Ki For Binding Research Articles

Offsetting Low-Affinity Carbohydrate Binding with Covalency to Engage Sugar-Specific Proteins for Tumor-Immune Proximity Induction.

Carbohydrate-binding receptors are often used by the innate immune system to potentiate inflammation, target endocytosis/destruction, and adaptive immunity (e.g., CD206, DC-SIGN, MBL, and anticarbohydrate antibodies). To access this class of receptors for cancer immunotherapy, a growing repertoire of bifunctional proximity-inducing therapeutics use high-avidity multivalent carbohydrate binding domains to offset the intrinsically low affinity associated with monomeric carbohydrate-protein binding interactions (Kd ≈ 10-3-10-6 M). For applications aimed at recruiting anticarbohydrate antibodies to tumor cells, large synthetic scaffolds are used that contain both a tumor-binding domain (TBD) and a multivalent antibody-binding domain (ABD) comprising multiple l-rhamnose monosaccharides. This allows for stable bridging between tumor cells and antibodies, which activates tumoricidal immune function. Problematically, such multivalent macromolecules can face limitations including synthetic and/or structural complexity and the potential for off-target immune engagement. We envisioned that small bifunctional "proximity-inducing" molecules containing a low-affinity monovalent ABD could efficiently engage carbohydrate-binding receptors for tumor-immune proximity by coupling weak binding with covalent engagement. Typical covalent drugs and electrophilic chimeras use high-affinity ligands to promote the fast covalent engagement of target proteins (i.e., large kinact/KI), driven by a favorably small KI for binding. We hypothesized the much less favorable KI associated with carbohydrate-protein binding interactions can be offset by a favorably large kinact for the covalent labeling step. In the current study, we test this hypothesis in the context of a model system that uses rhamnose-specific antibodies to induce tumor-immune proximity and tumoricidal function. We discovered that synthetic chimeric molecules capable of preorganizing an optimal electrophile (i.e., SuFEx vs activated ester) for protein engagement can rapidly covalently engage natural sources of antirhamnose antibody using only a single low-affinity rhamnose monosaccharide ABD. Strikingly, we observe chimeric molecules lacking an electrophile, which can only noncovalently bind the antibody, completely lack tumoricidal function. This is in stark contrast to previous work targeting small molecule hapten and peptide-specific antibodies. Our findings underscore the utility of covalency as a strategy to engage low-affinity carbohydrate-specific proteins for tumor-immune proximity induction.

Native CB1 receptor affinity, intrinsic activity and accumbens shell dopamine stimulant properties of third generation SPICE/K2 cannabinoids: BB-22, 5F-PB-22, 5F-AKB-48 and STS-135

In order to investigate the in vivo dopamine (DA) stimulant properties of selected 3rd generation Spice/K2 cannabinoids, BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, their in vitro affinity and agonist potency at native rat and mice CB1 receptors was studied. The compounds bind with high affinity to CB1 receptors in rat cerebral cortex homogenates and stimulate CB1-induced [35S]GTPγS binding with high potency and efficacy. BB-22 and 5F-PB-22 showed the lowest Ki of binding to CB1 receptors (0.11 and 0.13 nM), i.e., 30 and 26 times lower respectively than that of JWH-018 (3.38 nM), and a potency (EC50, 2.9 and 3.7 nM, respectively) and efficacy (Emax, 217% and 203%, respectively) as CB1 agonists higher than JWH-018 (EC50, 20.2 nM; Emax, 163%). 5F-AKB-48 and STS-135 had higher Ki for CB1 binding, higher EC50 and lower Emax as CB1 agonists than BB-22 and 5F-PB-22 but still comparatively more favourable than JWH-018.The agonist properties of all the compounds were abolished or drastically reduced by the CB1 antagonist/inverse agonist AM251 (0.1 μM). No activation of G-protein was observed in CB1-KO mice. BB-22 (0.003–0.01 mg/kg i.v.) increased dialysate DA in the accumbens shell but not in the core or in the medial prefrontal cortex, with a bell shaped dose–response curve and an effect at 0.01 mg/kg and a biphasic time-course. Systemic AM251 (1.0 mg/kg i.p.) completely prevented the stimulant effect of BB-22 on dialysate DA in the NAc shell. All the other compounds increased dialysate DA in the NAc shell at doses consistent with their in vitro affinity for CB1 receptors (5F-PB-22, 0.01 mg/kg; 5F-AKB-48, 0.1 mg/kg; STS-135, 0.15 mg/kg i.v.). 3rd generation cannabinoids can be even more potent and super-high CB1 receptor agonists compared to JWH-018. Future research will try to establish if these properties can explain the high toxicity and lethality associated with these compounds.

