<i>N</i>‑(7-Cyano-6-(4-fluoro-3-(2-(3-(tri­fluoro­methyl)­phenyl)­acet­amido)­phen­oxy)­benzo­[d]thi­azol-2-yl)­cyclo­pro­pane­carb­ox­amide (TAK632) Promotes Inhibition of BRAF through the Induction of Inhibited Dimers

BRAF^V600E is the most common activating mutation in melanoma and patients treated with BRAF^V600E inhibitors all develop resistance within one year. A significant resistance pathway is paradoxical activation (transactivation) involving BRAF dimers, whereby an inhibitor bound protein subunit allosterically activates the other subunit. We recently reported on dimeric BRAF^V600E -selective vemurafenib inhibitors that stabilize an inactive αC-out/αC-out homodimeric conformation with improved inhibitor potency and selectivity in vitro. We set out to extend this strategy to target RAF homo- and heterodimers with the pan-RAF inhibitor TAK632 in dimeric configuration. Surprisingly, we find that monomeric TAK632 induces an active αC-in/αC-in BRAF dimer conformation, while dimeric TAK inhibitors cannot promote BRAF dimers and have significantly compromised potency in vitro. These studies uncover the intimate connection between BRAF dimerization and TAK632 mode of inhibition and highlight the importance of understanding the impact of BRAF inhibitors on kinase dimerization

Design, Synthesis, and Biological Evaluation of Allosteric Effectors That Enhance CO Release from Carboxyhemoglobin

Carbon monoxide (CO) poisoning causes between 5,000−6,000 deaths per year in the US alone. The development of small molecule allosteric effectors of CO binding to hemoglobin (Hb) represents an important step toward making effective therapies for CO poisoning. To that end, we have found that the synthetic peptide IRL 2500 enhances CO release from COHb in air, but with concomitant hemolytic activity. We describe herein the design, synthesis, and biological evaluation of analogs of IRL 2500 that enhance the release of CO from COHb without hemolysis. These novel structures show improved aqueous solubility and reduced hemolytic activity and could lead the way to the development of small molecule therapeutics for the treatment of CO poisoning

NF449, NF110, and NM16 inhibit virus interactions with Jurkat cells.

<p>(A) Infection by EV-A71-1095, in the presence of inhibitors, measured by staining for VP1 expression. (B) Inhibition of virus attachment by NF449, NF110, and NM16. Results are indicated as the mean and S.D. for triplicate samples. Asterisks indicate <i>P</i> < 0.01 compared to the no inhibitor control.</p

Inhibition of EV-A71 attachment by newly-synthesized NF449 analogues.

<p>(A) Attachment of ³⁵S-labeled EV-A71-1095 to RD cells in the presence of compounds at 0.4 or 4 μM. Results from two experiments, one testing NM1-11 and one testing NM12-16, are combined, and are normalized to results for the no inhibitor control in each experiment. (B) Virus attachment to RD monolayers in the presence of NF449, NF110, and NM16. Results are indicated as the mean and S.D. for triplicate samples. Asterisks indicate <i>P</i> < 0.01 compared to the no inhibitor control.</p

NF449 and NF110 specifically prevent attachment of a monoclonal antibody to the viral 5-fold vertex.

<p>(A) An EV-A71 pentamer shown with the 5-fold vertex at the center. VP1-98 (red) was mutated in an NF449 escape mutant. VP1-242K (light blue) is involved in virus interaction with PSGL-1. VP1-244K (dark blue) is implicated both in virus interactions with both PSGL-1 and NF449. The MA28-7 footprint, as determined by cryo-electronmicroscopy, is outlined in black. The VP2 epitope recognized by mAb 10F0 is indicated in green. (B) NF449 inhibits immunoprecipitation of EV-A71-1095 by MA28-7 but not by 10F0. EV-A71-1095 concentrated from supernatants of infected cells was incubated with MA28-7 or 10F0 fixed to Protein G beads, in the presence of NF449 at the indicated concentrations, or 200 μM Pirodavir; beads were washed, and immunoprecipitated proteins were examined on Coomassie-stained gels. Molecular weight markers are shown at the left. Arrows at the right indicate capsid proteins VP0 and VP1, and brackets indicate antibody heavy (HC) and light chains (LC). (C) NF449 and NF110 inhibit attachment of MA28-7 to mature virions. Purified ³⁵S-labeled mature virions were incubated with MA28-7-coated beads, in the presence of inhibitors as indicated. (D) NF110 inhibits attachment of MA28-7, but not 10F0 or MA105, to procapsids. Purified ³⁵S-labeled procapsids were incubated with monoclonal antibodies fixed to Protein G beads, in the presence of 20 μM NF110. Results are indicated as the mean and S.D. for triplicate samples. Asterisks indicate <i>P</i> < 0.01 compared to the no inhibitor control.</p

Structures of newly-synthesized NF449 analogues.

<p>Compounds NM2-16 are new. NM1 was resynthesized, but is identical to NF110.</p

NF449, NF110, and NM16 inhibit interaction with RD cells of a PSGL-1-nonbinding EV-A71 isolate.

<p>(A) Infection by EV-A71-02363 in the presence of inhibitors, measured by staining for VP1 expression (B) Attachment of ³⁵S-labeled EV-A71-02363 in the presence of NF449 and NF110 (left), and NM16 (right). Results are indicated as the mean and S.D. for triplicate samples. Asterisks indicate <i>P</i> < 0.01 compared to the no inhibitor control.</p

NF449 inhibits EV-A71 interactions with RD cells.

<p>(A) RD cell monolayers were exposed to EGFP-expressing EV-A71-1095 in the presence of NF449 or the control compound fondaparinux at the indicated concentrations, then incubated at 37°C for 16 hr. Infected cells were identified by flow cytometry to detect EGFP expression. (B) ³⁵S-labeled EV-A71-1095 was exposed to NF449 in medium with or without 10% FBS, then attachment to RD monolayers was measured as described in Materials and Methods. (C) HeLa cell monolayers were exposed to ³⁵S-labeled CVB3-RD in the presence of NF449, the anti-DAF antibody IF7, or an isotype-matched control antibody, and attachment was measured. Results are indicated as the mean and S.D. for triplicate samples. Asterisks indicate <i>P</i> < 0.01 compared to results with the no inhibitor control.</p

Inhibition of EV-A71 attachment and infection by commercially-available NF449 analogues.

<p>(A) Structures of the tested compounds. (B) ³⁵S-labeled EV-A71-1095 was exposed to compounds at 0.4 or 4 μM, then virus attachment to RD monolayers was measured. (C) RD monolayers were exposed to EV-A71-1095 in the presence of inhibitors at the indicated concentrations. After 16 hrs at 37°C, infected cells were stained to detect intracellular VP1, and analyzed by flow cytometry. Results are indicated as the mean and S.D. of triplicate samples. Asterisks indicate <i>P</i> < 0.01 compared to the no inhibitor control.</p

Mutations at the 5-fold capsid vertex reduce the protective effect of NF449.

<p>Wild-type EV-A71-1095 and VP1 mutants were exposed to NF449 for 1 h, applied to RD cell monolayers and incubated at 37°C for 16 h. Cells were then stained to detect intracellular VP1 and analyzed by flow cytometry. Results are indicated as the mean and S.D. of triplicate samples. Asterisks indicate <i>P</i> < 0.01 compared to no inhibitor control.</p