Inhibition of Jumonji demethylases reprograms severe dilated cardiomyopathy and prolongs survival.

Hypertrophic/dilated cardiomyopathy, often a prequel to heart failure, is accompanied by maladaptive transcriptional changes that contribute to arrythmias and contractile misfunction. Transgenic mice constitutively expressing high levels of calcineurin are known to develop extreme heart hypertrophy, which progresses to dilated cardiomyopathy, and to die several weeks after birth. Here, we characterized aberrant transcriptional and epigenetic pathways in this mouse model and established a pharmacological approach to treat established cardiomyopathy. We found that H3K4me3 (trimethyl histone 3 lysine 4) and H3K9me3 (trimethyl histone 3 lysine 9) Jumonji histone demethylases are markedly increased at the protein level and show enhanced enzymatic activity in diseased hearts. These epigenetic regulators continued to increase with time, further affecting cardiac gene expression. Our findings parallel the lower H3K4me3 and H3K9me3 levels seen in human patients. Inhibition of Jumonji demethylase activities in vivo results in lower histone demethylase enzymatic function in the heart and higher histone methylation levels and leads to partial reduction of heart size, reversal of maladaptive transcriptional programs, improved heart function, and prolonged survival. At the molecular level, target genes of transcription factor myocyte enhancer factor 2 are specifically regulated in response to pharmacological or genetic inhibition of Jumonji demethylases. Similar transcriptional reversal of disease-associated genes is seen in a second disease model based on cardiac mechanical overload. Our findings validate pharmacological inhibitors of Jumonji demethylases as potential therapeutics for the treatment of cardiomyopathies across disease models and provide evidence of the reversal of maladaptive transcriptional reprogramming leading to partial restoration of cardiac function. In addition, this study defines pathways of therapeutic resistance upregulated with disease progression.

Bioinformatic analysis of RHO family of GTPases identifies RAC1 pharmacological inhibition as a new therapeutic strategy for hepatocellular carcinoma.

OBJECTIVE: The RHO family of GTPases, particularly RAC1, has been linked with hepatocarcinogenesis, suggesting that their inhibition might be a rational therapeutic approach. We aimed to identify and target deregulated RHO family members in human hepatocellular carcinoma (HCC). DESIGN: We studied expression deregulation, clinical prognosis and transcription programmes relevant to HCC using public datasets. The therapeutic potential of RAC1 inhibitors in HCC was study in vitro and in vivo. RNA-Seq analysis and their correlation with the three different HCC datasets were used to characterise the underlying mechanism on RAC1 inhibition. The therapeutic effect of RAC1 inhibition on liver fibrosis was evaluated. RESULTS: Among the RHO family of GTPases we observed that RAC1 is upregulated, correlates with poor patient survival, and is strongly linked with a prooncogenic transcriptional programme. From a panel of novel RAC1 inhibitors studied, 1D-142 was able to induce apoptosis and cell cycle arrest in HCC cells, displaying a stronger effect in highly proliferative cells. Partial rescue of the RAC1-related oncogenic transcriptional programme was obtained on RAC1 inhibition by 1D-142 in HCC. Most importantly, the RAC1 inhibitor 1D-142 strongly reduce tumour growth and intrahepatic metastasis in HCC mice models. Additionally, 1D-142 decreases hepatic stellate cell activation and exerts an anti-fibrotic effect in vivo. CONCLUSIONS: The bioinformatics analysis of the HCC datasets, allows identifying RAC1 as a new therapeutic target for HCC. The targeted inhibition of RAC1 by 1D-142 resulted in a potent antitumoural effect in highly proliferative HCC established in fibrotic livers.

Genetic heterogeneity within collective invasion packs drives leader and follower cell phenotypes.

