Figure 5.

IT2-144 inhibits NEF-mediated activity and potentially binds in the MKT-077 binding pocket. A, IT2-144 inhibits a wide range of Hsp70–NEF combinations in luciferase refolding assays. Hsp72 (1 μm) was used with all of the NEFs except for GrpE, which used DnaK (1 μm). Likewise, DnaJA2 (0.5 μm) was used for all of the NEFs except for GrpE, which used DnaJ (0.5 μm). Denatured luciferase (0.1 μm) and potassium phosphate (10 mm) were also added. Results are the average of triplicate values, and the error bars represent S.E. B, IT2-144 docked to the MKT-077-binding site of Hsc70 (3HSC). Top view of the binding pocket (residues within 5 Å of IT2-144) highlights IT2-144 as well as ADP and magnesium in the adjacent nucleotide-binding cassette. C, scanning fluorescence spectra for all di-fluoro derivatives and the negative compound IT3-70a, with excitation wavelength set at 310 nm. D, change in fluorescence intensity with increasing Hsc70 concentration. Compounds were held at 50 μm, and fluorescence emission was read at optimal wavelengths, determined in spectral scans (see C). E, full-length Hsc70 was incubated with or without ADP and then tested for ability to bind IT2-144 (excitation 310 nm and emission 430 nm). EC₅₀ was determined from two independent experiments in triplicate. Full-length Hsc70 with mutations at position Thr-222 was also tested for ability to bind IT2-144. Thr-222 is highlighted in the structure of IT2-144 docked to Hsc70.