4 research outputs found
Chasing Protons: How Isothermal Titration Calorimetry, Mutagenesis, and p<i>K</i><sub>a</sub> Calculations Trace the Locus of Charge in Ligand Binding to a tRNA-Binding Enzyme
Drug
molecules should remain uncharged while traveling through
the body and crossing membranes and should only adopt charged state
upon protein binding, particularly if charge-assisted interactions
can be established in deeply buried binding pockets. Such strategy
requires careful p<i>K</i><sub>a</sub> design and methods
to elucidate whether and where protonation-state changes occur. We
investigated the protonation inventory in a series of <i>lin</i>-benzoguanines binding to tRNA−guanine transglycosylase, showing
pronounced buffer dependency during ITC measurements. Chemical modifications
of the parent scaffold along with ITC measurements, p<i>K</i><sub>a</sub> calculations, and site-directed mutagenesis allow elucidating
the protonation site. The parent scaffold exhibits two guanidine-type
portions, both likely candidates for proton uptake. Even mutually
compensating effects resulting from proton release of the protein
and simultaneous uptake by the ligand can be excluded. Two adjacent
aspartates induce a strong p<i>K</i><sub>a</sub> shift at
the ligand site, resulting in protonation-state transition. Furthermore,
an array of two parallel H-bonds avoiding secondary repulsive effects
contributes to the high-affinity binding of the <i>lin</i>-benzoguanines
Beyond Affinity: Enthalpy–Entropy Factorization Unravels Complexity of a Flat Structure–Activity Relationship for Inhibition of a tRNA-Modifying Enzyme
Lead
optimization focuses on binding-affinity improvement. If a
flat structure–activity relationship is detected, usually optimization
strategies are abolished as unattractive. Nonetheless, as affinity
is composed of an enthalpic and entropic contribution, factorization
of both can unravel the complexity of a flat, on first sight tedious
SAR. In such cases, the binding free energy of different ligands can
be rather similar, but it can factorize into enthalpy and entropy
distinctly. We investigated the thermodynamic signature of two classes
of <i>lin</i>-benzopurines binding to tRNA−guanine
transglycosylase. While the differences are hardly visible in the
free energy, they involve striking enthalpic and entropic changes.
Analyzing thermodynamics along with structural features revealed that
one ligand set binds to the protein without inducing significant changes
compared to the apo structure; however, the second series provokes
complex adaptation, leading to a conformation similar to the substrate-bound
state. In the latter state, a cross-talk between two pockets is suggested
Targeting the Blind Spot of Polycationic Nanocarrier-Based siRNA Delivery
Polycationic nanocarriers attract increasing attention to the field of siRNA delivery. We investigated the self-assembly of siRNA <i>vs</i> pDNA with polycations, which are broadly used for nonviral gene and siRNA delivery. Although polyethyleneimine (PEI) was routinely adopted as siRNA carrier based on its efficacy in delivering pDNA, it has not been investigated yet why PEI efficiently delivers pDNA to cells but is controversially discussed in terms of efficacy for siRNA delivery. We are the first to investigate the self-assembly of PEI/siRNA <i>vs</i> PEI/pDNA and the steps of complexation and aggregation through different levels of hierarchy on the atomic and molecular scale with the novel synergistic use of molecular modeling, molecular dynamics simulation, isothermal titration calorimetry, and other characterization techniques. We are also the fist to elucidate atomic interactions, size, shape, stoichiometry, and association dynamics for polyplexes containing siRNA <i>vs</i> pDNA. Our investigation highlights differences in the hierarchical mechanism of formation of related polycation–siRNA and polycation–pDNA complexes. The results of fluorescence quenching assays indicated a biphasic behavior of siRNA binding with polycations where molecular reorganization of the siRNA within the polycations occurred at lower N/P ratios (nitrogen/phosphorus). Our results, for the first time, emphasize a biphasic behavior in siRNA complexation and the importance of low N/P ratios, which allow for excellent siRNA delivery efficiency. Our investigation highlights the formulation of siRNA complexes from a thermodynamic point of view and opens new perspectives to advance the rational design of new siRNA delivery systems
Targeting the Blind Spot of Polycationic Nanocarrier-Based siRNA Delivery
Polycationic nanocarriers attract increasing attention to the field of siRNA delivery. We investigated the self-assembly of siRNA <i>vs</i> pDNA with polycations, which are broadly used for nonviral gene and siRNA delivery. Although polyethyleneimine (PEI) was routinely adopted as siRNA carrier based on its efficacy in delivering pDNA, it has not been investigated yet why PEI efficiently delivers pDNA to cells but is controversially discussed in terms of efficacy for siRNA delivery. We are the first to investigate the self-assembly of PEI/siRNA <i>vs</i> PEI/pDNA and the steps of complexation and aggregation through different levels of hierarchy on the atomic and molecular scale with the novel synergistic use of molecular modeling, molecular dynamics simulation, isothermal titration calorimetry, and other characterization techniques. We are also the fist to elucidate atomic interactions, size, shape, stoichiometry, and association dynamics for polyplexes containing siRNA <i>vs</i> pDNA. Our investigation highlights differences in the hierarchical mechanism of formation of related polycation–siRNA and polycation–pDNA complexes. The results of fluorescence quenching assays indicated a biphasic behavior of siRNA binding with polycations where molecular reorganization of the siRNA within the polycations occurred at lower N/P ratios (nitrogen/phosphorus). Our results, for the first time, emphasize a biphasic behavior in siRNA complexation and the importance of low N/P ratios, which allow for excellent siRNA delivery efficiency. Our investigation highlights the formulation of siRNA complexes from a thermodynamic point of view and opens new perspectives to advance the rational design of new siRNA delivery systems