Design, Synthesis and Characterization of Covalent KDM5 Inhibitors.

Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub-family. The covalent binding to the targeted proteins was confirmed by MS and time-dependent inhibition. Additional competition assays show that compounds were non 2-OG competitive. Target engagement and ChIP-seq analysis showed that the compounds inhibited the KDM5 members in cells at nano- to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.

Intracellular Metabolomics Identifies Efflux Transporter Inhibitors in a Routine Caco-2 Cell Permeability Assay-Biological Implications.

Caco-2 screens are routinely used in laboratories to measure the permeability of compounds and can identify substrates of efflux transporters. In this study, we hypothesized that efflux transporter inhibition of a compound can be predicted by an intracellular metabolic signature in Caco-2 cells in the assay used to test intestinal permeability. Using selective inhibitors and transporter knock-out (KO) cells and a targeted Liquid Chromatography tandem Mass Spectrometry (LC-MS) method, we identified 11 metabolites increased in cells with depleted P-glycoprotein (Pgp) activity. Four metabolites were altered with Breast Cancer Resistance (BCRP) inhibition and nine metabolites were identified in the Multidrug Drug Resistance Protein 2 (MRP2) signature. A scoring system was created that could discriminate among the three transporters and validated with additional inhibitors. Pgp and MRP2 substrates did not score as inhibitors. In contrast, BCRP substrates and inhibitors showed a similar intracellular metabolomic signature. Network analysis of signature metabolites led us to investigate changes of enzymes in one-carbon metabolism (folate and methionine cycles). Our data shows that methylenetetrahydrofolate reductase (MTHFR) protein levels increased with Pgp inhibition and Thymidylate synthase (TS) protein levels were reduced with Pgp and MRP2 inhibition. In addition, the methionine cycle is also affected by both Pgp and MRP2 inhibition. In summary, we demonstrated that the routine Caco-2 assay has the potential to identify efflux transporter inhibitors in parallel with substrates in the assays currently used in many DMPK laboratories and that inhibition of efflux transporters has biological consequences.

Repurposing Vandetanib plus Everolimus for the Treatment of ACVR1-Mutant Diffuse Intrinsic Pontine Glioma.

Somatic mutations in ACVR1 are found in a quarter of children with diffuse intrinsic pontine glioma (DIPG), but there are no ACVR1 inhibitors licensed for the disease. Using an artificial intelligence-based platform to search for approved compounds for ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (K d = 150 nmol/L) and reduce DIPG cell viability in vitro but has limited ability to cross the blood-brain barrier. In addition to mTOR, everolimus inhibited ABCG2 (BCRP) and ABCB1 (P-gp) transporters and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination was well tolerated in vivo and significantly extended survival and reduced tumor burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Four patients with ACVR1-mutant DIPG were treated with vandetanib plus an mTOR inhibitor, informing the dosing and toxicity profile of this combination for future clinical studies. SIGNIFICANCE: Twenty-five percent of patients with the incurable brainstem tumor DIPG harbor somatic activating mutations in ACVR1, but there are no approved drugs targeting the receptor. Using artificial intelligence, we identify and validate, both experimentally and clinically, the novel combination of vandetanib and everolimus in these children based on both signaling and pharmacokinetic synergies.This article is highlighted in the In This Issue feature, p. 275.

Computation of the head-related transfer function via the fast multipole accelerated boundary element method and its spherical harmonic representation.

The head-related transfer function (HRTF) is computed using the fast multipole accelerated boundary element method. For efficiency, the HRTF is computed using the reciprocity principle by placing a source at the ear and computing its field. Analysis is presented to modify the boundary value problem accordingly. To compute the HRTF corresponding to different ranges via a single computation, a compact and accurate representation of the HRTF, termed the spherical spectrum, is developed. Computations are reduced to a two stage process, the computation of the spherical spectrum and a subsequent evaluation of the HRTF. This representation allows easy interpolation and range extrapolation of HRTFs. HRTF computations are performed for the range of audible frequencies up to 20 kHz for several models including a sphere, human head models [the Neumann KU-100 ("Fritz") and the Knowles KEMAR ("Kemar") manikins], and head-and-torso model (the Kemar manikin). Comparisons between the different cases are provided. Comparisons with the computational data of other authors and available experimental data are conducted and show satisfactory agreement for the frequencies for which reliable experimental data are available. Results show that, given a good mesh, it is feasible to compute the HRTF over the full audible range on a regular personal computer.

C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-ones: Studies towards the identification of potent, cell penetrant Jumonji C domain containing histone lysine demethylase 4 subfamily (KDM4) inhibitors, compound profiling in cell-based target engagement assays.

Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 µM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggests that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.

8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors.

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.