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, [(1)H,(15)N]-HSQC, and (19)F 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.
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.
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
5-HT(1A) serotonin and D1 dopamine receptor agonists have been postulated to be able to improve negative and cognitive impairment symptoms of schizophrenia, while partial agonists and antagonists of the D2 and 5-HT(2A) receptors have been reported to be effective in reducing positive symptoms. There is therefore a need for well-defined homology models for the design of more selective antipsychotic agents, since no three-dimensional (3D) crystal structures of these receptors are currently available. In this study, homology models were built based on the high-resolution crystal structure of the β(2)-adrenergic receptor (2RH1) and further refined via molecular dynamics simulations in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer system with the GROMOS96 53A6 united atom force field. Docking evaluations with representative agonists and antagonists using AutoDock 4.2 revealed binding modes in agreement with experimentally determined site-directed mutagenesis data and significant correlations between the computed and experimental pK (i) values. The models are also able to distinguish between antipsychotic agents with different selectivities and binding affinities for the four receptors, as well as to differentiate active compounds from decoys. Hence, these human 5-HT(1A), 5-HT(2A), D1 and D2 receptor homology models are capable of predicting the activities of novel ligands, and can be used as 3D templates for antipsychotic drug design and discovery.
Selective blockade of the serotonin 5-HT(2A) receptor is a useful therapeutic approach for a number of disorders, including schizophrenia, insomnia and ischaemic heart disease. A series of aporphines were docked into a homology model of the rat 5-HT(2A) receptor using AutoDock. Selected compounds with high in silico binding affinities were screened in vitro using radioligand-binding assays against rat serotonin (5-HT(1A) and 5-HT(2A)) and dopamine (D1 and D2) receptors. (R)-Roemerine and (±)-nuciferine were found to have high affinity for the 5-HT(2A) receptor (K(i) = 62 and 139 nM, respectively), with (R)-roemerine showing 20- to 400-fold selectivity for the 5-HT(2A) receptor over the 5-HT(1A), D1 and D2 receptors. Investigation into the ligand-receptor interactions suggested that the selectivity of (R)-roemerine is due to it having stronger H-bonding and dipole-dipole interactions with several of the key residues in the 5-HT(2A) receptor-binding site.
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.
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.
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 < −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.
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