Understanding Of Ligand-receptor Interactions Research Articles

A DNA origami device spatially controls CD95 signalling to induce immune tolerance in rheumatoid arthritis.

DNA origami is capable of spatially organizing molecules into sophisticated geometric patterns with nanometric precision. Here we describe a reconfigurable, two-dimensional DNA origami with geometrically patterned CD95 ligands that regulates immune cell signalling to alleviate rheumatoid arthritis. In response to pH changes, the device reversibly transforms from a closed to an open configuration, displaying a hexagonal pattern of CD95 ligands with ~10 nm intermolecular spacing, precisely mirroring the spatial arrangement of CD95 receptor clusters on the surface of immune cells. In a collagen-induced arthritis mouse model, DNA origami elicits robust and selective activation of CD95 death-inducing signalling in activated immune cells located in inflamed synovial tissues. Such localized immune tolerance ameliorates joint damage with no noticeable side effects. This device allows for the precise spatial control of cellular signalling, expanding our understanding of ligand-receptor interactions and is a promising platform for the development of pharmacological interventions targeting these interactions.

Determination of the Cannabinoid CB1 Receptor's Positive Allosteric Modulator Binding Site through Mutagenesis Studies.

Positive allosteric modulators (PAMs) of the cannabinoid CB1 receptor (CB1) offer potential therapeutic advantages in the treatment of neuropathic pain and addiction by avoiding the adverse effects associated with orthosteric CB1 activation. Here, molecular modeling and mutagenesis were used to identify residues central to PAM activity at CB1. Six putative allosteric binding sites were identified in silico, including novel sites previously associated with cholesterol binding, and key residues within each site were mutated to alanine. The recently determined ZCZ011 binding site was found to be essential for allosteric agonism, as GAT228, GAT229 and ZCZ011 all increased wild-type G protein dissociation in the absence of an orthosteric ligand; activity that was abolished in mutants F191A3.27 and I169A2.56. PAM activity was demonstrated for ZCZ011 in the presence of the orthosteric ligand CP55940, which was only abolished in I169A2.56. In contrast, the PAM activity of GAT229 was reduced for mutants R220A3.56, L404A8.50, F191A3.27 and I169A2.56. This indicates that allosteric modulation may represent the net effect of binding at multiple sites, and that allosteric agonism is likely to be mediated via the ZCZ011 site. This study underlines the need for detailed understanding of ligand receptor interactions in the search for pure CB1 allosteric modulators.

Differential interaction patterns of opioid analgesics with µ opioid receptors correlate with ligand-specific voltage sensitivity.

The µ opioid receptor (MOR) is the key target for analgesia, but the application of opioids is accompanied by several issues. There is a wide range of opioid analgesics, differing in their chemical structure and their properties of receptor activation and subsequent effects. A better understanding of ligand-receptor interactions and the resulting effects is important. Here, we calculated the respective binding poses for several opioids and analyzed interaction fingerprints between ligand and receptor. We further corroborated the interactions experimentally by cellular assays. As MOR was observed to display ligand-induced modulation of activity due to changes in membrane potential, we further analyzed the effects of voltage sensitivity on this receptor. Combining in silico and in vitro approaches, we defined discriminating interaction patterns responsible for ligand-specific voltage sensitivity and present new insights into their specific effects on activation of the MOR.

Novel Xanomeline-Containing Bitopic Ligands of Muscarinic Acetylcholine Receptors: Design, Synthesis and FRET Investigation

In the last few years, fluorescence resonance energy transfer (FRET) receptor sensors have contributed to the understanding of GPCR ligand binding and functional activation. FRET sensors based on muscarinic acetylcholine receptors (mAChRs) have been employed to study dual-steric ligands, allowing for the detection of different kinetics and distinguishing between partial, full, and super agonism. Herein, we report the synthesis of the two series of bitopic ligands, 12-Cn and 13-Cn, and their pharmacological investigation at the M1, M2, M4, and M5 FRET-based receptor sensors. The hybrids were prepared by merging the pharmacophoric moieties of the M1/M4-preferring orthosteric agonist Xanomeline 10 and the M1-selective positive allosteric modulator 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) 11. The two pharmacophores were connected through alkylene chains of different lengths (C3, C5, C7, and C9). Analyzing the FRET responses, the tertiary amine compounds 12-C5, 12-C7, and 12-C9 evidenced a selective activation of M1 mAChRs, while the methyl tetrahydropyridinium salts 13-C5, 13-C7, and 13-C9 showed a degree of selectivity for M1 and M4 mAChRs. Moreover, whereas hybrids 12-Cn showed an almost linear response at the M1 subtype, hybrids 13-Cn evidenced a bell-shaped activation response. This different activation pattern suggests that the positive charge anchoring the compound 13-Cn to the orthosteric site ensues a degree of receptor activation depending on the linker length, which induces a graded conformational interference with the binding pocket closure. These bitopic derivatives represent novel pharmacological tools for a better understanding of ligand-receptor interactions at a molecular level.

