Chemoproteomics Demonstrates Target Engagement and Exquisite Selectivity of the Clinical Phosphodiesterase 10A Inhibitor MP-10 in Its Native Environment

Abstract

Phosphodiesterases (PDEs) regulate the levels of the second messengers cAMP and cGMP and are important drug targets. PDE10A is highly enriched in medium spiny neurons of the striatum and is an attractive drug target for the treatment of basal ganglia diseases like schizophrenia, Parkinson's disease, or Huntington's disease. Here we describe the design, synthesis, and application of a variety of chemical biology probes, based on the first clinically tested PDE10A inhibitor MP-10, which were used to characterize the chemoproteomic profile of the clinical candidate in its native environment. A clickable photoaffinity probe was used to measure target engagement of MP-10 and revealed differences between whole cell and membrane preparations. Moreover, our results illustrate the importance of the linker design in the creation of functional probes. Biotinylated affinity probes allowed identification of drug-interaction partners in rodent and human tissue and quantitative mass spectrometry analysis revealed highly specific binding of MP-10 to PDE10A with virtually no off-target binding. The profiling of PDE10A chemical biology probes described herein illustrates a strategy by which high affinity inhibitors can be converted into probes for determining selectivity and target engagement of drug candidates in complex biological matrices from native sources.

Unambiguous Identification of β‐Tubulin as the Direct Cellular Target Responsible for the Cytotoxicity of Chalcone by Photoaffinity Labeling

“…These data suggest that this 52 kDa protein may be the direct target responsible for chalcone's cytotoxicity. To further explore this, a set of competitive labeling experiments were performed (Figure B) . In brief, A549 cells were co‐treated with C95 and either cytotoxic chalcone C8 (IC 50 =0.79 μ m ) or non‐cytotoxic chalcone C4 (IC 50 >60 μ m ), both of which are structurally similar to C95 but do not possess the PAL group (Figure ), and this was followed by the standard photoaffinity labeling procedures.…”

“…Our rationale was that if a trypsin‐digested peptide from β‐tubulin was modified by C95, which was not detected in our previous attempts, the relative abundance of this natural peptide detected should then decrease. The data analysis was performed by following the reported label‐free MS quantitation method . Briefly, the extracted ion chromatograms (EIC) of the peptides with high‐confidence MS 2 spectra were obtained from the full MS 1 chromatograms.…”

Phosphodiesterase 10A inhibitor, MP-10 (PF-2545920), produces greater induction of c-Fos in dopamine D2 neurons than in D1 neurons in the neostriatum

“…PDE10A is particularly relevant to the basal ganglia system because it is highly enriched in MSNs (Kleiman et al, 2011;Seeger et al, 2003). MP-10 (also known as PF-2545920; 2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)quinoline) is a potent and selective PDE10A inhibitor (PDE10i) both in vitro and in vivo (Schulke et al, 2014). Treatment with MP-10 increases intracellular concentrations of cAMP/cGMP, augmenting dopamine D2 receptor antagonist-and dopamine D1 receptor agonist-mediated effects (Grauer et al, 2009;Schmidt et al, 2008).…”

Hide and seek: Identification and confirmation of small molecule protein targets

“…For Pfizer's clinical phosphodiesterase 10A (PDE10A) inhibitor MP-10 (PF-2545920) chemical probes were developed to identify non-intended additional targets (''offtargets''), to evaluate selectivity profiles and to prove target engagement in various cellular contexts. 25 Such aspects could potentially reveal deeper insights into the pharmacology of this advanced clinical candidate. Thus, Schülke et al synthesized a set of optimized covalent and non-covalent chemical probes based on the co-crystal structure of PDE10A with MP-10 ( Table 1, entry 4).…”