Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 76
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The ATP molecule is immobilized in the protein kinaseorientation via its c-phosphate [43]. Microsequencing of the proteins that areeluted from the c-phosphate-linked ATP Sepharose column with free MgATP revealed that the nucleotide selectively recovered purine-binding proteins, includingall protein kinases, dehydrogenases, purine dependent metabolic enzymes, DNAligases, heat shock proteins and a variety of miscellaneous ATP-binding enzymes.This immobilized proteome is estimated to represent about 5% of the expressedeukaryotic genome.The “ATP-binding cassette proteome” can be utilized to test the selectivity of purine analogs that have been shown to inhibit proteins kinases in vitro.
Using proteome mining ATP affinity array apparatus constructed in our laboratory, sufficientbiomass is applied to ensure recovery of 1 fmol/column of any protein expressed at100 copies/cell (107 cells). After stringent washing, each column in the array iseluted in parallel with molecules from a purine-based iterative library and fractionscollected. Eluates are screened for protein, and positive fractions generally contain:(1) a single protein, (2) a small number of structurally related proteins, or (3) a complex mixture of unrelated proteins.
Only the first two categories are considered to beof use since the third category suggests elution with a non-selective molecule. Eluted15.4 Proteome Mining of the Smooth Muscle CellFig. 15.7 Proteome Mining Strategy. Proteinsfrom a cell line, organ, or animal source areisolated on affinity column arrays to removenon-specific adherents. Combichem librariesare passed over the array; any proteins thatare eluted are analyzed by protein electrophoresis. Protein sequence obtained by massspectrometry is then used to search DNA andprotein databases. If a relevant target is identified, a sub-library of compounds can be evaluated to refine the lead. In this manner, aprotein target, a drug lead, and any potentialside-targets can be simultaneously identified.(Redrawn from [62].)proteins from the first two situations are identified and their biological significanceare considered.
If a protein has no obvious use as a drug target then it is ignored,and if the protein is deemed relevant, one immediately has a lead molecule, a defined target protein, and any potential side-targets. In the cases where a single protein is eluted, the candidate drug molecule is likely to be selective since it had anequal opportunity to interact with the rest of the captured proteome. Selectivitycan be investigated by changing the concentration of the drug molecule during elution (i.e., from nano- to micromolar concentrations). Information concerning potential toxicity is gained upon sequencing of other proteins that are simultaneouslyeluted.
If these are undesirable then iterative substitutions can be made aroundthe lead molecular scaffold to improve sensitivity and selectivity.Screening combichem libraries with a proteome mining approach maximally exploits the serendipitous nature of drug discovery by accelerating the hit rate over aconventional screen by a factorial of the proteome that is bound.
In the case ofpurine-binding proteins this may be a factor of 103. Rational interpretation of theoutcome is enabled by protein microsequencing, use of genome project data andthe ability to instantaneously search the literature for relevance. We are currentlyusing proteome mining to refine new anti-hypertensive drugs that specifically target proteins identified by our functional proteomic screens.27327415 Investigations in Smooth Muscle Cell Physiology15.5AcknowledgementsThe authors wish to thank the past and present members of the Haystead laboratory. We also thank Applied Biosystems for their generous support of our laboratory.15.6References12345678Wasinger, V. C., Cordwell, S.
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