Figure1. Chk signaling pathway.
The role of the cell cycle checkpoint kinase is to maintain the genome's fidelity by blocking the cell cycle and repairing damaged DNA after DNA damage. Known cell cycle checkpoint kinases include: Chk1 (checkpoint kinase 1) and Chk2 (checkpoint kinase 2). Both of them have overlapping substrate profiles and similar structures but are not expressed equally in cells and tissues of different tumors and other diseases. At present, the applied research of the two is mainly embodied as an important target for tumor cells, using a single agent or a combination of radiotherapy and DNA damage agents to treat tumors and related diseases.
As stated above, the Chk family is mainly composed of Chk1 and Chk2. Structure and function of Chk1: The human Chk1 gene was found on chromosome 11q24. There are currently 7 known transcriptional variants with a molecular weight of 54 kD and 13 exons. The full-length cDNA is 1891 bp, consisting of 476 amino acids. The serine/threonine kinase protein is composed of a protein comprising an N-terminal kinase domain, a variable junction region, a SQ/TQ region, and a C-terminal inhibitory domain. The N-terminus catalyzes the phosphorylation of the substrate region of the kinase, and the C-terminus accepts a phosphorylation-regulated serine/glutamine region. As a very conserved protein kinase in the process of biological evolution, Chk1 can be expressed in the cytoplasm and nucleus of tissues and cells of humans and various organisms, but the main site of action is in the nucleus.
Chk1 plays an important role in DNA damage repair and cell cycle reactivation after damage repair. It participates in the assembly of chromosomes and spindles and is closely related to the structural stability of chromatin. In general, Chk1 has no significant effect on the normal growth of somatic cells, but the mutated Chk1 gene will affect DNA damage repair, such as incomplete DNA replication and apoptosis. Structure and function of Chk2: The human Chk2 gene is located on chromosome 22q12.1. Transcriptional variants with a molecular weight of 60 kD and 14 exons are currently known. The full length of the cDNA is 2061 bp.
The kinase protein acts as a coding protein comprising a serine-glutamate/serine-glutamine amino acid pair (SQ/TQ)-rich region, a fork-related domain (FHA), and a protein kinase domain near the C-terminus (KD). Like Chk1, Chk2 is widely expressed in normal tissue cells, especially in the nucleus, and can be found at various stages of the cell cycle and has significant tissue specificity. As another important signal transduction protein in addition to Chk1 in the DNA damage repair pathway, Chk2 regulates cell cycle by activating downstream molecules, allowing damaged DNA to be repaired and maintaining chromosome stability, thus in different sporadic and hereditary tumor pathogenesis it can play a role in the middle.
Chk signaling pathway
The conduction of Chk signaling pathway can be mainly divided into Chk1 and Chk2 mediated signaling pathways. Chk1 regulates the cell cycle: Complete cell cycle checkpoints include receptors, signaling pathways, and effectors. The mechanism of how DNA damage reacts to cell cycle checkpoints during the initial reaction is not clear yet, but most studies
indicated that the telangiectasia ataxia mutant gene (ATM) and the ATM/Rad3 associated protein gene (ATR) are the most important receptors. After detecting DNA damage, the signal can be transmitted to the core of the entire pathway through intermediaries. In this process, the stably expressed Chk1 protein causes a series of cascade effects such as cell cycle arrest, activation of the mismatch repair system, and recovery of the cell cycle after repair. The ATR-Chk1-Cdc25 reaction pathway is the most important pathway for cells to cope with ionizing radiation and replication stress. After DNA damage, especially in the presence of DNA double-strand breaks (DSBs), cells activate ATR after recognizing DNA damage signals, and phosphorylating multiple serine sites on Chk1 while signaling down, the latter being activated by the subsequent phosphorylation of Cdc25, which accelerates the ubiquitination of Cdc25 and ultimately arrests the cell cycle. Most of the time, ATR transmits the signal to Chk1 as the main sensor, but some studies have also found that ATM can also activate the entire signal pathway by phosphorylating the Ser317 site of Chk1. The current study confirms that after activation of Chk1, by activating DNA damage checkpoints, DNA replication checkpoints, or spindle assembly checkpoints, the cell cycle can be arrested in the G1/S phase, S phase, and G2/M phase, respectively. Different periods are not just fixed in one period. Normally expressed Chk1 ensures the stability and integrity of DNA replication throughout cell division. If the expression of Chk1 is altered, progeny cells carrying the wrong gene may continue to grow and proliferate across the detection site, increasing the probability of malignant transformation of the cell. Chk2 regulates cell cycle: Cell cycle detection is a kind of negative feedback regulation mechanism. In the absence of DNA damage, Chk2 is mostly dispersed in the nucleus in the state of inactive monomer, but when DNA is DDR, especially DNA double strand breakage damage (DSB), which can be activated by the ATM-Chk2-Cdc25 signaling pathway to activate the corresponding check sites in the G1/S phase, S phase and G2/M phase block cell cycle progression. If DNA damage is irreparable, Chk2 kinase also induces DNA damage in apoptosis, which is divided into P53-dependent and P53-independent types.
