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.
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