Low-affinity Ligands Research Articles

Cutting edge: Extracellular signal-related kinase is not required for negative selection of developing T cells.

Signals initiated through the TCR during development can result in either survival and differentiation or cell death. High affinity signals that induce death elicit a robust yet transient activation of signaling pathways, including Erk, whereas low affinity ligands, which promote survival, generate a gradual and weaker activation of the same pathways. It was recently demonstrated that Erk localizes to distinct cellular locations in response to high and low affinity ligands. Although a requirement for Erk in positive selection is well established, its role in negative selection is controversial and, thus, the importance of Erk relocalization during development is not understood. In this study, we examined the role of Erk in negative selection using mice that are genetically deficient in both Erk1 and Erk2 in T cells. Results from three different models reveal that thymocyte deletion remains intact in the absence of Erk.

Development of the terrestrial biotic ligand model for predicting nickel toxicity to barley (Hordeum Vulgare): Ion effects at low pH

The focus of the present study was to investigate the potential for Al3+, Mg2+, and H+ to influence Ni2+ toxicity for barley seedlings grown in acidic aqueous solutions and to assess the capacity of a two-site terrestrial biotic ligand model (tBLM) to accurately predict 50% effect activities (EA50s). To accomplish these objectives, 48-h EA50Ni2+ values were obtained for three sets of exposures in which the pH and activity of Al3+ and Mg2+ were varied. Exposures contained both Al alone and in combination with Mg so that compound ion effects could be investigated. A tBLM was then constructed to predict EA50Ni2+ values from the exposure solution chemistry. The results show a slight protective effect of H+ against Ni2+ toxicity and a strong protective effect of Mg2+, as indicated by a 4.6- and 8.0-fold increase in the measured EA50Ni2+ values corresponding to changes in pH from 6.0 to 4.5 and {Mg2+} from 0 to 1.40 mM, respectively. Increasing solution {Al3+} from 0 to 0.5 microM had no effect on Ni2+ toxicity, although Al itself negatively affected root elongation. Comparison of EA50 values calculated as both Ni2+ and measured concentration of total Ni in the root ([Root-Ni]T) showed [Root-Ni]T to be a more normalized measure of Ni bioavailability. The strong correlation between root growth inhibition and tBLM-predicted root-Ni accumulation suggests that toxicity was influenced by Ni2+ binding to low-affinity ligands within the cell wall, in addition to Ni2+ uptake through Mg2+ transporters. Predicted EA50Ni2+ values generated with the model were all within a factor of +/-1.5 from measured values--a result that emphasizes the advantage of using the tBLM for risk assessment.

Differential Signaling Activation by Insulin and Insulin-Like Growth Factors I and II upon Binding to Insulin Receptor Isoform A

A variety of human malignancies overexpresses isoform A of the insulin receptor (IR-A) and produces IGFs (IGF-I and/or IGF-II). IR-A binds IGF-II with high affinity (although 4-fold lower than that for insulin), whereas it binds IGF-I with low affinity (approximately 30-fold lower than that for insulin). However, in engineered cells expressing only the IR-A, but not IGF-I receptor (R(-)/IR-A cells), IGF-II is a more potent mitogen than insulin. Herein, we investigated downstream signaling of IGF-II, IGF-I, and insulin in R(-)/IR-A cells to better understand their role in cell growth. We found that despite inducing a lower IR-A autophosphorylation than insulin, IGF-II was more potent than insulin for activating p70S6 kinase (p70S6K) and approximately equally potent in activating the early peaks of ERK1/2 and Akt. However, ERK1/2 activation persisted longer after IGF-II, whereas Akt activation persisted longer after insulin. Therefore, cells stimulated with IGF-II had a higher p70S6K/Akt activation ratio than cells stimulated with insulin. Remarkably, IGF-I also elicited a similar signaling pattern as IGF-II, despite inducing minimal IR-A autophosphorylation. ERK1/2 and protein kinase C seem to be involved in the preferential stimulation of p70S6K by IGFs. In conclusion, our study has identified a novel complex role of IR-A, which not only elicits a unique signaling pattern after IGF-II binding but also induces substantial downstream signaling upon binding to the low-affinity ligand IGF-I. These results underline the role of IR-A in physiology and disease.

