Cytotoxicities of Tumor-Seeking Dyes: Impact on Future Clinical Trials.

Heptamethine (Cy7) dyes with meso-Cl substituents injected intravenously (iv) into mice accumulate in tumors and persist there over several days. We believe this occurs via meso-Cl displacement by the only free cysteine residues of albumin; therefore, conjugating tumor-seeking dyes with fragments can increase selective therapeutic delivery to tumors and drug residence. This strategy has elevated significance recently because the first tumor-seeking dye-drug conjugate has moved into clinical trials. Options for further clinical research include modifying the dye, and use of preformed albumin adducts instead of dyes alone. Herein we show correlations of cytotoxicities, lipophilicities, organelle localization, apoptosis, cell-cycle arrest, wound healing/migration assays, and reactivities/affinities with human serum albumin are difficult to observe. However, our studies arrived at an important conclusion: preformed dye-drug-HSA adducts are less cytotoxic, and therefore preferable for subsequent clinical work, relative to direct injection of meso-Cl-containing forms.

Piptides: New, Easily Accessible Chemotypes For Interactions With Biomolecules.

Small molecule probe development is pivotal in biomolecular science. Research described here was undertaken to develop a non-peptidic chemotype, piptides, that is amenable to convenient, iterative solid-phase syntheses, and useful in biomolecular probe discovery. Piptides can be made from readily accessible pip acid building blocks and have good proteolytic and pH stabilities. An illustrative application of piptides against a protein-protein interaction (PPI) target was explored. The Exploring Key Orientations (EKO) strategy was used to evaluate piptide candidates for this. A library of only 14 piptides contained five members that disrupted epidermal growth factor (EGF) and its receptor, EGFR, at low micromolar concentrations. These piptides also caused apoptotic cell death, and antagonized EGF-induced phosphorylation of intracellular tyrosine residues in EGFR.

Unconventional Secondary Structure Mimics: Ladder-Rungs.

Secondary structures tend to be recognizable because they have repeating structural motifs, but mimicry of these does not have to follow such well-defined patterns. Bioinformatics studies to match side-chain orientations of a novel hydantoin triazole chemotype (1) to protein-protein interfaces revealed it tends to align well across parallel and antiparallel sheets, like rungs on a ladder. One set of these overlays was observed for the protein-protein interaction uPA⋅uPAR. Consequently, chemotype 1 was made with appropriate side-chains to mimic uPA at this interface. Biophysical assays indicate these compounds did in fact bind uPAR, and elicit cellular responses that affected invasion, migration, and wound healing.

Small molecules targeting the NEDD8·NAE protein-protein interaction.

Ubiquitination is a major controller of protein homeostasis in cells. Some ubiquitination pathways are modulated by a NEDDylation cascade, that also features E1 - 3 enzymes. The E1 enzyme in the NEDDylation cascade involves a protein-protein interaction (PPI) between NEDD8 (similar to ubiquitin) and NAE (NEDD8 Activating Enzyme). A small molecule inhibitor of the ATP binding site in NAE is in clinical trials. We hypothesized a similar effect could be induced by disrupting the NEDD8·NAE PPI, though, to the best of our knowledge, no small molecules have been reported to disrupt this to date. In the research described here, Exploring Key Orientations (EKO) was used to evaluate several chemotype designs for their potential to disrupt NEDD8·NAE; specifically, for their biases towards orientation of side-chains in similar ways to protein segments at the interface. One chemotype design was selected, and a targeted library of 24 compounds was made around this theme via solid phase synthesis. An entry level hit for disrupting NEDD8·NAE was identified from this library on the basis of its ability to bind NAE (K i of 6.4 ± 0.3 µM from fluorescence polarization), inhibit NEDDylation, suppress formation of the corresponding E1 - 3 complexes as monitored by cell-based immunoblotting, and cytotoxicity to K562 leukemia cells via early stage apoptosis. The cell-based immunoblot assay also showed the compound caused NEDD8 to accumulate in cells, presumably due to inhibition of the downstream pathways involving the E1 enzyme. The affinity and cellular activities of the hit compound are modest, but is interesting as first in class for this mode of inhibition of NEDDylation, and as another illustration of the way EKO can be used to evaluate user-defined chemotypes as potential inhibitors of PPIs.