The Link of mRNA and rRNA Transcription by PUF60/FIR through TFIIH/P62 as a Novel Therapeutic Target for Cancer.

The interaction between mRNA and ribosomal RNA (rRNA) transcription in cancer remains unclear. RNAP I and II possess a common N-terminal tail (NTT), RNA polymerase subunit RPB6, which interacts with P62 of transcription factor (TF) IIH, and is a common target for the link between mRNA and rRNA transcription. The mRNAs and rRNAs affected by FUBP1-interacting repressor (FIR) were assessed via RNA sequencing and qRT-PCR analysis. An FIR, a c-myc transcriptional repressor, and its splicing form FIRΔexon2 were examined to interact with P62. Protein interaction was investigated via isothermal titration calorimetry measurements. FIR was found to contain a highly conserved region homologous to RPB6 that interacts with P62. FIRΔexon2 competed with FIR for P62 binding and coactivated transcription of mRNAs and rRNAs. Low-molecular-weight chemical compounds that bind to FIR and FIRΔexon2 were screened for cancer treatment. A low-molecular-weight chemical, BK697, which interacts with FIRΔexon2, inhibited tumor cell growth with rRNA suppression. In this study, a novel coactivation pathway for cancer-related mRNA and rRNA transcription through TFIIH/P62 by FIRΔexon2 was proposed. Direct evidence in X-ray crystallography is required in further studies to show the conformational difference between FIR and FIRΔexon2 that affects the P62-RBP6 interaction.

Combinational antibody detection approach increases the clinical validity of colorectal cancer screening.

BACKGROUND: At different stages of the disease, biomarkers can help to determine disease progression and recurrence and provide a personalized indicator of therapeutic effectiveness. The serological identification of antigens by recombinant cDNA expression cloning (SEREX) has identified five SEREX antigens. RESULTS: Compared with healthy donors, anti-FIRΔexon2 and anti-SOHLH antibodies (Abs) in the sera of patients with colorectal cancer (CRC) were markedly higher. Furthermore, no correlation was noted between five SEREX antigens and the three tumor markers (CEA, CA19-9, and anti-p53 Abs), indicating that anti-FIRΔexon2 Abs are an independent candidate marker for patients with CRC. Generally, the levels of anti-FIRΔexon2 Abs combined with clinically available tumor markers were determined to be significantly higher compared with CEA, CA19-9. Moreover, in early-stage CRC, the levels of anti-FIRΔexon2 Abs combined with existing tumor markers were higher than those of CEA, CA19-9. CONCLUSION: Due to the highly heterogeneous nature of CRC, a single tumor marker is unlikely to become a standalone diagnostic test due to its commonly insufficient sensitivity and/or specificity. Using a combination antibody detection approach of tumor markers for CRC diagnosis has the potential to be an effective approach. Therefore, the use of serum protein biomarker candidates holds promise for the development of inexpensive, noninvasive, and inexpensive tests for the detection of CRC.

The C-terminal tail of Rad17, iVERGE, binds the 9‒1‒1 complex independently of AAA+ ATPase domains to provide another clamp-loader interface.

The ATR pathway plays a crucial role in maintaining genome integrity as the major DNA damage checkpoint. It also attracts attention as a therapeutic target in cancer treatment. The Rad17-RFC2-5 complex loads the Rad9-Hus1-Rad1 (9-1-1) DNA clamp complex onto damaged chromatin to activate the ATR pathway. We previously reported that phosphorylation of a polyanionic C-terminal tail of human Rad17, iVERGE, is essential for the interaction between Rad17 and the 9-1-1 complex. However, the molecular mechanism has remained unclear. Here, we show that iVERGE directly interacts with the Hus1 subunit of the 9-1-1 complex through Rad17-S667 phosphorylation independently of the AAA+ ATPase domains. An exogenous iVERGE peptide interacted with the 9-1-1 complex in vivo. The binding conformation of the iVERGE peptide was analyzed by de novo modeling with docking simulation, simulated annealing-molecular dynamics simulation, and the fragment molecular orbital method. The in silico analyses predicted the association of the iVERGE peptide with the hydrophobic and basic patches on the Hus1 protein, and the corresponding Hus1 mutants were deficient in the interaction with the iVERGE peptide in vivo. The iVERGE peptide occupied the same position as the C-terminus of Saccharomyces cerevisiae RAD24 on MEC3. The interaction energy calculation suggested that the Rad17 KYxxL motif and the iVERGE peptide are the primary and secondary interaction surfaces between the Rad17-RFC2-5 and 9-1-1 complexes. Our data reveal a novel molecular interface, iVERGE, between the Rad17-RFC2-5 and 9-1-1 complexes in vertebrates and implicate that Rad17 utilizes two distinct molecular interfaces to regulate the 9-1-1 complex.

