Mitochondria-Targeted Prodrug Nanoassemblies for Efficient Ferroptosis-Based Therapy via Devastating Ferroptosis Defense Systems.

Ferroptosis is a form of regulated cell death accompanied by lipid reactive oxygen species (ROS) accumulation in an iron-dependent manner. However, the efficiency of tumorous ferroptosis was seriously restricted by intracellular ferroptosis defense systems, the glutathione peroxidase 4 (GPX4) system, and the ubiquinol (CoQH2) system. Inspired by the crucial role of mitochondria in the ferroptosis process, we reported a prodrug nanoassembly capable of unleashing potent mitochondrial lipid peroxidation and ferroptotic cell death. Dihydroorotate dehydrogenase (DHODH) inhibitor (QA) was combined with triphenylphosphonium moiety through a disulfide-containing linker to engineer well-defined nanoassemblies (QSSP) within a single-molecular framework. After being trapped in cancer cells, the acidic condition provoked the structural disassembly of QSSP to liberate free prodrug molecules. The mitochondrial membrane-potential-driven accumulation of the lipophilic cation prodrug was delivered explicitly into the mitochondria. Afterward, the thiol-disulfide exchange would occur accompanied by downregulation of reduced glutathione levels, thus resulting in mitochondria-localized GPX4 inactivation for ferroptosis. Simultaneously, the released QA from the hydrolysis reaction of the adjacent ester bond could further devastate mitochondrial defense and evoke robust ferroptosis via the DHODH-CoQH2 system. This subcellular targeted nanoassembly provides a reference for designing ferroptosis-based strategy for efficient cancer therapy through interfering antiferroptosis systems.

Phase separation of Nur77 mediates XS561-induced apoptosis by promoting the formation of Nur77/Bcl-2 condensates.

The orphan nuclear receptor Nur77 is a critical regulator of the survival and death of tumor cells. The pro-death effect of Nur77 can be regulated by its interaction with Bcl-2, resulting in conversion of Bcl-2 from a survival to killer. As Bcl-2 is overexpressed in various cancers preventing them from apoptosis and promoting their resistance to chemotherapy, targeting the apoptotic pathway of Nur77/Bcl-2 may lead to new cancer therapeutics. Here, we report our identification of XS561 as a novel Nur77 ligand that induces apoptosis of tumor cells by activating the Nur77/Bcl-2 pathway. In vitro and animal studies revealed an apoptotic effect of XS561 in a range of tumor cell lines including MDA-MB-231 triple-negative breast cancer (TNBC) and MCF-7/LCC2 tamoxifen-resistant breast cancer (TAMR) in a Nur77-dependent manner. Mechanistic studies showed XS561 potently induced the translocation of Nur77 from the nucleus to mitochondria, resulting in mitochondria-related apoptosis. Interestingly, XS561-induced accumulation of Nur77 at mitochondria was associated with XS561 induction of Nur77 phase separation and the formation of Nur77/Bcl-2 condensates. Together, our studies identify XS561 as a new activator of the Nur77/Bcl-2 apoptotic pathway and reveal a role of phase separation in mediating the apoptotic effect of Nur77 at mitochondria.

Effects of folic acid supplementation on cognitive impairment: A meta-analysis of randomized controlled trials.

