The Corrections and the Oprahgate

It is clear that AC isoforms are implicated in several disease states and it is hypothesized that AC isoform-selective small molecules could be utilized together with knockout and transgenic animal models to investigate the

physiological roles of AC isoforms and validate their therapeutic potential (Pavan et al., 2009; Pierre et al., 2009). However, small molecule AC modulators are generally non-selective and/or display low potency for AC modulation (Seifert et al., 2012). The current collection of AC modulators is discussed below.

Forskolin (Figure 1.7) is a labdane diterpene that was isolated from the roots of the Coleus forskohlii plant, and was found to be a reversible activator of

adenylyl cyclase activity in several tissues including the cerebral cortex, striatum, heart, and liver (Seamon et al., 1981). Subsequent studies with recombinant adenylyl cyclases suggest that forskolin directly binds AC and is capable of stimulating all AC isoforms with the exception of AC9 (Sadana and Dessauer, 2009; Tesmer et al., 1997). Though forskolin has been used extensively as a research tool for the study of ACs and cAMP signaling (Insel and Ostrom, 2003), several properties of forskolin are suboptimal. For example, forskolin is not water-soluble, modulates AC isoforms in a non-selective fashion, and is known to modulate other enzymes including glucose transporters (Laurenza et al., 1989).

To improve upon these shortcomings, much effort has been directed toward the synthesis and pharmacological characterization of forskolin analogs. For

example, NKH477 was identified as a water-soluble forskolin analog that has enhanced potency for AC5 as compared to AC3 and AC2 (Toya et al., 1998).

More recent studies identified forskolin derivatives that display a range of modulatory properties, including inactivity, partial activation, enhanced efficacy for AC isoforms, and inhibition of AC isoforms (Onda et al., 2001; Pinto et al., 2008). Notably, BODIPY-conjugated forskolin displays potent inhibition of AC2, while partially activating AC1 and AC5 (Pinto et al., 2008). Additional chemical modification of forskolin may yield derivatives with novel modulatory properties or more favorable AC isoform-selectivity profiles, but studies to date have only provided marginal improvements.

Figure 1.7 Chemical structures of small molecule adenylyl cyclase modulators.

Early approaches to the development of adenylyl cyclase inhibitors focused on compounds that are nucleotides that contain an adenine (or purine) ring, known as the “p-site” inhibitors (Dessauer et al., 1999). The p-site inhibitors are generally non-selective for inhibition of adenylyl cyclase isoforms (Johnson et al., 1997) and bind to the active site in the presence of pyrophosphate (Tesmer et al., 2000), but non-competitively with respect to ATP (Dessauer and Gilman, 1997; Tesmer et al., 2000). It is clear that the nucleotide phosphate groups confer potency for inhibition of ACs, as adenine nucleoside 3’-polyphosphate ligands are among the most potent p-site inhibitors (Johnson et al., 1997).

However, the polyphosphate groups are expected to be poorly membrane-permeable. Though site ligands are generally non-selective, ribose-modified p-site ligands such as SQ22,536 and 9-CP-Ade display some degree of AC isoform selectivity in vitro (i.e., both compounds have similar potency values for inhibition of AC1, AC6, and AC8, but little or no activity for inhibition of AC2), but are

relatively less potent than the adenine 3’-polyphosphate p-site ligands (Johnson et al., 1997). The p-site inhibitors identified to date appear to have a trade-off between potency and AC isoform selectivity. Furthermore, p-site inhibitors may have off-target effects such as inhibition of DNA replication or purine metabolism due to their adenine rings (Seifert et al., 2012).

Given concerns about an intact adenine ring facilitating off-target effects, a virtual screen was conducted for compounds that retained the pharmacophore of a ribose-substituted p-site inhibitor for AC, but without an adenine ring (Onda et al., 2001). This screen yielded the second generation p-site inhibitor, NKY80

(Onda et al., 2001). Though NKY80 displays selectivity for AC5 over AC2 and AC3, and binds non-competitively with ATP and forskolin, the compound generally lacks potency for inhibition of AC (Onda et al., 2001).

An additional strategy for the development of more potent and/or selective inhibitors of AC involved the fusion of a metal-coordinating hydroxamic acid moiety to an adenine ring to exploit the requirement of metal ions for AC catalytic activity (Levy et al., 2003; Levy et al., 2002a; Levy et al., 2002b; Tesmer et al., 1999). In in vitro Sf9 cell membrane assays, PMC-6 emerged as a selective AC5 inhibitor (over AC2 and AC3), and the most potent among the PMC class of inhibitors (Iwatsubo et al., 2004). Furthermore, PMC-6 has activity for inhibition of isoproterenol-stimulated cAMP in intact cardiac myocytes, where it also displays efficacy for inhibition of apoptosis (Iwatsubo et al., 2004), suggesting possible therapeutic utility.

More recent studies have identified NB001 as a potent inhibitor of AC type 1 in intact cell assays (Wang et al., 2011). Interestingly, in agreement with AC1 knockout mouse studies, inhibition of AC1 activity with NB001 has analgesic effects in animal models of neuropathic and inflammatory pain (Vadakkan et al., 2006; Wang et al., 2011). These studies suggest that targeting AC1 has

analgesic utility, but the activity of NB001 in in vitro AC assays and an

understanding of the structure-activity relationship surrounding NB001 remain to be reported.

2’(3’)-O-N-Methylanthraniloyl (MANT)-substituted fluorescent nucleotides were found to be a novel and potent class of competitive AC inhibitors (Gille and

Seifert, 2003). Subsequent crystallography studies with MANT-GTP suggest that the MANT moiety binds in a hydrophobic patch at the C1/C2 interface and

prevents a necessary rotation that is required for AC catalytic activity (Mou et al., 2005; Wang et al., 2007b). Furthermore, a series AC isoform-selectivity and structure-activity relationship studies on the MANT class of compounds revealed a general preference for inhibition of AC5 and AC6 over other AC isoforms and that the catalytic site can accommodate nucleotides including adenine, guanine, hypoxanthine, and uracil (Gille et al., 2004; Goettle et al., 2009; Huebner et al., 2011; Mou et al., 2006; Pinto et al., 2011; Suryanarayana et al., 2009).

Consistent with the latter observation, MANT-ITP was identified as the most potent AC inhibitor from this class of compounds. Though the MANT nucleotides are generally more potent than the p-site inhibitors for inhibition of AC in vitro, these compounds are also limited by poor membrane-permeability (Seifert et al., 2012). Thus, these studies are most useful for the structural information that they provide surrounding the binding of ligands in the catalytic site of ACs.

Efforts to develop potent and AC isoform-selective small molecule modulators have only modestly contributed to the current understanding of AC targeting and several limitations surrounding specificity, AC isoform-selectivity, and cell permeability remain. Furthermore, the current repertoire of AC

modulators has not been comprehensively studied with respect to each of the nine membrane-bound AC isoforms in both in vitro and intact cell models, making it difficult to fully assess the isoform-selectivity of these compounds (Seifert et al., 2012). The discovery and development of novel small molecule modulators of

AC that are potent and isoform-selective is expected to facilitate the in vivo study of individual AC isoforms and allow for the full evaluation of AC isoforms as potential therapeutic targets.