Research Progress and Prospect of Nitrogen-containing Heterocycle in Anti-gastric Cancer Drugs: A Review.

Gastric cancer was the fifth most common cancer, and its drug treatment mainly included chemotherapy, targeted therapy, and immunotherapy. With the rise of immunotherapy in gastric cancer, small-molecule anti-gastric cancer drugs still have irreplaceable places because of many advantages, such as high stability and mass-productivity, high efficiency, and low cost. At present, the small-molecule anti-gastric cancer drugs in the clinic are constrained by their side effects. So, developing more novel anti-gastric cancer drugs with better efficacy and fewer side effects is urgently needed. Nitrogen-containing heterocycle molecules have attracted much attention from researchers due to their high biocompatibility, activity, and bioavailability, and they even could act with a unique mechanism. This review summarized various types of nitrogen-containing heterocycle antigastric cancer lead compounds from 2017 to 2022 in the last five years. Compared with monocyclic nitrogen-containing heterocycle and bicyclic nitrogen-containing heterocycle, the thick nitrogen-containing heterocycle applied as the skeleton not only showed high efficiency and low toxicity but also, interestingly, may have had some unique mechanism such as inhibition of aurora A and B kinases, etc. We propose two prospective and valuable strategies to develop more efficient candidates for anti-gastric cancer. One strategy was further optimized for some lead compounds mentioned in this review. The other strategy involved utilizing the "pseudo-natural products" concept proposed by Professor Wilhelm Waldmann, combining different nitrogen-containing heterocycle fragments in two and three-dimensional spaces to obtain new thick nitrogen-containing heterocycle skeletons. The strategy will contribute to the expansion of the thick nitrogenous heterocycle's framework, and it was expected that more novel mechanisms and more effective antigastric drugs could be found. These two strategies are expected to help researchers develop more anti-gastric cancer drugs with better potency and lower side effects.

High affinity and low PARP-trapping benzimidazole derivatives as a potential warhead for PARP1 degraders.

PARPi have been explored and applied in the treatment of various cancers with remarkable efficacy, especially BRCA1/2 mutated ovarian, breast, prostate, and pancreatic cancers. However, PARPi renders inevitable drug resistance and showed high toxicity because of PARP-Trapping with long-term clinic tracking. To overcome the drug resistance and the high toxicity of PARPi, many novel methods have been developed including PROTACs. Being an event-driven technology, PROTACs needs a high affinity, low toxicity warhead with no steric hindrance in binding process. Veliparib shows the lowest PARP-Trapping effect but could hardly to be the warhead of PROTACs because of the strong steric hindrance. Other PARP1 inhibitors showed less steric hindrance but owns high PARP-Trapping effect. Thus, the development of novel warhead with high PARP1 affinity, low PARP1-Trapping, and no steric hindrance would be valuable. In this work, we reserved benzimidazole as the motif to reserve the low PARP1-Trapping effect and substituted the pyrrole by aromatic ring to avoiding the steric hindrance in PARP1 binding cave. Thus, a series of benzimidazole derivates were designed and synthesized, and some biological activities in vitro were evaluated including the inhibition for PARP1 enzyme and the PARP-Trapping effect using MDA-MB-436 cell line. Results showed that the compound 19A10 has higher PARP1 affinity(IC50 = 4.62 nM)) and similar low PARP-Trapping effect compared with Veliparib(IC50 (MDA-MB-436) >100 µM). Docking study showed that the compound 19A10 could avoiding the steric hindrance which was much better than Veliparib. So, the compound 19A10 could potentially be a perfect warhead for PARP1 degraders. Besides, because of the depletion of the PARP1 and the decreasing of the binding capability, we suppose that the PROTACs using 19A10 as the warhead would be no-PARP-Trapping effect. Furthermore, QSAR study showed that to develop novel compounds with high PARP1 binding affinity and low PARP-Trapping, we can choose the skeleton with substituent R1H, R2 = piperiazine, and R3 with large tPSA. And, if we want to develop the compounds with high PARP1 binding affinity and high PARP-Trapping which can possibly improve the lethality against tumor cells, we can choose the skeleton with substituent R1F, R2 = 3-methy-piperiazine, and R3 with large tPSA.

Loss- and gain-of-function analyses reveal the essential role of Cyp19a1 in ovarian determination of the red-eared slider turtle.

