Detection of CTNNB1 Hotspot Mutations in Cell-Free DNA from the Urine of Hepatocellular Carcinoma Patients.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. The beta-catenin gene, CTNNB1, is among the most frequently mutated in HCC tissues. However, mutational analysis of HCC tumors is hampered by the difficulty of obtaining tissue samples using traditional biopsy. Here, we explored the feasibility of detecting tumor-derived CTNNB1 mutations in cell-free DNA (cfDNA) extracted from the urine of HCC patients. Using a short amplicon qPCR assay targeting HCC mutational hotspot CTNNB1 codons 32-37 (exon 3), we detected CTNNB1 mutations in 25% (18/73) of HCC tissues and 24% (15/62) of pre-operative HCC urine samples in two independent cohorts. Among the CTNNB1-mutation-positive patients with available matched pre- and post-operative urine (n = 13), nine showed apparent elimination (n = 7) or severalfold reduction (n = 2) of the mutation in urine following tumor resection. Four of the seven patients with no detectable mutations in postoperative urine remained recurrence-free within five years after surgery. In contrast, all six patients with mutation-positive in post-operative urine recurred, including the two with reduced mutation levels. This is the first report of association between the presence of CTNNB1 mutations in pre- and post-operative urine cfDNA and HCC recurrence with implications for minimum residual disease detection.

Structural basis for allosteric PARP-1 retention on DNA breaks.

The success of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors (PARPi) to treat cancer relates to their ability to trap PARP-1 at the site of a DNA break. Although different forms of PARPi all target the catalytic center of the enzyme, they have variable abilities to trap PARP-1. We found that several structurally distinct PARPi drive PARP-1 allostery to promote release from a DNA break. Other inhibitors drive allostery to retain PARP-1 on a DNA break. Further, we generated a new PARPi compound, converting an allosteric pro-release compound to a pro-retention compound and increasing its ability to kill cancer cells. These developments are pertinent to clinical applications where PARP-1 trapping is either desirable or undesirable.

Nitric Oxide Attenuates the Inflammatory Effects of Air During Extracorporeal Circulation.

Cardiopulmonary bypass causes a systemic inflammatory response reaction that may contribute to postoperative complications. One cause relates to the air/blood interface from the extracorporeal circuit. The modulatory effects of blending nitric oxide (NO) gas into the ventilation/sweep gas of the membrane lung was studied in a porcine model of air-induced inflammation in which NO gas was added and compared with controls with or without an air/blood interface. Healthy swine were supported on partial bypass under four different test conditions. Group 1: no air exposure, group 2: air alone, group 3: air plus 50 ppm NO, and group 4: air plus 500 ppm NO. The NO gas was blended into the ventilation/sweep site of the membrane lung. The platelets and leucocytes were activated by air alone. Addition of NO to the sweep gas attenuated the inflammatory response created by the air/blood interface in this model.

Characterization of the hepatitis B virus DNA detected in urine of chronic hepatitis B patients.

BACKGROUND: Detection of human hepatitis B virus (HBV) DNA in the urine of patients with chronic hepatitis B infection (CHB) has been reported previously, suggesting urine could provide a potential route of horizontal HBV transmission. However, it is not clear whether the HBV DNA detected in urine is indeed full-length, infectious viral DNA. The aim of this study is to assess the potential infectivity of urine from patients with CHB and to correlate HBV DNA detection in urine with clinical parameters, such as serum viral load and HBeAg status. METHODS: Urine from 60 CHB patients with serum viral loads ranging from undetectable to 108 IU/mL were analyzed for HBV DNA and serum immune markers. HBV DNA was detected from total urine DNA and size-fractionated urine DNA (separated into ≤1 kb and > 1 kb fractions) by PCR analysis of six regions of the HBV genome. RESULTS: Twenty-seven of 59 (45.7%) patients with HBV serum viral load (≥20 IU/mL) contained at least 20 copies per mL of fragmented HBV DNA in urine detected in at least 1 of the 6 PCR assay regions. Only one patient contained HBV DNA detected by all six regions, and was found to have evidence of blood in the urine. Sixteen of 25 urine samples with high viral load (> 105 IU/mL) and 11 of 34 urine samples with low viral load (< 105 IU/mL) contained detectable HBV DNA. Twelve of 27 (44.44%) patients with detectable HBV DNA in urine were HBeAg positive, and only 5 of these HBeAg positive patients were in the group of 33 (15.15%) patients with no detectable HBV DNA in urine. By Fishers' exact test, HBV DNA in urine is significantly associated with high serum viral load (P = 0.0197) and HBeAg (P = 0.0203). CONCLUSIONS: We conclude that urine from CHB patients with healthy kidney function should not contain full-length HBV DNA, and therefore should not be infectious.

