GSK467

Demethyltransferase AlkBH1 substrate diversity and relationship to human diseases

Ying Zhang1 · Caiyan Wang1

Abstract

AlkBH1 is a member of the AlkB superfamily which are kinds of Fe (II) and α-ketoglutarate (α-KG)-dependent dioxygenases. At present, only demethyltransferases FTO and AlkBH5 have relatively clear substrate studies among these members, the types and mechanisms of substrates catalysis of other members are not clear, especially the demethyltransferase AlkBH1. AlkBH1, as a demethylase, has important functions of reversing DNA methylation and repairing DNA damage. And it has become a promising target for the treatment of many cancers, the regulation of neurological and genetic related diseases. Many scholars have made important discoveries in the diversity of AlkBH1 substrates, but there is no comprehensive sum- mary, which affects the design inhibitor target of AlkBH1. Herein, We are absorbed in the latest progress in the study of AlkBH1 substrate diversity and its relationship with human diseases. Besides, we also discuss future research directions and suggest other studies to reveal the specific catalytic effect of AlkBH1 on cancer substrates.

Keywords Demethyltransferase · AlkBH1 · Substrate diversity · Tumor progression

Introduction

The AlkB family was first discovered in E.coli in 1977 [1], and it’s a dioxygenase that relies on Fe (II) and α-ketoglutarate(α-KG), which oxidizes and catalyzes the demethylation of nucleic acid bases [2]. Many studies have shown that abnormal methylation of nucleic acids can lead to the accumulation of cytotoxicity and the generation of oncogenic mutations [3]. However, the three members of the AlkB family (AlkBH1, AlkBH5 and FTO) have a dem- ethylation effect and can reverse the phenomenon of nucleic acid methylation, thereby inhibiting the accumulation of cytotoxicity and the appearance of cancers [4]. AlkBH1 has been identified as a demethylase that can repair methylated DNA base damage in mammalian genomes. And some sci- entists pointed out that AlkBH1 as a potential target of can- cer chemotherapy is highly expressed in a variety of tumors [5, 6].
To date, there are nine mammalian AlkB homologs that can perform monovalent and divalent oxidative dealkyla- tion of double-stranded(ds-)DNA/RNA substrates to com- pletes the methylation or demethylation modification [2, 7, 8]. However, their catalytic goals are different. Such as, FTO catalyzes the removal of 6-methyladenosine (6 mA) [9, 10], 3-methyluracil (m3U) and 3-methylthymidine (m3T) in DNA and RNA [11], while AlkBH5 selects RNA N6-methy- ladenosine (m6A) demethylated [12, 13]. AlkBH3 has high repair activity for single-stranded(ss-) nucleic acid meth- ylation damage (3-methylcytidine, m3C), and hardly affects ds-nucleic acid substrates [14, 15]. In contrast, AlkBH2 showed good repair activity against endogenous ds-DNA methylation damage [16]. Different member substrates have different specificity and participate in gene regulation in dif- ferent ways [17, 18]. Studies have found that AlkBH1 has different types of catalytic substrates and different func- tions, which can directly catalyze the removal of m6A, 5-methylcytosine (m5C), m3C, N1-methyladenosine (m1A), N6-methyladenine(N6-mA) and amounting studies shown that the catalytic role of AlkBH1 with different substrates is associated with the emergence of human diseases [19–21]. According to the biochemical characterization analysis of researchers at the University of Chicago, AlkBH1 has dem- ethylation activity on m1A methylation in tRNA in vitro. It was also shown that AlkBH1 controls tRNAiMet to influence the initiation of translation, and that m1A methylated tRNAs are preferentially recruited to polysomes to promote transla- tion elongation [22]. AlkBH1, as m6A demethylase, has a regulatory effect on the metastasis of gastric cancer cells. In vivo and in vitro experiments have proved that AlkBH1 has the function of inhibiting the invasion and metastasis of gastric cancer cells [21]. On the other hand, the development of selective inhibitors related to AlkBH1 demethylase has become a new direction to clarify its detailed functions and the treatments of AlkBH1 related diseases. However, in the current studies, the mechanism of action of AlkBH1 cata- lytic substrates is still unclear. This will directly affect the research of AlkBH1 specific inhibitors. In this case, we sum- marize all types of AlkBH1 substrates found, further clarify the diversity of substrates and their relationship with human diseases, and explain the existing mechanisms of catalytic substrates. In addition, we found that there are currently no specific inhibitors of the demethyltransferase AlkBH1, only Fe(II) chelating agents or non-selective α-KG analogs. So we detail some of the latest AlkBH1 demethylase inhibitors and hope to provide ideas for the optimization of subsequent screening.
Taken together, AlkBH1 is one of the most widely studied demethylation transferases in the AlkB family. Further research on the relationship between the substrates diversity and specificity of AlkBH1 and human diseases will bring important physiological insights, which also provides essen- tial evidences for the screening and optimizating specific inhibitors for AlkBH1-related diseases.

AlkBH1 catalytic RNA substrate types

m3C

According to literature research, the demethyltransferase AlkBH1 has the function of catalyzing RNA m3C [23]. m3C is the methylation at position 3 of the N atom of RNA cytosine, which is mainly distributed in mRNA, tRNA and rRNA, and it’s essential in cancers development and epige- netic diseases [15, 24]. In the current scientific researches, it is found that methyltransferase METTL8 can undergo methylation transfer [25]. Biochemical studies have shown that METTL8 mainly drives tumorigenesis by affecting the genetic organization within the nucleolus, and regulates the formation of R-loop through its enzyme catalysis of m3C [24]. In addition, current studies have found that AlkBH1 has the effect of demethylating m3C of mRNA, and has relatively weak demethylation activity on ss-DNA and ss- RNA of the substrate m3C [23, 26]. The overexpression of AlkBH1 in cultured human cells leads to a decrease in m3C levels, and vice versa. This proves the erasure characteristics of AlkBH1 on m3C.