Hirudin Binding Reveals Key Determinants of Thrombin Allostery

Thrombin exists in two allosteric forms, slow (S) and fast (F), that recognize natural substrates and inhibitors with significantly different affinities. Because under physiologic conditions the two forms are almost equally populated, investigation of thrombin function must address the contribution from the S and F forms and the molecular origin of their differential recognition of ligands. Using a panel of 79 Ala mutants, we have mapped for the first time the epitopes of thrombin recognizing a macromolecular ligand, hirudin, in the S and F forms. Hirudin binding is a relevant model for the interaction of thrombin with fibrinogen and PAR1 and is likewise influenced by the allosteric S-->F transition. The epitopes are nearly identical and encompass two hot spots, one in exosite I and the other in the Na+ site at the opposite end of the protein. The higher affinity of the F form is due to the preferential interaction of hirudin with Lys-36, Leu-65, Thr-74, and Arg-75 in exosite I; Gly-193 in the oxyanion hole; and Asp-221 and Asp-222 in the Na+ site. Remarkably, no correlation is found between the energetic and structural involvements of thrombin residues in hirudin recognition, which invites caution in the analysis of protein-protein interactions in general.

Trypanosome Cyclic Nucleotide Phosphodiesterase 2B Binds cAMP through Its GAF-A Domain

Trypanosoma brucei, the causative agent of sleeping sickness in humans and livestock, expresses at least three cAMP-specific class I phosphodiesterases (PDEs), all of which are essential for survival of the parasite. These PDEs have either one or two N-terminal GAF domains, which in other proteins function as signaling domains. However, neither the functional roles nor ligands for these domains in trypanosome PDEs are known. The present study shows that TbPDE2B, which contains two tandem GAF domains, binds cAMP with high affinity through its GAF-A domain. A purified recombinant N terminus + GAF-A domain binds cAMP with an affinity (Ki) of approximately 16 nM. It also binds cGMP but with a 15-fold lower affinity of approximately 275 nM. The TbPDE2B holoenzyme has a somewhat lower affinity (approximately 55 nM) for cAMP but a greatly lower affinity (approximately 10 microM) for cGMP. This suggests that both the selectivity and affinity for a ligand can be determined not only by the nature of the binding domain but also by the adjacent domains. Additionally, binding of cAMP to the holoenzyme showed positive cooperativity, with a Hill coefficient value of 1.75. However, binding of cGMP to the holoenzyme did not show any cooperativity, suggesting differences in the conformational changes caused by binding of these two cyclic nucleotides with the protein. Point mutation of a key predicted binding site residue (T317A) resulted in a complete loss of high affinity cAMP binding. This mutation increased the apparent Km of the mutant enzyme for substrate without altering the Vmax. A truncated catalytic domain construct of TbPDE2B also exhibited an increased Km, strongly suggesting that cAMP binding to the GAF-A domain can regulate TbPDE2B by allowing the full activity of the enzyme to be expressed. These properties of the GAF-A domain of TbPDE2B thus suggest that it could be a new target for anti-trypanosomal drugs.

Protease Domain Sodium, Calcium, and Zinc Sites in Factor VIIa: Crystal Structures and Kinetic Studies.

