Background
Angelman syndrome (AS) is a severe neurodevelopmental disorder characterized by developmental delay, intellectual disability, speech impairment, seizures, and ataxia [
1‐
5]. AS has a prevalence of 1:15,000 [
6,
7], and these individuals need care across their full lifespan, yet no cure currently exists. Thus, it is of great importance to develop treatments for AS. AS is caused by mutation of the ubiquitin protein ligase E3A (
UBE3A) gene, which is genomically imprinted. Only the maternally inherited copy is expressed in neurons [
8], whereas
UBE3A is biallelically expressed in most other tissues. This neuron-specific imprinting provides insight into why deletions or mutations in the maternal copy of
UBE3A primarily impact brain function and cause AS. However, the paternal
UBE3A allele is intact, as demonstrated by biallelic expression in other tissues, raising the possibility that AS could be treated by unsilencing the dormant paternal
UBE3A allele in neurons.
This led us to try pharmacological approaches to identify small molecules capable of unsilencing the dormant copy of
UBE3A. In a previous study, we developed a high-content assay to identify small molecules that could unsilence paternal
Ube3a in mouse primary neurons. In that screen, we used knock-in mice carrying a yellow fluorescent protein (YFP)-tagged
Ube3a reporter, allowing us to visualize maternal- or paternal-specific expression of
Ube3a-
YFP in cultured neurons. As expected,
Ube3a-
YFP was expressed in cultured neurons when inherited maternally but was not expressed (silenced) when inherited paternally. We found that topoisomerase I (Top1) inhibitors (e.g., topotecan) could effectively unsilence paternal
Ube3a in mice [
9], raising the possibility that topotecan or similar compounds [
10] could become treatments for AS. The translational potential was supported by evidence that topotecan treatment biochemically rescued the function of UBE3A, unsilenced
Ube3a in vivo in mice, and unsilenced paternal
UBE3A in induced pluripotent stem cell-derived neurons of AS patients [
11].
Topotecan is FDA-approved for the treatment of cancer and is well tolerated in adult and pediatric cancer patients [
12‐
15]. It is also used to treat brain tumors [
16,
17]. Topotecan crosses the BBB more readily than many topoisomerase inhibitors [
18]. However, active pumps extrude topotecan from the brain, which limits its functional CNS bioavailability [
19,
20]. Moreover, topotecan can produce some toxicities [
21,
22]. These limitations prompted us to search for novel Top1 inhibitors with better CNS bioavailability and improved safety profiles.
Indenoisoquinoline-derived Top1 inhibitors offer a promising class of compounds for paternal
Ube3a unsilencing as many of these compounds produce particularly stable Top1 cleavage complexes [
23‐
25], which we have shown are critical for producing paternal
Ube3a unsilencing [
26]. Over 300 indenoisoquinoline derivatives have been tested, some of which are very potent Top1 poisons and show antitumor activity in mouse models [
10,
27‐
31]. These Top1 inhibitors work by blocking the enzymatic activity of Top1 by stabilizing cleavage complexes, which are compound-bound intermediates of Top1-DNA [
10,
23‐
25,
32]. More importantly, when compared to topotecan, indenoisoquinoline-derived Top1 inhibitors demonstrate improved characteristics such as greater chemical stability of these cleavage complexes. In addition, they target a unique DNA sequence for cleavage (indenoisoquinolines --G
↓C-- vs. topotecan --T
↓G--) [
23‐
25,
32].
The goal of this study was to establish indenoisoquinoline derivatives that could effectively unsilence paternal
Ube3a, with the expectation that some of these compounds might prove to be safe and have favorable CNS bioavailability. All of the tested compounds showed a capacity to unsilence the paternal
Ube3a allele, with several of the compounds exhibiting unsilencing efficacy similar to topotecan. Excitingly, two of the tested indenoisoquinoline derivatives, indotecan (LMP400) and indimitecan (LMP776), are already in clinical trials [
33,
34]. The results of our study suggest additional Top1 inhibitors that should be advanced for AS preclinical testing of safety and CNS efficacy.
