Binding of the hemopressin peptide to the cannabinoid CB1 receptor: structural insights

Hemopressin, a bioactive nonapeptide derived from the α1 chain of hemoglobin, was recently shown to possess selective antagonist activity at the cannabinoid CB(1) receptor [Heimann, A. S., et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 20588-20593]. CB(1) receptor antagonists have been extensively studied for their possible therapeutic use in the treatment of obesity, drug abuse, and heroin addiction. In particular, many compounds acting as CB(1) receptor antagonists have been synthesized and subjected to experiments as possible anti-obesity drugs, but their therapeutic application is still complicated by important side effects. Using circular dichroism and nuclear magnetic resonance spectroscopy, this work reports the conformational analysis of hemopressin and its truncated, biologically active fragment hemopressin(1-6). The binding modes of both hemopressin and hemopressin(1-6) are investigated by molecular docking calculations. Our conformational data indicate that regular turn structures in the central portion of hemopressin and hemopressin(1-6) are critical for an effective interaction with the receptor. The results of molecular docking calculations, indicating similarities and differences in comparison to the most accepted CB(1) pharmacophore model, suggest the possibility of new chemical scaffolds for the design of new CB(1) antagonist lead compounds.     

Structure-function analysis of the antiangiogenic ATWLPPR peptide inhibiting VEGF(165) binding to neuropilin-1 and molecular dynamics simulations of the ATWLPPR/neuropilin-1 complex

Heptapeptide ATWLPPR (A7R), identified in our laboratory by screening a mutated phage library, was shown to bind specifically to neuropilin-1 (NRP-1) and then to selectively inhibit VEGF₁₆₅ binding to this receptor. In vivo, treatment with A7R resulted in decreasing breast cancer angiogenesis and growth. The present work is focused on structural characterization of A7R. Analogs of the peptide, obtained by substitution of each amino acid with alanine (alanine-scanning) or by amino acid deletion, have been systematically assayed to determine the relative importance of the side chains of each residue with respect to the inhibitory effect of A7R on VEGF₁₆₅ binding to NRP-1. We show here the importance of the C-terminal sequence LPPR and particularly the key role of C-terminal arginine. In solution, A7R displays significant secondary structure of the backbone adopting an extended conformation. However, the functional groups of arginine are very flexible in the absence of NRP-1 pointing to an induced fit upon binding to the receptor. A MD trajectory of the A7R/NRP-1 complex in explicit water, based on the recent tuftsin/NRP-1 crystal structure, has revealed the hydrogen-bonding network that contributes to A7R's binding activity.     

Binding site analysis of full-length alpha1a adrenergic receptor using homology modeling and molecular docking

The recent availability of crystal structure of bovine rhodopsin offers new opportunities in order to approach the construction of G protein coupled receptors. This study focuses the attention on the modeling of full-length alpha(1a) adrenergic receptor (alpha(1a)-AR) due to its biological role and significant implications in pharmacological treatment of benign prostate hyperplasia. This work could be considered made up by two main steps: (a) the construction of full structure of alpha(1a)-AR, through homology modeling methods; (b) the automated docking of an endogenous agonist, norepinephrine, and of an antagonist, WB-4101, using BioDock program. The obtained results highlight the key residues involved in binding sites of both agonists and antagonists, confirming the mutagenesis data and giving new suggestions for the rational design of selective ligands.     

Binding of teicoplanin and vancomycin to bovine serum albumin in vitro: a multispectroscopic approach and molecular modeling

In this paper, the binding properties of teicoplanin and vancomycin to bovine serum albumin (BSA) were investigated using fluorescence quenching, synchronous fluorescence, Fourier transform infrared (FTIR), circular dichroism (CD) and UV–vis spectroscopic techniques and molecular docking under simulative physiological conditions. The results obtained from fluorescence quenching data revealed that the drug–BSA interaction altered the conformational structure of BSA. Meanwhile, the 3D fluorescence, CD, FTIR and UV–vis data demonstrated that the conformation of BSA was slightly altered in the presence of teicoplanin and vancomycin, with different reduced α‐helical contents. The binding distances for the drug–BSA system were provided by the efficiency of fluorescence resonance energy transfer (FRET). Furthermore, the thermodynamic analysis implied that hydrogen bond and van der Waals' forces were the main interaction for the drug–BSA systems, which agreed well with the results from the molecular modeling study. The results obtained herein will be of biological significance in future toxicological and pharmacological investigation. Copyright © 2013 John Wiley & Sons, Ltd.     