m5C

According to reports, AlkBH1 can catalyze RNA 5-methyl- cytosine (m5C) [27]. m5C is the methylation at position 5 of the N atom of cytosine. Studies have found that m5C modification of RNA is essentially ubiquitous and the bio- logical processes involved in RNA processing, stress and the like [28, 29]. m5C is modified in different RNAs and widely exists in tRNA and rRNA, and studies have found that the enzymes involved in catalyzing RNA m5C are the methyl- transferases METTL3 and METTL14, but the expression levels of these two enzymes in m5C are different [30–32]. However, recent studies have shown that sequencing the transcriptomes of seven human tissues and ten mouse tissues found that the number of mRNA m5C sites and methylation levels in different tissues are significantly different [33, 34]. There are hundreds of high-confidence m5C sites in human and mouse testis, liver, heart, and skeletal muscle [35]. In addition, these m5C sites are not conserved between humans and mice, indicating that their regulation may be dynamic regulation. At the same time, previous studies have shown that m5C on mRNA has unique sequence and structural features, and has a tendency to depend on the m5C site of NSUN2. Because it has a 3′G-rich motif, it is usually located at the bottom of the small neck loop structure, so it is highly consistent with the tRNA substrates [36]. m5C regulates mRNA stability through YBX1, a new binding protein in the cytoplasm, and further regulates the proliferation and metastasis of bladder cancer [34, 35]. The research on the regulation mechanism of m5C also lays the foundation for the study of specific inhibitors of AlkBH1.

m1A

As one of the catalytic substrates of AlkBH1, m1A is also a new type of RNA methylation [22, 37]. It is the meth- ylation modification of the N atom at position 1 of the adenine RNA molecule. Studies have shown that m1A is a post-transcriptional modification in eukaryotes, in which the content of tRNA and rRNA is high [33, 38]. As a new type of RNA methylation, m1A modification needs to be explored urgently for its function and mechanism. And m1A is a reversible and dynamic modification revealed by full transcriptome mapping [39]. And revealed an important feature of gather in the 5′-untranslated regions (5′UTRs) of mRNA, which is similar to the N6-methy- ladenosine of internal mammalian mRNA modification [40]. AlkBH1 regulates the methylation process of m1A modified by the methylases METTL3, METTL14 and WTAP in RNA, so that the demethylation process of m1A occurs (Fig. 1). It has been demonstrated that m1A has a dynamic response to facilitated and induced m1A sites have been identified [22]. In summary, there are several methods available to provide comprehensively analyze m1A modification and study the potential functions of epigenetic regulation through m1A. Initially, it was discov- ered that m1A is a chemical modification widely present in tRNA and rRNA, and has important regulatory effects on the biological function of RNA [41]. Co-immunoprecipi- tation with specific m1A antibodies combined with more than 1000 m1A modification sites on human cell mRNA [42]. m1A is specifically distributed on the mRNA at the start of the translation region and near the first splice [43]. Furthermore, this m1A modification can change the sec- ondary structure of mRNA. For example, m1A located at 5′UTRs can increase the translation efficiency by reducing the stability of mRNA secondary structure [40].
In the study of AlkBH1-mediated tRNA demethylation to regulate translation, AlkBH1 can catalyze tRNA m1A demethylation [22, 37]. By CLIP sequencing and tRNA sequencing analysis, the researchers found that AlkBH1 binds to tRNA containing the m1A58 site. And biochemical characterization analysis shows that AlkBH1 has demethyla- tion activity on tRNA m1A in vitro [37]. Besides, AlkBH1 controls the initial phase of tRNAiMet affecting translation, and tRNA methylated by m1A is preferentially recruited into polysomes to accelerate translation extension [37, 44]. The control of tRNA demethylation involved in the regulation of AlkBH1 may affect protein synthesis [37]. In this study, it was discovered that the mechanism that AlkBH1 participates in the regulation of proteins can be used in the response of human cells to glucose deficiency.

m6A

m6A is the most abundant modification on AlkBH1 RNA. The modification of m6A refers to the demethylation modi- fication of the N atom at position 6 of the adenine RNA mol- ecule m6A [45]. Demethylation was changed from 6-methy- ladenosine to adenosine by AlkBH1 (Fig. 2). Among the demethylation modifications of AlkBH1, it is found that RNA methylation modifications account for more than 60% of all RNA modifications, and m6A is often found in mRNA and LncRNA modifications in high organism. Besides, microRNA, rRNA and tRNA also have m6A modification [21, 45]. The modification of m6A mainly occurs on adenine in the RRACH sequence, and its function is determined by “Writer”, “Eraser” and “Reader”. “Writer” is methyltrans- ferase and currently known components of it are METTL3, METTL14, WTAP and KIAA1429 [46, 47]. AlkBH5 and FTO as “Erasers” are demethylases that can reverse meth- ylated. m6A is bound by m6A binding protein recognition (Readers), including YTH domain proteins and nuclear het- erogeneous proteins HNRNP family (Fig. 3).
Increasing evidence have shown that the modification of m6A plays a vital biological function in mammals. The modification of m6A involves almost all aspects of RNA metabolism and transcriptome analysis of m6A gene showed that there was a certain connection between the pathway and RNA metabolism [48]. And based on the localization of methyltransferase and demethylase, m6A can be introduced or removed in the nucleus or cytoplasm. m6A may have dif- ferent effects on RNA depending on whether it present in the nucleus or cytoplasm. Currently, several functions of m6A have been confirmed. For example, affect the shearing of mRNA precursors, regulate the nuclear export of RNA, reg- ulate the translation of mRNA, affect the stability of mRNA and so on [49, 50]. Some research data have found that the methylation of pri-miRNA relies on METTL3 to promote the recognition and processing of DGCR, thereby promot- ing the maturation of miro-RNA [51]. In addition, the m6A recognition protein HNNP2B1 promotes the processing of pri-miRNA into pre-miRNA (Fig. 4). The m6A modifica- tion has important significance in gene expression, and its abnormal regulation mechanism may be related to the devel- opment of many diseases [52]. In addition, m6A may affect sperm development, UV-induced DNA damage response, tumor formation or metastasis, stem cells self-renewal, fat differentiation, biological rhythms, cell division and other life processes [47, 53].
In the entire enzyme system of m6A, its methyltrans- ferase METTL3 is the earliest identified enzyme that binds to S-adenosyl methionine [54, 55]. Besides, the deletion of METTL3 caused a decrease in the m6A peak in mouse embryonic stem cells and Hela cells. METTL3 and its homologous protein METTL14 were located on subcellular organelle-nuclear plaques in the nucleus rich in splicing fac- tor, showing that m6A modification may be related to RNA splicing [56, 57]. Under the action of the demethyltrans- ferase AlkBH1, the expression level of m6A is related to the metastasis of head and neck cancers [58]. Therefore, m6A as a substrate of AlkBH1 is expected to become a target for cancer treatment.

