An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states.

Deep Learning-Driven Transformation: A Novel Approach for Mitigating Batch Effects in Diffusion MRI Beyond Traditional Harmonization.

BACKGROUND: "Batch effect" in MR images, due to vendor-specific features, MR machine generations, and imaging parameters, challenges image quality and hinders deep learning (DL) model generalizability. PURPOSE: We aim to develop a DL model using contrast adjustment and super-resolution to reduce diffusion-weighted images (DWIs) diversity across magnetic field strengths and imaging parameters. STUDY TYPE: Retrospective. SUBJECTS: The DL model was built using an open dataset from one individual. The MR machine identification model was trained and validated on a dataset of 1134 adults (54% females, 46% males), with 1050 subjects showing no DWI abnormalities and 84 with conditions like stroke and tumors. The 21,000 images were divided into 80% for training, 20% for validation, and 3500 for testing. FIELD STRENGTH/SEQUENCE: Seven MR scanners from four manufacturers with 1.5 T and 3 T magnetic field strengths. DWIs were acquired using spin-echo sequences and high-resolution T2WIs using the T2-SPACE sequence. ASSESSMENT: An experienced, board-certified radiologist evaluated the effectiveness of restoring high-resolution T2WI and harmonizing diverse DWI with metrics such as PSNR and SSIM, and the texture and frequency attributes were further analyzed using gray-level co-occurrence matrix and 1-dimensional power spectral density. The model's impact on machine-specific characteristics was gauged through the performance metrics of a ResNet-50 model. Comprehensive statistical tests were employed for statistical robustness, including McNemar's test and the Dice index. RESULTS: Our DL protocol reduced DWI contrast and resolution variation. ResNet-50 model's accuracy decreased from 0.9443 to 0.5786, precision from 0.9442 to 0.6494, recall from 0.9443 to 0.5786, and F1 score from 0.9438 to 0.5587. The t-SNE visualization indicated more consistent image features across multiple MR devices. Autoencoder halved learning iterations; Dice coefficient >0.74 confirmed signal reproducibility in 84 lesions. CONCLUSION: This study presents a DL strategy to mitigate batch effects in diffusion MR images, improving their quality and generalizability. EVIDENCE LEVEL: 3 TECHNICAL EFFICACY: Stage 1.

DGCR8-dependent efficient pri-miRNA processing of human pri-miR-9-2.

Microprocessor complex, including DiGeorge syndrome critical region gene 8 (DGCR8) and DROSHA, recognizes and cleaves primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical miRNAs. The study of DGCR8 haploinsufficiency reveals that the efficiency of this activity varies for different miRNA species. It is thought that this variation might be associated with the risk of schizophrenia with 22q11 deletion syndrome caused by disruption of the DGCR8 gene. However, the underlying mechanism for varying action of DGCR8 with each miRNA remains largely unknown. Here, we used in vivo monitoring to measure the efficiency of DGCR8-dependent microprocessor activity in cultured cells. We confirmed that this system recapitulates the microprocessor activity of endogenous pri-miRNA with expression of a ratiometric fluorescence reporter. Using this system, we detected mir-9-2 as one of the most efficient targets. We also identified a novel DGCR8-responsive RNA element, which is highly conserved among mammalian species and could be regulated at the epi-transcriptome (RNA modification) level. This unique feature between DGCR8 and pri-miR-9-2 processing may suggest a link to the risk of schizophrenia.

Gene regulatory network analysis defines transcriptome landscape with alternative splicing of human umbilical vein endothelial cells during replicative senescence.

BACKGROUND: Endothelial cell senescence is the state of permanent cell cycle arrest and plays a critical role in the pathogenesis of age-related diseases. However, a comprehensive understanding of the gene regulatory network, including genome-wide alternative splicing machinery, involved in endothelial cell senescence is lacking. RESULTS: We thoroughly described the transcriptome landscape of replicative senescent human umbilical vein endothelial cells. Genes with high connectivity showing a monotonic expression increase or decrease with the culture period were defined as hub genes in the co-expression network. Computational network analysis of these genes led to the identification of canonical and non-canonical senescence pathways, such as E2F and SIRT2 signaling, which were down-regulated in lipid metabolism, and chromosome organization processes pathways. Additionally, we showed that endothelial cell senescence involves alternative splicing. Importantly, the first and last exon types of splicing, as observed in FLT1 and ACACA, were preferentially altered among the alternatively spliced genes during endothelial senescence. We further identified novel microexons in PRUNE2 and PSAP, each containing 9 nt, which were altered within the specific domain during endothelial senescence. CONCLUSIONS: These findings unveil the comprehensive transcriptome pathway and novel signaling regulated by RNA processing, including gene expression and splicing, in replicative endothelial senescence.