OBJECTIVE: With the increasing number of patients with cognitive impairment, nonpharmacological ways to delay cognitive impairment have attracted people's attention, such as lifestyle changes and nutritional supplementation. Folic acid supplementation appears to be a promising treatment option. However, it remains controversial whether folic acid supplementation is effective in delaying adult's cognitive impairment. Therefore, we conducted a meta-analysis to analyze the effects of folic acid supplementation on different cognitive impairments. METHODS: We systematically searched PubMed, Web of Science, EMbase, Cochrane Central Register of Controlled Trials, China National Knowledge Infrastructure (CNKI), WanFang and VIP databases for randomized controlled trials on January 22, 2024. The included population comprised those diagnosed with cognitive impairment. We included trials that compared folic acid treatment with placebo, other dosing regimens, or other intervention controls. Conducting quality evaluation of included studies according to the Cochrane Risk of Bias tool. Statistical analyses were performed using Review Manager software. RESULTS: Twenty-two trials, including 3604 participants, met inclusion criteria. Compared with controls, the cognitive function of Alzheimer's disease (AD) patients showed improvement with folic acid supplementation: supplementation with < 3 mg (standardized mean differences (SMD) = 0.15, 95% confidence interval (CI) -0.10 to 0.41), and supplementing with ≥ 3 mg folic acid could improve cognitive function in AD patients (SMD = 1.03, 95% CI 0.18 to 1.88). Additionally, it reduced homocysteine (HCY) levels (mean differences (MD) = -4.74, 95% CI -8.08 to -1.39). In mild cognitive impairment (MCI) patients, cognitive function improved with folic acid supplementation: supplementation with > 400 µg (SMD = 0.38, 95% CI 0.13 to 0.63), and supplementation with ≤ 400 µg (SMD = 1.10, 95% CI 0.88 to 1.31). It also reduced HCY levels at intervention ≤ 6 months (MD = -3.93, 95% CI -5.05 to -2.82) and intervention > 6 months (MD = -4.38, 95% CI -5.15 to -3.61). However, supplementing with folic acid did not improve cognitive function in vascular cognitive impairment (VCI) patients, with folic acid supplements < 3 mg (SMD = -0.07, 95% CI -0.23 to -0.08), folic acid supplements ≥ 3 mg (SMD = 0.46, 95% CI -0.57 to 1.49), however, it reduced HCY levels at intervention > 6 months (MD = -5.91, 95% CI -7.13 to -4.69) and intervention ≤ 6 months (MD = -11.15, 95% CI -12.35 to -9.95). CONCLUSIONS: Supplement folic acid is beneficial to the cognitive profile of patients with MCI, supplementation with ≥ 3 mg folic acid can improve cognitive function in AD patients.

Discovery of 5-(Pyrimidin-2-ylamino)-1H-indole-2-carboxamide Derivatives as Nur77 Modulators with Selective and Potent Activity Against Triple-Negative Breast Cancer.

The orphan nuclear receptor Nur77 has been validated as a potential drug target for treating breast cancer. Therefore, focusing on the SAR study of the lead 8b (KDSPR(Nur77) = 354 nM), we found the active compound ja which exhibited improved Nur77-binding capability (KDSPR(Nur77) = 91 nM) and excellent antiproliferative activities against breast cancer cell lines. Interestingly, ja acted as a potent and selective Nur77 antagonist, displaying good potency against triple-negative breast cancer (TNBC) cell lines but did not inhibit human normal breast cancer cell line MCF-10A (SI > 20). Exceptionally, ja Nur77-dependently caused mitochondria dysfunction and induced the caspase-dependent apoptosis by mediating the TP53 phosphorylation pathway. Moreover, ja significantly suppressed MDA-MB-231 xenograft tumor growth but had no apparent side effects in mice and zebrafish. Overall, ja demonstrated to be the first Nur77 modulator mediating the TP53 phosphorylation pathway that has the potential as a novel anticancer agent for TNBC.

The novel CDK9 inhibitor, XPW1, alone and in combination with BRD4 inhibitor JQ1, for the treatment of clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is a highly lethal malignancy with few therapeutic options. Cyclin­dependent kinase 9 (CDK9), a potential therapeutic target of many cancers, has been recently observed to be upregulated in ccRCC patients. Therefore, we aimed to investigate the therapeutic potential of CDK9 in ccRCC and develop a novel CDK9 inhibitor with low toxicity for ccRCC treatment. METHODS: The expression of CDK9 in ccRCC was checked using the online database and tissue microarray analysis. shRNA-mediated CDK9 knockdown and CDK inhibitor were applied to evaluate the effect of CDK9 on ccRCC. Medicinal chemistry methods were used to develop a new CDK9 inhibitor with drugability. RNA-seq and ChIP-seq experiments were conducted to explore the mechanism of action. MTS, western blotting, and colony formation assays were performed to evaluate the anti-ccRCC effects of CDK9 knockdown and inhibition in vitro. The in vivo anti-tumour efficacy was evaluated in a xenograft model. RESULTS: CDK9 is overexpressed and associated with poor survival in ccRCC. Knockdown or inhibition of CDK9 significantly suppressed ccRCC cells. XPW1 was identified as a new potent and selective CDK9 inhibitor with excellent anti-ccRCC activity and low toxicity. In mechanism, XPW1 transcriptionally inhibited DNA repair programmes in ccRCC cells, resulting in an excellent anti-tumour effect. CDK9 and BRD4 were two highly correlated transcriptional regulators in ccRCC patients, and the BRD4 inhibitor JQ1 enhanced XPW1's anti-ccRCC effects in vitro and in vivo. CONCLUSIONS: This work provides valuable insights into the therapeutic potential of CDK9 in ccRCC. The CDK9 inhibitor XPW1 would be a novel therapeutic agent for targeting ccRCC, alone or in rational combinations.

