Identification of drug-target interactions (DTIs) has great practical importance in the drug discovery process for known diseases. However, only a small proportion of DTIs in these databases has been verified experimentally, and the computational methods for predicting the interactions remain challenging. As a result, some effective computational models have become increasingly popular for predicting DTIs. In this work, the authors predict potential DTIs from the local structure of drug-target associations' network, which is different from the traditional global network similarity methods based on structure and ligand. A novel method called PPDTS is proposed to predict DTIs. First, according to the DTIs' network local structure, the known DTIs are converted into a binary network. Second, the Resource Allocation algorithm is used to obtain a drug-drug similarity network and a target-target similarity network. Third, a Collaborative Filtering algorithm is used with the known drug-target topology information to obtain similarity scores. Fourth, the linear combination of drug-target similarity model and the target-drug similarity model are innovatively proposed to obtain the final prediction results. Finally, the experimental performance of PPDTS has proved to be higher than that of the previously mentioned four popular network-based similarity methods, which is validated in different experimental datasets. Some of the predicted results can be supported in UniProt and DrugBank databases.

Tepotinib is a novel tyrosine kinase inhibitor recently approved for the treatment of non-small cell lung cancer (NSCLC). In this study, we evaluated the tepotinib's potential to perpetrate pharmacokinetic drug interactions and modulate multidrug resistance (MDR). Accumulation studies showed that tepotinib potently inhibits ABCB1 and ABCG2 efflux transporters, which was confirmed by molecular docking. In addition, tepotinib inhibited several recombinant cytochrome P450 (CYP) isoforms with varying potency. In subsequent drug combination experiments, tepotinib synergistically reversed daunorubicin and mitoxantrone resistance in cells with ABCB1 and ABCG2 overexpression, respectively. Remarkably, MDR-modulatory properties were confirmed in ex vivo explants derived from NSCLC patients. Furthermore, we demonstrated that anticancer effect of tepotinib is not influenced by the presence of ABC transporters associated with MDR, although monolayer transport assays designated it as ABCB1 substrate. Finally, tested drug was observed to have negligible effect on the expression of clinically relevant drug efflux transporters and CYP enzymes. In conclusion, our findings provide complex overview on the tepotinib's drug interaction profile and suggest a promising novel therapeutic strategy for future clinical investigations.

Alisertib (MLN8237), a novel Aurora A kinase inhibitor, is currently being clinically tested in late-phase trials for the therapy of various malignancies. In the present work, we describe alisertib's potential to perpetrate pharmacokinetic drug-drug interactions (DDIs) and/or to act as an antagonist of multidrug resistance (MDR). In accumulation assays, alisertib potently inhibited ABCC1 transporter, but not ABCB1 or ABCG2. The results of molecular modeling suggested a bifunctional mechanism for interaction on ABCC1. In addition, alisertib was characterized as a low- to moderate-affinity inhibitor of recombinant CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2D6 isoenzymes, but without potential clinical relevance. Drug combination studies revealed the capability of alisertib to synergistically antagonize ABCC1-mediated resistance to daunorubicin. Although alisertib exhibited substrate characteristics toward ABCB1 transporter in monolayer transport assays, comparative proliferation studies showed lack of its MDR-victim behavior in cells overexpressing ABCB1 as well as ABCG2 and ABCC1. Lastly, alisertib did not affect the expression of ABCC1, ABCG2, ABCB1 transporters and CYP1A2, CYP3A4, CYP2B6 isozymes on mRNA level in various systemic and tumoral models. In conclusion, our study suggests that alisertib is a drug candidate with negligible potential for perpetrating systemic pharmacokinetic DDIs on ABCB1, ABCG2 and cytochromes P450. In addition, we introduce alisertib as an effective dual-activity chemosensitizer whose MDR-antagonistic capacities are not impaired by efflux or effect on MDR phenotype. Our in vitro findings provide important pieces of information for clinicians when introducing alisertib into the clinical area.