CTL Activation Using the Natural Low-Affinity Epitope 222-229 from Tyrosinase-Related Protein 1 Leads to Tumor Rejection

Vaccine strategies for cancer immunotherapy have focused on peptide ligands with high affinity for MHC class I. Largely, these vaccines have not been therapeutic. We have examined the peptide specificity of a strongly protective T-cell response that eradicates established B16 melanoma and find that the recognized epitope is generated by a low-affinity MHC class I ligand from tyrosinase-related protein 1 (TRP1). Cytotoxic T-cell responses are induced against TRP1(222-229) by several vaccination schemes using a Toll-like receptor agonist, T regulatory cell depletion, or the immune modulator B7-DCXAb to drive immunity. TRP1(222) CTL are generated from multiple antigen sources, including antigens expressed by tumors growing in situ, tumor cell lysates, and peptide vaccines. The key finding in this study is that protection from freshly implanted or established B16 tumors is primarily mediated by TRP1(222)-specific CTL and not by CTL specific for more traditional melanoma antigens such as TRP2 or gp100. This finding challenges the assumption that the optimal peptide antigens for cancer vaccines are high-affinity MHC ligands. We propose that when administered appropriately, native low-affinity MHC ligands are optimal inducers of immunotherapeutic CTL.

The structural basis for peptide selection by the transport receptor OppA

Oligopeptide-binding protein A (OppA) from Lactococcus lactis binds peptides of an exceptionally wide range of lengths (4-35 residues), with no apparent sequence preference. Here, we present the crystal structures of OppA in the open- and closed-liganded conformations. The structures directly explain the protein's phenomenal promiscuity. A huge cavity allows binding of very long peptides, and a lack of constraints for the position of the N and C termini of the ligand is compatible with binding of peptides with varying lengths. Unexpectedly, the peptide's amino-acid composition (but not the exact sequence) appears to have a function in selection, with a preference for proline-rich peptides containing at least one isoleucine. These properties can be related to the physiology of the organism: L. lactis is auxotrophic for branched chain amino acids and favours proline-rich caseins as a source of amino acids. We propose a new mechanism for peptide selection based on amino-acid composition rather than sequence.

Stereocontrolled Synthesis and Pharmacological Evaluation of Azetidine‐2,3‐Dicarboxylic Acids at NMDA Receptors

The four stereoisomers of azetidine-2,3-dicaroxylic acid (L-trans-ADC, L-cis-ADC, D-trans-ADC, and D-cis-ADC) were synthesized in a stereocontrolled fashion following two distinct strategies: one providing the two cis-ADC enantiomers and one giving access to the two trans-ADC enantiomers. The four azetidinic amino acids were characterized in a radioligand binding assay ([(3)H]CGP39653) at native NMDA receptors: L-trans-ADC showed the highest affinity (K(i)=10 microM) followed by the D-cis-ADC stereoisomer (21 microM). In contrast, the two analogues L-cis-ADC and D-trans-ADC were low-affinity ligands (>100 and 90 microM, respectively). Electrophysiological characterization of the ADC compounds at the four NMDA receptor subtypes NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D expressed in Xenopus oocytes showed that L-trans-ADC displayed the highest agonist potency at NR1/NR2D (EC(50)=50 microM), which was 9.4-, 3.4-, and 1.9-fold higher than the respective potencies at NR1/NR2A-C. D-cis-ADC was shown to be a partial agonist at NR1/NR2C and NR1/NR2D with medium-range micromolar potencies (EC(50)=720 and 230 microM, respectively). A subsequent in silico ligand-protein docking study suggested an unusual binding mode for these amino acids in the agonist binding site.

Pre-docking filter for protein and ligand 3D structures.