ChimericSeq: An open-source, user-friendly interface for analyzing NGS data to identify and characterize viral-host chimeric sequences.

Identification of viral integration sites has been important in understanding the pathogenesis and progression of diseases associated with particular viral infections. The advent of next-generation sequencing (NGS) has enabled researchers to understand the impact that viral integration has on the host, such as tumorigenesis. Current computational methods to analyze NGS data of virus-host junction sites have been limited in terms of their accessibility to a broad user base. In this study, we developed a software application (named ChimericSeq), that is the first program of its kind to offer a graphical user interface, compatibility with both Windows and Mac operating systems, and optimized for effectively identifying and annotating virus-host chimeric reads within NGS data. In addition, ChimericSeq's pipeline implements custom filtering to remove artifacts and detect reads with quantitative analytical reporting to provide functional significance to discovered integration sites. The improved accessibility of ChimericSeq through a GUI interface in both Windows and Mac has potential to expand NGS analytical support to a broader spectrum of the scientific community.

Detection of urine DNA markers for monitoring recurrent hepatocellular carcinoma.

AIM: This study aimed to explore the potential of detecting hepatocellular carcinoma (HCC)-associated DNA markers, TP53 249T mutations and aberrant methylation of RASSF1A and GSTP1 genes, for monitoring HCC recurrence. HCC remains a leading cause of death worldwide, with one of the fastest growing incidence rates in the US. While treatment options are available and new ones emerging, there remains a poor prognosis of this disease mostly due to its late diagnosis and high recurrence rate. Although there are no specific guidelines addressing how HCC recurrence should be monitored, recurrence is usually monitored by serum-alpha fetal protein and imaging methods such as magnetic resonance imaging (MRI). However, early detection of recurrent HCC remains limited, particularly at the site of treated lesion. METHODS: Here, the authors followed 10 patients that were treated for a primary HCC, and monitored for months or years later. At these follow-up visits, urine was collected and tested retrospectively for 3 DNA biomarkers that associate with HCC development. RESULTS: This 10-patient study compared detection of urine DNA markers with MRI for monitoring HCC recurrence. Five patients were confirmed by MRI for recurrence, and all 5 had detectable DNA biomarkers up to 9 months before recurrence confirmation by MRI. CONCLUSION: Overall, this suggests that detection of HCC-associated DNA markers in urine could provide a promising tool to complement detection of recurrent HCC by imaging.

Purification of DNA Damage-Dependent PARPs from E. coli for Structural and Biochemical Analysis.

Human PARP-1, PARP-2, and PARP-3 are key players in the cellular response to DNA damage, during which their catalytic activities are acutely stimulated through interaction with DNA strand breaks. There are also roles for these PARPs outside of the DNA damage response, most notably for PARP-1 and PARP-2 in the regulation of gene expression. Here, we describe a general method to express and purify these DNA damage-dependent PARPs from E. coli cells for use in biochemical assays and for structural and functional analysis. The procedure allows for robust production of PARP enzymes that are free of contaminant DNA that can interfere with downstream analysis. The described protocols have been updated from our earlier reported methods, most importantly to introduce PARP inhibitors in the production scheme to cope with enzyme toxicity that can compromise the yield of purified protein.

Fluorescent sensors of PARP-1 structural dynamics and allosteric regulation in response to DNA damage.

Poly(ADP-ribose) (PAR) is a posttranslational modification predominantly synthesized by PAR polymerase-1 (PARP-1) in genome maintenance. PARP-1 detects DNA damage, and damage detection is coupled to a massive increase PAR production, primarily attached to PARP-1 (automodification). Automodified PARP-1 then recruits repair factors to DNA damage sites. PARP-1 automodification eventually leads to release from DNA damage thus turning off catalytic activity, although the effects of PAR on PARP-1 structure are poorly understood. The multiple domains of PARP-1 are organized upon detecting DNA damage, creating a network of domain contacts that imposes a major conformational transition in the catalytic domain that increases PAR production. Presented here are two novel fluorescent sensors that monitor the global and local structural transitions of PARP-1 that are associated with DNA damage detection and catalytic activation. These sensors display real-time monitoring of PARP-1 structural transitions upon DNA damage detection, and their reversal upon PARP-1 automodification. The fluorescent sensors are further used to investigate intramolecular and intermolecular PARP-1 activation, followed by the observation that intramolecular activation of PARP-1 is the predominant response to DNA strand breaks in cells. These results provide a unique perspective on the interplay between PARP-1 DNA damage recognition, allosteric regulation, and catalytic activity.