Salmonella enteritidis (SE) is a pathogen that can readily infect ovarian tissues and colonize the granulosa cell layer such that it can be transmitted via eggs from infected poultry to humans in whom it can cause food poisoning. Ducks are an important egg-laying species that are susceptible to SE infection, yet the host-pathogen interactions between SE and ducks have not been thoroughly studied to date. Herein, we performed dual RNA-sequencing analyses of these two organisms in a time-resolved infection model of duck granulosa cells (dGCs) by SE. In total, 10,510 genes were significantly differentially expressed in host dGCs, and 265 genes were differentially expressed in SE over the course of infection. These differentially expressed genes (DEGs) of dGCs were enriched in the cytokine-cytokine receptor interaction pathway via KEGG analyses, and the DEGs in SE were enriched in the two-component system, bacterial secretion system, and metabolism of pathogen factors pathways as determined. A subsequent weighted gene co-expression network analysis revealed that the cytokine-cytokine receptor interaction pathway is mostly enriched at 6 h post-infection (hpi). Moreover, a number of pathogenic factors identified in the pathogen-host interaction database (PHI-base) are upregulated in SE, including genes encoding the pathogenicity island/component, type III secretion, and regulators of systemic infection. Furthermore, an intracellular network associated with the regulation of SE infection in ducks was constructed, and 16 cytokine response-related dGCs DEGs (including IL15, CD40, and CCR7) and 17 pathogenesis-related factors (including sseL, ompR, and fliC) were identified, respectively. Overall, these results not only offer new insights into the mechanisms underlying host-pathogen interactions between SE and ducks, but they may also aid in the selection of potential targets for antimicrobial drug development.

Recent antitumor drug development has included investigation of a wide variety of anti-angiogenesis therapies. Because cancer cells in tumors require new blood vessels to grow and spread, they stimulate capillary proliferation from existing vessels as well as new vessel formation from endothelial precursor cells. Our previous findings suggested that drug resistance in mouse endothelial cells supported tumor growth, but the relationship between endothelial cells (ECs) and nasopharyngeal carcinoma (NPC) cells remained unclear. Exosomes are small membrane vesicles that are released by several cell types, including human microvascular ECs (HMECs). Exosomes carrying membrane and cytoplasmic constituents have been described as participants in a novel mechanism of cell-to-cell communication. In the present study, we investigated the mechanisms underlying the interactions between HMECs and NPC cells. We found that drug-resistant HMECs secreted small heterogeneous 40-100 nm vesicles, defined as exosomes. Co-incubation of NPC cells with doxorubicin-resistant (R-DOX) HMEC-derived exosomes resulted in promotion of their proliferation, migration, and chemoresistance, as well as changes in the expression of epithelial-mesenchymal transition (EMT) markers. These effects were significantly inhibited by treatment with GW4869 (an exosome inhibitor). We also found that GW4869 inhibited the stimulation of drug-resistant HMECs on NPC progression by modulating EMT in vivo. These data suggest that exosomes participate in a novel mechanism by which drug-resistant ECs enhance NPC progression.

Interactions between oncogenic proteins contribute to the phenotype and drug resistance. Here, EZH2 (enhancer of zest homolog 2) is identified as a crucial factor that mediates HIF-1 (hypoxia-inducible factor) inhibitor resistance. Mechanistically, targeting HIF-1 enhanced the activity of EZH2 through transcription activation of SUZ12 (suppressor of zest 12 protein homolog). Conversely, inhibiting EZH2 increased HIF-1α transcription, but not the transcription of other HIF family members. Additionally, the negative feedback regulation between EZH2 and HIF-1α is confirmed in lung cancer patient tissues and a database of cell lines. Moreover, molecular prediction showed that a newly screened dual-target compound, DYB-03, forms multiple hydrogen bonds with HIF-1α and EZH2 to effectively inhibit the activity of both targets. Subsequent studies revealed that DYB-03 could better inhibit migration, invasion, and angiogenesis of lung cancer cells and HUVECs in vitro and in vivo compared to single agent. DYB-03 showed promising antitumor activity in a xenograft tumor model by promoting apoptosis and inhibiting angiogenesis, which could be almost abolished by the deletion of HIF-1α and EZH2. Notably, DYB-03 could reverse 2-ME2 and GSK126-resistance in lung cancer. These findings clarified the molecular mechanism of cross-regulation of HIF-1α and EZH2, and the potential of DYB-03 for clinical combination target therapy.

