FK506-binding protein (FKBP) partitions a modified HIV protease inhibitor into blood cells and prolongs its lifetime in vivo.

HIV protease inhibitors are a key component of anti-retroviral therapy, but their susceptibility to cytochrome P(450) metabolism reduces their systemic availability and necessitates repetitive dosing. Importantly, failure to maintain adequate inhibitor levels is believed to provide an opportunity for resistance to emerge; thus, new strategies to prolong the lifetime of these drugs are needed. Toward this goal, numerous prodrug approaches have been developed, but these methods involve creating inactive precursors that require enzymatic processing. Using an alternative strategy inspired by the natural product FK506, we have synthetically modified an HIV protease inhibitor such that it acquires high affinity for the abundant, cytoplasmic chaperone, FK506-binding protein (FKBP). This modified protease inhibitor maintains activity against HIV-1 protease (IC(50) = 19 nM) and, additionally, it is partitioned into the cellular component of whole blood via binding to FKBP. Interestingly, redistribution into this protected niche reduces metabolism and improves its half-life in mice by almost 20-fold compared with the unmodified compound. Based on these findings, we propose that addition of FKBP-binding groups might partially overcome the poor pharmacokinetic properties of existing HIV protease inhibitors and, potentially, other drug classes.

A Non-immunogenic Bivalent d-Protein Potently Inhibits Retinal Vascularization and Tumor Growth.

We report a general approach to engineering multivalent d-proteins with antibody-like activities in vivo. Mirror-image phage display and structure-guided design were utilized to create a d-protein that uses receptor mimicry to antagonize vascular endothelial growth factor A (VEGF-A). Selections against the d-protein form of VEGF-A using phage-displayed libraries of two different domain scaffolds yielded two proteins that bound distinct receptor interaction sites on VEGF-A. X-ray crystal structures of the d-protein/VEGF-A complexes were used to guide affinity maturation and to construct a heterodimeric d-protein VEGF-A antagonist with picomolar activity. The d-protein VEGF-A antagonist prevented vascular leakage in a rabbit eye model of wet age-related macular degeneration and slowed tumor growth in the MC38 syngeneic mouse tumor model with efficacies comparable to those of approved antibody drugs, and in contrast with antibodies, the d-protein was non-immunogenic during treatment and following subcutaneous immunizations.

Synthesis of orthogonally reactive FK506 derivatives via olefin cross metathesis.

Chemical inducers of dimerization (CIDs) are employed in a wide range of biological applications to control protein localization, modulate protein-protein interactions and improve drug lifetimes. These bifunctional chemical probes are assembled from two synthetic modules, which each provide affinity for a distinct protein target. FK506 and its derivatives are often employed as modules in the syntheses of these bifunctional constructs, owing to the abundance and favorable distribution of their target, FK506-binding protein (FKBP). However, the structural complexity of FK506 necessitates multi-step syntheses and/or multiple protection-deprotection schemes prior to installation into CIDs. In this work, we describe an efficient, one-step synthesis of FK506 derivatives through a selective, microwave-accelerated, cross metathesis diversification step of the C39 terminal alkene. Using this approach, FK506 is modified with an array of functional groups, including primary amines and carboxylic acids, which make the resulting derivatives suitable for the modular assembly of CIDs. To illustrate this idea, we report the synthesis of a heterobifunctional HIV protease inhibitor.

Chemical control over protein-protein interactions: beyond inhibitors.

Protein-protein interactions have become attractive drug targets and recent studies suggest that these interfaces may be amenable to inhibition by small molecules. However, blocking specific interactions may not be the only way of manipulating the extensive network of interacting proteins. Recently, several approaches have emerged for promoting these interactions rather than inhibiting them. Typically, these strategies employ a bifunctional ligand to simultaneously bind two targets, forcing their juxtaposition. Chemically "riveting" specific protein contacts can reveal important aspects of regulation, such as the consequences of stable dimerization or the effects of prolonged dwell time. Moreover, in some cases, entirely new functions arise when two proteins, which normally do not interact, are brought into close proximity with one another. Together with inhibitors, bifunctional molecules are part of a growing toolbox of chemical probes that can be used to reversibly and selectively control the interact-ome. Using these reagents, new insights into the dynamics of protein-protein interactions and their importance in biology are beginning to emerge. Future hurdles in this area lie in the development of robust synthetic platforms for rapidly generating compounds to meet the challenges of diverse protein-protein interfaces.

Methylthioninium chloride (methylene blue) induces autophagy and attenuates tauopathy in vitro and in vivo.

More than 30 neurodegenerative diseases including Alzheimer disease (AD), frontotemporal lobe dementia (FTD), and some forms of Parkinson disease (PD) are characterized by the accumulation of an aggregated form of the microtubule-binding protein tau in neurites and as intracellular lesions called neurofibrillary tangles. Diseases with abnormal tau as part of the pathology are collectively known as the tauopathies. Methylthioninium chloride, also known as methylene blue (MB), has been shown to reduce tau levels in vitro and in vivo and several different mechanisms of action have been proposed. Herein we demonstrate that autophagy is a novel mechanism by which MB can reduce tau levels. Incubation with nanomolar concentrations of MB was sufficient to significantly reduce levels of tau both in organotypic brain slice cultures from a mouse model of FTD, and in cell models. Concomitantly, MB treatment altered the levels of LC3-II, cathepsin D, BECN1, and p62 suggesting that it was a potent inducer of autophagy. Further analysis of the signaling pathways induced by MB suggested a mode of action similar to rapamycin. Results were recapitulated in a transgenic mouse model of tauopathy administered MB orally at three different doses for two weeks. These data support the use of this drug as a therapeutic agent in neurodegenerative diseases.