Collective invasion, the coordinated movement of cohesive packs of cells, has become recognized as a major mode of metastasis for solid tumors. These packs are phenotypically heterogeneous and include specialized cells that lead the invasive pack and others that follow behind. To better understand how these unique cell types cooperate to facilitate collective invasion, we analyzed transcriptomic sequence variation between leader and follower populations isolated from the H1299 non-small cell lung cancer cell line using an image-guided selection technique. We now identify 14 expressed mutations that are selectively enriched in leader or follower cells, suggesting a novel link between genomic and phenotypic heterogeneity within a collectively invading tumor cell population. Functional characterization of two phenotype-specific candidate mutations showed that ARP3 enhances collective invasion by promoting the leader cell phenotype and that wild-type KDM5B suppresses chain-like cooperative behavior. These results demonstrate an important role for distinct genetic variants in establishing leader and follower phenotypes and highlight the necessity of maintaining a capacity for phenotypic plasticity during collective cancer invasion.

Clinical characteristics and outcomes of thymoma-associated myasthenia gravis.

BACKGROUND AND PURPOSE: Prognosis of myasthenia gravis (MG) in patients with thymoma is not well established. Moreover, it is not clear whether thymoma recurrence or unresectable lesions entail a worse prognosis of MG. METHODS: This multicenter study was based on data from a Spanish neurologist-driven MG registry. All patients were aged >18 years at onset and had anti-acetylcholine receptor antibodies. We compared the clinical data of thymomatous and nonthymomatous patients. Prognosis of patients with recurrent or nonresectable thymomas was assessed. RESULTS: We included 964 patients from 15 hospitals; 148 (15.4%) had thymoma-associated MG. Median follow-up time was 4.6 years. At onset, thymoma-associated MG patients were younger (52.0 vs. 60.4 years, p < 0.001), had more generalized symptoms (odds ratio [OR]: 3.02, 95% confidence interval [CI]: 1.95-4.68, p < 0.001) and more severe clinical forms according to the Myasthenia Gravis Foundation of America (MGFA) scale (OR: 1.6, 95% CI: 1.15-2.21, p = 0.005). Disease severity based on MGFA postintervention status (MGFA-PIS) was higher in thymomatous patients at 1 year, 5 years, and the end of follow-up. Treatment refractoriness and mortality were also higher (OR: 2.28, 95% CI: 1.43-3.63, p = 0.001; hazard ratio: 2.46, 95% CI: 1.47-4.14, p = 0.001). Myasthenic symptoms worsened in 13 of 27 patients with recurrences, but differences in long-term severity were not significant. Fifteen thymomatous patients had nonresectable thymomas with worse MGFA-PIS and higher mortality at the end of follow-up. CONCLUSIONS: Thymoma-associated MG patients had more severe myasthenic symptoms and worse prognosis. Thymoma recurrence was frequently associated with transient worsening of MG, but long-term prognosis did not differ from nonrecurrent thymoma. Patients with nonresectable thymoma tended to present severe forms of MG.

A comprehensive study of epigenetic alterations in hepatocellular carcinoma identifies potential therapeutic targets.