Discussion
The goal of this study was to explore indenoisoquinoline derivatives as possible AS therapeutics by characterizing their effects on Ube3a unsilencing in mouse cortical neurons in vitro. Here, we identify indotecan (LMP400) as a potential AS therapeutic agent that warrants further examination in vivo for CNS bioavailability and safety.
The unique expression of
UBE3A governed by genomic imprinting provides a therapeutic opportunity for AS by reactivating the paternal
UBE3A allele [
8,
9,
11]. Our research team previously reported that topoisomerase I inhibitors can reactivate the dormant
UBE3A allele, providing the first proof of concept of pharmacological reactivation of paternal
UBE3A as a potential therapeutic intervention for AS [
9]. Because of the anticancer activity of Top1 inhibitors, many derivatives that overcome the limitations of camptothecins [
10,
41,
42] have been synthesized for clinical development. One of these, topotecan, is FDA-approved for ovarian and lung cancers [
43], while another, irinotecan, is approved for colon cancers [
44]. However, these compounds may have limited clinical potential for treating AS. For example, topotecan has several flaws such as decreased bioavailability due to plasma protein binding of the lactone hydrolysis product, removal from cells by drug efflux transporters, and long infusion times necessitated by relatively low stability of the ternary drug-DNA-enzyme cleavage complexes [
10,
18]. These limitations prompted us to search for novel Top1 inhibitors as potential AS therapeutics, with the expectation that lead candidates could then be vetted for having improved CNS bioavailability and better safety profiles. For these studies, we focused on indenoisoquinoline-derived Top1 inhibitors that might overcome the limitations of topotecan [
10]. Of many indenoisoquinoline-derived Top1 inhibitors, indotecan and indimitecan were selected to examine their unsilencing effects on paternal
Ube3a because of their similar ability to effectively inhibit almost 100% of Top1 enzymatic activity [
33] and recent completion of phase I clinical trials (
ClinicalTrials.gov ID: NCT01051635).
Here, we demonstrate that indenoisoquinoline-derived Top1 inhibitors are potent
Ube3a unsilencers, with different unsilencing properties. Of the compounds we tested in vitro, indotecan (LMP400) [
10,
34] appears to have more favorable paternal
Ube3a unsilencing properties than topotecan. While both topotecan and indotecan exhibit CNS penetrance [
18,
40], there are not yet data available to directly compare their relative CNS bioavailability; there is a need to carefully establish the CNS bioavailability of indotecan. One potential advantage of indotecan is that it is not a substrate for the transporters ABCG2 and MDR1 [
32], suggesting that it may stay longer in the CNS than topotecan because transporters extrude topotecan from the brain [
19]. Moreover, although the cytotoxicity profile of indotecan is similar to that of topotecan, its efficacy and potency are better than topotecan. Indimitecan (LMP776) [
10] appears to be more toxic than topotecan, while the three other indenoisoquinoline derivatives (indotecan, DB-IV-58, and DB-V-37) showed similar cytotoxicity to topotecan in our cultured cortical neurons. In addition, the efficacy of indimitecan is lower than that of topotecan. The structural differences in indenoisoquinoline-derived compounds are responsible for their different unsilencing effects. The only structural difference between indotecan and indimitecan is in the side chain which is appended to the heterocyclic system that intercalates in the DNA break generated by Top1 [
10,
23]. These characteristics will be important considerations for the future design of paternal
UBE3A unsilencers. Although indotecan exhibits better efficacy and potency than topotecan, the similar cytotoxicity of the two drugs must be considered for in vivo applications. Importantly, the DNA cleavage complex patterns of indotecan are different from topotecan in a manner that may confer some important advantages for clinical use. Indenoisoquinolines such as indotecan produce more stable cleavage complexes than camptothecins such as topotecan [
45], which, based on the mechanism of
Ube3a unsilencing [
26], should enhance
Ube3a unsilencing as we observed. Moreover, after drug removal, the Top1-DNA complexes induced by indenoisoquinolines persist under conditions where camptothecin-induced Top1-DNA complexes completely reverse [
45]. This observation further suggests that the similar cytotoxicity of indotecan might be offset by the potential for a briefer treatment regimen in vivo, which remains to be addressed. Known off-target effects for topoisomerase inhibitors generally lead to the transient downregulation of long genes [
11,
46,
47]. Genome-wide analyses are necessary to reveal all potential off-target effects for indenoisoquinolines (e.g., indotecan). Alternatively, an in silico analysis using SEA (similarity ensemble approach;
http://sea.bkslab.org) enables us to predict off-targets. SEA analysis revealed that indotecan possesses 55 potential off-targets, including aurora kinase A, aurora kinase B, and acetylcholinesterase. Regardless of off-target effects, clinical trials have demonstrated that, at least at the concentrations examined, indotecan is well tolerated in a clinical population [
34]. However, it still remains to be addressed whether off-target effects arise at the concentration at which indotecan is effective, as we reported that indotecan has a very low EC
50 of ~ 26 nM to produce
Ube3a unsilencing.