Virtual drug screening using protein-ligand docking techniques is a time-consuming process, which requires high computational power for binding affinity calculation. There are millions of chemical compounds available for docking. Eliminating compounds that are unlikely to exhibit high binding affinity from the screening set should speed-up the virtual drug screening procedure. We performed docking of 6353 ligands against twenty-one protein X-ray crystal structures. The docked ligands were ranked according to their calculated binding affinities, from which the top five hundred and the bottom five hundred were selected. We found that the volume and number of rotatable bonds of the top five hundred docked ligands are similar to those found in the crystal structures and corresponded with the volume of the binding sites. In contrast, the bottom five hundred set contains ligands that are either too large to enter the binding site, or too small to bind with high specificity and affinity to the binding site. A pre-docking filter that takes into account shapes and volumes of the binding sites as well as ligand volumes and flexibilities can filter out low binding affinity ligands from the screening sets. Thus, the virtual drug screening procedure speed is increased.

Comparative Analysis of LFA-1 Activation State and Functional Involvement in Enhanced Cytotoxicity of NK Cell Lines KHYG-1 and NK-92

Background: KHYG-1 and NK-92 are highly cytotoxic IL-2 dependent NK cell lines. NK cytotoxicity is regulated through an array of receptors with activating and inhibitory functions for spontaneous elimination of pathogen infected or tumor cells. Important roles have been described for the leukocyte function-associated antigen (LFA)- 1 adhesion receptor (αLβ2) in NK cytotoxicity. In T cells, the integrin LFA-1 occurs in a non-functional, bent form and requires activation, through divalent cations e.g., for ligand binding. In marked contrast, IL-2 stimulated NK cells could directly engage the LFA-1 ligand intercellular adhesion molecules (ICAM)-1, thereby inducing conjugate formation, granule polarization, degranulation, and tumor cell lysis (Barber DF et al., 2004. JI 173:3653–9). Strikingly, in the NK cell line KHYG-1 granules were constitutively polarized (clustered around the MTOC; Suck G et al., 2006. Int Immunol 18:1347–54) and LFA-1 downstream signaling molecules, the spleen tyrosine kinase (Syk) and extracellular signal-regulated MAP kinase (ERK) constitutively phosphorylated (Suck G et al., 2005. Exp Hem. 33:1160–71). These previous findings prompted us to investigate LFA-1 activation state and functional involvement in cytotoxicity in KHYG-1 and NK- 92.Methods: Adhesion assays were performed on immobilized ICAMs and % adherence determined in a fluorescence plate reader (Tang XY et al., 2008 JI, 180:4793–804). LFA-1 was activated with Mg/EGTA (5 mM MgCl2 and 1.5 mM EGTA), monoclonal antibody (mAb) KIM185 (10 mg/mL) or both; LFA-1 mAb, clone MHM24, was used for blocking studies, 1–10 mg/ml and mAb KIM127 for immunoprecipitation; cytotoxicity, conjugate formation, and degranulation (CD107a) were measured by Flow cytometry and cell morphology imaged by phase contrast (Olympus, 1X70) or confocal microscopy (Zeiss, LSM510 Meta/Nikon A1). Excel software was employed for statistical analyses.Results: In cell binding assays KHYG-1 and NK-92 showed constitutive adhesion to the LFA1-1 ligand ICAM-1, 61% +/− 5.8% SD and 55% +/− 7.5% SD, respectively. However, only 22% NK-92 and 10.5% KHYG-1 cells adhered to the lower affinity ligand ICAM-3 and activation with Mg/EGTA or KIM185 was required to increase binding to 73% in KHYG- 1 and to 62% in NK-92. Immunoprecipitation experiments with the activation reporter mAb KIM127 revealed an activated extended LFA-1 conformation in both cell lines. Together these results suggested an intermediate affinity activation state for the LFA- 1 population in KHYG-1 and NK-92. Despite such similarities, ICAM-1 engagement triggered pronounced cell spreading in KHYG-1, similar to phorbol ester stimulated T cells, but not in NK-92 or primary NK cells. It is conceivable that the capacity to undergo cytoskeletal remodeling in KHYG-1 may be impaired as potentially also reflected in the constitutively polarized granule state. In addition, LFA-1 blocking studies with LFA- 1 antibody MHM24 did not inhibit target conjugate formation in KHYG-1, compared to partial inhibition in NK-92, and cytotoxicity against K562 was only about 40% diminished in KHYG-1, compared to almost complete abrogation (maximum 98%) in NK-92. Nevertheless, overall cytolytic potential against K562 was comparable among the two cell lines, which implicated important functions for other receptors at least in KHYG-1.Conclusion: Results suggested a constitutively activated intermediate affinity state for LFA-1 in KHYG-1 and NK-92. It is conceivable that a similar activated state occurs in primary NK cells. Interestingly, although cytoskeletal dysfunction was indicated in KHYG-1, cytotoxicity of the cell line was unaffected, rendering it a valuable model for the study of alternative pathways.