Identification of hub molecules of FUS-ALS by Bayesian gene regulatory network analysis of iPSC model: iBRN.

Fused in sarcoma/translated in liposarcoma (FUS) is a causative gene of amyotrophic lateral sclerosis (ALS). Mutated FUS causes accumulation of DNA damage and cytosolic stress granule (SG) formation, thereby motor neuron (MN) death. However, key molecular aetiology remains unclear. Here, we applied a novel platform technology, iBRN, "Non- biased" Bayesian gene regulatory network analysis based on induced pluripotent stem cell (iPSC)-derived cell model, to elucidate the molecular aetiology using transcriptome of iPSC-derived MNs harboring FUSH517D. iBRN revealed "hub molecules", which strongly influenced transcriptome network, such as miR-125b-5p-TIMELESS axis and PRKDC for the molecular aetiology. Next, we confirmed miR-125b-5p-TIMELESS axis in FUSH517D MNs such that miR-125b-5p regulated several DNA repair-related genes including TIMELESS. In addition, we validated both introduction of miR-125b-5p and knocking down of TIMELESS caused DNA damage in the cell culture model. Furthermore, PRKDC was strongly associated with FUS mis-localization into SGs by DNA damage under impaired DNA-PK activity. Collectively, our iBRN strategy provides the first compelling evidence to elucidate molecular aetiology in neurodegenerative diseases.

Clinicopathologic Study of Placentas From Women With a Fontan Circulation.

BACKGROUND: Pregnant women with a Fontan circulation have a high risk of obstetric complications, such as preterm delivery and small for gestational age (SGA), which may be affected by low blood flow to the placenta and hypoxia. This study investigated placental pathology in a Fontan circulation.Methods and Results:Eighteen pregnancies in 11 women with a Fontan circulation were reviewed. Pregnancy outcomes showed 9 miscarriages and 9 live births, with 4 preterm deliveries. Five neonates were SGA (<5th percentile). Eight placentas from live births in 7 women were available for the study. Five placentas had low weight placenta for gestational age, and 7 grossly showed a chronic subchorionic hematoma. Histological examination revealed all placentas had some form of histological hypoxic lesions: maternal vascular malperfusion in 7, fetal vascular malperfusion in 1, and other hypoxia-related lesions in 8. Quantitative analyses, including immunohistochemistry (CD31, CD68, and hypoxia inducible factor-1α antibodies) and Masson's trichrome staining, were also performed and compared with 5 control placentas. Capillary density and the area of fibrosis were significantly greater in placentas from women with a Fontan circulation than in control placentas. CONCLUSIONS: Placentas in a Fontan circulation were characterized by a high frequency of low placental weight, chronic subchorionic hematoma, and constant histological hypoxic changes, which could reflect altered maternal cardiac conditions and lead to poor pregnancy outcomes.

Identification of a selective DDX3X inhibitor with newly developed quantitative high-throughput RNA helicase assays.

The DEAD-box family of RNA helicases plays essential roles in both transcriptional and translational mRNA degradation; they unwind short double-stranded RNA by breaking the RNA-RNA interactions. Two DEAD-box RNA helicases, eukaryotic translation initiation factor 4A3 (eIF4A3) and DEAD-box helicase 3 (DDX3X), show high homology in the ATP-binding region and are considered key molecules for cancer progression. Several small molecules that target eIF4A3 and DDX3X have been reported to inhibit cancer cell growth; however, more potent compounds are required for cancer therapeutics, and there is a critical need for high-throughput assays to screen for RNA helicase inhibitors. In this study, we developed novel fluorescence resonance energy transfer-based high-throughput RNA helicase assays for eIF4A3 and DDX3X. Using these assays, we identified several eIF4A3 allosteric inhibitors whose inhibitory effect on eIF4A3 ATPase showed a strong correlation with inhibitory effect on helicase activity. From 102 compounds that exhibited eIF4A3 ATPase inhibition, we identified a selective DDX3X inhibitor, C1, which showed stronger inhibition of DDX3X than of eIF4A3. Small-molecule helicase inhibitors can be valuable for clarifying the molecular machinery of DEAD-box RNA helicases. The high-throughput quantitative assays established here should facilitate the evaluation of the helicase inhibitory activity of compounds.