Computational Screening of Inhibitory Compounds for SARS-Cov-2 3CL Protease with a Database Consisting of Approved and Investigational Chemicals.

Computational screening is one of the fundamental techniques in drug discovery. Each compound in a chemical database is bound to the target protein in virtual, and candidate compounds are selected from the binding scores. In this work, we carried out combinational computation of docking simulation to generate binding poses and molecular mechanics calculation to estimate binding scores. The coronavirus infectious disease has spread worldwide, and effective chemotherapy is strongly required. The viral 3-chymotrypsin-like (3CL) protease is a good target of low molecular-weight inhibitors. Hence, computational screening was performed to search for inhibitory compounds acting on the 3CL protease. As a preliminary assessment of the performance of this approach, we used 51 compounds for which inhibitory activity had already been confirmed. Docking simulations and molecular mechanics calculations were performed to evaluate binding scores. The preliminary evaluation suggested that our approach successfully selected the inhibitory compounds identified by the experiments. The same approach was applied to 8820 compounds in a database consisting of approved and investigational chemicals. Hence, docking simulations, molecular mechanics calculations, and re-evaluation of binding scores including solvation effects were performed, and the top 200 poses were selected as candidates for experimental assays. Consequently, 25 compounds were chosen for in vitro measurement of the enzymatic inhibitory activity. From the enzymatic assay, 5 compounds were identified to have inhibitory activities against the 3CL protease. The present work demonstrated the feasibility of a combination of docking simulation and molecular mechanics calculation for practical use in computational virtual screening.

Identification of antibody against wingless­type MMTV integration site family member 7B as a biliary cancer tumor marker.

Biliary cancer has a poor prognosis due to a lack of specific biomarkers and difficulty in diagnosis. The present study aimed to identify serum tumor markers for the diagnosis of biliary cancer via serological identification of antigens by recombinant cDNA expression cloning. Wingless­type MMTV integration site family, member 7 (WNT7B) was identified as a target antigen, suggesting the presence of serum antibodies against this antigen. Deletion mutants were then prepared to evaluate the response to serum antibodies. When serum antibody levels against WNT7B deletion mutants (WNT7B-92­2, -92­260, 2-260 and 184-260) were examined using amplified luminescence proximity homogeneous assay­linked immunosorbent assay, the levels of the antibody against WNT7B with amino acids 184­260 were higher in patients with biliary cancer than in healthy donors. Therefore, the region covering residues 184­260 of WNT7B was decomposed to generate seven peptides, and the levels of antibodies against these peptides were measured. Among them, the levels of antibodies against WNT7B234­253 and WNT7B244­260 were higher in patients with biliary cancers than in healthy donors (WNT7B234­253, P=0.0009; WNT7B244­260, P=0.0005). The levels of the antibody against the former were specifically high in patients with biliary cancer but not in those with esophageal, gastric, colorectal, pancreatic, or breast cancer. Furthermore, analysis by the cutoff value of WNT7B234­253 defined by ROC showed a high sensitivity of 70% in patients with biliary cancer. Therefore, the serum levels of the antibody against WNT7B234­253 may be useful as a marker for biliary cancer diagnosis.

Assessing the Potency of ß-Lactamase Inhibitors with Diverse Inactivation Mechanisms against the PenA1 Carbapenemase from Burkholderia multivorans.