We tested 11 structural analogues of indotecan/indimitecan for their ability to unsilence paternal
Ube3a, and these compounds could be roughly categorized based on their ability to inhibit Top1 in cell-free assays: those compounds (DB-IV-26, DB-IV-50, DB-IV-56, DB-IV-58, DB-V-37, and DB-V-41) that have 100% of the ability of camptothecin to stabilize the ternary drug-DNA-Top1 cleavage complexes [
37,
48], those compounds (DB-V-46, DB-V-47, and MNR-IV-64) that inhibit between 50 and 75% of Top1 [
37], and those compounds (MJ-II-66A) that inhibit between 20 and 50% of Top1 [
30]. We also tested DB-III-17, as this is an intermediate compound for synthesizing or modifying the analogues. The compounds with lower Top1 inhibitory activities (DB-III-17, DB-V-46, DB-V-47, and MJ-II-66A) showed a very limited therapeutic index. Their ambiguous EC
50 values were mainly due to limited effective dose ranges. Although we did not test their cytotoxicities in our cultured cortical neurons, we expect that they are more toxic than topotecan because we could not measure the fluorescence intensity in unsilenced UBE3A-YFP protein, possibly due to cell death produced at concentrations > 1 μM. On the other hand, we observed similar efficacy and potency of three compounds (DB-IV-50, DB-IV-56, and MNR-IV-64) with between 75 and ~ 100% Top1 inhibitory activities relative to camptothecin, but their effectiveness seems to be less than those of indotecan or topotecan. Interestingly, although DB-IV-26 and DB-V-41 have ~ 100% Top1 inhibitory activity, their EC50 values were also ambiguous due to limited effective dose ranges. Since the cleavage complexes are critical for producing paternal
Ube3a unsilencing [
26], we suspect that their cleavage complexes may not be stable enough to produce paternal
Ube3a unsilencing. More importantly, the various hydroxylated side chains contribute to differences in the pharmacological action in
Ube3a unsilencing. For example, the compounds with lower Top1 inhibitory activities (DB-III-17, DB-V-46, DB-V-47, and MJ-II-66A) either lack the hydroxylated side chains that potentially serve as hydrogen-bond acceptors/donors that enable Top1 inhibitory activities and cytotoxicity at physiological pH [
49] or the hydroxylated side chain is cyclic. On the other hand, other compounds possessing dimethoxy or methylenedioxy groups, and/or straight hydroxylated side chains, which appear to be the main contributors to Top1 inhibitory activity and cytotoxicity, effectively unsilence paternal
Ube3a.
Taken together, our study suggests that clinical development of paternal Ube3a unsilencers will require optimization of Top1 inhibition and cytotoxicity through modulating chemical characteristics, including the length of the side chains. Although in vivo assays are necessary to further evaluate the unsilencing effects of indenoisoquinolines, this study provides a framework for developing novel AS therapies using different classes of Top1 inhibitors.