Inhibitors of SPSB-iNOS interactions as a potential novel class of anti-infectives

The SPRY-domain of the SOCS box protein 2 (SPSB2) plays an important role in the proteasomal degradation of inducible nitric oxide synthase (iNOS). SPSB2 knockout mice show prolonged expression of iNOS and enhanced killing of persistent pathogens such as Mycobacterium tuberculosis and Leishmania major, suggesting that inhibitors of the SPSB2-iNOS interaction represent a potential novel class of anti-infectives. In this study, attempts to discover small molecule inhibitors of SPSB2-iNOS interaction were performed using in silico guided fragment-based drug design (FBDD) approach. The best fragment hit STK441224, however, was found to bind promiscuously to SPSB2 by saturation transfer difference spectroscopy (STD), Carr-Purcell-Meiboom-Gill (CPMG), ¹⁹ F and [¹ H,¹⁵N]-HSQC NMR experiments, with an estimated KD of 1.8 mM by surface plasmon resonance (SPR). Further predictions by SiteMap and FTMap revealed that the iNOS binding site of SPSB2 is less druggable, explaining the poor outcome from the current FBDD campaign. Thus, other approaches to discover potent and specific inhibitors of the SPSB2-iNOS interaction were explored. Utilising an in silico structure-based drug design approach, a disulphide-bridged cyclic peptide Ac-c[CVDINNNC]-NH2 was designed and synthesised. It was found to bind to the iNOS binding site on SPSB2 with a KD of 4.4 nM, as shown by SPR, [¹H,¹⁵N]-HSQC and ¹⁹F NMR experiments, with approximately 70-fold improvement in affinity, compared to the linear peptide DINNN (KD ≈ 318 nM). An in vitro assay on macrophage cell lysates further showed a complete inhibition of SPSB2-iNOS interactions by the cyclic peptide. In addition, the solution structure of the cyclic peptide was found to closely match that of the crystal structure of SPSB2-bound linear peptide DINNN with a backbone RMSD of 1.21 Å. The designed peptide was also found to be stable against pepsin, trypsin and α-chymotrypsin, and in human plasma. The disulphide-bridged cyclic peptide, however, is reductively labile. To generate redox-stable inhibitors of SPSB2-iNOS interaction that would retain activity in the cell cytoplasm, two cyclic peptide analogues, one containing a thioether bridge (CP1) and the other a lactam bridge (CP2), as well as four cyclic peptidomimetics (M1-M4), incorporating organic moieties as cyclisation linkers, were generated. All analogues were able to bind to the iNOS binding site of SPSB2, with five of the six analogues binding with stronger affinities (3-15 fold) than the linear peptide DINNN (CP1, KD 31 nM; CP2, KD 21 nM; M1, KD 29 nM; M2, KD 99 nM; M3, KD 54 nM; M4, KD 465 nM), as determined by 19F NMR and SPR, respectively. All analogues were able to compete with full-length iNOS for binding to SPSB2 in macrophage cell lysates. As CP2 is the most potent redox-stable analogue, with more sites for derivatisation and is easier to synthesise compared to other analogues, CP2 is being used as the template to generate analogues for macrophage-targeted delivery studies. Binding studies by SPR and 19F NMR revealed an approximately 5-fold improvement in binding affinity of rhodamine B isothiocyanate (RBITC)-conjugated cyclic peptide analogue CP4 to the iNOS binding site of SPSB2 (KD ≈ 4 nM), while the oligohistidine-conjugated analogues CP5 and CP6 showed a modest 1-2 fold drop in their binding affinities to SPSB2 compared to CP2 (KD ≈ 33-51 nM). Imaging studies of mannose or GalNAc glycopolymer-conjugated analogues of CP4 (i.e. CP7 and CP8, respectively) by confocal laser scanning microscopy revealed that both analogues were taken up by bone marrow-derived macrophages but not HEK 293 cells. On the other hand, oligohistidine-conjugated analogue CP6 showed signs of endosomal escape after 9 h of incubation, although coincident signs of cell necrosis or apoptosis were observed in some of these macrophages. In summary, several potent and stable cyclic peptide and peptidomimetic inhibitors of SPSB2-iNOS interactions were identified in this study. One of these analogues was able to be derivatised for macrophage-targeted delivery studies without negatively affecting their binding to SPSB2. This study also showed that both mannose and GalNAc glycopolymers are viable choices for macrophage-targeted delivery although more work to improve the endosomal escape of this new class of cyclic peptide inhibitors of SPSB2-iNOS interaction to the cytoplasm of macrophages without causing cell injury is needed