Study on the molecular mechanism of inhibiting HIV-1 integrase by EBR28 peptide via molecular modeling approach

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the HIV-1 lifecycle which aids the integration of viral DNA into the host chromosome. Recently synthesized 12-mer peptide EBR28, which can strongly bind to IN, is one of the most potential small peptide leading compounds inhibiting IN binding with viral DNA. However, the binding mode between EBR28 peptide with HIV-1 IN and the inhibition mechanism remain uncertain. In this paper, the binding modes of EBR28 with HIV-1 IN monomer core domain (IN(1)) and dimmer core domain (IN(2)) were investigated by using molecular docking and molecular dynamics (MD) simulation methods. The results indicated that EBR28 bound to the interfaces of the IN(1) and IN(2) systems mainly through the hydrophobic interactions with the beta3, alpha1 and alpha5 regions of the proteins. The binding free energies for IN(1) with a series of EBR28 mutated peptides were calculated with the MM/GBSA model, and the correlation between the calculated and experimental binding free energies is very good (r=0.88). Thus, the validity of the binding mode of IN(1) with EBR28 was confirmed. Based on the binding modes, the inhibition mechanism of EBR28 was explored by analyzing the essential dynamics (ED), energy decomposition and the mobility of EBR28 in the two docked complexes. The proposed inhibition mechanism is represented that EBR28 binds to the interface of IN(1) to form the IN(1)_EBR28 complex and preventes the formation of IN dimmer, finally leads to the partial loss of binding potency for IN with viral DNA. All of the above simulation results agree well with experimental data, which provide us with some helpful information for designing anti-HIV small peptide drugs.     

Refinement of glucagon-like peptide 1 docking to its intact receptor using mid-region photolabile probes and molecular modeling

The glucagon-like peptide 1 (GLP1) receptor is an important drug target within the B family of G protein-coupled receptors. Its natural agonist ligand, GLP1, has incretin-like actions and the receptor is a recognized target for management of type 2 diabetes mellitus. Despite recent solution of the structure of the amino terminus of the GLP1 receptor and several close family members, the molecular basis for GLP1 binding to and activation of the intact receptor remains unclear. We previously demonstrated molecular approximations between amino- and carboxyl-terminal residues of GLP1 and its receptor. In this work, we study spatial approximations with the mid-region of this peptide to gain insights into the orientation of the intact receptor and the ligand-receptor complex. We have prepared two new photolabile probes incorporating a p-benzoyl-l-phenylalanine into positions 16 and 20 of GLP1(7-36). Both probes bound to the GLP1 receptor specifically and with high affinity. These were each fully efficacious agonists, stimulating cAMP accumulation in receptor-bearing CHO cells in a concentration-dependent manner. Each probe specifically labeled a single receptor site. Protease cleavage and radiochemical sequencing identified receptor residue Leu(141) above transmembrane segment one as its site of labeling for the position 16 probe, whereas the position 20 probe labeled receptor residue Trp(297) within the second extracellular loop. Establishing ligand residue approximation with this loop region is unique among family members and may help to orient the receptor amino-terminal domain relative to its helical bundle region.     

Structure-based design of a novel peptide inhibitor of HIV-1 integrase: a computer modeling approach

The insertion of viral DNA into the host chromosome is an essential step in the replication of HIV-1, and is carried out by an enzyme, HIV-1 integrase (IN). Since the latter has no human cellular counterpart, it is an attractive target for antiviral drug design. Several IN inhibitors having activities in the micromolar range have been reported to date. However, no clinically useful inhibitors have yet been developed. Recently reported diketo acids represent a novel and selective class of IN inhibitors. These are the only class which appear to selectively target integrase and two of the inhibitors, L-708,906 and L-731,988, are the most potent inhibitors of preintegration complexes described to date.The X-ray crystal structure of the IN catalytic domain complexed with a diketo acid derivative inhibitor, 5CITEP, has recently been determined. Although the structure is of great value as a platform for drug design, experimental data suggest that crystal packing effects influence the observed inhibitor position. This has been confirmed by computational docking studies using the latest version (3.0) of the AutoDock program, which has been shown to give results largely consistent with available experimental data. Using AutoDock 3.0 and SYBYL6.6 we have modeled the complexes of IN with the diketo acid inhibitors so as to identify the enzyme binding site. In the quest for novel, potent and selective small molecule inhibitors, we present here a new approach to peptide inhibitor design using a, b- unsaturated (dehydro) residues, which confer a unique conformation on a peptide sequence. Based on the above models, we selected a tetrapeptide sequence containing a dehydro-Phe residue, which was found to have an open conformation as ascertained from its X-ray crystal structure. Docking results on this peptide led us to propose a modification at the C-terminal end. The modified peptide was found to dock in a similar position as the diketo acid inhibitors and was predicted to have a comparable potency.     