Estrogen signaling exerts a decisive role in female sex determination and differentiation in chicken and fish. Aromatase encoded by Cyp19a1 is the key enzyme that catalyzes the conversion of androgen to estrogen. Correlative analyses implicate the potential involvement of aromatase in reptilian sexual development, however, the direct genetic evidence is lacking. Herein, we found that Cyp19a1 exhibited temperature-dependent sexually dimorphic expression, and located in the medullary somatic cells in early female embryos of the red-eared slider turtle (Trachemys scripta elegans), before the gonad is distinct. To determine the functional role of Cyp19a1 in turtle ovarian determination, we established loss- and gain-of-function models through in ovo lentivirus-mediated genetic manipulation. At female-producing temperature, inhibition of aromatase or knockdown of Cyp19a1 in turtle embryos resulted in female-to-male sex reversal, with the formation of a testis-like structure and a male distribution pattern of germ cells, as well as ectopic expression of male-specific markers (SOX9 and AMH) and disappearance of ovarian regulator FOXL2. On the contrary, overexpression of Cyp19a1 at male-producing temperature led to male-to-female sex reversal. In conclusion, our results suggest that Cyp19a1 is both necessary and sufficient for ovarian determination in the red-eared slider turtle, establishing causality and a direct genetic link between aromatase and reptilian sex determination and differentiation.

Design, synthesis, and evaluation of 1H-benzo[d]imidazole-4-carboxamide PARP-1 inhibitors using different saturated nitrogen-contained heterocycle as linker group.

Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors have been successfully applied in the clinical treatment of various cancer. Side effects and drug resistant cases were reported, and more effective PARP-1 inhibitors were required. However, studies on the AD site of PARP-1 inhibitors are currently incomplete. Therefore, to synthesize more potential candidate PARP-1 inhibitors and disclose some AD site SAR of the PARP-1 inhibitors, herein, a series of 2-phenyl-benzimidazole-4-carboxamide derivatives using different saturated nitrogen-contained heterocycles as linker group (6a-6t) have been designed, synthesized, and evaluated PARP-1 inhibitory activity and proliferation inhibitory against BRCA-1 mutant MDA-MB-436 cell line in vitro. The results showed 6b (IC50 = 8.65 nM) exhibited the most PARP-1 enzyme inhibitory activity comparable with Veliparib (IC50 = 15.54 nM) and Olaparib (IC50 = 2.77 nM); 6m exhibited the strongest MDA-MB-436 cell anti-proliferation activity (IC50 = 25.36 ± 6.06 µM) comparable with Olaparib (IC50 = 23.89 ± 3.81 µM). The compounds 6b, 6r, and 6m could be potential candidates for effective PARP-1 inhibitors and valuable for further optimization. The analysis of activity data also showed that the holistically anti-proliferation activity of the 1,4-diazepane group was about~twofold than that of the piperazine group. Meanwhile, the terminal 3-methyl-furanyl group exhibited the most robust PARP-1 inhibitory and anti-proliferation activity. It is hoped that the results could benefitable for further optimization of PARP-1 inhibitors. Furthermore, we note that some compounds (6d,6g,6n,6p,6s) showed poor PARP-1 inhibitory (>500 nM) but relatively good anti-proliferation activity, which indicates the proliferation inhibitory mechanism against MDA-MB-436 cell line was worth investigating in-depth.

Antibacterial and ß-amyloid precursor protein-cleaving enzyme 1 inhibitory polyketides from the fungus Aspergillus chevalieri.

One new prenylated benzenoid, (±)-chevalieric acid (1), and four new anthraquinone derivatives, (10S,12S)-, (10S,12R)-, (10R,12S)-, and (10R,12R)-chevalierone (2-5), together with ten previously described compounds (6-15), were isolated from the fungus Aspergillus chevalieri (L. Mangin) Thom and Church. The structures of new compounds were elucidated by extensive 1D and 2D nuclear magnetic resonance (NMR), and HRESIMS spectroscopic analysis. The absolute configurations of 2-5 were determined by experimental and calculated electronic circular dichroism (ECD) and DP4+ analysis. Compound 10 showed weak cytotoxicity against human lung cancer cell line A549 with IC50 39.68 µM. Compounds 2-5 exhibited antibacterial activities against the methicillin-resistant Staphylococcus aureus (MRSA) and opportunistic pathogenic bacterium Pseudomonas aeruginosa. The MIC value for compound 6 against MRSA is 44.02 µM. Additionally, Compounds 8, 10, 11 showed weak to moderate inhibitory activities against the ß-secretase (BACE1), with IC50 values of 36.1, 40.9, 34.9 µM, respectively.