BACKGROUND & AIMS: A causal link has recently been established between epigenetic alterations and hepatocarcinogenesis, indicating that epigenetic inhibition may have therapeutic potential. We aimed to identify and target epigenetic modifiers that show molecular alterations in hepatocellular carcinoma (HCC). METHODS: We studied the molecular-clinical correlations of epigenetic modifiers including bromodomains, histone acetyltransferases, lysine methyltransferases and lysine demethylases in HCC using The Cancer Genome Atlas (TCGA) data of 365 patients with HCC. The therapeutic potential of epigenetic inhibitors was evaluated in vitro and in vivo. RNA sequencing analysis and its correlation with expression and clinical data in the TCGA dataset were used to identify expression programs normalized by Jumonji lysine demethylase (JmjC) inhibitors. RESULTS: Genetic alterations, aberrant expression, and correlation between tumor expression and poor patient prognosis of epigenetic enzymes are common events in HCC. Epigenetic inhibitors that target bromodomain (JQ-1), lysine methyltransferases (BIX-1294 and LLY-507) and JmjC lysine demethylases (JIB-04, GSK-J4 and SD-70) reduce HCC aggressiveness. The pan-JmjC inhibitor JIB-04 had a potent antitumor effect in tumor bearing mice. HCC cells treated with JmjC inhibitors showed overlapping changes in expression programs related with inhibition of cell proliferation and induction of cell death. JmjC inhibition reverses an aggressive HCC gene expression program that is also altered in patients with HCC. Several genes downregulated by JmjC inhibitors are highly expressed in tumor vs. non-tumor parenchyma, and their high expression correlates with a poor prognosis. We identified and validated a 4-gene expression prognostic signature consisting of CENPA, KIF20A, PLK1, and NCAPG. CONCLUSIONS: The epigenetic alterations identified in HCC can be used to predict prognosis and to define a subgroup of high-risk patients that would potentially benefit from JmjC inhibitor therapy. LAY SUMMARY: In this study, we found that mutations and changes in expression of epigenetic modifiers are common events in human hepatocellular carcinoma, leading to an aggressive gene expression program and poor clinical prognosis. The transcriptional program can be reversed by pharmacological inhibition of Jumonji enzymes. This inhibition blocks hepatocellular carcinoma progression, providing a novel potential therapeutic strategy.

Next-generation sequencing reveals a new mutation in the LTBP2 gene associated with microspherophakia in a Spanish family.

BACKGROUND: Microspherophakia is a rare autosomal recessive eye disorder characterized by small spherical lens. It may present as an isolated finding or in association with other ocular and/or systemic disorders. This clinical and genetic heterogeneity requires the study of large genes (ADAMTSL4, FBN1, LTBP2, ADAMTSL-10 and ADAMTSL17). The purpose of the present study is to identify the genetic cause of this pathology in a consanguineous Spanish family. METHODS: A clinical exome sequencing experiment was executed by the TruSight One® Sequencing Panel (TSO) from Illumina©. Sanger sequencing was used to validate the NGS results. RESULTS: Only the insertion of an adenine in exon 36 of the LTBP2 gene (c.5439_5440insA) was associated with pathogenicity. This new mutation was validated by Sanger sequencing and segregation analysis was also performed. Haplotype analyses using the polymorphic markers D14S1025, D14S43 and D14S999 close to the LTBP2 gene indicated identity by descent in this family. CONCLUSION: We describe the first case of a microspherophakia phenotype associated with a novel homozygous mutation in the LTBP2 gene in a consanguineous Caucasian family by means of NGS technology.

Effects of low-intensity laser therapy over mini-implants success rate in pigs.

The success rate of miniscrews when used as temporary orthodontic anchorage is relatively high, but some factors could affect its clinical success such as inflammation around the miniscrew. Low-intensity laser therapy has been widely used for biostimulation of tissue and wound healing specially for its anti-inflammatory effects. The purpose of this study was to evaluate the effect of low-intensity laser therapy over the miniscrew success rate. Five Landrace's pigs received 50 miniscrews on the buccal side of the mandible and on the palate of the maxilla. All the miniscrews were immediately loaded with 250 gf. The laser group were irradiated with a 780-nm diode laser with 70 mWs for 1 min (dose = 34 J/cm(2)); the contralateral side was used as the control group. The miniscrews were photographed and analyzed clinically every week to determine their stability and presence of local inflammation. After 3 weeks, histological analysis and fluorescent microscopy were performed to compare the laser and the control side. Clinical results showed a success rate of 60% for the control group and 80% for the laser-treated group. The histological analysis and fluorescent microscopy demonstrated that the laser group had less inflammatory cells than the control group and the bone neoformation around the miniscrew was more intense. Low-intensity laser therapy increased the success rate of orthodontic miniscrews, probably due to anti-inflammatory effect and bone stimulation.

