The free energy profile of tubulin straight-bent conformational changes, with implications for microtubule assembly and drug discovery.

αß-tubulin dimers need to convert between a 'bent' conformation observed for free dimers in solution and a 'straight' conformation required for incorporation into the microtubule lattice. Here, we investigate the free energy landscape of αß-tubulin using molecular dynamics simulations, emphasizing implications for models of assembly, and modulation of the conformational landscape by colchicine, a tubulin-binding drug that inhibits microtubule polymerization. Specifically, we performed molecular dynamics, potential-of-mean force simulations to obtain the free energy profile for unpolymerized GDP-bound tubulin as a function of the ∼12° intradimer rotation differentiating the straight and bent conformers. Our results predict that the unassembled GDP-tubulin heterodimer exists in a continuum of conformations ranging between straight and bent, but, in agreement with existing structural data, suggests that an intermediate bent state has a lower free energy (by ∼1 kcal/mol) and thus dominates in solution. In agreement with predictions of the lattice model of microtubule assembly, lateral binding of two αß-tubulins strongly shifts the conformational equilibrium towards the straight state, which is then ∼1 kcal/mol lower in free energy than the bent state. Finally, calculations of colchicine binding to a single αß-tubulin dimer strongly shifts the equilibrium toward the bent states, and disfavors the straight state to the extent that it is no longer thermodynamically populated.

Effects of solution concentration on the physicochemical properties of a polymeric anticancer therapeutic.

Poly(γ-glutamyl-glutamate) paclitaxel (PGG-PTX) is a series of eighteen semiflexible polymer-drug constructs varying in PTX loading fraction (f(PTX) of 0.18, 0.24, and 0.37) and spatial PTX arrangement (uniform, "even" and "random"; clustered, "clusters" and "ends"; concentrated, "ends" and "side"). Structural properties of PGG-PTX in dilute and concentrated conditions are determined from coarse-grained molecular dynamics (MD) simulations. Since PGG-PTX does not have a specific conformation, MD simulations were run until minimal structural deviations persisted. Root-mean-square deviation (RMSD) clustering was then used to determine the significant, unique characteristic conformations. Results show that dilute PGG-PTX undergoes a globule-to-filament transition with respect to increasingly denser PTX arrangements. While a similar transition is apparent in concentrated conditions, PGG-PTX tends to be more filamentous on the whole. PGG-PTX is also more rigid in concentrated conditions, and a higher PTX loading fraction leads to decreased flexibility. In general, the dilute "ends", "middle", and "side" PGG-PTX molecules at f(PTX) = 0.18 and 0.24 prove to be the most efficaciously promising and are recommended for future biological testing. This study demonstrates the practicality of molecular modeling toward understanding structural behavior of an anticancer therapeutic in different solution concentrations.

Aggregation properties of a polymeric anticancer therapeutic: a coarse-grained modeling study.

The effects of paclitaxel (PTX) loading fraction and spatial PTX arrangement on poly(γ-glutamyl-glutamate) paclitaxel (PGG-PTX) aggregation were explored using coarse-grained molecular dynamics. Results show that the PTX loading fraction does not significantly impact aggregation, and the spatial PTX arrangement only affects aggregation at more concentrated PTX arrangements. Overall, the f(PTX) = 0.37 'ends' and f(PTX) = 0.18 'even' PGG-PTX systems exhibit the highest aggregation and the 'middle' and 'side' systems the lowest.

Coarse-grained modeling study of nonpeptide RGD ligand density and PEG molecular weight on the conformation of poly(γ-glutamyl-glutamate) paclitaxel conjugates.

Molecular shape, flexibility, and surface hydrophilicity are thought to influence the ability of nanoparticles to cross biological barriers during drug delivery. In this study, coarse-grained (CG) molecular dynamics (MD) simulations were used to study these properties of a polymer-drug construct in potential clinical development: poly(γ-glutamyl-glutamate)-paclitaxel-poly(ethylene glycol) nonpeptide RGD (PGG-PTX-PEG-npRGD), a linear glutamyl-glutamate polymer with paclitaxel and poly(ethylene glycol)-nonpeptide RGD side groups. It was hypothesized that the PEG molecular weight (MW) (500 Da; 1,000 Da; and 2,000 Da) and nonpeptide RGD ligand density (4, 8, 12, and 16 per molecule), respectively, may have advantageous effects on the shape, flexibility, and surface hydrophilicity of PGG-PTX-PEG-npRGD. Circular dichroism spectroscopy was used to suggest initial structures for the all-atom (AA) models of PGG-PTX-PEG-npRGD, which were further converted to CG models using a commercially available mapping algorithm. Due to its semi-flexibility, PGG-PTX-PEG-npRGD is not limited to one specific conformation. Thus, CG MD simulations were run until statistical equilibrium, at which PGG-PTX-PEG-npRGD is represented as an ensemble of statistically similar conformations. The size of a PGG-PTX-PEG-npRGD molecule is not affected by the PEG MW or the nonpeptide RGD density, but higher PEG MW results in increased surface density of a PGG-PTX-PEG-npRGD molecule. Most PGG-PTX-PEG-npRGD shapes are globular, although filamentous shapes were also observed in the PEG500 and PEG1000 molecules. PEG500 and PEG1000 molecules are more flexible than PEG2000 systems. A higher presence of npRGD ligands results in decrease surface hydrophilicity of PGG-PTX-PEG-npRGD. These results indicate that the PGG-PTX-PEG1000-npRGD(4) and PGG-PTX-PEG1000-npRGD(8) molecules are the most efficacious candidates and are further recommended for experimental preclinical studies.

