang 2021). For the protein’s target modulation confirmation, such as neddylation inhibition or accumulation of CRLs substrates, the western blotting technique is performed (Busche et al. 2021). The anticancer activity or biological activity can be analyzed via IC50-based assays like MTT, whereas the growth and suppression are evaluated via apoptosis, cell arrest, and autophagy (Zhao et al. 2021). Broadly, the identification of neddylation blockers is a long and complex process involving HTS processing, applicant’s SAR virtualization, in vivo and in vitro experimental verification, the measure of physiological potency and ideal sensitivity and pharmacokinetic properties of the drugs in animal models till the suitable candidates are identified, that achieves established standards for every stage, as a lead compound (Zhang et al. 2020). A schematic representation for the steps of the discovery of the neddylation inhibitor is shown in Fig. 6.

Fig. 6.

A schematic representation of the steps of the discovery of neddylation inhibitor. Showing steps from in vitro enzymatic assay up to conformation of Lead compounds

Conclusion

The extensive applications and mechanism revisions of the MLN4924 inhibitor in countless carcinoma cells, melanoma xenografts models, and a variety of clinical experiments have largely expanded our perception of neddylation and its involvement in melanoma biology. Since the last decade, medicinal chemistry has been focused on targeting neddylation and has exposed various fundamental mechanisms at the cellular level of the neddylation sequence. After the discovery of MLN4924, several drug discovery techniques were conducted to search for more discriminating and less toxic compounds for inhibition of neddylation. Whereas MLN4924 appears the most dynamic neddylation suppressor designed for chemotherapy and new inhibitors of neddylation are still developing. Though various enzymes or PPIs in the neddylation reaction persist mainly unexploited for specific small-molecule inhibitors, besides NAE and the interaction site DCN1-UBE2M. Additional researches are carried out to get further reliable crystal or co-crystal configurations for high-resolution UBE2M-UBE2F, CLRs-RBX1/RBX2, and the interactions of substrates of neddylation with PPIs alone or with conjunction to small molecules characterized via specific HTS. Our perception of neddylation complexity is growing rapidly and as an important regulator, neddylation is emerging for several pathways associated with cancer either on the pro-or anti-tumor prospects. A simple assessment of the variety of regulated NEDD8 pathway control methods and approaches will assist the eventual use of neddylation inhibitors in treating various diseases. The validation and structural optimization of drugs will gradually lead to the development and discovery of effective neddylation sequence-targeted blockers for efficient treatment of a wide range of diseases, especially cancer, which is characterized by an over-activated neddylation pathway. Future studies can provide additional input into areas that will selectively restrict NEDD8 and how it interacts accurately with the pathogenesis and maintenance of cancer regulation needs to understand.

Author contributions

IB, LG, MM, MZ, HS and MK delineated and conducted the literature survey. All listed authors wrote, read, and approved the manuscript.

Funding

This research did not receive any specific Grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declarations

Conflict of interest

The authors declare no conflict of interest could be perceived as prejudicial to the impartiality of the reported research.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.