Development of antibacterial agents and materials

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

In one Aim, we have developed hydrogel networks displaying dAla-dAla via self-assembly using one beta-hairpin scaffolds, which were functionalized at their C-terminus with the dipepetide via a variable linker resulting in gelators MA1-3 CD, TEM, and rheology indicated that each gelator undergoes triggered self-assembly to form morphologically well-defined fibrils comprising viscoelastic gels. The copy number of target dipeptide can be controlled by doping in various amount of unmodified hairpin during gelation. Resulting materials are loaded via direct binding of vancomycin to dAla-dAla displayed from the fibril network. The solution phase association constants of monomeric MA1-3 determined by isothermal calorimetry (ITC) inversely correlate with the release rates of vancomycin from gels prepared from each of the gelators. For example, MA-3 shows the tightest binding and its corresponding gel displays the slowest release rate. Conversely, a negative control hairpin void of dAla-dAla shows no binding and burst release kinetics from its corresponding gel. Additional biophysical experiments and release simulations using mathematical models of mass transport dynamics indicate that drug release is largely dependent on the on/off-rates defining the reversible binding event between drug and target. Further, bioluminescence experiments using fluorescently-labelled vancomycin shows that the MA-3 gel offers sustained release of drug over months in vivo as compared to control gel. Lastly, we demonstrated that the gel can be loaded multiple times by simple tail-vein injection after releasing its cargo. In the second Aim, we designed a new class of CPPs whose ability to perturb cellular membranes is coupled to an enzyme-mediated shift in the folding potential of the peptide into its bioactive conformation. Cancer cells rich in negatively-charged surface components that also highly express alkaline phosphatase (ALP) are susceptible to the action of these peptides. Our peptide contains an N-terminal region with an (AB)n repeat of hydrophilic and hydrophobic residues followed by a central -DPP- motif that reverses the direction of the main chain Importantly, the C-terminal region contains a negatively-charged phosphorylated Tyr that disrupts an otherwise ideal second (AB)n repeat. This disruption in amphiphilicity keeps the peptide unfolded and inactive until it is presented to dephosphorylating enzymes. Removal of the phosphate restores amphiphilicity and shifts the conformational bias of the peptide towards hairpin folding, which is ultimately achieved when it binds to negatively-charged components displayed from the surface of cells. We showed that DVD-1P preferentially lyses cells with efficacies that match the cellular expression levels of ALP. The mechanistic fate of the peptide can be further tuned by peptide concentration to affect cell-penetrating properties. When cells are treated with sub-lytic concentrations of DOX-funtionalized. The peptide delivers the drug into cells with efficacies dependent on ALP expression levels. Overall, this work shows that it is possible to affect selective MLPs action by directing their folding with spatial resolution. During the course of these studies we have developed new synthetic protocols impacting the synthesis of peptides.