14-3-3σ and Its Modulators in Cancer

14-3-3σ is an acidic homodimer protein with more than one hundred different protein partners associated with oncogenic signaling and cell cycle regulation. This review aims to highlight the crucial role of 14-3-3σ in controlling tumor growth and apoptosis and provide a detailed discussion on the structure–activity relationship and binding interactions of the most recent 14-3-3σ protein-protein interaction (PPI) modulators reported to date, which has not been reviewed previously. This includes the new fusicoccanes stabilizers (FC-NAc, DP-005), fragment stabilizers (TCF521-123, TCF521-129, AZ-003, AZ-008), phosphate-based inhibitors (IMP, PLP), peptide inhibitors (2a–d), as well as inhibitors from natural sources (85531185, 95911592). Additionally, this review will also include the discussions of the recent efforts by a different group of researchers for understanding the binding mechanisms of existing 14-3-3σ PPI modulators. The strategies and state-of-the-art techniques applied by various group of researchers in the discovery of a different chemical class of 14-3-3σ modulators for cancer are also briefly discussed in this review, which can be used as a guide in the development of new 14-3-3σ modulators in the near future

Identification of peptide binding sequence of TRIM25 on 14-3-3σ by bioinformatics and biophysical techniques

14-3-3σ protein is one of the seven isoforms from the highly conserved eukaryotic 14-3-3 protein family. Downregulation of 14-3-3σ expression has been observed in various tumors. TRIM25 is responsible for the proteolytic degradation of 14-3-3σ, in which abrogation of TRIM25 suppressed tumor growth through 14-3-3σ upregulation. However, to date, the exact 14-3-3σ interacting residues of TRIM25 have yet to be resolved. Thus, this study attempts to identify the peptide binding sequence of TRIM25 on 14-3-3σ via both bioinformatics and biophysical techniques. Multiple sequence alignment of the CC domain of TRIM25 revealed five potential peptide binding sequences (Peptide 1–5). Nuclear magnetic resonance (NMR) assay (1H CPMG) identified Peptide 1 as an important sequence for binding to 14-3-3σ. Competition NMR assay suggested that Peptide 1 binds to the amphipathic pocket of 14-3-3σ with an estimated KD of 116.4 µM by isothermal titration calorimetry. Further in silico docking and molecular dynamics simulations studies proposed that Peptide 1 is likely to interact with Lys49, Arg56, Arg129, and Tyr130 residues at the amphipathic pocket of 14-3-3σ. These results suggest that Peptide 1 may serve as a biological probe or a template to design inhibitors of TRIM25-14-3-3σ interaction as a potentially novel class of anticancer agents. Communicated by Ramaswamy H. Sarma</p

Heterocyclic Substitutions Greatly Improve Affinity and Stability of Folic Acid towards FRα. an In Silico Insight

Folate receptor alpha (FRα) is known as a biological marker for many cancers due to its overexpression in cancerous epithelial tissue. The folic acid (FA) binding affinity to the FRα active site provides a basis for designing more specific targets for FRα. Heterocyclic rings have been shown to interact with many receptors and are important to the metabolism and biological processes within the body. Nineteen FA analogs with substitution with various heterocyclic rings were designed to have higher affinity toward FRα. Molecular docking was used to study the binding affinity of designed analogs compared to FA, methotrexate (MTX), and pemetrexed (PTX). Out of 19 FA analogs, analogs with a tetrazole ring (FOL03) and benzothiophene ring (FOL08) showed the most negative binding energy and were able to interact with ASP81 and SER174 through hydrogen bonds and hydrophobic interactions with amino acids of the active site. Hence, 100 ns molecular dynamics (MD) simulations were carried out for FOL03, FOL08 compared to FA, MTX, and PTX. The root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of FOL03 and FOL08 showed an apparent convergence similar to that of FA, and both of them entered the binding pocket (active site) from the pteridine part, while the glutamic part was stuck at the FRα pocket entrance during the MD simulations. Molecular mechanics Poisson-Boltzmann surface accessible (MM-PBSA) and H-bond analysis revealed that FOL03 and FOL08 created more negative free binding and electrostatic energy compared to FA and PTX, and both formed stronger H-bond interactions with ASP81 than FA with excellent H-bond profiles that led them to become bound tightly in the pocket. In addition, pocket volume calculations showed that the volumes of active site for FOL03 and FOL08 inside the FRα pocket were smaller than the FA–FRα system, indicating strong interactions between the protein active site residues with these new FA analogs compared to FA during the MD simulations