Controlled Modular Multivalent Presentation of the CD40 Ligand on P22 Virus-like Particles Leads to Tunable Amplification of CD40 Signaling

Ligands of the tumor necrosis factor superfamily (TNFSF) are appealing targets for immunotherapy research due to their integral involvement in stimulation or restriction of immune responses. TNFSF-targeted therapies are currently being developed to combat immunologically based diseases and cancer. A crucial determinant of effective TNFSF receptor binding and signaling is the trimeric quaternary structure of the ligand. Additionally, ligand multivalency is essential to propagate strong signaling in effector cells. Thus, designing a synthetic platform to display trimeric TNFSF ligands in a multivalent manner is necessary to further the understanding of ligand-receptor interactions. Viral nanocages have architectures that are amenable to genetic and chemical modifications of both their interior and exterior surfaces. Notably, the exterior surface of virus-like particles can be utilized as a platform for the modular multivalent presentation of target proteins. In this study, we build on previous efforts exploring the bacteriophage P22 virus-like particle for the exterior multivalent modular display of a potent immune-stimulating TNFSF protein, CD40 ligand (CD40L). Using a cell-based reporter system, we quantify the effects of tunable avidity on CD40 signaling by CD40L displayed on the surface of P22 nanocages. Multivalent presentation of CD40L resulted in a 53.6-fold decrease of the half maximal effective concentration (EC50) compared to free CD40L, indicating higher potency. Our results emphasize the power of using P22-based biomimetics to study ligand-receptor interactions within their proper structural context, which may contribute to the development of effective immune modulators.

A Review of Molecular Modelling Studies of Dihydrofolate Reductase Inhibitors Against Opportunistic Microorganisms and Comprehensive Evaluation of New Models

Dihydrofolate reductase (DHFR) has been used as a target for antimicrobial drug discovery against a variety of pathogenic microorganisms, including opportunistic microorganisms; Pneumocystis carinii (pc), Toxoplasma gondii (tg) and Mycobacterium avium complex (ma). In this regard, several DHFR inhibitors are reported against pc and tg and ma. However, selectivity issue of these inhibitors over human DHFR often preclude their development and clinical use. In the first part of this work, various computational approaches including available crystallographic structures, binding affinity prediction, pharmacophore mapping, QSAR, homology modelling used for design of DHFR inhibitors against opportunistic microorganisms are reviewed, to understand specific interactions required for inhibition of microbial DHFR. Secondly, comprehensive molecular modelling techniques were used, to establish structure-chemical-feature-based pharmacophore models for pcDHFR, tgDHFR and mammalian DHFR. The results show that, the information encoded by ligand based approaches like pharmacophore mapping and 3D-QSAR methods are in well agreement with the information coded in the receptor structure. A combination of ligand and structure based approaches provides understanding of ligand-receptor interactions. The study indicated that the value of small alkyl moieties at position 5 of the bicyclic nitrogen containing nucleus along with a bulky group attached at the C-6 via suitable linker could optimize activity, with regard to both potency and selectivity.

HIV protease inhibition: limited recent progress and advances in understanding current pitfalls.

The identification of HIV-1 protease (HIVp) as a target for therapeutic intervention against AIDS was soon followed by major efforts to understand its substrate specificity, reaction kinetics and three-dimensional structure, both in the free state and in complex with a number of ligands including substrate mimics, products, and inhibitors. On the whole these studies have been extremely successful and have had a major impact on our understanding of ligand-receptor interactions and enzyme inhibition mechanisms. HIVp has also become a paradigm for the development and testing of new drug-design methodologies both in vitro and in silico. Even though thousands of potential HIVp inhibitors exhibiting amazing chemical diversity have been synthesized or identified from natural sources, only a few have turned out to be useful for human therapy. Although the alternative goal of preventing enzyme dimerization has been achieved as a proof of concept, this approach has not yet yielded a clinical candidate. The review covers the general strategies that led to some of the most useful inhibitors, the reasons for our limited success in effectively inhibiting this retroviral target in a clinical setting, current progress with second-generation inhibitors, and new avenues for research.

Flow cytometric measurement of kinetic and equilibrium binding parameters of arginine-glycine-aspartic acid ligands in binding to glycoprotein IIb/IIIa on platelets.