PM2.5-Induced Programmed Myocardial Cell Death via mPTP Opening Results in Deteriorated Cardiac Function in HFpEF Mice.

PM2.5 exposure can induce or exacerbate heart failure and is associated with an increased risk of heart failure hospitalization and mortality; however, the underlying mechanisms remain unclear. This study focuses on the potential mechanisms underlying PM2.5 induction of cardiomyocyte programmed necrosis as well as its promotion of cardiac function impairment in a mouse model of heart failure with preserved ejection fraction (HFpEF). HFpEF mice were exposed to concentrated ambient PM2.5 (CAP) (CAP group) or filtered air (FA) (FA group) for 6 weeks. Changes in myocardial pathology and cardiac function were documented for comparisons between the two groups. In vitro experiments were performed to measure oxidative stress and mitochondrial permeability transition pore (mPTP) dynamics in H9C2 cells following 24 h exposure to PM2.5. Additionally, co-immunoprecipitation was conducted to detect p53 and cyclophilin D (CypD) interactions. The results showed exposure to CAP promoted cardiac function impairment in HFpEF mice. Myocardial pathology analysis and in vitro experiments demonstrated that PM2.5 led to mitochondrial damage in cardiomyocytes and, eventually, their necrosis. Moreover, our experiments also suggested that PM2.5 increases mitochondrial reactive oxygen species (ROS), induces DNA oxidative damage, and decreases the inner mitochondrial membrane potential (ΔΨm). This indicates the presence of mPTP opening. Co-immunoprecipitation results showed a p53/CypD interaction in the myocardial tissue of HFpEF mice in the CAP group. Inhibition of CypD by cyclosporin A was found to reverse PM2.5-induced mPTP opening and H9C2 cell death. In conclusion, PM2.5 induces mPTP opening to stimulate mitochondria-mediated programmed necrosis of cardiomyocytes, and it might exacerbate cardiac function impairment in HFpEF mice.

Recent Progress in the Research on Benzimidazole PARP-1 Inhibitors.

Poly (ADP-ribose) polymerase-1 (PARP-1) is a multifunctional protein that plays an important role in DNA repair and genome integrity. PARP-1 inhibitors can be used as effective drugs not only to treat BRCA-1/2 deficient cancers because of the synthetic lethality effect but also to treat non- BRCA1/2 deficient tumours because of the effect of PARP capture. Therefore, PARP inhibitors have become a focus of compelling research. Among these inhibitors, substituted benzimidazole derivatives were mainly concerned as lead compounds. However, the commercially available benzimidazole PARP-1 inhibitors have some shortcomings, such as serious toxicity in combination with chemotherapy drugs and in vivo cardiovascular side effects such as anemia. Therefore it is crucial for scientists to explore more structure-activity relationships of the benzimidazole PARP-1 inhibitors and access safer and more effective PARP inhibitors. As the binding regions of PARP-1 and the substrates are usually characterized by NI site and AD site, the modification of benzimidazoles mainly occurs on the benzimidazole skeleton (NI site) and the side chain of benzimidazole in the 2-C position (AD site). Herein, the recent progress in the research on benzamides PARP inhibitors was introduced. We noticed that even though many efforts were made to the modification of NI sites, there was still a lack of optimistic and impressive results. However, the structure-activity relationships of the modification of AD sites have not been thoroughly discovered yet. We hope that enlightened by the previous research, more research on AD sites should be carried out, and more effective benzimidazole PARP-1 inhibitors could be designed, synthesized, and applied to clinics.

Recent progress of research on anti-tumor agents using benzimidazole as the structure unit.

With the development of exploration for disease-related proteins or receptors, more and more novel structural lead compounds are required to designed and synthesized. The benzimidazole is an effective structural unit in which the benzene ring is fused at the 4 and 5 positions of the imidazole ring and wildly used in drug design. Here, we introduce some recent progress of research for anti-tumor agents which was target to various target proteins such as DNA topoisomerase, angiogenesis, serine/threonine protein kinase, and tyrosine protein kinase. These anti-tumor agents are all introduced benzimidazole as the structure unit. Further docking study showed that the benzimidazole group was not only act as a skeleton to expand the structure of molecule but also as an excellent ligand unit to form hydrogen bond or π-π conjugation and hydrophobic interaction with target proteins or receptors. We expect that introducing benzimidazole in the chemical structure could be a reasonable and priority strategy in novel anti-tumor agents' design.

