Article pubs.acs.org/jmc
A Potent Cyclic Peptide Targeting SPSB2 Protein as a Potential Antiinfective Agent Beow Keat Yap,† Eleanor W. W. Leung,† Hiromasa Yagi,† Charles A. Galea,† Sandeep Chhabra,† David K. Chalmers,† Sandra E. Nicholson,‡,§ Philip E. Thompson,† and Raymond S. Norton*,† †
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Victoria, Australia § The Department of Medical Biology, University of Melbourne, Parkville, Victoria 3052, Australia ‡
S Supporting Information *
ABSTRACT: The protein SPSB2 mediates proteosomal degradation of inducible nitric oxide synthase (iNOS). Inhibitors of SPSB2−iNOS interaction may prolong the lifetime of iNOS and thereby enhance the killing of persistent pathogens. We have designed a cyclic peptide, Ac-c[CVDINNNC]-NH2, containing the key sequence motif mediating the SPSB2−iNOS interaction, which binds to the iNOS binding site on SPSB2 with a Kd of 4.4 nM, as shown by SPR, [1H,15N]-HSQC, and 19F NMR. An in vitro assay on macrophage cell lysates showed complete inhibition of SPSB2−iNOS interactions by the cyclic peptide. Furthermore, its solution structure closely matched (backbone rmsd 1.21 Å) that of the SPSB2-bound linear DINNN peptide. The designed peptide was resistant to degradation by the proteases pepsin, trypsin, and chymotrypsin and stable in human plasma. This cyclic peptide exemplifies potentially a new class of anti-infective agents that acts on the host innate response, thereby avoiding the development of pathogen resistance.
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INTRODUCTION Nitric oxide (NO), which is produced biosynthetically from Larginine by inducible nitric oxide synthase (iNOS) in cells such as macrophages and dendritic cells, plays an important role in combating infections.1 Several studies have reported that the expression of iNOS was significantly enhanced in animal models during infections such as salmonellosis,2 tuberculosis,3 and leishmaniasis.4 In addition, iNOS knockout mice were found to succumb to infection by intracellular pathogens within 3−4 weeks.5 These observations indicate that iNOS and NO play an important role in host immunity. Recently, Kuang et al.6 identified SPRY domain-containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of Leishmania major parasites. It was also shown that a linear peptide from the disordered N-terminal region of iNOS, KEEKDINNNVKKT, bound to SPSB2 with a Kd of 13 nM, © XXXX American Chemical Society
and that the most important residues mediating this interaction were DINNN, in particular the first, third, and fifth residues of this sequence motif. Structural studies by X-ray crystallography revealed that the SPRY domain of SPSB2 binds to Asp184, Asn186, and Asn188 of the DINNN sequence of the VASA peptide, the same motif as that in the N-terminal region of iNOS (where the corresponding residues are Asp23, Asn25, and Asn27).7 In SPSB2, Tyr120 of the SPRY domain was found to be critical for binding to the iNOS peptide,6 with Arg100, Gly101, Thr102, His103, Leu123, Leu124, Leu125, Ser126, Asn127, Ser128, Val206, and Trp207 also contributing to binding.6 In addition to SPSB2, SPSB1 and SPSB4 were also found to be important in the negative regulation of iNOS expression in macrophages.8 Both SPSB1 and SPSB4 bound to the same DINNN sequence in iNOS as SPSB2, suggesting that inhibitors that mimic the iNOS peptide and target the SPRY domain of the SPSB proteins would be advantageous in disrupting the Received: April 17, 2014
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dx.doi.org/10.1021/jm500596j | J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Article
Figure 1. Ac-c[CVDINNNC]-NH2 interacts with SPSB2. (A) SPR sensorgram of immobilized SPSB2 exposed to increasing concentrations of cyclic peptide Ac-c[CVDINNNC]-NH2 in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20, pH 7.4, at 25 °C. (B) kon and koff of the cyclic peptide Ac-c[CVDINNNC]-NH2, its reduced counterpart, and the linear peptide Ac-DINNN-NH2 estimated from the reference subtracted sensorgrams fitted to a steady state 1:1 interaction model. (C) ITC raw thermogram data (upper panel) and binding isotherm (lower panel) for the cyclic peptide in complex with SPSB2 protein in 50 mM HEPES, 50 mM NaCl, pH 7.4, at 25 °C. The data were fitted using a single-site binding model.
highly stable against a range of proteolytic enzymes (pepsin, trypsin, and α-chymotrypsin) as well as in human plasma.
iNOS interaction with all of these SPSB proteins, thus prolonging iNOS expression in cells where iNOS is being produced in response to infection. As SPSB proteins were found to bind only to iNOS, but not endothelial NOS (eNOS) and neuronal NOS (nNOS),9 this strategy would limit any potential toxicity associated with excessive systemic NO. Linear peptides with free carboxyl and amino terminals are likely to be unstable in vivo as they are susceptible to degradation by both endo- and exopeptidases. Moreover, they are generally unstructured and flexible in solution, rendering them entropically unfavorable for receptor binding and biological activity. Consistent with this, cyclic peptides have been found to be more resistant to enzymatic degradation and proteolytic digestion by proteases than their linear counterparts, with improved potency as well as high selectivity and binding affinity.10 In this study, we describe the design and synthesis of a cyclic peptide mimicking the SPSB2-bound conformation of the linear sequence DINNN. Structural analysis by NMR indicated that the solution structure of the designed peptide closely matched the bound state, and binding studies with SPR and ITC showed that it bound with high affinity to SPSB2. An inhibition assay with macrophage lysate confirmed that the cyclic peptide is a potent inhibitor of the SPSB2−iNOS interaction. The peptide was also found to be
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RESULTS AND DISCUSSION Design of Cyclic Peptide Ac-c[CVDINNNC]-NH2. Cyclic peptides were designed based on the SPSB2-bound conformation of DINNN,7 which is a Type I β-turn conformation with the distance between Cα(i) and Cα(i + 3) being 50% of the calculated structures and supported by low temperature coefficient (magnitude