AlkBH1 catalytic DNA substrate types

6mA

N6-methyladenine (6mA), as one of the new catalytic substrates of demethyltransferase AlkBH1, mainly exists in DNA for methylation modification [59]. 6mA refers to the process of methylation at position 6 of the N atom of adenine, which occupies an important position in DNA methylation modification. The methylation modification of eukaryotic DNA is mainly 5mC, and the 6mA of DNA in prokaryotes is more. As common, it is used as a restriction repair system to maintain mismatch repair in prokaryotic DNA replication. DNA methylation plays a significant role in epigenetics, and is involved in the regulation of genome imprinting, X chromosome inactivation, transposon sup- pression, gene expression, maintenance of genetic memory, embryonic development, and tumor production [60]. In the study of 6mA related enzymes, the study of methyltrans- ferase N6AMT1 and methyltransferase AlkBH1 found that the 6mA modification of DNA by N6AMT1 and AlkBH1 is related to the occurrence of tumors [59]. The 6mA modifi- cation of DNA was significantly reduced in tumors, accom- panied by low expression of N6AMT1 and high expression of AlkBH1. Interference with N6AMT1 or overexpression of AlkBH1 in human tumor cells can promote tumor devel- opment, and AlkBH1 can repair 6mA DNA methylation. However, the 6mA modification accounts for 0.051% of the total adenine in the genome. [G/C] AGG [C/T] is the most relevant area for 6mA modification [59]. The 6mA sites are concentrated in the coding region and greatly activate gene transcription. In the past few years, sequencing technology has found 6mA DNA modifications in eukaryotes, including Chlamydomonas, Drosophila melanogaster, Mus musculus [61, 62].

N6‑mA

N6-methyladenine(N6-mA) is another DNA methylation modification base besides 5mC in eukaryotic genome [63]. The level of genomic N6-mA shows dramatic fluctuations during the development of early embryonic and cancers. The regulatory mechanism of his new modified base is the key to decoding the biological function of it. Previously, AlkBH1 was reported as a DNA N6-mA demethylase. However, in the latest research, the demethylase activity of various nucleic acid substrates of AlkBH1 was systemati- cally detected by establishing a quantitative enzyme activ- ity detection system based on LC–MS/MS. Further more, through the study of the structure of AlkBH1 and DNA complex, it was found that AlkBH1 prefers to catalyze the demethylation of N6-mA in bubbled DNA rather than ss- DNA [63]. Further experiments show that AlkBH1 can also efficiently catalyze a variety of local unpaired nucleic acids demethylation of N6-mA in bulge, R-loop, D-loop, and stem loop, etc. It clearly revealed that AlkBH1 highly relies on the secondary structure of the substrates [64, 65].
The study found that the unique substrate preference of AlkBH1 is based on its unique structural basis, while other members of the AlkB family and the Tet family did not show preference for local unpaired nucleic acids. Through analyzing the monomer structure of AlkBH1, it was found that AlkBH1 is composed of a catalytic domain (DSBH) at the C-terminus, an adjacent substrate nucltide recogni- tion lid (NRL), and an N-terminal extension region (NTE) [65]. The NRL subdomain is thinly divided into Flip1 and Flip2. The study found that the Flip1 was far away from the center of enzyme activity and completely turned out, which is very different members of same family. Based on structural analysis and binding experiments, the researchers speculated that the flipped Flip1 may cause AlkBH1 to fail to act on double strended substrates, but played an impor- tant role in identifying the double-stranded regions of local unpaired substrates [66].
After analyzing the structure of the DNA complex of AlkBH1, it is found that when AlkBH1 binds to the sub- strate, its Flip1 still remains completely eversion, resulting in its inability to actively eversion N6-mA in the substrate. Unable to catalyze the double-stranded demethylation of the substrate. The flipped Flip1 and α1 are located on both sides of the catalytic center, interacting with the double- stranded zones on the left and right sides of the protrusion, respectively. The researchers further performed DNA Immu- noprecipitation Sequencing (DIP-seq) and single-stranded DNA-seq analysis on mouse early embryonic developmen- tal cells. The results showed that N6-mA colocated sig- nificantly with unpaired regions of the genome, suggesting that the regulation of N6-mA may be related to eukaryotic chromosomes [67]. The dynamic changes of the high level structure of chromosomes are closely related and provide a basis for explaining the occurrence and development of many AlkBH1 modified N6-mA related diseases [63].
Taken together, although AlkBH1 is the first member of its family, the substrates diversity and specificity of AlkBH1 have been controversial since its extensive sub- strate spectrum exceeds the damage repair reported in researches. Recently, AlkBH1 was reported as a N6-mA erasing agent for ss-DNA substrates. Biochemical analy- sis and structural analysis provide key informations for the new features of the AlkBH1 substrate, which is character- ized by a local unpaired structure that contains N6-mA base flips with flanking double helices [68, 69]. To sum up, the current research indicates that AlkBH1 can directly cata- lyze five substrates: m6A, m1A, m3C, m5C, and N6-mA. Even though the research on the catalytic mechanism of the above substrates is not very clear, even AlkBH1 has little research on substrate specificity, but more and more studies will provide the basis for exploring the catalytic substrate mechanism.

