ID: 229 Rational design of cysteine mutants of PAI-1 with very long half-lives

Abstract

Medical University of Ohio, Toledo, Ohio, USA Proteolytic activity initiated by uPA is commonly recognized as a critical factor in angiogenesis and metastasis. Many cancers overexpress uPA and the reduction of proteolytic activity has been proposed as a cancer treatment option. Indeed, uPA inhibitors have been shown to reduce angiogenesis and tumor growth. Thus, we want to identify novel inhibitors of uPA suitable for cancer treatment. We have chosen PAI-1, which inhibits the urokinase plasminogen activator. However, PAI-1 is not a stable molecule and converts itself into the latent form with a half-life in the range of t1/2~2 hours. Based on the known structure of active PAI-1, we have identified amino acids that can be substituted with a cysteine residue to produce disulfide bridges linking the top and bottom parts of strands A3 and A5 as well as sites within the helix D region in hopes of preventing a conversion into the latent PAI-1. We have created a total of seven cysteine mutants via point mutation (two to six point mutations) generating possible sites for a disulfide bridge formation at the top and bottom parts of A3 and A5, within the helix D region, or by a combination thereof. Desired mutations were introduced by PCR using appropriate primers. The mutant forms of PAI-1 containing the chitin binding intein tag were then purified using affinity chromatography wherein the intein tag is cleaved leaving the mutant PAI-1 protein. Cysteine mutations resulted in proteins with an extended half-life of PAI-1 from 2 to over 700 hours depending on the mutant. Novel PAI-1s were fully functional against uPA and showed activity in the in vitro model of angiogenesis e.g. inhibition of sprout formation. The mutant with the longest half-life (one that produced a disulfide bridge linking the top part of strands A3 and A5) was chosen for further study. VLHL PAI-1 expressed in the E. Coli vector produced a modest yield of 1 mg-purified protein from 1L of cell culture. Thus, we expressed it in the baculovirus vector, with an 6His purification tag that produced ~18 mg of PAI-1/1L. Two different forms were made: fully active VLHL PAI-1 and VLHLNS PAI-1 with an Arg369?Ala mutation in the P1 position, which will be used in future anticancer study as a negative control. VLHLNS PAI-1 mutation (Arg369?Ala) was introduced by PCR and gene was transferred into the baculovirus vector in the same way as VLHL PAI-1. We assume that the VLHLNS construct will remain in an active conformation as VLHL PAI-1 does, but will not have any inhibitory activity toward uPA. Our study suggests that both proteins are in an active conformation, however they convert into the latent form after treatment with reducing agents, which break the disulfide bridge. Fully active VLHL PAI-1 inhibits uPA as demonstrated by chromogenic assay (SPEC-TROZYME®) and forms the uPA/PAI-1 complex as shown on PAGE gel while VLHLNS PAI-1 does not. We believe that PAI-1s with extended half-lives are therapeutically desired in cancer treatment and Cys mutated PAI-1s could launch a new class of novel anti-cancer agents. Keywords: PAI-1, cysteine mutants. 170