Generation of PKT16, a N-methylated TSP-1 mimetic peptide with improved efficiency in inducing PCD in the malignant CLL B cells. (A, left) Table enclosing the sequence of peptides generated by replacing the amino acids of the PKHB1 sequence with its N-methyl derivatives. (Right) After accounting for spontaneous PCD, cell viability was measured by Annexin-V/PI colabeling in the OSU-CLL cell line treated with the PKHB1-derived peptides described in the table (200 µM, 6 hours). The percentages, expressed as a histogram, refer to the Annexin-V-positive cells and are presented at mean ± standard deviation (SD). (B) The OSU-CLL cells were preincubated with PKHB1 or the derivatives PKHB1-SO and PKHB1-SO₂, and cell viability was assessed by flow cytometry by Annexin-V/PI costaining. After accounting for spontaneous PCD, the percentages of Annexin-V-positive cells were graphed as a mean ± SD. (C) Primary structure of PKHB1, peptide 1, and PKT16. Compared with PKHB1, Arg2 and Met7 of PKHB1 were respectively replaced by N-methyl Arg (N-MeArg) and Nor-Leucine (NLe) (highlighted in red). (D, left) Cytotoxicity, measured by Annexin-V/PI colabeling in a panel of CLL B lymphocytes treated over the course of 6 hours with PKHB1 (n = 29) or the optimized peptide PKT16 (n = 42) at the indicated concentrations. The data, referring to Annexin-V positivity, are presented at mean ± standard error of the mean (SEM). (Right) The EC50 was calculated with the GraphPad Prism software by a nonlinear regression curve. Statistical significance was calculated by the Student t test. ns, not significant.