Lysine Demethylase 4A is a Centrosome Associated Protein Required for Centrosome Integrity and Genomic Stability.

Centrosomes play a fundamental role in nucleating and organizing microtubules in the cell and are vital for faithful chromosome segregation and maintenance of genomic stability. Loss of structural or functional integrity of centrosomes causes genomic instability and is a driver of oncogenesis. The lysine demethylase 4A (KDM4A) is an epigenetic 'eraser' of chromatin methyl marks, which we show also localizes to the centrosome with single molecule resolution. We additionally discovered KDM4A demethylase enzymatic activity is required to maintain centrosome homeostasis, and is required for centrosome integrity, a new functionality unlinked to altered expression of genes regulating centrosome number. We find rather, that KDM4A interacts with both mother and daughter centriolar proteins to localize to the centrosome in all stages of mitosis. Loss of KDM4A results in supernumerary centrosomes and accrual of chromosome segregation errors including chromatin bridges and micronuclei, markers of genomic instability. In summary, these data highlight a novel role for an epigenetic 'eraser' regulating centrosome integrity, mitotic fidelity, and genomic stability at the centrosome.

Therapeutic targeting of histone lysine demethylase KDM4B blocks the growth of castration-resistant prostate cancer.

Epigenetics is an emerging mechanism for tumorigenesis. Treatment that targets epigenetic regulators is becoming an attractive strategy for cancer therapy. The role of epigenetic therapy in prostate cancer (PCa) remains elusive. Previously we demonstrated that upregulation of histone lysine demethylase KDM4B correlated with the appearance of castration resistant prostate cancer (CRPC) and identified a small molecular inhibitor of KDM4B, B3. In this study, we further investigated the role of KDM4B in promoting PCa progression and tested the efficacy of B3 using clinically relevant PCa models including PCa cell line LNCaP and 22Rv1 and xenografts derived from these cell lines. In loss and gain-functional studies of KDM4B in PCa cells, we found that overexpression of KDM4B in LNCaP cells enhanced its tumorigenicity whereas knockdown of KDM4B in 22Rv1 cells reduced tumor growth in castrated mice. B3 suppressed the growth of 22Rv1 xenografts and sensitized tumor to anti-androgen receptor (AR) antagonist enzalutamide inhibition. B3 also inhibited 22Rv1 tumor growth synergistically with rapamycin, leading to cell apoptosis. Comparative transcriptomic analysis performed on KDM4B knockdown and B3-treated 22Rv1 cells revealed that B3 inhibited both H3K9me3 and H3K27me3 demethylase activities. Our studies establish KDM4B as a target for CRPC and B3 as a potential therapeutic agent. B3 as monotherapy or in combination with other anti-PCa therapeutics offers proof of principle for the clinical translation of epigenetic therapy targeting KDMs for CRPC patients.

Results in mediastinal lymph node staging of surgical lung cancer: Data from the prospective cohort of the Spanish Video-Assisted Thoracic Surgery Group.

OBJECTIVES: The objective of this study was to assess the diagnostic performance of combined computerised tomography (CT) and positron emission tomography (PET) in mediastinal staging of surgical lung cancer based on data obtained from the prospective cohort of the Spanish Group for Video-Assisted Thoracic Surgery (GEVATS). METHODS: A total of 2782 patients underwent surgery for primary lung carcinoma. We analysed diagnostic success in mediastinal lymph node staging (cN2) using CT and PET. Bivariate and multivariate analyses were performed of the factors involved in this success. The risk of unexpected pN2 disease was analysed for cases in which an invasive testing is recommended: cN1, the tumour centrally located or the tumour diameter >3 cm. RESULTS: The overall success of CT together with PET was 82.9% with a positive predictive value of 0.21 and negative predictive value of 0.93. If the tumour was larger than 3 cm and for each unit increase in mediastinal SUVmax, the probability of success was lower with OR 0.59 (0.44-0.79) and 0.71 (0.66-0.75), respectively. In the video-assisted thoracic surgery (VATS) approach, the probability of success was higher with OR 2.04 (1.52-2.73). The risk of unexpected pN2 increased with the risk factors cN1, the tumour centrally located or the tumour diameter >3 cm: from 4.5% (0 factors) to 18.8% (3 factors) but did not differ significantly as a function of whether invasive testing was performed. CONCLUSIONS: CT and PET together have a high negative predictive value. The overall success of the staging is lower in the case of tumours >3 cm and high mediastinal SUVmax, and it is higher when VATS is performed. The risk of unexpected pN2 is higher if the disease is cN1, the tumour centrally located or the tumour diameter >3 cm but does not vary significantly as a function of whether patients have undergone invasive testing.

