Discovery of 6-Phenylhexanamide Derivatives as Potent Stereoselective Mitofusin Activators for the Treatment of Mitochondrial Diseases

Abstract

Mutations in the mitochondrial fusion protein mitofusin (MFN) 2 cause the chronic neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A), for which there is currently no treatment. Small-molecule activators of MFN1 and MFN2 enhance mitochondrial fusion and offer promise as therapy for this condition, but prototype compounds have poor pharmacokinetic properties. Herein, we describe a rational design of a series of 6-phenylhexanamide derivatives whose pharmacokinetic optimization yielded a 4-hydroxycyclohexyl analogue, 13, with the potency, selectivity, and oral bioavailability of a preclinical candidate. Studies of 13 cis- and trans-4-hydroxycyclohexyl isostereomers unexpectedly revealed functionality and protein engagement exclusively for the trans form, 13B. Preclinical absorption, distribution, metabolism, and excretion (ADME) and in vivo target engagement studies of 13B support further development of 6-phenylhexanamide derivatives as therapeutic agents for human CMT2A.

Burst mitofusin activation reverses neuromuscular dysfunction in murine CMT2A

“…To determine whether benefits of mitofusin activation in cultured neurons would translate to therapeutic effects on neuromuscular dysfunction in CMT2A we contemplated an in vivo trial in our CMT2A mouse. However, Chimera C is rapidly degraded by the liver and undergoes first-pass metabolism, making it impractical for in vivo studies ( Dang et al, 2020 ). We therefore evaluated in vivo efficacy of mitofusin activation in CMT2A using MiM111, a structurally distinct compound having a mitofusin activation profile similar to Chimera C ( Figure 1—figure supplement 3 ), but which is metabolically stable with good nervous system bioavailability ( Dang et al, 2020 ).…”

“…However, Chimera C is rapidly degraded by the liver and undergoes first-pass metabolism, making it impractical for in vivo studies ( Dang et al, 2020 ). We therefore evaluated in vivo efficacy of mitofusin activation in CMT2A using MiM111, a structurally distinct compound having a mitofusin activation profile similar to Chimera C ( Figure 1—figure supplement 3 ), but which is metabolically stable with good nervous system bioavailability ( Dang et al, 2020 ). We hypothesized that intermittent or ‘burst’ mitofusin activation (a dosing schedule that reversed mitochondrial dysfunction for <12 hr each day) ( Figure 3—figure supplement 1 ) would confer therapeutic benefits by cyclically enhancing mitochondrial fitness and transport, while minimizing the possibility of mitofusin toxicity that might occur with constant mitofusin activation ( El Fissi et al, 2018 ; Meyer et al, 2017 ).…”

“…Based on a minimum effective MiM111 plasma concentration of 30 ng/ml ( Dang et al, 2020 ) and a plasma t 1/2 of 2.3 hr with C max of 24,000 ng/ml after intramuscular administration of 30 mg/kg ( Figure 3—figure supplement 1 ), we estimated that daily IM dosing would reverse mitochondrial dysmotility in CMT2A mice for ~12 hr out of every 24 hr. Indeed, Figure 3—figure supplement 1 show that mitochondrial motility in sciatic nerve axons of MFN2 T105M mice was normalized 4 hr after a single intramuscular injection of MiM111 (30 mg/kg), declined by approximately half after 12 hr, and returned to CMT2A baseline after 24 hr.…”

“…For this reason, the specific functional benefits accruing from mitofusin activation in CMT2A cannot unambiguously be defined. Mitochondrial fusion and motility are impaired in CMT2A ( Chen and Chan, 2006 ) and allosteric mitofusin activation corrects both of these parameters ( Franco et al, 2016 ; Rocha et al, 2018 ; Dang et al, 2020 ). Mitochondrial respiratory dysfunction, measured here as loss of inner membrane polarization, is a consequence of diminished fusion-mediated homeostatic repair ( Chen and Chan, 2006 ), and its improvement can therefore also be explained by enhanced fusogenicity.…”

“…Here, we studied two structurally distinct but functionally similar allosteric mitofusin activators, a new class of drug that is the first to directly enhance mitochondrial fusion and transport. Although the prototype compounds were found not to be ‘druggable’, a new generation of mitofusin activators have addressed pharmaceutical limitations of the initial chemical series ( Dang et al, 2020 ). As described previously ( Rocha et al, 2018 ; Dang et al, 2020 ), mitofusin activators have minimal effects in normal cells, likely because increasing the probability that mitofusins are in their open/ ‘active’ conformation is a subtle intervention that can be readily compensated for in the absence of a pre-existing imbalance between mitochondrial fusion and fission.…”