Binding properties of palmatine to DNA: spectroscopic and molecular modeling investigations

Palmatine, an isoquinoline alkaloid, is an important medicinal herbal extract with diverse pharmacological and biological properties. In this work, spectroscopic and molecular modeling approaches were employed to reveal the interaction between palmatine and DNA isolated from herring sperm. The absorption spectra and iodide quenching results indicated that groove binding was the main binding mode of palmatine to DNA. Fluorescence studies indicated that the binding constant (K) of palmatine and DNA was ~ 10⁴ L·mol^?1. The associated thermodynamic parameters, ΔG, ΔH, and ΔS, indicated that hydrogen bonds and van der Waals forces played major roles in the interaction. The effects of chemical denaturant, thermal denaturation and pH on the interaction were investigated and provided further support for the groove binding mode. In addition to experimental approaches, molecular modeling was conducted to verify binding pattern of palmatine–DNA. Copyright © 2015 John Wiley & Sons, Ltd.     

Coarse-grained peptide modeling using a systematic multiscale approach

A systematic new approach to derive multiscale coarse-grained (MS-CG) models has been recently developed. The approach employs information from atomistically detailed simulations to derive CG forces and associated effective potentials. In this work, the MS-CG methodology is extended to study two peptides representing distinct structural motifs, alpha-helical polyalanine and the beta-hairpin V(5)PGV(5). These studies represent the first known application of this approach to peptide systems. Good agreement between the MS-CG and atomistic models is achieved for several structural properties including radial distribution functions, root mean-square deviation, and radius of gyration. The new MS-CG models are able to preserve the native states of these peptides within approximately 1 A backbone root mean-square deviation during CG simulations. The MS-CG approach, as with most coarse-grained models, has the potential to increase the length and timescales accessible to molecular simulations. However, it is also able to maintain a clear connection to the underlying atomistic-scale interactions.     

Binding mode of ecdysone agonists to the receptor: comparative modeling and docking studies

Three-dimensional structure models of the ligand-binding domain of the ecdysone receptor of Heliothis virescens were built by the homology modeling technique from the crystal structures of nuclear receptors. Two models were created based both on known ligand-binding domain structures of the receptors with the highest sequence identity to the ecdysone receptor, and on those of steroid hormone receptors. The latter model, which was found to have better stereochemical quality and be in good agreement with the binding of the steroidal framework of the endogenous agonist 20-hydroxyecdysone, was used for docking studies. The docking of 20-hydroxyecdysone to the receptor model revealed that the ligand molecule can interact with the receptor in a similar manner to other steroid hormone-receptor complexes. The docking of a dibenzoylhydrazine agonist, chromafenozide, was performed based on the correspondences between the molecule and 20-dydroxyecdysone expected by molecular comparison. The interactions of the ligands with the receptor in the complexes modeled were investigated and found to be consistent with known structure-activity relationships.     

Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1

Neuropilin-1 (NRP-1), a non-tyrosine kinase receptor of vascular endothelial growth factor-165 (VEGF165), was found expressed on endothelial and some tumor cells. Since its overexpression is correlated with tumor angiogenesis and progression, the targeting of NRP-1 could be a potential anti-cancer strategy. To explore this hypothesis, we identified a peptide inhibiting the VEGF165 binding to NRP-1 and we tested whether it was able to inhibit tumor growth and angiogenesis. To prove the target of peptide action, we assessed its effects on binding of radiolabeled VEGF165 to recombinant receptors and to cultured cells expressing only VEGFR-2 (KDR) or NRP-1. Antiangiogenic activity of the peptide was tested in vitro in tubulogenesis assays and in vivo in nude mice xenotransplanted in fat-pad with breast cancer MDA-MB-231 cells. Tumor volumes, vascularity and proliferation indices were determined. The selected peptide, ATWLPPR, inhibited the VEGF165 binding to NRP-1 but not to tyrosine kinase receptors, VEGFR-1 (flt-1) and KDR; nor did it bind to heparin. It diminished the VEGF-induced human umbilical vein endothelial cell proliferation and tubular formation on Matrigel and in co-culture with fibroblasts. Administration of ATWLPPR to nude mice inhibited the growth of MDA-MB-231 xenografts, and reduced blood vessel density and endothelial cell area but did not alter the proliferation indices of the tumor. In conclusion, ATWLPPR, a previously identified KDR-interacting peptide, was shown to inhibit the VEGF165 interactions with NRP-1 but not with KDR and to decrease the tumor angiogenesis and growth, thus validating, in vivo, NRP-1 as a possible target for antiangiogenic and antitumor agents.     