PARP-1 Activation Requires Local Unfolding of an Autoinhibitory Domain.

Poly(ADP-ribose) polymerase-1 (PARP-1) creates the posttranslational modification PAR from substrate NAD(+) to regulate multiple cellular processes. DNA breaks sharply elevate PARP-1 catalytic activity to mount a cell survival repair response, whereas persistent PARP-1 hyperactivation during severe genotoxic stress is associated with cell death. The mechanism for tight control of the robust catalytic potential of PARP-1 remains unclear. By monitoring PARP-1 dynamics using hydrogen/deuterium exchange-mass spectrometry (HXMS), we unexpectedly find that a specific portion of the helical subdomain (HD) of the catalytic domain rapidly unfolds when PARP-1 encounters a DNA break. Together with biochemical and crystallographic analysis of HD deletion mutants, we show that the HD is an autoinhibitory domain that blocks productive NAD(+) binding. Our molecular model explains how PARP-1 DNA damage detection leads to local unfolding of the HD that relieves autoinhibition, and has important implications for the design of PARP inhibitors.

Discovery and structure-activity relationship of novel 2,3-dihydrobenzofuran-7-carboxamide and 2,3-dihydrobenzofuran-3(2H)-one-7-carboxamide derivatives as poly(ADP-ribose)polymerase-1 inhibitors.

Novel substituted 2,3-dihydrobenzofuran-7-carboxamide (DHBF-7-carboxamide) and 2,3-dihydrobenzofuran-3(2H)-one-7-carboxamide (DHBF-3-one-7-carboxamide) derivatives were synthesized and evaluated as inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1). A structure-based design strategy resulted in lead compound 3 (DHBF-7-carboxamide; IC50 = 9.45 µM). To facilitate synthetically feasible derivatives, an alternative core was designed, DHBF-3-one-7-carboxamide (36, IC50 = 16.2 µM). The electrophilic 2-position of this scaffold was accessible for extended modifications. Substituted benzylidene derivatives at the 2-position were found to be the most potent, with 3',4'-dihydroxybenzylidene 58 (IC50 = 0.531 µM) showing a 30-fold improvement in potency. Various heterocycles attached at the 4'-hydroxyl/4'-amino of the benzylidene moiety resulted in significant improvement in inhibition of PARP-1 activity (e.g., compounds 66-68, 70, 72, and 73; IC50 values from 0.718 to 0.079 µM). Compound 66 showed selective cytotoxicity in BRCA2-deficient DT40 cells. Crystal structures of three inhibitors (compounds (-)-13c, 59, and 65) bound to a multidomain PARP-1 structure were obtained, providing insights into further development of these inhibitors.

Targeting PARP-1 allosteric regulation offers therapeutic potential against cancer.

PARP-1 is a nuclear protein that has important roles in maintenance of genomic integrity. During genotoxic stress, PARP-1 recruits to sites of DNA damage where PARP-1 domain architecture initiates catalytic activation and subsequent poly(ADP-ribose)-dependent DNA repair. PARP-1 inhibition is a promising new way to selectively target cancers harboring DNA repair deficiencies. However, current inhibitors target other PARPs, raising important questions about long-term off-target effects. Here, we propose a new strategy that targets PARP-1 allosteric regulation as a selective way of inhibiting PARP-1. We found that disruption of PARP-1 domain-domain contacts through mutagenesis held no cellular consequences on recruitment to DNA damage or a model system of transcriptional regulation, but prevented DNA-damage-dependent catalytic activation. Furthermore, PARP-1 mutant overexpression in a pancreatic cancer cell line (MIA PaCa-2) increased sensitivity to platinum-based anticancer agents. These results not only highlight the potential of a synergistic drug combination of allosteric PARP inhibitors with DNA-damaging agents in genomically unstable cancer cells (regardless of homologous recombination status), but also signify important applications of selective PARP-1 inhibition. Finally, the development of a high-throughput PARP-1 assay is described as a tool to promote discovery of novel PARP-1 selective inhibitors.

Structural Implications for Selective Targeting of PARPs.