Blocking Inhibitory KIR Is Insufficient for Optimal Killing of AML and ALL Targets: Additional Requirements for NKG2A and LIR-1 Blockade

NK cell alloreactivity is determined by the interaction between inhibitory NK cell receptors and their MHC class I cognate ligands. The best characterized NK cell receptor that recognizes MHC I is KIR. Mismatching donors and recipients for KIR ligands has led to transplant strategies that can result in less relapse and better survival in myeloid leukemia patients. However, other MHC recognizing receptors may also be involved in leukemia eradication. We developed a model using primary AML and ALL targets to explore the hierarchy of different class I MHC recognizing inhibitory NK cell receptors. In addition to KIR, NKG2A is expressed on 54±14% of PB NK cells and is the dominant NK cell receptor expressed in the first six months after transplant. LIR-1 is expressed on 35±17% of PB NK cells and is more likely to be co-expressed on KIR+ NK cells than KIR- NK cells. The goal of our study was to determine optimal conditions to enhance NK alloreactivity against primary AML and ALL targets. A pan-HLA blocking antibody (HP-1F7, binding to HLA, B, C, E and G) was used as a positive control to interrupt all signals from MHC class I. Receptor blockade with anti-KIR (1-7F9, blocking KIR 2DL1/L2/L3, Novo Nordisk), anti-NKG2A and anti-LIR-1 was tested either alone or in combination to understand the hierarchy of inhibitory receptors that determine NK cell reactivity of leukemia targets. Resting NK cells were tested fresh or activated with low concentrations of IL-2 (5 IU for 42 hours) to mimic conditions achievable by pharmacologic IL-2 administration. Six of ten primary AML targets were sensitive to allogeneic NK cell killing only when HLA-A, B, C, E and G were blocked (7.8±5.7 vs. 24±9.6, n=6; p<0.005). Six of seven primary ALL targets also responded to resting allogeneic NK cells but only after class I blockade (8.2±5.7 vs. 25±9.7, n=6; p<0.005). Low concentrations of IL-2 enhanced mean killing with class I blockade by approximately 10% for AML and ALL targets. CD107a degranulation yielded similar results. We next evaluated the class I recognizing inhibitory receptors on NK cells responsible for the enhanced killing of primary leukemia targets. Experiments were performed with KIR, NKG2A and LIR-1 blockade. In both cytotoxicity and CD107a degranulation assays, single inhibitory receptor blockade resulted in enhanced killing slightly. However, addition of LIR-1 blockade to either KIR blockade or NKG2A blockade consistently increased killing of all targets sensitive to pan-HLA blockade. Purification of KIR- NK cells, expressing NKG2A or LIR-1, showed they were potently alloreactive against primary leukemia targets only upon dual blockade with NKG2A and LIR-1 mAbs. Blocking of more than one receptor was always more effective than single receptor blockade. All leukemia targets expressed HLA-E (the ligand for NKG2A) but none expressed HLA-G (the high affinity ligand for LIR-1) by Western Blot analysis, suggesting that other HLA class I (low affinity ligands for LIR-1) are involved. Our data show that:Both KIR+ and KIR- NK cells exhibit significant potential for cytotoxicity of killing primary AML and ALL blasts but only when more than one class I receptor was blocked,KIR- NK cells can be licensed through receptors other than KIR, andMore than half of primary AML and ALL targets tested are susceptible to allogeneic NK cell killing following multiple inhibitory receptor blockade.Therefore, it is not surprising that the current strategies of adoptive NK cell therapy may be limited. Clinical trials with mAb blocking of multiple MHC class I inhibitor receptors (KIR, NKG2A, or LIR-1) to enhance NK cell killing of AML and ALL is warranted.

