ABSTRACT
The budding of HIV from infected cells is driven by the protein-protein interaction between the p6 domain of the HIV Gag protein and the UEV domain of the human TSG101 protein. We report the development of a cyclic peptide inhibitor of the p6/UEV interaction, from a non cell-permeable parent that was identified in a SICLOPPS screen. Amino acids critical for the activity of the parent cyclic peptide were uncovered using alanine-scanning, and a series of non-natural analogues synthesized and assessed. The most potent molecule disrupts the p6/UEV interaction with an IC50 of 6.17 ± 0.24 µM by binding to UEV with a Kd of 11.9 ± 2.8 µM. This compound is cell permeable and active in a cellular virus-like particle budding assay with an IC50 of â¼2 µM. This work further demonstrates the relative simplicity with which the potency and activity of cyclic peptides identified from SICLOPPS libraries can be optimized.
Subject(s)
DNA-Binding Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Peptides, Cyclic/pharmacology , Protein Binding/drug effects , Transcription Factors/metabolism , gag Gene Products, Human Immunodeficiency Virus/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Drug Development , Endosomal Sorting Complexes Required for Transport/chemistry , Endosomal Sorting Complexes Required for Transport/genetics , Escherichia coli/genetics , HEK293 Cells , HIV/chemistry , HIV/drug effects , HeLa Cells , Humans , Peptides, Cyclic/toxicity , Protein Domains , Transcription Factors/chemistry , Transcription Factors/genetics , Virus Release/drug effectsABSTRACT
In this article we describe the production and screening of a genetically encoded library of 106 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.
Subject(s)
DNA-Binding Proteins/chemistry , Endosomal Sorting Complexes Required for Transport/chemistry , Escherichia coli/metabolism , Peptide Synthases/chemistry , Peptides/chemistry , Transcription Factors/chemistry , gag Gene Products, Human Immunodeficiency Virus/chemistry , Crystallography, X-Ray , ErbB Receptors/metabolism , Escherichia coli Proteins/chemistry , Ethylmaleimide/chemistry , Gene Library , HEK293 Cells , HIV , HeLa Cells , Humans , Inhibitory Concentration 50 , Iodoacetamide/chemistry , Peptide Hydrolases/chemistry , Plasmids , Protein Domains , Protein Interaction Mapping , Substrate SpecificityABSTRACT
The identification of initial hits is a crucial stage in the drug discovery process. Although many projects adopt high-throughput screening of small-molecule libraries at this stage, there is significant potential for screening libraries of macromolecules created using chemical biology approaches. Not only can the production of the library be directly interfaced with a cell-based assay, but these libraries also require significantly fewer resources to generate and maintain. In this context, cyclic peptides are increasingly viewed as ideal scaffolds and have proven capability against challenging targets such as protein-protein interactions. Here we discuss a range of methods used for the creation of cyclic peptide libraries and detail examples of their successful implementation.
Subject(s)
Drug Discovery/methods , Peptide Library , Peptides, Cyclic , Cell Surface Display Techniques , In Vitro Techniques , Protein BiosynthesisABSTRACT
Cyclic peptides are an emerging class of molecular therapeutics that are increasingly viewed as ideal backbones for modulation of protein-protein interactions. A split-intein based method, termed SICLOPPS, enables the rapid generation of genetically encoded cyclic peptide libraries of around a hundred million members. Here we review recent approaches using SICLOPPS for the discovery of bioactive compounds.