Synthesis and evaluation of 2-(4-[4-acetylpiperazine-1-carbonyl] phenyl)-1H-benzo[d]imidazole-4-carboxamide derivatives as potential PARP-1 inhibitors and preliminary study on structure-activity relationship.

Although 1H-benzo[d]imidazole-4-carboxamide derivatives have been explored for a long time, the structure-activity relationship of the substituents in the hydrophobic pocket (AD binding sites) has not thoroughly discovered. Here in, a series of 2-(4-[4-acetylpiperazine-1-carbonyl]phenyl)-1H-benzo[d]imidazole-4-carboxamide derivatives have been designed, synthesized, and successful characterization as novel and effective poly ADP-ribose polymerases (PARP)-1 inhibitors to improve the structure-activity relationships about the substituents in the hydrophobic pocket. These derivatives were evaluated for their PARP-1 inhibitory activity and cellular inhibitory against BRCA-1 deficient cells (MDA-MB-436) and wild cells (MCF-7) using PARP kit assay and MTT method. The results indicated that compared with other heterocyclic compounds, furan ring-substituted derivatives 14n-14q showed better PARP-1 inhibitory activity. Among this derivatives, compound 14p displayed the strongest inhibitory effects on PARP-1 enzyme (IC50  = 0.023 µM), which was close to that of Olaparib. 14p (IC50  = 43.56 ± 0.69 µM) and 14q (IC50  = 36.69 ± 0.83 µM) displayed good antiproliferation activity on MDA-MB-436 cells and inactivity on MCF-7 cells, indicating that 14p and 14q have high selectivity and targeting. The molecular docking method was used to explore the binding mode of compound 14p and PARP-1, and implied that the formation of hydrogen bond was essential for PARP-1 inhibition activities. This study also showed that in the hydrophobic pocket (AD binding sites), the introduction of strong electronegative groups (furan ring, e.g.) or halogen atoms in the side chain of benzimidazole might improve its inhibitory activity and this strategy could be applied in further research.

Theaflavin-3,3'-digallate attenuates cigarette smoke extract-induced pulmonary emphysema in mice by suppressing necroptosis.

Pulmonary emphysema is one of the most important pathological manifestations of chronic obstructive pulmonary disease and is commonly associated with cigarette smoking. Previous studies have indicated that necroptosis, a novel non-apoptotic cell death mechanism associated with inflammation and oxidative stress, may contribute to the development of pulmonary emphysema. Theaflavin-3,3'-digallate (TF-3), one of the theaflavins present in black tea, is known to possess several bioactive properties. In the present study, it was demonstrated that TF-3 significantly reduced the generation of reactive oxygen species and the mRNA expression levels of TNF-α, IL-1ß and IL-6 in CSE-treated human normal lung epithelial BEAS-2B cells. To further explore the role of TF-3 in necroptosis, the necroptotic rates of BEAS-2B cells were examined via flow cytometry and immunofluorescence assays. The results demonstrated that TF-3 may suppress necroptosis in CSE-treated BEAS-2B cells. Furthermore, it was determined that TF-3 significantly inhibited the CSE-induced phosphorylation of p38 MAPK, receptor-interacting serine/threonine-protein kinase three (RIPK3) and mixed lineage kinase domain-like (MLKL) in BEAS-2B cells. Another experiment demonstrated that a pharmacological inhibitor of the p38 MAPK pathway, SB203580, significantly reduced the protein expression levels of phosphorylated (p)-RIPK3 and phosphorylated (p-)MLKL, which indicated that TF-3 suppressed necroptosis via the p38 MAPK/RIPK3/MLKL signaling pathways. In vivo, it was observed that TF-3 treatment significantly attenuated morphological lung injury in mice with CSE-induced emphysema. Moreover, TF-3 significantly reduced the levels of proinflammatory cytokines, TNF-α and IL-1ß and significantly enhanced the antioxidant capacity of the lung tissues in mice with emphysema. TF-3 also significantly inhibited the levels of p-RIPK3 and p-MLKL in the lungs of mice with emphysema. Therefore, the present study indicated that TF-3 may attenuate CSE-induced emphysema in mice by inhibiting necroptosis.