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD(+) as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fueled the pursuit of small molecule PARP inhibitors. The design of PARP inhibitors has matured over the past several years resulting in several lead candidates in clinical trials. PARP inhibitors are mainly used in clinical trials to treat cancer, particularly as sensitizing agents in combination with traditional chemotherapy to reduce side effects. An exciting aspect of PARP inhibitors is that they are also used to selectivity kill tumors with deficiencies in DNA repair proteins (e.g., BRCA1/2) through an approach termed "synthetic lethality." In the midst of the tremendous efforts that have brought PARP inhibitors to the forefront of modern chemotherapy, most clinically used PARP inhibitors bind to conserved regions that permits cross-selectivity with other PARPs containing homologous catalytic domains. Thus, the differences between therapeutic effects and adverse effects stemming from pan-PARP inhibition compared to selective inhibition are not well understood. In this review, we discuss current literature that has found ways to gain selectivity for one PARP over another. We furthermore provide insights into targeting other domains that make up PARPs, and how new classes of drugs that target these domains could provide a high degree of selectivity by affecting specific cellular functions. A clear understanding of the inhibition profiles of PARP inhibitors will not only enhance our understanding of the biology of individual PARPs, but may provide improved therapeutic options for patients.

Discovery and structure-activity relationships of modified salicylanilides as cell permeable inhibitors of poly(ADP-ribose) glycohydrolase (PARG).

The metabolism of poly(ADP-ribose) (PAR) in response to DNA strand breaks, which involves the concerted activities of poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolase (PARG), modulates cell recovery or cell death depending upon the level of DNA damage. While PARP inhibitors show high promise in clinical trials because of their low toxicity and selectivity for BRCA related cancers, evaluation of the therapeutic potential of PARG is limited by the lack of well-validated cell permeable inhibitors. In this study, target-related affinity profiling (TRAP), an alternative to high-throughput screening, was used to identify a number of druglike compounds from several chemical classes that demonstrated PARG inhibition in the low-micromolar range. A number of analogues of one of the most active chemotypes were synthesized to explore the structure-activity relationship (SAR) for that series. This led to the discovery of a putative pharmacophore for PARG inhibition that contains a modified salicylanilide structure. Interestingly, these compounds also inhibit PARP-1, indicating strong homology in the active sites of PARG and PARP-1 and raising a new challenge for development of PARG specific inhibitors. The cellular activity of a lead inhibitor was demonstrated by the inhibition of both PARP and PARG activity in squamous cell carcinoma cells, although preferential inhibition of PARG relative to PARP was observed. The ability of inhibitors to modulate PAR metabolism via simultaneous effects on PARPs and PARG may represent a new approach for therapeutic development.

Pharmacological blockade of serotonin 5-HT7 receptor reverses working memory deficits in rats by normalizing cortical glutamate neurotransmission.

The role of 5-HT7 receptor has been demonstrated in various animal models of mood disorders; however its function in cognition remains largely speculative. This study evaluates the effects of SB-269970, a selective 5-HT7 antagonist, in a translational model of working memory deficit and investigates whether it modulates cortical glutamate and/or dopamine neurotransmission in rats. The effect of SB-269970 was evaluated in the delayed non-matching to position task alone or in combination with MK-801, a non-competitive NMDA receptor antagonist, and, in separate experiments, with scopolamine, a non-selective muscarinic antagonist. SB-269970 (10 mg/kg) significantly reversed the deficits induced by MK-801 (0.1 mg/kg) but augmented the deficit induced by scopolamine (0.06 mg/kg). The ability of SB-269970 to modulate MK-801-induced glutamate and dopamine extracellular levels was separately evaluated using biosensor technology and microdialysis in the prefrontal cortex of freely moving rats. SB-269970 normalized MK-801 -induced glutamate but not dopamine extracellular levels in the prefrontal cortex. Rat plasma and brain concentrations of MK-801 were not affected by co-administration of SB-269970, arguing for a pharmacodynamic rather than a pharmacokinetic mechanism. These results indicate that 5-HT7 receptor antagonists might reverse cognitive deficits associated with NMDA receptor hypofunction by selectively normalizing glutamatergic neurotransmission.

Indole- and benzothiophene-based histamine H3 antagonists.

Previous research on histamine H(3) antagonists has led to the development of a pharmacophore model consisting of a central phenyl core flanked by two alkylamine groups. Recent investigation of the replacement of the central phenyl core with heteroaromatic fragments resulted in the preparation of novel 3,5-, 3,6- and 3,7-substituted indole and 3,5-substituted benzothiophene analogs that demonstrate good to excellent hH(3) affinities. Select analogs were profiled in a rat pharmacokinetic model.

Heterocyclic replacement of the central phenyl core of diamine-based histamine H3 receptor antagonists.

