Oxetane Grafts Installed Site‐Selectively on Native Disulfides to Enhance Protein Stability and Activity In Vivo

Abstract A four‐membered oxygen ring (oxetane) can be readily grafted into native peptides and proteins through site‐selective bis‐alkylation of cysteine residues present as disulfides under mild and biocompatible conditions. The selective installation of the oxetane graft enhances stability and activity, as demonstrated for a range of biologically relevant cyclic peptides, including somatostatin, proteins, and antibodies, such as a Fab arm of the antibody Herceptin and a designed antibody DesAb‐Aβ against the human Amyloid‐β peptide. Oxetane grafting of the genetically detoxified diphtheria toxin CRM197 improves significantly the immunogenicity of this protein in mice, which illustrates the general utility of this strategy to modulate the stability and biological activity of therapeutic proteins containing disulfides in their structures.

Abstract: Af our-membered oxygen ring (oxetane) can be readily grafted into native peptides and proteins through siteselective bis-alkylation of cysteine residues present as disulfides under mild and biocompatible conditions.T he selective installation of the oxetane graft enhances stability and activity, as demonstrated for ar ange of biologically relevant cyclic peptides,including somatostatin, proteins,and antibodies,such as aF ab arm of the antibody Herceptin and ad esigned antibody DesAb-Ab against the human Amyloid-b peptide. Oxetane grafting of the genetically detoxified diphtheria toxin CRM 197 improves significantly the immunogenicity of this protein in mice,w hich illustrates the general utility of this strategy to modulate the stability and biological activity of therapeutic proteins containing disulfides in their structures.
The rational modification of the structure of peptides and proteins offers aw ide range of opportunities for the modulation of their biological activity. [1] Many efforts have been made to develop strategies that induce such conforma-tional changes and modulation. Towards this end, macrocyclization and stapling have emerged as useful tactics to chemically manipulate peptides and proteins,increasing their proteolytic stability,cell permeability,and producing changes in polarity,binding activity,and pharmacokinetic properties. [2] During the last years,d ifferent approaches have been developed for the covalent tethering of the side chains of natural or non-canonical amino acids. [2] Considering natural residues,c ysteine (Cys) has been the residue of choice for stapling through alkylation, [3] arylation, [4] cycloaddition, [4b] and disulfide forming reactions both at native [5] or engineered [6] Cys residues.M ore recently,n itrogen arylation has also been shown to be au seful strategy for macrocyclization of lysine residues on peptides. [7] Otherwise,e fficient macrocyclization of linear peptides through the formation of an oxadiazole has also been reported. [8] However,alarge number of stapling/macrocyclization/re-bridging strategies consist of the introduction of non-canonical amino acids and their subsequent ligation by ring-closing metathesis, [9] lactamization, [10] or cycloaddition reactions. [2c, 4b] Common to many of these strategies is either the requirement for complicated orthogonal protection procedures,s equence engineering,the appendage of bulky/constrained linkers between the two residues,orthe use of organic solvents.These conditions have limited, for instance,t he application of such methods for the stapling of residues on intact, full-length proteins to impart structural conformational constraints leading to enhanced stability and activity.T hus,t here remains an eed for simple and robust strategies for stapling native peptides and proteins.