Redefining Tumor-Associated Macrophage Subpopulations and Functions in the Tumor Microenvironment.

The immunosuppressive status of the tumor microenvironment (TME) remains poorly defined due to a lack of understanding regarding the function of tumor-associated macrophages (TAMs), which are abundant in the TME. TAMs are crucial drivers of tumor progression, metastasis, and resistance to therapy. Intra- and inter-tumoral spatial heterogeneities are potential keys to understanding the relationships between subpopulations of TAMs and their functions. Antitumor M1-like and pro-tumor M2-like TAMs coexist within tumors, and the opposing effects of these M1/M2 subpopulations on tumors directly impact current strategies to improve antitumor immune responses. Recent studies have found significant differences among monocytes or macrophages from distinct tumors, and other investigations have explored the existence of diverse TAM subsets at the molecular level. In this review, we discuss emerging evidence highlighting the redefinition of TAM subpopulations and functions in the TME and the possibility of separating macrophage subsets with distinct functions into antitumor M1-like and pro-tumor M2-like TAMs during the development of tumors. Such redefinition may relate to the differential cellular origin and monocyte and macrophage plasticity or heterogeneity of TAMs, which all potentially impact macrophage biomarkers and our understanding of how the phenotypes of TAMs are dictated by their ontogeny, activation status, and localization. Therefore, the detailed landscape of TAMs must be deciphered with the integration of new technologies, such as multiplexed immunohistochemistry (mIHC), mass cytometry by time-of-flight (CyTOF), single-cell RNA-seq (scRNA-seq), spatial transcriptomics, and systems biology approaches, for analyses of the TME.

ATP reduces mitochondrial MECR protein in liver of diet-induced obese mice in mechanism of insulin resistance.

Mitochondrial 2-enoyl-acyl-carrier protein reductase (MECR) is an enzyme in the mitochondrial fatty acid synthase (mtFAS) pathway. MECR activity remains unknown in the mechanism of insulin resistance in the pathogenesis of type 2 diabetes. In the present study, MECR activity was investigated in diet-induced obese (DIO) mice. Mecr mRNA was induced by insulin in cell culture, and was elevated in the liver of DIO mice in the presence hyperinsulinemia. However, MECR protein was decreased in the liver of DIO mice, and the reduction was blocked by treatment of the DIO mice with berberine (BBR). The mechanism of MECR protein regulation was investigated with a focus on ATP. The protein was decreased in the cell lysate and DIO liver by an increase in ATP levels. The ATP protein reduction was blocked in the liver of BBR-treated mice by suppression of ATP elevation. The MECR protein reduction was associated with insulin resistance and the protein restoration was associated with improvement of insulin sensitivity by BBR in the DIO mice. The data suggest that MECR protein is regulated in hepatocytes by ATP in association with insulin resistance. The study provides evidence for a relationship between MECR protein and insulin resistance.

Cigarette smoke extract increases mitochondrial membrane permeability through activation of adenine nucleotide translocator (ANT) in lung epithelial cells.

Cigarette smoke is one of major risk factors in the pathogenesis of chronic obstructive pulmonary disease (COPD). It is generally believed that cigarette smoke induces mitochondrial damage in the alveolar epithelial cells to contribute to COPD. However, the exact molecular mechanism remains unknown for the mitochondrial damage. In this study, cigarette smoke extract (CSE) was found to induce the mitochondrial membrane permeability (MMP), which promoted proton leakage leading to the reduction in mitochondrial potential and ATP production. ANT in the mitochondrial inner membrane was activated by CSE for the alteration of MMP. The activation was observed without an alteration in the protein level of ANT. Inhibition of the ANT activity with ADP or bongkrekic acid prevented the MMP alteration and potential drop upon CSE exposure. The ANT activation was observed with a rise in ROS production, inhibition of the mitochondrial respiration, decrease in the complex III protein and rise in mitophagy activity. The results suggest that ANT may mediate the toxic effect of cigarette smoke on mitochondria and control of ANT activity is a potential strategy in intervention of the toxicity.

Precise and Programmable Detection of Mutations Using Ultraspecific Riboregulators.

