E-FTMap: A Protein Structure Based Pharmacophore Identification Server for Guiding Fragment Expansion.

Although integrin alpha subunit I domains exist in multiple conformations, it is controversial whether integrin beta subunit I-like domains undergo structurally analogous movements of the alpha7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the beta(3) integrin I-like domain to lock its beta6-alpha7 loop and alpha7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor alpha(IIb)beta(3)(T329C/A347C) was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded alpha(IIb)beta(3)(V332C/M335C) was locked in a high affinity state. The results suggest that activation of the beta subunit I-like domain is analogous to that of the alpha subunit I domain, i.e. that axial movement in the C-terminal direction of the alpha7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation.

The present PhD work aimed to contribute to the knowledge available on the antitumor properties of flavonoids through the in vitro evaluation of the bioactivity of some uncommon semi-synthetic derivatives against a panel of breast and cervical cancer cell lines that are well-established models for certain types of gynecological cancer. The work included the semi-synthetic preparation of seven naringenin oxime and oxime ether derivatives prior to their bioactivity testing, and the in vitro evaluation of nine protoflavone-chalcone hybrid compounds and their reference fragments against breast (hormone-dependent MCF-7 and triple-negative MDA-MB-231), and cervical (SiHa & HeLa) cancer cell lines in addition to human HL-60 leukemia cells and non-cancerous mouse embryonic fibroblasts (NIH/3T3). The work included cytotoxicity, cell cycle distribution, caspase-3 activity, and antioxidant activity evaluations. Further, the cytotoxic activity of the hybrid compounds was evaluated in comparison with that of their corresponding fragments in a virtual and experimental combination study. In summary, our work led to the following results.  Two naringenin oxime isomers and five oxime ether derivatives were synthesized, purified and characterized. Four of these compounds, such as the minor product naringenin Z-oxime, and the ethyl, allyl, and tert-butyl ethers of naringenin E-oxime were prepared as new compounds.  When evaluating the in vitro cytotoxicity of the prepared derivatives of naringenin, tert-butyl oxime ether (6) showed the most potent effects on different gynecological cancer cells, with significant activity against MCF-7 and HeLa cells.  The flow cytometric analysis of compound (6) on gynecological cancer cells revealed significant accumulation of cells in the hypodiploid (subG1) phase in HeLa & SiHa cell lines, indicating the apoptosis induction effect, and induced cycle suppression at G2/M stage in MCF-7 cancer cells. Further, the proapoptotic activity of this compound was confirmed in HeLa cells by detecting the increased activity of caspase-3.  To our surprise, naringenin methyl oxime ether was more potent in the ORAC assay than its parent compound, while all other analogs were up to an order of magnitude less active. This suggests good peroxyl radical scavenging capacity for this compound. There was no apparent correlation between the in vitro cytotoxic and antioxidant activities of the tested compounds, suggesting that their anticancer effects are likely not related to their antioxidant properties.  Four protoflavone hybrid compounds were identified as promising antitumor lead compounds based on their prominent in vitro cytotoxic effects and their selectivity on different breast and cervical cancer cells with antiproliferative effects better than cisplatin.  The most potent compounds have an intense proapoptotic effect on TNBC, as evidenced by flow cytometric investigation and caspase-3 activity. It was shown that compound 10c induces a considerable expansion in caspase-3 activity in a concentration-time dependent manner with a significant increase in the sub G1 phase.  A novel approach was used to evaluate the bioactivity of the hybrid compounds in comparison with that of their corresponding fragments. A virtual combination study was performed by using the Chou-Talalay method as a mathematical tool, and results were compared to the corresponding experimental combinations of the cells with non-coupled fragments. This gave valuable extra information as virtual combination index values and confirmed that the studied hybrid compounds are much more potent than what would be expected by a mathematical sum of the bioactivity of their fragments.  Based on the above, we demonstrated the use of a novel approach to evaluate the bioactivity of hybrid compounds in general, and suggested an extension of the applicability of the Chou-Talalay method, one of the currently available most popular platforms for drug-drug combination studies. Altogether, our present study provided valuable information about the antitumor potential of two series of unusual semi-synthetic flavonoid derivatives, and made a significant contribution to identifying a set of highly potent hybrid lead compounds obtained through fragment-based drug design. Further, by providing a novel use for an existing convenient and very widely used mathematical platform, we believe our study may have contributed to the research of hybrid compounds also in a more general manner.

