Lacosamide isothiocyanate-based agents: novel agents to target and identify lacosamide receptors

Abstract

(R)-Lacosamide ((R)-2, (R)-N-benzyl 2-acetamido-3-methoxypropionamide) has recently gained regulatory approval for the treatment of partial-onset seizures in adults. Whole animal pharmacological studies have documented that (R)-2 function is unique. A robust strategy is advanced for the discovery of interacting proteins associated with function and toxicity of (R)-2 through the use of (R)-2 analogues, 3, which contain "affinity bait (AB)" and "chemical reporter (CR)" functional groups. In 3, covalent modification of the interacting proteins proceeds at the AB moiety, and detection or isolation of the selectively captured protein occurs through the bioorthogonal CR group upon reaction with an appropriate probe. We report the synthesis, pharmacological evaluation, and interrogation of the mouse soluble brain proteome using 3 where the AB group is an isothiocyanate moiety. One compound, (R)-N-(4-isothiocyanato)benzyl 2-acetamido-3-(prop-2-ynyloxy)propionamide ((R)-9), exhibited excellent seizure protection in mice, and like (R)-2, anticonvulsant activity principally resided in the (R)-stereoisomer. Several proteins were preferentially labeled by (R)-9 compared with (S)-9, including collapsin response mediator protein 2.

Scheme 1

“Affinity Bait” and “Chemical Reporter” (AB&CR) Strategy to Scan the Brain Proteome for (R)-Lacosamide Target(s)

Scheme 2

General Synthetic Pathway to Prepare Lacosamide AB&CR Agents 9–11

Figure 1

Chemical evaluation of the AB and the CR units in 3

Figure 2

Proteome reactivity profiles of three different isothiocyanate (NCS) agents 9–11. Figure 2A; AB&CR agent-labeled proteins were detected by in-gel fluorescence scanning after Cu(I)-mediated cycloaddition to either a rhodamine-azide reporter probe 39 or a rodamine-alkyne reporter probe 40. Figure 2B; CRMP2 protein was detected by western blot using anti-CRMP2. Figure 2C; All proteins in lysate were visualized by Coomassie blue staining after in-gel fluorescence scanning. (All images shown in gray scale).

Figure 3

In vitro labeling of externally added GST-CRMP2 and endogenous CRMP2 in mixture of mouse lysate and overexpressed GST-CRMP2. Figure 3A; AB&CR agent-labeled proteins were detected by in-gel fluorescence scanning after Cu(I)-mediated cycloaddition to rhodamine-azide reporter probe 39. Figure 3B; CRMP2 and GST proteins were detected by western blot using anti-CRMP2 and anti-GST antibodies. Figure 3C; All proteins in lysate were visualized by Coomassie blue staining after in-gel fluorescence scanning. (All images shown in gray scale).