Prediction of Water Distributions and Displacement at Protein-Ligand Interfaces.

The retention and displacement of water molecules during formation of ligand-protein interfaces play a major role in determining ligand binding. Understanding these effects requires a method for positioning of water molecules in the bound and unbound proteins and for defining water displacement upon ligand binding. We describe an algorithm for water placement and a calculation of ligand-driven water displacement in >9000 protein-ligand complexes. The algorithm predicts approximately 38% of experimental water positions within 1.0 Å and about 83% within 1.5 Å. We further show that the predicted water molecules can complete water networks not detected in crystallographic structures of the protein-ligand complexes. The algorithm was also applied to solvation of the corresponding unbound proteins, and this allowed calculation of water displacement upon ligand binding based on differences in the water network between the bound and unbound structures. We illustrate use of this approach through comparison of water displacement by structurally related ligands at the same binding site. This method for evaluation of water displacement upon ligand binding may be of value for prediction of the effects of ligand modification in drug design.

Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo.

Recombinant protein-polymer scaffolds such as elastin-like polypeptides (ELPs) offer drug-delivery opportunities including biocompatibility, monodispersity, and multifunctionality. We recently reported that the fusion of FK-506 binding protein 12 (FKBP) to an ELP nanoparticle (FSI) increases rapamycin (Rapa) solubility, suppresses tumor growth in breast cancer xenografts, and reduces side effects observed with free-drug controls. This new report significantly advances this carrier strategy by demonstrating the coassembly of two different ELP diblock copolymers containing drug-loading and tumor-targeting domains. A new ELP nanoparticle (ISR) was synthesized that includes the canonical integrin-targeting ligand (Arg-Gly-Asp, RGD). FSI and ISR mixed in a 1:1 molar ratio coassemble into bifunctional nanoparticles containing both the FKBP domain for Rapa loading and the RGD ligand for integrin binding. Coassembled nanoparticles were evaluated for bifunctionality by performing in vitro cell-binding and drug-retention assays and in vivo MDA-MB-468 breast tumor regression and tumor-accumulation studies. The bifunctional nanoparticle demonstrated superior cell target binding and similar drug retention to FSI; however, it enhanced the formulation potency, such that tumor growth was suppressed at a 3-fold lower dose compared to an untargeted FSI-Rapa control. This data suggests that ELP-mediated scaffolds are useful tools for generating multifunctional nanomedicines with potential activity in cancer.

Elastin-like polypeptide switches: A design strategy to detect multimeric proteins.

Elastin-Like Polypeptides (ELPs) reversibly phase separate in response to changes in temperature, pressure, concentration, pH, and ionic species. While powerful triggers, biological microenvironments present a multitude of more specific biological cues, such as antibodies, cytokines, and cell-surface receptors. To develop better biosensors and bioresponsive drug carriers, rational strategies are required to sense and respond to these target proteins. We recently reported that noncovalent association of two ELP fusion proteins to a "chemical inducer of dimerization" small molecule (1.5 kDa) induces phase separation at physiological temperatures. Having detected a small molecule, here we present the first evidence that ELP multimerization can also detect a much larger (60 kDa) protein target. To demonstrate this strategy, ELPs were biotinylated at their amino terminus and mixed with tetrameric streptavidin. At a stoichiometric ratio of [4:1], two to three biotin-ELPs associate with streptavidin into multimeric complexes with an apparent Kd of 5 nM. The increased ELP density around a streptavidin core strongly promotes isothermal phase separation, which was tuned to occur at physiological temperature. This phase separation reverses upon saturation with excess streptavidin, which only favors [1:1] complexes. Together, these findings suggest that ELP association with multimeric biomolecules is a viable strategy to deliberately engineer ELPs that respond to multimeric protein substrates.

Image-driven pharmacokinetics: nanomedicine concentration across space and time.

Clinical pharmacokinetics (PK) primarily measures the concentration of drugs in the blood. For nanomedicines it may be more relevant to determine concentration within a target tissue. The emerging field of image-driven PK, which utilizes clinically accepted molecular imaging technology, empirically and noninvasively, measures concentration in multiple tissues. Image-driven PK represents the intersection of PK and biodistribution, combining to provide models of concentration across space and time. Image-driven PK can be used both as a research tool and in the clinic. This review explores the history of pharmacokinetics, technologies used in molecular imaging (especially positron emission tomography) and research using image-driven pharmacokinetic analysis. When standardized, image-driven PK may have significant implications in preclinical development as well as clinical optimization of targeted nanomedicines.