Environment-dependent introgression from Quercus dentata to a coastal ecotype of Quercus mongolica var. crispula in northern Japan.

Introgression from one species in a specific environment to another may facilitate colonization of the environment by the recipient species. However, such environment-dependent introgression has been clarified in limited plant taxa. In northern Japan, there are two interfertile oak species: Quercus dentata (Qd) in coastal areas and Q. mongolica var. crispula (Qc) in inland areas. However, at higher latitudes where Qd is rare, a coastal Qc ecotype with Qd-like traits is distributed in the coastal areas. We distinguished inland Qc, coastal Qc, and coastal Qd populations based on genome-wide genotypes and multitrait phenotypes and verified introgression from coastal Qd to coastal Qc using reduced library sequencing. Genotypes and phenotypes differed among the populations, and coastal Qc was intermediate between inland Qc and coastal Qd. The ABBA-BABA test showed introgression from coastal Qd to coastal Qc. In coastal Qc, we found various stages of introgression after the first generation of backcross but detected no genomic regions where introgression was enhanced. Overall, we show evidence for introgression from a coastal species to an ecotype of an inland species, which has colonized the coastal environment. It remains unclear whether introgressed alleles are selected in the coastal environment.

Spatial variation in bird pollination and its mitigating effects on the genetic diversity of pollen pools accepted by Camellia japonica trees within a population at a landscape level.

Bird pollination can vary spatially in response to spatial fluctuations in flowering even within plant populations. In this study, we examined the hypothesis that the spatial variation in bird pollination may induce mitigating effects, which maintains or increases genetic diversity of pollen pools at local sites with low flowering densities. To test this hypothesis, we analyzed the landscape-level genetic effects within a population of Camellia japonica on the pollen pools accepted by individuals in two reproductive years by using genotypes at eight microsatellite loci of 1323 seeds from 19 seed parents. Regression analyses using the quadratic models of correlated paternity between pollen pools against spatial distances between the seed-parent pairs revealed not only local pollination but also some amount of long-distance pollen dispersal. The genetic diversity of pollen pools accepted by seed parents tended to be negatively related to the densities of flowering individuals near the seed parents during winter (when the effective pollination of C. japonica is mediated mostly by Zosterops japonica). We show that the low density of flowering individuals may induce the expansion of the foraging areas of Z. japonica and consequently increase the genetic diversity of pollen pools. This spatial variation in bird pollination may induce the mitigating effects on the C. japonica population. The comparisons between the two study years indicate that the overall pattern of bird pollination and the genetic effects described here, including the mitigating effects, may be stable over time.

Terahertz optical properties of wood-plastic composites.

Terahertz (THz) optical materials containing polymeric materials have been useful for terahertz technologies. We investigated the THz optical properties of wood-plastic composites (WPCs), which are composed of polystyrene and wood powder, and their suitability as THz optical materials. We found that the refractive indexes and absorption coefficients of the WPCs increased with increasing wood powder content. WPCs are inexpensive and have tunable THz optical properties.

Detection of single human hairs with a terahertz nonlinear quantum cascade laser.

We report the demonstration of imaging of a single human hair with a terahertz quantum cascade laser (THz-QCL) source based on intracavity difference-frequency generation. A single human hair whose diameter was about 100 µm was detected using the THz-QCL source operating at 240 K, of which the THz beam had a linear polarization. The results show that the THz image of a human hair clearly depends on the polarization direction of the THz beam. The THz QCL sources that are capable of room temperature operation will be useful for detection of small foreign objects like human hairs.

Appropriate delivery method for cardiac disease pregnancy based on noninvasive cardiac monitoring.