Burkholderia cepacia complex (Bcc) poses a serious health threat to people with cystic fibrosis or compromised immune systems. Infections often arise from Bcc strains, which are highly resistant to many classes of antibiotics, including ß-lactams. ß-Lactam resistance in Bcc is conferred largely via PenA-like ß-lactamases. Avibactam was previously shown to be a potent inactivator of PenA1. Here, we examined the inactivation mechanism of PenA1, a class A serine carbapenemase from Burkholderia multivorans using ß-lactamase inhibitors (ß-lactam-, diazabicyclooctane-, and boronate-based) with diverse mechanisms of action. In whole cell based assays, avibactam, relebactam, enmetazobactam, and vaborbactam restored susceptibility to piperacillin against PenA1 expressed in Escherichia coli. The rank order of potency of inactivation in vitro based on kinact/KI or k2/K values (range: 3.4 × 102 to 2 × 106 M-1 s-1) against PenA1 was avibactam > enmetazobactam > tazobactam > relebactam > clavulanic acid > vaborbactam. The contribution of selected amino acids (S70, K73, S130, E166, N170, R220, K234, T237, and D276) in PenA1 toward inactivation was evaluated using site-directed mutagenesis. The S130A, R220A, and K234A variants of PenA1 were less susceptible to inactivation by avibactam. The R220A variant was purified and assessed via steady-state inhibition kinetics and found to possess increased Ki-app values and decreased kinact/KI or k2/K values against all tested inhibitors compared to PenA1. Avibactam was the most affected by the alanine replacement at 220 with a nearly 400-fold decreased acylation rate. The X-ray crystal structure of the R220A variant was solved and revealed loss of the hydrogen bonding network between residues 237 and 276 leaving a void in the active site that was occupied instead by water molecules. Michaelis-Menten complexes were generated to elucidate the molecular contributions of the poorer in vitro inhibition profile of vaborbactam against PenA1 (k2/K, 3.4 × 102 M-1 s-1) and was compared to KPC-2, a class A carbapenemase that is robustly inhibited by vaborbactam. The active site of PenA1 is larger than that of KPC-2, which impacted the ability of vaborbactam to form favorable interactions, and as a result the carboxylate of vaborbactam was drawn toward K234/T235 in PenA1 displacing the boronic acid from approaching the nucleophilic S70. Moreover, in PenA1, the tyrosine at position 105 compared to tryptophan in KPC-2, was more flexible rotating more than 90°, and as a result PenA1's Y105 competed for binding with the cyclic boronate vs the thiophene moiety of vaborbactam, further precluding inhibition of PenA1 by vaborbactam. Given the 400-fold decreased k2/K for the R220A variant compared to PenA1, acyl-enzyme complexes were generated via molecular modeling and compared to the PenA1-avibactam crystal structure. The water molecules occupying the active site of the R220A variant are unable to stabilize the T237 and D276 region of the active site altering the ability of avibactam to form favorable interactions compared to PenA1. The former likely impacts the ability of all inhibitors to effectively acylate this variant enzyme. Based on the summation of all evidence herein, the utility of these newer ß-lactamase inhibitors (i.e., relebactam, enmetazobactam, avibactam, and vaborbactam) in combination with a ß-lactam against B. multivorans producing PenA1 and the R220A variant is promising.

Anti-FIRΔexon2 autoantibody as a novel indicator for better overall survival in gastric cancer.