Tuo-Min-Ding-Chuan Decoction Alleviates Airway Inflammations in the Allergic Asthmatic Mice Model by Regulating TLR4-NLRP3 Pathway-Mediated Pyroptosis: A Network Pharmacology and Experimental Verification Study.

Background: Tuo-Min-Ding-Chuan Decoction (TMDCD) is an effective traditional Chinese medicine (TCM) formula granule for allergic asthma (AA). Previous studies proved its effects on controlling airway inflammations, while the specific mechanism was not clear. Methods: We conducted a network pharmacology study to explore the molecular mechanism of TMDCD against AA with the public databases of TCMSP. Then, HUB genes were screened with the STRING database. DAVID database performed GO annotation and KEGG functional enrichment analysis of HUB genes, and it was verified with molecular docking by Autodock. Then, we built a classic ovalbumin-induced allergic asthma mice model to explore the mechanism of anti-inflammation effects of TMDCD. Results: In the network pharmacology study, we found out that the potential mechanism of TMDCD against AA might be related to NOD-like receptor (NLR) signaling pathway and Toll-like receptor (TLR) signaling pathway. In the experiment, TMDCD showed remarkable effects on alleviating airway inflammations, airway hyperresponsiveness (AHR), and airway remodeling in the asthmatic mice model. Further molecular biology and immunohistochemistry experiments suggested TMDCD could repress TLR4-NLRP3 pathway-mediated pyroptosis-related gene transcriptions to inhibit expressions of target proteins. Conclusion: TMDCD could alleviate asthmatic mice model airway inflammations by regulating TLR4-NLRP3 pathway-mediated pyroptosis.

Discovery of 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole-2-carboxamide derivatives as novel anti-cancer agents targeting Nur77.

Encouraged by our previous findings and in continuation of our ongoing study project in designing and synthesis of novel Nur77-targeting anti-cancer agents, a series of 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole-2-carboxamide derivatives were designed, synthesized and biologically evaluated as potent Nur77 modulators. Among synthesized compounds, 8b maintained good potency against different liver cancer cell lines and other types of cancer cell lines while exhibiting lower toxicity than the positive compound celastrol. Moreover, 8b displayed excellent Nur77-binding activity, superior to the lead compound 10g and comparable to the reference compound celastrol. The cytotoxic action of 8b towards cancer cells was associated with its induction of Nur77-mitochondrial targeting and Nur77-dependent apoptosis. Notably, 8b has good in vivo safety and anti-hepatocellular carcinoma (HCC) activity. Altogether, this study reveals that 8b is a novel Nur77 modulator with great promise for further research.

MicroRNA-506 ameliorates breast cancer-induced osteolytic bone metastasis via the NFATc-1 signaling pathway.

Breast cancer is becoming a common life-threatening disease, especially in women, along with higher incidence and mortality. MicroRNA (miR)-506 was reported to participate in breast cancer progression, while the role of miR-506 in breast cancer-induced osteolytic bone metastasis is unclear. In the present study, we found significant downregulation of miR-506 in breast cancer tissues and cell lines. Overexpression of miR-506 notably reduced the proliferative, migratory and invasive rates of MCF7 and MDA-MB-231 cells, and reduced the production of inflammatory factors IL-6 and TNF-α in MCF7 cells. Moreover, overexpression of miR-506 obviously inhibited tumor growth in an in vivo animal model. In addition, overexpression of miR-560 efficiently attenuated breast cancer-induced osteolysis in vivo, which was characterized by increased bone volume/total volume (BT/TV), trabecular number (Tb. N), and trabecular thickness (Tb. Th), as well as the reduced trabecular separation (Tb. Sp). The nuclear factor of activated T cell cytoplasmic 1 (NFATc1) was identified as a downstream target of miR-506, and overexpression of miR-506 could inhibit breast cancer progression by targeting NFATc1. Furthermore, our results showed that NFATc-1 might participate in the inhibition of miR-506 on breast cancer-induced osteolysis. In conclusion, our findings provide insights into understanding the pathogenesis of breast cancer and breast cancer-induced osteolytic bone metastasis, and miR-506 might serve as a novel biomarker for this disease.

