MediQuest Therapeutics, Inc.

We have recently confirmed that lipoteichoic acid (LTA), a major constituent of the gram-positive bacterial surface, is the endotoxin of gram-positive bacteria that induces proinflammatory molecules in a Toll-like receptor 2 (TLR2)-dependent manner. LTA is an anionic amphipath whose physicochemical properties are similar to those of lipopolysaccharide (LPS), which is found on the outer leaflet of the outer membranes of gram-negative organisms. Hypothesizing that compounds that sequester LPS could also bind to and inhibit LTA-induced cellular activation, we screened congeneric series of polyamine sulfonamides which we had previously shown effectively neutralized LPS both in vitro and in animal models of endotoxemia. We observed that these compounds do bind to and neutralize LTA, as reflected by the inhibition of TLR2-mediated NF-κB induction in reporter gene assays. Structure-activity studies showed a clear dependence of the acyl chain length on activity against LTA in compounds with spermine and homospermine scaffolds. We then sought to examine possible correlations between the neutralizing potency toward LTA and antimicrobial activity in Staphylococcus aureus . A linear relationship between LTA sequestration activity and antimicrobial activity for compounds with a spermine backbone was observed, while all compounds with a homospermine backbone were equally active against S. aureus , regardless of their neutralizing potency toward LTA. These results suggest that the number of protonatable charges is a key determinant of the activity toward the membranes of gram-positive bacteria. The development of resistance to membrane-active antibiotics has been relatively slower than that to conventional antibiotics, and it is possible that compounds such as the acylpolyamines may be useful clinically, provided that they have an acceptable safety profile and margin of safety. A more detailed understanding of the mechanisms of interactions of these compounds with LPS and LTA, as well as the gram-negative and -positive bacterial cell surfaces, will be instructive and should allow the rational design of analogues which combine antisepsis and antibacterial properties.

Background Antizyme is a critical regulator of cellular polyamine levels due to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, through dependence on an unusual +1 translational frameshift mechanism that responds to polyamine levels.Results HEK293 cells were depleted of polyamines by treatment with an ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and grown in the presence or absence of exogenous polyamines prior to the analysis of ribosomal frameshifting levels. Results obtained using an optimized dual luciferase assay system reveal a 10‐fold dynamic range of frameshifting, which correlates positively with polyamine addition. Polyamine addition to cells, which have not been pre‐treated with DFMO, also resulted in an increase in antizyme frameshifting but to a lesser degree (1.3 to 1.5‐fold). In addition, the constructs with the 3′ deletion were more responsive to stimulation by polyamine addition than those with the 5′ deletion.Conclusions The observed regulation of antizyme frameshifting demonstrates the efficiency of a polyamine homeostatic mechanism, and illustrates the utility of a quantifiable cell‐based assay for the analysis of polyamines or their analogues on translational frameshifting.

It has been hypothesized that one method whereby cancer cells can circumvent the ability of polyamine biosynthesis inhibitors from completely depleting their internal polyamines is via the import of these mols. from external sources.We have developed a group of polyamine analogs that potently inhibit this polyamine uptake system in a series of cancer cell lines.Modification of various nitrogen atom substituents together with the stereochem. of the amino acid portion of our earlier lead compound, L-Lys-Spm, has produced candidates with several orders of magnitude higher potency.Compounds were evaluated by their ability to inhibit the uptake of radiolabeled 3H-spermidine into MDA-MB-231 breast cancer cells.Addnl., the analogs′ ability to increase the growth inhibitory effects of the polyamine biosynthesis inhibitor DFMO, even in the presence of added external spermidine, was evaluated.Finally, their ability to deplete polyamine levels in these cells, when used in combination with DFMO, was measured.