Molecular mechanisms involved in substrate catalysis

AlkB family proteins have important functions to repair cer- tain diseases caused by DNA or RNA damage. For example, the level of 6mA in the genome exhibits dramatic fluctua- tions during early embryonic development and cancer devel- opment, leading to the occurrence of some diseases [70, 71]. Interestingly, it is reported that AlkBH1 has demethylating activity on four other substrates, such as histone H2A, m3C on DNA and RNA, and m5C or m1A on tRNA [72]. In February 2020, related studies with AlkBH1 revealed the structural basis of AlkBH1’s preference for locally unpaired nucleic acid substrates, and provided new research prospects for the regulatory biology of the controversial 6mA modifi- cation in mammalian genomes. These findings include that it helps to further understand the process of DNA apparent modification and the development of drugs for related dis- eases. However, the controversy surrounding the biologi- cal significance and function of DNA 6mA modification in mammals and other lower organisms such as nematodes and fruit flies has not been resolved. The current methodology surrounding DNA 6mA whole genome detection still faces considerable challenges. This field still urgently needs more research to clarify the true face of science.
At present, it is found that AlkBH2 and AlkBH3, members of the AlkB family, have obvious catalytic effects on the substrate. AlkBH2 and AlkBH3 can effectively eliminate the damage of m1A and m3C in the genome, and avoid the accumulation of cytotoxicity caused by blocked gene tran- scription and replication. Even though AlkBH1 has little research on the catalytic mechanism of substrates and the specificity of substrates.

Demethyltransferase AlkBH1 and human diseases

Glioblastoma

According to researches, the AlkBH1 gene has an important relationship with epigenetic [21, 64]. However, this epige- netic signature implies human diseases, especially highly malignant brain cancer and glioblastoma. AlkBH1 plays an important role in gliomas. Studies have shown that the level of N6-mA in glioblastoma is significantly increased, which coexisted with heterochromatic histone modifications (mainly H3K9me3) [64]. And N6-mA levels are dynamically regulated by the DNA demethylase AlkBH1 which depletion results in transcriptional silencing of the oncogenic pathway by reducing access to chromatin. Targeting N6-mA modula- tor AlkBH1 in patient-derived human glioblastoma models inhibits tumor cells proliferation and prolongs survival of tumor mice, supporting this new DNA modification as a glioblastoma potentialtherapeutic target [73]. It is proved that human glioma cells have abundant N6-mA modifica- tions in the genome, and N6-mA modifications are involved in the development of cancers, and it was found that target- ing N6-mA demethylase AlkBH1 is expected to become a new strategy for treating gliomas [64]. Collectively, many results about AlkBH1 will uncover a novel epigenetic node in cancer through the DNA modification N6-mA.

Relationship between AlkBH1 and other cancers

After reducing the expression level of AlkBH1 gene in the tumor tissues of gastric cancer patients, it was found that the proliferation ability and migration rate of gastric cancer related cell lines were improved, and reduces the survival rate of gastric cancer patients [21, 74]. These studies indi- cate that the low expression of AlkBH1 in gastric cancer tissue has a certain correlation with the development and prognosis of gastric cancer patients. In other AlkBH related cancer studies, researchers have constructed liver and ovar- ian cancer cell lines with AlkBH1 gene overexpression/ silence, compared with the control group, its DNA 6 mA modification level, proliferation ability, cell migration rate and cell invasion ability were all significant changes have occurred [74]. More and more studies show that AlkBH1 gene and its expression products are used as tumor mark- ers to make tumor diagnosis more accurate and faster [23, 58, 75]. As potential anti-tumor target genes, they provide new therapeutic approaches for tumor treatment. With the deepening of tumor researches, it has been discovered that epigenetic modification, especially DNA methylation, plays an key role in tumorigenesis and development. DNA meth- ylation refers to a process in which an organism transfers a methyl group to a specific base under the catalysis of a DNA methyltransferase [76]. However, the AlkB superfam- ily has a demethylation function, which can reverse DNA methylation, thereby repairing DNA damage and treating certain methylation-modified diseases [77]. Overexpression of N6AMT1 or AlkBH1 in liver cancer cells and ovarian cancer cells can reduce the 6 mA level of genomic DNA, thereby promoting cell proliferation,colony formation, migration, invasion and tumor formation in nude mice [59]. In short, the abnormal expression of AlkBH1 is related to the occurrence of many cancers and the prognosis of the diseases [59].

Neurological diseases

Research suggests that AlkBH1 has an important relation- ship with neurological diseases [64]. And m6A is one of the most abundantly modified substrates of AlkBH1, it can play an irreplaceable regulatory role in the development of the nervous system. Previous studies have found that m6A is more abundant in D. melanogaster [78]. The D. mela- nogaster knockout of the m6A modified gene Imet4 can be born and survive, but the D. melanogaster have a short lifespan and exhibit obvious behavioral abnormalities. In addition, the overexpression or silencing of the AlkBH1 will also affect the expression level of m6A, and then promote or inhibit the development of mouse cerebellum [79]. This indicates that m6A is used as a substrate for modification, even in different genes and different species. Another exam- ple the knockout of the methyltransferase METTL14, which interacts with the demethyltransferase AlkBH1 in the nerv- ous system of mice, will severely affect the development of mouse cerebral cortex [80]. Deletion of the AlkBH1 bound YTHDF2 gene in mice led to an increase in overall m6A levels, which prevented participation in neural stem cell differentiation and neuronal axon dendrites from entering the RNA degradation pathway [81]. This RNA degradation pathway seriously affects the differentiation of neurons, leading to slow development of the mouse forebrain and cerebral cortex [82]. In addition to the cerebral cortex, the methylation modification and level of cerebellar RNA m6A are more prominent. The dynamic process of m6A methyla- tion and demmethylation runs through the whole process of postpartum cerebellar development. The deletion of AlkBH1 gene under hypotension and hypoxia causes m6A to par- ticipate in disorderly cerebellar development and accelerate RNA nucleation [26]. This process directly leads to a signifi- cant delay in cerebellar development. All the above research results indicate that the AlkBH1 gene has an essential func- tion in the mammalian nervous system, and its deletion and overexpression may cause abnormal nervous system diseases [83].