Molecular Mechanism Underlying Effects of Wumeiwan on Steroid-Dependent Asthma: A Network Pharmacology, Molecular Docking, and Experimental Verification Study.

Background: Steroid-dependent asthma (SDA) is characterized by oral corticosteroid (OCS) resistance and dependence. Wumeiwan (WMW) showed potentials in reducing the dose of OCS of SDA patients based on our previous studies. Methods: Network pharmacology was conducted to explore the molecular mechanism of WMW against SDA with the databases of TCMSP, STRING, etcetera. GO annotation and KEGG functional enrichment analysis were conducted by metascape database. Pymol performed the molecular docking. In the experiment, the OVA-induced plus descending dexamethasone intervention chronic asthmatic rat model was conducted. Lung pathological changes were analyzed by H&E, Masson, and IHC staining. Relative expressions of the gene were performed by real-time PCR. Results: A total of 102 bioactive ingredients in WMW were identified, as well as 191 common targets were found from 241 predicted targets in WMW and 3539 SDA-related targets. The top five bioactive ingredients were identified as pivotal ingredients, which included quercetin, candletoxin A, palmidin A, kaempferol, and beta-sitosterol. Besides, 35 HUB genes were obtained from the PPI network, namely, TP53, AKT1, MAPK1, JUN, HSP90AA1, TNF, RELA, IL6, CXCL8, EGFR, etcetera. GO biological process analysis indicated that HUB genes were related to bacteria, transferase, cell differentiation, and steroid. KEGG pathway enrichment analysis indicated that the potential mechanism might be associated with IL-17 and MAPK signaling pathways. Molecular docking results supported these findings. H&E and Masson staining proved that WMW could reduce airway inflammation and remodeling of model rats, which might be related to the downward expression of IL-8 proved by IHC staining and real-time PCR. Conclusion: WMW could be a complementary and alternative therapy for SDA by reducing airway inflammation.

Tuo-Min-Ding-Chuan Decoction Alleviate Ovalbumin-Induced Allergic Asthma by Inhibiting Mast Cell Degranulation and Down-Regulating the Differential Expression Proteins.

Allergic asthma is a stubborn chronic inflammatory disease, and is considered a co-result of various immune cells, especially mast cells, eosinophils and T lymphocytes. At present, the treatment methods of allergic asthma are limited and the side effects are obvious. Traditional Chinese medicine has been used to treat diseases for thousands of years in China. One such example is the treatment of allergic asthma, which take the characteristics of less adverse reactions and obvious curative effect. Tuo-Min-Ding-Chuan Decoction (TMDCD) is a traditional Chinese medicine compound for the treatment of allergic asthma optimized from Ma-Xing-Gan-Shi Decoction (MXGSD), which was put forward in Treatise on Febrile Diseases by Zhang Zhongjing in the Eastern Han Dynasty. The compound shows a significant clinical effect, but the mechanism of its influence on the immune system is still unclear. The purpose of this study was to observe whether TMDCD could alleviate the symptoms of ovalbumin (OVA) challenged allergic asthma mice, and to explore its immune regulatory mechanism, especially on mast cell (MC) degranulation. The results showed TMDCD could not only reduce the airway hyperresponsiveness (AHR), inflammatory cell infiltration and mucus secretion in the lung tissue of OVA challenged mice, but also decrease the levels of total IgE, OVA-specific IgE, histamine and LTC4 in serum. We found that TMDCD can downregulate the expression of Fractalkine, Tryptase ε, IL-25, CCL19, MCP-1, OX40L, Axl, CCL22, CD30, G-CSF, E-selectin, OPN, CCL5, P-selectin, Gas6, TSLP in OVA challenged mice serum by using mouse cytokines antibody array. It has been reported in some literatures that these differentially expressed proteins are related to the occurrence of allergic asthma, such as tryptase ε, MCP-1, CCL5, etc. can be released by MC. And the results of in vitro experiments showed that TMDCD inhibited the degranulation of RBL-2H3 cells stimulated by DNP-IgE/BSA. Taken together, we made the conclusion that TMDCD could reduce the infiltration of inflammatory cells in lung tissue and alleviate airway remodeling in mice with allergic asthma, showed the effects of anti-inflammatory and antiasthmatic. TMDCD could also reduce the levels of IgE, histamine, LTC4, Tryptase ε, and other MC related proteins in the serum of allergic asthma mice, and the in vitro experiments showed that TMDCD could inhibit IgE mediated degranulation and histamine release of RBL-2H3 cells, proved its anti allergic effect.

