Hyperpolarized ¹³C magnetic resonance detection of carboxypeptidase G2 activity

Abstract

Carboxypeptidase G2 (CPG2) is a bacterial enzyme that is currently employed in a range of targeted cancer chemotherapy strategies such as gene-directed enzyme prodrug therapy (GDEPT). Employing dynamic nuclear polarization (DNP) and natural abundance 13 C magnetic resonance spectroscopy (MRS), we observed the CPG2-mediated conversion of a novel hyperpolarized reporter probe 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu) to 3,5-difluorobenzoic acid (3,5-DFBA) and L-glutamic acid (L-Glu) in vitro. Isotopic labeling of the relevant nuclei with 13 C in 3,5-DFBGlu or related substrates will yield a further factor of 100 increase in the signal-to-noise. We discuss the feasibility of translating these experiments to generate metabolic images of CPG2 activity in vivo. New frontiers in metabolic imaging have recently been realized in vivo through a combination magnetic resonance spectroscopy (MRS) and novel hyperpolarization techniques employing dynamic nuclear polarization (DNP). The significant enhancement of the MR signal by more than a factor of 10,000 has transformed insensitive techniques such as 13 C and 15 N MRS into versatile strategies that afford a window on the dynamics of endogenous enzymatic processes by generating high spatial resolution and real-time maps of the metabolism of hyperpolarized substrates noninvasively (1,2). These techniques have primarily focused on hyperpolarized 13 C pyruvate as a substrate due to its favorable relaxation characteristics and central role in cellular energy metabolism (3,4). Other hyperpolarized imaging reporters have emerged in recent years to probe a range of key endogenous metabolic reactions (5-8), and further applications such as the measurement of pH in vivo (9).The bacterial enzyme carboxypeptidase G2 (CPG2, EC 3.4.17.11) and other exogenous enzymes have been utilized in promising targeted chemotherapeutic strategies to activate selectively nontoxic prodrugs into cytotoxic drugs in tumors (10). Several strategies have been developed to target the enzyme to a tumor, including the use of CPG2-antibody conjugates in antibody-directed enzyme prodrug therapy (ADEPT) (11), viral vectors that carry the gene encoding for CPG2 in gene-directed enzyme prodrug therapy (GDEPT) (12), and more recently the use of bacteria engineered to express CPG2 (13). These therapeutic strategies would benefit from robust imaging strategies that afford high spatial and temporal resolution images of the biodistribution of CPG2 activity.We have focused on developing MRI reporters for CPG2, a Zn 2ϩ -dependent exopeptidase that activates relatively nontoxic prodrugs into activated DNA alkylating agents by removing their glutamate moiety. 3,5-Difluorobenzoyl-Lglutamic acid (3,5-DFBGlu) is a reporter probe that can be used to detect CPG2 activity in vivo using 19 F MRS, utilizing a 1.4 ppm chemical shift difference associated with the CPG2-mediated conversion of 3,5-DFBGlu to 3,5-DFBA and L-glutamic acid (Fig. 1) (14). In this study we investigated the MR properties of the relevant 13 C nuclei (na...

Chemistry and biochemistry of13C hyperpolarized magnetic resonance using dynamic nuclear polarization

“…3,5-Diflurobenzoyl-L-glutamate was HP at natural abundance and reacted with carboxypetidase G2 (CPG2) as a reporter probe, with cleavage to glutamate indicating CPG2 activity. 185 Interestingly, the amide carbonyl is not observed; its lifetime shortening is attributed to depolarization via an exchange effect with zinc and is reversed by addition of a chelator ( i.e. , EDTA).…”

Detecting Enzyme Activities with Exogenous MRI Contrast Agents

“…Similarly, the 13 C-hyperpolarized metabolites lactate, alanine, fumarate, [5- 13 C]glutamine, 1-keto[1- 13 C]isocaproate, diethyl[1- 13 C]succinate, [2- 13 C]fructose, and 3,5-diflurorbenzoylglutamic acid potentially detect multiple enzyme activities, and therefore should be considered as methods to monitor biochemical pathways rather than specific enzymes. [73] …”

Imaging Mouse Cancer Models In Vivo Using Reporter Transgenes

“…Positive contrast (a hyperintense signal) can be produced by β-galactosidase in T 1 -weighted imaging (Louie et al 2000), which in many cases is preferable to the generation of negative contrast. Furthermore, hyperpolarized MRI has already demonstrated its value in the study of cancer (Day et al 2007;Gallagher et al 2008) and offers a potential way to increase the MR signal radically, but thus far potential reporters have only been demonstrated in vitro (Jamin et al 2009;Chen et al 2011).…”