No abstract

Protein kinase inhibitors are a well-established class of clinically useful drugs, particularly for the treatment of cancer. Achieving inhibitor selectivity for particular protein kinases often remains a significant challenge in the development of new small molecules as drugs or as tools for chemical biology research. This review summarises the methodologies available for measuring kinase inhibitor selectivity, both in vitro and in cells. The interpretation of kinase inhibitor selectivity data is discussed, particularly with reference to the structural biology of the protein targets. Measurement and prediction of kinase inhibitor selectivity will be important for the development of new multi-targeted kinase inhibitors.

In this study we present a method for defining the binding modes of a set of structurally related isoindolinone inhibitors of the MDM2-p53 interaction. This approach derives the location and orientation of isoindolinone binding, based on an analysis of the patterns of magnitude and direction of chemical shift perturbations for a series of inhibitors of the MDM2-p53 interaction. The MDM2-p53 complex is an attractive target for therapeutic intervention in cancer cells with intact tumor suppressor p53, as it offers the possibility of releasing p53 by blocking the MDM2-p53 binding site with a small molecule antagonist to promote apoptosis. Isoindolinones are a novel class of MDM2-antagonists of moderate affinity, which still require the development of more potent candidates for clinical applications. As the applicability of conventional structural methods to this system is limited by a number of fundamental factors, the exploitation of the information contained in chemical shift perturbations has offered a useful route to obtaining structural information to guide the development of more potent compounds. For a set of 12 structurally related isoindolinones, the data suggests 4 different orientations of binding, caused by subtle changes in the chemical structure of the inhibitors.

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...