Genetic related diseases

In recent years, the correlation between epigenetic modifica- tion and the occurrence of complex human diseases has been newly recognized. Epigenetic reversibility provides a new therapeutic direction for treating diseases. The researchers point out that AlkBH1 is a gene encoding m6A demethylase. In cells deficient in the AlkBH1 gene, increased m6A lev- els lead to transcriptional silencing [84]. m6A deposition is inversely related to the evolutionary period of LINE-1 trans- poson. When m6A is deposited, m6A deposition is related to the epigenetic silencing of LINE-1 transposons and neigh- boring enhancers and genes, thereby resisting gene activa- tion signals during stem cell differentiation. Because the full-length LINE-1 transposons are abundant on the X chro- mosome, genes on the X chromosome are usually silenced. Researchers have found that even though m6A’s effect on epigenetic silencing is different from other genes, and the effect of m6A on the evolution of mammalian silenced is dif- ferent from the effect of gene activation in other organisms [78]. Studies have shown that m6A is one of the important components of epigenetic regulation. The effect of m6A on epigenetic silencing is closely related to the occurrence and development of many human diseases, especially cancers and genetic diseases with high morbidity and mortality [85].

Inhibitor design and target therapy

AlkBH1 has been shown to be involved in the occurrence and development of a variety of human diseases, especially different types of cancer and neurological diseases [21, 64, 86]. However, the research on using enzymes as small mol- ecule inhibitors to repair alkylation damage is still in its infancy. If some purposeful chemical motifs can be found to increase or decrease the activity of alkylation injury, the potential clinical value can be explored by changing the expression of genetic information. Based on this view, we will summarize some of the latest AlkBH1 inhibitors, and hope to provide an idea for the optimization of subsequent screening. In recent years, the research on specific inhibitors of AlkBH1 has not made breakthrough progress. However, the inhibitors that have been found against AlkBH1 are also Fe (II) chelator and analogues of α-KG [87]. Substances in the AlkB superfamily that have a relative inhibitory effect on AlkBH1 will be summarized.
Studies have found that 4-[N′-(4-Benzyl-pyridine-3- carbonyl)-hydrazino]-4-oxo-but-2-enoicacid is a special “two-component” possibility it also occupies substrate bind- ing inhibitors [88]. However, it has high selective inhibitory activity against FTO in the AlkB superfamily compared to other AlkB subfamily members. The analysis of the struc- tural activity commonality of AlkB shows that the pyridine ring is connected to Glu234, and the acyl tail is similar in structure to NOG and occupies the α-KG site to complete the inhibitory effect of the two components [89, 90]. In 2018, researchers reported another selective inhibitor that uses a multi-protein dynamic combinatorial chemistry strat- egy with an acylsulfur-like backbone [91]. In the calcula- tion model, the two 11 N atoms coordinate with Fe (II) in a bidentate manner, mimicking the α-KG bonding mode. However, this inhibitor also has a certain inhibitory effect on AlkBH1 family members, but the inhibitory effect is stronger than FTO [87].
Different modifications of nucleic acid epigenetics play a vital role in gene regulation and the normal function of human body [92]. Therefore, it will have a certain impact on the occurrence and development of human diseases. Studies have found that the AlkB family affects diseases by repair- ing reversible oxidative dealkylation. Today, several AlkB related enzymes are being targeted for the treatment of cer- tain diseases. However, apart from the successful examples of FTO, AlkBH3 and AlkBH5 demethylases, no specific inhibitors of AlkBH1 have been found. And for some poten- tial clients the selectivity of enzymes is still poor, and drug screening clues for AlkBH1 are still in the experimental stage. In summary, even though a variety of inhibitors have been found in the AlkB family, the inhibitors of AIkBH1 currently found are mostly α-KG analogs, there are no spe- cific inhibitors, and the inhibitory effect is not particularly obvious. Therefore, finding and optimizing specific inhibi- tors are essential for the prevention and treatment of related clinical diseases.

Conclusion

In recent years, study on AlkB protein’s function has achieved many results. The relationship between mamma- lian AlkB family and related diseases in revealing impor- tant epigenetic regulatory mechanisms of embryogenesis and differentiation. And found that the substrates catalyzed by various members of the AlkB superfamily have impor- tant links with many human diseases. As current research has shown that AlkBH1, is used as tRNA and DNA dem- ethylase and has been linked to a variety of diseases such as diabetes and various cancers. At the same time, AlkBH1 DNA demethylase may be a potential marker of cancer. However, the catalytic mechanism of AlkBH1 substrate is limited, and some reporting mechanisms are controver- sial or unclear. Therefore, related research is necessary to improve our current understanding and more in vivo studies are needed. In a word, the exact function and mechanism of the comprehensive study of the AlkBH1 family has high clinical value and is worth establishing in the future.

