Biology subjects

Moran, T.

Publications and source records attributed to Moran, T..

6 recordsLinked to original sources

bioRxiv2024

N-Acetyl-2-Aminofluorene (AAF): In vivo temporal expression patterns of growth cycle-dependent macromolecular binding constants KD are revealed by primary cultures of premalignant hepatocytes derived from a multi-cyclic hepatocarcinogenic feeding regimen

Biochemical investigations of the hepatoprocarcinogen N-acetyl-2-aminofluorene (AAF) have shown that normal adult rat hepatocytes in primary culture express two sets of pharmacokinetic constants - designated Systems I and II, and Sites I and II - associated respectively with the metabolism (System I [high-affinity Km[APPARENT] and low-velocity VMAX[APPARENT]] and System II [low-affinity Km APPARENT] and high-velocity VMAX[APPARENT]]), and the macromolecular binding (Site I [high-affinity KD[APPARENT] and low capacity BMAX[APPARENT]] and Site II ([low-affinity KD[APPARENT] and high-capacity BMAX[APPARENT]]) of AAF. Additional findings - that genomically saturating levels of AAF-DNA adducts form far below reported extracellular AAF concentrations required to block replicative and repair DNA synthesis; and, that biphasic Site I and Site II BMAX[APPARENT] and KD[APPARENT] expression curves varied inversely with respect to time and magnitude during hepatocyte growth - led us to wonder how macromolecular binding constants are expressed during chemical hepatocarcinogenesis. These questions were addressed by Scatchard analysis measurements through five consecutive AAF feeding cycles. Notably, cultured premalignant hepatocytes displayed reduced and elevated BMAX[APPARENT] and KD[APPARENT] levels, respectively, akin to the Site I and Site II expression curves observed during hepatocyte growth transitions in vitro. In contrast, prominent hepatocellular functions such as N-OH-AAF production, DNA replication, cell aggregation and resistance to AAF toxicity displayed different temporal trajectories. Impact StatementStriking similarities are observed between both Site I and Site II BMAX and KD expression curves during in vitro and in vivo premalignant growth transitions. These new findings fit earlier ones that hepatocytes growing during carcinogen exposure manifest fewer intranuclear AAF-DNA adducts. How this phenomenon leads to malignancy remains unclear.

pharmacology and toxicology↗

bioRxiv2021

A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening

Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have put forth, only few compounds remain in late stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies (MAbs) conjugated to donor and acceptor fluorophores that produces a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for Remdesivir of 9.3 M against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, {beta}), Brazilian/Japanese variant P.1 (Gamma, {gamma}), and Californian (Epsilon, {varepsilon}), variants of concern or interest (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.

pharmacology and toxicology↗

bioRxiv2020

Development of a high-throughput homogeneous AlphaLISA drug screening assay for the detection of SARS-CoV-2 Nucleocapsid

The coronavirus disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is in urgent need of therapeutic options. High-throughput screening (HTS) offers the research field an opportunity to rapidly identify such compounds. In this work, we have developed a homogeneous cell-based HTS system using AlphaLISA detection technology for the SARS-CoV-2 nucleocapsid protein (NP). Our assay measures both recombinant NP and endogenous NP from viral lysates and tissue culture supernatants (TCS) in a sandwich-based format using two monoclonal antibodies against the NP analyte. Viral NP was detected and quantified in both tissue culture supernatants and cell lysates, with large differences observed between 24 hours and 48 hours of infection. We simulated the viral infection by spiking in recombinant NP into 384-well plates with live Vero-E6 cells and were able to detect the NP with high sensitivity and a large dynamic range. Anti-viral agents that inhibit either viral cell entry or replication will decrease the AlphaLISA NP signal. Thus, this assay can be used for high-throughput screening of small molecules and biologics in the fight against the COVID-19 pandemic.

microbiology↗

bioRxiv2020

Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2

Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), emerged in Wuhan, Hubei province of China, and has been responsible of coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2, and the high economical and health impact of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.

microbiology↗

bioRxiv2017

N-acetyl-2-aminofluorene (AAF) processing in adult rat hepatocytes in primary culture occurs by high-affinity low-velocity and low-affinity high-velocity AAF metabolite-forming systems

N-acetyl-2-aminofluorene (AAF) is a procarcinogen used widely in physiological investigations of chemical hepatocarcinogenesis. Its metabolic pathways have been described extensively, yet little is known about its biochemical processing, growth cycle expression and pharmacological properties inside living hepatocytes - the principal cellular targets of this hepatocarcinogen. In this report, primary monolayer adult rat hepatocyte cultures and high specific-activity [ring G-3H]-N-acetyl-2-aminofluorene were used to extend previous observations of metabolic activation of AAF by highly differentiated, proliferation-competent hepatocytes in long-term cultures. AAF metabolism proceeded by zero-order kinetics. Hepatocytes processed significant amounts of procarcinogen ({approx}12 g AAF/106 cells/day). Five ring-hydroxylated and one deacylated species of AAF were secreted into the culture media. Extracellular metabolite levels varied during the growth cycle (days 0-13), but their rank quantitative order was time invariant: 5-OH-AAF > 7-OH-AAF > 3-OH-AAF > N-OH-AAF > aminofluorene (AF) > 1-OH-AAF. Lineweaver-Burk analyses revealed two principal classes of metabolism: System I (high-affinity and low-velocity), Km[APPARENT] = 1.64 x 10-7 M and VMAX[APPARENT] = 0.1 nmols/106 cells/day; and, System II (low-affinity and high-velocity), Km[APPARENT] = 3.25 x 10-5 M and VMAX[APPARENT] = 1000 nmols/106 cells/day. A third system of metabolism of AAF to AF, with Km[APPARENT] and VMAX[APPARENT] constants of 9.6 x 10-5 M and 4.7 nmols/106 cells/day, was also observed. Evidence provided in this report and its companion paper suggests selective roles and intracellular locations for System I- and System II-mediated AAF metabolite formation during hepatocarcinogenesis, although some of the molecules and mechanisms responsible for multi-system processing remain to be fully defined.

cancer biology↗

bioRxiv2017

High-affinity low-capacity and low-affinity high-capacity N-acetyl-2-aminofluorene (AAF) macromolecular binding sites are revealed during the growth cycle of adult rat hepatocytes in primary culture

Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites - a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II) - associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins and lipids from isolated nuclei, Site IDAY 4 targets (KD[APPARENT] {approx} 2-4 x 10-6 M and BMAX[APPARENT] {approx} 6 pmols/106 cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4 targets (KD[APPARENT] {approx} 1.5 x 10-3 M and BMAX[APPARENT] {approx} 350 pmols/106 cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.

cancer biology↗