ABSTRACT
Polyamine biosynthesis is regulated by ornithine decarboxylase (ODC), which is transcriptionally activated by c-Myc. A large library was screened to find molecules that potentiate the ODC inhibitor, difluoromethylornithine (DFMO). Anthranilic acid derivatives were identified as DFMO adjunct agents. Further studies identified the far upstream binding protein 1 (FUBP1) as the target of lead compound 9. FUBP1 is a single-stranded DNA/RNA binding protein and a master controller of specific genes including c-Myc and p21. We showed that 9 does not inhibit 3H-spermidine uptake yet works synergistically with DFMO to limit cell growth in the presence of exogenous spermidine. Compound 9 was also shown to inhibit the KH4 FUBP1-FUSE interaction in a gel shift assay, bind to FUBP1 in a ChIP assay, reduce both c-Myc mRNA and protein expression, increase p21 mRNA and protein expression, and deplete intracellular polyamines. This promising hit opens the door to new FUBP1 inhibitors with increased potency.
Subject(s)
Eflornithine , Spermidine , Eflornithine/pharmacology , RNA, Messenger/genetics , RNA-Binding Proteins , Spermidine/metabolismABSTRACT
Targeting polyamine metabolism is a proven anticancer strategy. Cancers often escape the polyamine biosynthesis inhibitors by increased polyamine import. Therefore, there is much interest in identifying polyamine transport inhibitors (PTIs) to be used in combination therapies. In a search for new PTIs, we serendipitously discovered a LAT-1 efflux agonist, which induces intracellular depletion of methionine, leucine, spermidine, and spermine, but not putrescine. Because S-adenosylmethioninamine is made from methionine, a loss of intracellular methionine leads to an inability to biosynthesize spermidine, and spermine. Importantly, we found that this methionine-depletion approach to polyamine depletion could not be rescued by exogenous polyamines, thereby obviating the need for a PTI. Using 3H-leucine (the gold standard for LAT-1 transport studies) and JPH-203 (a specific LAT-1 inhibitor), we showed that the efflux agonist did not inhibit the uptake of extracellular leucine but instead facilitated the efflux of intracellular leucine pools.
Subject(s)
Biological Transport/drug effects , Large Neutral Amino Acid-Transporter 1/metabolism , Polyamines/metabolism , Small Molecule Libraries/pharmacology , Animals , Benzoxazoles/pharmacology , CHO Cells , Cricetulus , Drug Discovery , Humans , Leucine/metabolism , Methionine/metabolism , Putrescine/metabolism , Small Molecule Libraries/chemistry , Spermidine/metabolism , Spermine/metabolism , Tyrosine/analogs & derivatives , Tyrosine/pharmacologyABSTRACT
Pancreatic cancers are currently the fourth leading cause of cancer-related death and new therapies are desperately needed. The most common pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). This report describes the development of therapies, which effectively deplete PDAC cells of their required polyamine growth factors. Of all human tissues, the pancreas has the highest level of the native polyamine spermidine. To sustain their high growth rates, PDACs have altered polyamine metabolism, which is reflected in their high intracellular polyamine levels and their upregulated import of exogenous polyamines. To understand how these cancers respond to interventions that target their specific polyamine pools, L3.6pl human pancreatic cancer cells were challenged with specific inhibitors of polyamine biosynthesis. We found that pancreatic cell lines have excess polyamine pools, which they rebalance to address deficiencies induced by inhibitors of specific steps in polyamine biosynthesis (e.g., ornithine decarboxylase (ODC), spermidine synthase (SRM), and spermine synthase (SMS)). We also discovered that combination therapies targeting ODC, SMS, and polyamine import were the most effective in reducing intracellular polyamine pools and reducing PDAC cell growth. A combination therapy containing difluoromethylornithine (DFMO, an ODC inhibitor) and a polyamine transport inhibitor (PTI) were shown to significantly deplete intracellular polyamine pools. The additional presence of an SMS inhibitor as low as 100 nM was sufficient to further potentiate the DFMO + PTI treatment.
ABSTRACT
3,3-Dimethylcyclopentanes (neopentylenes) are ubiquitous in Nature but largely absent from synthetic pharmaceutical libraries. Neopentylenes define a hydrophobic and rigid 3-D topology with distinct molecular pharmacology, as exemplified here with two neopentylene-fused analogues of the synthetic anti-inflammatory drug, ibuprofen.