RecA Protein as Therapeutic Target Using Peptides & PNAs

描述（由申请人提供）：recA蛋白在细菌中具有双重功能，导致抗生素耐药性的发展，这使其成为减缓耐药病原体发病率的理想靶标。首先，RecA诱导细胞SOS系统响应DNA损伤，这可能导致易于出错的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，这可以将抗性基因从一个生物体传播到另一个生物体。在这两种情况下，RecA作为核蛋白丝是有活性的，其中多个单体沿着单链DNA从头到尾沿着聚合.我们的目标是抑制RecA的结构肽，模拟二聚化界面，并与肽核酸（PNA）设计抑制RecA翻译通过反义干扰。为此，我们设计的第一个肽抑制剂具有3 μ M的IC 50。在此资助下，界面残基将使用噬菌体展示技术随机化，以提高结合和特异性。已经设计了几种反义PNA，并将测试其体外抑制翻译。肽和PNA系统都将在大肠杆菌中测试体内活性。大肠杆菌中使用生物素或肽易位标签。

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.