Lipophilic lysine-spermine conjugates are potent polyamine transport inhibitors for use in combination with a polyamine biosynthesis inhibitor.

Cancer cells can overcome the ability of polyamine biosynthesis inhibitors to completely deplete their internal polyamines by the importation of polyamines from external sources. This paper discusses the development of a group of lipophilic polyamine analogues that potently inhibit the cellular polyamine uptake system and greatly increase the effectiveness of polyamine depletion when used in combination with DFMO, a well-studied polyamine biosynthesis inhibitor. The attachment of a length-optimized C(16) lipophilic substituent to the epsilon-nitrogen atom of an earlier lead compound, D-Lys-Spm (5), has produced an analogue, D-Lys(C(16)acyl)-Spm (11) with several orders of magnitude more potent cell growth inhibition on a variety of cultured cancer cell types including breast (MDA-MB-231), prostate (PC-3), melanoma (A375), and ovarian (SK-OV-3), among others. These results are discussed in the context of a possible membrane-catalyzed interaction with the extracellular polyamine transport apparatus. The resulting novel two-drug combination therapy targeting cellular polyamine metabolism has shown exceptional efficacy against cutaneous squamous cell carcinomas (SCC) in a transgenic ornithine decarboxylase (ODC) mouse model of skin cancer. A majority (88%) of large, aggressive SCCs exhibited complete or nearly complete remission to this combination therapy, whereas responses to each agent alone were poor. The availability of a potent polyamine transport inhibitor allows, for the first time, for a real test of the hypothesis that starving cells of polyamines will lead to objective clinical response.

Polyamine analogs with xylene rings induce antizyme frameshifting, reduce ODC activity, and deplete cellular polyamines.

Numerous studies have correlated elevated polyamine levels with abnormal or rapid cell growth. One therapeutic strategy to treat diseases with increased cellular proliferation rates, most obviously cancer, has been to identify compounds which lower cellular polyamine levels. An ideal target for this strategy is the protein antizyme-a negative regulator of polyamine biosynthesis and import, and a positive regulator of polyamine export. In this study, we have optimized two tissue-culture assays in 96-well format, to allow the rapid screening of a 750-member polyamine analog library for compounds which induce antizyme frameshifting and fail to substitute for the natural polyamines in growth. Five analogs (MQTPA1-5) containing xylene (1,4-dimethyl benzene) were found to be equal to or better than spermidine at stimulating antizyme frameshifting and were inefficient at rescuing cell growth following polyamine depletion. These compounds were further characterized for effects on natural polyamine levels and enzymes involved in polyamine metabolism. Finally, direct measurements of antizyme induction in cells treated with two of the lead compounds revealed an 8- to 15-fold increase in antizyme protein over untreated cells. The impact of the xylene moiety and the distance between the positively charged amino groups on antizyme frameshifting and cell growth are discussed.

Induction of apoptosis by aryl-substituted diamines: role of aromatic group substituents and distance between nitrogens.

A series of aromatic substituted diamines was synthesized and characterized for their cytotoxic profiles against human breast and prostate tumor cell lines. Following a structure function analysis of the effects of changes of the benzyl substituents and the distance between amino groups the most potent analogues were analyzed biologically and were shown to induce apoptosis. These compounds do not induce the enzyme SSAT or deplete intracellular polyamine levels, mechanisms demonstrated by other cytotoxic polyamine analogues.

Synthesis of bis-spermine dimers that are potent polyamine transport inhibitors.

A series of novel spermine dimer analogues was synthesized and assessed for their ability to inhibit spermidine transport into MDA-MB-231 breast carcinoma cells. Two spermine molecules were tethered via their N(1) primary amines with naphthalenedisulfonic acid, adamantanedicarboxylic acid and a series of aliphatic dicarboxylic acids. The linked spermine analogues were potent polyamine transport inhibitors and inhibited cell growth cytostatically in combination with a polyamine synthesis inhibitor. Variation in the linker length did not alter polyamine transport inhibition. The amount of charge on the molecule may influence the molecular interaction with the transporter since the most potent spermidine transport inhibitors contained 5-6 positive charges.

Tools for Investigating Functional Interactions Between Lipid-Derived Autacoids and their Receptors.

A method for rapidly evaluating functional interactions between ligands and G-protein--coupled receptors has been developed. The technology is based on the ability of animals to change color by controlling the position of pigmented organelles within skin cells called melanophores. cDNA coding for a receptor to be studied is expressed in immortalized frog melanophores. Stimulation of a receptor that normally functions to activate either adenyl cyclase or phospholipase C induces centrifugal melanosome translocation and cell darkening. Conversely, application of an agonist to cells expressing a receptor that operates to inhibit adenyl cyclase induces centripetal pigment movement and cell lightening. The simple optical change can be used to investigate ligand-receptor interactions at several levels, including single-cell analysis and high-throughput chemical screening. Current efforts are focused on (1) identifying small peptides that activate or block thromboxane. A(2) and platelet-activating factor (PAF) receptors and (2) cloning eicosanoid receptors.