Pharmacophore-Based Design of Phenyl-[hydroxycyclohexyl] Cycloalkyl-Carboxamide Mitofusin Activators with Improved Neuronal Activity

Abstract

Mitochondrial fragmentation from defective fusion or unopposed fission contributes to many neurodegenerative diseases. Small molecule mitofusin activators reverse mitochondrial fragmentation in vitro, promising a novel therapeutic approach. The first-in-class mitofusin activator, 2, has a short plasma t 1/2 and limited neurological system bioavailability, conferring “burst activation”. Here, pharmacophore-based rational redesign generated analogues of 2 incorporating cycloalkyl linker groups. A cyclopropyl-containing linker, 5, improved plasma and brain t 1/2, increased nervous system bioavailability, and prolonged neuron pharmacodynamic effects. Functional and single-crystal X-ray diffraction studies of stereoisomeric analogues of 5 containing sulfur as a “heavy atom”, 14A and 14B, showed that 5 biological activity resides in the trans-R/R configuration, 5B. Structural analysis revealed stereoselective interactions of 5 associated with its mimicry of MFN2 Val372, Met376, and His380 side chains. Modification of murine ALS phenotypes in vitro and in vivo supports advancement of 5B for neurological conditions that may benefit from sustained mitofusin activation.

Piperine Derivatives Enhance Fusion and Axonal Transport of Mitochondria by Activating Mitofusins

“…Piperine's fusogenic potency (EC 50 ) and efficacy (E max ; maximum mitochondrial aspect ratio) were comparable to those of 2 studied in parallel (Figure 2B). Piperine's isomer chavicine (Supplemental Figure S12) lacked fusogenic activity (Figure 2), recapitulating stereoisomer-specific mitofusin activation reported for other small molecule mitofusin activators [15,16].…”

“…Chemistry 2022, 4, FOR PEER REVIEW 5 Published mitofusin activator structure-activity studies suggest that a cyclohexyl or heterocyclic carboxamide, a defined linker length, and an aromatic group are required for mitofusin activation. Within limits of the defining pharmacophore model, compounds with substitutions at the cyclohexyl, linker or phenyl groups retained functional potency while exhibiting different pharmacokinetic properties [15,16]. It is not known if piperine's piperidine amide structure can confer mitofusin stimulating activity.…”

“…Mitochondrial staining and imaging. Mitochondrial fusogenicity was measured using standard methods [15,16,21]. Briefly, mitochondrial elongation was assessed in Mfn2deficient MEFs unless otherwise stated after adding compounds at concentrations ranging from 0.5 nM-10 µM in DMSO, overnight.…”

“…Mitochondrial aspect ratio (mitochondrial length/width) was measured from images acquired at room temperature on a Nikon Ti Confocal microscope using a 60 × 1.3 NA oil-immersion objective and analyzed using ImageJ. Compound fusogenicity, quantified as mitochondrial aspect ratio (mitochondrial length/width), was indexed to the maximal response elicited by the prototype mitofusin activator 2 [15,16]. Concentration-response curves were generated using a sigmoidal model in Prism 8 software.…”

“…We wondered if previously described effects of piperine on mitochondrial function [10,11] might result from an interaction with an as-yet-unrecognized pharmacological target. Piperine has chemical similarities with some recently described novel small molecules that promote mitochondrial fusion by activating mitofusin (MFN) proteins [15,16]. Mitofusins initiate and mediate reparative mitochondrial fusion, and can regulate mitochondrial motility and quality control [17].…”