Design, Synthesis, and Characterization of Cyclic Peptidomimetics of the Inducible Nitric Oxide Synthase Binding Epitope That Disrupt the Protein–Protein Interaction Involving SPRY Domain-Containing Suppressor of Cytokine Signaling Box Protein (SPSB) 2 and Inducible Nitric Oxide Synthase

Abstract

SPRY domain-containing suppressor of cytokine signaling box protein (SPSB) 2-deficient macrophages have been found to exhibit prolonged expression of inducible nitric oxide synthase (iNOS) and enhanced killing of persistent pathogens, suggesting that inhibitors of the SPSB2-iNOS interaction have potential as novel anti-infectives. In this study, we describe the design, synthesis, and characterization of cyclic peptidomimetic inhibitors of the SPSB2-iNOS interaction constrained by organic linkers to improve stability and druggability. SPR, ITC, and (19)F NMR analyses revealed that the most potent cyclic peptidomimetic bound to the iNOS binding site of SPSB2 with low nanomolar affinity (KD 29 nM), a 10-fold improvement over that of the linear peptide DINNN (KD 318 nM), and showed strong inhibition of SPSB2-iNOS interaction in macrophage cell lysates. This study exemplifies a novel approach to cyclize a Type II β-turn linear peptide and provides a foundation for future development of this group of inhibitors as new anti-infectives.

Molecular design opportunities presented by solvent‐exposed regions of target proteins

“…Importantly, cPTs exhibited high stability to trypsin and chymotrypsin, in contrast with the significant susceptibility of the corresponding linear PTs. 93,94 This structure-guided conformational restriction approach is also nicely illustrated by the identification of cyclic peptidomimetic inhibitors of inducible nitric oxide synthase, 95 inhibitors of tricyclic indole diazepinone myeloid cell leukemia-1 96 and the WD repeat domain 5 protein inhibitors (as exemplified by 124 and 125) ( Figure 19E). 97,98 Based on the linear peptidomimetic 123, structure-based design resulted in the identification of macrocyclic (Figure 19).…”

“…This structure‐guided conformational restriction approach is also nicely illustrated by the identification of cyclic peptidomimetic inhibitors of inducible nitric oxide synthase, inhibitors of tricyclic indole diazepinone myeloid cell leukemia‐1 and the WD repeat domain 5 protein inhibitors (as exemplified by 124 and 125 ) (Figure E) . Based on the linear peptidomimetic 123 , structure‐based design resulted in the identification of macrocyclic peptidomimetics 124 and 125 (MM‐589), which could bind to WD repeat domain 5 (WDR5) and inhibit the WDR5‐mixed lineage leukemia (MLL) protein‐protein interaction.…”

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

“…The power of 19 F-NMR is well illustrated by our recent studies on the development of peptide mimetics that also target the iNOS binding site on SPSB2 [32]. As shown in Figure 2, the binding of mimetics M1 and M2 to 5-F-Trp-SPSB2 caused similar downfield shifts of the W207 resonance to that caused by c[CVDINNNC]-NH 2 (not shown), although the chemical shift changes were slightly larger and the peak widths were broader for M1 and M2 (Figure 2).…”

Neue Modalitäten für schwierige Zielstrukturen in der Wirkstoffentwicklung

“…[118] Die DINNN-Sequenz und das Disulfidbrückenanalogon banden SPSB2 mit Bindungsaffi-nitäten von 318 bzw.4 .4 nm.U mm çglichen Stabilitätsproblemen der Disulfidbrücke vorzubeugen, wurde eine Reihe von organischen Gerüststrukturen entwickelt, die die N-und C-Termini der linearen Bindungssequenz verknüpfen sollten. [118] Die DINNN-Sequenz und das Disulfidbrückenanalogon banden SPSB2 mit Bindungsaffi-nitäten von 318 bzw.4 .4 nm.U mm çglichen Stabilitätsproblemen der Disulfidbrücke vorzubeugen, wurde eine Reihe von organischen Gerüststrukturen entwickelt, die die N-und C-Termini der linearen Bindungssequenz verknüpfen sollten.…”

“…Durch die Nachahmung des N-terminalen Te trapeptids des Smac-Proteins,e ines natürlichen IAP-Inhibitors,w urden über ein Tr iazol cyclisierte Peptidsequenzen entdeckt, indem man DNA-kodierte Bibliotheken gegen die BIR-2-und BIR-3-Domänen von XIAP durchmusterte [117]. Diese Tr effer wurden optimiert, um eine gute AffinitätzuBIR-2 und BIR-3 zu erreichen (139 bzw.1 60 nm ;A bbildung 7c), wiesen aber eine zehnfach geringere funktionale Aktivitäta uf,w ie durch die Wiederherstellung der Caspase-3-Aktivitätgezeigt wurde.Füre inen neuartigen hybriden Makrocyclus,d er gegen die PPI zwischen der induzierbaren Stickstoffmonoxid-Synthase (iNOS) und dem SOCS-Box-Protein 2( SPSB2) gerichtet ist, wurde ein lineares Peptid mit einem DINNN-Bindungsmotiv aus der N-terminalen Region von iNOS in einer b-Schleife cyclisiert [118]. Die DINNN-Sequenz und das Disulfidbrückenanalogon banden SPSB2 mit Bindungsaffi-nitäten von 318 bzw.4 .4 nm.U mm çglichen Stabilitätsproblemen der Disulfidbrücke vorzubeugen, wurde eine Reihe von organischen Gerüststrukturen entwickelt, die die N-und C-Termini der linearen Bindungssequenz verknüpfen sollten.…”