Salmonella is a globally distributed zoonotic pathogen. Among them, S. Pullorum is a host-specific pathogen that seriously affects the development of the poultry breeding industry in China. It mainly infects chickens and can cause white scabs, and the mortality rate after infection is almost 100%. As antibiotics are widely used in animal feed and other production processes, Salmonella resistance has gradually increased. Therefore, there is an increasing need to develop new technologies to control multi-drug resistant (MDR) pathogens and confirm their actual effectiveness in the target food matrix. Bacteriophage can efficiently and specifically lyse bacteria, and will be a potential bactericide to replace antibiotics. In this study, 34 strains of Salmonella bacteriophages were isolated from environmental resources. Therein, phage Pu20 with the widest host spectrum had the strongest ability to lyse tested Salmonella strains. Further studies showed that Pu20 had high pH tolerance and heat resistance, short incubation period. Pu20 can effectively inhibit the growth of two strains of MDR Salmonella in liquid egg white and yolk at 4 ℃ and 25 ℃, respectively. According to morphological and phylogenetic analysis, Pu20 belongs to the Podoviridae family. Genomic analysis of Pu20 indicates a linear 59435 bp dsDNA sequence with no homology to virulence or antibiotic resistance-related genes. Together, these results sheds light on the potential biocontrol application value of Pu20 in food products.

Talazoparib (Talzenna) is a novel poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitor that is clinically used for the therapy of breast cancer. Furthermore, the drug has shown antitumor activity against different cancer types, including non-small cell lung cancer (NSCLC). In this work, we investigated the possible inhibitory interactions of talazoparib toward selected ATP-binding cassette (ABC) drug efflux transporters and cytochrome P450 biotransformation enzymes (CYPs) and evaluated its position in multidrug resistance (MDR). In accumulation studies, talazoparib interacted with the ABCC1 and ABCG2 transporters, but there were no significant effects on ABCB1. Furthermore, incubation assays revealed a negligible capacity of the tested drug to inhibit clinically relevant CYPs. In in vitro drug combination experiments, talazoparib synergistically reversed daunorubicin and mitoxantrone resistance in cells with ABCC1 and ABCG2 expression, respectively. Importantly, the position of an effective MDR modulator was further confirmed in drug combinations performed in ex vivo NSCLC patients-derived explants, whereas the possible victim role was refuted in comparative proliferation experiments. In addition, talazoparib had no significant effects on the mRNA-level expressions of MDR-related ABC transporters in the MCF-7 cellular model. In summary, our study presents a comprehensive overview on the pharmacokinetic drug-drug interactions (DDI) profile of talazoparib. Moreover, we introduced talazoparib as an efficient MDR antagonist.

GRS is a drug combination of three components including ginsenoside Rb1, ruscogenin and schisandrin. It derived from the well-known TCM formula Sheng MaiSan, a widely used traditional Chinese medicine for the treatment of cardiovascular diseases in clinic. The present study illuminates its underlying mechanisms against myocardial ischemic diseases based on the combined methods of bioinformatic prediction and experimental verification. A protein database was established through constructing the drug-protein network. And the target-pathway interaction network clustered the potential signaling pathways and targets of GRS in treatment of myocardial ischemic diseases. Several target proteins, such as NFKB1, STAT3 and MAPK14, were identified as the candidate key proteins, and MAPKs and JAK-STAT signaling pathway were suggested as the most related pathways, which were in accordance with the gene ontology analysis. Then, the predictive results were further validated and we found that GRS treatment alleviated hypoxia/reoxygenation (H/R)-induced cardiomyocytes injury via suppression of MDA levels and ROS generation, and potential mechanisms might related to the suppression of activation of MAPKs and JAK2-STAT3 signaling pathways. Conclusively, our results offer the evidence that GRS attenuates myocardial ischemia injury via regulating oxidative stress and MAPKs and JAK2-STAT3 signaling pathways, which supplied some new insights for its prevention and treatment of myocardial ischemia diseases.