References:

1. Mishina Y, He C (2006) Oxidative dealkylation DNA repair medi- ated by the mononuclear non-heme iron AlkB proteins. J Inorg Biochem 100:670–678
2. Fedeles BI, Singh V, Delaney JC, Li D, Essigmann JM (2015) The AlkB family of Fe(II)/alpha-ketoglutarate-dependent dioxy- genases: repairing nucleic acid alkylation damage and beyond. J Biol Chem 290:20734–20742
3. Guengerich FP (2015) Introduction: metals in biology: alpha- ketoglutarate/Iron-dependent dioxygenases. J Biol Chem 290:20700–20701
4. Welford RW, Kirkpatrick JM, McNeill LA, Puri M, Oldham NJ, Schofield CJ (2005) Incorporation of oxygen into the succinate co-product of iron(II) and 2-oxoglutarate dependent oxygenases from bacteria, plants and humans. FEBS Lett 579:5170–5174
5. Rudkjobing LA, Eiberg H, Mikkelsen HB, Binderup ML, Bis- gaard ML (2015) The analysis of a large Danish family supports the presence of a susceptibility locus for adenoma and colorectal cancer on chromosome 11q24. Fam Cancer 14:393–400
6. Shivange G, Kodipelli N, Anindya R (2014) A nonradioactive restriction enzyme-mediated assay to detect DNA repair by Fe(II)/2-oxoglutarate-dependent dioxygenase. Anal Biochem 465:35–37
7. Brickner JR, Soll JM, Lombardi PM et al (2017) A ubiquitin- dependent signalling axis specific for ALKBH-mediated DNA dealkylation repair. Nature 551:389–393
8. Li Q, Huang Y, Liu X, Gan J, Chen H, Yang CG (2016) Rhein inhibits AlkB repair enzymes and sensitizes cells to methylated DNA damage. J Biol Chem 291:11083–11093
9. Martin Carli JF, LeDuc CA, Zhang Y, Stratigopoulos G, Leibel RL (2018) FTO mediates cell-autonomous effects on adipogen- esis and adipocyte lipid content by regulating gene expression via 6mA DNA modifications. J Lipid Res 59:1446–1460
10. Zhang X, Wei LH, Wang Y et al (2019) Structural insights into FTO’s catalytic mechanism for the demethylation of multiple RNA substrates. Proc Natl Acad Sci 116:2919–2924
11. Zhu Y, Zhou G, Yu X et al (2017) LC-MS–MS quantitative analy- sis reveals the association between FTO and DNA methylation. PLoS ONE 12:e0175849
12. Jin D, Guo J, Wu Y et al (2020) m(6)A demethylase ALKBH5 inhibits tumor growth and metastasis by reducing YTHDFs-medi- ated YAP expression and inhibiting miR-107/LATS2-mediated YAP activity in NSCLC. Mol Cancer 19:40
13. Chen S, Zhou L, Wang Y (2020) ALKBH5-mediated m(6)A dem- ethylation of lncRNA PVT1 plays an oncogenic role in osteosar- coma. Cancer Cell Int 20:34
14. Woo HH, Chambers SK (2019) Human ALKBH3-induced m(1)A demethylation increases the CSF-1 mRNA stability in breast and ovarian cancer cells. Biochim Biophys Acta Gene Regul Mech 1862:35–46
15. Chen Z, Qi M, Shen B et al (2019) Transfer RNA demethylase ALKBH3 promotes cancer progression via induction of tRNA- derived small RNAs. Nucleic Acids Res 47:2533–2545
16. Yi C, Chen B, Qi B et al (2012) Duplex interrogation by a direct DNA repair protein in search of base damage. Nat Struct Mol Biol 19:671–676
17. Ji N, Wang X, Yin C, Peng W, Liang R (2019) CrgA protein represses AlkB2 monooxygenase and regulates the degradation of medium-to-long-chain n-alkanes in pseudomonas aeruginosa SJTD-1. Front Microbiol 10:400
18. Nilsen A, Fusser M, Greggains G, Fedorcsak P, Klungland A (2014) ALKBH4 depletion in mice leads to spermatogenic defects. PLoS ONE 9:e105113
19. Ougland R, Rognes T, Klungland A, Larsen E (2015) Non-homol- ogous functions of the AlkB homologs. J Mol Cell Biol 7:494–504
20. Pilzys T, Marcinkowski M, Kukwa W et al (2019) ALKBH over- expression in head and neck cancer: potential target for novel anticancer therapy. Sci Rep 9:13249
21. Li Y, Zheng D, Wang F, Xu Y, Yu H, Zhang H (2019) Expres- sion of demethylase genes, FTO and ALKBH1, is associated with prognosis of gastric cancer. Dig Dis Sci 64:1503–1513
22. Kawarada L, Suzuki T, Ohira T, Hirata S, Miyauchi K, Suzuki T (2017) ALKBH1 is an RNA dioxygenase responsible for cytoplas- mic and mitochondrial tRNA modifications. Nucleic Acids Res 45:7401–7415
23. Ma CJ, Ding JH, Ye TT, Yuan BF, Feng YQ (2019) AlkB homo- logue 1 demethylates N(3)-methylcytidine in mRNA of mammals. ACS Chem Biol 14:1418–1425
24. Xu L, Liu X, Sheng N et al (2017) Three distinct 3-methylcytidine (m(3)C) methyltransferases modify tRNA and mRNA in mice and humans. J Biol Chem 292:14695–14703
25. Zhang LH, Zhang XY, Hu T et al (2020) The SUMOylated METTL8 induces R-loop and tumorigenesis via m3C. iScience 23:100968
26. Wagner A, Hofmeister O, Rolland SG et al (2019) Mitochondrial Alkbh1 localizes to mtRNA granules and its knockdown induces the mitochondrial UPR in humans and C. elegans. J Cell Sci. https://doi.org/10.1242/jcs.223891
27. Haag S, Sloan KE, Ranjan N et al (2016) NSUN3 and ABH1 modify GSK467 the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation. EMBO J 35:2104–2119
28. Auxilien S, Guerineau V, Szweykowska-Kulinska Z, Golinelli- Pimpaneau B (2012) The human tRNA m (5) C methyltransferase Misu is multisite-specific. RNA Biol 9:1331–1338