A series of small molecules consisting of a heterocyclic core flanked by two basic functionalities were synthesized and screened for in vitro affinity at the human histamine H(3) receptor (hH(3)R). Nine of the twenty-eight compounds tested were found to possess a hH(3)R K(i) of less than 5 nM and consisted of a diverse range of central hetero-aromatic linkers (pyridine, pyrazine, oxazole, isoxazole, thiazole, furan, thiophene, and pyrrole). One member of this series, (4-isopropyl-piperazin-1-yl)-(6-piperidin-1-ylmethyl-pyridin-3-yl)-methanone (37), was found to be a high affinity, selective antagonist that crosses the blood-brain barrier and occupies H(3) receptors after oral administration in the rat.

JNJ-10181457, a selective non-imidazole histamine H(3) receptor antagonist, normalizes acetylcholine neurotransmission and has efficacy in translational rat models of cognition.

Histamine 3 (H(3)) receptors are distributed throughout the brain and regulate histamine as well as the activity of other neurotransmitters including acetylcholine (ACh). Impaired ACh neurotransmission is associated with deficits of cognitive-related functioning in many species including humans. The goal of these studies was to evaluate the behavioral and neurochemical effects of JNJ-10181457, a selective non-imidazole histamine H(3) receptor antagonist, in rats. The pharmacokinetic profile and receptor occupancy of JNJ-10181457 were tested. The efficacy of JNJ-10181457 was evaluated, acutely, in the imetit-induced water licking model, delayed non-matching to position (DNMTP) task and microdialysis studies. In addition, the effects of repeated administration of JNJ-10181457 were evaluated in the reversal learning task. A single administration of JNJ-10181457 (10 mg/kg, i.p.) resulted in significant plasma and brain exposure and maximal H(3) receptor occupancy. In addition, JNJ-10181457 reversed imetit-induced water licking, similarly to thioperamide (10 mg/kg, i.p.). In the DNMTP task, scopolamine (0.06 mg/kg, i.p.) significantly decreased percentage correct responding. These effects were significantly reversed by JNJ-10181457 (10 mg/kg, i.p.) and also by donepezil (1 mg/kg, i.p.), an acetylcholinesterase inhibitor, and were associated with normalization of ACh neurotransmission in the cortex. Repeated administration of JNJ-10181457 (10 mg/kg, i.p.) significantly increased percentage correct responding in the reversal learning task. Treatment discontinuation was not associated with rebound effects on cognition. These results indicate that selective blockade of histamine H(3) receptors might have therapeutic utility for the treatment of working memory deficits and learning disorders, especially those in which ACh neurotransmission is compromised.

Novel imidazole-based histamine H3 antagonists.

A novel series of imidazole containing histamine H(3) receptor ligands were investigated and found to be potent functional antagonists. After improving the stability of these molecules towards liver microsomes, these compounds were found to have no appreciable affinity for CYP P450s. Subsequent in vivo experiments showed significant brain uptake of (4-chloro-phenyl)-[2-(1-isopropyl-piperidin-4-ylmethoxy)-3-methyl-3H-imidazol-4-yl]-methanone 22.

Effects of SB-269970, a 5-HT7 receptor antagonist, in mouse models predictive of antipsychotic-like activity.

5-HT7 receptors have been linked to a number of psychiatric disorders including anxiety and depression. The localization of 5-HT7 receptors in the thalamus, a key sensory processing center, and the high affinity of many atypical antipsychotic compounds for these receptors have led to the speculation of the utility of 5-HT7 antagonists in schizophrenia. The goal of these studies was to examine the effects of pharmacologic blockade and genetic ablation of 5-HT7 receptors in animal models predictive of antipsychotic-like activity. We evaluated the effects of SB-269970, a selective 5-HT7 receptor antagonist, on amphetamine and ketamine-induced hyperactivity and prepulse inhibition (PPI) deficits. In addition, sensorimotor gating function and locomotor activity were evaluated in 5-HT7 knockout mice. Locomotor activity was measured for up to 180 min using an automated infrared photobeam system, and PPI was evaluated in startle chambers. SB-269970 (3, 10 and 30 mg/kg, intraperitoneally) significantly blocked amphetamine [3 mg/kg, subcutaneously (s.c.)] and ketamine (30 mg/kg, s.c.)-induced hyperactivity and reversed amphetamine (10 mg/kg, s.c.)-induced but not ketamine (30 mg/kg, s.c.)-induced PPI deficits, without changing spontaneous locomotor activity and startle amplitude. The largest dose of SB-269970 did not block the effects of amphetamine in 5-HT7 knockout mice. Collectively, these results indicate that blockade of 5-HT7 receptors partially modulates glutamatergic and dopaminergic function and could be clinically useful for the treatment of positive symptoms of schizophrenia.