Chemotherapy is an effective anti-tumor treatment. Some anticancer chemotherapeutic drugs can not only induce cell death, but can also elicit antitumor immune responses. Here, the stability of cisplatin-loaded polymeric micelles (CDDP-PMs), pharmacokinetic drug-drug interactions of CDDP and anti-PD-L1 antibody (aPD-L1) in vivo and the alteration of the tumor microenvironment by combination of CDDP-PMs and aPD-L1 were evaluated. CDDP-PMs were fabricated by coordinated complexation and self-assembly method for tumor targeting. CDDP-PMs with higher mass ratio of copolymer have higher thermodynamic stability. The pharmacokinetic study showed that the CDDP and aPD-L1 were metabolized and cleared by two different pathways, suggesting that there is almost no risk of potential drug interactions between CDDP and aPD-L1 and the combination of aPD-L1 and CDDP- PMs may not alter the tissue distribution of CDDP. In vivo antitumor test showed that the tumor growth inhibition rates of CDDP-PMs combined with medium-dose aPD-L1 and CDDP-PMs combined with high-dose PD-L1 were 89.41% and 93.16%, respectively and therapeutic efficacy can be further increased by increasing the dose of aPD-L1 in co-administration group. This therapeutic system by combining chemotherapy and immunotherapy further increases the link between them and holds great potential to offer better safety and antitumor efficacy profiles.

A growing body of research shows that drug monomers from traditional Chinese herbal medicines have antineuroinflammatory and neuroprotective effects that can significantly improve the recovery of motor function after spinal cord injury (SCI). Here, we explore the role and molecular mechanisms of Alpinetin on activating microglia-mediated neuroinflammation and neuronal apoptosis after SCI.

Radiotherapy, chemotherapy, autologous/allogeneic stem cell transplantation, and targeted drug therapy are currently available therapeutic options for multiple myeloma (MM), but the clinical outcome remains unsatisfactory owing to frequent occurrence of drug resistance. Anti apoptosis is one of the main mechanisms to mediate drug resistance. Studies have shown that MCL-1 plays a key role in the growth of cancer cells "escaping" drug attacks, but the underlying mechanism remains unclear. Our previous study demonstrated that lncRNA H19 was highly expressed in the serum of MM patients. Bioinformatics predicts that miR-29b-3p is the downstream target gene, and MCL-1 is the downstream target protein of miR-29b-3p. Therefore, we speculated that MCL-1 may be involved in the occurrence of drug resistance through epigenetics. On the basis of these previous findings, the present study was intended to explore the biological function of H19, interactions between the downstream target genes, and the effect of H19 on BTZ resistance of myeloma cells. In addition, in vivo experiments we have also confirmed that H19 promoted tumor growth and may develop resistance to bortezomib partly. It was found that H19 reduced cell sensitivity to the chemotherapeutic drug BTZ by working as a miRNA sponge to inhibit the expression of miR-29b-3p, enhance MCL-1 transcriptional translation and inhibit apoptosis. These findings may help gain insights into the molecular mechanism of acquired BTZ resistance and develop new drug targets for the clinical treatment of MM.

Deficiency in DNA double-strand break (DSB) repair mechanisms has been widely exploited for the treatment of different malignances, including homologous recombination (HR)-deficient breast and ovarian cancers. Here we demonstrate that diffuse large B cell lymphomas (DLBCLs) expressing LMO2 protein are functionally deficient in HR-mediated DSB repair. Mechanistically, LMO2 inhibits BRCA1 recruitment to DSBs by interacting with 53BP1 during repair. Similar to BRCA1-deficient cells, LMO2-positive DLBCLs and T cell acute lymphoblastic leukemia (T-ALL) cells exhibit a high sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Furthermore, chemotherapy and PARP inhibitors synergize to inhibit the growth of LMO2-positive tumors. Together, our results reveal that LMO2 expression predicts HR deficiency and the potential therapeutic use of PARP inhibitors in DLBCL and T-ALL.

Encorafenib (LGX818, trade name Braftovi), a novel BRAF inhibitor, has been approved for the treatment of melanoma and colorectal cancer. In the present work, we evaluated encorafenib's possible antagonistic effects on the pharmacokinetic mechanisms of multidrug resistance (MDR), as well as its perpetrator role in drug interactions. Firstly, encorafenib potently inhibited the efflux function of the ABCC1 transporter in drug accumulation assays, while moderate and null interaction levels were recorded for ABCB1 and ABCG2, respectively. In contrast, the mRNA expression levels of all the tested transporters were not altered by encorafenib. In the drug combination studies, we found that daunorubicin and topotecan resistances were synergistically attenuated by the encorafenib-mediated interaction in A431-ABCC1 cells. Notably, further experiments in ex vivo patient-derived explants confirmed the MDR-modulating ability of encorafenib. Advantageously, the overexpression of tested drug efflux transporters failed to hinder the antiproliferative activity of encorafenib. In addition, no significant modulation of the CYP3A4 enzyme's activity by encorafenib was observed. In conclusion, our work indicated that encorafenib can act as an effective chemosensitizer targeting the ABCC1-induced MDR. Our in vitro and ex vivo data might provide valuable information for designing the novel effective scheme applicable in the clinical pharmacotherapy of BRAF-mutated/ABCC1-expressing tumors.

