title: Computational and biochemical design of a nanopore cleavable by a cancer-secreted enzyme.
abstract: Many proteinaceous macromolecules selectively transport substrates across lipid bilayers and effectively serve as gated nanopores. Here, we engineered cleavage-site motifs for human matrix metalloprotease 7 (MMP-7) into the extracellular and pore-constricting loops of OprD, a bacterial substrate-specific transmembrane channel. Concurrent removal of two extracellular loops allowed MMP-7 to access and hydrolyze a cleavage-site motif engineered within the pore's major constricting loop, in both membrane-incorporated and detergent-solubilized OprDs. Import of antibiotics by the engineered OprDs into living bacteria pointed to their proper folding and integration in biological membranes. Purified engineered OprDs were also found to be properly folded in detergent. Hence, this study demonstrates the design of nanopores with a constriction cleavable by tumor-secreted enzymes (like MMP-7) for their potential incorporation in lipid-based nanoparticles to accelerate drug release at the tumor site.
authors.affiliation: Laboratory of Ion Channels, The Mina and Everard Goodman Faculty of Life Sciences, The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 52900 (Israel).
authors.forename: Ilya
authors.initials: I
authors.lastname: Pittel
authors.forename: Naomi
authors.initials: N
authors.lastname: Alper
authors.forename: Shiran
authors.initials: S
authors.lastname: Yonai
authors.forename: Shani
authors.initials: S
authors.lastname: Basch
authors.forename: Leah
authors.initials: L
authors.lastname: Blum
authors.forename: Ayelet
authors.initials: A
authors.lastname: Bachur
authors.forename: Yoav
authors.initials: Y
authors.lastname: Paas
mesh_terms: D020816:Amino Acid Motifs; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D016503:Drug Delivery Systems; D006801:Humans; D015378:Imipenem / Q000494:pharmacology; D020783:Matrix Metalloproteinase 7 / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D009154:Mutation; D058608:Nanopores; D018272:Porins / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
chemical_list: D003902:Detergents; D018272:Porins; D011994:Recombinant Proteins; C077157:OprD protein, Pseudomonas aeruginosa; D015378:Imipenem; D020783:Matrix Metalloproteinase 7
keywords: channel; drug release; membrane proteins; nanopores; protein engineering