The L3MBTL3 Methyl-Lysine Reader Domain Functions As a Dimer

L3MBTL3 recognizes mono- and dimethylated lysine residues on histone tails. The recently reported X-ray cocrystal structures of the chemical probe UNC1215 and inhibitor UNC2533 bound to the methyl-lysine reading MBT domains of L3MBTL3 demonstrate a unique and flexible 2:2 dimer mode of recognition. In this study, we describe our <i>in vitro</i> analysis of L3MBTL3 dimerization via its MBT domains and additionally show that this dimerization occurs within a cellular context in the absence of small molecule ligands. Furthermore, mutations to the first and second MBT domains abrogated L3MBTL3 dimerization both <i>in vitro</i> and in cells. These observations are consistent with the hypothesis that L3MBTL3 engages methylated histone tails as a dimer while carrying out its normal function and provides an explanation for the presence of repeated MBT domains within L3MBTL3

The docking poses of UNC10225498 and UNC10112646 with PPIP5K.

<p>(A) UNC10225498 (thick sticks; orange carbons) (B) UNC10112646 (thick sticks; green carbons). The ATP pocket is outlined as a gray transparent surface and ATP itself is depicted by thin magenta sticks. Residues predicted to interact with docked compounds are highlighted (see text for details).</p

A High-Throughput Screening-Compatible Strategy for the Identification of Inositol Pyrophosphate Kinase Inhibitors

<div><p>Pharmacological tools—‘chemical probes’—that intervene in cell signaling cascades are important for complementing genetically-based experimental approaches. Probe development frequently begins with a high-throughput screen (HTS) of a chemical library. Herein, we describe the design, validation, and implementation of the first HTS-compatible strategy against any inositol phosphate kinase. Our target enzyme, PPIP5K, synthesizes ‘high-energy’ inositol pyrophosphates (PP-InsPs), which regulate cell function at the interface between cellular energy metabolism and signal transduction. We optimized a time-resolved, fluorescence resonance energy transfer ADP-assay to record PPIP5K-catalyzed, ATP-driven phosphorylation of 5-InsP₇ to 1,5-InsP₈ in 384-well format (Z’ = 0.82 ± 0.06). We screened a library of 4745 compounds, all anticipated to be membrane-permeant, which are known—or conjectured based on their structures—to target the nucleotide binding site of protein kinases. At a screening concentration of 13 μM, fifteen compounds inhibited PPIP5K >50%. The potency of nine of these hits was confirmed by dose-response analyses. Three of these molecules were selected from different structural clusters for analysis of binding to PPIP5K, using isothermal calorimetry. Acceptable thermograms were obtained for two compounds, UNC10112646 (Kd = 7.30 ± 0.03 μM) and UNC10225498 (Kd = 1.37 ± 0.03 μM). These Kd values lie within the 1–10 μM range generally recognized as suitable for further probe development. <i>In silico</i> docking data rationalizes the difference in affinities. HPLC analysis confirmed that UNC10225498 and UNC10112646 directly inhibit PPIP5K-catalyzed phosphorylation of 5-InsP₇ to 1,5-InsP₈; kinetic experiments showed inhibition to be competitive with ATP. No other biological activity has previously been ascribed to either UNC10225498 or UNC10112646; moreover, at 10 μM, neither compound inhibits IP6K2, a structurally-unrelated PP-InsP kinase. Our screening strategy may be generally applicable to inhibitor discovery campaigns for other inositol phosphate kinases.</p></div

Structures and dose-response relationships for three inhibitors of PPIP5Ks.

<p>Structures for (A) UNC10112646 (B) UNC10225354 and (C) UNC10225498 (D) Dose-response curves for the inhibition of PPIP5K by UNC10225354 (IC₅₀ = 5.24 ± 0.18 μM), UNC10225498 (IC₅₀ = 2.14 ± 0.07 μM), and UNC10112646 (IC₅₀ = 6.96 ± 0.03 μM). (E) Counterscreen results for the three inhibitors performed in the absence of PPIP5K and 5-InsP₇ show that these inhibitors do not interfere with the detection reagents and assay signal. In these experiments, 100% activity is equivalent to consumption of 19.5 ± 0.8% of the ATP.</p

Analysis of the mechanism of inhibition of PPIP5K by UNC10112646 and UNC10225498.

<p>Assays were performed in HTS format with 2-fold serial dilutions from 100 μM of either UNC10112646 (squares) and UNC10225498 (circles) and varying concentrations of ATP as indicated. Data represent means and standard errors from 3 experiments. The mean Ki values for inhibition of PPIP5K by UNC10225498 and UNC10112646 were 2.0 ± 0.6 μM and 3.6 ± 1.3 μM, respectively. In these experiments, the uninhibited PPIP5K activity is equivalent to consumption of 18.9 ± 0.9% of the ATP.</p

HPLC analysis of the effects of UNC10225498 and UNC10112646 upon the kinase activities of PPIP5K and IP6K.

<p>Kinase reactions contained either vehicle (0.5% DMSO) or inhibitor in 0.5% DMSO. Reactions were quenched and analyzed by HPLC as described in the methods section. Representative HPLC data are shown for both (A) PPIP5K and (C) IP6K, including no enzyme control (open circles), vehicle control (closed circles), 10 μM UNC10225498 (‘5498’, light gray circles), or UNC10112646 (‘2646’, dark gray circles). Panels B and D show the means and SEM for the percentage of product formed in 3 experiments.</p

Steps of the PPIP5K HTS Assay.

<p>Steps of the PPIP5K HTS Assay.</p

HTS of PPIP5K against a kinase-focused library of potential nucleotide antagonists.

<p>(A) Representative technical replicates measured on the same day. (B) Comparison of the mean values of biological replicates (black and white circles) obtained on two different days. R² = 0.99. (C) Z’ Factor (0.82 ± 0.06) (D) %CV (8.6 ± 1.3) (E) Signal:background ratio (16.4 ± 1.1) (F) These plates included negative controls (0.5% DMSO; gray circle, broken line) and positive controls (20 μM UNC10225354; gray square, broken line). Each plate also contained one 10-point titration for UNC10225354; data (black circles) depict means and SEMs (n = 5). IC₅₀ = 5.2 ± 0.2 μM. In these experiments, 100% activity is equivalent to consumption of 18.9 ± 1.5% of the ATP.</p

Schematic depicting the three phases of the HTS Assay for PPIP5K.

<p>The schematic describes how the degree of PPIP5K activity is inversely proportional to the magnitude of the HTRF signal. During the kinase reaction 5-InsP₇ phosphorylation to 1,5-InsP₈ is coupled to ATP conversion to ADP. After 60 minutes the kinase reactions are quenched with EDTA (not shown) and the ADP detection reagents are added. The HTRF signal is measured after another 30 minutes. (A) In the absence of inhibitor there is production of ADP, which competes with the ADP tracer for the ADP antibody, resulting in a low HTRF signal. (B) In the presence of inhibitor, ADP production is decreased thereby allowing ADP tracer to bind to the ADP antibody, resulting in a high HTRF signal. The hypothetical examples shown in (A) and (B) represent two extreme assay outcomes of 100% and 0% phosphorylation respectively.</p

Analysis by ITC of the interaction of UNC10225498 and UNC10112646 with PPIP5K The upper panels show the raw data for heat output from the ligand/protein titrations; the lower panels show the least squares fitting of the titration data assuming a single site binding model.

<p>(A) UNC10225498; Kd = 1.37 ± 0.03 μM (B) UNC10112646; Kd = 7.30 ± 0.03 μM. Representative data are shown. Kd values represent means and standard deviations from two experiments</p