Background There are numerous significant physiological changes occurring in circulation during labor. To detect these rapid hemodynamic changes, invasive and intermittent measurement techniques are not reliable. To suggest a suitable delivery method for pregnancy with cardiac disease, this study analyzed how each delivery method influences cardiac function using a noninvasive and continuous measurement technique. Methods A prospective study was accomplished at the National Cerebral and Cardiovascular Center in Japan from October 1, 2014, to November 30, 2018. The classification of the healthy heart pregnant women was according to the delivery method: vaginal delivery (VD) without epidural anesthesia, VD with epidural anesthesia, and caesarean section (CS). The hemodynamic parameters cardiac index (CI), stroke volume index (SI), and heart rate (HR) were evaluated regularly throughout delivery by noninvasive electrical cardiometry monitor. Results Ten cases were examined for each group. CI and HR were significantly increased before VD, while the increase in CI and HR was mild in the epidural group in comparison to the nonepidural group. SI was increased toward the delivery in the epidural group, and it was constant in the nonepidural group. However, there was no alteration in the level of outcomes of the two groups. In CS, SI increased and HR decreased before delivery. After delivery, SI continued to increase, while HR did not change but CI increased. Conclusion In VD, the increase in venous circulation according to the autotransfusion is managed by increasing HR. By epidural anesthesia, the increase in HR was suppressed and SI was increased. However, as epidural anesthesia increases the vascular capacity, the level of SI outcome was comparable. In CS, the HR was decreased because of the spinal anesthesia and the SI was increased because of many factors like hydration. As there are many factors to control in CS, VD with epidural anesthesia will be the first preference for most cardiac patients.

Terahertz imaging with room-temperature terahertz difference-frequency quantum-cascade laser sources.

We demonstrate high-quality non-destructive imaging using a broadband terahertz quantum cascade laser source based on Cerenkov difference-frequency generation. The source exhibited ultra-broadband terahertz emission spectra, as well as a single-lobed Gaussian-like far-field pattern at -30 °C. These features allowed us to build a compact imaging system with a high spatial resolution, from which a nearly theoretical minimum beam spot size was obtained. As a result, we achieve well-resolved, high-contrast images of objects obscured by opaque materials. We also achieved terahertz imaging with the THz DFG-QCL operated at room temperature.

An admixture of Quercus dentata in the coastal ecotype of Q. mongolica var. crispula in northern Hokkaido and genetic and environmental effects on their traits.

In northern Japan, coastal oak forests consist of Quercus dentata (Qd) on the coastal side and Q. mongolica var. crispula (Qc) on the inland side. In the forests of northern Hokkaido, Qd is rare, and a coastal ecotype of Qc with some Qd-like traits grows on the coastal side. To reveal the genetic background of this ecotype, nuclear microsatellite genotypes in closely related oak taxa were obtained from the Eurasian continent, Sakhalin, and Hokkaido. The clustering of these genotypes suggests an admixture of Qd in the coastal ecotype of Qc. Next, we evaluated the effects of admixture and coastal stress on the leaf and shoot traits of Qc and Qd along coastal-inland gradients in northern Hokkaido. The admixture of Qd in Qc was quantified by the Qd ancestry proportions. Coastal stress causes bud mortality in the upper parts of shoots and was quantified by the survival patterns of buds in shoots. The genetic and environmental effects on the traits at Qd-abundant and Qd-rare sites were estimated using linear mixed models. The genetic effect was detected in all traits. Both genetic and environmental effects were detected in most traits. Some traits differed between Qd-abundant and Qd-rare sites in addition to these effects, indicating more Qd-like traits at Qd-rare sites. The findings suggest that an admixture of Qd characterizes the genetic background of the coastal ecotype of Qc and that not only the coastal stress but also the genetic background is responsible for the leaf and shoot traits of Qc and Qd in northern Hokkaido.

Impact of Pregnancy on Aortic Root in Women with Repaired Conotruncal Anomalies.