There is no clinically available biomarker for efficiently indicating the overall survival or therapy response of gastric cancer (GC). The autoantibodies (Abs) in the sera of anti-far-upstream element-binding protein-interacting repressor-lacking exon2 (FIRΔexon2), anti-sorting nexin 15, and anti-spermatogenesis and oogenesis-specific basic helix-loop-helix 1 were markedly higher in GC patients than in healthy donors (HDs). These Abs were identified by large-scale serological identification of antigens by recombinant cDNA expression cloning screenings and their expression levels were evaluated by amplified luminescence proximity homogeneous assay. In particular, compared with age-matched HDs, the level of anti-FIRΔexon2 Abs in GC patients was significantly higher (P < .001). The Spearman's rank correlation analysis between anti-FIRΔexon2 Abs and clinically available tumor markers such as carcinoembryonic antigen (CEA) was statistically insignificant, indicating that FIRΔexon2 Abs is an independent biomarker. We performed receiver-operating curve analysis to evaluate the anti-FIRΔexon2 Ab as a candidate biomarker with CEA and carbohydrate antigen 19-9 (CA19-9). The overall survival of GC patients with high anti-FIRΔexon2 Abs titer was significantly favorable (P = .04) than that of GC patients who were below detection level of anti-FIRΔexon2 Abs. However, clinical stages were not apparently correlated with the levels of anti-FIRΔexon2 Ab, CEA, and CA19-9. In conclusion, anti-FIRΔexon2 Abs detected in GC patients is a potential biomarker for monitoring a better prognosis. Hence, anti-FIRΔexon2 Abs is a promising biomarker for indicating better overall survival of gastric cancer patients.

Post-transcriptional regulation of BRG1 by FIRΔexon2 in gastric cancer.

Brahma-related gene 1 (BRG1), an ATPase subunit of the SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex controls multipotent neural crest formation by regulating epithelial-mesenchymal transition (EMT)-related genes with adenosine triphosphate-dependent chromodomain-helicase DNA-binding protein 7 (CHD7). The expression of BRG1 engages in pre-mRNA splicing through interacting RNPs in cancers; however, the detailed molecular pathology of how BRG1and CHD7 relate to cancer development remains largely unveiled. This study demonstrated novel post-transcriptional regulation of BRG1 in EMT and relationship with FIRΔexon2, which is a splicing variant of the far-upstream element-binding protein (FUBP) 1-interacting repressor (FIR) lacking exon 2, which fails to repress c-myc transcription in cancers. Previously, we have reported that FIR complete knockout mice (FIR-/-) was embryonic lethal before E9.5, suggesting FIR is crucial for development. FIRΔexon2 acetylated H3K27 on promoter of BRG1 by CHIP-sequence and suppressed BRG1 expression post-transcriptionally; herein BRG1 suppressed Snai1 that is a transcriptional suppressor of E-cadherin that prevents cancer invasion and metastasis. Ribosomal proteins, hnRNPs, splicing-related factors, poly (A) binding proteins, mRNA-binding proteins, tRNA, DEAD box, and WD-repeat proteins were identified as co-immunoprecipitated proteins with FIR and FIRΔexon2 by redoing exhaustive mass spectrometry analysis. Furthermore, the effect of FIRΔexon2 on FGF8 mRNA splicing was examined as an indicator of neural development due to impaired CHD7 revealed in CHARGE syndrome. Expectedly, siRNA of FIRΔexon2 altered FGF8 pre-mRNA splicing, indicated close molecular interaction among FIRΔexon2, BRG1 and CHD7. FIRΔexon2 mRNA was elevated in human gastric cancers but not in non-invasive gastric tumors in FIR+/ mice (K19-Wnt1/C2mE x FIR+/-). The levels of FIR family (FIR, FIRΔexon2 and PUF60), BRG1, Snai1, FBW7, E-cadherin, c-Myc, cyclin-E, and SAP155 increased in the gastric tumors in FIR+/- mice compared to those expressed in wild-type mice. FIR family, Snai1, cyclin-E, BRG1, and c-Myc showed trends toward higher expression in larger tumors than in smaller tumors in Gan-mice (K19-Wnt1/C2mE). The expressions of BRG1 and Snai1 were positively correlated in the gastric tumors of the Gan-mice. Finally, BRG1 is a candidate substrate of F-box and WD-repeat domain-containing 7 (FBW7) revealed by three-dimensional crystal structure analysis that the U2AF-homology motif (UHM) of FIRΔexon2 interacted with tryptophan-425 and asparate-399 (WD)-like motif in the degron pocket of FBW7 as a UHM-ligand motif. Together, FIRΔexon2 engages in multi-step post-transcriptional regulation of BRG1, affecting EMT through the BRG1/Snai1/E-cadherin pathway and promoting tumor proliferation and invasion of gastric cancers.