Characterization of a clinical polymer-drug conjugate using multiscale modeling.

The molecular conformation of certain therapeutic agents has been shown to affect the ability to gain access to target cells, suggesting potential value in defining conformation of candidate molecules. This study explores how the shape and size of poly-γ-glutamyl-glutamate paclitaxel (PGG-PTX), an amphiphilic polymer-drug with potential chemotherapeutic applications, can be systematically controlled by varying hydrophobic and hydrophilic entities. Eighteen different formulations of PGG-PTX varying in three PTX loading fractions (f(PTX)) of 0.18, 0.24, and 0.37 and six spatial arrangements of PTX ('clusters', 'ends', 'even', 'middle', 'random', and 'side') were explored. Molecular dynamics (MD) simulations of all-atom (AA) models of PGG-PTX were run until a statistical equilibrium was reached at 100 ns and then continued as coarse-grained (CG) models until a statistical equilibrium was reached at an effective time of 800 ns. Circular dichroism spectroscopy was used to suggest initial modeling configurations. Results show that a PGG-PTX molecule has a strong tendency to form coil shapes, regardless of the PTX loading fraction and spatial PTX arrangement, although globular shapes exist at f(PTX) = 0.24. Also, less uniform PTX arrangements such as 'ends', 'middle', and 'side' produce coil geometries with more curvature. The prominence of coil shapes over globules suggests that PGG-PTX may confer a long circulation half-life and high propensity for accumulation to tumor endothelia. This multiscale modeling approach may be advantageous for the design of cancer therapeutic delivery systems. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 936-951, 2010.

Systems analysis of PKA-mediated phosphorylation gradients in live cardiac myocytes.

Compartmentation and dynamics of cAMP and PKA signaling are important determinants of specificity among cAMP's myriad cellular roles. Both cardiac inotropy and the progression of heart disease are affected by spatiotemporal variations in cAMP/PKA signaling, yet the dynamic patterns of PKA-mediated phosphorylation that influence differential responses to agonists have not been characterized. We performed live-cell imaging and systems modeling of PKA-mediated phosphorylation in neonatal cardiac myocytes in response to G-protein coupled receptor stimuli and UV photolysis of "caged" cAMP. cAMP accumulation was rate-limiting in PKA-mediated phosphorylation downstream of the beta-adrenergic receptor. Prostaglandin E1 stimulated higher PKA activity in the cytosol than at the sarcolemma, whereas isoproterenol triggered faster sarcolemmal responses than cytosolic, likely due to restricted cAMP diffusion from submembrane compartments. Localized UV photolysis of caged cAMP triggered gradients of PKA-mediated phosphorylation, enhanced by phosphodiesterase activity and PKA-mediated buffering of cAMP. These findings indicate that combining live-cell FRET imaging and mechanistic computational models can provide quantitative understanding of spatiotemporal signaling.

A high-throughput quantitative multiplex kinase assay for monitoring information flow in signaling networks: application to sepsis-apoptosis.

To treat complex human diseases effectively, a systems-level approach is needed to understand the interplay of environmental cues, intracellular signals, and cellular behaviors that underlie disease states. This approach requires high-throughput, multiplex techniques that measure quantitative temporal variations of multiple protein activities in the intracellular signaling network. Here, we describe a single microtiter-based format that simultaneously quantifies protein kinase activities in the phosphatidylinositol 3-kinase pathway (Akt), nuclear factor-kappaB pathway (IKK), and three core mitogen-activated protein kinase pathways (ERK, JNK1, MK2). These parallel high-throughput assays are stringently linear, redundantly specific, reproducible, and sensitive compared with classical low-throughput techniques. When applied to a model of sepsis-induced colon epithelial apoptosis, this approach identified a late phase of Akt activity as a critical mediator of cell survival that quantitatively contributed to the efficacy of insulin as an anti-apoptotic cue. Thus, sampling parallel nodes in the intracellular signaling network identified part of the molecular mechanism underlying the efficacy of insulin in the treatment of human sepsis.