During physiologic coagulation, the factor VIIa (FVIIa)/tissue factor (TF) complex activates FIX and FX. FVIIa consists of a N-terminal γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like (EGF) domains, and a C-terminal serine protease domain. We obtained crystals of FVIIa/soluble TF in the presence of Na+, Rb+, or Choline+ (Ch+) under conditions containing micromolar concentrations of Zn2+. Rb+ is a large monovalent ion and has been used to identify Na+-sites in several proteins; whereas, Ch+ cannot substitute for Na+. The various crystals diffracted from 2.0 to 2.4 Å and belonged to the space group P212121. In the crystal structures, Na+ or Rb+ in FVIIa coordinates to the carbonyl groups of residues 185 (chymotrypsin numbering), 185A, 221, and 224 as well as to two water molecules. Thus, the Na+-site in FVIIa is similar to that of FXa and activated protein C but not to that of thrombin. Ca2+ in the protease domain of FVIIa is coordinated to the carboxylates of Glu70 and Glu80 as seen earlier by Banner and coworkers. Additionally, the crystal structures also showed two Zn2+-sites, one involving His71 and the other involving His117. The Zn2+-sites are unique to FVIIa since the His residues are not present in other proteases. To investigate the role of Na+, Ca2+, and Zn2+-sites in the protease domain of FVIIa, a series of biochemical and kinetic studies were performed. Na+ increased the kcat for hydrolysis of S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) ~22-fold by FVIIaWT whereas Ca2+ increased it ~by 230-fold. In the presence of Ca2+, Na+ had virtually no effect on the hydrolysis of S-2288; however, in the presence of Na+, Ca2+ increased the kcat ~12-fold. Thus, the increase in kcat by Ca2+ in the presence or absence of Na+ was similar (~250-fold). Further, Na+ had no effect on Km whereas Ca2+ increased it ~3.5-fold. However, the increase by in Km is biologically not pertinent since the Gla and EGF1 domains of FVIIa determine the Km for activation of FIX and FX. Moreover, FVIIaF225P (Na+-site mutant) showed little response to Na+ and FVIIaE80V (Ca2+-site mutant) showed no response to Ca2+ in hydrolyzing S-2288. These data indicate that the Na+ and Ca2+ effects observed are due to the occupancy of the protease domain Na+ and Ca2+ sites. Consistent with the Km data, Na+ had no effect on the binding of p-aminobenzamidine (pAB, S1 site probe) to FVIIaWT. Interestingly, Ca2+ decreased the Ki for pAB binding by ~5-fold indicating that the increase in Km for S-2288 caused by Ca2+ is not related to the S1 site but rather to the S2 and/or S3/S4 sites in FVIIa. In further studies, Zn2+ inhibited the potentiation of S-2288 hydrolysis by FVIIaWT with Ki ~1 of μM in the absence and ~30 μM in the presence of Ca2+. We conclude that the Na+-site in FVIIa is not linked to the synthetic substrate binding site(s), and that the Ca2+-site is linked to the substrate binding site(s). These observations are in contrast to what has been previously observed for FXa and activated protein C. Thus, in the absence of TF, Na+ and Ca2+ are positive regulators for catalysis by FVIIa; whereas, Zn2+ exerts a negative effect. Conceivably, occupancy of the Na+-site and the protease domain Ca2+-site may render FVIIa in a conformation suitable for TF binding and substrate hydrolysis. The local Zn2+ concentration following release by activated platelets at the site of hemostasis could dampen coagulation as a regulatory mechanism.

Effects of high pressure on glucose transport in the human erythrocyte.

The effects of raised hydraulic pressure on D-glucose exit from human red cells at 25 degrees C were determined using light scattering measurements in a sealed pressurized spectrofluorimeter cuvette. The reduction in the rates of glucose exit with raised pressure provides an index of the activation volume, deltaV++ (delta ln k/deltaP)(T) = -deltaV++/RT. Raised pressure decreased the rate constant of glucose exit from 0.077 +/- 0.003 s(-1) to 0.050 +/- 0.002 s(-1) (n = 5, P < 0.003). The Ki for glucose binding to the external site was 2.7 +/- 0.4 mm (0.1 MPa) and was reduced to 1.45 +/- 0.15 mm (40 MPa), (P < 0.01, Student's t test). Maltose had a biphasic effect on deltaV++. At [maltose] <250 microM, deltaV++ of glucose exit increased above that with [maltose = 0 mM], at >1 mm maltose, deltaV++ was reduced below that with [maltose = 0 mM]. Pentobarbital (2 mM) decreased the deltaV++ of net glucose exit into glucose-free solution from 30 +/- 5 ml mol(-1) (control) to 2 +/- 0.5 ml mol(-1) (P < 0.01). Raised pressure had a negligible effect on L-sorbose exit. These findings suggest that stable hydrated and liganded forms of GLUT with lower affinity towards glucose permit higher glucose mobilities across the transporter and are modelled equally well with one-alternating or a two-fixed-site kinetic models.