Anthraquinones from a marine-derived Streptomyces spinoverrucosus.

Four new anthraquinone analogues including galvaquinones A-C (1-3) and an isolation artifact, 5,8-dihydroxy-2,2,4-trimethyl-6-(3-methylbutyl)anthra[9,1-de][1,3]oxazin-7(2H)-one (4), were isolated from a marine-derived Streptomyces spinoverrucosus based on activity in an image-based assay to identify epigenetic modifying compounds. The structures of 1-4 were elucidated by comprehensive NMR and MS spectroscopic analysis. Galvaquinone B (2) was found to show epigenetic modulatory activity at 1.0 µM and exhibited moderate cytotoxicity against non-small-cell lung cancer (NSCLC) cell lines Calu-3 and H2887.

Host Cell Redox Alterations Promote Latent HIV-1 Reactivation through Atypical Transcription Factor Cooperativity.

Immune cell state alterations rewire HIV-1 gene expression, thereby influencing viral latency and reactivation, but the mechanisms are still unfolding. Here, using a screen approach on CD4+ T cell models of HIV-1 latency, we revealed Small Molecule Reactivators (SMOREs) with unique chemistries altering the CD4+ T cell state and consequently promoting latent HIV-1 transcription and reactivation through an unprecedented mechanism of action. SMOREs triggered rapid oxidative stress and activated a redox-responsive program composed of cell-signaling kinases (MEK-ERK axis) and atypical transcription factor (AP-1 and HIF-1α) cooperativity. SMOREs induced an unusual AP-1 phosphorylation signature to promote AP-1/HIF-1α binding to the latent HIV-1 proviral genome for its activation. Consistently, latent HIV-1 reactivation was compromised with pharmacologic inhibition of oxidative stress sensing or of cell-signaling kinases, and transcription factor's loss of expression, thus functionally linking the host redox-responsive program to viral transcriptional rewiring. Notably, SMOREs induced the redox program in primary CD4+ T cells and reactivated latent HIV-1 in aviremic patient samples alone and in combination with known latency-reversing agents, thus providing physiological relevance. Our findings suggest that manipulation of redox-sensitive pathways could be exploited to alter the course of HIV-1 latency, thus rendering host cells responsive to help achieve a sterilizing cure.

JIB-04 Has Broad-Spectrum Antiviral Activity and Inhibits SARS-CoV-2 Replication and Coronavirus Pathogenesis.

Pathogenic coronaviruses are a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified small-molecule inhibitors that potently block the replication of severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with a 50% effective concentration of 695 nM, with a specificity index of greater than 1,000. JIB-04 showed in vitro antiviral activity in multiple cell types, including primary human bronchial epithelial cells, against several DNA and RNA viruses, including porcine coronavirus transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved weight gain and survival. These results highlight the potential utility of JIB-04 as an antiviral agent against SARS-CoV-2 and other viral pathogens. IMPORTANCE The coronavirus disease 2019 (COVID-19), the disease caused by SARS-CoV-2 infection, is an ongoing public health disaster worldwide. Although several vaccines are available as a preventive measure and the FDA approval of an orally bioavailable drug is on the horizon, there remains a need for developing antivirals against SARS-CoV-2 that could work on the early course of infection. By using infectious reporter viruses, we screened small-molecule inhibitors for antiviral activity against SARS-CoV-2. Among the top hits was JIB-04, a compound previously studied for its anticancer activity. Here, we showed that JIB-04 inhibits the replication of SARS-CoV-2 as well as different DNA and RNA viruses. Furthermore, JIB-04 conferred protection in a porcine model of coronavirus infection, although to a lesser extent when given as therapeutic rather than prophylactic doses. Our findings indicate a limited but still promising utility of JIB-04 as an antiviral agent in the combat against COVID-19 and potentially other viral diseases.

