Statistics of crystallographic analysis.

a<p>Completeness and <i>R-</i>merge, are given for overall data and for the highest resolution shell.</p><p>The highest resolution shells for the dataset were 2.54–2.50 Å.</p>b<p><i>R</i>merge = ∑ | <i>I_i</i> – <<i>I</i>> |/∑|<i>I_i</i>|; where <i>I_i</i> is intensity of an observation and <<i>I</i>> is the mean value of that reflection and the summations are over all equivalents.</p>c<p><i>R-work</i> = ∑<i>_h</i> || <i>Fo(h)</i> | – | <i>Fc(h)</i> ||/∑<i>_hFo(h)</i>; where <i>Fo</i> and <i>Fc</i> are the observed and calculated structure factor amplitudes, respectively. The <i>R-free</i> was calculated with 5% of the data excluded from the refinement.</p

Inhibition of 20 S proteasome activity by homopiperazine derivatives.

<p>A. Purified erythrocyte-derived 20 S proteasome was incubated in the absence (control) or presence of the indicated HPDS at 5 µM. Chymotrypsin-like, caspase-like and trypsin-like activities were determined by measuring fluorescence generated from the cleavage of specific substrates. Results are represented as relative fluorescence units (RFU) with control set at 100%. The means ± S.D. (bars) of three independent experiments are shown. <i>P</i>-values were calculated by one-way ANOVA with the Student-Newman-Keuls multiple comparisons test. Asterisks indicate p<0.05 against corresponding controls. B. RPMI8226 cells were treated with or without 5 µM HPDs, and analyzed for proteolytic activities as described above. C. RPMI8226 cells were cultured in the absence (control) or presence of 10 µM HPDs for 24 hours, and subjected to immunoblotting for ubiquitinated proteins and GAPDH (internal control).</p

Comparison of K-7174 and bortezomib in cytotoxic activity and proteasome binding.

<p>A. Cell proliferation was measured by MTT assays after culturing with serially diluted K-7174, K-10487 and bortezomib for 72 hours. Absorbance at 450 nm was analyzed with a microplate reader, and expressed as a percentage of the value of corresponding untreated cells. The IC₅₀ value was defined as the concentration of each drug that produces 50% inhibition of cell growth. The means ± S.D. (bars) of three independent experiments are shown. Asterisks indicate “not determined”. B. Overall crystallographic structures showing the folds of ß1 to ß7 subunits in the proteasome bound with K-7174 (left panel) and bortezomib (right panel). Mutation sites observed in bortezomib-resistant cells are circled. C. Structure of the proteasome in complex with bortezomib (PDB cord 2F16) overlapped that with K-7174 described here. Only the protein atoms of the bortezomib-bound form are shown. Bortezomib-resistant mutant residues (Ala49, Thr21, Cys52 and Met45) are colored red and shown as a space-filling diagram.</p

Cytotoxic effects of K-7174 on bortezomib-resistant MM cells.

<p>We established wild-type (WT) and mutant (mutant) sublines from RPMI8226 by transducing with wild-type and mutated <i>PSMB5</i> cDNA, respectively, and analyzed the expression of VENUS by flow cytometry (A) and proteasome ß5 subunit by immunoblotting (B). The signal intensities of ß5 subunit (PSMB5) were quantified, normalized to those of the corresponding GAPDH, and shown as relative values in the panel B. C. Cell proliferation was measured by MTT assays after culturing each subline with either K-7174 or bortezomib at the indicated doses for 72 hours. Results are represented as relative absorbance with untreated control set at 100%. The means ± S.D. (bars) of three independent experiments are shown. <i>P</i>-values were calculated by one-way ANOVA with the Student-Newman-Keuls multiple comparisons test. Asterisks indicate p<0.01 against the WT subline. D. Each subline was cultured with either K-7174 or bortezomib (Bort) at the indicated doses for 48 hours. Whole cell lysates were subjected to immunoblotting for cellular protein ubiquitination, proteasome ß5 subunit (PSMB5) and GAPDH (internal control).</p