29. Van Haute L, Dietmann S, Kremer L et al (2016) Deficient meth- ylation and formylation of mt-tRNA(Met) wobble cytosine in a patient carrying mutations in NSUN3. Nat Commun 7:12039
30. Li Q, Li X, Tang H et al (2017) NSUN2-mediated m5C methyla- tion and METTL3/METTL14-mediated m6A methylation coop- eratively enhance p21 translation. J Cell Biochem 118:2587–2598
31. Dong Z, Cui H (2020) The emerging roles of RNA modifications in glioblastoma. Cancers 12:736
32. Sibbritt T, Patel HR, Preiss T (2013) Mapping and significance of the mRNA methylome. Wiley Interdiscip Rev RNA 4:397–422
33. Zhao BS, Roundtree IA, He C (2017) Post-transcriptional gene regulation by mRNA modifications. Nat Rev Mol Cell Biol 18:31–42
34. Yang Y, Wang L, Han X et al (2019) RNA 5-methylcytosine facili- tates the maternal-to-zygotic transition by preventing maternal mRNA decay. Mol Cell 75:1188-1202 e1111
35. Chen X, Li A, Sun BF et al (2019) 5-Methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs. Nat Cell Biol 21:978–990
36. Yang X, Yang Y, Sun BF et al (2017) 5-Methylcytosine promotes mRNA export—NSUN2 as the methyltransferase and ALYREF as an m(5)C reader. Cell Res 27:606–625
37. Liu F, Clark W, Luo G et al (2016) ALKBH1-mediated tRNA demethylation regulates translation. Cell 167:816-828 e816
38. Oerum S, Degut C, Barraud P, Tisne C (2017) m1A Post-tran- scriptional modification in tRNAs. Biomolecules 7:20
39. Li X, Xiong X, Wang K et al (2016) Transcriptome-wide mapping reveals reversible and dynamic N(1)-methyladenosine methylome. Nat Chem Biol 12:311–316
40. Grozhik AV, Olarerin-George AO, Sindelar M, Li X, Gross SS, Jaffrey SR (2019) Antibody cross-reactivity accounts for wide- spread appearance of m(1)A in 5′UTRs. Nat Commun 10:5126
41. Xiong X, Li X, Yi C (2018) N(1)-methyladenosine methylome in messenger RNA and non-coding RNA. Curr Opin Chem Biol 45:179–186
42. Safra M, Sas-Chen A, Nir R et al (2017) The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution. Nature 551:251–255
43. Peer E, Rechavi G, Dominissini D (2017) Epitranscriptomics: regulation of mRNA metabolism through modifications. Curr Opin Chem Biol 41:93–98
44. Rashad S, Han X, Sato K et al (2020) The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation. RNA Biol 17:1092–1103
45. Wu L, Pei Y, Zhu Y et al (2019) Association of N(6)-methylad- enine DNA with plaque progression in atherosclerosis via myo- cardial infarction-associated transcripts. Cell Death Dis 10:909
46. Lin S, Choe J, Du P, Triboulet R, Gregory RI (2016) The m(6)A methyltransferase METTL3 promotes translation in human cancer cells. Mol Cell 62:335–345
47. Chen M, Wei L, Law CT et al (2018) RNA N6-methyladeno- sine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2. Hepatology 67:2254–2270
48. Dai D, Wang H, Zhu L, Jin H, Wang X (2018) N6-methyladeno- sine links RNA metabolism to cancer progression. Cell Death Dis 9:124
49. Yu J, Chen M, Huang H et al (2018) Dynamic m6A modification regulates local translation of mRNA in axons. Nucleic Acids Res 46:1412–1423
50. Linder B, Grozhik AV, Olarerin-George AO, Meydan C, Mason CE, Jaffrey SR (2015) Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods 12:767–772
51. Erson-Bensan AE, Begik O (2017) m6A modification and impli- cations for microRNAs. Microrna 6:97–101
52. Liu ZX, Li LM, Sun HL, Liu SM (2018) Link between m6A modi- fication and cancers. Front Bioeng Biotechnol 6:89
53. Chen X, Yu C, Guo M et al (2019) Down-regulation of m6A mRNA methylation is involved in dopaminergic neuronal death. ACS Chem Neurosci 10:2355–2363
54. Wang P, Doxtader KA, Nam Y (2016) Structural basis for coop- erative function of Mettl3 and Mettl14 methyltransferases. Mol Cell 63:306–317
55. Bedi RK, Huang D, Eberle SA, Wiedmer L, Sledz P, Caflisch A (2020) Small-molecule inhibitors of METTL3, the major human epitranscriptomic writer. ChemMedChem 15:744–748
56. Sun T, Wu R, Ming L (2019) The role of m6A RNA methylation in cancer. Biomed Pharmacother 112:108613
57. Tang C, Klukovich R, Peng H et al (2018) ALKBH5-dependent m6A demethylation controls splicing and stability of long 3′-UTR mRNAs in male germ cells. Proc Natl Acad Sci 115:E325–E333
58. Pilzys T, Marcinkowski M, Kukwa W, Garbicz D, Dylewska M, Ferenc K (2019) ALKBH overexpression in head and neck cancer: potential target for novel anticancer therapy. Sci Rep 9:13249
59. Xiao CL, Zhu S, He M et al (2018) N(6)-methyladenine DNA modification in the human genome. Mol Cell 71:306-318.e307
60. Fernandes GFS, Silva GDB, Pavan AR, Chiba DE, Chin CM, Dos Santos JL (2017) Epigenetic regulatory mechanisms induced by resveratrol. Nutrients 9:1201
61. Luo GZ, Blanco MA, Greer EL, He C, Shi Y (2015) DNA N(6)- methyladenine: a new epigenetic mark in eukaryotes? Nat Rev Mol Cell Biol 16:705–710
