Enhanced helicity, protease resistance, and serum stability of hydrocarbon-stapled BID BH3 compounds. (A and B) α,α-disubstituted non-natural amino acids containing olefinic side chains of varying length were synthesized as previously reported (16, 31, 32). Non-natural amino acid substitutions were made to flank three (substitution positions i and i+4) or six (i and i+7) amino acids within the BID BH3 peptide, so that reactive olefinic residues would reside on the same face of the α helix. (C) Circular dichroism was used to measure the percentages of SAHB maintained in helical configuration when dissolved in aqueous potassium phosphate solution (pH7) (supporting online material). (D) Fluoresceinated SAHB_A and BID BH3 peptide were incubated at 37°C in mouse serum or injected intravenously (10 mg/kg) into NOD SCID mice. Serum concentrations of SAHB_A and BID BH3 peptide were measured at the indicated time points with a fluorescence-based high-performance liquid chromatography detection assay. Both assays demonstrated enhanced serum stability of SAHB_A.

SAHB_A targets the binding pocket of BCL-X_L, displays enhanced BCL-2 binding affinity, and specifically activates cytochrome c release from mitochondria in vitro. (A) HSQC experiments show similar spectral changes in ¹⁵N-BCL-X_L upon binding SAHB_A or BID BH3 peptide. (B) K_d's for binding of individual peptides to glutathione S-transferase-BCL-2 were determined by fluorescence polarization. (C) Mouse liver mitochondria (wild-type or Bak^-/-, 0.5 mg/ml) were incubated for 40 min with 25 to 200 nM concentrations of BID BH3 peptide, SAHB_A, or SAHB_A(G→E), and cytochrome c was measured in the supernatant and sedimented mitochondria by an enzyme-linked immunosorbent assay.

SAHB_A penetrates Jurkat leukemia cells by fluid-phase endocytosis and localizes to the mitochondrial membrane. Jurkat leukemia cells were incubated with FITC-labeled peptides for 4 hours at 37°C, followed by FACS analysis (A). FITC-SAHB_A uptake occurred in a time-dependent manner at 37°C (B), but no FITC-SAHB_A labeling was evident by 4 hours, when the experiment was performed at 4°C (C). Live confocal images demonstrated a colocalization of FITC-SAHB_A with 4.4-kD dextran-labeled endosomes (D) but not transferrin-labeled endosomes (E) at 4 hours. A mitochondrial colocalization was evident by 24 hours, as demonstrated by the merged images of FITC-SAHB_A and MitoTracker in live cells (F) and those of FITC-SAHB_A and Tom20 (a mitochondrial outer-membrane marker) in fixed cells (G). Arrows highlight sites of colocalization corresponding to the surface of mitochondria cut in cross section (G).

SAHB_A triggers apoptosis in Jurkat cells and inhibits a panel of human leukemia cells. FACS analysis of annexin V-treated cells was used to monitor apoptosis of Jurkat cells treated with 0.5 to 5 μM concentrations of BID BH3 peptide, SAHB_A, or SAHB_A(G→E) for 20 hours (A). Jurkat, REH, MV4;11, SEMK2, and RS4;11 leukemia cells were treated with serial dilutions of SAHB_A (B), BID BH3 peptide (C), or SAHB_A(G→E) (D), and MTT assays were performed at 48 hours to measure viability.

SAHB_A suppresses growth of human leukemia cells in vivo, prolonging the survival of leukemic mice. (A) Leukemic SCID beige mice [with a day-1 natural logarithm (ln) bioluminescence range of 14.4 to 15.9] were treated with intravenous injections of 10 mg/kg SAHB_A or vehicle (5% DMSO in D5W) daily for 7 days and were monitored for survival; leukemia burden was quantified by total body luminescence (photons/s/mouse) on days 1, 3, and 5. The disease course from days 3 to 5 differed between SAHB_A-treated animals and controls (P = 0.016, Fisher's exact test [box in (A)], as illustrated by representative Xenogen images of bioluminescent leukemic mice (B); red signal represents the highest level of leukemia on the colorimetric scale. (C) Median survival was prolonged in SAHB_A-treated animals as compared to controls (P = 0.004, log rank test). (D) To compare SAHB_A with SAHB_A(G→E), leukemic mice (with a day 1 ln bioluminescence range of 17.1 to 17.9) were treated daily with SAHB (10 mg/kg) or vehicle, and animals were imaged on days 1 and 3 to measure total body luminescence.