Anti-FIRΔexon2, a splicing variant form of PUF60, autoantibody is detected in the sera of esophageal squamous cell carcinoma.

Anti-PUF60 autoantibodies are reportedly detected in the sera of patients with dermatomyositis and Sjögren's syndrome; however, little is known regarding its existence in the sera of cancer patients. FIR, a splicing variant of the PUF60 gene, is a transcriptional repressor of c-myc. In colorectal cancer, there is an overexpression of the dominant negative form of FIR, in which exon 2 is lacking (FIRΔexon2). Previously, large-scale SEREX (serological identification of antigens by recombinant cDNA expression cloning) screenings have identified anti-FIR autoantibodies in the sera of cancer patients. In the present study, we revealed the presence and significance of anti-FIR (FIR/FIRΔexon2) Abs in the sera of patients with esophageal squamous cell carcinoma (ESCC). Our results were validated by an amplified luminescence proximity homogeneous assay using sera of patients with various cancer types. We revealed that anti-FIRΔexon2 Ab had higher sensitivity than anti-FIR Ab. Receiver operating characteristic (ROC) analysis was applied for evaluating the use of anti-FIRΔexon2 Ab as candidate markers such as anti-p53 Ab and carcinoembryonic antigen, and the highest area under the ROC curve was observed in the combination of anti-FIRΔexon2 Ab and anti-p53 Ab. In summary, our results suggest the use of anti-FIRΔexon2 Ab in combination with the anti-p53 Ab as a predictive marker for ESCC. The area under the ROC curve was further increased in the advanced stage of ESCC. The value of anti-FIRΔexon2 autoantibody as novel clinical indicator against ESCC and as a companion diagnostic tool is discussed.

Disturbed alternative splicing of FIR (PUF60) directed cyclin E overexpression in esophageal cancers.

Overexpression of alternative splicing of far upstream element binding protein 1 (FUBP1) interacting repressor (FIR; poly(U) binding splicing factor 60 [PUF60]) and cyclin E were detected in esophageal squamous cell carcinomas (ESCC). Accordingly, the expression of FBW7 was examined by which cyclin E is degraded as a substrate via the proteasome system. Expectedly, FBW7 expression was decreased significantly in ESCC. Conversely, c-myc gene transcriptional repressor FIR (alias PUF60; U2AF-related protein) and its alternative splicing variant form (FIRΔexon2) were overexpressed in ESCC. Further, anticancer drugs (cis-diaminedichloroplatinum/cisplatin [CDDP] or 5-fluorouracil [5-FU]) and knockdown of FIR by small interfering RNA (siRNA) increased cyclin E while knockdown of FIRΔexon2 by siRNA decreased cyclin E expression in ESCC cell lines (TE1, TE2, and T.Tn) or cervical SCC cells (HeLa cells). Especially, knockdown of SAP155 (SF3b1), a splicing factor required for proper alternative splicing of FIR pre-mRNA, decreased cyclin E. Therefore, disturbed alternative splicing of FIR generated FIR/FIRΔexon2 with cyclin E overexpression in esophageal cancers, indicating that SAP155 siRNA potentially rescued FBW7 function by reducing expression of FIR and/or FIRΔexon2. Remarkably, Three-dimensional structure analysis revealed the hypothetical inhibitory mechanism of FBW7 function by FIR/FIRΔexon2, a novel mechanism of cyclin E overexpression by FIR/FIRΔexon2-FBW7 interaction was discussed. Clinically, elevated FIR expression potentially is an indicator of the number of lymph metastases and anti-FIR/FIRΔexon2 antibodies in sera as cancer diagnosis, indicating chemical inhibitors of FIR/FIRΔexon2-FBW7 interaction could be potential candidate drugs for cancer therapy. In conclusion, elevated cyclin E expression was, in part, induced owing to potential FIR/FIRΔexon2-FBW7 interaction in ESCC.

Anti-FIRs (PUF60) auto-antibodies are detected in the sera of early-stage colon cancer patients.

