Comparison of panitumumab and cetuximab binding.

<p>(A) Surface plasmon resonance of EGFR binding to panitumumab and cetuximab. Increasing concentrations of EGFR construct (1.23, 3.7, 11.1, 33.3 and 100 nM) were injected over immobilized antibody. In black and blue are the raw binding traces of the two different EGFR constructs and in red is data fit to a 1:1 binding model. (B) No binding was observed for cetuximab and EGFRD3 ND2 (S468R), blue line. (C) Overlay of panitumumab and EGFRD3 ND2 is shown in orange, panitumumab and EGFRD3 ND2(S468R) in magenta and cetuximab and EGFR domain III in cyan. (PDB: 1YY9). Only EGFR domain III (not domains I, II, and IV) is shown in the cetuximab complex for clarity. Panitumumab and cetuximab recognize the same epitope.</p

Comparison of residue 468 binding sites.

<p>(A) Binding site of panitumumab with EGFRD3 ND2. The S468 interaction with D100 is mediated through a water molecule. (B) Binding site of panitumumab with EGFRD3 ND2(S468R). R468 makes hydrogen bond interactions with D100, S440 and the main chain carbonyls of R101 and V102. (C) Binding site of cetuximab with S468 (PDB: 1YY9). Y104 packs against the S468 position and would not tolerate a large side chain in that position. (D) Model of EGFR S468R bound to cetuximab. The EGFR S468R mutation would severely clash with Y104. Model created by overlaying EGFRD3 ND2(S468R) onto EGFR domain III in the cetuximab structure without further refinement.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Paratope surfaces and CDR interactions.

<p>(A) Electrostatic surface of the CDR region of panitumumab. Large negatively charged cavity in center of CDR regions is clearly visible. (B) Electrostatic surface of CDR region for cetuximab reveals that Y104 fills the central cavity. (C) Light chain CDR interactions with the final β-strand of EGFR domain III. EGFR in tan with L1, L2 and L3 colored gray, purple, and yellow, respectively. (D) Heavy chain CDR interactions with the β-sheet surface of EGFR domain III that binds EGF. EGFR in tan with H1, H2, and H3 in cyan, gold and blue, respectively.</p

EGFR domain III mutants.

<p>(A) Panitumumab epitope on EGFR domain III of EGFR is shown in yellow. EGF binding surface on EGFR domain III is shown in red (PDB:1IVO). Mutations are shown on surface relative to panitumumab epitope and EGF binding interface. EGF and panitumumab have significant overlap in the domain III binding surfaces. S468R as well as many additional mutations are located on the perimeter of the EGF binding site but are centrally located in the panitumumab epitope. (B-E) Panitumumab is shown in magenta and cetuximab is shown in cyan. Red lettering represents mutation and the number in parenthesis represents numbering based on mature protein sequence used in the crystal structure. The remaining residues are numbered to be consistent with crystal structure numbering. (B)The K467T mutation will result in a clash with W94 in cetuximab but rotamer conformations exist that would minimize the steric clash in panitumumab, as a result cetuximab is sensitive to this mutation but panitumumab remains active. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163366#pone.0163366.ref013" target="_blank">13</a>]. (C) I491M would result in steric clashes with D103 and Y102 in cetuximab and F91 and the main chain of T103 in panitumumab. (D) G465R would result in steric clashes with W52 and His35 in cetuximab and H52 and Y35 in panitumumab. (E) S464L is in a tightly packed region and would clash with Y102 and Y104 in cetuximab and V102 and Y35 in panitumumab.</p

Systematic Study of the Glutathione (GSH) Reactivity of <i>N</i>‑Arylacrylamides: 1. Effects of Aryl Substitution

Success in the design of targeted covalent inhibitors depends in part on a knowledge of the factors influencing electrophile reactivity. In an effort to further develop an understanding of structure–reactivity relationships among <i>N</i>-arylacrylamides, we determined glutathione (GSH) reaction rates for a family of <i>N</i>-arylacrylamides independently substituted at ortho-, meta-, and para-positions with 11 different groups common to inhibitor design. We find that substituent effects on reaction rates show a linear Hammett correlation for ortho-, meta-, and para-substitution. In addition, we note a correlation between ¹H and ¹³C NMR chemical shifts of the acrylamide with GSH reaction rates, suggesting that NMR chemical shifts may be a convenient surrogate measure of relative acrylamide reactivity. Density functional theory calculations reveal a correlation between computed activation parameters and experimentally determined reaction rates, validating the use of such methodology for the screening of synthetic candidates in a prospective fashion

Unfolded Protein Response in Cancer: IRE1α Inhibition by Selective Kinase Ligands Does Not Impair Tumor Cell Viability

The kinase/endonuclease inositol requiring enzyme 1 (IRE1α), one of the sensors of unfolded protein accumulation in the endoplasmic reticulum that triggers the unfolded protein response (UPR), has been investigated as an anticancer target. We identified potent allosteric inhibitors of IRE1α endonuclease activity that bound to the kinase site on the enzyme. Structure–activity relationship (SAR) studies led to <b>16</b> and <b>18</b>, which were selective in kinase screens and were potent against recombinant IRE1α endonuclease as well as cellular IRE1α. The first X-ray crystal structure of a kinase inhibitor (<b>16</b>) bound to hIRE1α was obtained. Screening of native tumor cell lines (>300) against selective IRE1α inhibitors failed to demonstrate any effect on cellular viability. These results suggest that IRE1α activity is not essential for viability in most tumor cell lines, in vitro, and that interfering with the survival functions of the UPR may not be an effective strategy to block tumorigenesis

Oxopyrido[2,3‑<i>d</i>]pyrimidines as Covalent L858R/T790M Mutant Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors

In nonsmall cell lung cancer (NSCLC), the threonine⁷⁹⁰–methionine⁷⁹⁰ (T790M) point mutation of EGFR kinase is one of the leading causes of acquired resistance to the first generation tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Herein, we describe the optimization of a series of 7-oxopyrido­[2,3-<i>d</i>]­pyrimidinyl-derived irreversible inhibitors of EGFR kinase. This led to the discovery of compound <b>24</b> which potently inhibits gefitinib-resistant EGFR^L858R,T790M with 100-fold selectivity over wild-type EGFR. Compound <b>24</b> displays strong antiproliferative activity against the H1975 nonsmall cell lung cancer cell line, the first line mutant HCC827 cell line, and promising antitumor activity in an EGFR^L858R,T790M driven H1975 xenograft model sparing the side effects associated with the inhibition of wild-type EGFR