Herein we report amethod for site-selective peptide and protein stapling through the bis-alkylation of the sulfhydryl side chain of Cys residues resulting from disulfide reduction, using commercially available 3,3-bis(bromomethyl)oxetane 1 (Figure 1). Oxetanes have become common motifs in drug design due to their ability to modulate parameters including solubility,b asicity,l ipophilicity,a nd metabolic stability. [11] While there are examples of the modification of small peptides with oxetanes, [12] their incorporation and modulation of the structure and activity of complex biomolecules [13] remain mostly unexplored. Ar equirement for ad irect method to graft Cys residues present as disulfides on proteins is the compatibility of the reagent with ar educing agent, such as tris(2-carboxyethyl)phosphine (TCEP). Importantly,w ef ound that am odel pentapeptide bearing two Cys residues reacted with 1 in the presence of TCEP to afford the corresponding stapled cyclic peptide in 75 %y ield (see the Supporting Information, Figures S2-S5, S34-S39, 48-49 for characterization and discussion of structural features). We then explored the use of 1 to graft two cyclic and biologically relevant peptides: somatostatin 2 and its analogue octreotide 4,w hich can be used for imaging and treating neuroendocrine tumors. [14] Peptides 2 and 4 were reacted simultaneously with TCEP and oxetane 1 in a1:9 mixture of DMF/H 2 Oat258 8Cfor 12 h. After HPLC purification, the grafted cyclic peptides 3 and 5 were obtained in 76 %a nd 81 %y ield, respectively (Figure 2a). Thea ffinity of these derivatives to the natural somatostatin receptor 2( SSTR2) was experimentally determined by tryptophan fluorescence spectroscopy (Supporting Information, Figure S1). While octreotide 4 and surrogate peptide 5 showed as imilar affinity against this receptor, grafted somatostatin 3 displayed improved binding properties,s howing a4 -fold enhancement in K D value (Figure 2b). Thei mprovement of binding activity is ac onsiderable advantage of the incorporation of the oxetane graft when compared, for instance,with the recently reported methylene thioacetal that led to ad ecrease in binding affinity to SSTR2. [3b] Interestingly,0 .5 msM Ds imulations performed on these derivatives in explicit water and using ff14SB amber force-field [15] suggested that octreotide 4 and its stapled derivative 5 presented as imilar conformational behavior in solution (Figure 2d;S upporting Information, Figure S8), [16] displaying an equilibrium between antiparallel b-sheet structures and conformations in which the C-terminal residues form a3 10 helix-like fold, as reported in DMSO solution. In contrast, stapled somatostatin 3 was more rigid and displayed am ore defined conformation in solution than 2 (Figure 2c; Supporting Information, Figures S6 and S7). [17] In fact, 3 showed ac losely related b-sheet arrangement in solution stabilized by at ypical hydrogen bond network, which is apparently ideal for amore efficient binding to the receptor. Finally,a nalysis of the stability of 3 and 5 both in human plasma as well in the presence glutathione (GSH) showed that the oxetane grafted peptides remain intact under these conditions (Supporting Information, Figures S13-S18).
To initially test the potential of using this one-pot, siteselective bis-alkylation oxetane stapling method directly on proteins,w ec hose thioredoxin (Trx) as am odel protein that features an aturally occurring,s olvent-exposed disulfide bond. We could reduce and staple the disulfide bond in as traightforward manner through selective bis-alkylation with 1 in the presence of TCEP and 10 %D MF in sodium phosphate buffer at pH 8.5. Complete conversion was achieved after 24 hat378 8C, as confirmed by HPLC-MS analysis (Supporting Information, Figures S19 and S20). Furthermore, analysis of the CD spectra of the native and stapled Trx-1 (Supporting Information, Figure S22) indicated that both molecules present very similar conformational preferences in solution. Although this is supported by MD simulations performed on both proteins in explicit water, the calculations indicate as mall increase in flexibility for the peptide backbone of stapled Tr x-1 (Supporting Information, Figure S11). This result may be explain attending to the greater SÀS distance in Thrx-1 when compared to the native Tr x(4.18 and 2.04 ,respectively). Finally,weconfirmed the suppression of Tr xr edox activity [18] through the selective and covalent disulfide stapling (Supporting Information, Figure S21).
Next, we demonstrated the utility of the oxetane graft to build stapled antibodies.F irst, the exposed disulfide bond tethering the heavy and light chains of aF ab fragment of Herceptin (Fab-Her), an antibody currently used to treat Her2 + breast cancer patients, [19] was readily stapled using 1 under aqueous buffered conditions in the presence of TCEP at pH 8.5 and at 37 8 8C (Figures 3a,b;Supporting Information, Figures S23 and S24). Theoxetane stapled Fab-Her-1,unlike the disulfide native antibody,w as stable under reducing conditions and in human plasma (Supporting Information, Figures S25 and S26). This stability is akey aspect of antibody therapeutics design as thiol-exchange reactions in plasma lowers efficacya nd adds side-toxicity. [20] Importantly,arelatively small but significant increase in binding affinity to the Her2 receptor,a sd etermined by bio-layer interferometry (BLI) experiments (Figure 3d;S upporting Information, Figure S27), was observed for Fab-Her-1 when compared with the native antibody.N ext, we extended our stapling strategy to the antibody DesAb-Ab 3-9 ,w hich was designed to target the region 3-9 of human Amyloid-b (Ab42) peptide,t he aggregation of which is ah allmark of Alzheimersdisease. [21] This antibody features ac hallenging,h indered intra-domain disulfide typical of VH domains.O fn ote,c omplete conversion into the oxetane grafted antibody DesAb-Ab 3-9 -1 was   Figures S28 and S29). Owing to the fact that the disulfide is deeply buried, an excess of TCEP (40 equiv) and longer reaction times were required (Supporting Information). Unlike the reduced antibody that readily reacts with thiolspecific Elmansr eagent, the stapled DesAb-Ab 3-9 -1 did not react suggesting complete consumption of the reduced Cys during stapling (Supporting Information). Finally,analysis of secondary structural content by CD showed no significant differences between the original and stapled antibodies (Figure 3e). MD simulations performed on a3 Dm odel of DesAb-Ab 3-9 ,p reviously generated using ABodyBuilder, [22] and on the stapled derivative DesAb-Ab 3-9 -1,s uggest that, although the 3D structure is maintained upon the chemical modification, the oxetane motif provokes as lightly increase in the degree of flexibility ( Supporting Information, Figure S31). Collectively,t hese data demonstrate the suitability of the oxetane motif to staple solvent accessible disulfide bonds on proteins with minimal secondary structure alterations.