Differences between High- and Low-Affinity Complexes of Enzymes and Nonenzymes

Physical differences in small molecule binding between enzymes and nonenzymes were found through mining the protein-ligand database, Binding MOAD (Mother of All Databases). The data suggest that divergent approaches may be more productive for improving the affinity of ligands for the two classes of proteins. High-affinity ligands of enzymes are much larger than those with low affinity, indicating that the addition of complementary functional groups is likely to improve the affinity of an enzyme inhibitor. However, this process may not be as fruitful for ligands of nonenzymes. High- and low-affinity ligands of nonenzymes are nearly the same size, so modest modifications and isosteric replacement might be most productive. The inherent differences between enzymes and nonenzymes have significant ramifications for scoring functions and structure-based drug design. In particular, nonenzymes were found to have greater ligand efficiencies than enzymes. Ligand efficiencies are often used to indicate druggability of a target, and this finding supports the feasibility of nonenzymes as drug targets. The differences in ligand efficiencies do not appear to come from the ligands; instead, the pockets yield different amino acid compositions despite very similar distributions of amino acids in the overall protein sequences.

Crystallography‐Independent Determination of Ligand Binding Modes

Pass the spin: The internuclear NOE interactions for pharmacophore mapping (INPHARMA) can be used to derive the structure of receptor/ligand complexes for low-affinity lead compounds identified in the early stages of drug discovery. The relative binding mode and, in favorable cases, the absolute binding mode of pairs of competitive low-affinity ligands can be identified with INPHARMA. This is demonstrated for a system comprising protein kinase A (T in the schematic representation) and two activity inhibitors with known structures (LA and LB).

Characterisation of bioactive compounds in infant formulas using immobilised recombinant estrogen receptor-α affinity columns

In this study, the use of recombinant estrogen receptor alpha (ERα)-based affinity columns was reported, for the isolation and the identification of estrogenic substances present in complex matrices, focusing on bioactive compounds present in foodstuff. The capability of affinity columns to trap high, but also low-affinity radio-labelled ligands (17β-estradiol, genistein and bisphenol A) was demonstrated. Three pooled samples of infant formulas (milk-based, hypoallergenic and soy-based formulas for infants aged 0–4 months) from a EU market basket were prepared by the CASCADE Network of Excellence. After determining the estrogenic activity of these food samples, human recombinant ERα ligand binding domain (LBD) based affinity columns combined with suitable analytical methods (high resolution LC–MS/MS) were used to identify the bioactive compounds present in the soy-based formula extract, namely phytoestrogens (genistein and daidzein) involved in the agonistic activity measured. Incubations of genistein with liver microsomes were carried out and the extracts analysed following the same protocol, demonstrating that hERα affinity columns can also be used for trapping active metabolites. This approach combining bioluminescent cell lines with this useful tool based on hERα-LBD affinity columns thus allowed the purification and the concentration of both known and unknown estrogenic ligands prior to investigation of their structure using LC–MS.

