RecA Protein as Therapeutic Target Using Peptides & PNAs

描述(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产生，这使其成为减缓耐药病原体发病率的理想靶点。首先，RecA诱导细胞SOS系统对DNA损伤做出反应，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。第二，RecA催化同源重组，可以将耐药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都是一种核蛋白细丝，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界面的结构肽和旨在通过反义干扰抑制RecA翻译的肽核酸(PNA)来抑制RecA。为此，我们第一个设计的多肽抑制剂的IC50为3MU M。在这笔资金下，将使用噬菌体展示技术对界面残基进行随机化，以提高结合和特异性。已经设计了几种反义PNA，并将测试它们对翻译的体外抑制作用。将使用生物素或多肽易位标签在大肠杆菌中测试多肽和PNA系统的体内活性。

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.