¹⁹F‑NMR-Based Dual-Site Reporter Assay for the Discovery and Distinction of Catalytic and Allosteric Kinase Inhibitors

In modern kinase drug discovery, allosteric inhibitors have become a focus of attention due to their potential selectivity, but such compounds are difficult to identify. Here we describe an NMR-based competition assay using ¹⁹F-containing reporter molecules, which allows for rapid identification and discrimination between ATP-competitive and allosteric kinase inhibitors. We illustrate the principle of such a dual-site competition assay with the example of catalytic and allosteric ABL1 kinase inhibitors. The assay can also be used to identify and characterize mixed binding modes of well-known drugs, as shown for crizotinib and fingolimod

Intrinsically Disordered Regions in the Transcription Factor MYC:MAX Modulate DNA Binding via Intramolecular Interactions

The basic helix–loop–helix leucine zipper (bHLH-LZ) transcription factor (TF) MYC is in large part an intrinsically disordered oncoprotein. In complex with its obligate heterodimerization partner MAX, MYC preferentially binds E-Box DNA sequences (CANNTG). At promoters containing these sequence motifs, MYC controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. A vast network of proteins in turn regulates MYC function via intermolecular interactions. In this work, we establish another layer of MYC regulation by intramolecular interactions. We used nuclear magnetic resonance (NMR) spectroscopy to identify and map multiple binding sites for the C-terminal MYC:MAX DNA-binding domain (DBD) on the intrinsically disordered regions (IDRs) in the MYC N-terminus. We find that these binding events in trans are driven by electrostatic attraction, that they have distinct affinities, and that they are competitive with DNA binding. Thereby, we observe the strongest effects for the N-terminal MYC box 0 (Mb0), a conserved motif involved in MYC transactivation and target gene induction. We prepared recombinant full-length MYC:MAX complex and demonstrate that the interactions identified in this work are also relevant in cis, i.e., as intramolecular interactions. These findings are supported by surface plasmon resonance (SPR) experiments, which revealed that intramolecular IDR:DBD interactions in MYC decelerate the association of MYC:MAX complexes to DNA. Our work offers new insights into how bHLH-LZ TFs are regulated by intramolecular interactions, which open up new possibilities for drug discovery

Identification of Two Secondary Ligand Binding Sites in 14-3‑3 Proteins Using Fragment Screening

Proteins typically interact with multiple binding partners, and often different parts of their surfaces are employed to establish these protein–protein interactions (PPIs). Members of the class of 14-3-3 adapter proteins bind to several hundred other proteins in the cell. Multiple small molecules for the modulation of 14-3-3 PPIs have been disclosed; however, they all target the conserved phosphopeptide binding channel, so that selectivity is difficult to achieve. Here we report on the discovery of two individual secondary binding sites that have been identified by combining nuclear magnetic resonance-based fragment screening and X-ray crystallography. The two pockets that these fragments occupy are part of at least three physiologically relevant and structurally characterized 14-3-3 PPI interfaces, including those with serotonin <i>N</i>-acetyltransferase and plant transcription factor FT. In addition, the high degree of conservation of the two sites implies their relevance for 14-3-3 PPIs. This first identification of secondary sites on 14-3-3 proteins bound by small molecule ligands might facilitate the development of new chemical tool compounds for more selective PPI modulation

Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor

The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit <b>1</b>, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as <b>2</b> and <b>3</b>. Compound <b>2</b> was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key interactions. Compound <b>3</b> modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate biological functions of the PAK1 kinase

Inhibition of prenylated KRAS in a lipid environment

<div><p>RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRAS^G12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRAS^G12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRAS^G12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the <i>in vitro</i> assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.</p></div

BLI experiments assess dependence of K_d on prenylation, PS, and HVR.

<p>(A) Response data at various concentrations of compound <b>3</b> with different protein preparations (with or without prenylation, with or without PS). Error bars represent assay method variability (three standard deviations for buffer). (B) Dose-response data for compounds <b>3</b> and <b>4</b> with prenylated HVR (light blue & yellow) and with full-length prenylated KRAS^G12V (dark blue & orange); all in PS. Error bars represent assay method variability (three standard deviations for buffer).</p

Chemical structures of compounds 1–6.

<p>Chemical structures of compounds 1–6.</p

Coupled KRAS-BRAF-MEK assay recapitulates known KRAS biology.

<p>(A) Schematic of the assay components: GTP-loaded full-length, prenylated KRAS^G12V (purple); full-length BRAF (red); biotinylated MEK1^K97R (blue); phosphatidylserine (negatively-charged lipid head-groups in green). Readout happens at the pMEK level using alphascreen technology with streptavidin donor beads, protein A acceptor beads, and an anti-phospho-MEK antibody. (B) AlphaScreen response units measuring phospho-MEK levels resulting from KRAS activation of BRAF in the presence of different phospholipids; the KRAS preparation is wildtype, prenylated protein, loaded with GTPγS. (C) AlphaScreen response units measuring phospho-MEK levels resulting from KRAS activation of BRAF, assessing dependence of the coupled assay on nucleotide, prenylation, PS, and KRAS mutation status (WT = wildtype); nucleotides are GDPβS and GTPγS, non-hydrolyzable analogues of GDP and GTP respectively. (D) Dose-response curve for titrating the RAS-Binding domain of CRAF in a coupled assay with GTPγS-loaded, prenylated KRAS^G12V in the presence of PS; IC₅₀ = 0.013 μM ± 0.002 μM (geomean ± standard deviation, N = 4) (E) Correlation plot of IC₅₀ values in the BRAF^V600E counter-screen <i>vs</i> IC₅₀ values in the coupled assay (with GTPγS-loaded, prenylated KRAS^G12V in the presence of PS) for a diverse subset of RAF inhibitors from historical programs, including (in yellow circle) compound C from reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174706#pone.0174706.ref033" target="_blank">33</a>]; magenta are Type-I inhibitors and blue are Type-II inhibitors. R² of the regression line is 0.83.</p

NMR experiments probe mechanism and binding site.

<p>(A) ³¹P-NMR spectrum of non-prenylated GMPPNP-loaded KRAS^G12V shows two environments for the γ-phosphate (top) with a shift to a single inactive population with 1 mM compound <b>2</b> (middle) and a single active population with 0.5 mM of the CRAF-RBD (bottom); α and β phosphate have single environments and the KRAS^G12V is present at 5 mg/ml. (B) ¹⁵N,¹H-HSQC spectrum for non-prenylated GDP-KRAS^G12V in absence (black) or presence (blue) of 400 μM compound <b>2</b> (protein concentration is 0.5 mg/ml). The residues with major chemical shift changes are indicated with circles. (C) Residues engaged in nOe’s between non-prenylated GDP-KRAS^G12V and compound <b>2</b> shown in blue stick model with labels using the crystal structure for compound <b>6</b> (PDB-ID 4EPY) as reference; compound <b>6</b> in orange, GDP and RAS protein in green, switch-I in magenta and switch-II in yellow.</p

Summary of biochemical, cellular, and biophysical measures of potency in μM.

<p>Summary of biochemical, cellular, and biophysical measures of potency in μM.</p