Binding of tetracycline and chlortetracycline to the enzyme trypsin: spectroscopic and molecular modeling investigations

Tetracycline (TC) and chlortetracycline (CTC) are common members of the widely used veterinary drug tetracyclines, the residue of which in the environment can enter human body, being potentially harmful. In this study, we establish a new strategy to probe the binding modes of TC and CTC with trypsin based on spectroscopic and computational modeling methods. Both TC and CTC can interact with trypsin with one binding site to form trypsin-TC (CTC) complex, mainly through van der Waals' interactions and hydrogen bonds with the affinity order: TC>CTC. The bound TC (CTC) can result in inhibition of trypsin activity with the inhibition order: CTC>TC. The secondary structure and the microenvironment of the tryptophan residues of trypsin were also changed. However, the effect of CTC on the secondary structure content of trypsin was contrary to that of TC. Both the molecular docking study and the trypsin activity experiment revealed that TC bound into S1 binding pocket, competitively inhibiting the enzyme activity, and CTC was a non-competitive inhibitor which bound to a non-active site of trypsin, different from TC due to the Cl atom on the benzene ring of CTC which hinders CTC entering into the S1 binding pocket. CTC does not hinder the binding of the enzyme substrate, but the CTC-trypsin-substrate ternary complex can not further decompose into the product. The work provides basic data for clarifying the binding mechanisms of TC (CTC) with trypsin and can help to comprehensively understanding of the enzyme toxicity of different members of tetracyclines in vivo.     

Binding of polylysine to charged bilayer membranes: molecular organization of a lipid.peptide complex.

The interaction between a positively charged peptide (poly-L-lysine) and model membranes containing charged lipids has been investigated. Conformational changes of the polypeptide as well as changes in the membrane lipid distribution were observed upon lipid-protein agglutination: 1. The strong binding of polylysine is shown directly by the use of spinlabelled polypeptide. Upon binding to phosphatidic acid a shift in the hyperfine coupling constant from 16.5 to 14.6 Oe is observed. The spectrum of the lipid-bound peptide is superimposed on the spectrum of polylysine in solution. Half of the lysine groups are bound to the charged membranes. A change in the conformation of polylysine from a random coil to a partially ordered configuration is suggested. 2. Spin labelling of the lipid component gives evidence concerning the molecular organization of a lipid mixture containing charged phosphatitid acid. Addition of polylysine induces the formation of crystalline patches of bound phosphatidic acid. 3. Excimer forming pyrene decanoic acid has been employed. Addition of positively charged polylysine (pH 9.0) to phosphatidic acid membranes increases the transition temperature of the lipid from Tt = 50 to Tt = 62 degrees C. Thus, a lipid segregation of lipid into regions of phosphatidic acid bound to the peptide which differ in their microviscosity from the surrounding membrane is induced. One lysine group binds one phosphatidic acid molecule, but only half of the phosphatidic acid is bound. 4. Direct evidence for charge induced domain formation in lipid mixtures containing phosphatidic acid is given by electron microscopy. Addition of polylysine leads to a change in the surface curvature of the bound charged lipid. The domain size is estimated from the electron micrographs. The number of domains present is dependent on both the ratio of charged to uncharged lipids as well as on the amount of polylysine added to the vesicles. The size of the domains is not dependent on membrane composition. However, the size seems to increase in a stepwise manner that is correlated with a multiple of the area covered by one polylysine molecule.     

From cluster calculations to molecular materials: a mixed pseudopotential approach to modeling mixed-valence systems

In this paper we present a technique for finding an appropriate parameterization of ultrasoft pseudopotentials for modeling mixed-valence materials. For the example of hexacyanometallate molecular building blocks, we show how ionic cluster calculations can be used to determine a set of parameters for the metal centers. Pseudopotentials chosen in such a way are then shown to be suitable for periodic calculations of the corresponding mixed-valence materials (e.g., Prussian Blue).This work was originally presented at the Modelling and Design of Molecular Materials conference in Wrocaw, Poland.     