The effect of temperature on tight metal binding by peat and soil derived solid humic acids

The brown biomaterials called humic acids (HA) in peats and soils retain water and bind metal cations and other solutes. Studies of the interactions of purified solid peat and soil-derived HA from different countries with metal cations in water probe HA microstructures and help to characterize the metal binding sites. Labile cations such as Caaq2+, Co aq2+, Cu aq2+, Fe aq3+, Mg aq2+ and Mn aq2+ tightly bind to solid HAs in sequential steps. The isotherms A vs. c are well fitted with the Langmuir model and plots of 1/A vs. 1/c are linear for each step. Here, A is mmol bound metal g–1 HA and c is the equilibrium cation concentration (M). This paper compares the stoichiometric site capacities vi and equilibrium constants Ki for tight binding of Ca aq2+, Co aq2+, Cu aq2+, Fe aq3+, Mg aq2+ and Mn aq2+ at different solid HA sites. Measurements at different temperatures give linearly correlated metal binding enthalpy and entropy changes, indicating that conformational changes and cation/HA hydration/dehydration are important factors in metal binding and release by solid HAs. Key words: Humic acids; metal binding; isotherms; thermodynamics

Primary spontaneous coronary artery dissections in atherosclerotic patients

Clonidine, clinically used in the treatment of hypertension, is a central α2-adrenergic agonist that reduces blood pressure and slows heart rate by reducing sympathetic stimulation. Considering the structural similarity between clonidine and hydrophobic heterocyclic nitric oxide synthase (NOS) inhibitors, the effect of clonidine on the nitric oxide (NO) pathway was investigated. This was verified by determination of NOS activity in vitro and by analysis of inducible Ca2+-independent NOS (NOS-II) mRNA expression and measurement of nitrite levels in rat C6 glioma cells, taken as a cellular model. Clonidine inactivated neuronal Ca2+-dependent NOS (NOS-I) competitively without affecting NOS-II and endothelial Ca2+-dependent NOS (NOS-III) activity. However, the value of Ki for clonidine binding to NOS-I depended on tetrahydrobiopterin (BH4) concentration, as reported for NOS inhibition by other nitrogen heterocyclic compounds. In particular, the value of Ki for clonidine binding to NOS-I increased (from [7.9 ± 0.4] × 10−5 M to [8.0 ± 0.4] × 10−3 M) as BH4 concentration was increased (between 3.0 × 10−7 M and 1.0 × 10−3 M), at pH 7.5 and 37.0°. In addition, clonidine (1.0 × 10−4 M) enhanced NOS-II mRNA expression in rat C6 glioma cells, as induced by Escherichia coli lipopolysaccharide (LPS) plus interferon-γ (IFN-γ). Finally, clonidine (1.0 × 10−4 M to 1.0 × 10−3 M) dose dependently increased the levels of LPS/IFN-γ-induced nitrites, the breakdown product of NO, in supernatants of rat C6 glioma cells. As reported for other NOS inhibitors, clonidine was also able to regulate NOS-I and NOS-II inversely.

