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

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

MAP4K4 Is a Threonine Kinase That Phosphorylates FARP1

Mitogen-activated protein kinase 4 (MAP4K4) regulates the MEK kinase cascade and is implicated in cytoskeletal rearrangement and migration; however, identifying MAP4K4 substrates has remained a challenge. To ascertain MAP4K4-dependent phosphorylation events, we combined phosphoproteomic studies of MAP4K4 inhibition with <i>in vitro</i> assessment of its kinase specificity. We identified 235 phosphosites affected by MAP4K4 inhibition in cells and found that pTP and pSP motifs were predominant among them. In contrast, <i>in vitro</i> assessment of kinase specificity showed that MAP4K4 favors a pTL motif. We showed that MAP4K4 directly phosphorylates and coimmunoprecipitates with FERM, RhoGEF, and pleckstrin domain-containing protein 1 (FARP1). MAP4K4 inhibition in SH-SY5Y cells increases neurite outgrowth, a process known to involve FARP1. As FARP1 and MAP4K4 both contribute to cytoskeletal rearrangement, the results suggest that MAP4K4 exerts some of its effects on the cytoskeleton via phosphorylation of FARP1

Deconstruction of Activity-Dependent Covalent Modification of Heme in Human Neutrophil Myeloperoxidase by Multistage Mass Spectrometry (MS⁴)

Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1<i>H</i>-purin-6­(9<i>H</i>)-one (<b>1</b>), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by <b>1</b> in a labile peptide–heme–peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS⁴) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that <b>1</b> was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of <b>1</b>. The final orientation of <b>1</b> perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of <b>1</b> by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain

PF-06446846 targets the human ribosome, inducing stalling during proprotein convertase subtilisin/kexin type 9 (PCSK9) translation.

<p>(A) Structure of PF-06446846. (B) Luciferase activity of HeLa-based cell-free translation reactions programmed with mRNAs encoding PCSK9-luciferase, PCSK9(1–35)-luciferase, and PCSK9(1–33)-luciferase fusions and luciferase alone in the absence (black bars) or presence (grey bars) of 50 μM PF-06446846. All error bars represent one standard deviation of three replicates. (C) PF-06446846 sensitivity dependence on the amino acid sequence of PCSK9(1–33). PCSK9-luciferase fusions encode the native PCSK9 amino acid sequence with common codons or rare codons or a native, double-frameshifted mRNA sequence that results in a changed amino acid sequence (See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#pbio.2001882.s001" target="_blank">S1E Fig</a> for sequences). All error bars represent one standard deviation of three replicates. (D) ³H-PF-06446846 binding to purified human ribosomes, K_d: 7.0 μM (95% CI: 5.5–8.4) B_max: 28.7 pmol/mg (95% CI: 26.5–30.8). The symbols within the graph represent the individual measurements obtained from three independent experiments. B_max and K_d values were calculated using GraphPad PRISM, in which the complete (<i>n</i> = 3) dataset was fit to the one site-specific binding equation. (E–F) Electrophoreograms of toeprints of stalled ribosomes on the (E) PCSK9(1–35)-luciferase fusion construct and (F) full-length PCSK9-luciferase fusion. The nucleotide (nt) positions from the “A” of the ATG initiation codon of the first and last of the group of toeprinting peaks are indicated. The expected position of the P-site of the stalled ribosome from ribosome profiling is also indicated. (G) Schematic of ribosomal toeprinting assays. 5ʹ 6-FAM labeled primers are extended by reverse transcriptase, which terminates when blocked by a ribosome. In this case, we also hypothesize that additional factors may be bound to the stalled ribosome, obstructing the reverse transcriptase at more 3ʹ positions and over a broader range of positions then what is normally observed [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#pbio.2001882.ref013" target="_blank">13</a>]. (H) Sucrose density gradient profiles of cell-free translation reactions programmed with an mRNA encoding an N-terminally extended PCSK9 in the presence of 100 μM PF-06446846 (grey) and vehicle (blue). (I) Tris-Tricine SDS-PAGE gels showing ³⁵S-Met-labelled peptides that sediment in the polysome region of the gradient. (J) Model of the species isolated by density gradient centrifugation containing one stalled ribosome and two queued ribosomes. The individual quantitative observations that underlie Fig 1B–D are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#pbio.2001882.s029" target="_blank">S14 Table</a>.</p

Features of PF-06446846–sensitive transcripts.

<p>(A) Changes in the mean read position or center of density [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#pbio.2001882.ref022" target="_blank">22</a>] plotted against expression levels. Genes with a local Z-score greater than 3 are highlighted in red. (B–D) Center-of-density analysis with first (B) 50, (C) 100, and (D) 150 codons omitted, showing that stalling preferentially occurs before codon 50. (E) Expression changes for PF-06446846–sensitive transcripts occur at the step of translation. Plot of Z-score–transformed [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#pbio.2001882.ref022" target="_blank">22</a>] read counts for mRNA-seq and ribo-seq. PF-06446846–sensitive nascent chains are highlighted in red. (F) Alignment of PF-06446846–sensitive sequences. The sequences are aligned according to the stall position, and the residues predicted to reside in the ribosome exit tunnel, P-site, and A-site are indicated.</p

Identification and validation of PF-06446846–sensitive nascent chains.

