The use of liposomal carriers and the modification of therapeutic molecules through the attachment of poly(ethylene glycol) [PEG] moieties ('pegylation') are the most common approaches for enhancing the delivery of parenteral agents. Although 'classical' liposomes (i.e. phospholipid bilayer vehicles) have been effective in decreasing the clearance of encapsulated agents and in passively targeting specific tissues, they are associated with considerable limitations. Pegylation may be an effective method of delivering therapeutic proteins and modifying their pharmacokinetic properties, in turn modifying pharmacodynamics, via a mechanism dependent on altered binding properties of the native protein. Pegylation reduces renal clearance and, for some products, results in a more sustained absorption after subcutaneous administration as well as restricted distribution. These pharmacokinetic changes may result in more constant and sustained plasma concentrations, which can lead to increases in clinical effectiveness when the desired effects are concentration-dependent. Maintaining drug concentrations at or near a target concentration for an extended period of time is often clinically advantageous, and is particularly useful in antiviral therapy, since constant antiviral pressure should prevent replication and may thereby suppress the emergence of resistant variants. Additionally, PEG modification may decrease adverse effects caused by the large variations in peak-to-trough plasma drug concentrations associated with frequent administration and by the immunogenicity of unmodified proteins. Pegylated proteins may have reduced immunogenicity because PEG-induced steric hindrance can prevent immune recognition. Two PEG-modified proteins are currently approved by the US Food and Drug Administration; several others, including cytokines such as interferon-alpha (IFNalpha), growth factors and free radical scavengers, are under development. Careful assessment of various pegylated IFNalpha products suggests that pegylated molecules can be differentiated on the basis of their pharmacokinetic properties and related changes in pharmacodynamics. Because the size, geometry and attachment site of the PEG moiety play a crucial role in determining these properties, therapeutically optimised agents must be designed on a protein-by-protein basis.

Synthesis of the vinyl sulfone and chloroethyl sulfone derivatives of poly(ethylene glycol) (PEG) is described. The chloroethyl sulfone (CES-PEG) is rapidly converted to the vinyl sulfone (VS-PEG) in the presence of base but is stable in water at neutral pH. Reactions with small molecules such as beta-mercaptoethanol and N alpha-acetyllysine show that the vinyl sulfone derivative is highly selective for reaction with sulfhydryl groups relative to reaction with amino groups. Also, VS-PEG is stable in water. These properties indicate that VS-PEG should be useful for selective attachment of PEG to protein cysteine groups. This hypothesis was verified by reacting VS-PEG with cysteine groups of reduced ribonuclease (RNase); the reaction is rapid and selective at pH 7-9. Reaction at lysine sites of unreduced RNase occurs slowly at pH 9.3 and is essentially complete after 100 h. Amino acid residues other than lysine and cysteine are not reactive toward VS-PEG. The covalent linkage between VS-PEG and lysine or cysteine groups is shown to be stable.

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The acquisition, maintenance and modulation of dendritic architecture are critical to neuronal form, plasticity and function. Morphologically, dendritic shape impacts functional connectivity and is largely mediated by organization and dynamics of cytoskeletal fibers that provide the underlying scaffold and tracks for intracellular trafficking. Identifying molecular factors that regulate dendritic cytoskeletal architecture is therefore important in understanding mechanistic links between cytoskeletal organization and neuronal function. In a neurogenomic-driven genetic screen of cytoskeletal regulatory molecules, we identified Formin3 (Form3) as a critical regulator of cytoskeletal architecture in Drosophila nociceptive sensory neurons. Form3 is a member of the conserved Formin family of multi-functional cytoskeletal regulators and time course analyses reveal Form3 is cell-autonomously required for maintenance of complex dendritic arbors. Cytoskeletal imaging demonstrates form3 mutants exhibit a specific destabilization of the dendritic microtubule (MT) cytoskeleton, together with defective dendritic trafficking of mitochondria, satellite Golgi and the TRPA channel Painless. Biochemical studies reveal Form3 directly interacts with MTs via FH1-FH2 domains and promotes MT stabilization via acetylation. Neurologically, mutations in human Inverted Formin 2 (INF2; ortholog of form3) have been causally linked to Charcot-Marie-Tooth (CMT) disease. CMT sensory neuropathies lead to impaired peripheral sensitivity. Defects in form3 function in nociceptive neurons results in a severe impairment in noxious heat evoked behaviors. Expression of the INF2 FH1-FH2 domains rescues form3 defects in MT stabilization and nocifensive behavior revealing conserved functions in regulating the cytoskeleton and sensory behavior thereby providing novel mechanistic insights into potential etiologies of CMT sensory neuropathies.Significance StatementMechanisms governing cytoskeletal architecture are critical in regulating neural function as aberrations are linked to a broad spectrum of neurological and neurocognitive disorders. Formins are important cytoskeletal regulators however their mechanistic roles in neuronal architecture are poorly understood. We demonstrate mutations in Drosophila formin3 lead to progressive destabilization of the dendritic microtubule cytoskeleton resulting in severely reduced arborization coupled to impaired organelle and ion channel trafficking, as well as nociceptive sensitivity. INF2 mutations are implicated in CMT sensory neuropathies, and INF2 expression can rescue microtubule and nociceptive behavioral defects in form3 mutants. While CMT sensory neuropathies have been linked to defects in axonal development and myelination, our studies connect dendritic cytoskeletal defects with peripheral insensitivity suggesting possible alternative etiological bases.