Due to the complexity of the Chk1 and Chk2 signaling pathways, many protein factors can regulate the activity of the Chk signaling pathway. Since Chk1 and Chk2 can specifically regulate the cell cycle, many studies are investigating inhibitors of Chk1 and Chk2 to find cancer therapeutic targets, and the recent research on Chk1 and Chk2 inhibitors is now organized as follows: Chk1 inhibitors: tumor cells with missing Chk1 expression often show multiple defects, such as slow cell proliferation, stagnation of cell cycle checkpoints and increased sensitivity to DNA damaging agents. This mechanism has become a new hotspot in tumor research by relieving the blocking effect of Chk1 on cell cycle detection, and it is also the basis for many scholars to find potential Chk1 inhibitors. Tumor cell proliferation can be stopped not only using Chk1 inhibitors in combination with chemotherapy or radiotherapy, but also using Chk1 inhibitor alone. Chk1 inhibitors combined with DNA damage agents can make DNA damage reagents to be selective for tumor cells, release G2 and S phase arrest of damaged naked p53 cancer cells, allowing them to continue to develop, eventually through mitotic mutations or cell death. The death pathway induces premature death. However, normal cells are less affected by the G2 phase detection point, and the damaged DNA can be repaired by p53 at the G1 detection site. The use of Chk1 inhibitors to remove S and G2 detection sites increases the sensitivity and selectivity of DNA damage agents to cancer cells. However, only a small amount of information on Chk1 inhibitors and Chk1/2 dual inhibitors is expected to enter clinical applications, such as Sch 900776 (MK-8776), and AZD7762 and LY2603618 are still in clinical trials. Other inhibitors such as UCN-01, CBP-501 and XL-844 (EXEL-9844) only enter preclinical assessment and clinical evaluation. Because these inhibitors inhibit other targets involved in the carcinogenesis process, such as PDK-1, CDK2, MAPKAPK2, FLT-3, PDGF, and KDR, they are also toxic to normal cells and therefore have poor selectivity. Due to cytotoxicity and controversial mode of administration, clinical advances in the first generation of Chk1 inhibitors have ceased. In recent years, a series of thioquinazolinones have been clinically used as Chk1 allosteric inhibitors. Substituting S- and/or G2-phase checkpoint-regulated anti-metabolite and DNA-damaging drugs, including gemcitabine, irinotecan (SN-38), cisplatin, docetaxel, etc., are drugs in the p53-deficient colon cancer and have obvious effects in tumors such as breast cancer, prostate cancer and leukemia. In the past, Chk1 inhibitors only exerted anti-cancer effects when combined with chemotherapy or radiotherapy, but Wang et al found that artificial activation of Chk1 is enough to kill cancer cells, which makes Chk1 inhibitor a single treatment for tumors. At present, there are reports on single-agent anti-tumor of Chk1 inhibitors such as SAR020106, CCT-244747, S-024, S-144, D-501036, V-158411 and LY2606368, and only LY2606368 is yet to enter phase III. Advances in Chk2 inhibitors: Based on the repair of DNA damage by Chk2, studies on the sensitivity of Chk2 inhibitors towards tumor cells to DNA damage agents have been widely concerned. Chk2 inhibitors can enhance the damage of cancer cells by radiotherapy and chemotherapy, while also protecting normal cells from the side effects of radiotherapy. The current major Chk2 inhibitors include: PV1019, CCT241533, AZD7762, XL9844, VRX0466617, C3742, ABI, Debromohymenialdisine (DBH), DBH-derived Indoloazepine, and NCS109555. Some of these Chk2 inhibitors have entered Phase I clinical studies, such as NSC109555, which potentiates the cytotoxic effects of cisplatin on pancreatic cancer MIA PACA-2 cells. The small molecule Chk2 inhibitor C3742 can enhance the killing effect of cisplatin on P53-deficient tumor cells, and XL9844 combined with gemcitabine can inhibit tumor cells. At the same time, Chk2 inhibitors can also enhance the apoptosis of tumor cells by radiotherapy. However, it is disappointing that most of the Chk2 inhibitors, due to their lack of specificity, cannot increase the anti-tumor effect, but show resistance to chemoradiotherapy. Therefore, in the future, research on high specificity Chk2 inhibitors will become an important direction for anti-tumor therapy.
Some studies have found that Chk1 expression is low in normal cells, but is high in some malignant tumors. Tumor cells with missing expression of Chk1 often exhibit multiple defects, such as slow cell proliferation, loss of stagnation reaction at the cell cycle checkpoint, and increased sensitivity to DNA damaging agents, and mutations in the Chk1 gene also contribute to tumor development. Haruki et al found that the short isomer of Chk1 is clearly expressed in small cell lung cancer. At the same time, Chk1 gene mutation will also have an important impact on the occurrence and development of tumors.
Mendoza et al found that Chk1 may participate in microtubule-associated proteins (tau) by regulating cell cycle arrest due to the accumulation of large amounts of DNA damage in brain cells of patients with Alzheimer's disease (AD). Phosphorylation and toxic expression will lead to the onset of AD.
By studying the expression of Chk2 in mouse oocyte maturation and early embryonic development, Dai et al. found that Chk2 exhibits a dynamic localization pattern, and interference with Chk2 activity leads to defects in cell cycle progression. Such defects can lead to birth defects, miscarriage, and even infertility.