دانلود رایگان مقاله طراحی منطقی آنالوگ مقاوم در برابر AKR1C3 از PR-104

abstract

The clinical stage anti-cancer agent PR-104 has potential utility as a cytotoxic prodrug for exogenous bacterial nitroreductases expressed from replicating vector platforms. However substrate selectivity is compromised due to metabolism by the human one- and two-electron oxidoreductases cytochrome P450 oxidoreductase (POR) and aldo-keto reductase 1C3 (AKR1C3). Using rational drug design we developed a novel mono-nitro analog of PR-104A that is essentially free of this off-target activity in vitro and in vivo. Unlike PR-104A, there was no biologically relevant cytotoxicity in cells engineered to express AKR1C3 or POR, under aerobic or anoxic conditions, respectively. We screened this inert prodrug analog, SN34507, against a type I bacterial nitroreductase library and identified E. coli NfsA as an efficient bioactivator using a DNA damage response assay and recombinant enzyme kinetics. Expression of E. coli NfsA in human colorectal cancer cells led to selective cytotoxicity to SN34507 that was associated with cell cycle arrest and generated a robust ‘bystander effect’ at tissue-like cell densities when only 3% of cells were NfsA positive. Anti-tumor activity of SN35539, the phosphate pre-prodrug of SN34507, was established in ‘mixed’ tumors harboring a minority of NfsA-positive cells and demonstrated marked tumor control following heterogeneous suicide gene expression. These experiments demonstrate that off-target metabolism of PR-104 can be avoided and identify the suicide gene/prodrug partnership of E. coli NfsA/SN35539 as a promising combination for development in armed vectors.

4. Discussion

An ideal GDEPT prodrug is substantially less cytotoxic than its corresponding active metabolite(s) and is a unique substrate for the chosen prodrug activating enzyme under physiological conditions. PR-104 is a clinical stage nitroaromatic prodrug that is demonstrably more active than the prototypical NTR prodrug CB1954 in gene therapy models [24]. Nevertheless, several mechanisms of PR-104A activation including hypoxia-selective metabolism [10], aerobic activation by AKR1C3 [15] and oxygeninsensitive non-CYP450 hepatic enzymes [36], indicate this prodrug lacks selectivity for NTRs in the systemic setting. In order to generate an NTR-specific prodrug for gene therapy applications, the chemical structure of PR-104A was modified to minimize activation by endogenous human enzymes. Rational design led to the synthesis of the prodrug SN34507, an analog of PR-104A lacking the ancillary ortho nitro moiety predicted to reduce recognition by the AKR1C3 active site. Additionally, the potential for ortho nitro reduction leading to intramolecular cyclization of the activate metabolite to form non-cytotoxic by-products (as seen for PR-104A [10]) is negated. Loss of AKR1C3-dependent cytotoxicity was confirmed in 2D anti-proliferative IC50 assays, 3D multicellular layer assays and tumor bearing murine models (Fig. 3). Suggestion of residual activity in low cell density assays did not result in biologically meaningful activity in high cell density MCL and tumor models, with no evidence of AKR1C3 driven antitumor activity.