Identification of four potential epigenetic modulators from the NCI structural diversity library using a cell-based assay.

Epigenetic pathways help control the expression of genes. In cancer and other diseases, aberrant silencing or overexpression of genes, such as those that control cell growth, can greatly contribute to pathogenesis. Access to these genes by the transcriptional machinery is largely mediated by chemical modifications of DNA or histones, which are controlled by epigenetic enzymes, making these enzymes attractive targets for drug discovery. Here we describe the characterization of a locus derepression assay, a fluorescence-based mammalian cellular system which was used to screen the NCI structural diversity library for novel epigenetic modulators using an automated imaging platform. Four structurally unique compounds were uncovered that, when further investigated, showed distinct activities. These compounds block the viability of lung cancer and melanoma cells, prevent cell cycle progression, and/or inhibit histone deacetylase activity, altering levels of cellular histone acetylation.

Characterizing heterogeneous cellular responses to perturbations.

Cellular populations have been widely observed to respond heterogeneously to perturbation. However, interpreting the observed heterogeneity is an extremely challenging problem because of the complexity of possible cellular phenotypes, the large dimension of potential perturbations, and the lack of methods for separating meaningful biological information from noise. Here, we develop an image-based approach to characterize cellular phenotypes based on patterns of signaling marker colocalization. Heterogeneous cellular populations are characterized as mixtures of phenotypically distinct subpopulations, and responses to perturbations are summarized succinctly as probabilistic redistributions of these mixtures. We apply our method to characterize the heterogeneous responses of cancer cells to a panel of drugs. We find that cells treated with drugs of (dis-)similar mechanism exhibit (dis-)similar patterns of heterogeneity. Despite the observed phenotypic diversity of cells observed within our data, low-complexity models of heterogeneity were sufficient to distinguish most classes of drug mechanism. Our approach offers a computational framework for assessing the complexity of cellular heterogeneity, investigating the degree to which perturbations induce redistributions of a limited, but nontrivial, repertoire of underlying states and revealing functional significance contained within distinct patterns of heterogeneous responses.

Epigenetic modulation reveals differentiation state specificity of oncogene addiction.

Hyperactivation of the MAPK signaling pathway motivates the clinical use of MAPK inhibitors for BRAF-mutant melanomas. Heterogeneity in differentiation state due to epigenetic plasticity, however, results in cell-to-cell variability in the state of MAPK dependency, diminishing the efficacy of MAPK inhibitors. To identify key regulators of such variability, we screen 276 epigenetic-modifying compounds, individually or combined with MAPK inhibitors, across genetically diverse and isogenic populations of melanoma cells. Following single-cell analysis and multivariate modeling, we identify three classes of epigenetic inhibitors that target distinct epigenetic states associated with either one of the lysine-specific histone demethylases Kdm1a or Kdm4b, or BET bromodomain proteins. While melanocytes remain insensitive, the anti-tumor efficacy of each inhibitor is predicted based on melanoma cells' differentiation state and MAPK activity. Our systems pharmacology approach highlights a path toward identifying actionable epigenetic factors that extend the BRAF oncogene addiction paradigm on the basis of tumor cell differentiation state.

JIB-04 has broad-spectrum antiviral activity and inhibits SARS-CoV-2 replication and coronavirus pathogenesis.