Bifunctional molecules evade cytochrome P(450) metabolism by forming protective complexes with FK506-binding protein.

Despite their large size and complexity, the macrolide natural products rapamycin and FK506 have excellent pharmacological characteristics. We hypothesize that these unexpected properties may arise from protective, high affinity interactions with the cellular FK506-binding protein, FKBP. In this model, the drug-FKBP complex might sequester the small molecule and limit its degradation by restricting access to metabolic enzymes. In support of this idea, we found that adding FKBP blocks binding of FK506 to the common cytochrome P(450) enzyme CYP3A4 in vitro. To further test this idea, we have systematically modified a small collection of otherwise unrelated compounds, such that they acquire affinity for FKBP. Strikingly, we found that many of these synthetic derivatives, but not the unmodified parent compounds, are also protected from CYP3A4-mediated metabolism. Depending on the properties of the linker, the bifunctional molecules exhibited up to a 3.5-fold weaker binding to CYP3A4, and this protective effect was observed in the presence of either purified FKBP or FKBP-expressing cells. Together, these results suggest that the surprising pharmacology of rapamycin and FK506 might arise, in part, from binding to their abundant, intracellular target, FKBP. Furthermore, these findings provide a framework by which other small molecules might be systematically modified to impart this protective effect.

The D1 dopamine receptor is constitutively phosphorylated by G protein-coupled receptor kinase 4.

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate agonist-activated GPCRs, initiating their homologous desensitization. In this article, we present data showing that GRK4 constitutively phosphorylates the D1 receptor in the absence of agonist activation. This constitutive phosphorylation is mediated exclusively by the alpha isoform of GRK4; the beta, gamma, and delta isoforms are ineffective in this regard. Mutational analysis reveals that the constitutive phosphorylation mediated by GRK4alpha is restricted to the distal region of the carboxyl terminus of the receptor, specifically to residues Thr428 and Ser431. Phosphorylation of the D1 receptor by GRK4alpha results in a decrease in cAMP accumulation, an increase in receptor internalization, and a decrease in total receptor number--all of which are abolished in a D1 receptor mutant containing T428V and S431A. The increase in internalized D1 receptors induced by GRK4alpha phosphorylation is due to enhanced receptor internalization rather than retarded trafficking of newly synthesized receptors to the cell surface. The constitutive phosphorylation of the D1 receptor by GRK4alpha does not alter agonist-induced desensitization of the receptor because dopamine pretreatment produced a similar decrease in cAMP accumulation in control cells versus cells expressing GRK4alpha. These observations shift the attenuation of D1 receptor signaling from a purely agonist-driven process to one that is additionally modulated by the complement of kinases that are coexpressed in the same cell. Furthermore, our data provide direct evidence that, in contrast to current dogma, GRKs can (at least in some instances) constitutively phosphorylate GPCRs in the absence of agonist activation resulting in constitutive desensitization.

The role of phosphorylation in D1 dopamine receptor desensitization: evidence for a novel mechanism of arrestin association.

Homologous desensitization of D(1) dopamine receptors is thought to occur through their phosphorylation leading to arrestin association which interdicts G protein coupling. In order to identify the relevant domains of receptor phosphorylation, and to determine how this leads to arrestin association, we created a series of mutated D(1) receptor constructs. In one mutant, all of the serine/threonine residues within the 3rd cytoplasmic domain were altered (3rdTOT). A second construct was created in which only three of these serines (serines 256, 258, and 259) were mutated (3rd234). We also created four truncation mutants of the carboxyl terminus (T347, T369, T394, and T404). All of these constructs were comparable with the wild-type receptor with respect to expression and adenylyl cyclase activation. In contrast, both of the 3rd loop mutants exhibited attenuated agonist-induced receptor phosphorylation that was correlated with an impaired desensitization response. Sequential truncation of the carboxyl terminus of the receptor resulted in a sequential loss of agonist-induced phosphorylation. No phosphorylation was observed with the most severely truncated T347 mutant. Surprisingly, all of the truncated receptors exhibited normal desensitization. The ability of the receptor constructs to promote arrestin association was evaluated using arrestin-green fluorescent protein translocation assays and confocal fluorescence microscopy. The 3rd234 mutant receptor was impaired in its ability to induce arrrestin translocation, whereas the T347 mutant was comparable with wild type. Our data suggest a model in which arrestin directly associates with the activated 3rd cytoplasmic domain in an agonist-dependent fashion; however, under basal conditions, this is sterically prevented by the carboxyl terminus of the receptor. Receptor activation promotes the sequential phosphorylation of residues, first within the carboxyl terminus and then the 3rd cytoplasmic loop, thereby dissociating these domains and allowing arrestin to bind to the activated 3rd loop. Thus, the role of receptor phosphorylation is to allow access of arrestin to its receptor binding domain rather than to create an arrestin binding site per se.