Tight metal binding by humic acids and its role in biomineralization †

Analytical and thermodynamic data, EPR, FTIR, solution 1H and solid-state 13C cross polarization magic angle spinning NMR and solid-state extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) spectra have been recorded for purified humic acids (HAs) isolated from a German peat (GHA), an Irish peat (IHA), an unpolluted New Hampshire bog soil (NHA) and their tightly bound copper(II), iron(III) and manganese(II) forms. Brief water washing of partly or fully metal-loaded HAs leaves ‘tightly’ bound metal in the isolated freeze-dried solids. Most of this metal is removed by washing with 0.1 M HCl, indicating acidic HA functional groups as principal metal binding sites. The number of nearest-neighbour atoms co-ordinated to tightly bound CuII (four), FeIII (six, probably with distorted geometry) and MnII (six, undistorted) in solid GHA, IHA and NHA were determined by XANES and EXAFS spectroscopy with reference standards. Isotherms measured at 20.0 °C and pH 2.4–3.2 with [M]total = 0.18–25.8 mM for tight, reversible Cu2+(aq), Fe3+(aq), and Mn2+(aq) binding by solid IHA and NHA fit the Langmuir model and give the pH-independent stoichiometric site capacities νi and equilibrium constants Ki for metal binding at specific HA sites i = A, B and C. Tight binding sites A, B and C of IHA are occupied by CuII, sites A and B by FeIII and site A by MnII, while only identical metal binding site A in NHA is tight enough to resist metal removal by brief water washing. A new helical HA molecular model based on the empirical formula C36H30N2O15·xH2O visualizes metal binding and the likely roles of HAs in biomineralization. Site A is suggested to be carboxylate, mixed ligands probably constitute site B, and site C is tentatively assigned as the interior of the HA helix. Binding free energies and EPR evidence suggest that Cu2+(aq), Fe3+(aq) and Mn2+(aq) rapidly transfer between specific HA binding sites. This affects rates of metal release and transfer to minerals.

Glutamine 161 of Glut1 glucose transporter is critical for transport activity and exofacial ligand binding.

The possible role of 5 transmembrane amino acid residues in the function of the Glut1 glucose transporter was investigated by site-directed mutagenesis. The residues were chosen based on their containing hydroxyl or amide side chains capable of hydrogen bonding to glucose and their complete conservation in Glut1 through Glut5. Asn100, Gln161, Gln200, Tyr292, and Tyr293 were individually replaced by hydrophobic and/or polar residues, and the mutants were analyzed by expression in Xenopus oocytes. Substitution of leucine or asparagine for Gln161 reduced the relative transport activity of Glut1 by 50- and 10-fold, respectively, as measured by uptake of 2-deoxyglucose normalized to the plasma membrane content of the mutant transporters. Substitutions at the other residues had either a modest (approximately 2-fold) effect or no effect on the relative transport activity. The Ki for binding of the exofacial ligand 4,6-O-ethylidene-alpha-D-glucose to Glut1 was increased by 18-fold upon substitution of asparagine for Gln161. The Km for zero-trans influx of 2-deoxyglucose was not affected by the substitution of asparagine for Gln161, but the catalytic turnover was decreased by 7.5-fold. These data, combined with the high degree of conservation of Gln161 among the specific members of the 12-helix membrane transporter superfamily that transport hexoses, support the contention that Gln161 forms part of the exofacial substrate-binding site of these molecules. However, the decreased transport activity resulting from mutations at Gln161 is likely due to an additional effect on the rate of a conformational change involved in net substrate uptake. This constitutes the first evidence that a residue within the NH2 terminal half of Glut1 is critical for transport function.

Inactivation of Tetrahymena rRNA self-splicing byc/s-platin proceeds through dissociable complexes

The anti-cancer drug cis-diamminedichloroplatinum (II) (cis-DDP) reacted with Tetrahymena self-splicing rRNA ribozyme, causing loss of self-splicing activity and formation of a number of platinated RNA species. The formation of one distinct platinated product, migrating at an apparent size of 2400 nt, was closely associated with ribozyme inactivation. This platinated RNA was resistant to T1 ribonuclease digestion, suggesting the presence of inter-strand Pt cross-links. The reaction rate of cis-DDP with the ribozyme followed first order kinetics and showed a saturation effect with increasing cis-DDP concentration, characteristic of an affinity-label type of interaction rather than bimolecular collision. The apparent KI for binding of cis-DDP to the ribozyme was 62 microM. Ribozyme treated with urea was not inactivated by cis-DDP, indicating that the native structure of the RNA is required for reaction with cis-DDP. Mg++, which binds to the ribozyme and causes conformational changes in the molecule, protected the ribozyme from inactivation by cis-DDP and also prevented the formation of platinated RNA. These results suggest that binding of cis-DDP to sites formed by certain secondary or tertiary structural elements of the RNA enhance the rate and the specificity of reaction of the reagent with the ribozyme.

