Many metalloenzyme inhibitors consist of two chemical

Many metalloenzyme inhibitors consist of two chemical components: the MBG, the portion of the inhibitor designed to bind to the metal, and the scaffold, the portion of the inhibitor recognized by the amino cb 839 sale residues that form the substrate-binding site of the metalloenzyme. The MGB is often a major contributor to the overall potency of the inhibitor (though it is acknowledged that many examples of metalloenzyme inhibitors have been reported that do not utilize a MBG). The attraction of the MBG to the metal ion is governed by electronic factors. The metal ion is generally electron-deficient, while the MBG is nucleophilic. The magnitude of the MBG’s interaction with the metal, and therefore inhibitor potency, can be ‘tuned’ by modulating its electronic character. If the metal/MBG interaction is strong the inhibitor will certainly be effective against the intended target but may also inhibit unintended related metalloenzymes. Our approach has been to attenuate the magnitude of the MGB/metal interaction in order to initially improve target selectivity while retaining residual potency. Inhibitor potency and selectivity are then further improved through modifications to the scaffold that increase the magnitude of interactions within the substrate binding pocket. MBGs were initially selected using an in silico method that rank ordered them by affinity for heme–iron. Emphasis was placed on MBGs with lower affinity than the current high-potency imidazoles and pyridines that appear in inhibitors that respectively show modest (TAK-700) or no selectivity (abiraterone) for CYP17 lyase compared to hydroxylase. Due to lack of lyase selectivity, these inhibitors must be administered with prednisone to address MES-associated side-effects (hyperkalemia, hypertension). CYP17, a steroidogenic enzyme that is required for androgen biosynthesis, catalyzes two sequential chemical steps. CYP17 hydroxylase catalyzes the conversion of progesterone and pregnenolone to their 17-hydroxy analogs and is required for the synthesis of glucocorticoids (). CYP17 lyase breaks the covalent bond between C and C of 17-hydroxypregnenolone, forming the androgen, dehydroepiandrosterone (DHEA), and thus represents the first committed step in sex steroid biosynthesis. Though inhibition of either CYP17 hydroxylase or lyase should result in decreased androgen biosynthesis, lyase is the preferred point of intervention since hydroxylase inhibition also prevents glucocorticoid synthesis and causes the accumulation of mineralocorticoids. Since cortisol is the most abundant steroid synthesized and secreted by the adrenal gland, mineralocorticoids get pulled by mass action into the glucocorticoid pathway and will unlikely accumulate as a result of CYP17 lyase-selective inhibition. A key challenge to the design of lyase-selective inhibitors is that the single-chain CYP17 protein uses the same active site to catalyze both the lyase and hydroxylase reactions. Both reactions require heme–iron and oxidoreductase cofactors, as well as NADPH and molecular oxygen as co-substrates. However, CYP17 has an additional cofactor, cytochrome b5, that enhances lyase activity. The crystal structure of the CYP17 hydroxylase conformation has been solved but not the CYP17-cytochrome b5 complex. Thus, there is little structural information to guide the design of CYP17 lyase-selective inhibitors and their optimization has been empirical to date. Not surprisingly, given the dearth of structural information and protein target similarity constraints, all CYP17 inhibitors reported to date affect both the hydroxylase and lyase enzyme functions to some extent. Our design and discovery strategy focused on the investigation of alternative, lower-affinity MBGs on the premise that lyase-selective CYP17 inhibitors may provide safer and more effective clinical agents. Herein, we disclose a unique series of inhibitors, including the Phase 2 clinical agent (VT-464), which exhibit selective potency for CYP17 lyase.