Options for the stabilization of well-defined helical peptide drugs and basic

Options for the stabilization of well-defined helical peptide drugs and basic research tools have received considerable attention in the last decade. attachment to a target cell HIV entry is effected by fusion of the respective cell membranes mediated by gp41.[6] In the prefusogenic state gp41 Coumarin 7 is trimerized via an N-terminal heptad repeat (NHR) and an N-terminal fusion peptide inserts into the target membrane. The protein then undergoes a conformational rearrangement in which the C-peptide packs as Coumarin 7 an antiparallel helix against the surface of the NHR trimer.[7] Anchoring of the respective protein termini into viral and cell membranes ensures that this rearrangement requires membrane juxtaposition and it thus provides a mechanism for promoting fusion (Figure 1).[8] Peptides derived from this C-peptide helix (some as short as 12-16 residues) have been shown to bind the coiled coil and inhibit membrane fusion by HIV in human cells.[8-9] The best known of these Enfuvirtide (Fuzeon?) consists of 36-residues and is an FDA-approved treatment.[10] However like other short peptide drugs its chemical synthesis is extraordinarily expensive and it exhibits poor serum stability (t1/2 ~3.8 hours).[11] We reasoned that a helix-grafted alternative might retain similar specificity but have improved stability solubility and availability. Figure 1 (values of ~77 °C ~79 °C ~83 °C for 5-helix GLUE-Cpep and the complex respectively). The melting transition for the 1:1 sample is also highly cooperative further supporting a well-defined assembly. Figure 4 (were co- transformed with plasmids encoding 5-helix fused to the N- terminal half of spGFP (N-spGFP-5-helix) and one of two C-spGFP fusions: GLUE-Cpep or the gp41 C-peptide by itself. Interaction-dependent reassembly of GFP fragments (to Coumarin 7 generate a fluorescent signal) was measured by flow cytometry. Cells expressing either ligand construct are highly fluorescent in contrast to a control with nothing fused to C-spGFP (Figure 4D). We further characterized this interaction using an Enzyme-Linked Immunosorbant Assay (ELISA). The grafted GLUE binds 5-helix with slightly better affinity than the native C-peptide (Figure 4E columns 3 and 4 respectively) while the wild type GLUE exhibits no appreciable affinity (Figure 4E column 2) confirming the need for the grafted domain. This ELISA signal is observed even for a GLUE-Cpep sample that was pre-incubated with human serum (Supporting Information) confirming that the degradation-resistant form of the protein remains functional. Taken together these experiments show that the helix-grafted GLUE binds 5-helix in the context of a complex cellular milieu in a manner comparable to the native ligand and with improved serum longevity. Binding selectivity was assessed by measuring the amount of protein that is co-purified from expressing an untagged GLUE-Cpep (~17.1 kDa) and His6x-tagged 5-helix Mouse monoclonal to ENO2 (~25.4 kDa). Coumarin 7 As seen in Figure 4F the tagged 5-helix co-purifies with a single protein which was identified as GLUE-Cpep by mass spectrometry (Supporting Information Figure S2). The similar amounts of each co-purified protein (as determined by densitometry measurements of each protein band) further indicates that the complex involves a 1:1 ratio of proteins. The relatively miniscule levels of other co-purified cellular proteins indicates excellent selectivity for this interaction even in a complex cellular environment suggesting a reasonably strong mutual affinity. In conclusion we have demonstrated that the solvent exposed C-terminal alpha helix of the GLUE protein scaffold can be dramatically modified and extended so as to mimic the function of the gp41 C-peptide. ELISA and Coumarin 7 co-purification data indicate that GLUE-Cpep selectively binds 5-helix a protein that mimics the native C-peptide receptor. Unlike the isolated C-helix of Enfuvirtide GLUE-Cpep is soluble and well-folded in aqueous solution at room temperature (~25 °C) and is resistant to degradation in human serum at physiological temperature (~37 °C). Thus this protein drug lead overcomes challenges faced by traditional peptide reagents and may represent a new reagent for inhibition of HIV entry. Additionally helix-grafting onto PH and PH-like domains such as GLUE may be a general approach to the development of new reagents of interest to a diverse set of diseases that rely on helix-driven assembly. Finally GLUE-Cpep serves as a starting point for the generation of higher affinity and more selective mutants through the application of high-throughput screening or selection methods. Such experiments are currently underway and.