Abstract B258: Investigating PERK biological pathway using protein/peptide microarrays and SAR with small molecule inhibitors

Abstract Accumulation of unfolded proteins in the endoplasmic occurs when the cell is subject to stress caused by various pathological conditions such as hypoxia, viral infection, and glucose depravation. Under such stress the cell will initiate an unfolded protein response (UPR), a protective mechanism that is specifically designed to re-establish homeostasis and normal endoplasmic reticulum (ER) function. This adaptive mechanism inhibits overall protein translation, but enhances the translation of a small number of key stress response proteins that will clear the ER of unfolded proteins and send them to the cytoplasm for degradation. The UPR is initiated by several proteins such as IRE1a, ATF6a and PERK, the later being a key Ser/Thr protein kinase in UPR signaling. In response to inducers of ER stress, BiP (GRP78) dissociates from the luminal ER domain of PERK, resulting in the oligomerization, autophosphorylation, and activation of PERK which in turn phosphorylates eIF-2a on Ser51 and Nrf2 (unknown site of phosphorylation). In order to further identify potential substrates for PERK, this enzyme was tested against large protein and peptide microarrays. A significant number of proteins and peptides in the microarrays were found to be phosphorylated by PERK. After additional selection, several of these substrates were further characterized using a microfluidic mobility-shift assay and submitted to LC/MS analysis to identify the site of phosphorylation by PERK. These newly identified peptide substrates were used to develop a robust biochemical assay for the testing of small molecule inhibitors of PERK activity. A limited SAR was established for a set of compounds against PERK and two other related protein kinases, GCN2 and PKR. One of the most potent and selective PERK inhibitors was found to modulate PERK cellular signaling as evidenced by blocking the phosphorylation of (Ser51)eIF-2a as judged by Elisa and Western blot analysis. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B258.</jats:p

Engineering of an isolated p110α subunit of PI3Kα permits crystallization and provides a platform for structure-based drug design

PI3Kα remains an attractive target for the development of anticancer targeted therapy. A number of p110α crystal structures in complex with the nSH2-iSH2 fragment of p85 regulatory subunit have been reported, including a few small molecule co-crystal structures, but the utilization of this crystal form is limited by low diffraction resolution and a crystal packing artifact that partially blocks the ATP binding site. Taking advantage of recent data on the functional characterization of the lipid binding properties of p110α, we designed a set of novel constructs allowing production of isolated stable p110α subunit missing the Adapter Binding Domain and lacking or featuring a modified C-terminal lipid binding motif. While this protein is not catalytically competent to phosphorylate its substrate PIP2, it retains ligand binding properties as indicated by direct binding studies with a pan-PI3Kα inhibitor. Additionally, we determined apo and PF-04691502 bound crystal structures of the p110α (105-1048) subunit at 2.65 and 2.85 Å, respectively. Comparison of isolated p110α(105-1048) with the p110α/p85 complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p110α for iterative SBDD. Importantly, this crystal form of p110α readily accommodates the binding of noncovalent inhibitor by means of a fully accessible ATP site. The strategy presented here can be also applied to structural studies of other members of PI3KIA family

Abstract 2327: Structural and kinetic characterization of crizotinib with wild-type and mutant anaplastic lymphoma kinase

Abstract Dysregulation of Anaplastic Lymphoma Kinase (ALK), primarily through gene translocations, has been shown to be involved in a variety of cancers. Crizotinib, an orally available small molecule inhibitor of the ALK tyrosine kinase has demonstrated marked efficacy in clinical trials of NSCLC patients harboring the EML4-ALK oncogenic gene rearrangement. Mutation of some residues within the ALK kinase domain have been reported to confer acquired or de novo resistance to crizotinib. To understand the binding of crizotinib to ALK and the mechanism of resistance of specific mutations we generated and kinetically characterized wild-type (WT) and mutant ALK kinase domains (KD). Additionally, ALK KD crystal structures were determined of the WT nonphosphorylated apoenzyme and complexes with crizotinib bound to WT and a L1196M gatekeeper mutation. No large protein conformational changes are necessary for crizotinib to bind to unliganded ALK. The interactions which crizotinib makes with ALK are similar to its binding to c-Met with the exception of notable differences in the position of the activation loop between ALK and c-Met. Mutation of the L1196 gatekeeper residue to methionine results in a ∼8-fold increase in catalytic efficiency of phosphorylation of an activation loop peptide and also more rapid enzyme auto-phosphorylation. In addition, inhibition of L1196M ALK by crizotinib was reduced ∼9-fold, compared to wild-type enzyme, from Ki determinations. The crystal structures show that L1196 or M1196 make direct contact with crizotinib. The diminished activity of crizotinib against L1196M ALK is therefore likely due to both higher intrinsic kinase activity and a subtle change in the ALK-crizotinib binding interactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2327. doi:10.1158/1538-7445.AM2011-2327</jats:p

Design, Synthesis and In Vitro

In order to find novel cyclooxygenase (COX)-2 inhibitors for treating inflammatory-based diseases such as Alzheimer's disease (AD), an ethyl carboxylate side chain was added to 5-(4-chlorophenyl)-6-(4-(methylsulfonyl)phenyl)-3-(methylthio)-1,2,4-triazine (lead compound II) to maintain residual inhibition of COX-1 through interacting with Arg120. A preliminary molecular docking study on both the COX-1/COX-2 active sites truly confirmed our hypothesis. Accordingly, a series of ethyl 5,6-diaryl-1,2,4-triazine-3-ylthioacetate derivatives were synthesized and their chemical structures were confirmed by NMR, IR and MS spectra. Further in vitro COX-1/COX-2 evaluations revealed that compound 6c (COX-2 IC50=10.1M, COX-1 IC50=88.8M) is the most selective COX-2 inhibitor while maintaining residual inhibition of COX-1. In order to evaluate their potential use against AD, an in vitro evaluation of -amyloid fibril formation was performed. The results indicated that the prototype compounds 6 are effective -amyloid destabilizing agents while compound 6c could inhibit 94% of the -amyloid fibril formation after 48h. Finally, the in silico assessment results of their blood-brain barrier permeability were satisfactory