A Chemical Biology Approach to Discover the Biological Targets of the Antiepileptic Drug Lacosamide

Lacosamide (Vimpat[registered trademark]) is a potent antiepileptic drug that received market approval for the adjunctive treatment of partial-onset seizures in adults in Europe and the United States. The pharmacological studies document that lacosamide has a unique profile of activity that differentiates it from existing antiepileptic agents. Based on these findings, we hypothesized that lacosamide binds to different proteins, with low-to-modest affinity. This research project aims to identify the lacosamide biological targets that modulate function and toxicity. We propose a novel target identification approach where an affinity bait (AB) and a chemical reporter (CR) group are strategically placed within the lacosamide framework. These compounds are termed lacosamide AB&CR agents. The AB moiety leads to permanent capture of the binding protein while the bioorthogonal CR unit is used for either detection or isolation of the complex upon reaction with a probe. The understanding of lacosamide's mechanism(s) of action will help increase our understanding of epileptic disorders, and permit the rational development of new clinical agents. In the first part of our study, we explored the structure-activity relationship (SAR) for the 3-oxy site in lacosamide. We showed that incorporation of non-bulky, hydrophobic groups at this site provided lacosamide derivatives with excellent activities in animal seizure models. This information was used to design and stereospecifically synthesize a series of lacosamide AB&CR agents where either the AB or the CR group was installed at the 3-oxy site. Most lacosamide AB&CR agents were evaluated for anticonvulsant activity in animal models. In the second part of our study, the lacosamide AB&CR agents were utilized to interrogate the rat brain soluble and membrane-bound proteome for potential binding partners of lacosamide. We used several subcellular fractionation methods to deconvolute the rat brain proteome. Within each subcellular fraction, different protein purification methods were employed to partition the lysate and aid the identification process. Several potential proteins were selectively targeted by the lacosamide AB&CR agents. Further analysis did not confirm that these proteins were directly linked to lacosamide function. These studies documented the strengths and limitations of the AB&CR strategy for receptor identification and are discussed

Synthesis and anticonvulsant activities of N-benzyl (2R)-2-acetamido-3-oxysubstituted propionamide derivatives

Lacosamide has been submitted for regulatory approval in the United States and Europe for the treatment of epilepsy. Previous synthetic methods did not permit the elaboration of the structure–activity relationship (SAR) for the 3-oxy site in lacosamide. We report an expedient five-step stereospecific synthesis for N-benzyl (2R)-2-acetamido-3-oxysubstituted propionamide analogs beginning with d-serine methyl ester. The procedure incorporated alkyl (e.g. methyl, primary, secondary, and tertiary) and aryl groups at this position. The SAR for the 3-oxy site showed maximal activity in animal seizure models for small 3-alkoxy substituents

The Structure−Activity Relationship of the 3-Oxy Site in the Anticonvulsant ( R )- N -Benzyl 2-Acetamido-3-methoxypropionamide

Lacosamide ((R)-N-benzyl 2-acetamido-3-methoxypropionamide, (R)-1) is a low molecular weight anticonvulsant recently introduced in the United States and Europe for adjuvant treatment of partial-onset seizures in adults. In this study, we define the structure-activity relationship (SAR) for the compound's 3-oxy site. Placement of small non-polar, non-bulky substituents at the 3-oxy site provided compounds with pronounced seizure protection in the maximal electroshock (MES) seizure test with activities similar to (R)-1. The anticonvulsant activity loss that accompanied introduction of larger moieties at the 3-oxy site in (R)-1 was offset, in part, by including unsaturated groups at this position. Our findings were similar to a recently reported SAR study of the 4′-benzylamide site in (R)-1 (J. Med. Chem.2010, 53, 1288–1305). Together, these results indicate that both the 3-oxy and 4′-benzylamide positions in (R)-1 can accommodate non-bulky, hydrophobic groups and still retain pronounced anticonvulsant activities in rodents in the MES seizure model

Synthesis and Anticonvulsant Activities of ( R )- N -(4′-Substituted)benzyl 2-Acetamido-3-methoxypropionamides

The structure-activity relationship (SAR) for the N-benzyl group in the clinical antiepileptic agent (R)-lacosamide ((R)-N-benzyl 2-acetamido-3-methoxypropionamide, (R)-3) has been explored. Forty-three compounds were prepared and then evaluated at the National Institute of Neurological Disorders and Stroke Anticonvulsant Screening Program for seizure protection in the maximal electroshock (MES) and subcutaneous Metrazol models. Comparing activities for two series of substituted aryl regioisomers (2′, 3′, 4′) showed that 4′-modified derivatives had the highest activity. Significantly, structural latitude existed at the 4′-site. The SAR indicated that non-bulky 4′-substituted (R)-3 derivatives exhibited superb activity, independent of their electronic properties. Activities in the MES test of several compounds were either comparable with or exceeded that of (R)-3, and surpassed the activities observed for the traditional antiepileptic agents phenytoin, phenobarbital, and valproate

DNA display of fragment pairs as a tool for the discovery of novel biologically active small molecules

Fragment-based lead discovery has proven to be a powerful method in the drug discovery process. The combinatorial output that is accessible by combining fragments is very attractive; however, identifying fragment pairs that bind synergistically and linking them productively can be challenging. Several technologies have now been established to prepare and screen nucleic acid-encoded libraries (ssDNA, dsDNA, PNA), and it has been shown that pairs of molecules combined by hybridization can bind synergistically to a target. Herein we apply this concept to combinatorially pair two libraries of small molecule fragments, use the fittest fragments supplemented with closely related analogs to build a focused library covalently linking the fragments with different spacers, and apply this strategy to the discovery of a potent ligand for Hsp70

Novel PTP1B inhibitors identified by DNA display of fragment pairs

DNA display of PNA-encoded libraries was used to pair fragments containing different phosphotyrosine surrogates with diverse triazoles. Microarray-based screening of the combinatorially paired fragment sets (62,500 combinations) against a prototypical phosphatase, PTP1B, was used to identify the fittest fragments. A focused library (10,000 members) covalently pairing identified fragments with linkers of different length and geometry was synthesized. Screening of the focused library against PTP1B and closely related TCPTP revealed orthogonal inhibitors. The selectivity of the identified inhibitors for PTP1B versus TCPT was confirmed by enzymatic inhibition assay

Lacosamide Isothiocyanate-Based Agents: Novel Agents To Target and Identify Lacosamide Receptors

(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 analogs, 3, that 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