ENDOR Spectroscopic Evidence for the Geometry of Binding of retro-inverso-N^ω-Nitroarginine-Containing Dipeptide Amides to Neuronal Nitric Oxide Synthase

Abstract

We describe in detail the use of 35 GHz Mims pulsed 15N and 1,2H electron−nuclear double resonance (ENDOR) spectroscopy to study the binding of substrates and inhibitors to nitric oxide synthase (NOS). We show that reliable distance estimates, and limited orientation information, can be derived from a small set of data taken near the peak of the absorption mode EPR signal, while more precise orientations require a more extensive data set. The ENDOR approach is then applied to the binding of isoform-selective and non-selective nitroarginine inhibitors. Recently, we reported a family of N ω-nitroarginine-containing dipeptide amides as highly selective inhibitors of nNOS (Huang, H. et al. J. Med. Chem. 1999, 42, 3147−3153). Two of the most potent analogues were the retro-inverso-dipeptide amides l-ArgNO 2 -l-Lys-NH2 (LL) and d-Lys-d-ArgNO 2 −NH2 (DD). To rationalize the common selectivities of LL and DD, it was proposed that in both cases the nitroarginine group binds at the heme binding site, therefore requiring one of these molecules to undergo a 180° flip to accommodate such an interaction. The present studies confirm that the dipeptides indeed bind to holo-nNOS quite similarly from the point of view of the nitroguanidine functionality, supporting the earlier interpretation. The data further suggest that a substantial fraction of the DD epimer is distributed among other binding geometries.

The partial retro–inverso modification: A road traveled together

“…49 Electron-nuclear double resonance (ENDOR) spectroscopy confirmed the hypothe-sis that the nitroarginine group binds at the heme-binding site that requires a 180°flip to accommodate such interaction ( Figure 9). 50 Reckless and colleagues developed NR58-3.14.3, a disulfide-bridged RI analog of a tridecapeptide derived from mature human monocyte chemoattractant protein-1 (MCP-1) that is a pan-selective chemokine inhibitor. 51 This peptide, which was ϳ1000-fold more potent than the parent peptide in inhibiting leukocyte migration, induced by a wide range of chemokines in vitro, was also a functional chemokine inhibitor in vivo, and therefore was a powerful anti-inflammatory agent.…”

“…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.…”