Pathogenic coronaviruses represent a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified several small-molecule inhibitors that potently block the replication of the newly emerged severe acute respiratory syndrome virus 2 (SARS-CoV-2). Among them, JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with an EC50 of 695 nM, with a specificity index of greater than 1,000. JIB-04 showed in vitro antiviral activity in multiple cell types against several DNA and RNA viruses, including porcine coronavirus transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved weight gain and survival. These results highlight the potential utility of JIB-04 as an antiviral agent against SARS-CoV-2 and other viral pathogens.

A prospective randomized controlled trial of an interpersonal violence prevention program with a Mexican American community.

Using methods of community-based participatory research, a prospective randomized controlled trial of a violence prevention program based on Latino cultural values was implemented with elementary school children in a Mexican American community. Community members participated in intervention program selection, implementation, and data collection. High-risk students who participated in the program had greater nonviolent self-efficacy and demonstrated greater endorsement of program values than did high-risk students in the control group. This collaborative partnership was able to combine community-based participatory research with a rigorous study design and provide sustained benefit to community partners.

A UHPLC-MS/MS method for the quantification of JIB-04 in rat plasma: Development, validation and application to pharmacokinetics study.

Methylation of lysine by histone methyltransferases can be reversed by lysine demethylases (KDMs). Different KDMs have distinct oncogenic functions based on their cellular localization, stimulating cancer cell proliferation, reducing the expression of tumor suppressors, and/or promoting the development of drug resistance. JIB-04 is a small molecule that pan-selectively inhibits KDMs, showing maximal inhibitory activity against KDM5A, and as secondary targets, KDM4D/4B/4A/6B/4C. Recently, it was found that JIB-04 also potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines via the inhibition of a new target, serine hydroxymethyltransferase 2. Pharmacokinetic characterization and an analytical method for the quantification of JIB-04 are necessary for the further development of this small molecule. Herein, a sensitive, specific, fast and reliable UHPLC-MS/MS method for the quantification of JIB-04 in rat plasma samples was developed and fully validated using a SCIEX 6500+ triple QUAD LC-MS system equipped with an ExionLC UHPLC unit. The chromatographic separation was achieved on a reverse phase ACE Excel 2 Super C18 column with a flow rate of 0.5 mL/min under gradient elution. The calibration curves were linear (r2 > 0.999) over concentrations from 0.5 to 1000 ng/mL. The accuracy (RE%) was between -7.4% and 3.7%, and the precision (CV%) was 10.2% or less. The stability data showed that no significant degradation occurred under the experimental conditions. This method was successfully applied to the pharmacokinetic study of JIB-04 in rat plasma after intravenous and oral administration and the oral bioavailability of JIB-04 was found to be 44.4%.

Disruption of the Plasmodium falciparum Life Cycle through Transcriptional Reprogramming by Inhibitors of Jumonji Demethylases.

Little is known about the role of the three Jumonji C (JmjC) enzymes in Plasmodium falciparum (Pf). Here, we show that JIB-04 and other established inhibitors of mammalian JmjC histone demethylases kill asexual blood stage parasites and are even more potent at blocking gametocyte development and gamete formation. In late stage parasites, JIB-04 increased levels of trimethylated lysine residues on histones, suggesting the inhibition of P. falciparum Jumonji demethylase activity. These epigenetic defects coincide with deregulation of invasion, cell motor, and sexual development gene programs, including gene targets coregulated by the PfAP2-I transcription factor and chromatin-binding factor, PfBDP1. Mechanistically, we demonstrate that PfJmj3 converts 2-oxoglutarate to succinate in an iron-dependent manner consistent with mammalian Jumonji enzymes, and this catalytic activity is inhibited by JIB-04 and other Jumonji inhibitors. Our pharmacological studies of Jumonji activity in the malaria parasite provide evidence that inhibition of these enzymatic activities is detrimental to the parasite.