Anti-PUF60, poly(U)-binding-splicing factor, autoantibodies are reported to be detected in the sera of dermatomyositis and Sjogren's syndrome that occasionally associated with malignancies. PUF60 is identical with far-upstream element-binding protein-interacting repressor (FIR) that is a transcriptional repressor of c-myc gene. In colorectal cancers, a splicing variant of FIR that lacks exon2 (FIRΔexon2) is overexpressed as a dominant negative form of FIR. In this study, to reveal the presence and the significance of anti-FIRs (FIR/FIRΔexon2) antibodies in cancers were explored in the sera of colorectal and other cancer patients. Anti-FIRs antibodies were surely detected in the preoperative sera of 28 colorectal cancer patients (32.2% of positive rates), and the detection rate was significantly higher than that in healthy control sera (Mann-Whitney U test, p < 0.01). The level of anti-FIRs antibodies significantly decreased after the operation (p < 0.01). Anti-FIRs antibodies were detected in the sera of early-stage and/or recurrent colon cancer patients in which anti-p53 antibodies, CEA, and CA19-9 were not detected as well as in the sera of other cancer patients. Furthermore, the area under the curve of receiver operating characteristic for anti-FIRs antibodies was significantly larger (0.85) than that for anti-p53 antibodies or CA19-9. In conclusions, the combination of anti-FIRs antibodies with other clinically available tumor markers further improved the specificity and accuracy of cancer diagnosis.

Effects of novel small compounds targeting TrkB on neuronal cell survival and depression-like behavior.

Brain-derived neurotrophic factor (BDNF) and its high affinity receptor tyrosine kinase receptor B (TrkB) are involved in neuronal survival, maintenance, differentiation and synaptic plasticity. Deficiency of BDNF was reported to be associated with psychological disorders such as depression. Hence we examined proliferative effect of 11 candidate TrkB agonistic compounds in TrkB-expressing SH-SY5Y cells, via a hypothesis that some candidate compounds identified in our previous in silico screening for a small molecule targeting the BDNF binding domain of TrkB should activate TrkB signaling. In the present study, two promising compounds, 48 and 56, were identified and subsequently assessed for their ability to induce TrkB phosphorylation in vitro and in vivo. Likewise those seen in BDNF, the compounds mediated TrkB phosphorylation was blocked by the Trk inhibitor, K252a. Since BDNF-TrkB signaling deficiency is associated with the pathogenesis of depression and reactivation of this signaling by antidepressants is a cause of the pathogenic state recovery, the compounds were subjected to the assessment for forced swim test, which is a mouse model of depression. We found that compound 48 significantly reduced mouse immobility time compared with the control vehicle injection, suggesting the confirmation of hypothetical antidepressant-like efficacy of 48 compound in vivo. Thus, our present study demonstrated that compound 48, selected through in silico screening, is a novel activator of TrkB signaling and a potential antidepressant molecule.

Structural insight of DNA topoisomerases I from camptothecin-producing plants revealed by molecular dynamics simulations.

DNA topoisomerase I (Top1) catalyzes changes in DNA topology by cleaving and rejoining one strand of the double stranded (ds)DNA. Eukaryotic Top1s are the cellular target of the plant-derived anticancer indole alkaloid camptothecin (CPT), which reversibly stabilizes the Top1-dsDNA complex. However, CPT-producing plants, including Camptotheca acuminata, Ophiorrhiza pumila and Ophiorrhiza liukiuensis, are highly resistant to CPT because they possess point-mutated Top1. Here, the adaptive convergent evolution is reported between CPT production ability and mutations in their Top1, as a universal resistance mechanism found in all tested CPT-producing plants. This includes Nothapodytes nimmoniana, one of the major sources of CPT. To obtain a structural insight of the resistance mechanism, molecular dynamics simulations of CPT- resistant and -sensitive plant Top1s complexed with dsDNA and topotecan (a CPT derivative) were performed, these being compared to that for the CPT-sensitive human Top1. As a result, two mutations, Val617Gly and Asp710Gly, were identified in O. pumila Top1 and C. acuminata Top1, respectively. The substitutions at these two positions, surprisingly, are the same as those found in a CPT derivative-resistant human colon adenocarcinoma cell line. The results also demonstrated an increased linker flexibility of the CPT-resistant Top1, providing an additional explanation for the resistance mechanism found in CPT-producing plants. These mutations could reflect the long evolutionary adaptation of CPT-producing plant Top1s to confer a higher degree of resistance.

