Redox‐stable cyclic peptide inhibitors of the SPSB2–iNOS interaction

Abstract

Edited by Barry HalliwellSPSB2 mediates the proteasomal degradation of iNOS. Inhibitors of SPSB2-iNOS interaction are expected to prolong iNOS lifetime and thereby enhance killing of persistent pathogens. Here, we describe the synthesis and characterization of two redox-stable cyclized peptides containing the DINNN motif required for SPSB2 binding. Both analogues bind with low nanomolar affinity to the iNOS binding site on SPSB, as determined by SPR and 19 F NMR, and efficiently displace full-length iNOS from binding to SPSB2 in macrophage cell lysates. These peptides provide a foundation for future development of redox-stable, potent ligands for SPSB proteins as a potential novel class of anti-infectives.Keywords: anti-infective; cyclic peptide; inducible nitric oxide synthase; redox-stable; SPRY domain of the SOCS-box protein 2 SPSB2 protein plays a key role in modulating the lifetime of iNOS in macrophages during infection [1]. It acts as a negative regulator that recruits an E3 ubiquitin ligase complex that polyubiquitinates iNOS and thereby mediates its proteasomal degradation [1]. SPSB2-deficient macrophages exhibit prolonged iNOS expression, NO production and ultimately enhanced killing of persistent pathogens such as Leishmania major parasites and Mycobacterium tuberculosis, suggesting that inhibitors of the SPSB2-iNOS interaction have potential as a new class of anti-infectives [1].Kuang et al. [1] showed that a linear peptide from the disordered N-terminal region of iNOS, KEEK-DINNNVKKT, bound to SPSB2 with a K D of 13 nM, and that the most important residues mediating this interaction were DINNN, in particular the first, third and fifth residues of this sequence motif. In addition, 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 in the N-terminal region of iNOS (where the corresponding residues are Asp23, Asn25Abbreviations SPSB2, SPRY domain of the SOCS-box protein 2; iNOS, inducible nitric oxide synthase; SPR, surface plasmon

Applications of 19F-NMR in Fragment-Based Drug Discovery

“…The distance between the side chain nitrogen of W207 and one of the Asn residues of M1 was found to fluctuate between 3.1 and 10.3 Å throughout the simulations. These observations suggest that although mimetic M1 is bound to SPSB2, it did not lock the iNOS binding site of SPSB2 into a single bound conformation, which is consistent with the observation of a broader 19 F resonance for W207 in the presence of M1 compared with the cyclic peptides [29,30].…”

“…In support of this, 19 F-NMR has been used to confirm the binding sites of cyclic peptides containing the DINNN sequence [29,30]. The 19 F resonance of W207 shifted significantly downfield upon binding to the cyclic peptide, Ac-c[CVDINNNC]-NH 2 [29], confirming its binding to the iNOS binding site.…”

Nitric Oxide Synthase Inhibitors

“…Since this complex is blocked, the NO production from iNOS is prolonged, increasing the killing activity against pathogen microorganisms, making it an interesting anti-infective target [32]. Some cyclic peptidomimetic compounds were designed using this strategy, and the most potent compound 7 ( Figure 5) showed strong inhibition of SPSB2-iNOS complex in macrophage cell lysates and potent affinity value…”

Anti-Infective Peptides to Enhance the Host Innate Response: Design, Development and Delivery

“…As the bound conformation of the peptide has a reverse turn across the DINN sequence [18], we designed small peptides in which the native conformation was stabilized by bridging tethers [24,25]. Computer modelling confirmed the potential of both the disulfide-containing octapeptide Acc[CVDINNNC]-NH 2 (1) (Figure 3) [26] and the head to tail lactam-bridged heptapeptide c[WDINNNβA] (2) [27], both of which were readily synthesized. [26].…”

“…The disulfide-containing peptide 1 is susceptible to reduction [27]. While disulfide bridges can form in the cytoplasm under oxidative stress [29], for example during infection, cyclic peptide analogues that are stable to the normally reducing conditions of the cell cytoplasm would be likely to be more effective.…”