Knowing the ligand or peptide binding site in proteins is highly important to guide drug discovery, but experimental elucidation of the binding site is difficult. Therefore, various computational approaches have been developed to identify potential binding sites in protein structures. However, protein and ligand flexibility are often neglected in these methods due to efficiency considerations despite the recognition that protein-ligand interactions can be strongly affected by mutual structural adaptations. This is particularly true if the binding site is unknown, as the screening will typically be performed based on an unbound protein structure. Herein we present DynaBiS, a hierarchical sampling algorithm to identify flexible binding sites for a target ligand with explicit consideration of protein and ligand flexibility, inspired by our previously presented flexible docking algorithm DynaDock. DynaBiS applies soft-core potentials between the ligand and the protein, thereby allowing a certain protein-ligand overlap resulting in efficient sampling of conformational adaptation effects. We evaluated DynaBiS and other commonly used binding site identification algorithms against a diverse evaluation set consisting of 26 proteins featuring peptide as well as small ligand binding sites. We show that DynaBiS outperforms the other evaluated methods for the identification of protein binding sites for large and highly flexible ligands such as peptides, both with a holo or apo structure used as input.

High density lipoprotein (HDL3) binds with high affinity to many types of cells, but controversy exists concerning the nature and biological significance of the binding. We have recently demonstrated that HDL and apoproteins (apo)-AI, -AII, and -CI stimulate a specific and dose-dependent increase in placental lactogen (hPL) release from human trophoblast cells. To examine the possible relationship between HDL3 binding and stimulation of hPL release, we have characterized the binding of [125I]HDL3 to an enriched fraction of hPL-producing trophoblast cells. Binding studies were performed on trophoblast cells isolated by isopycnic centrifugation of collagenase/hyaluronidase-dispersed placental tissue and apo-E free-HDL3 (density, 1.125-1.215 g/ml). Scatchard analysis of binding studies performed at 37 C for 2 h revealed two classes of binding sites: 1) high affinity binding sites with a Kd of 9.7 +/- 2.2 micrograms/ml (1.3 x 10(-7) M) and 9.8 +/- 3.2 x 10(5) binding sites/trophoblast cell, and 2) low affinity binding sites with a Kd of 172.8 +/- 64.8 micrograms/ml (2.3 x 10(-6) M) and an estimated 3.2 x 10(6) sites/cell. As has been found in hepatocytes and other cells, the number of HDL3-binding sites per trophoblast cell (but not the binding affinity) decreased at lower incubation temperatures. In addition, HDL3 binding to trophoblasts cells did not require calcium and was not affected by prior treatment of the cells with pronase or trypsin. HDL3-binding sites on trophoblast cells, however, were not specific for HDL3. Low density lipoprotein (density, 1.063-1.055 g/ml), which does not stimulate hPL release, was nearly as potent on a molar basis as HDL3 in binding to the high and low affinity binding sites on trophoblast cells. Furthermore, nitrated HDL3, which does not compete for high affinity binding to trophoblast cells, stimulated hPL release. Although the characteristics of HDL3 binding to trophoblast cells are similar to those of other cells, these results strongly suggest that the binding of HDL3 to high affinity binding sites is not essential for HDL-mediated hPL release.