The labile iron pool in human erythroid cells

Although most cellular iron is firmly bound (e.g. in haemoglobin), some, the labile iron pool (LIP), is bound to low-affinity ligands. The LIP is regarded as the crossroads of cellular iron traffic. Using multi-parameter flow-cytometry of cells treated with the metal-sensitive sensor calcein and the cell-permeable chelator deferiprone, we studied LIP in various human erythroid cell populations in peripheral blood, bone marrow and culture. Erythroid maturation was found to be associated with a decrease in the LIP. In the peripheral blood, nucleated erythrocytes (normoblasts) had 5.8-fold and 8.8-fold greater LIP than reticulocytes and erythrocytes respectively. Early reticulocytes had 2.5-fold more LIP than late reticulocytes. In the bone-marrow and in culture, LIP decreased by c. 30-fold as early erythroid precursors matured to late precursors. Adding holo-transferrin to iron-depleted cultures elevated LIP by 3.9-fold. We also show that in beta-thalassaemia, a disease associated with iron-overload, erythrocytes and reticulocytes in the blood and erythroid precursors in culture have a significantly greater LIP than their normal counterparts. In conclusion, the LIP in erythroid cells is altered under physiological (maturation) and pathological (thalassaemia) conditions. The methodology presented might be useful for evaluating the LIP in various diseases and for studying the efficacy of iron-chelators.

Fetal Exposure to High-Avidity TCR Ligand Enhances Expansion of Peripheral T Regulatory Cells

Lately, it has become clear that regulatory T cells (Tregs) play a major role in the maintenance of peripheral tolerance and control of autoimmunity. Despite these critical functions, the process underlying the development of Tregs remains largely undefined. Herein, altered peptide ligand (APL) variants derived from the proteolipid protein-1 (PLP1) epitope were expressed on immunoglobulins (Igs) and the resulting Ig-APLs were used to deliver the APLs from mother to fetus through the maternal placenta to influence thymic T cell selection. This delivery system was then adapted to the SJL/J mouse, a strain that expresses only the DM20 form of PLP, which lacks the dominant PLP1 epitope in the thymus during fetal and neonatal development. This model, which restores thymic T cell selection for PLP1, was then used to determine whether affinity plays a role in the development of Tregs. The findings show that fetal exposure to low-affinity peptide ligand was unable to drive development of Tregs while variants with higher affinity to the TCR resulted in significant seeding of the periphery with mature, naive Tregs. Thus, contrary to pathogenic T cells, Tregs require avid TCR-ligand interaction to undergo thymic development and maturation.

Monitoring of influenza virus hemagglutinin in process samples using weak affinity ligands and surface plasmon resonance

Surface plasmon resonance (SPR) was used to screen the interaction between a variety of affinity ligands and hemagglutinin (HA) from human influenza virus, with the aim of identifying low affinity ligands useful for the development of a rapid bioanalytical sensor. Three sialic acid-based structures and four lectins were evaluated as sensor ligands. The sialic acid-based ligands included a natural sialic acid-containing glycoprotein, human α1-acid glycoprotein (α1-AGP), and two synthetic 6′-sialyllactose-conjugates, with varying degree of substitution. The interaction of HA with the four lectin-based ligands, concanavalin A (Con A), wheat germ agglutinin (WGA), Maackia amurensis lectin (MAL), and Sambucus nigra agglutinin (SNA), showed a wide variation of affinity strengths. Affinity and kinetics data were estimated. Strong affinities were observed for Con A, WGA, α1-AGP, and a 6′-sialyllactose-conjugate with a high substitution degree, and low affinities were observed for MAL and a 6′-sialyllactose-conjugate with low substitution. The main objective, to identify a low affinity ligand which could be used for on-line monitoring and product quantification, was met by a 6′-sialyllactose–ovalbumin conjugate that had 0.6 mol ligand per mol carrier protein. The apparent affinity of this ligand was estimated to be 1.5 ± 0.03 μM ( K D) on the SPR surface. Vaccine process samples containing HA were analyzed in the range 10–100 μg HA mL −1 and correlated with single-radial immunodiffusion. The coefficient of variation on the same chip was between 0.010 and 0.091.