Conjugated β-Cyclodextrin Enhances the Affinity of Folic Acid towards FRα: Molecular Dynamics Study

Drug targeting is a progressive area of research with folate receptor alpha (FRα) receiving significant attention as a biological marker in cancer drug delivery. The binding affinity of folic acid (FA) to the FRα active site provides a basis for recognition of FRα. In this study, FA was conjugated to beta-cyclodextrin (βCD) and subjected to in silico analysis (molecular docking and molecular dynamics (MD) simulation (100 ns)) to investigate the affinity and stability for the conjugated system compared to unconjugated and apo systems (ligand free). Docking studies revealed that the conjugated FA bound into the active site of FRα with a docking score (free binding energy &lt; −15 kcal/mol), with a similar binding pose to that of unconjugated FA. Subsequent analyses from molecular dynamics (MD) simulations, root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg) demonstrated that FA and FA–βCDs created more dynamically stable systems with FRα than the apo-FRα system. All systems reached equilibrium with stable RMSD values ranging from 1.9–2.4 Å and the average residual fluctuation values of the FRα backbone atoms for all residues (except for terminal residues ARG8, THR9, THR214, and LEU215) were less than 2.1 Å with a consistent Rg value of around 16.8 Å throughout the MD simulation time (0–100 ns). The conjugation with βCD improved the stability and decreased the mobility of all the residues (except residues 149–151) compared to FA–FRα and apo-FRα systems. Further analysis of H-bonds, binding free energy (MM-PBSA), and per residue decomposition energy revealed that besides APS81, residues HIS20, TRP102, HIS135, TRP138, TRP140, and TRP171 were shown to have more favourable energy contributions in the holo systems than in the apo-FRα system, and these residues might have a direct role in increasing the stability of holo systems

A cyclic peptide inhibitor of the iNOS–SPSB protein–protein interaction as a potential anti-infective agent

SPRY domain- and SOCS box-containing proteins SPSB1, SPSB2, and SPSB4 interact with inducible nitric oxide synthase (iNOS), causing the iNOS to be polyubiquitinated and targeted for degradation. Inhibition of this interaction increases iNOS levels, and consequently cellular nitric oxide (NO) concentrations, and has been proposed as a potential strategy for killing intracellular pathogens. We previously described two DINNN-containing cyclic peptides (CP1 and CP2) as potent inhibitors of the murine SPSB-iNOS interaction. In this study, we report the crystal structures of human SPSB4 bound to CP1 and CP2 and human SPSB2 bound to CP2. We then used these structures to design a new inhibitor in which an intramolecular hydrogen bond was replaced with a hydrocarbon linkage to form a smaller macrocycle while maintaining the bound geometry of CP2 observed in the crystal structures. This resulting pentapeptide SPSB-iNOS inhibitor (CP3) has a reduced macrocycle ring size, fewer nonbinding residues, and includes additional conformational constraints. CP3 has a greater affinity for SBSB2 ( KD = 7 nM as determined by surface plasmon resonance) and strongly inhibits the SPSB2-iNOS interaction in macrophage cell lysates. We have also determined the crystal structure of CP3 in complex with human SPSB2, which reveals the structural basis for the increased potency of CP3 and validates the original design

A Potent Cyclic Peptide Targeting SPSB2 Protein as a Potential Anti-infective Agent

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 <i>K</i>_d 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

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

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 ¹⁹F NMR analyses revealed that the most potent cyclic peptidomimetic bound to the iNOS binding site of SPSB2 with low nanomolar affinity (<i>K</i>_D 29 nM), a 10-fold improvement over that of the linear peptide DINNN (<i>K</i>_D 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