Haploinsufficiency of the c-myc transcriptional repressor FIR, as a dominant negative-alternative splicing model, promoted p53-dependent T-cell acute lymphoblastic leukemia progression by activating Notch1.

FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR⁺/⁻) C57BL6 mice were generated. FIR complete knockout (FIR⁻/⁻) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR⁺/⁻ mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR⁺/⁻TP53⁻/⁻ generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR⁺/⁻TP53⁻/⁻ compared with that in FIR⁺/⁺TP53⁻/⁻ mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion, the alternative splicing of FIR, which generates FIRΔexon2, may contribute to both colorectal carcinogenesis and leukemogenesis.

Molecular simulation analysis of the structure complex of C2 domains of DKK family members and ß-propeller domains of LRP5/6: explaining why DKK3 does not bind to LRP5/6.

Dickkopf (DKK) proteins interact with low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to modulate WNT signaling. The interaction is mediated by a cysteine-rich domain (C2) in the DKK protein and beta-propeller domains (PD) of LRP5/6. However, the third member of the DKK family (DKK3) does not bind to LRP5/6. To determine why DKK3 does not bind to the receptor domains, we performed a molecular modeling simulation study including homology modeling, protein-protein docking and molecular dynamics (MD). The computed affinities (ΔGbinding) between the C2 and PD models were consistent with the previously reported experimental results. The C2 model of DKK3 showed the lowest affinity for PD models. Multiple sequence alignment of C2 domains revealed that the DKK3 genes have a unique 7-amino-acid insertion (L249-E255 in human DKK3) and P258 in a finger loop 1 (FL1). Interestingly, the insertion sequence is evolutionally conserved. MD simulations of high-affinity complex models of C2 and PD showed that FL1 directly interacts with the PD models and stabilizes the complex models. We also built a 7-amino-acid-deletion/P258G mutant model of DKK3C2 and estimated its affinities for the PD models. The affinity for human LRP5PD2 was increased by the substitution (ΔGbinding＝－48.9kcal/mol) and the affinity was compatible with that of high-affinity ligands. The results suggested that the lack of affinity between human DKK3 and human LRP5/6 results from: i) insertion of the 7 amino acids, and ii) P258 in human DKK3. The sequence differences thus suggest an explanation for this unique property of DKK3.

Identification of novel candidate compounds targeting TrkB to induce apoptosis in neuroblastoma.

Neuroblastoma (NB) is one of the most frequent solid tumors in children and its prognosis is still poor. The neurotrophin receptor TrkB and its ligand brain-derived neurotrophic factor (BDNF) are expressed at high levels in high-risk NBs and are involved in defining the poor prognosis of the patients. However, the TrkB targeting therapy has never been realized in the clinic. We performed an in silico screening procedure utilizing an AutoDock/grid computing technology in order to identify novel small chemical compounds targeting the BDNF-binding domain of TrkB. For the first screening, a library of three million synthetic compounds was screened in silico and was ranked according to the Docking energy. The top-ranked 37 compounds were further functionally screened for cytotoxicity by using NB cell lines. We have finally identified seven compounds that kill NB cells with the IC50 values of 0.07-4.6 µmol/L. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed that these molecules induce apoptosis accompanied by p53 activation in NB cell lines. The candidate compounds and BDNF demonstrated an antagonistic effect on cell growth, invasion, and colony formation, possibly suggesting competition at the BDNF-binding site of TrkB. The candidate compounds had tumor-suppressive activity in xenograft and in vivo toxicity tests (oral and intravenous administrations) using mice, and did not show any abnormal signs. Using in silico Docking screening we have found new candidate TrkB inhibitors against high-risk NBs, which could lead to new anti-cancer drugs.