<p>(A) Outline of the approach to identify PF-06446846–targeted mRNAs. (B) Example readplot and (C) example cumulative fractional read (CFR) plot for proprotein convertase subtilisin/kexin type 9 (PCSK9). A CFR plot depicts at each codon the percentage of reads aligning at or 5ʹ to that codon. In all plots, data from 1.5 μM PF-06446846 treatments are shown in red and vehicle treatments are shown in blue. The major stall and the position of D_max is marked. (D) Scatterplot showing the distribution of D_max values as a function of read counts; red indicates D_max Z-score > 3 (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#sec007" target="_blank">Materials and methods</a> for Z-score calculations) and green indicates 2 < Z-score < 3. (E) Scatterplot of fold change versus expression level when reads mapping 3ʹ to D_max position (for Z-score > 2) or codon 50 (for Z-score < 2) are used. Genes for which D_max Z-score > 2 and DeSeq fasle discovery rate (FDR) < 10% are highlighted in red, in green for Dmax Z-score > 2 but FDR > 10%, and in purple for D_max Z-score < 2 with FDR < 10%. (F–I) Example readplots for PF-06446846–sensitive proteins (F) HSD17B11, (G) RPL27, (H) PCBP1, and (I) cadherin-1 (CDH1). Bars representing the treatment dataset are red and go upwards and bars representing the vehicle datasets are blue and go downwards. All graphs are derived from the 1-h treatment time in the first study. (J) Cell-free translation assays showing inhibition of translation by 50 μM PF-06446846 when the stall sites identified by ribosome profiling are fused to the N-terminus of luciferase. (K) Inhibition of in vitro translation of full-length Midikine- and BCAP31-luciferase fusions in the cell-free translation system. (L) In vitro translation of control constructs not predicted to be inhibited by PF-06446846 from cell-based experiments. (M) In vitro translation of constructs with PF-06446846–induced stalls identified only at the 10-min treatment time. The individual quantitative observations that underlie Fig 5J–M are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#pbio.2001882.s029" target="_blank">S14 Table</a>.</p

Oral administration of PF-06446846 reduces plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) and total cholesterol levels in rats.

<p>(A–B) Plasma PCSK9 levels following (A) a single and (B) 12 daily oral doses of PF-06446848. Rats were administered the indicated dose of PF-06446846, and plasma concentrations of PCSK9 were measured by commercial ELISA at 1, 3, 6, and 24 h after dosing (A) or the 12th daily dose (B). Symbols represent mean concentration ± standard error and were jittered to provide a clearer graphical representation. Data were analyzed using a mixed model repeated measure (MMRM) with treatment, day, and hour as fixed factors; treatment by day and hour as an interaction term; and animal as a random factor. The significance level was set at a level of 5%. No adjustment for multiple comparisons was used. *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001. (C–E) Total plasma (C), low-density lipoprotein (LDL) (D), and high-density lipoprotein (HDL) (E) cholesterol levels in rats measured 24 h following 14 daily oral doses of PF-06446846. Symbols represent individual animal values. The middle horizontal bar represents the group mean ± standard deviation. Difference between group means relative to vehicle was performed by a one-way ANOVA followed by a Dunnett’s multiple comparisons test; * <i>p</i> ≤ 0.05, **** <i>p</i> ≤ 0.0001. The individual quantitative observations that underlie Fig 2 are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#pbio.2001882.s029" target="_blank">S14 Table</a>.</p

The PF-06446846–induced stall site is revealed by ribosome profiling.

<p>(A–D) Ribosome footprint density plots displaying the number of reads aligning to a given codon per million total reads for the proprotein convertase subtilisin/kexin type 9 (PCSK9) coding region from Huh7 cells treated for (A) 1 h and (B) 10 min. (C) Ribo-seq datasets from the second study and (D) mRNA-seq datasets from the second study. The upward red bars indicate readmaps from cells treated with 1.5 μM PF-06446846 and the blue downward bars represent vehicle. In panels A–D, read positions are mapped according to inferred location of the ribosome P-site [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#pbio.2001882.ref019" target="_blank">19</a>]. (E) The footprint density downstream from the stall in the 10-min treatment (black) and 1-h treatment (light grey) compared with PCSK9 expression as measured by ELISA (middle grey). Error bars represent one standard deviation of three replicates. The individual quantitative observations that underlie Fig 4E are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#pbio.2001882.s029" target="_blank">S14 Table</a>.</p

Discovery of a Selective Covalent Inhibitor of Lysophospholipase-like 1 (LYPLAL1) as a Tool to Evaluate the Role of this Serine Hydrolase in Metabolism

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization

Discovery of a JAK3-Selective Inhibitor: Functional Differentiation of JAK3-Selective Inhibition over pan-JAK or JAK1-Selective Inhibition

PF-06651600, a newly discovered potent JAK3-selective inhibitor, is highly efficacious at inhibiting γc cytokine signaling, which is dependent on both JAK1 and JAK3. PF-06651600 allowed the comparison of JAK3-selective inhibition to pan-JAK or JAK1-selective inhibition, in relevant immune cells to a level that could not be achieved previously without such potency and selectivity. <i>In vitro</i>, PF-06651600 inhibits Th1 and Th17 cell differentiation and function, and <i>in vivo</i> it reduces disease pathology in rat adjuvant-induced arthritis as well as in mouse experimental autoimmune encephalomyelitis models. Importantly, by sparing JAK1 function, PF-06651600 selectively targets γc cytokine pathways while preserving JAK1-dependent anti-inflammatory signaling such as the IL-10 suppressive functions following LPS treatment in macrophages and the suppression of TNFα and IL-1β production in IL-27-primed macrophages. Thus, JAK3-selective inhibition differentiates from pan-JAK or JAK1 inhibition in various immune cellular responses, which could potentially translate to advantageous clinical outcomes in inflammatory and autoimmune diseases