In vitro, in vivo, and in silico analyses of the antitumor activity of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazoles

Abstract

Phortress is a novel, potent, and selective experimental antitumor agent. Its mechanism of action involves induction of CYP1A1-catalyzed biotransformation of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) to generate electrophilic species, which covalently bind to DNA, exacting lethal damage to sensitive tumor cells, in vitro and in vivo. Herein, we investigate the effects of DNA adduct formation on cellular DNA integrity and progression through cell cycle and examine whether a relevant pharmacodynamic end point may be exploited to probe the clinical mechanism of action of Phortress and predict tumor response. Single cell gel electrophoresis (SCGE) was applied to quantify DNA damage and cell cycle analyses conducted upon 5F 203 treatment of benzothiazole-sensitive MCF-7 and inherently resistant MDA-MB-435 breast carcinoma cells. Following treatment of xenograft-bearing mice and mice possessing hollow fiber implants containing MCF-7 or MDA-MB-435 cells with Phortress (20 mg/kg, i.p., 24 hours), tumor cells and xenografts were recovered for analyses by SCGE. Dose- and time-dependent DNA single and double strand breaks occurred exclusively in sensitive cells following treatment with 5F 203 in vitro (10 nmol/L–10 μmol/L; 24–72 hours). In vivo, Phortress-sensitive and Phortress-resistant tumor cells were distinct; moreover, DNA damage in xenografts, following treatment of mice with Phortress, could be determined. Interrogation of the mechanism of action of 5F 203 in silico by self-organizing map-based cluster analyses revealed modulation of phosphatases and kinases associated with cell cycle regulation, corroborating observations of selective cell cycle perturbation by 5F 203 in sensitive cells. By conducting SCGE, tumor sensitivity to Phortress, an agent currently undergoing clinical evaluation, may be determined.

Bioactivation of Fluorinated 2-Aryl-benzothiazole Antitumor Molecules by Human Cytochrome P450s 1A1 and 2W1 and Deactivation by Cytochrome P450 2S1

“…5F 203 showed antiproliferative potency in sensitive human breast and ovarian cancer cell lines (GI 50 < 1 nM for MCF-7, MDA 468, and IGROV-1 cells) and emerged as a lead compound, currently in phase 1 clinical trials in the United Kingdom. , The results of coincubation of 2-4(-amino-3-methyl-phenyl)benzothiazole (DF 203, an anticancer agent discovered earlier, yielding inactive metabolites that led to the discovery of 5F 203) with α-naphthoflavone, a P450 1A1 inhibitor, showed inhibition of anticancer activity and revealed a role for P450 1A1 in the anticancer activity of the benzothiazoles . The involvement of P450 1A1 was reported for 5F 203 bioactivation (to generate reactive intermediates to form protein and DNA adducts) in the antitumor process within sensitive cells. , P450 1A1 is expressed in fetal liver and in some extrahepatic adult human tissues but not at appreciable levels in adult liver. , 5F 203 acts as both a substrate and an inducer of P450 1A1, the latter process involving binding to the aryl hydrocarbon receptor …”

“…16,17 5F 203 acts as both a substrate and an inducer of P450 1A1, the latter process involving binding to the aryl hydrocarbon receptor. 18 Although P450 1A1 has been shown to be involved in the bioactivation of 5F 203, the products are still uncharacterized. In a prior study of the metabolism of 5F 203 in human Blymphoblastoid cells expressing P450 1A1, 1B1, and 1A2, respectively, 13 P450 1A1 exhibited the highest level of metabolic capacity.…”

“…19 Even less information has been reported on the metabolism of GW 610. GW 610, 19 like 5F 203, 18 can induce P450 1A1 in breast cancer cell lines (MCF-7 and MDA 468). However, P450 1A1 was neither highly expressed nor could be induced in the sensitive colon cancer cell lines KM12 and HCC2998.…”

Aryl Hydrocarbon Receptor Ligand 5F 203 Induces Oxidative Stress That Triggers DNA Damage in Human Breast Cancer Cells

“…The investigational agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) displays selective and potent anticancer activity in vitro , and its prodrug lysylamide derivative (Phortress) retains this selectivity and potency profile in vivo. − 5F 203 is an AhR agonist that activates the AhR signaling pathway and causes DNA damage, cell cycle arrest, and apoptosis in breast cancer cells that are sensitive to its cytotoxic actions. − Similar to the mechanism of other AhR agonists, 5F 203 binds to AhR, and the 5F 203–AhR complex translocates from the cytosol to the nucleus. Once within the nucleus, the 5F 203–AhR complex binds to xenobiotic response elements within the respective promoters of target genes, such as cytochrome P450 1A1 (CYP1A1) , to activate their transcription.…”

Putative tumor suppressor cytoglobin promotes aryl hydrocarbon receptor ligand–mediated triple negative breast cancer cell death

“…We previously discovered that CYGB gene expression is restored when cells are treated with the aryl hydrocarbon receptor (AhR) partial agonist 2‐(4‐amino‐3‐methylphenyl)‐5‐fluorobenzothiazole (5F 203) . 5F 203 displays selective and potent anticancer activity in vitro, and its prodrug lysylamide derivative (Phortress) is readily bioconverted into 5F 203 to confer anticancer actions in vivo . 5F 203 activates AhR signaling to induce DNA damage, cell cycle arrest, and promote apoptotic body formation in MDA‐MB‐468 cells .…”