The T Cell Receptor’s α-Chain Connecting Peptide Motif Promotes Close Approximation of the CD8 Coreceptor Allowing Efficient Signal Initiation

The CD8 coreceptor contributes to the recognition of peptide-MHC (pMHC) ligands by stabilizing the TCR-pMHC interaction and enabling efficient signaling initiation. It is unclear though, which structural elements of the TCR ensure a productive association of the coreceptor. The alpha-chain connecting peptide motif (alpha-CPM) is a highly conserved sequence of eight amino acids in the membrane proximal region of the TCR alpha-chain. TCRs lacking the alpha-CPM respond poorly to low-affinity pMHC ligands and are unable to induce positive thymic selection. In this study we show that CD8 participation in ligand binding is compromised in T lineage cells expressing mutant alpha-CPM TCRs, leading to a slight reduction in apparent affinity; however, this by itself does not explain the thymic selection defect. By fluorescence resonance energy transfer microscopy, we found that TCR-CD8 association was compromised for TCRs lacking the alpha-CPM. Although high-affinity (negative-selecting) pMHC ligands showed reduced TCR-CD8 interaction, low-affinity (positive-selecting) ligands completely failed to induce molecular approximation of the TCR and its coreceptor. Therefore, the alpha-CPM of a TCR is an important element in mediating CD8 approximation and signal initiation.

Competitive Inhibitors of Helicobacter pylori Type II Dehydroquinase: Synthesis, Biological Evaluation, and NMR Studies

Several 3-heteroaryl analogs of the known dehydroquinase inhibitor (1R,4R,5R)-1,4,5-trihydroxy-2-cyclohexene-1-carboxylic acid (4) were synthesized and tested as inhibitors of Helicobacter pylori type II dehydroquinase, the third enzyme of the shikimic acid pathway. All of these compounds proved to be reversible competitive inhibitiors of this enzyme and proved to be, with the exception of nitrile 8 e, more potent than the parent inhibitor 4 (K(i)=370 microM). The 2-thienyl derivative 8 b was found to be the most potent inhibitor of the series and has a K(i) value of 540 nM, which is almost seven hundred times lower than that of the parent inhibitor. The 3-nitrothienyl derivative 8 d and 2-furanyl derivative 8 a also had a good affinity of 1 microM. The conformation of the potent competitive inhibitor 8 b, when bound in the active site of the H. pylori enzyme, was elucidated by 1D-selective inversion NOE, Saturation Transfer Difference (STD) and transferred NOESY NMR experiments. One of the conformations that exists in solution for the potent competitive inhibitor 2-thienyl derivative 8 b is selected when it is bound to the active site of the enzyme. In the bound conformation derivative 8 b has the sulfur atom of its thienyl group oriented towards the double bond of the cyclohexene moiety. The large STD effects observed for the aromatic protons of 8 b show that it is the thiophene side of the ligand that makes closest contact with enzyme protons. Docking studies using GOLD3.0.1 suggest that the conformation determined by NMR allows strong lipophilic interactions with the enzyme residues Pro9, Asn10, Ile11, Gly78 and Ala 79. Competitive STD experiments carried out with high-, medium- and low-affinity ligands 8 b, 5 d and 5 f show that they all bind in the same site of Helicobacter pylori dehydroquinase.

Two-State Allosteric Modeling Suggests Protein Equilibrium as an Integral Component for Cyclic AMP (cAMP) Specificity in the cAMP Receptor Protein ofEscherichia coli

Activation of the cAMP receptor protein (CRP) from Escherichia coli is highly specific to its allosteric ligand, cAMP. Ligands such as adenosine and cGMP, which are structurally similar to cAMP, fail to activate wild-type CRP. However, several cAMP-independent CRP variants (termed CRP*) exist that can be further activated by both adenosine and cGMP, as well as by cAMP. This has remained a puzzle because the substitutions in many of these CRP* variants lie far from the cAMP-binding pocket (>10 A) and therefore should not directly affect that pocket. Here we show a surprising similarity in the altered ligand specificity of four CRP* variants with a single substitution in D53S, G141K, A144T, or L148K, and we propose a common basis for this phenomenon. The increased active protein population caused by an equilibrium shift in these variants is hypothesized to preferentially stabilize ligand binding. This explanation is completely consistent with the cAMP specificity in the activation of wild-type CRP. The model also predicts that wild-type CRP should be activated even by the lower-affinity ligand, adenosine, which we experimentally confirmed. The study demonstrates that protein equilibrium is an integral factor for ligand specificity in an allosteric protein, in addition to the direct effects of ligand pocket residues.