The F-domain of rat HNF-4alpha1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4alpha. (i) Fluorescent binding assays indicate that wild-type, full-length HNF-4alpha (amino acids 1-455) has high affinity (Kd=0.06-12 nm) for long chain fatty acyl-CoAs (LCFA-CoA) and low affinity (Kd=58-296 nm) for unesterified long chain fatty acids (LCFAs). LCFA-CoA binding was due to close molecular interaction as shown by fluorescence resonance energy transfer (FRET) from full-length HNF-4alpha tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor), which yielded an intermolecular distance of 33 A, although no FRET to cis-parinaric acid was detected. (ii) Deleting the N-terminal A-D-domains, comprising the AF1 and DNA binding functions, only slightly affected affinities for LCFA-CoAs (Kd=0.9-4 nm) and LCFAs (Kd=93-581 nm). (iii) Further deletion of the F-domain robustly reduced affinities for LCFA-CoA and reversed ligand specificity (i.e. high affinity for LCFAs (Kd=1.5-32 nm) and low affinity for LCFA-CoAs (Kd=54-302 nm)). No FRET from HNF-4alpha-E (amino acids 132-370) tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor) was detected, whereas an intermolecular distance of 28 A was calculated from FRET between HNF-4alpha-E and cis-parinaric acid. (iv) Circular dichroism showed that LCFA-CoA, but not LCFA, altered the secondary structure of HNF-4alpha only when the F-domain was present. (v) cis-Parinaric acid bound to HNF-4alpha with intact F-domain was readily displaceable by S-hexadecyl-CoA, a nonhydrolyzable thioether analogue of LCFA-CoAs. Truncation of the F-domain significantly decreased cis-parinaric acid displacement. Hence, the C-terminal F-domain of HNF-4alpha regulated ligand affinity, ligand specificity, and ligand-induced conformational change of HNF-4alpha. Thus, characteristics of F-domain-truncated mutants may not reflect the properties of full-length HNF-4alpha.

High-affinity binding sites for oxygenated sterols in rat liver microsomes: possible identity with antiestrogen binding sites

Relationship between lectin monosaccharide specificity and binding to the plasma membrane of human fibroblasts

Cdc25B is a key regulator of entry into mitosis, and its activity and localization are regulated by binding of the 14-3-3 dimer. There are three 14-3-3 binding sites on Cdc25B, with Ser(323) being the highest affinity binding and is highly homologous to the Ser(216) 14-3-3 binding site on Cdc25C. Loss of 14-3-3 binding to Ser(323) increases cyclin/Cdk substrate access to the catalytic site, thereby increasing its activity. It also affects the localization of Cdc25B. Thus, phosphorylation and 14-3-3 binding to this site is essential for down-regulating Cdc25B activity, blocking its mitosis promoting function. The question of how this inhibitory signal is relieved to allow Cdc25B activation and entry into mitosis is yet to be resolved. Here, we show that Ser(323) phosphorylation is maintained into mitosis, but phosphorylation of Ser(321) disrupts 14-3-3 binding to Ser(323), mimicking the effect of inhibiting Ser(323) phosphorylation on both Cdc25B activity and localization. The unphosphorylated Ser(321) appears to have a role in stabilizing 14-3-3 binding to Ser(323), and loss of the Ser hydroxyl group appears to be sufficient to significantly reduce 14-3-3 binding. A consequence of loss of 14-3-3 binding is dephosphorylation of Ser(323). Ser(321) is phosphorylated in mitosis by Cdk1. The mitotic phosphorylation of Ser(321) acts to maintain full activation of Cdc25B by disrupting 14-3-3 binding to Ser(323) and enhancing the dephosphorylation of Ser(323) to block 14-3-3 binding to this site.

High and low affinity membrane binding sites for fibroblast growth factors in the developing chick brain

The extracellular, ligand binding regions of ErbB receptors consist of four domains that can assume at least two alternative conformations, extended and locked. The locked conformation, observed in several crystal structures, is held together by a noncovalent intramolecular tether and is incompatible with current models for receptor dimerization and ligand activation. Based on structures of ligand-receptor complexes in the extended conformation, the high affinity ligand binding pocket between domains I and III is disrupted in the locked conformation. Therefore the biological role of the locked conformation is not clear. To address the impact of the locked conformation on ligand binding, we compared extracellular domains of wild-type ErbB3, mutant domains in a constitutively locked or extended conformation and partial extracellular domain constructs. We found that the constitutively locked receptor domains and truncated constructs carrying only domains I-II or III-IV strongly bind ligand, albeit with reduced affinity compared to wild-type receptor. This suggests that the locked conformation cannot be discounted for ligand binding. The significant binding by both partial interfaces in domains I and III also suggests that "partial bivalency" may be the reason for the low nanomolar and high picomolar binding observed for ErbB3 in the respective "low" and high affinity states. In contrast to EGFR (ErbB1), ErbB3 retains high ligand binding affinity at an endosome-comparable pH in both the extended and locked conformations. Ligand affinity for the locked conformation even improves at low pH. For ErbB3, the contribution of domain I to ligand binding is strong and increases at low pH while its contribution is thought to be minimal for EGFR, regardless of pH. This shift in domain contribution and pH dependency provides a mechanistic explanation for some of the divergent properties of EGFR and ErbB3.

