Patent Review

James      E. McGee; Anuradha      Roy

Abstract

The section on patent review will be focused in the areas of interest to the readers of CCHTS. The search was conducted using the following key words: combinatorial chemistry, high throughput screening, drug repurposing, chemical library, high content screening, drug discovery and natural products. All patents highlighted here are identified by the patent number issued either by the World Intellectual Property Organization or by a regional patent office. HIGH THROUGHPUT SCREENING US 7972803 B2: Centrosomal proteins and secretion. (Jurczyk, A., Bortell, R., Rossini, A., Doxsey, S., US). The patent is based on the discovery of role of centrosomal proteins in cellular secretion and encompasses methods for identifying candidate modulators of cellular secretion. Centrosomes are cytosolic organelles comprising of two centrioles, surrounded by the pericentriolar material (PCM) comprising of proteins like pericentrin and γ-tubulin, which are required for microtubule nucleation, organization and cell cycle progression. Centrosome has also been implicated in diverse functions like cell polarization, protein localization in certain signaling pathways and cilia/flagella formation. The inventors identified a new role of centrosome in protein secretion in pancreatic islets through its ability to interface with the secretory vesicles. The centrosome proteins were shown to co-localize with insulin granules in insulinoma cell lines and in isolated mouse islets. A siRNA knockdown of centrosome proteins was accompanied by loss of intracellular insulin granules from beta cells as well as hypersecretion of insulin into the media in vitro and in plasma in vivo, thus suggesting a critical role for centrosome proteins in regulation of insulin storage and secretion. The inventors hypothesize that centrosome is a novel drug target for treatment of diseases associated with abnormal secretion such as diabetes, Huntingtons disease, Alzheimers disease etc. The patent includes methods for screening compound libraries to fins modulators of activity and levels of centrosome proteins including reporter assays, ELISAs and subcellular localization changes using fluorescence microscopy. US 7935493 B2: Fragments of fluorescent proteins for protein fragment complementation Assays. (Michnick, S., MacDonald M., Lamerdin, J., US). The invention provides methods for generating fragments of known reporter fluorescent proteins and creating mutant fragments with improved spectral characteristics for use in Protein-Fragment Complementation Assays (PCAs). The methodology has widespread use in establishing assays for drug discovery, target validation, high-throughput/content screening, pathwaymapping, mechanism-of-action studies, biosensors, and diagnostics. The fragments are generated based on detailed structural information for fluorescent proteins and are designed such that the basic chromophore formation and folding of the protein are not affected. The fragmentation is selected in areas that have little or no impact on the two complementing halves to fold and reconstitute active structures. The fragmentation design is explained in detail for the GFP, a protein in which a α-helix threads through the center of a rigid β-barrel and is attached to the chromophore, p-hydroxybenzylideneimidazolinone formed from residues 65-67. Fragmentation is made in β-turns at extreme ends orany one of six regions of amino-acids spanning the β-loops to preserve the barrel structure. The two fragments, F1 and F2 are amplified by PCR or synthesized as oligonucleotides are fused in frame at 5 or at 3 end with genes encoding target proteins, A and B, in two different expression vectors. If proteins A & B interact, F1 and F2 are brought in close proximity and are capable of refolding and reconstituting a functional fluorescent protein. The structural homology between known fluorescent proteins from Aequorea, Anemonia and Anthozoa allowed ease of designing complementing fragments. In addition to generating fragments capable for complementation, the inventors created mutant fragments with specific spectral properties with variable intensities and signal shifts in green, yellow, cyan, orange-red and red region. The complementation of fragments and use of the technology for high throughput screening was demonstrated using a number of fusion proteins that are known to have protein-protein interactions like p53 dimerization, PI3kinase and PKA/PKC. The adaptation of the PCA to high throughput assays was shown for interactions between FK506 binding proteins, FKBP and FRAPS (FKBP-rapamycin-Associated protein). Rapamycin induced complex formation between the two proteins and was visualized by fluorescence microscopy and quantified spectrofluormetrically. The methodology also encompasses using multi-color multiplexing as well as combining fluorescent or luminescent PCA......

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