The effect of pregnancy on aortic root in women with repaired conotruncal anomalies (CTA) has not been clarified. This study examined aortic diameters during and after pregnancy in women with repaired CTA. A retrospective review of consecutive pregnant women with repaired CTA was performed for results of echocardiography from 1 year before pregnancy to 3 years after delivery and compared with findings from healthy pregnant volunteers. Participants comprised 42 subjects and 49 deliveries with repaired CTA (CTA group), and 47 healthy pregnant women (control group). Although no maternal aortic events were encountered, aortic diameters during pregnancy increased by 1.0 ± 2.2 mm (maximum, 7.0 mm) in the CTA group and 0.6 ± 1.3 mm (maximum, 3.4 mm) in the control group (p = 0.13). The CTA subgroup with increase in aortic diameter ≥ 3.5 mm during pregnancy showed no reversion to baseline diameter at follow-up > 6 months after delivery. Significant risk factors for increased aortic diameter and no reversal included pulmonary atresia, history of aortopulmonary shunt, older age at repair, and smaller left ventricular end-diastolic diameter pre-pregnancy. Women with repaired CTA tolerated pregnancy and delivery well. However, the aortic root progressively dilated during pregnancy and 1/4 of them, especially those with longstanding high aortic blood flow before repair, showed an increase of aortic root size of ≥ 3.5 mm during the peripartum period; this dilatation tended not to revert to the pre-pregnancy diameter. Therefore, serial measurement of the aortic root during pregnancy and after delivery is recommended in these women.

Recurrent Congenital Heart Diseases Among Neonates Born to Mothers with Congenital Heart Diseases.

The frequency of newborns with congenital heart disease (CHD) is approximately 1% in the general population; however, the recurrence rate of CHD in mothers with CHD differs in ethnicity and reports. We therefore aimed to determine the prevalence of CHD among neonates born to mothers with CHD in our institute in Japan. We reviewed the medical charts of 803 neonates delivered by 529 women with CHD at the National Cerebral and Cardiovascular Center from 1982 to 2016. They included isolated ventricular septal defect (VSD,31.4%), isolated atrial septal defect (ASD, 23.3%), tetralogy of Fallot (TOF,10.6%). We defined CHD in neonates as being diagnosed within 1 month of birth. We estimated that the average rate of the CHD recurrence was 3.1%. The recurrence ratios in each maternal CHD were 8.6%, 7.1%, 6.2%, 4.8%, 3.6%, and 1.5% for PS, CoA, TOF, atrioventricular septal defect, VSD, and ASD, respectively. The rate of CHD in offsprings whose mothers have CHD was 3 times greater than that of mothers with healthy hearts. Almost half of neonates with CHD had the same phenotype as their mother in our series. Especially, PS and CoA were closely related to the type of maternal CHD.

Radiologist involvement is associated with reduced use of MRI in the acute period of low back pain in a non-elderly population.

PURPOSE: To test the hypothesis that "acute-period" lumbar MRI in non-elderly patients with low back pain is less frequently performed at clinics/hospitals with greater involvement of full-time radiologists in the imaging workflow. METHODS: In a national-level claims database, we identified 14,819 non-elderly patients (mean age: 38.7±8.0 years) who visited clinics/hospitals for low back pain in 2013-2015. We classified the clinics/hospitals into four groups based on the level of full-time radiologist involvement and MRI ownership, and compared the frequency of acute-period lumbar MRI using hierarchical logistic regression analysis. RESULTS: Patients visiting facilities without a full-time radiologist (n=2105) were significantly (p<0.001) more likely to undergo acute-period MRI than those visiting facilities with ≥1 radiologist partially managing imaging workflow (level-1, n=491) or ≥1 radiologist intensively involved in imaging workflow (level-2, n=1190) (15.7% vs. 6.9% and 7.3%; adjusted odds ratio of no-radiologist versus level-2: 2.93, p=0.018). No difference was observed between level-1 and level-2 involvement. CONCLUSIONS: Facilities with no full-time radiologist were more likely to perform acute-period MRI to assess for low back pain, while no difference was seen between facilities with varying levels of radiologist involvement in the imaging workflow. Radiologist involvement may contribute to optimal utilisation of medical imaging. KEY POINTS: • Lumbar MRI was more frequently performed at facilities without full-time radiologists. • Full-time radiologists may play an important role in appropriate utilisation of imaging. • Frequency of MRI was similar between moderate and intensive radiologist involvement.

Cardiomyopathy Phenotypes and Pregnancy Outcomes with Left Ventricular Noncompaction Cardiomyopathy.