Synthesis, structure, and aromaticity of the nickel(II) complex of pyricorrole, a molecular hybrid of porphyrin and corrole.

Formal migration of one meso-carbon atom in the porphyrin ring into the pyrrole moiety results in an isomer "pyricorrole", a pyridine-containing corrole macrocycle. We prepared the nickel(II) complex of pyricorrole by the nickel(II)-induced cyclization of a linear precursor. Electronic absorption and proton NMR spectra of this compound revealed the presence of an 18π-electron circuit over the macrocycle, suggesting that aromaticity was retained after intensive rearrangement of the porphyrin core. X-ray crystallography of the nickel(II) complex confirmed the planar structure and demonstrated that it possesses hybrid properties of porphyrin and corrole.

Computational analysis on the binding of epitope peptide to human leukocyte antigen class I molecule A*2402 subtype.

Immunological response induced by small amino peptide has attracted much recent attention in the field of immunotherapy. Wilms' tumor (WT1) protein is one of the potent tumor antigens inducing immunological response in mouse and human, because WT1 is over expressed in many types of leukemia and various kinds of solid tumors. A 9-mer peptide encoded in WT1 protein (CMTWNQMNL; amino acid 235-243) is known to serve as antigenic peptide for human leukocyte antigen (HLA)-A*2402 molecule. It was reported that the replacement of the second amino residue, which is deeply responsible for the peptide binding to HLA, induced strong immunological response compared to the natural peptide. In this study, 19 kinds of single amino substitutions were introduced at position 2 of this 9-mer WT1 peptide. We performed molecular dynamics simulation on the complex of each of WT1 epitope peptides and HLA-ß2 micro globulin (ß2m) molecule, and subsequently estimated the binding affinity using molecular mechanics/generalized-Born surface area method combined with normal mode analysis. Our computation indicated that the peptide containing M2Y or M2W mutation showed high binding affinity to the HLA-ß2m molecule as well as the natural peptide. We have also examined the role of the residue at position 2 in peptide binding to HLA-ß2m. The calculation showed that van der Waals interaction between the side chain of the residue at position 2 and hydrophobic residues inside B-pocket of HLA are important. These findings will be helpful to search other potent peptides that will enhance strong immunological response specific to HLA-A*2402 molecule.

Exploring coumarin egress channels in human cytochrome P450 2A6 by random acceleration and steered molecular dynamics simulations.

The kinetic analysis of coumarin oxidation by CYP2A6 suggested that substrate binding and release occurred in the multiple steps and such events proceeded rapidly. However, the crystal structure of the CYP2A6-coumarin complex reveals that no obvious channel is open enough to allow coumarin to pass through. Thus, an intriguing and important question arises: how coumarin enters and exits the active site, which is deeply buried at the center of CYP2A6 fold. In this study, geometric analysis of the potential openings was first performed on all the available crystal structures of CYP2A6. And then, random acceleration molecular dynamics simulations were used to explore the possible substrate egress channels in CYP2A6. Two channels were most frequently observed. Afterwards, steered molecular dynamics simulations were performed and potentials of mean force were constructed to compare the preference of the two channels serving as the substrate egress channel. The results showed that channel 2c, which is located between helices I and G and the helix B'-C region, was the most likely channel for coumarin egress. The opening of channel 2c was characterized by a rotation of Phe111 together with a bending of helix B'. Our findings will not only be helpful for understanding the unbinding mechanism of coumarin and for identifying structural determinants related to the biological function of CYP2A6, but also provide further insight into the channel selectivity of P450s.

Peptide HIV-1 integrase inhibitors from HIV-1 gene products.

Anti-HIV peptides with inhibitory activity against HIV-1 integrase (IN) have been found in overlapping peptide libraries derived from HIV-1 gene products. In a strand transfer assay using IN, inhibitory active peptides with certain sequential motifs related to Vpr- and Env-derived peptides were found. The addition of an octa-arginyl group to the inhibitory peptides caused a remarkable inhibition of the strand transfer and 3'-end-processing reactions catalyzed by IN and significant inhibition against HIV replication.