Effect of Knowledge/Practice of COVID-19 Prevention Measures on Return-to-Work Concerns; Attitudes About the Efficacy of Traditional Chinese Medicine: Survey on Supermarket Staff in Huanggang, China.

Objective: The objective of this study was to investigate how knowledge and practice of coronavirus disease 2019 (COVID-19) prevention measures affected concerns about returning to work among supermarket staff. Attitudes about the ability of traditional Chinese medicine (TCM) to prevent COVID-19 were also assessed. Methods: A cross-sectional study was conducted in Huanggang, Hubei Province, China from April 23 to 25, 2020. Participants were invited to fill out an electronic questionnaire on their cell phones. Results: The results showed that from 2,309 valid questionnaires, 61.5% of participants were concerned about resuming work. Major concerns included asymptomatic infection (85.01%) and employees gathering in the workplace (78.96%). Multivariate logistic regression indicated that the female gender, having school-aged children and pregnancy were risk factors for being concerned about resuming work, while good knowledge and practice of preventive measures were protective factors. Knowledge and practice of preventive measures were positively correlated. Among preventive measures, the highest percentage of participants knew about wearing masks and washing hands. Meanwhile, 65.8% of participants expressed confidence in the ability of TCM to prevent COVID-19, where 74 and 51.3% thought there was a need and a strong need, respectively, for preventive TCM-based products. Among them, 71.5% preferred oral granules. Regarding TCM as a COVID-19 preventative, most were interested in information about safety and efficacy. Conclusion: These findings suggested that promoting knowledge and practices regarding COVID-19 prevention can help alleviate concerns about returning to work. Meanwhile, TCM can feasibly be accepted to diversify COVID-19 prevention methods. Clinical Trial Registration:http://www.chictr.org.cn/, identifier: ChiCTR2000031955.

Development of MIF/IL-1ß biosensors for discovery of critical quality attributes and potential allergic rhinitis targets from clinical real-world data by intelligent algorithm coupled with in vitro and vivo mechanism validation.

There are still huge challenges from clinical real-world data to accurate targets and critical quality attributes (CQAs) for effective treatment of allergic rhinitis (AR). Here, we present a novel integrated strategy that biosensors and intelligent algorithms were used to angle AR targets and CQAs from clinical real world. Firstly, bagging and boosting partial least squares discrimination analysis (PLS-DA) and Monte-Carlo sampling were proposed to screen accurate AR targets. Macrophage migration inhibitory factor (MIF) and Interleukin-1beta (IL-1ß) potential targets were obtained based on large-scale analysis of one thousand proteins and in-depth precise screening of seventy proteins. Furthermore, high electron mobility transistor (HEMT) biosensors were fabricated and successfully modified by MIF and IL-1ß potential targets with a low detection concentration as 1 pM and quantitative range from 1 pM to 10 nM. Surprisingly, through MIF/IL-1ß biosensors, we angled 5-O-methylvisammioside, amygdalin, and cimicifugoside three CQAs. The strong interaction was discovered among three CQAs and MIF/IL-1ß biosensors with almost all KD up to 10-11 M. Finally, interaction among three CQAs and MIF/IL-1ß biosensors were evaluated by in vitro and vivo experiments. In this paper, two critical potential targets and three effective CQAs for AR treatment were discovered and validated by biosensor and advanced algorithms. It provides a superior integrated idea for angling critical targets and CQAs from clinical real-world data by biosensors and informatics.

Design, synthesis, and biological evaluation of 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-Indole-2-Carbohydrazide derivatives: the methuosis inducer 12A as a Novel and selective anticancer agent.

This study describes the synthesis and vacuole-inducing activity of 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole-2-carbohydrazide derivatives, including five potent derivatives 12c, 12 g, 12i, 12n, and 12A that exhibit excellent vacuole-inducing activity. Remarkably, 12A effectively induces methuosis in tested cancer cells but not human normal cells. In addition, 12A exhibits high pan-cytotoxicity against different cancer cell lines but is hardly toxic to normal cells. It is found that the 12A-induced vacuoles are derived from macropinosomes but not autophagosomes. The 12A-induced cytoplasmic vacuoles may originate from the endoplasmic reticulum (ER) and be accompanied by ER stress. The MAPK/JNK signalling pathway is involved in the 12A-induced methuotic cell death. Moreover, 12A exhibits significant inhibition of tumour growth in the MDA-MB-231 xenograft mouse model. The excellent potency and selectivity of 12A prompt us to select it as a good lead compound for further development of methuosis inducers and investigation of the molecular and cellular mechanisms underlying methuosis.

