Pharmacokinetics and target engagement of novel FKBP12
ligands
The newly designed 4-arylthiomethyl-1-carboxyalkyl-1,2,3-triazoles were
synthesized with the aim of serving as potential ligands that can
concurrently bind to both the ionic and hydrophobic pockets of the
FKBP12/RyR1 complex . These compounds demonstrated in vitro target
engagement with FKBP12 through the rapamycin-inducible FKBP/FRP binding
domain protein fragment complementation assay, thereby stabilizing
cytosolic Ca2+ levels in human myotubes under
nitro-oxidative stress . Within this class of compounds, MP-001 (Fig.
1A) demonstrated superior binding affinity to FKBP12 and elicited
co-localization of FKBP12/RyR1 in human myotubes under nitro-oxidative
stress conditions . MP-001 not only attenuated Ca2+ ER
leakage through the RyR induced by nitro-oxidative stress but also
demonstrated the capacity to rectify the heightened susceptibility to
spontaneous Ca2+ release observed in conditions of ER
Ca2+ overload resulting from the RyR2 R2474S mutation,
which disrupts the interaction with the FKBP12.6 subunit . This
manifestation is evidenced by the increased Ca2+content released upon stimulation with caffeine (Suppl. Fig. 1).
Polar group exchange from carboxylic acid to N,N-dimethylamino moiety
yielded compound MP-002 (Fig. 1A) with improved blood-brain-brain
barrier (BBB) permeability. However, MP-002 exhibits very rapid
metabolism, which results in low circulating levels, making it
unsuitable for chronic treatment of the central nervous system (CNS).
Concretely, MP-002 displayed a Cmax of 5.1 ± 2.4 ng/mL
with an inappreciable half-life in serum after an oral dosing of 30
mg/kg (data not shown).
Subsequently, for chronic treatment in the SOD1G93Amouse model, we selected compound MP-010 (Fig. 1A) as an improved
version of MP-002, incorporating a sulfoxide functional group to enhance
its pharmacokinetic properties. MP-010 demonstrated favorable oral
absorption, with a mean Cmax of 1144.4 ± 397.2 ng/mL and
a half-life of 3.0 h in serum (Fig. 1B), a Cmax of
1173.1 ± 279.6 ng/g and a half-life of 2.5 h in muscle (Fig. 1C), and a
Cmax of 340.8 ± 166.9 ng/g and a half-life of 3.7 h in
the brain after a dosing of 30 mg/kg (Fig. 1D). The brain-to-serum
concentration ratio (Cb:Cs) of MP-010 was significantly greater than
0.04, even after 18 hours following dosing (Fig. 1D). This value is the
threshold at which a molecule is commonly considered to be ”brain
penetrant,” as blood brain volume approximates 4% of total brain volume
.
With respect to the stabilizing properties of RyR, both compounds MP-002
and MP-010 showed a complete rectification of the Ca2+ER leak caused by the RyR2 R2474S mutation, as evidenced by the
increased Ca2+ content released upon stimulation with
caffeine (Suppl. Fig. 1).
Furthermore, we sought to evaluate another compound with in vivo
stabilities comparable to those of MP-010 but with reduced or
insignificant tissue distribution. Our aim was to determine that any
positive effects of MP-010 are attributable to its mechanisms of action
within the CNS. Based on this assumption, we chose MP-001 as an
alternative compound, with a Cmax and half-life of
2179.8 ± 305.9 ng/mL and 2.5 hours in serum (Fig. 1B), 163.0 ± 22.1 ng/g
and 2.5 hours in muscle (Fig. 1C), and 30.3 ± 5.6 ng/g and 1.2 hours in
the brain (Fig. 1D), respectively. Its Cb:Cs ratio was found to be less
than 0.04 (Fig. 1D).
Therefore, we used MP-010 and MP-001 to investigate the hypothesis that
modulating intracellular Ca2+ levels by facilitating
the interaction between FKBP12 and RyR may confer neuroprotective
effects in ALS.