For many years, D-amino acids were considered minor contributors to biological processes compared with their L-enantiomers. Although free D-amino acids were known to occur in bacterial cell-wall peptidoglycans and in various invertebrates, amphibians, and plants, they were initially thought to be absent in mammals. Over the past two decades, however, several D-amino acids have been identified in mammalian tissues and investigated for their biological roles [1,2]. Among them, D-serine (D-Ser) and D-aspartate (D-Asp) are the most abundant. While D-Ser is well characterized as a neuromodulator in the central nervous system, the role of D-Asp in different cellular contexts has only recently begun to be thoroughly investigated [3,4]. In brain, D-Asp has been demonstrated to act as a co-agonist at the N-methyl D-aspartate receptor (NMDAR) and mGlu5 receptors [5,6], enhancing glutamatergic neurotransmission and influencing synaptic plasticity and cognition [7]. Notably, D-Asp levels in the mammalian brain are transient: they are high during early postnatal development and decrease thereafter [8]. Altered D-Asp metabolism has therefore been linked to neuropsychiatric conditions, including autism spectrum disorder, associated with NMDAR hyperactivation [9], and schizophrenia (SCZ), related to NMDAR hypoactivation [10]. In particular, reduced D-Asp levels have been reported in post-mortem brains of SCZ patients [8,10,11]. Current SCZ treatments involve antipsychotic drugs combined with psychosocial interventions, yet adverse side effects remain significant [12]. Thus, modulating D-amino acid levels, particularly D-Asp, has emerged as a promising alternative strategy to restore NMDAR function.

D-Asp homeostasis depends on a balance between biosynthetic and degradative pathways. Although D-Asp biosynthesis is still unclear, its catabolism is mediated by D-aspartate oxidase (DASPO, EC 1.4.3.1), a peroxisomal flavoprotein that oxidatively deaminates D-Asp to oxaloacetate and ammonia [13]. During catalysis, the flavin adenine dinucleotide (FAD) cofactor of DASPO is reduced to FADH2 and is subsequently reoxidized by molecular oxygen, yielding hydrogen peroxide (Fig. 1A). Structural studies of hDASPO have revealed a stable ∼40 kDa monomer consisting of a FAD-binding domain (FBD) and a substrate-binding domain (SBD), with the active site located at their interface [14]. Key residues, including His54 and Arg237, display conformational flexibility, suggesting a gating mechanism for substrate entry and stabilization (Fig. 1B).

The postnatal decline in brain D-Asp levels is related to progressive increase in DASPO expression [6,15]. Eventually, in the adult brain, D-Asp distribution is inversely correlated with DASPO expression [16]. Notably, the markedly reduced D-Asp levels observed in SCZ patients correlate with upregulated human DASPO (hDASPO) mRNA expression and increased hDASPO enzymatic activity [10,17]. At present times, clinically validated inhibitors of hDASPO do not exist, although several groups have attempted to identify small-molecule modulators. Early efforts focused on carboxylic acid analogues of the natural substrate, such as malonic acid, meso-tartaric acid, and aminooxyacetic acid, which showed modest activity [14,18]. Katane and co-workers later reported thiolactomycin as a mixed-type inhibitor [19] and subsequently identified 5-aminonicotinic acid as a more potent scaffold [20]. Additional chemical series, including amino-thiophenes, quinolines, and triazole–pyrimidines, have also been explored [20]. More recently, olanzapine, a widely used second-generation antipsychotic, was unexpectedly found to inhibit hDASPO (Fig. 2) [21].

Given the limited potency of available hDASPO inhibitors and the lack of clinically viable candidates, we sought an alternative strategy based on Targeted Protein Degradation (TPD) [[22], [23], [24]]. TPD enables modulation of protein function through selective degradation rather than enzymatic inhibition [25]. Among TPD strategies, proteolysis targeting chimeras (PROTACs), heterobifunctional molecules linking a protein-binding ligand to an E3 ubiquitin ligase recruiter, have proven effective in degrading previously undruggable targets through the ubiquitin-proteasome system (UPS) [26]. By inducing formation of a ternary complex between the protein of interest (POI) and the E3 ligase, PROTACs promote POI ubiquitination and subsequent proteasomal degradation. Because degradation is “event-driven,” PROTACs do not require high-affinity inhibition or sustained binding, allowing catalytic turnover of multiple POI molecules [27].

We previously demonstrated that hDASPO is a highly stable protein (estimated half-life of approx. 48 h), degraded through the UPS [28]. In vitro and cellular studies revealed that hDASPO is ubiquitinated, thus it should be retro-translocated to the cytosol for degradation: peroxisomal matrix proteins may be exported to cytosol and thus can be degraded by the proteasome [29]. Furthermore, proteins destined for the peroxisome are synthesized on cytosolic free ribosomes and transported post-translationally by means of two types of peroxisomal targeting signals and the association to cytosolic receptors [30,31]. Notably, stably folded proteins are substrates for peroxisomal import [32]. In this work, based on the mentioned knowledge, we designed a series of bifunctional compounds targeting the cytosolic fraction of hDASPO (i.e., the neo-synthesized and/or the retrotranslocated proteins), selecting suitable POI ligands, E3 ligase recruiters and linkers. The resulting molecules were synthesized and evaluated for binding affinity and degradation efficiency through biochemical and cellular assays. To date, no PROTACs directed against hDASPO, or against any peroxisomal enzyme, have been reported. Here, we present the first bifunctional degrader, PROTAC 17, capable of reducing hDASPO abundance through UPS-mediated degradation. This result not only demonstrates for the first time that a peroxisomal enzyme can be successfully targeted by TPD, but also expands the conceptual boundaries of PROTAC technology, opening the way to the modulation of previously inaccessible metabolic pathways.