Hepatocyte growth factor (HGF) signaling through its receptor Met has been implicated in hepatocellular carcinoma tumorigenesis and progression. Met interaction with integrins is shown to modulate the downstream signaling to Akt and ERK (extracellular-regulated kinase). In this study, we developed a mechanistically detailed systems biology model of HGF/Met signaling pathway that incorporated specific interactions with integrins to investigate the efficacy of integrin-binding peptide, AXT050, as monotherapy and in combination with other therapeutics targeting this pathway. Here we report that the modeled dynamics of the response to AXT050 revealed that receptor trafficking is sufficient to explain the effect of Met-integrin interactions on HGF signaling. Furthermore, the model predicted patient-specific synergy and antagonism of efficacy and potency for combination of AXT050 with sorafenib, cabozantinib, and rilotumumab. Overall, the model provides a valuable framework for studying the efficacy of drugs targeting receptor tyrosine kinase interaction with integrins, and identification of synergistic drug combinations for the patients.

The application of pesticides in mixtures often exerts multiple pressures on agricultural soils in the short term. Therefore, it is necessary to assess the effects of mixed application on the environmental behavior and ecotoxicity of pesticides in soil. In this study, we assessed the effects of three common pesticides through mixed application on soil degradation and toxicity toward the earthworm Eisenia fetida. Compared with the degradation half-lives (DT50) the single pesticide, the DT50 values of avermectin, imidacloprid and carbendazim in the binary mixtures were similar. However, their DT50 values in the ternary mixtures were approximately 1.5 times longer than those in the individual applications, enhancing their stable in soil after two or three applications. The ternary mixtures of the pesticides showed significantly synergistic toxicity toward E. fetida, while their binary mixtures exhibited a changing interaction throughout the entire effect level range. The ternary mixtures activated higher SOD and CAT activities in E. fetida than the individual treatments, confirming their synergistic effects. By conducting avoidance tests with E. fetida, ternary toxic interactions were effectively assessed within a relatively short testing period. In summary, the three pesticides in ternary mixtures exhibited longer degradation half-lives and synergistic toxicity toward earthworms compared to individual or binary mixtures.

Sphingolipid metabolism affects prognosis and resistance to immunotherapy in patients with cancer and is an emerging target in cancer therapy with promising diagnostic and prognostic value. Long noncoding ribonucleic acids (lncRNAs) broadly regulate tumour-associated metabolic reprogramming. However, the potential of sphingolipid metabolism-related lncRNAs in pancreatic adenocarcinoma (PAAD) is poorly understood. In this study, coexpression algorithms were employed to identify sphingolipid metabolism-related lncRNAs. The least absolute shrinkage and selection operator (LASSO) algorithm was used to develop a sphingolipid metabolism-related lncRNA signature (SMLs). The prognostic predictive stability of the SMLs was validated using Kaplan-Meier. Univariate and multivariate Cox, receiver operating characteristic (ROC) and clinical stratification analyses were used to comprehensively assess the SMLs. Gene set variation analysis (GSVE), gene ontology (GO) and tumor mutation burden (TMB) analysis explored the potential mechanisms. Additionally, single sample gene set enrichment analysis (ssGSEA), ESTIMATE, immune checkpoints and drug sensitivity analysis were used to investigate the potential predictive function of the SMLs. Finally, an SMLs-based consensus clustering algorithm was utilized to differentiate patients and determine the suitable population for immunotherapy. The results showed that the SMLs consists of seven sphingolipid metabolism-related lncRNAs, which can well determine the clinical outcome of individuals with PAAD, with high stability and general applicability. In addition, the SMLs-based consensus clustering algorithm divided the TCGA-PAAD cohort into two clusters, with Cluster 1 showing better survival than Cluster 2. Additionally, Cluster 1 had a higher level of immune cell infiltration than Cluster 2, which combined with the higher levels of immune checkpoints in Cluster 1 suggests that Cluster 1 is more consistent with an immune 'hot tumor' profile and may respond better to immune checkpoint inhibitors (ICIs). This study offers new insights regarding the potential role of sphingolipid metabolism-related lncRNAs as biomarkers in PAAD. The constructed SMLs and the SMLs-based clustering are valuable tools for predicting clinical outcomes in PAAD and provide a basis for clinical selection of individualized treatments.

