Immunostimulation by a partially modified retro-inverso-tuftsin analog containing Thr1.sum..psi.[NHCO](R,S)Lys2 modification

Abstract

The tuftsin retro-inverso analogue H-Thr psi[NHCO](R,S)Lys-Pro-Arg-OH was synthesized through a novel procedure for the high-yield incorporation of isolated retro-inverso bonds into peptide chains and the use of the new Meldrum's acid derivative (CH3)2C(OCO)2CH(CH2)4NHCOCF3 followed by its efficient coupling in solution to trimethylsilylated H-D-Thr(t-Bu)NH2. Closely related peptide impurities were eliminated both from the crude final peptide and the fully protected tetrapeptide amide precursor via ion-exchange and reversed-phase displacement chromatography, respectively. The tuftsin retro-inverso analogue proved to be completely resistant to enzymatic degradation in vitro, either against isolated aminopeptidases or human plasma proteolytic enzymes. When administered either orally or intravenously, it was significantly more active than normal tuftsin in increasing the number of specific antibody secreting cells in spleen of mice immunized with sheep erythrocytes. Furthermore, the analogue exerted an enhanced stimulatory effect on the cytotoxic activity of splenocytes against YAC-1 tumor cells. Finally, retro-inverso-tuftsin was about 10-fold more potent than the native peptide in reducing rat adjuvant arthritis. The resistance of the retro-inverso analogue to peptidases might explain the increased in vivo activities and allows its further immunopharmacological characterization.

The partial retro–inverso modification: A road traveled together

“…47 Recently, attention has been directed to tissuespecific isoforms of nitric oxide synthase (NOS) that are responsible for the production of NO, an important signaling molecule, and to their role in different physiological and pathophysiological processes. Modulation of NOS activity is an important target for the development of anti-inflammatory agents.…”

“…Fusion of membrane-penetrating peptides such as polyArg, the hexadecapeptide derived from the Drosophila Antennapedia homeobox (Antp), and the undecapeptide derived from the HIV-TAT [47][48][49][50][51][52][53][54][55][56][57] to the cargo molecules frequently enables their internalization. Very often the proteolytic susceptibility of these membrane-penetrating peptides compromises their successful application as molecular tools and drug carriers.…”

“…54 Therefore, a combination of resistance to proteolysis, good membrane-penetrating capacity, and lack of immunogenicity and antigenicity makes the RI membrane-penetrating peptides into important potential molecular delivery vehicles. Linking the RI protein transduction domain (PTD)-4, an optimized analog of HIV-TAT [47][48][49][50][51][52][53][54][55][56][57], to a PNA antisense for the AUG translation initiation region of prepro-oxytocin mRNA generated a powerful antisense reagent. 55 This conjugate was effectively internalized by cultured cerebral cortex neurons and markedly reduced levels of mRNA coding for prepro-oxytocin and the expression of prepro-oxytocin in oxytocin-producing magnocellular neurons from the hypothalamic supraoptic nucleus.…”

“…Internalization and inhibition of growth in mammary cancer cells MCF-7, colon cancer cells (HCT-116), and human peripheral blood lymphocytes stimulated by phytohemoglobin, and reduction in c-Myc transcriptional activity was achieved by a RI Antp-H1-S6A,F8A. This hybrid was com- Recently, Snyder and coworkers reported the restoration of endogenous p53 activity by a RI hybrid of p53CЈ (human p53[361-382]) and TAT-HIV [47][48][49][50][51][52][53][54][55][56][57] linked through their C-and N-terminals, respectively, to a spacing Gly residue ( Figure 11A). 58 In cancer cells, p53CЈ peptide can induce apoptosis by activating endogenous p53 and by restoring function to several p53 DNA contact mutants.…”

“…This study reported that RI p53[361-382]-Gly-TAT-HIV [47][48][49][50][51][52][53][54][55][56][57] permeates into the cells, induces cell cycle arrest ( Figure 11B), transcriptionally activates p53-responsive genes, inhibits tumor cell proliferation in a p53-dependent manner, inhibits the growth of distant solid tumors in immune competent mice ( Figure 11C), and extends the survival of mice harboring either terminal peritoneal carcinomatosis or terminal peritoneal lymphoma ( Figure 11D). In summary, the delivery of permeable proteolytically stable RI p53[361-382]-Gly-TAT-HIV [47][48][49][50][51][52][53][54][55][56][57] is potently activating the p53 tumor suppressor pathway, thus offering an effective treatment for multiple preclinical cancer models. 58 Ramanathan and coworkers reported an interesting strategy to enhance intestinal absorption of large peptides by targeting the sodium-dependent multivitamin transporter (SDMT) as a cellular entry site for oral delivery of biotinylated RI peptides.…”