62. Zhang G, Huang H, Liu D et al (2015) N6-methyladenine DNA modification in drosophila. Cell 161:893–906
63. Zhou C, Liu Y (2016) DNA N(6)-methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs. Bone Res 4:16033
64. Xie Q, Wu TP, Gimple RC et al (2018) N(6)-methyladenine DNA modification in glioblastoma. Cell 175:1228-1243.e1220
65. Zhang M, Yang S, Nelakanti R et al (2020) Mammalian ALKBH1 serves as an N(6)-mA demethylase of unpairing DNA. Cell Res 30:197–210
66. Tian LF, Liu YP, Chen L et al (2020) Structural basis of nucleic acid recognition and 6mA demethylation by human ALKBH1. Cell Res 30:272–275
67. Mielecki D, Zugaj DL, Muszewska A et al (2012) Novel AlkB dioxygenases–alternative models for in silico and in vivo studies. PLoS ONE 7:e30588
68. Wu TP, Wang T, Seetin MG et al (2016) DNA methylation on N(6)-adenine in mammalian embryonic stem cells. Nature 532:329–333
69. Xiong J, Ye TT, Ma CJ, Cheng QY, Yuan BF, Feng YQ (2019) N 6-hydroxymethyladenine: a hydroxylation derivative of N6-meth- yladenine in genomic DNA of mammals. Nucleic Acids Res 47:1268–1277
70. Gillingham D, Shahid R (2015) Catalysts for RNA and DNA modification. Curr Opin Chem Biol 25:110–114
71. Wu Y, Zhou C, Yuan Q (2018) Role of DNA and RNA N6-adenine methylation in regulating stem cell fate. Curr Stem Cell Res Ther 13:31–38
72. Zhang C, Jia G (2018) Reversible RNA modification N(1)-meth- yladenosine (m(1)A) in mRNA and tRNA. Genom Proteom Bio- inform 16:155–161
73. Zhou C, Liu Y, Li X, Zou J, Zou S (2016) DNA N(6)-methylad- enine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs. Bone Res 4:16033
74. Wang C, Huang Y, Zhang J, Fang Y (2020) MiRNA-339–5p sup- presses the malignant development of gastric cancer via targeting ALKBH1. Exp Mol Pathol 115:104449
75. Liu Y, Yuan Q, Xie L (2018) The AlkB family of Fe (II)/alpha- ketoglutarate-dependent dioxygenases modulates embryogen- esis through epigenetic regulation. Curr Stem Cell Res Ther 13:136–143
76. Zhou Z, Li HQ, Liu F (2018) DNA methyltransferase inhibi- tors and their therapeutic potential. Curr Top Med Chem 18:2448–2457
77. Roy TW, Bhagwat AS (2007) Kinetic studies of Escherichia coli AlkB using a new fluorescence-based assay for DNA demethyla- tion. Nucleic Acids Res 35:e147
78. Widagdo J, Anggono V (2018) The m6A-epitranscriptomic signa- ture in neurobiology: from neurodevelopment to brain plasticity. J Neurochem 147:137–152
79. Muller TA, Struble SL, Meek K, Hausinger RP (2018) Characteri- zation of human AlkB homolog 1 produced in mammalian cells and demonstration of mitochondrial dysfunction in ALKBH1- deficient cells. Biochem Biophys Res Commun 495:98–103
80. Weng H, Huang H, Wu H et al (2018) METTL14 inhibits hemat- opoietic stem/progenitor differentiation and promotes leukemo- genesis via mRNA m(6)A modification. Cell Stem Cell 22:191- 205 e199
81. Li M, Zhao X, Wang W et al (2018) Ythdf2-mediated m(6)A mRNA clearance modulates neural development in mice. Genome Biol 19:69
82. Paris J, Morgan M, Campos J et al (2019) Targeting the RNA m(6) A reader YTHDF2 selectively compromises cancer stem cells in acute myeloid leukemia. Cell Stem Cell 25:137–148 e136
83. Wakisaka KT, Muraoka Y, Shimizu J et al (2019) Drosophila alpha-ketoglutarate-dependent dioxygenase AlkB is involved in repair from neuronal disorders induced by ultraviolet damage. NeuroReport 30:1039–1047
84. Muller TA, Tobar MA, Perian MN, Hausinger RP (2017) Bio- chemical characterization of AP lyase and m(6)A demethylase activities of human AlkB homologue 1 (ALKBH1). Biochemistry 56:1899–1910
85. Valastyan S, Weinberg RA (2011) Tumor metastasis: molecular insights and evolving paradigms. Cell 147:275–292
86. Ougland R, Jonson I, Moen MN et al (2016) Role of ALKBH1 in the core transcriptional network of embryonic stem cells. Cell Physiol Biochem 38:173–184
87. Welford RW, Schlemminger I, McNeill LA, Hewitson KS, Schof- ield CJ (2003) The selectivity and inhibition of AlkB. J Biol Chem 278:10157–10161
88. Toh JDW, Sun L, Lau LZM et al (2015) A strategy based on nucleotide specificity leads to a subfamily-selective and cell-active inhibitor of N(6)-methyladenosine demethylase FTO. Chem Sci 6:112–122
89. Bian K, Chen F, Humulock ZT, Tang Q, Li D (2017) Copper inhibits the AlkB family DNA repair enzymes under Wilson’s disease condition. Chem Res Toxicol 30:1794–1796
90. Xie LJ, Liu L, Cheng L (2020) Selective inhibitors of AlkB family of nucleic acid demethylases. Biochemistry 59:230–239
91. Das M, Yang T, Dong J et al (2018) Multiprotein dynamic com- binatorial chemistry: a strategy for the simultaneous discovery of subfamily-selective inhibitors for nucleic acid demethylases FTO and ALKBH3. Chem Asian J 13:2854–2867
92. Chen Y, Hong T, Wang S, Mo J, Tian T, Zhou X (2017) Epige- netic modification of nucleic acids: from basic studies to medical applications. Chem Soc Rev 46:2844–2872

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