Objectives: To evaluate the pharmacodynamical effects and pharmacological mechanism of Ginsenoside H dripping pills (GH) in chronic unpredictable mild stress (CUMS) model rats. Methods: First, the CUMS-induced rat model was established to assess the anti-depressant effects of GH (28, 56, and 112 mg/kg) by the changes of the behavioral indexes (sucrose preference, crossing score, rearing score) and biochemical indexes (serotonin, dopamine, norepinephrine) in Hippocampus. Then, the components of GH were identified by ultra-performance liquid chromatography-iron trap-time of flight-mass spectrometry (UPLC/IT-TOF MS). After network pharmacology analysis, the active ingredients of GH were further screened out based on OB and DL, and the PPI network of putative targets of active ingredients of GH and depression candidate targets was established based on STRING database. The PPI network was analyzed topologically to obtain key targets, so as to predict the potential pharmacological mechanism of GH acting on depression. Finally, some major target proteins involved in the predictive signaling pathway were validated experimentally. Results: The establishment of CUMS depression model was successful and GH has antidepressant effects, and the middle dose of GH (56 mg/kg) showed the best inhibitory effects on rats with depressant-like behavior induced by CUMS. Twenty-eight chemical components of GH were identified by UPLC/IT-TOF MS. Subsequently, 20(S)-ginsenoside Rh2 was selected as active ingredient and the PPI network of the 43 putative targets of 20(S)-ginsenoside Rh2 containing in GH and the 230 depression candidate targets, was established based on STRING database, and 47 major targets were extracted. Further network pharmacological analysis indicated that the cAMP signaling pathway may be potential pharmacological mechanism regulated by GH acting on depression. Among the cAMP signaling pathway, the major target proteins, namely, cAMP, PKA, CREB, p-CREB, BDNF, were used to verify in the CUMS model rats. The results showed that GH could activate the cAMP-PKA-CREB-BDNF signaling pathway to exert antidepressant effects. Conclusions: An integrative pharmacology-based pattern was used to uncover that GH could increase the contents of DA, NE and 5-HT, activate cAMP-PKA-CREB-BDNF signaling pathway exert antidepressant effects.

Triptolide is a potent anti-inflammatory agent that also possesses anticancer activity, including against colorectal cancer (CRC), one of the most frequent cancers around the world. In order to clarify how triptolide may be effective against CRC, we analyzed the proteome and phosphoproteome of CRC cell line HCT116 after incubation for 48 h with the drug (40 nM) or vehicle. Tandem mass tagging led to the identification of 403 proteins whose levels increased and 559 whose levels decreased in the presence of triptolide. We also identified 3,110 sites in proteins that were phosphorylated at higher levels and 3,161 sites phosphorylated at lower levels in the presence of the drug. Analysis of these differentially expressed and/or phosphorylated proteins showed that they were enriched in pathways involving ribosome biogenesis, PI3K-Akt signaling, MAPK signaling, nucleic acid binding as well as other pathways. Protein-protein interactions were explored using the STRING database, and we identified nine protein modules and 15 hub proteins. Finally, we identified 57 motifs using motif analysis of phosphosites and found 16 motifs were experimentally verified for known protein kinases, while 41 appear to be novel. These findings may help clarify how triptolide works against CRC and may guide the development of novel treatments.

The protein α-hemolysin (α-HL) is a self-assembling exotoxin that binds to the membrane of a susceptible host cell. In this paper, experimental studies show that cyrtominetin (CTM) can inhibit the hemolytic activity of α-HL. To understand how CTM can affect hemolytic activity, molecular dynamics simulations were carried out for α-HL-CTM complex and these results were compared with the crystal structure of monomeric α-HL. With this approach, the analysis revealed that the inhibition of CTM involves CTM directly binding to α-HL. Due to the binding of CTM, the conformation of the critical "Loop" region was restrained. This mechanism was confirmed by the experimental data. These findings indicate that CTM hinders the lysis activity of α-HL through a novel mechanism.