Expression, Purification and Characterization of Inactive and Active Forms of ERK2 from Insect Expression System

Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies

Structural and biophysical comparisons of the pomalidomide- and CC-220-induced interactions of SALL4 with cereblon.

The design of cereblon-binding molecular glues (MGs) that selectively recruit a desired protein while excluding teratogenic SALL4 is an area of significant interest when designing therapeutic agents. Previous studies show that SALL4 is degraded in the presence of IKZF1 degraders pomalidomide, and to a lesser extent by CC-220. To expand our understanding of the molecular basis for the interaction of SALL4 with cereblon, we performed biophysical and structural studies demonstrating that SALL4 zinc finger domains one and two (ZF1-2) interact with cereblon (CRBN) in a unique manner. ZF1 interacts with the N-terminal domain of cereblon and ZF2 binds as expected in the C-terminal IMiD-binding domain. Both ZF1 and ZF2 contribute to the potency of the interaction of ZF1-2 with CRBN:MG complexes and the affinities of SALL4 ZF1-2 for the cereblon:CC-220 complex are less potent than for the corresponding pomalidomide complex. Structural analysis provides a rationale for understanding the reduced affinity of SALL4 for cereblon in the presence of CC-220, which engages both ZF1 and ZF2. These studies further our understanding of the molecular glue-mediated interactions of zinc finger-based proteins with cereblon and may provide structural tools for the prospective design of compounds with reduced binding and degradation of SALL4

Structural and biophysical comparisons of the pomalidomide- and CC-220-induced interactions of SALL4 with cereblon

The design of cereblon-binding molecular glues (MGs) that selectively recruit a desired protein while excluding teratogenic SALL4 is an area of significant interest when designing therapeutic agents. Previous studies show that SALL4 is degraded in the presence of IKZF1 degraders pomalidomide, and to a lesser extent by CC-220. To expand our understanding of the molecular basis for the interaction of SALL4 with cereblon, we performed biophysical and structural studies demonstrating that SALL4 zinc finger domains one and two (ZF1-2) interact with cereblon (CRBN) in a unique manner. ZF1 interacts with the N-terminal domain of cereblon and ZF2 binds as expected in the C-terminal IMiD-binding domain. Both ZF1 and ZF2 contribute to the potency of the interaction of ZF1-2 with CRBN:MG complexes and the affinities of SALL4 ZF1-2 for the cereblon:CC-220 complex are less potent than for the corresponding pomalidomide complex. Structural analysis provides a rationale for understanding the reduced affinity of SALL4 for cereblon in the presence of CC-220, which engages both ZF1 and ZF2. These studies further our understanding of the molecular glue-mediated interactions of zinc finger-based proteins with cereblon and may provide structural tools for the prospective design of compounds with reduced binding and degradation of SALL4

Rationally Designed PI3Kα Mutants to Mimic ATR and Their Use to Understand Binding Specificity of ATR Inhibitors

ATR, a protein kinase in the PIKK family, plays a critical role in the cell DNA-damage response and is an attractive anticancer drug target. Several potent and selective inhibitors of ATR have been reported showing significant antitumor efficacy, with most advanced ones entering clinical trials. However, due to the absence of an experimental ATR structure, the determinants contributing to ATR inhibitors' potency and specificity are not well understood. Here we present the mutations in the ATP-binding site of PI3Kα to progressively transform the pocket to mimic that of ATR. The generated PI3Kα mutants exhibit significantly improved affinity for selective ATR inhibitors in multiple chemical classes. Furthermore, we obtained the X-ray structures of the PI3Kα mutants in complex with the ATR inhibitors. The crystal structures together with the analysis on the inhibitor affinity profile elucidate the roles of individual amino acid residues in the binding of ATR inhibitors, offering key insights for the binding mechanism and revealing the structure features important for the specificity of ATR inhibitors. The ability to obtain structural and binding data for these PI3Kα mutants, together with their ATR-like inhibitor binding profiles, makes these chimeric PI3Kα proteins valuable model systems for structure-based inhibitor design

LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors

Purpose: Targeting RAF for anti-tumor therapy in RAS-mutant tumors holds promise. Herein we describe in detail novel properties of the type II RAF inhibitor LXH254. Experimental Design: LXH254 was profiled in biochemical, in vitro, and in vivo assays including examining the activities of the drug in a large panel of cancer-derived cell lines, and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF. Results: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations co-expressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS mutant lines loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Lastly, in vivo, LXH254 drove complete regressions of isogenic variants of RAS mutant cells lacking ARAF expression, while parental lines were only modestly sensitive. Conclusions: LXH254 is a novel RAF-inhibitor able to inhibit dimerized BRAF and CRAF as well as monomeric BRAF while largely sparing ARAF

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

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

Cellular activity of ligands on signaling in KRAS and BRAF mutant tumor cells.

<p>Dose-response data for all compounds from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174706#pone.0174706.g003" target="_blank">Fig 3</a> on SW1990 cells (A) and A375 cells (B), assessing impact on pAKT and pERK, together with their total protein controls and a GAPDH control.</p

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