Recent progress in mitochondria-targeting-based nanotechnology for cancer treatment.

Mitochondria play critical roles in the regulation of the proliferation and apoptosis of cancerous cells. Nanosystems for targeted delivery of cargos to mitochondria for cancer treatment have attracted increasing attention in the past few years. This review will summarize the state of the art of design and construction of nanosystems used for mitochondria-targeted delivery. The use of nanotechnology for cancer treatment through various pathways such as energy metabolism interference, reactive oxygen species (ROS) regulation, mitochondrial protein targeting, mitochondrial DNA (mtDNA) interference, mitophagy inducing, and combination therapy will be discussed. Finally, the major challenges and an outlook in this field will also be provided.

Synthesis and biological evaluation of (3/4-(pyrimidin-2-ylamino)benzoyl)-based hydrazine-1-carboxamide/carbothioamide derivatives as novel RXRα antagonists.

Abnormal alterations in the expression and biological function of retinoid X receptor alpha (RXRα) have a key role in the development of cancer. Potential modulators of RXRα as anticancer agents are explored in growing numbers of studies. A series of (4/3-(pyrimidin-2-ylamino)benzoyl)hydrazine-1-carboxamide/carbothioamide derivatives are synthesised and evaluated for anticancer activity as RXRα antagonists in this study. Among all synthesised compounds, 6A shows strong antagonist activity (half maximal effective concentration (EC50) = 1.68 ± 0.22 µM), potent anti-proliferative activity against human cancer cell lines HepG2 and A549 cells (50% inhibition of cell viability (IC50) values < 10 µM), and low cytotoxic property in normal cells such as LO2 and MRC-5 cells (IC50 values > 100 µM). Further bioassays indicate that 6A inhibits 9-cis-RA-induced activity in a dose-dependent manner, and selectively binds to RXRα-=LΒD with submicromolar affinity (Kd = 1.20 × 10-7 M). 6A induces time-and dose-dependent cleavage of poly ADP-ribose polymerase, and significantly stimulates caspase-3 activity, leading to RXRα-dependent apoptosis. Finally, molecular docking studies predict the binding modes for RXRα-LBD and 6A.

Insights into the stereoselectivity of human SETD7 methyltransferase.

Human SETD7 methyltransferase (hSETD7) is involved in a wide range of physiological processes, and has been considered as a significant target to develop new drugs. (R)-PFI-2, one hSETD7 inhibitor, could bind to the pocket of substrates with potent (low nanomolar) activity and high selectivity, while its enantiomer (S)-PFI-2 showed 500-fold less activity in IC50 determination. Why do this pair of enantiomers, with nearly identical structures, exert tremendously different inhibitory activity? We performed a total of 900 ns long-timescale molecular dynamics (MD) simulations and 80 ps hybrid quantum mechanics/molecular mechanics (QM/MM) MD simulations to understand the molecular mechanism of the stereoselectivity of hSETD7. For each SAM/hSETD7/PFI-2 system, we characterized and compared the residual fluctuation of hSETD7, and generated molecular interaction fingerprints (IFP) to exemplify the propensities of SAM/hSETD7-inhibitor interactions. Based on the QM/MM MD, we accurately captured the difference of hydrogen bonds between the SAM/hSETD7/(R)-PFI-2 and SAM/hSETD7/(S)-PFI-2 systems. Especially the strength of the hydrogen bond between G336 and two inhibitors, which determines the stability of the post-SET loop. The energy barrier for (S)-PFI-2 was much bigger than (R)-PFI-2 from global minimum to bioactive conformation as the potential energy surface scanning (PES) showed. Moreover, by estimating the binding affinity and phylogenetic tree analysis, we discovered 16 hotspots were essential for binding both enantiomers but the specific mode of interaction between these hotspots and enantiomorphs is different. Our findings reveal the effect of chirality on the inhibition activity of hSETD7 in detail, and provide valuable information for hSETD7 structure-based drug development.