Little is known about pregnancies of left ventricular noncompaction cardiomyopathy (LVNC), much less cases in which LVNC was definitively diagnosed prepregnancy. We report the cases of three pregnant Japanese women definitively diagnosed with LVNC prepregnancy. Case 1 presented LVNC with restrictive phenotype. Her pregnancy was terminated due to exacerbated pulmonary hypertension and low output status at 30 weeks' gestation. Case 2 presented isolated LVNC with nonsustained ventricle tachycardia. A cesarean section was performed at 36 weeks' gestation because of placenta previa. Case 3 presented dilated LVNC. Labor induction was performed because of decreased left ventricular ejection fraction, leading to a vaginal delivery at 37 weeks' gestation. In all cases, no thromboembolic event was identified during pregnancy; two patients received anticoagulants. We reviewed all English-literature cases of pregnant women definitively diagnosed with LVNC prepregnancy to analyze causes of adverse pregnancy outcomes and the necessity of anticoagulation. Four of the six pregnancies identified were terminated due to exacerbated cardiomyopathy phenotypes and not complications due to noncompaction itself, resulting in three cases' preterm deliveries. No thromboembolic event was identified by maintenance of the anticoagulation strategy determined prepregnancy. In pregnancies with LVNC, the possibility of a severe cardiac event and the indications for termination of the pregnancy can depend on the cardiomyopathy phenotypes, not noncompaction itself. Anticoagulation only because of the pregnancy itself may be redundant. In the management of LVNC during pregnancy, close monitoring of the condition of different phenotypes and reassessment of the necessity of anticoagulation can contribute to the pregnancy outcome.

The RNA helicase DDX39B and its paralog DDX39A regulate androgen receptor splice variant AR-V7 generation.

Mounting evidence suggests that constitutively active androgen receptor (AR) splice variants, typified by AR-V7, are associated with poor prognosis and resistance to androgen deprivation therapy in prostate cancer patients. However, mechanisms governing the generation of AR splice variants are not fully understood. In this study, we aimed to investigate the dynamics of AR splice variant generation using the JDCaP prostate cancer model that expresses AR splice variants under androgen depletion. Microarray analysis of JDCaP xenografts before and after expression of AR splice variants suggested that dysregulation of RNA processing pathways is likely involved in AR splice variant generation. To explore factors contributing to generation of AR-V7 mRNA, we conducted a focused RNA interference screen in AR-V7-positive JDCaP-hr cells using an shRNA library targeting spliceosome-related genes. This screen identified DDX39B as a regulator of AR-V7 mRNA expression. Simultaneous knockdown of DDX39B and its paralog DDX39A drastically and selectively downregulated AR-V7 mRNA expression in multiple AR-V7-positive prostate cancer cell lines. DDX39B was upregulated in relapsed JDCaP xenografts expressing AR splice variants, suggesting its role in expression of AR splice variants. Taken together, our findings offer insight into the mechanisms of AR splice variant generation and identify DDX39 as a potential drug target for the treatment of AR splice variant-positive prostate cancer.

Discovery of spiro[indole-3,2'-pyrrolidin]-2(1H)-one based inhibitors targeting Brr2, a core component of the U5 snRNP.

Bad response to refrigeration 2 (Brr2) is a member of the Ski2-like RNA helicases, and an essential component of the U5 small nuclear ribonucleoprotein (snRNP). A particularly important role of Brr2 is the ATP-dependent unwinding of the U4/U6 RNA duplex, which is a critical step in spliceosomal activation. Despite its biological importance, selective inhibitor for Brr2 had not been reported until our recent report. Here, we describe novel and structurally distinct spiro[indole-3,2'-pyrrolidin]-2(1H)-one based Brr2 inhibitors with superior activity to the previously reported 4,6-dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione series. Using an RNA dependent ATPase assay as a guide, high-throughput screening, hit validation by structure-activity relationship (SAR) study, and subsequent chemical optimization to increase the ATPase inhibitory activity were performed. Thereafter, selectivity and helicase inhibitory activity of optimized compounds were confirmed. In the course of the study, compounds were synthesized using a three-component reaction, which accelerated the optimization process. All these efforts finally culminated in the discovery of the potent and selective Brr2 inhibitors (32a and 33a) exhibiting helicase inhibitory activity at submicromolar concentrations. Thus, compounds 32a and 33a could be valuable molecular probes to study the functions of Brr2 and molecular machinery of RNA splicing.