Antagonists of platelet glycoprotein IIb/IIIa (GPIIb/IIIa) represent a new therapeutic approach in inhibiting platelet aggregation, thus providing a powerful form of antithrombotic therapy. The measurement of binding of arginine-glycine-aspartic acid (RGD) peptidomimetics to GPIIb/IIIa on platelets is a key for the further understanding of ligand-receptor interactions and, thus, the design of new antagonists. The flow cytometric measurement of dynamic and equilibrium binding parameters of two new potent RGD peptidomimetics, L-762,745 and L-769,434, containing a fluorescein moiety is described in this paper. Kinetic binding measurements with these fluorescent ligands indicate a two-step binding mechanism that involves a conformational rearrangement of the receptor-ligand complex. The overall second-order binding constants are for both fluorescent ligands several orders of magnitude slower than for diffusion-controlled processes. The values of k(-1) and K(D) obtained by fitting the kinetic binding data in a two-step model are in good agreement with directly detected values of k(off)(L-762,745) = (1.9 +/- 0.6) 10(-3) s(-1), k(off)(L-769,434) = (5.1 +/- 0.7) 10(-3) s(-1), KD(L-762,745) = 12 +/- 0.5 nM, and K(D)(L-769,434) = 8 +/- 0.3 nM. Equilibrium binding measurements of fluorescent ligands with an orally active nonfluorescent antagonist, L-738,167, provided apparent dissociation binding constant K(D) of this ligand in the range from 0.1 to 0.2 nM. The kinetic dissociation measurement of L-738,167 using the binding of the fluorescent ligand L-762,745 as a reporting method yielded a k(off) for L-738,167 of (4.1 +/- 0.1) x 10(-4) s(-1) (t1/2 = 28 min).

Molecular structure and granulocyte/macrophage colony-stimulating factor activity.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) plays a critical role in myeloid differentiation and in several immune and inflammatory processes. GM-CSF binds to specific cellular receptors (GM-CSFR) which belong to a recently described supergene family. These receptors are potential targets for pharmacologic design and such design depends on a molecular understanding of ligand-receptor interactions. We present our initial studies evaluating the potential active sites of the molecule. The sites on the GM-CSF molecule that were studied represent two alpha-helices predicted to be critical for GM-CSF activity, as implicated by human-murine chimeric molecule studies. These helices are predicted to be adjacent in native GM-CSF. Peptides corresponding to amino acids 17-31 and 78-99 of GM-CSF were synthesized and cross-linked to one another in two different orientations. The ability of anti-GM-CSF to bind the individual and complexed peptides was evaluated by both ELISA and radioimmunoassay. Significant binding to all peptides was demonstrated. A preferred orientation of the two peptides was apparent, and this agreed with the predicted model structures. Antibodies were developed against the coupled peptides, and these demonstrated significant cross-reactivity with recombinant human GM-CSF. Additionally, analyses of anti-peptide antisera binding studies predict these two amino acid sequences to lie in parallel planes to one another in the native human GM-CSF molecule.

Conformational perturbation of interleukin‐2: A strategy for the design of cytokine analogs

Interleukin-2 (IL-2) is a representative of a growing family of small proteins termed lymphokines which are responsible for mediating cell differentiation, growth and function in the immune system. Many of these proteins are being evaluated for their clinical potential. From the perspective of drug development, structure-function analysis of these molecules and their receptors require the use methodologies different than those traditionally employed for small peptides and other natural products. However, similar pharmacologic principles apply and an understanding of ligand-receptor interactions and the associated responses is required in order to efficiently pursue agonist and antagonist design. Although IL-2 is a protein of only 133 amino acid residues for which a low resolution X-ray structure does exist, the complexity of its receptor system has provided an added challenge to structure-function studies. Consequently, little is known concerning the receptor contact residues for this protein. We have attempted to utilize established principles of protein and peptide structure to manipulate the conformation of IL-2 in a manner which has provided analogs helpful for receptor interaction studies. These proteins have not only providing useful information on the nature of the IL-2 receptor but have also revealed potential strategies for the design of IL-2 agonists and antagonists.

Homosexuality and Prostitution

Previously, a novel series of 1<i>H</i>-4-substituted imidazole compounds were described as potent and selective histamine (HA) H₃ receptor ligands (Yates et al., 1999). The present studies extend the structure-activity relationships for optimal HA H₃ receptor affinity and central nervous system penetration by incorporation of a conformationally restricted cyclopropane nucleus. Moreover, the current studies extend our understanding of ligand-receptor interactions at the HA H₃ receptor with the development of high affinity HA H₃ receptor antagonists containing a stereochemical presentation. Structure-activity relationships were established from in vitro HA H₃receptor-binding affinities using [³H]N^α-methylhistamine and rat cortical tissue homogenates. Systematic optimization of multiple structural features critical for HA H₃ receptor affinity provided some of the most potent HA H₃ receptor agents described. For example, GT-2331 was determined to bind to a single population of HA H₃ receptors with a <i>K</i>_i of 0.125 nM. In vivo, GT-2331 has a favorable central nervous system penetration profile with an ED₅₀ of 0.08 mg/kg (i.p.) in rats and a long duration of action (<i>T</i>_1/2 &gt; 4 h). In addition, GT-2331 was extremely selective for the HA H₃receptor versus other HA receptors and a battery of neurotransmitter, neuropeptide, hormone, or enzyme systems. Several compounds were tested in vitro which suggested HA H₃ receptor heterogeneity and are discussed in terms of structure-activity relationships for the HA H₃ receptor.