The ability to identify single-nucleotide mutations is critical for probing cell biology and for precise detection of disease. However, the small differences in hybridization energy provided by single-base changes makes identification of these mutations challenging in living cells and complex reaction environments. Here, we report a class of de novo-designed prokaryotic riboregulators that provide ultraspecific RNA detection capabilities in vivo and in cell-free transcription-translation reactions. These single-nucleotide-specific programmable riboregulators (SNIPRs) provide over 100-fold differences in gene expression in response to target RNAs differing by a single nucleotide in E. coli and resolve single epitranscriptomic marks in vitro. By exploiting the programmable SNIPR design, we implement an automated design algorithm to develop riboregulators for a range of mutations associated with cancer, drug resistance, and genetic disorders. Integrating SNIPRs with portable paper-based cell-free reactions enables convenient isothermal detection of cancer-associated mutations from clinical samples and identification of Zika strains through unambiguous colorimetric reactions.

Downregulation of Sfrp5 in insulin resistant rats promotes macrophage-mediated pulmonary inflammation through activation of Wnt5a/JNK1 signaling.

BACKGROUND: Insulin resistance (IR), a common co-morbidity of chronic obstructive pulmonary disease (COPD), aggravates airway inflammation in COPD patients, but its mechanism is unclear. Sfrp5, a novel anti-inflammatory adipocytokine, inhibits macrophage-mediated inflammation of adipose tissue and abrogates IR. However, few studies have been conducted on the regulatory role of Sfrp5 in lung inflammation. METHODS: In the present study, 30 SD rats were divided into two groups: the normal food (NF) group and the high-fat diet (HFD) group. Oral glucose tolerance test (OGTT) and insulin release test were performed to assess whether a successful IR rat model was established. The expression of Sfrp5 and key downstream moleculars of Wnt5a/JNKl signaling was detected. Lung tissue pathomorphology and macrophage activation were observed. In addition, we counted the number of inflammatory cells and measured inflammatory cytokines in bronchoalveolar lavage fluid (BALF). In vitro, rat lung macrophages were isolated and treated with Wnt5a, Sfrp5, and/or JNK inhibitor SP600125. JNK activity and inflammatory cytokines expression were examined. RESULTS: We found that in a rat model of IR, Sfrp5 expression of lung tissue was downregulated, while the Wnt5a/JNKl pathway was activated and the lung inflammatory response was enhanced. Meanwhile, Sfrp5 significantly suppressed Wnt5a/JNKl-induced macrophage activation. CONCLUSIONS: Collectively, IR reduces Sfrp5 expression of lung tissue and activates the Wnt5a/JNK1 pathway, promoting macrophage activation and contributing to the lung's inflammatory response. In contrast, Sfrp5 suppresses the inflammatory response by inhibiting the Wnt5a/JNKl pathway, which could be a target of treatment of COPD.

Ring finger protein 145 (RNF145) is a ubiquitin ligase for sterol-induced degradation of HMG-CoA reductase.

Cholesterol biosynthesis is tightly regulated in the cell. For example, high sterol concentrations can stimulate degradation of the rate-limiting cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, HMGCR). HMGCR is broken down by the endoplasmic reticulum membrane-associated protein complexes consisting of insulin-induced genes (Insigs) and the E3 ubiquitin ligase gp78. Here we found that HMGCR degradation is partially blunted in Chinese hamster ovary (CHO) cells lacking gp78 (gp78-KO). To identify other ubiquitin ligase(s) that may function together with gp78 in triggering HMGCR degradation, we performed a small-scale short hairpin RNA-based screening targeting endoplasmic reticulum-localized E3s. We found that knockdown of both ring finger protein 145 (Rnf145) and gp78 genes abrogates sterol-induced degradation of HMGCR in CHO cells. We also observed that RNF145 interacts with Insig-1 and -2 proteins and ubiquitinates HMGCR. Moreover, the tetrapeptide sequence YLYF in the sterol-sensing domain and the Cys-537 residue in the RING finger domain were essential for RNF145 binding to Insigs and RNF145 E3 activity, respectively. Of note, amino acid substitutions in the YLYF or of Cys-537 completely abolished RNF145-mediated HMGCR degradation. In summary, our study reveals that RNF145, along with gp78, promotes HMGCR degradation in response to elevated sterol levels and identifies residues essential for RNF145 function.