To demonstrate the practical application of our method to therapeutic proteins,w ei nvestigated the effects of the selective introduction of the oxetane staple into the genetically detoxified diphtheria toxin CRM 197 ,which features four Cys residues in the form of two disulfides.R ecently,i th as been shown that antibodies against CRM 197 neutralized diphtheria toxin in HIV infected young individuals. [23] Furthermore,C RM 197 is ac linically used carrier in many glycoconjugate vaccines. [24] Previous structural studies showed that only the disulfide C186-C201 connecting the fragment A( Cd omain) and B( T/R domain) of CRM 197 is selectively reduced in the presence of the highly hindered C461-C471 disulfide upon treatment with dithiothreitol. [24b] Addition of aslight excess of TCEP to CRM 197 under aqueous buffered conditions at pH 8.5 and 37 8 8C, followed by an excess of 1,led to the introduction of one oxetane graft (Figure 4a,b and the Supporting Information, Figure S32 for mass spectrometry analysis), presumably at C186-C201 according to our previous findings. [25] Theimpact of the installation of the oxetane moiety into CRM 197 on its structure and thermal stability was studied by CD and differential scanning calorimetry (DSC) analysis,r espectively,a nd compared with the native protein (Figure 4c-e). We found that both the far and near UV CD spectra of CRM 197 -1 were nearly identical to those of CRM 197 ,w hich indicates that the 3D structure is preserved upon the chemical stapling. TheD SC curves also corroborate this finding.A lthough CRM 197 -1 exhibited ab roader DSC peak when compared to the sharp change in  To evaluate the biological effects resulting from the introduction of the oxetane staple into CRM 197 ,w ef irst assayed the capacity of competing with the binding of anti-CRM 197 serum to the proteins.W ef ound that the stapled CRM 197 -1 induces an inhibition that was slightly lower compared to the unmodified protein (Figure 4f). In contrast, amuch better inhibition of the binding to acommercial antidiphtheria toxoid human recombinant monoclonal antibody was observed for CRM 197 -1 compared to the unmodified protein (Supporting Information, Figure S33a). To ascertain that protein epitopes were not impaired by the chemical modification of the disulfide bond, groups of 8B ALB/c mice were immunized with both unmodified CRM 197 and the stapled CRM 197 -1 (Figure 4g) Figure S33b). These data, together with 200 ns MD simulations performed on both proteins (Supporting Information, Figure S12), suggest that the oxetane bridging of the disulfide bond does not cause relevant structural modifications on the protein but results in improved immunogenic activity in vivo,m ost likely through chemical stabilization of the antigen against proteases and/or other degradation factors.
In summary,w eh ave presented an efficient method for oxetane stapling of Cys residues present as native disulfides on peptides and proteins under mild and biocompatible aqueous conditions.T he four-membered oxetane ring has an ideal distance to enable direct stapling of native disulfides on several protein scaffolds,including antibodies.T his approach is however dependent on solvent accessibility of the disulfide within the protein of interest. Furthermore,a nd unlike current protocols,t his method does not require prior sequence engineering neither purification after the disulfide reduction step.The selective installation of the oxetane motif enables stabilization of folded structures and results in disulfide-grafted products with enhanced bioactivity that are stable under biological conditions.W edemonstrate the value of oxetane graft installation on protein through the regioselective disulfide stapling of the protein carrier CRM 197 that showed as ignificant increase in its immunogenicity in vivo. Because many therapeutic proteins feature Cys residues in the form of disulfide bonds,w ea nticipate that their direct modulation through oxetane grafting can, in principle,b e used as ag eneral strategy to enhance their in vivo stability and to fine-tune their structure for optimal pharmacokinetics and activity.