Uncoupling Neuroprotection from Immunosuppression: the Discovery of ILS-920 - Accounts in Drug Discovery Case Studies in Medicinal Chemistry - page 326

experiments. We were particularly interested in testing the hypothesis, men-
tioned earlier, that immunophilin ligand-induced neurite outgrowth is mediated
by immunophilins other than FKBP12, notably FKBP52 and/or FKBP38-
Ca 21 -calmodulin (CaM). 24,48 Specifically, FKBP52 was proposed to mediate
the neurite outgrowth action of FK506 through activation of steroid receptor
complexes that mediate downstream responses to estrogen, androgen, and
glucocorticoid hormones. However, rapamycin is a potent inhibitor of
FKBP52, has some neuroprotective activity, but lacks e cacy in an animal
model. Alternately, an FKBP38-calmodulin-Ca 21 complex formed at high
Ca 21 concentrations was recently proposed to mediate Ca 21 overload-induced
cell death, through its interaction as a negative effector of the anti-apoptotic
Bcl-2 protein.
Anity purification from F-11 cell lysates using resin with covalently bound
WYE-592 and ILS-920 identified FKBP52 and the a1 subunit of L-type Ca 21
channels (CACNB1) as the major binding partners for the compounds.
Moreover, ILS-920 demonstrated binding selectivity for FKBP52 over
FKBP12. The observed magnitude difference in binding anities for ILS-920
among homologous immunophilins appears consistent with the reported dif-
ferences in binding anities for FKBP25 between rapamycin and FK506, 53 and
with previous observations that modification distant to the FKBP binding
domain of rapamycin can affect immunophilin binding.
Having demonstrated that ILS-920 is a binding partner for FKBP52, we
further investigated whether a functional correlation between FKBP52 inhi-
bition and neuroprotection could be established. The observation that neurite
outgrowth was essentially unchanged in CACNB1 siRNA-treated neurons, but
significantly increased in FKBP52 siRNA-treated neurons, suggested that
inhibition of FKBP52 was a mediator of neurite outgrowth of the compounds,
given that neurite outgrowth of this magnitude is consistent with the reported
activity of an FKBP52 antibody. 48 Preliminary transcriptional analysis of
cortical neuronal cultures treated with WYE-592 or ILS-920 suggested up-
regulation of genes involved in cholesterol biosynthesis, consistent with acti-
vation of glucocorticoid and other steroid receptors, an effect that can be
attributed to the immunophilin ligand-induced dissociation of FKBP52 from
steroid receptor complexes. Stimulation of cholesterol biosynthesis is a com-
mon feature of agents that promote neurite outgrowth, including FK506, sterol
hormones, and geldanamycin. 48
ILS-920 was also observed to bind the b1 subunit of L-type calcium channels
and partially inhibit L-type voltage-gated calcium channel (VGCC) current.
Interestingly, the in vivo ecacy, or lack thereof, of ILS-920, WYE-592,
FK506, and rapamycin correlated with their capacity to inhibit L-type VGCC-
mediated Ca 21 signaling pathways. Note that ILS-920 inhibits the L-type
VGCC directly, whereas FK506 inhibits calcineurin, a key component of
L-type VGCC signaling. We hypothesized that attenuation of Ca 21 influx
might be mechanistically important, since Ca 21 overload is generally con-
sidered to be a critical event in excitotoxic mediated neuronal death, 54 although
additional work to test this further is needed.