RecA Protein as Therapeutic Target Using Peptides & PNAs

描述（由申请人提供）：recA蛋白在细菌中具有导致抗生素耐药性发展的双重功能，这使其成为减缓耐药病原体发生率的理想靶点。首先，RecA诱导细胞SOS系统响应DNA损伤，这可能导致容易出错的DNA修复和有利的蛋白质突变，以逃避抗生素的挑战。其次，RecA催化同源重组，使抗性基因在生物间传播。在这两种情况下，RecA作为一个核蛋白丝是活跃的，其中多个单体沿着单链DNA从头到尾聚合。我们的目标是用模拟二聚化界面的结构肽和设计用于通过反义干扰抑制RecA翻译的肽核酸（PNAs）来抑制RecA。为此，我们设计的第一个肽抑制剂的IC50为3 μ m。在这笔资金的支持下，界面残基将随机化，使用噬菌体展示技术来提高结合和特异性。已经设计了几种反义PNAs，并将对它们的体外翻译抑制作用进行测试。肽和PNA系统都将使用生物素或肽易位标签在大肠杆菌中进行体内活性测试。

DESCRIPTION (provided by applicant): The recA protein has dual functions in bacteria leading to the development of antibiotic resistance, which makes it an ideal target for slowing the incidence of drug resistant pathogens. First, RecA induces the cellular SOS system in response to DNA damage, which can lead to error-prone DNA repair and advantageous protein mutations to escape the antibiotic challenge. Second, RecA catalyzes homologous recombination, which can spread resistance genes from organism to organism. In both instances, RecA is active as a nucleoprotein filament in which multiple monomers are polymerized head to tail along single- stranded DNA. We aim to inhibit RecA with structured peptides that mimic the dimerization interface, and with peptide nucleic acids (PNAs) designed to inhibit RecA translation through antisense interference. To these ends, our first designed peptide inhibitor has an IC50 of 3 mu M. Under this funding, interfacial residues will be randomized Using phage display techniques to improve the binding and specificity. Several antisense PNAs have been designed and will be tested for their in vitro inhibition of translation. Both peptide and PNA systems will be tested for in vivo activity in E. coli using either biotin or peptide translocation tags.