Transcription Factor Inhibition
Zinc-finger Transcription Factor Inhibition: Gli Proteins in the Hedgehog Pathway
The Hedgehog signaling pathway is responsible for the formation and persistence of Basal Cell Carcinomas, as well as the growth and metastasis of Medulloblastoma, a lethal childhood brain cancer. As ultimate regulators of his pathway, the Cis2His2 Zn(II) binding Gli proteins are a sought-after therapeutic target. Traditionally difficult to target with organic therapeutics, herein we present efforts towards inhibiting Gli proteins with Co(III)-DNA conjugates. Specificity for Gli over other Cis2His2 proteins is conferred by conjugating the consensus DNA sequence of Gli to Co(III)-Schiff base. This brings the active Co(III)-Schiff base inhibitor in close enough proximity to the Cis2His2 binding sites to irreversibly displace the structural zinc.
Within this strategy, we are further exploring delivery vehicles to overcome the barrier of limited cellular uptake of Co(III)-DNA conjugates. We explore graphene oxide nanoplatelets coated with polyethyleneimine to impart positive charge into the nanocarrier. This permits adsorption of the Co(III)-DNA inhibitor onto the surface as well as rapid cellular internalization.
Inhibiting Amyloid-β Aggregation - Experimental Approach
The aggregation of Aβ is believed to be foundational to the pathogenesis of Alzheimer’s disease (AD). In vitro aggregation kinetics have been shown to correlate with rates of disease progression in both AD patients and animal models, thus proving to be a useful metric for testing Aβ-targeted therapeutics. Here we present evidence of Cobalt(III) Schiff base complex (Co(III)-sb) modulation of Aβ aggregation kinetics by a variety of complementary techniques. These include Thioflavin T (ThT) fluorescence, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). Our data was fitted to kinetic rate laws using a mathematical model developed by Knowles et al. in order to extract mechanistic information about the effect of Co(III)-sb on aggregation kinetics. Our analysis revealed that Co(III)-sb significantly decreases the kinetic parameter k+, and significantly increases the polymerization rate kn, suggesting that Co(III)-sb causes Aβ to rapidly form stable oligomeric species that are unable to elongate into mature fibrils. This result was corroborated by TEM and AFM of Aβ aggregates in vitro. We also demonstrate that Aβ aggregate mixtures produced in the presence of Co(III)-sb exhibit decreased cytotoxicity compared to untreated samples.
Inhibiting Amyloid-β Aggregation - Computational Approach
Coordination complexes have emerged as prominent modulators of amyloid aggregation via their interaction with the N-terminal histidine residues of amyloid-β (Aβ). Herein, we report the synthesis and characterization of a novel cobalt(III) Schiff base complex with methylamine axial ligands, and we present both computational and experimental data demonstrating the reduction of β-sheet formation by this complex. The computations include molecular dynamics simulations of both monomeric and pentameric Aβ, which demonstrate decreased formation of β-sheet structures, destabilization of preformed β-sheets, and suppression of aggregation. Replica Exchange Molecular Dynamics (REMD) has been employed to further study the computational landscape of AB bound to Co(III)-sb complexes.
Light-Activated Cobalt Inhibition
The Meade Lab’s Co(III)-sb complexes can be modified to include an electron donor component, in this case Ruthenium tris(bipyridine). When the complex is irradiated, photoinduced electron transfer (PET) occurs, initiating displacement of the inert axial ligands and subsequent coordination to active site histidine residues. This allows the complex to be used as a prodrug, which is administered as an inactive compound and triggered by a controllable stimulus to become active in the body.