A fragment-based drug design paradigm has been successfully applied in the discovery of lead series of ketohexokinase inhibitors. The paradigm consists of three iterations of design, synthesis, and X-ray crystallographic screening to progress low molecular weight fragments to leadlike compounds. Applying electron density of fragments within the protein binding site as defined by X-ray crystallography, one can generate target specific leads without the use of affinity data. Our approach contrasts with most fragment-based drug design methodology where solution activity is a main design guide. Herein we describe the discovery of submicromolar ketohexokinase inhibitors with promising druglike properties.

The plasma clearance of intestinally derived remnant lipoproteins by the liver is a process that likely involves three steps. Our model suggests that the initial rapid clearance by the liver begins with sequestration of the remnants within the space of Disse, where apolipoprotein E secreted by hepatocytes enhances remnant binding and uptake. Heparan sulfate proteoglycans (HSPG), which are also abundant in the space of Disse, mediate this enhanced binding. Next, the remnants undergo further processing in the space of Disse by hepatic and lipoprotein lipases, which may also serve as ligands mediating remnant uptake. The final step, endocytosis by hepatocytes, appears to be mediated, at least in part, by the low density lipoprotein (LDL) receptor and by the LDL receptor-related protein (LRP). Cell-surface HSPG play a critical role in remnant uptake, not only in the important initial sequestration or capture step in the space of Disse, but also as an essential or integral component of the HSPG-LRP pathway. In addition, HSPG appear to function alone as a receptor and display unique handling properties for specific isoforms of apolipoprotein E.

Multiple binding sites for phencyclidine on the nicotinic acetylcholine receptor from Torpedo ocellata electric organ

Topology of ligand binding sites on the nicotinic acetylcholine receptor

BackgroundHIV-1 gene expression is driven by the long terminal repeat (LTR), which contains many binding sites shown to interact with an array of host and viral factors. Selective pressures within the host as well as the low fidelity of reverse transcriptase lead to changes in the relative prevalence of genetic variants within the HIV-1 genome, including the LTR, resulting in viral quasispecies that can be differentially regulated and can potentially establish niches within specific cell types and tissues.MethodsUtilizing flow cytometry and electromobility shift assays, specific single-nucleotide sequence polymorphisms (SNPs) were shown to alter both the phenotype of LTR-driven transcription and reactivation. Additional studies also demonstrated differential loading of transcription factors to probes derived from the double-variant LTR as compared to probes from the wild type.ResultsThis study has identified specific SNPs within CCAAT/enhancer binding protein (C/EBP) site I and Sp site III (3 T, C-to-T change at position 3, and 5 T, C-to-T change at position 5 of the binding site, respectively) that alter LTR-driven gene transcription and may alter the course of viral latency and reactivation. The HIV-1 LAI LTRs containing the SNPs of interest were coupled to a plasmid encoding green fluorescent protein (GFP), and polyclonal HIV-1 LTR-GFP stable cell lines utilizing bone marrow progenitor, T, and monocytic cell lines were constructed and utilized to explore the LTR phenotype associated with these genotypic changes.ConclusionsAlthough the 3 T and 5 T SNPs have been shown to be low-affinity binding sites, the fact that they can still result in effective HIV-1 LTR-driven gene expression, particularly within the TF-1 cell line, has suggested that the low binding site affinities associated with the 3 T C/EBP site I and 5 T Sp site III are potentially compensated for by the interaction of nuclear factor-κB with its corresponding binding sites under selected physiological and cellular conditions. Additionally, tumor necrosis factor-α and Tat can enhance basal transcription of each SNP-specific HIV-1 LTR; however, differential regulation of the LTR is both SNP- and cell type-specific.