An affordable approach to interactive desktop molecular modeling

The amazing revolution in computer hardware performance and cost reduction has yet to be carried over to computer software. In fact, application software today is often more expensive and less reliable than the hardware. New enhancements in software development techniques, such as object oriented programming and interactive graphics based user interface design, finally may be having a significant impact on the time-to-market and reliability of these application programs. We discuss our experiences using one such set of software development tools available on the NeXT workstation and describe the effort required to port our MidasPlus molecular modeling package to the NeXT workstation.     

How neuropilin-1 regulates receptor tyrosine kinase signalling: the knowns and known unknowns

Essential roles of NRP1 (neuropilin-1) in cardiovascular development and in neuronal axon targeting during embryogenesis are thought to be mediated primarily through binding of NRP1 to two unrelated types of ligands: the VEGF (vascular endothelial growth factor) family of angiogenic cytokines in the endothelium, and the class 3 semaphorins in neurons. A widely accepted mechanism for the role of NRP1 in the endothelium is VEGF binding to NRP1 and VEGFR2 (VEGF receptor 2) and VEGF-dependent formation of complexes or NRP1-VEGFR2 holoreceptors with enhanced signalling activity and biological function. However, although some basic features of this model are solidly based on biochemical and cellular data, others are open to question. Furthermore, a mechanistic account of NRP1 has to accommodate research which emphasizes the diversity of NRP1 functions in different cell types and particularly an emerging role in signalling by other growth factor ligands for RTKs (receptor tyrosine kinases) such as HGF (hepatocyte growth factor) and PDGF (platelet-derived growth factor). It is uncertain, however, whether the model of NRP1-RTK heterocomplex formation applies in all of these situations. In the light of these developments, the need to explain mechanistically the role of NRP1 in signalling is coming increasingly to the fore. The present article focuses on some of the most important unresolved questions concerning the mechanism(s) through which NRP1 acts, and highlights recent findings which are beginning to generate insights into these questions.     

Semaphorin 3A lytic hybrid peptide binding to neuropilin-1 as a novel anti-cancer agent in pancreatic cancer

We previously reported that novel targeted “hybrid peptide” in which epidermal growth factor receptor (EGFR) binding peptide was conjugated with lytic-type peptide had selective cytotoxic activity to EGFR expressing cancer cells. In this study, we have generated a novel type hybrid peptide, semaphorin 3A lytic (Sema3A-lytic), which is composed of two functional amino acid domains: a sequence derived from Sema3A that binds to neuropilin-1 (NRP1) and a cytotoxic lytic peptide. We found that this hybrid peptide had cytotoxic activity against NRP1-positive pancreatic cancer cell lines such as BxPC-3 and Panc-1, whereas the peptide did not affect the viability of normal cells in vitro. It was also found by affinity analysis that Sema3A peptide binds to NRP1, and two arginines (372R and 377R) in Sema3A peptide are involved in the interaction with NRP1 protein. In addition, confocal microscopy analysis revealed that Sema3A-lytic peptide could not penetrate normal cells regardless of the presence of NRP1 mRNA, suggesting that the ability of Sema3A-lytic peptide to concentrate adjacent to the cell membrane by binding to NRP1 with the target-binding moiety contributes to its selective cytotoxic activity. These results indicate that Sema3A-lytic hybrid peptide would be a possible anti-cancer agent for treatment of human pancreatic cancer.     

Human thrombospondin's (TSP-1) C-terminal domain opens to interact with the CD-47 receptor: a molecular modeling study

Thrombospondin-1 (TSP-1) interaction with the membranous receptor CD-47 involves the peptide RFYVVMWK (4N-1) located in its C-terminal domain. However, the available X-ray structure of TSP-1 describes this peptide as completely buried into a hydrophobic pocket, preventing any interaction. Where classical standard methods failed, an appropriate approach combining normal mode analysis and an adapted protocol of energy minimization identified the large amplitude motions responsible of the partial solvent exposure of 4N-1. In agreement, the obtained model of the open TSP-1 was further used for protein-protein docking experiments against a homology model generated for CD-47. Considering the multiple applications of the CD-47 receptor as a target, our results open new pharmacological perspectives for the design of TSP-1:CD-47 inhibitors and CD-47 antagonists. We also suggest a common opening mechanism for proteins sharing the same fold as TSP-1. This work also suggests the usefulness of our approach in other topics in which predictions of protein-protein interactions are of importance.