Flow cytometry measurement of the labile iron pool in human hematopoietic cells

Iron is important for many biological processes, and its deficiency or excess is involved in pathological conditions. Although most iron is firmly bound (e.g., in hemoglobin), some, the labile iron pool (LIP), is bound to low-affinity ligands. The level of LIP is regulated to meet the cell's requirements for iron but prevent excess. We describe herein a multiparameter flow cytometry procedure for measuring LIP in various human hematopoietic cells. Peripheral blood and bone marrow (BM) cells were loaded with calcein-AM, washed, and then incubated with or without the high-affinity iron-chelator Deferiprone (L1). Specific cell subpopulations were identified based on side-light scattering and expression of surface antigens. LIP was determined based on the ability of L1 to bind and remove iron from calcein and thereby increase the fluorescence emitted by the cells. Blood cells differ in their LIP content in the order monocytes > PMN > RBC > lymphocytes. Analysis of BM cells indicated a similar tendency among precursors of the different lineages. The results also showed that among myeloid precursors, LIP increases along cell maturation. Flow cytometry might be useful for evaluating LIP in various diseases and for studying the efficacy of iron-chelators.

Low Affinity Interaction with CLL B-Cell Receptors Induces Apoptosis by Down-Regulating Mcl-1 and Up-Regulating BimEL.

The B-cell receptor (BCR) engages antigens and triggers divergent signaling pathways, inducing survival, proliferation, and differentiation or anergy and apoptosis. The decision as to which pathway is activated and which functional outcome ensues is regulated by a series of receptor and cellular interactions. In particular, signal transduction via the BCR is influenced by the affinity of receptor-ligand binding and by the number of antigen-binding sites engaged. To address the role of BCR-antigen-binding affinity and valency in CLL, we used a series of anti-μ chain mAbs that differ in their affinities for the ligand as surrogates of antigen binding to the BCR (HB57: Ka = 5x108, “high”; Mu53: Ka = 2x107, “intermediate”‘ and P24: Ka = ∼2x106 M−1, “low”). We also mimicked the valency of the surrogate antigens (and hence the number of binding sites occupied) by preparing these mAbs in either their native dimeric state or in multimeric forms by conjugating them to dextran and to DYNA beads M-450. The native mAbs and the mAbs bound to dextran are soluble; the mAbs bound to beads are insoluble. The functional effects on CLL B cells of each mAb, in all three forms (i.e., soluble dimeric, soluble multimeric on dextran, and insoluble multimeric on beads), were then assessed in vitro. We noted striking and consistent differences in CLL cell responses when the mAb of different affinities for the BCR were used in the soluble, native dimeric state. Whereas the higher affinity anti-μ mAbs (HB57 and Mu53) induced minimal to modest proliferation, the low affinity mAb P24 drastically reduced proliferation. Similarly the soluble, dimeric forms of the same three mAbs had striking and consistently different effects on CLL cell survival. In particular, the low affinity mAb induced significant apoptosis as measured by Annexin V/propidium iodide immunofluorescent analyses (mean 61.2%; range 32–99.2%) as well as by PARP cleavage. Western blotting studies of cell lysates from CLL cells stimulated with the low affinity mAb vs. the higher affinity mAbs or isotype matched control mAbs revealed downregulation of the anti-apoptotic protein Mcl-1 and upregulation of the pro-apoptotic protein BimEL. The multimeric forms of the three anti-μ mAbs, both soluble and insoluble, increased CLL cell survival and proliferation, irrespective of affinities, by maintaining Mcl-1 levels and failing to upregulate Bim. This study illustrates the importance of the “quality” of BCR-antigen interaction for CLL cell survival and growth. Soluble low affinity interactions with antigens of reduced valency lead to CLL cell death, whereas soluble low affinity interactions with multivalent antigens lead to cell survival and growth. The use of soluble, monomeric, low affinity ligands reactive with shared stereotypic BCRs, therefore, should be considered as an immunotherapeutic approach in CLL.