Immunoaffinity-purified DNA polymerase alpha displays novel properties.

The purification and characterization of a novel and more intact form of the DNA polymerase alpha-primase complex from calf thymus are described. The polymerase-primase was enriched 10,000-fold to apparent homogeneity by chromatography on phosphocellulose, heparin-Sepharose, and an immobilized anti-human DNA polymerase alpha monoclonal antibody [SJK287-38; Tanaka, S., Hu, S., Wang, T. S.-F., & Korn, D. (1982) J. Biol. Chem. 257, 8386-8390]. A quantitative elution from the antibody column was achieved by shifting the pH from neutrality to between 12.5 and 13. From 1 kg of calf thymus, the procedure yields 1-2 mg of polymerase-primase with a specific activity of 30,000-40,000 units/mg for the polymerase and 15,000-20,000 units/mg for the primase. The complex sediments at 9 S through a sucrose gradient and exhibits a Stokes radius of 6.0 nm, yielding a native molecular mass of 335,000. Denaturing gel electrophoresis of the complex gives bands of Mrs 180,000, 155,000, 148,000, 73,000, 59,000, and 48,000 with a relative abundance of the two smallest subunits. Primase activity was partially resolved from the complex by centrifugation through sucrose gradients. The primer-forming activity was found to be associated with the Mr 59,000 and 48,000 polypeptides. In contrast to conventional preparations, the immunopurified polymerase displays several features which show it is the most intact form of the enzyme known to date. The deoxynucleoside triphosphate Km values are all within the range of 0.6-0.9 microM. The Km for binding to a single RNA primer on M13 DNA is 3.5 nM; the Ki for nonspecific binding to unprimed DNA is 70 microM (nucleotide).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of serine proteinases by tetra-p-amidinophenoxy-neo-pentane: thermodynamic and molecular modeling study.

The inhibitory effect of the aromatic tetra-benzamidine derivative tetra-p-amidinophenoxy-neo-pentane (TAPP) on the catalytic properties of beta-trypsin (EC 3.4.21.4), alpha-thrombin (EC 3.4.21.5), factor Xa (EC 3.4.21.6), Lys77-plasmin (EC 3.4.21.7) and beta-kallikrein-B (EC 3.4.21.35) was investigated (between pH 2 and 8, I = 0.1 M; T = 37 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Over the whole pH range explored, TAPP and benzamidine show the same values of the dissociation inhibition constant (Ki) for beta-trypsin; at variance with the affinity of TAPP for alpha-thrombin, factor Xa, Lys77-plasmin and beta-kallikrein-B which is higher than that found for benzamidine association around neutrality, but tends to converge in the acidic pH limb. On lowering the pH from 5.5 to 3.0, values of Ki for TAPP binding to beta-trypsin as well as for benzamidine association to all the enzymes investigated decreased thus reflecting the pK-shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of Ki for TAPP binding to alpha-thrombin, factor Xa, Lys77-plasmin and beta-kallikrein-B may be described as depending on the pK-shift, upon inhibitor association, of two equivalent proton-binding amino acid residues. Considering the X-ray three-dimensional structures and the computer-generated molecular models of serine proteinases: TAPP and :benzamidine adducts, the observed binding behaviour of TAPP and benzamidine to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).

Distinct atrial natriuretic factor receptor sites on cultured bovine aortic smooth muscle and endothelial cells

Cultured bovine aortic smooth muscle and endothelial cells each display distinct specific binding sites for radiolabeled atrial natriuretic peptide (ANF). 125I-pro-rANF (103-126)I binding to both cell types is rapid, reversible and competitive. Scatchard plots of the binding data show Bmax values of 5.5 and 0.1 - 2.1 X 10(5) sites/cell and Kd values of 2.1 and 0.3 nM for smooth muscle and endothelial cells, respectively. In addition, ANF elevates levels of cGMP substantially in both cell types at concentrations of ANF close to its Kd and Ki for binding. Sodium nitroprusside, however, has essentially no effect on cGMP levels in either cell type. These results show that distinct functionally active receptor sites for ANF exist on both vascular smooth muscle and endothelial cells.